Dynamic Surface Tensiometry in Medicine

S T U D I E S IN I N T E R F A C E SCIENCE Dynamic Surface Tensiometry in Medicine STUDIES IN I N T E R F A C E SE...

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S T U D I E S IN I N T E R F A C E SCIENCE

Dynamic Surface Tensiometry in Medicine

STUDIES

IN I N T E R F A C E

SERIES D. M 6 b i u s

SCIENCE

EDITORS and R. M i l l e r

Vol. I Dynamics of Adsorption at Liquid Interfaces

Theory, Experiment, Application by S.S. Dukhin, G. Kretzschmar and R. Miller Vol. 2

An Introduction to Dynamics of Colloids by J.K.G. Dhont Vol. 3 Interfacial Tensiometry by A.I. Rusanov and V.A. Prokhorov Vol. 4 New Developments in Construction and Functions of Organic Thin Films edited by T. Kajiyama and M. Aizawa Vol. 5 Foam and Foam Films by D. Exerowa and P.M. Kruglyakov Vol. 6 Drops and Bubbles in Interfacial Research edited by D. M6bius and R. Miller Vol. 7 Proteins at Liquid Interfaces edited by D. M6bius and R. Miller Vol. 8

Dynamic Surface Tensiometry in Medicine by V.N. Kazakov, O.V. Sinyachenko, V.B. Fainerman, U. Pison and R. Miller

Dynamic Surface Tensiometry in Medicine VALERY N. K A Z A K O V and OLEG V. S I N Y A C H E N K O

Medical University, Donetsk, Ukraine VALENTIN B. FAINERMAN

Institute of Technical Ecology, Donetsk, Ukraine ULRICH PISON

Virchow Klinik, CharitY, Humboldt Universit~it, Berlin, Germany REINHARD MILLER

Max-Planck-lnstitut f~r Kolloid- und Grenzfi6chenforschung Berlin-Adlershof, Germany

2000 ELSEVIER Amsterdam

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2000 Elsevier Science B.V. All rights reserved.

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First edition 2000

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ISBN: 0 444 50411 7

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Preface The dynamic and equilibrium properties of thin layers between two fluids, e.g., adsorption, interface tension, viscosity and elasticity, are primarily determined by the composition of the respective interracial layers. This composition, in turn, can be essentially different from that characteristic for the bulk phases, if substances of special amphiphilic structure, so called surfactants, are present in one or both adjacent fluids. Human biological liquids contain numerous low- and high-molecular weight surfactants. The human organism contains

interfaces with enormous surfaces.

The physicochemical and

biochemical processes taking place at these interfaces are extremely important for the vital functions of the organism as a whole, and the interracial properties may reflect peculiarities of age and sex, health and disease. The present book is the first attempt to systematically present the results of dynamic and equilibrium surface tensions measurements of serum and urine samples that were obtained from healthy human of various sex and age (more than 150 volunteers), and to compare these results with measurements of biological liquids obtained from patients suffering from various diseases (more than 1300 patients) or withmeasurements of amniotic fluid obtained from women at various stages of pregnancy. The work of M. Polfinyi in 1911 is probably one of the earliest research of surface tension of human biological liquids (cerebrospinal liquid). O. Ktinzel in 1941 has published the first systematic surface tension studies of serum and r surface tension behaviour of blood, r expired r

liquid. Only recently, studies of and amniotic liquid, gastric juice, saliva,

air and other human biologic liquids have been published; the most recent

examples are Hrncir & Rosina (1997), Brydon et al. (1995), Efentakis & Dressman (1998), Joura et al. (1995), Fell & Mohammad (1995), Adamczyk et al. (1997), Boda et al. (1997) and Manalo et al. (1996). However, such studies are still incomprehensive, and the methods used are often not reliable enough. One branch of medical science, pulmonary medicine especially

vi neonatology, has systematically used interfacial tensiometry for studying pulmonary surfactant. In this particular area, significant progress was achieved in the treatment of diseases related to alterations of the lung surfactant system (Pulmonary Surfactant: From Molecular Biology to Clinical Practice 1992, Surfactant Therapy for Lung Disease 1995, Pison et al. 1996). We believe that, similar to the progress in pulmonary medicine attributed to surface chemical studies of lung surfactant, progress in other medical branches could be expected through studies of interfacial characteristics of other human biological liquids. Such studies, however, will be successful only by using standardised techniques of dynamic and equilibrium surface tension measurements. For several years the authors of this book have been engaged in studies aimed at the improvement of the maximum bubble pressure method (Fainerman & Miller 1998a). These studies resulted in the development of the computer controlled tensiometers MPT 1 and MPT 2, commercially manufactured by LAUDA (Lauda, Germany), since 1993 and widely used in various areas of science and technology. The device is capable of measuring dynamic surface tensions within a wide range of surface lifetime (1 ms to 100 s for the standard version of the MPT 2 tensiometer). Therefore the initial intention was to apply just this apparatus to studies of dynamic and equilibrium surfacetymsion of biological liquids. However, already at the primary stage of the investigations it became apparent that the properties of serum and other biological liquids are essentially different from those characteristic to solutions of lowmolecular weight surfactants, for which the tensiometer MPT 1/2 were initially developed. Therefore, to achieve a sufficient reproducibility of the results, additional studies of the effect produced by the geometrical shape and surface properties of the capillaries had become inevitable. The results demonstrated that the capillaries for measurements of biological liquids should be essentially different from the standard developed for surfactants. The main theoretical and experimental issues related to the maximum bubble pressure technique as applied to biological liquids are presented in Chapter 2. We believe that one of the most significant achievements in this respect is the possible modification of the commercially

vii available instrument MPT 2, so that physicians, either practical or engaged in fundamental researches, can obtain relevant data and compare them with the values presented in this book. Therefore, the tensiometric parameters of biologic liquids taken from both healthy persons and patients suffering from various diseases, and the correlations between tensiometric and other data, i.e. clinical or biochemical parameters reported here, can be easily used, verified and extended by other scientists. A single example may illustrate how important selecting the right method is for studying of biological liquids. In the studies performed by Hrncir & Rosina (1997), the drop volume method was used to measure the surface tension of healthy persons' blood. The number of volunteers (adult males and females) was similar to that employed in our studies as reported in Chapter 3. It should be noted here that the drop volume method, as simple as it is on a first glance, is in fact rather complicated. This is due to the static and dynamic corrections to be used, and the fact that the viscosity of the liquid and the adhesive properties and geometric shape of the capillaries significantly affect the results (Miller & Fainerman 1998). For this method, only modern automated devices, e.g., TVT 1 (also manufactured by LAUDA, Germany) are capable of producing results, which can be compared with data obtained by other methods. Moreover, the application of this method for studies of surface tension of protein solutions (note that proteins are the main surface active constituents of blood) shows that in addition to significant scattering the results differ essentially from data measured by the Wilhelmy plate method (Tornberg & Lundh 1981). The average surface tension measured by Hrncir & Rosina (1997) for all samples of blood was 56 mN/m, which is comparable to the equilibrium surface tension of serum reported in Chapter 3. However, the standard deviation of data obtained in the drop volume study by Hrncir & Rosina (1997) was 19 raN/m, whereas the standard deviation of data obtained by the maximum bubble pressure method in Chapter 3 was 4 mN/m. This is significantly better. A more detailed discussion of the differences of the various methods in use for measuring dynamic surface tension of biological fluids will be provided in Chapter 3.

viii An important distinctive feature of the present experimental studies is that complete dynamic tensiograms of biological liquids have been measured. This makes the tensiometric studies more comprehensive and informative. Here again a single example will be given. In the study performed by Brydon et al. (1995) the equilibrium surface tension of cerebrospinal liquid was measured using the Wilhelmy plate method. Similarly to a number of earlier studies (Ktinzel 1936, 1941), the equilibrium surface tension values were found in the interval 59-64 mN/m. These results agree perfectly well with our data for equilibrium surface tension in the control group of patients

(60.4mN/m,

see Chapter7).

Moreover,

with

increasing protein

concentration, which is observed during some diseases, our studies support a trend revealed by earlier investigations that equilibrium surface tension decreases. While the Wilhelmy plate method provides only a single value for each liquid studied at a time, the dynamic tensiometry gives equilibrium surface tension, the shape of the tensiogram, characteristic slopes in particular intervals, and surface tension values at definite surface lifetimes. These dynamic characteristics are much more informative and exhibit better correlation with the pathology studied and the composition of the liquid, than an equilibrium surface tension value alone does. In addition to the measurement techniques, a correct interpretation and analysis of the tensiometric data obtained is extremely important. The kinetic theory of adsorption from solutions, and the theory of equilibrium adsorption layers of surfactant/protein mixtures (Miller et al. 1994, Dukhin et al. 1995, Fainerman & Miller 1998b) provide the basis for both the choice of the most characteristic parameters of tensiograms and the analysis of the results. Some theoretical models describing the adsorption of proteins are presented in Chapter 1. Chapters 4 to 8 will summarise dynamic surface tension data measured in biological samples that were obtained from patients with various diseases. Chapter 4 will give data from patients with kidney disease, chapter 5 from patients with rheumatic diseases, chapter 6 with pulmonary diseases, chapter 7 with diseases of the central nervous system, and chapter 8 with neoplasms. The authors of this book are indebted to a number of colleagues from the Donetsk Medical University (Donetsk, Ukraine), the Institute of Colloid Chemistry and Chemistry of Water

ix (Ukrainian National Academy of Sciences, Kiev, Ukraine), the Institute of Technical Ecology (Donetsk, Ukraine), the Max-Planck-Institut ftir Kolloid- und Grenzfl~ichenforschung (Golm, Germany), the University of Antwerp (Belgium), and LAUDA Dr. R. Wobser GmbH & CO. KG. (Lauda-KOnigshofen, Germany). We express our thanks to everyone who contributed to the development of experimental methods for performing these studies, the evaluation of theoretical models for discussing the experiments, and finally the preparation of the manuscript. A great tribute is given to the late Professor Paul Joos, one of the most distinguished scientists in the area of physical chemistry of surface phenomena. Professor Joos gave active support at the very beginning of our studies and he was among the authors of our first joint publication devoted to the tensiometry of human biological liquids (Kazakov et al.,

1995). The

comprehensive theoretical analysis of the hydrodynamic problems related to the maximum bubble pressure method, performed by Prof. S.S. Dukhin and Dr. V.I. Koval'chuk, showed us how to adjust the parameters of the MPT 1/2 tensiometers to modify this device for reliable measurements of biological liquids. We express our special thanks also to Ms. S.V. Lylyk for her technical assistance in performing the tensiometric studies. Invaluable help during the preparation of the manuscript was rendered by Dr. E.V. Aksenenko and D.V. Trukhin. The authors understand the deficiencies that are associated with the proposed publication. Sampling periods of biological liquids during the course of some of the diseases studied were quite narrow. In the discussion of revealed trends, we were unable sometimes to explain new results unambiguously in the framework of known mechanisms. Nevertheless, we hope that the readers will pay less attention to these drawbacks but rather keeping in mind the pioneering character of this book. The authors believe that dynamic interface tensiometry of human biological liquids is a fascinating new method and deserves a broad use for prospective studies of various diseases.

The authors

Donetsk/Berlin, 1999

References

Adamczyk, E., Amebrant, T., Glantz, P.O., Acta Odontologica Scandinavica, 55(1997)384. Boda, D., Eck, E., Boda, K., J. Perinat. Med., 25(1997)146. Brydon, H.L., Hayward, R., Harkness, W., Bayston, R., British J. Neurosurgery, 9(1995)645. Dukhin, S.S., Kretzschmar, G., Miller, R. Dynamic of Adsorption at Liquid Interfaces. Theory, Experiments, Application, in "Studies in Interface Science". Vol. 1, Elsevier, 1995 Efentakis, M., Dressman, J.B., European J. Drug Metabolism and Pharmacokinetics, 23(1998)97 Fainerman, V.B., Miller, R., In "Drops and Bubbles in Interfacial Research", in "Studies of Interface Science", D. MObius and R. Miller (Eds.), Vol. 6, Elsevier, Amsterdam, 1998a, p. 279-326 Fainerman, V.B., Miller, R., In "Proteins at Liquid Interfaces", in "Studies of Interface Science", D. M6bius and R. Miller (Eds.), Vol. 7, Elsevier, Amsterdam, 1998b, p. 51-102 Fell, J.T., Mohammad, H.A.H., International Journal of Pharmaceutics. 125(1995)327. Hmcir, E., Rosina, J., Physiological Research. 46(1997)319. Joura, E.A., Kainz, C., Joura, E.M., Bohm, R., Gruber, W., Gitsch, G., Zeitschrift ftir Geburtshilfe und Neonatologie. 199(1995)78. Kazakov, V.N., Fainerman, V.B., Sinyachenko, O.V., Miller, R., Joos, P., Lylyk, S.V., Ayko, A.E., Trukhin, D.V., Ermolayeva, M.N., Arch. Clin. Exp. Med. (Ukraine), 5(1995)3. Kazakov, V.N., Sinyachenko, O.V., Trukhin D.V., Pison, U., Colloids Surfaces A, 1998. Ktinzel, O., Deut. Zeitsch. Nervenheilkunde, 139(1936)265. Ktinzel, O., Ergeb. Inneren Med. Kinderheil, 60(1941)565. Manalo, E., Merritt, T.A., Kheiter, A., Amirkhanian, J., Cochrane, C., Pediatr. Res., 39(1996)947. Miller, R., Fainerman, V.B., In "Drops and Bubbles in Interfacial Research", in "Studies of Interface Science", D. M6bius and R. Miller (Eds.), Vol. 6, Elsevier, Amsterdam, 1998, p. 139-186 Miller, R., Joos, P., Fainerman, V.B., Adv. Colloid Interface Sci., 49(1994)249. Pison, U., Herold, R., Schtirch, S., Colloid Surfaces A, 114(1996)165. Pol~inyi, M., Biochem. Zeitsch., 34(1911)205. Pulmonary Surfactant: From Molecular Biology to Clinical Practice, Eds. B. Robertson, L.M.G. Van Golde and J.J. Batenburg, Elsevier, Amsterdam, 1992. Surfactant Therapy for Lung Disease, Eds. B. Robertson and H.W. T~iusch, Marcel Dekker Inc., New York, 1995. Tomberg, E., Lundh, G., J. Colloid Interface Sci., 79(1981)76.

Contents

xi

Preface

Chapter 1 - Theory of protein adsorption and model experiments

1.1.

Thermodynamics of protein adsorption at the liquid/fluid interfaces

1.2.

Adsorption kinetics

16

1.3.

Experimental studies of model biological liquids

20

1.4.

Influence of additives

26

1.5.

Summary

36

1.6.

References

37

Chapter 2 - Experimental technique and analysis of tensiograms

41

2.1.

Experimental methods

41

2.2.

The design of maximum bubble pressure tensiometer

43

2.3.

Theory of the maximum bubble pressure method

45

2.4.

Experimental technique

55

2.5.

Analysis of tensiograms

59

2.6.

Summary

64

2.7.

References

64

Chapter 3 - Dynamic interfacial tensiometry of biological liquids for healthy

68

persons

3.1.

Dependence of dynamic surface tension on sex and age of patients

68

3.2.

Dynamic surface tension of serum and amniotic liquid for pregnant women

81

3.3.

Summary

96

3.4.

References

96

Chapter 4 - Application of Surface Tensiometry in Nephrology

99

4.1.

Glomerulonephrites

100

4.1.1

Variation in surface tensiometric parameters for various forms of the disease

100

xii 4.1.2. Influence of particular serum and urine components on dynamic surface

118

tension 4.1.3. Effect of treatment on variations in surface tensiometric parameters

142

4.2.

Primary pyelonephritis and urolithiasis

152

4.3.

Diabetic nephropathy

163

4.4.

Other renal diseases

175

4.5.

Summary

183

4.6.

References

183

Chapter 5 - Surface tensiometry in rheumatology

191

5.1.

Pathogenesis of rheumatic diseases

191

5.2.

Systemic lupus erythematosus

195

5.3.

Rheumatism

207

5.4.

Sclerodermia systematica

215

5.5.

Rheumatoid arthritis

216

5.6.

Reiter's disease

228

5.7.

Psoriasis

229

5.8.

Gout

231

5.9.

Osteoarthrosis

236

5.10.

Effect of glucocorticoid therapy and plasmapheresis

237

5.11.

Summary

241

5.12.

References

241

Chapter 6 - Surface tensiometry in pulmonology

245

6.1.

Pathogenesis of respiratory diseases

245

6.2.

Bronchitis

258

6.3.

Bronchial asthma and other pulmonary diseases

264

6.4.

Dust pathology of respiratory organs

269

6.5.

Summary

281

xiii

6.6.

References

Chapter 7 - Surface tensiometry in neurology

282 286

7.1.

Tensiogram parameters for diseases of the nervous system

286

7.2.

Influence of the nosological form of an infection disease

297

7.3.

Role of patients age and duration of a disease

298

7.4.

Correlation between surface tension parameters and amount of proteins and

303

other components 7.5.

Role of tensiometry in therapy, diagnosis and prognosis

313

7.6.

Summary

321

7.7.

References

322

Chapter 8 - Interfacial tensiometry in oncology

324

8.1.

Pathogenesis of oncological disease

324

8.2.

Serum tensiograms for different tumour localisations

328

8.3.

Correlation between surface tensions and biological liquid's composition

335

8.4.

Influence of ~,-therapy on dynamic surface tensions

347

8.5.

Effects of operative treatments

353

8.6.

Summary

355

8.7.

References

358

List Of symbols

359

Subject Index

365

0

10.

This Page Intentionally Left Blank

Chapter 1

Theory of protein adsorption and model experiments In order to understand key parameters under discussion in this book, the dynamic surface tension characteristics of biological liquids, it is suitable to give a short introduction into the physical processes of adsorption of molecules like proteins and short-chain surface active molecules at liquid interfaces. This survey allows then to understand the role the dynamic surface tension characteristics can play in the analysis of correlations between these values related to the adsorption of all surface active component and medical findings related to particular diseases. The thermodynamics as well as the dynamics and mechanics of adsorption layers formed at liquid interfaces will be presented and discussed on the basis of up-to-date theoretical models.

1.1. Thermodynamics of protein adsorption at the liquid/fluid interfaces Human biologic liquids contain various surfactants capable of adsorbing at liquid interfaces and changing the surface (interfacial) tension. Adsorption processes involve proteins, phospholipids, and low molecular weight surfactants, which play a significant role in vital functions of the human organism, in respiratory processes and haematogenesis. The practical importance of the adsorption process of surfactants and polyelectrolytes, and in particular, proteins at liquid interfaces has stimulated the development of various theoretical models to describe the equilibrium and dynamic behaviour of this process. In most cases the adsorbed monolayers of surfactants, proteins and lipids exhibit non-ideal behaviour. To account for the non-ideality of surfactant monolayers in the equation of state and adsorption isotherm, the regular solution theory is generally used (Lucassen-Reynders 1966, 1972, 1982). Recently new theoretical models have been proposed considering actual physical phenomena within surfactant monolayers, in particular, the reorientation of adsorbed molecules (Fainerman et al. 1997), and the formation of dimers, trimers and larger clusters (Fainerman & Miller 1996a). The abundant surface active component in human blood is human serum albumin (HSA). Its concentration in the serum is 35 to 50 g/1. The properties of protein adsorption layers differ in a

2 number of aspects from those characteristic to surfactant monolayers. With protein adsorption surface denaturation takes place, leading to the unfolding of protein molecules within the surface layer, at least at low surface pressures. The partial molar surface area for proteins, in contrast to surfactants, is large and variable. This property, and also the large number of configurations possible for an adsorbed protein molecule, significantly exceeding that in the bulk, leads to an increased non-ideality in the surface layer entropy. This makes it impossible to apply the most simple models (Henry, Langmuir) for the description of protein adsorption layers. Various thermodynamic models for the protein adsorption at liquid interfaces were proposed. The interrelation between protein denaturation processes at the surface and the activity of the solvent (water) molecules was shown to exist by Ter-Minassian-Saraga (1981), while Joos (1975) had shown that the degree of surface denaturation decreases with increasing surface pressure. Lucassen-Reynders (1994) had analysed the effect of the size of mixed molecules on the entropy of protein surface layers. Joos & Serrien (1991) were the first to derive an equation for the adsorption of proteins possessing two modifications with different partial molar area. From this relation it follows that the surface pressure controls both the composition and the thickness of a protein surface layer. In particular, the part of molecules possessing the minimal surface area increases with increasing surface pressure FI. The concept proposed by Joos & Serrien (1991) was further developed for an arbitrary but discrete number of different configurations of protein molecules within the surface layer. Fainerman et al. (1996a) and Makievski et al. (1998) derived equations of state for the surface layer and isotherms of protein adsorption at liquid/fluid interface. These new relationships reflect the main feature of high molecular electrolytes possessing flexible chains: the capability of changing the partial molar surface area in response to a variation in surface pressure. The new equations describe the case of a non-ideal surface layer, that is, the non-ideality of enthalpy and entropy of mixing resulting from the differences in size of protein and solvent molecules. The effect of the electric charge of a protein molecule is considered, and contribute significantly to the surface pressure. The model of multiple discrete states of protein molecules within the surface layer was even generalised to the case of an infinite number of infinitesimal states (the continuum model). Recently the adsorption behaviour of concentrated protein solutions was considered (Fainerman & Miller 1998b).

Surface pressure and adsorption isotherms for proteins at a liquid/fluid interface can be derived from Butler's (1932) equation for the chemical potential ~t~ of ith state of a protein molecule within the surface layer: lt.t~ = bt~s + RTlnf.Sx~

--0"(1) i

(1.1)

and the corresponding equation for the chemical potential g~ within the solution bulk, g~ = l.ti~ + RTlnfi~x~

(1.2)

where g0s and g ~ are the standard chemical potentials, R is the gas constant, T is the absolute temperature, o is the surface tension, o3i are the partial molar areas, fi are the activity coefficient, x i =

N i /2N

i are the molar fractions, and

Ni are

the number of moles of the

i th

state. Here the superscripts 's' and 'a' refer to the surface (interface) and the bulk. For ideal bulk phases it follows from Eqs. (1.1) and (1.2) that

1 - I = - ~R| T [I I ( 1- ki_>~l0 i) +Inf~

(1.3)

0)0

Kic=(1 0i/n

(1.4)

where FI = o0- o is the surface pressure, c0 is the surface tension of the solvent (i = 0), 0 i = Fio3i , Fi are the adsorptions of component i, 00 = 1- ~-'~0i , ni = coi/o30, c is the total protein i>__l

bulk concentration. The coefficients K i = (x~/X~)xr__,0 for i_> 1 are the distribution coefficients of states at infinite dilution. It can be assumed that the value of o30 is close to the area of a water molecule, and therefore the adsorption of a protein molecule in the ith state leads to the desorption of ni = o3i/o30 water molecules. This is however only true when the adsorption layer comprises of water molecules, thus the adsorption layer is about 0.3 nm thick. Real adsorption layers of proteins are much thicker. Moreover, their thickness increases with the protein adsorption. Thus, from the

theoretical point of view, the procedure employed by Douillard et al. (1994), where the real thickness of the protein layer was taken into account, seems to be more reliable. In this case the portion of water molecules within the surface increases, while the number of desorbed water molecules per protein molecule becomes significantly larger than coiAo0. Fainerman et al. (1996a) assumed that COo- co~, coincides with the choice of the dividing surface defined by Lucassen-Reynders (1966, 1972, 1982)

2.r~ - 1/~z

(1.5)

i=0

Here ma is the mean partial molar area of all states

This choice of the dividing surface ensures that for each state the relation 0i = Fizz holds. As mr is the same for all states, Fimr is the surface molar fraction of the respective adsorption state. Therefore the transformation of Eqs.(1.1) and (1.2) into Eqs.(1.3) and (1.4) with the introduction of 0i instead of x~ is a rigorous procedure. Another important advantage which follows from the choice of the dividing surface according to Eq. (1.5) and mr~ according to Eq. (1.6) is the fact that there is no contribution due to the non-ideality of entropy of mixing to the solvent activity coefficient. Finally, using the Lucassen-Reynders' dividing surface one can exclude the adsorption layer thickness from further consideration so that the actual number of water molecules displaced from the adsorption layer during the adsorption of protein molecules needs not to be accounted for. It is seen from Eq. (1.5) that for ~ F~ = 0 the Lucasseni=1

Reynders'

dividing

surface

is shifted towards the

solution bulk

by the

distance

zX= (m0/mr).dH2o as compared to the Gibbs' dividing surface for which F0=0. Here dH2o is the diameter of a water molecule. For a saturated monolayer however, these two areas coincide with one another. Note that for proteins (mr~~ m0) the value of A becomes negligibly small, and therefore for any adsorption the Lucassen-Reynders' dividing surface coincides with the Gibbs' dividing surface.

The activity coefficients in Eqs. (1.3) and (1.4) can be represented in a form which accounts for the enthalpy and entropy of mixing, denoted by superscripts H and E, respectively (Lucassen-Reynders 1994, Makievski et al. 1998), In fs = In fisH + In fisE, i > _0

(1.7)

lnfi sH = a(1 -Fxo~x) 2, i>_ 1

(1.8)

lnfi sE = 1-co~ ~~ Fj - 1 - ni,i >1

(1.9)

j>o

In f~H = aF~o~x, 2 2

(1.10)

lnf~ E = 1 - c o 0 ~ F j = 0 j__0

(1.11) n

Here a is the intermolecular interaction constant, and Fz = )--'~Fi . For simplicity it can be i=l

assumed that the non-ideality of enthalpy of the surface layer is independent of the state of molecules within the surface, and therefore depends only on the total adsorption. Proteins are polyelectrolytes, i.e., they contain ionised groups. At the isoelectric point both hydroxyl groups and amino groups possess equal degree of ionisation, and thus the whole molecule is electro-neutral. In strong acidic media the hydroxyl groups become neutral and the molecule acquires an excess of positive charges, while a neutralisation of the amino groups in strong alkaline media results in a negative net charge of the protein molecule. Therefore the maximal total charge of a protein molecule in acidic or alkaline media is equal to the number of amino or hydroxyl groups, while at the isoelectric point, i.e., at complete ionisation of hydroxyl and amino groups, the charge is equal to the total number of both groups. Thus the charges of a protein molecule is more or less bound by counterions. A polyelectrolyte molecule in a semidilute solution can be regarded as a random walk of electrostatic blobs (Dobrynin et al. 1995). The blob charge of a polyelectrolyte not botmd by counterions usually is in the amount of several units. It can be presumed that at the isoe~ectric point the charges of different blobs possess opposite signs. As the total number of blol~s is rather high, the entire protein molecule

6 appears electro-neutral. The counterion bounding both in separate blobs and in the whole protein molecule is about 90 %, which corresponds to ionic micelles. Thus, the number of unbound charges of a protein molecule remains sufficiently large, and counts to tens or hundreds. The interaction between unbound charges has to result in strong repulsion between polyelectrolyte chains as shown by Klein & Luckham (1982, 1984). Based on the Gouy-Chapman theory Davies (1951, 1958) had derived an adsorption isotherm and an equation of state for charged surfactant molecules using an electric double layer model. The same model (DEL) was used by Borwankar & Wasan (1988), but they took the nonideality of the surface layer into account. Combining the results of Davies and Borwankar & Wasan with Eqs. (1.7), (1.10), (1.11), (1.13), and using the condition co0=c0z, one can transform Eq. (1.3) into

FI = -

RT[In(1- Fzcoz) + a(Fzcoz)2]+ ~4[RT,2 g R T c z } ,'/2rtchq~-l] p

it) E

-

-

(1.12)

where F is the Faraday constant, e is the dielectric permittivity of the medium, cz is the total concentration of ions within the solution, q~= zFt~/2RT, z is the number of unbound unit charges in the protein molecule, and W is the electric potential. Substituting the chemical potential by the electrochemical potential, the following expression can be obtained instead of the adsorption isotherm (1.4)

Kc(

0ifis

1-

0i

exp(2q~)

(1.13)

(f~)n,

_

The electric potential is determined by the surface charge density zFzF sh~ = (8eRTcz)1/2

(1.14)

Analysis of Eq.(1.12) has shown that for a usual 1:1 ionic surfactant at low bulk ion concentration, the approximate relation tp )~ 1 is valid (Fainerman 1991). This approximation leads to a linear dependence of H on F z in the electrostatic term of Eq. (1.12). For protein solutions, however, the situation is quite different. At high ion concentrations the Debye length

ae = (sRT/F2cx) in is small; e.g., for c x = 0.1 mol/1 the value of ae = 1.3 nm. This means that for protein solutions the DEL thickness can be smaller than the adsorption layer thickness. Therefore the concentration of ions in Eqs. (1.12) and (1.14) is just their concentration within the adsorption layer, which can exceed 1 mol/1 due to the ionisation of hydroxyl and amino groups, and the contribution of counterions. It follows from Eqs. (1.12) and (1.14) that for large c x the approximation tp <<1 can be used. Thus, introducing the series expansions shq) = q~ + q)3/3 ! + ...

(1.15)

chq~ = 1 + q)2/2! + ...,

(1.16)

into Eqs. (1.12) and (1.16), one obtains an equation of state for non-ideal charged surface layers of a protein

.T[

1-I = - - - l n ( 1 -

Fx0)x) +

<11 )

0) 2

where a~l = z2F/cox(8sRTcx)1/2. Using expressions (1.7)-(1.9), (1.15), (1.16), and the relation exp(2q~) = (chq~ + shq))2

(1.18)

one obtains from Eq. (1.13) the protein adsorption isotherm for any i th state of the molecule

Fiox exp _ aFxo 2 x2 i -% -cox bzc =

1 - i - -o, 2aFx0) x + 2 __ ae~ Fxcox + ~ z i~(l_ Fxox)i~i/~

Fxox (1 19) "

where st is a constant which determines the variation in surface activity of the protein molecule in the ith state with respect to state 1 characterised by a minimum partial molar area col; b~ = bl i~ . Here i can be either integer or fractional and the increment of i is defined by Ai -- A0)/Ol. For st = 0 one obtains bi = bl = const, while for st > 0 the value of bi increases with increasing oi. Let us now evaluate the electrostatic constant ael: Assuming cox = 6.10 6 m2/mol (i.e., 10

nm 2

per

one protein molecule) and z = 30, for c x = 2 mol/1 one obtains ael = 100. In the Flory (1941,

1942)-Huggins' (1942)

theory of amorphous polymer solutions the constant a (otherwise called

the Flory parameter Z) is of the order of unity. Thus one can neglect this term in Eq. (1.17) as compared to ael. Moreover, for high ae:values the logarithmic term in Eq. (1.17) can also be neglected which leads to the approximation FI ~ F2 . This is in good agreement with the scaling analysis FI ~ F}9/4 (de Gennes 1987, Douillard et al. 1994). Taking into account the protein molecular charge the quadratic dependence of the osmotic pressure on z and the protein concentration can be obtained (Franks 1988). Considering the molecular charge of the protein leads to a significant simplification of the adsorption isotherm of Eq. (1.19). First of all, for all states of the protein molecule the parameter bl is constant and i = 1 can be assumed. Moreover, COl -< COY:and a = Z and a d z has values of the same order but are of opposite signs. It will be

shown below that for large ael the surface layer coverage Fzcoz remains low even for large H. In conclusion, the exponential term in Eq. (1.19) is close to unity, and does not depend on Fzcoz and thus can be omitted. Simultaneously, the two insufficiently defined parameters z and a are excluded. These simplifications allow the transformation of the equation of state for protein surface layers and the adsorption isotherm into the following simple form

RT[ln(1

COy

blc =

l-'ycoz)aell-'2co 2]

[-'if.or.

(1.20)

(1.21)

The total adsorption in these equations can be expressed via the adsorption in state 1 Fz = Fl'~-"i'~ exp co~(i - 1) exp,_ (i - 1)I7~ I i=l CO~E---RT

(1.22)

The first exponential factor arises due to the non-ideality of entropy of mixing. This factor, and also i~ correspond to a relative increase in adsorption in the states with coi > COl" TO simplify Eq. (1.22) and subsequent expressions, this factor can be omitted by using a value of ~=0.5 which for the present protein systems compensates its effect. In addition, we consider that the use of the ratio i~=bi/bl as pre-exponential factor is more general. Finally, the value of mean partial molar area for all states, and the adsorption in any ith state can

be expressed respectively by ~" i(a+1) exp(_ iHc~ / i-1

--~J

( il_Io,/ ~--'~iaexp - - ~ )

O)E=(.O1

(1.23)

n

i=l

i~ex~ - ( i - ]1)HO1 RT

r~=v Z ~ ] ~-~i- ex~_ ( i - 1)l-IOl i=l RT

(1.24)

J

The adsorption model described above assumes different discrete states of protein molecules within the surface layer, and neighbouring states differ from one another by the molar area increment Ao. From the viewpoint of scaling analysis, (Ao) in has to be close to the size of an electrostatic blob. In the adsorption layers of proteins the flexibility of chains increases due to the high concentration of both protein and inorganic electrolyte (Dobrynin et al. 1995). This allows one to consider an infinitesimal change in the molar areas by do instead of discrete states. One of the advantages of this continuous state model is that Ao can be excluded from the equations. Note that an increment Ao = Ol also leads to the elimination of this parameter from the equations which then describe the behaviour of rather inflexible chains. To perform the transformation from the discrete to the continuum model one has to replace formally the summations in Eqs. (1.22)-(1.24) by an integration. For example, the sum in Eq. (1.22) and (1.24) transforms into

1 O)max --C01

~" ~ 4 O1 e

(01

FI(~176 do -

(1.25)

RT

In some cases simple analytical expressions can be obtained instead of the cumbersome Eqs. (1.22)-(1.24). For example, for a =0 and

(0max))0)1

(in

fact, ~max----)oo)

instead of

Eq. (1.23) one obtains

O)z --

(D1 1+

(1.26)

10 For I-l~0 the results obtained from Eq. (1.26) agree well with data calculated from Eq. (1.23). For the continuous state model instead of Eq. (1.22) at ot = 0 one obtains

F~: = H(comax _co,) 1 -

RT

'

(1.27)

To illustrate the theory for protein adsorption, and to compare the results with experimental data, numerical calculations according to Eqs. (1.20)-(1.24) have been performed (Fainerman & Miller 1998a, Makievski et al. 1998). The transition from the discrete to the continuum model was made by decreasing the increment Aco until the calculation results became independent o f Aco. As an example, the surface pressure of a protein solution (molecular mass M = 24000 g/mol, col = 2 n m 2, comax-----60 n m 2) as a function of the surface layer coverage 0 = F~.coz is shown in Fig. 1.1.

25t

/I

2O 15

!_ O0,00

e'*F ,- - - - - 0,05

) 0,10

--t 0,15

0,20

Fig. 1.1. Dependence of surface pressure H on the adsorption layer coverage 0, calculated for a protein solution with M = 24000,

O 1 = 2 nIl'l 2

and r

= 60 nm2, ot = 2 and a~ = 400; curve 1 calculated for n = 1,

curve 2 - for Ao = o~, curve 3 - continuum model (Ao < 0.02o0. For ACO= col the curve differs from that calculated for the continuum model. However, already for Aco = 0.1 col the two results are close to each other. For Aco = col Eq. (1.24) predicts that at H > 10 m N / m all states with i > 1 are r e m o v e d from the surface layer, and the curve H = H(0) coincides with that calculated for n = 1, that is, when only one adsorption state with coi = col

11 exists. The effect of the coefficient ct, which accounts for the adsorption activities of different states and the non-ideality of entropy of the surface layer, is not so strong. An increase of cz results only in an increase in surface pressure and adsorption at very low protein concentrations. This is the result of the high adsorption activity of the states possessing large COl. In this case, however, the sharp increase of Fx and 17 within a narrow concentration range, characteristic of proteins, disappears. It seems that the actual value of the coefficient (z should not exceed 1 (restricted to the case of non-ideality of entropy, which corresponds to ~ ~ 0.5). As the curves for cz = 0 and cz = 1 (see, Makievski et al. 1998) are hardly distinguishable from one another, one can assume for proteins cx = 0. This reduces the number of parameters involved in Eqs. (1.20) and (1.21). Thus the adsorption of proteins in the framework of the continuum model can be described by a set of four independent parameters: o31, fDmax, ael and bl. Note that in the framework of the discrete model for

CO1 =

Ao) the number of independent parameters is

also four. 35

--

302520-

1

r,=..n

15E

10-

///

50 0,00

r

0,10

0,20

0,30

0,,

Fig. 1.2. Dependence of surface pressure on the adsorption layer coverage for a protein solution (M = 24000, tol = 2 nm2, COmax = 60 n m 2 and tx = 2), ael-" 400 (curve 1) and ael = 100 (curve 2). Decreasing ael leads to sharper dependencies of Fz on blc, while in contrast the 17 dependence on Fz, becomes more pronounced with increasing ael. For constant surface pressure the increase of a~ leads to a decrease in the total adsorption because the interion repulsion becomes

12 stronger. The intermolecular repulsion of chains leads to a decrease of the surface layer coverage so that the layer remains less packed even at high surface pressure. The dependence shown in Fig. 1.2 indicates that for ae~ = 400 the adsorption layer coverage does not exceed 20%. The parameters COl and (0max also significantly effect the shape of the dependencies of Fz on blC and I7 on F~. With the increase in col, the dependencies of FI and F~ on blc become less steep. A similar effect is observed also for COmax. For a fixed I-I the adsorption increases with increasing COl. The values of o~l and (Omax are directly related to the molecular mass of the protein, its physico-chemical characteristics, and to the properties of the solvent. It can be argued that col/2 cannot be smaller than the dimension of the electrostatic blob or the correlation length (de Gennes 1987). The parameter (Omax is defined as the maximum area which a denatured protein molecule can occupy at the surface. The main feature of the theoretical model is the self-regulation of both the state of the adsorbed molecules and the adsorption layer thickness via the surface pressure. The theory is based on the concept first formulated by Joos and Serrien (1991) and differs essentially from the known thermodynamic, statistical and scaling models in the self-regulation mechanism. This mechanism is inherent in the Butler (1932) equation, from which all main equations are derived. Of course, the surface pressure cannot be regarded as the only self-regulation parameter, but for the solution/fluid interface this factor is possibly the main one. From Eq. (1.24) one can calculate the part of adsorbed molecules existing in state c0i. This part is expressed by the ratio Fi/F~, the distribution function Fi/F~ which gives the probability density of Fi as a function of the partial molar area O)i at a given FI (cf. Fig. 1.3). For very low FI (0.1 mN/m) all states are represented in the protein adsorption layer; however the part of molecules with maximum area c0i = (Omax--60 nrn 2 is higher than that for all other states (a = 2 was assumed). At I-I = 0.5 mN/m the maximum probability density is achieved for the molecules with o~i ~ 17 nm 2, while at FI = 1 mN/m a maximum is obtained at o3i ~ 10 nm 2. With a further increase in FI, the amount of molecules occupying a minimum area increases continuously. For I7 >_ 10 mN/m, only a small number of adsorbed molecules occupy an area exceeding (Oi "-O)min----2 nm 2. Therefore, the equilibrium adsorption layer is formed by almost

13 completely denatured proteins at low surface pressure, while for large surface pressure it is built by molecules in a natural state with a minimum surface area demand.

0,08 0,07 0,06 r

0,05 0,04 ._

0,03 0,02 0,01 0 0

10

20

30

40

50

60

70

2 C0 i , n m

Fig. 1.3. Dependence of the distribution function Fi/Fz on c0i for a protein solution (M = 24000, c01= 2 nm2, C0ma x -" 6 0

am2, (X- "

2

and ael = 600) at 1-I = 0.1 (1), 0.5 (2), 1 (3) and 5 mN/m (4).

The protein adsorption layer coverage remains very low for surface pressures FI around 20 to 30 mN/m if ael is sufficiently large (Fig. 1.2). The theoretical model of Eqs. (1.20)-(1.24) predicts a subsequent unrealistic sharp increase of surface pressure with weak increase of protein concentration, and a simultaneous slight increase of the adsorption. This contradicts experimental data which show that, starting from some protein bulk concentration, FI remains almost constant, while the adsorption continues to increase. This results in an increase in surface coverage which in turn leads to an almost complete saturation of the adsorption layer at high'protein concentration (Graham & Phillips, 1979b). Graham & Phillips had attributed these results to the formation of a second adsorption layer towards the solution bulk. Subsequently some attempts were made to apply this hypothesis to explain the fact that the surface pressure is independent of the adsorption in concentrated surface layers. A theoretical adsorption isotherm which agrees with the experimental data was derived by Guzman et al. (1986). Douillard & Lefebvre (1990) also employed the two-layer model of protein adsorption, which assumes that the composition of the first layer only affects

14 the surface pressure. It can be argued that a multilayer adsorption model is quite appropriate to describe protein adsorption at a solid surface. The self-consistent field theory developed by Leermakers et al. (1996) can be referred to as an example. For the water/air interface, however, at least for the globular HSA-type protein, statistical models do not indicate the possibility for the formation of a second layer, see e.g. Uraizee & Narsimhan (1991). The phenomenon discovered by Graham & Phillips was explained by Makievski et al. (1998) in the framework of monolayer adsorption of proteins, assuming that the inter-ion interaction parameter of the surface layer equation of state decreases with increasing adsorption, i.e., with increasing ionic concentration in the surface layer. Adsorption increase in the concentrated protein adsorption layer does not lead to an increase in the surface pressure. We believe that this effect is related to the formation of two-dimensional aggregates rather than a second layer, which, however, cannot be completely excluded. The results of Graham & Phillips (1997c), plotted as surface pressure FI versus area per adsorbed protein molecule (protein mass) A, show the characteristic behaviour of an insoluble monolayer which exhibits a transition region from a liquid-expanded to a liquid-condensed state, i.e., an inflection point and almost horizontal portion between the inflection point and the collapse point exist. It is therefore quite natural to explain the phenomenon by a 2D transition in the protein adsorption layer. Equations (1.20), (1.21) and (1.23) can be generalised for the case when a 2D aggregation of the proteins in the monolayer lakes place. To do so, we proceed first with a simplification of these equations, noting that for large surface pressures only state 1 exists as it follows from Eq. (1.24). Then, it follows from Eqs. (1.23) and (1.24) that F z = F 1 and coy = col. Theoretical models which assume aggregation in adsorbed and spread (insoluble) monolayers were proposed by Ruckenstein & Bhakta (1994), Israelachvili (1994), Ruckenstein & Li (1995), Fainerman et al. (1996b) and Fainerman & Miller (1996a). One can easily modify Eqs. (1.20) and (1.21) for monolayers comprised of monomers and aggregates by expressing the protein adsorption F z as the sum of the adsorption of monomers in state 1 (F 1) and of aggregates (mmers) Fz =

F m.

F 1+

Therefore, the adsorption of protein expressed in terms of kinetic entities is

F m.

On the other hand, the measured total adsorption recalculated in terms of

15 monomers is defined as Fs = F 1 + mF m . The equilibrium between aggregates and monomers in the surface layer can be described by the relation

F m = Fl ~ , ~ )

(1.28)

which has been derived by Fainerman & Miller (1996a) from the mass action law in the framework of a quasi-chemical aggregation model. Here F c is the critical adsorption of protein aggregation in the surface layer, that is, the value of adsorption at which the surface pressure attains the value 1-I~. For the isotherm plotted in I-I- A co-ordinates, this adsorption value corresponds to the inflection point. Therefore, for the total adsorption defined above, one can write: m-1

(1.29)

I-'; = r 1 + m F m = r 1 d- m F 1

(1.30)

For m ~ 1 (which seems to be the case) very simple relations follow for F 1, F m and cox, namely F 1 _---Fc, F m -- 0 and cox

= (o)11-";/1-"c ).

Note that mF m r 0. It is clear that

1-"m =

0 also for

I-" 1 < F c.

Therefore, for F x > F c the equation of state for the surface layer and the adsorption isotherm of the protein solution can be presented in the form"

l-I= -~,RT[F~ l n 0 - F: co, ) - a ~,co~F~ ]

(1.31)

bc =

(1.32)

Fzcol 1 - Fxco~

It is seen that the adsorption isotherm (1.32) predicts an increase in protein adsorption for F z > F c. For example, the values F c - 2.0-2.5 mg/m 2 found for HSA and 13-casein correspond to a monolayer coverage of Fcco1 =0.5-0.6. Therefore, the adsorption for the monolayer at

16 maximum coverage (provided that minimum area per protein molecule remains unchanged) counts towards 4-5 mg/m 2. Retaining only the leading term in the expansion of the logarithm in Eq. (1.31), one obtains H = const for Fr

> r c.

Retaining two terms in this expansion, one can

show that the difference between the surface pressure H at F z > Fr and that at Fr = F c (i.e., when H = Hc) can be expressed as

H=H

+

RTFco~ 1 2

~1 x - F + )

(1.33)

Noting that the area per protein molecule in the surface layer can be expressed as A = 1/F~, and using (1.31) one obtains

H=

__RT A

co~ _

- c~ A-~-ln 1 - ~ -

col ael

(1.34)

or approximately (again retaining two leading terms in the expansion of the logarithm)

H=Hr

2A~-

-1

(1.35)

where A c = 1/Ft.

1.2. Adsorption kinetics The equilibrium states of adsorbed protein molecules as described above may change under certain conditions.

In fact, an evolution of the equilibrium states occure if the adsorption

process is extremely slow. In addition, the reconstruction process of the molecular states within the surface will influence the adsorption kinetics of protein. The state of the protein molecule within the solution bulk depends on the structure of the molecule and properties of the solvent, such as pH value and ionic strength. It can be assumed generally that a certain set of molecular conformations in the bulk exist, which differ from one another in the coi values at the moment of initial contact with the surface. Therefore the total bulk concer,tration c of a protein is the sum of concentrations ci (c = Zci), which correspond to

17 various conformations of the molecules in the bulk. The equilibrium composition of the adsorption layer (Fi/Fz), the surface layer, is controlled by the surface pressure. In general, the composition of the surface layer does not coincide with that of the bulk phase; therefore the mi values in the surface layer will differ from the corresponding bulk values. This will lead to a reconformation of states within the adsorption layer. We can consider Fig. 1.3 as an example. Assume that the flow of protein molecules from the solution bulk is comprised mainly of the states possessing mi = 20 nm z. At I-I = 0.5 mN/m the most probable state for the equilibrium composition of the surface layer is also the one with mi = 20 nm 2. Therefore at 1-I - 0.5 mN/m the conformation of the adsorbed molecules within the surface layer will actually remain unchanged. However due to the subsequent increase of the adsorption and corresponding increase of surface pressure, both the relative and absolute number of the equilibrium states with ( O i - - 2 0 n m 2 will be continuously decreased. For example, at 1-I = 1 mN/m the most probable state will be the one possessing mi = 10 nm 2. Therefore both the molecules adsorbed earlier, and the new molecules with mi = 20 nm 2 which had just approached the surface, will undergo a reconformation within the surface layer: some portion of their segments will have to desorb. It is to be noted that for the initial state of the protein molecule within the surface layer a more realistic value of mi would be between.m1 and 2ml. This means that according to the model for small FI values all adsorbed molecules will undergo a denaturation within the surface layer. The reconformation of states of adsorbed molecules which initially possess, i.e., the i th state, can be represented schematically as: k; k;, ri_ 1 ~ r i ~ ri+ 1

(1.36)

where the superscript '+' or '-' at the kinetic rate constants k denote the forward or backward reaction, respectively. The mass balance equation for the ith state of the adsorbed molecules can be given in the form:

dr~ dt

- -Fi(k 7 + ki+,)+ Fi_,k + + Fi+,kT+, + I i

(1.37)

18 where Ii is the diffusion flux of the ith state molecules from the solution bulk. Therefore the variation rate of the adsorption for this ith state depends on its reconformation rate due to the decrease of O)i by Ao~, i.e. the rate for the closest conformations which differ from the considered one by Ao~, and the diffusion flux of this state from the solution bulk. For the description of the adsorption kinetics, the model of discrete molecular states within the surface layer seems to be more suitable. According to Fig. 1.3, the process of surface denaturation of proteins, that is, the increase of o~i with respect to the initial value, takes place for very low surface pressures. At low FI the process of protein adsorption seems to be controlled by diffusion (Miller 1991). The experimental data presented by Benjamins et al. (1978), Paulsson & Dejmek (1992), Ghosh & Bull (1963), Graham & Phillips (1979a), Kalischewski & SchOgerl (1979) and de Feijter et al. (1987) support the diffusion model for at least up to values of FI < 2 mN/m. From the results published by Ghosh & Bull (1963), Kalischewski & Schtigerl (1979) and de Feijter et al. (1987) it could be deduced that the time t* at which ~ begins to decrease, and the protein bulk concentration in the range from 0.001 to 0.05 g/l, are related to each other by the expression c2t*= const. The latter follows from the simplest diffusion kinetics equation valid for FI ~ 0 (Miller 1991):

FE(r~__,0) = 2

(1.38)

where D is the diffusion coefficient, and t is the time. It can thus be supposed that in low concentrated protein solutions the surface denaturation process has enough time to be completed, and therefore the composition of the adsorption layer at FI < 2 mN/m corresponds approximately to the equilibrium composition. Further reconformation processes of the states within the surface layer depends, according to Fig. 1.3, on the desorption of segments which were adsorbed previously. One can assume as a first approximation that only backward reactions in Eq. (1.36) affect the value of dFi/dt

dr~

dt-= Fi+lk~-+l-Fik ~-+ I i

(1.39)

19 Assuming that Fi = Fi~ AFi and Fi+1 = Fi+l~+ AFi+l, where the Fi values with superscript '0' refer to the equilibrium state (at equilibrium the relations dFi/dt = 0 and Ii = 0 hold), and for small deviations from equilibrium we obtain from Eq. (1.39) dAFi dt

_

A17"i+lk~-+l-Al-'ik~-+ I i

(1.40)

An important result of the theory of equilibrium adsorption of proteins is the fact that the kinetic constant of the backward reaction for any i th state can be expressed via the kinetic constant for any particular state, say, i = n. It follows from Eqs. (1.24) and (1.40) that ia ( (n-1)YIcol] k~- = - - k ~ e x n RT

(1.41)

The kinetic constants for the forward reactions can be expressed similarly. As the constants k~ and k~- are interrelated via the adsorption equilibrium constant bi, and all bi in turn are related to b~, it follows that to describe the adsorption kinetics in the framework of the proposed model, in addition to the equilibrium adsorption characteristics (col, COma• a and bl) only one extra kinetic constant, say, k~, and the coefficient of the bulk diffusion of protein D would be required. An important practical result follows immediately from Eq. (1.41). One can see from Fig. 1.3 that for FI > 5 mN/m the adsorption layer is comprised mainly of the states with col < 2oi. In this case the adsorption rate will be determined by the transition of F2 (with o2 = 2ol) into F1, that is, the molecules from the solution can occupy the place at the surface if some molecules being in state 2 would transform into state 1, therefore making room in the adsorption layer. Thus if the adsorption is controlled by the process I-'2 ~ Fl, then assuming FI-- Fz (which is true within a narrow FI range), from Eqs. (1.40) and (1.41) one obtains dYI p( YICOl/ d t = k~ ex - - ~ )

(1.42)

where k0 is a constant. This equation is the well-known MacRitchie relation (1977, 1989, 1991), derived from experimental data. For a number of proteins the COl value in Eq. (1.42)

20 varies in the range of 0.5 to 2.5 nm 2 (MacRitchie 1991), which agrees with the estimates of o)1 as the minimum surface area occupied by a protein molecule in the adsorption layer, or the increment of molar surface area Am for the chains possessing limited flexibility. It is clear that protein adsorption in high concentrated solvents differs significantly from protein adsorption in low concentrated solvents as described above. During protein adsorption in high concentrated solvents surface denaturation cannot be completed because the rate of any increase in 0~i is limited, and thus there is not enough room in the surface layer (W0stneck et al. 1996a). In contrast to low concentrated solvents where unfolding of the molecule within the surface is followed by a refolding process, almost no surface denaturation takes place in high concentrated solvents, and the composition of the dynamic surface layer is similar to the initial conformation distribution of the adsorbed molecules. This view explains why the shear elasticity and viscosity for 13-1actoglobulin adsorption layers formed at low protein concentrations were found to exceed those measured at larger concentrations, while the surface tension of the solutions remains constant (Kr~igel et al. 1995). One can expect that many unusual properties of the dynamic adsorption layers of proteins can be explained by protein molecule processing during the reconformation at the surface.

1.3. Experimental studies of model biological liquids The surface tension isotherms and dynamic surface tensions of HSA and bovine serum albumin (BSA) are essentially the same and were studied in a number of publications (Peters 1985, Graham & Philips 1979a, 1979b, 1979c, Lassen & Malmsten 1996, Serrien et al. 1992, Suttisprasit et al. 1992, Paulsson & Dejmek 1992, Dussaud et al. 1994, Turro et al. 1995, Hansen & Myrvold 1995, Boury et al. 1995, Miller et al. 1993 and Tripp et al. 1995). The dynamic surface tensions for other protein solutions (lysozyme, myoglobin, 13-1actoglobulin, 13-casein, ribonuclease, etc.) were investigated by Graham & Philips (1979b), Serrien et al. (1992), Paulsson & Dejmek (1992), Douillard et al. 1994), Xu & Damodaran (1994), Kr~igel et al. (1995), WOstneck et al. (1996b), Hermel & Miller (1995), MacRitche (1989) and Clark et al. (1995). Very low HSA or BSA concentrations (ca. 0.01 g/l) decrease the equilibrium surface tension at pH 7 to values of 50- 52 mN/m. Increasing the concentration of HSA or BSA from 0.01 to 10 g/1 decreases the equilibrium surface tension only by an additional 2 to 4 mN/m. It

21 has to be noted that for low-concentrated HSA solutions, the time required to attain the adsorption equilibrium is 20 to 30 hours, while for concentrated solutions this time is only a tens of a second or a few minutes . Most studies of dynamic surface tension with HSA solutions were done at concentrations of less than 1 g/l, while no systematic studies were carried out at concentrations reassembling blood (approximately 35 to 50 g/l).

73[] [] O DD

68-

9

~

9

63-

9 AA o

~ 9

[]

%

9 O0

AA

0(30

%

% c~ 9A OQ

58-

53 1

I

r

I

I

10

1 O0

1000

10000

I

100000

t, [s] Fig. 1.4. Dynamic surtace tension c tor HSA solutions at various concentrations: 2.10"8mol/l (m), 5.10.8 mol/1(+), 10 -7 mol/1(O), 5.10 -7 mol/1(A),10"6mol/l (D), 10.5 mol/l (o). To verify the equations of state and adsorption isotherms as derived above, experimental studies on both dynamic and equilibrium surface tensions for HSA solutions were performed using the ADSA method (Rotenberg et al. 1983, Cheng et al. 1990). The dynamic surface tensions for HSA solutions at various concentrations are shown in Fig. 1.4 (Makievski et al. 1998). It is seen that within the time range of up to 4 hours, equilibrium is achieved only with HSA concentrations > 10 -6 mol/1. This is in agreements with data presented by Gonzalez & MacRitchie (1970) who studied the BSA whose structure and properties are similar to those of HSA. To obtain estimates for the equilibrium surface tensions for less concentrated HSA solutions the curves cy = o(t) were extrapolated to t ~ oo. The dependencies of o as functions of t 1/2 are presented in Fig. 1.5.

22 For the mixed adsorption mechanism the derivative do/dt -1/2 is defined by the relationship obtained by Fainerman et al. (1994)

do dt -'/2 -

RTF~ c

n 4-D

)~ RW_____~

(1.43)

+ cJ3t1/~

where 13 is the adsorption rate constant. For 13 ~

oo which corresponds to the diffusion

adsorption mechanism, or for t ~ o o the second term in the right hand side of Eq. (1.43) vanishes, and therefore this expression transforms into the known relationship presented by Joos and Hansen (see Chapter 2). In fact, the curves presented in Fig. 1.5 possess a linear part in the large t region (which corresponds to small t ~/2 values), and therefore the point of intersection with the abscissa axis corresponds to the equilibrium surface tension. However, the experimental values of (do/dtl/2)t_,|

estimated from Fig. 1.5 are approximately 100 times

higher than those calculated from the Joos-Hansen formula.

" " 70

ee

9

AAA

;~ 60

**** DO

o

9

0

9

9

[]

[]

O

[]

55 |

50 ~0

,

,

~

0,01

0,02

0,03

t

~ 0,04

9 0,05

-I12 -I12 ,s

Fig. 1.5. Dynamic surface tensions g of HSA solutions as a function of tm for different bulk concentrations, c = 2.10.8 mol/l (11), 5.10"s mol/l (o), 9.10.8 mol/l (A), 2.10-7 mol/l (@), 10.6 mol/l (D). Thus in case of HSA the range within which a diffusion adsorption mechanism takes place, can be estimated from Eq. (2.19) as t ~/2 < 10 .4 S"1/2, that is, t > 108 s. In this t range a considerable decrease of the slope of o = o (t "1/2) is observed. However, this does not affect significantly the estimated position of the intersection point with the abscissa as this range is rather narrow. On

23 the other hand, if the second term in the right hand side of Eq. (1.43) contributes mainly to the derivative, the first term can be neglected, and therefore an extrapolation dcr/dt 1 is more justified. The data presented in Fig. 1.5 are replotted in Fig. 1.6 in ~ versus t l coordinates. One can see that also in this plot a linear part exists for t---~.

73

"jim

II

o

9

68

63

g-

[]

[]

58

53

--

0

1

0,001

[]

[]

1

0,002

T

0,003

Y

-

-

0,004

I

0,005

Fig. 1.6. Dynamic surface tensions of liSA solutions plotted as a function oft]: c = 2.10.8 mol/l (11), 5.10.8 mol/1 (A), 9.10.8 mol/1 (o),

2 . 1 0 -7 mol/l (O), 10 -6 mol/l (D).

The equilibrium surface tensions obtained from the two extrapolation procedures discussed above (t -1/2 and t -1 at t ~oo) were found to be close to one another, with differences generally smaller than 0.5 mN/m. In the following the average values between these two extrapolations were used. In Fig. 1.7 the experimental equilibrium surface pressure isotherm of HSA at pH 7 at the solution/air interface is plotted versus the initial HSA bulk concentration. It is to be noted that for HSA concentrations > 10 -7 mol/1 our data agree well with data for BSA presented by Gonzalez & MacRitchie (1970), Feijter et al. (1978), Ward & Regan (1980), ~'ornberg & Lundh (1981) and Graham & Phillips (1979b). In contrast, for HSA concentrations < 10 -7 mol/1 the values obtained from the ADSA experiments were

lower compared to the

results reported by Graham & Phillips (1979b). One likely explanation for this discrepancy is a potential decrease of the protein concentration within the drop due to the adsorption of protein molecules at the drop surface (Makievski et al. 1998).

24 The theoretical curves presented in Fig. 1.7 were calculated for the following set of parameters using Eqs. (1.20)-(1.24), (1.32) and (1.33)" o~l 03max - - 8 0

nm 2, Ao3 - -

~1,

ael--

-

(Omin -- 4 0

nm 2

(area per HSA molecule),

320, c~ - 0 and bl = 2.107 l/mol. These parameter values agree

remarkably well with the data presented by MacRitchie (1977, 1991), Murray & Nelson (1996) and Peters (1985). In particular, the minimum area per BSA (or HSA) molecule within the monolayer is in fact 40 to 50 nm 2. In spread BSA monolayers the increase in surface pressure becomes appreciable when the area per protein molecule decreases to about 150 nm 2, which corresponds to a monolayer coverage of approximately 20 %, see Fig. 1.2.

2520-

_t~

9

600

IO-L 5 -

1,00E- 10

1,00E-08

1,00E-06

1,00E-04

c [mol/l] Fig. 1.7. Experimental equilibrium surface pressure isotherm of HSA for pH 7 plotted as a function of the initial HSA concentration c, solid line - theoretical adsorption isotherm calculated from Eqs. (1.20)(1.24), (1.32) and (1.33). It has to be noted that for Aco - col a variation of

03max

in the range from 40 to 200 nm 2 does not

effect the theoretical dependencies of FI and Fz on c. For HSA at approximately 1 mg/m 2 and a total concentration of ions within the surface layer of 2 mol/1, with the total number of aminoacid groups in the HSA molecule equal to 585 (Peters 1985)the adsorption layer thickness is 4 rim.. Assuming the minimum free charge of an albumin molecule with z ~ 20 (Peters 1985), one can estimate the value of ael theoretically, see Eq. (1.17), which agrees with the value obtained from fitting the isotherm to the experimental data. The minimum area of an adsorbed HSA molecule corresponds to the three-domain molecular structure, with each domain being

25 comprised of 9 loops connected by sulfide bridges. At pH 7 the size of such molecule is 14 x 4 x 4 nm. This configuration is possibly independent of H and Fz, meaning that HSA molecules do not undergo denaturation at a liquid/gas interface. The FI - A isotherm for a BSA adsorption layer, as reproduced from Graham & Philips (1979c), is shown in Fig. 1.8. Theoretical

curves were calculated

from Eqs. (1.20)-(1.24)

for

A > A c = 0.5 m2/mg, and from Eq. (1.35) for A < A c. Again, the parameters of these equations were

O)max =

80 nm 2

(per protein molecule), (Omin =

At0 = 40

nm 2, (z = 0 and aei = 320. It is

obvious that the theoretical curves agree well with the experimental 1-I - A isotherm.

2520-

"''-..

lO

0,0

0,5

1,0

1,5

2,0

A [m2/mg]

Fig. 1.8. Dependence of surface pressure on the area per 1 mg of BSA in the surface layer. A - data from Graham & Phillips (1997c) for adsorbed layers, dotted line - data from Murray (1997) for spread layers, solid line - calculated for A > 0.5 mVmg from Eqs. (I.20)-(I.24), and for A < 0.5 mVmg from Eq. (1.35). Moreover, for the same parameter values and concentrations lower than the critical value, c < cc = 1.4.10 .3 g/1 (2.10 -8 mol/1), {cc is the bulk concentration at which the aggregation in the surface begins} the model corresponding to Eqs. (1.20)-(1.24) describes satisfactorily the experimental H - c and F z - c isotherms of BSA as reported by Graham & Philips (1979b). Figure 1.8 also shows the experimental isotherm for a spread monolayer of BSA (Murray, 1997). This dependence is seen to be rather similar in its details to that characteristic for the adsorption layer. There exists a kink point at H c = 18 mN/m, and an increasing portion for area

26 values lower than A c. Other conclusions can be drawn for the protein [3-casein. Graham & Phillips (1979b) have generated isotherms for surface pressure and adsorption independently using the Wilhelmy plate method in a Langmuir trough for measuring surface tensions and radioactivity and ellipsometry methods for measuring adsorption of aqueous solutions of i3-casein. They found that the two experimental isotherms correspond satisfactorily to the theoretical model when using the following parameters: et al. 1996). The value about

8min =

(-OI =mo,)=

f-Omax ---- 8 0 nlTl 2

6 n l I l 2,

O,)max =

80 nm 2, Ot = 0 and a~ = 100 (Fainerman

corresponds to a minimum adsorption layer thickness of

0.75 nm for a completely denatured [5-casein molecule. These results agree with

measurements of the mass of adsorbed protein from the concentration decrease inside a drop at the moment when the decrease of surface tension begins to occur (Miller et al. 1998a). The value o~l = 6

nm 2

corresponds to the maximum adsorption layer thickness of

8max --

6.7 nm that

agrees well with experimental data of Atkinson et al. (1995). It can be stressed that all three independent experimental data sets obtained by Graham & Phillips, i.e. Fx = Fx(c), FI = H(c) and 8 = 8(c), and also the corresponding derived dependencies, e.g., 1-I = H(Fx) or 8 = 8(Fx), agree satisfactorily with the multiple molecular state model for protein molecules at the surface for the same set of four main parameters of Eqs. (1.20)-(1.24).

1.4. Influence of additives

The addition of inorganic electrolytes, urea, simple carbohydrates, ionic or non-ionic lowmolecular surfactants, and variations in the pH of a solution, affect significantly both equilibrium and dynamic surface tensions. These additives, including the surface active molecules, influence not only the properties of the solution itself, but they mainly effect the properties and structure of HSA molecules, resulting in binding to or ionisation of amino acid groups, interaction within polypeptide chains, variation of the HSA molecular conformation in the bulk and in the surface layer. It has to be noted here that data concerning these effects are still scarce and often contradictory. It is known that the addition of inorganic ions (K +, Li +, Na +, 2+

2+

2+

3+

Ca , Mg , Fe , Fe , CI, F, HPO42-, PO43-, etc.), usually surface inactive substances, results in an increase of the surface tension. This increase of surface tension of biologic liquids due to

27 the increase in concentration of inorganic salts can be significant in the short time range, when the adsorption of proteins and other surfactants is relatively small or even negligible. In contrast, for medium and long surface lifetimes adsorption results in a decrease of surface tension. For example, the addition of 0.1 mol/1 NaC1 to a BSA solution decreases the surface tension in the medium and long time range (Kalischewski & Scht~gerl 1979). The effect of the solvent properties on dynamic surface tensions of BSA solution was illustrated by Paulsson & Dejmek (1992). When distilled water was replaced by synthetic milk ultrafiltrate (SMUF, pH 6.6, ionic strength 0.08), the surface tension of a BSA solution at a concentration of 0.1 g/1 after a surface lifetime of approximately 50 s had decreased to 72 mN/m for water and to 60 mN/m for the SMUF solution. It was shown by Fainerman & Miller (1996b) and Joos & Serrien (1989) that fructose (and, similarly, glucose and saccharose) promotes the structuring of water molecules, while urea destroys this structure. These effects influence significantly the adsorption activity of lowmolecular weight surfactants. For example, the addition of fructose leads to a decrease of the dynamic surface tension for both short and long surface lifetimes, while the addition of urea results in an increase of cr(t). On the other hand, the effect of these additives on dynamic surface tension of protein solutions is not restricted to the variation in the structure of the solvent. The addition of urea leads to a denaturation of BSA or HSA both in the bulk and at the surface, which results in a significant decrease of the surface tensions (Serrien et al. 1992). The pH of the solution influences the secondary structure of protein molecules (Peters 1985), and can directly affect its adsorption activity (Hermel & Miller 1995). The decrease of pH with respect to its initial value of 7.5 leads to an appreciable decrease in surface tension of HSA (Hansen & Myrvold 1995) and other proteins (W~stneck et al. 1996b). The relative increase of the pH values also results in an increase of adsorption activity for concentrated HSA solutions, but this effect is less pronounced than that corresponding to a similar decrease of the pH value. The effect of low molecular weight non-ionic surfactants (alcohols, acids, oxyethylated ethers, etc.), on dynamic surface tensions of HSA and other proteins depends on their concentration and adsorption activity. For example, the effect of ethanol on the conformation of HSA or BSA

28 in solution leads to a significant decrease in its adsorption activity (Dussaud et al. 1994); this effect, however, can be overcompensated by the adsorption of ethanol, and the surface tension can therefore decrease. Oxyethylated surfactants, for example TWEEN 20 which possess higher surface activity (Kr/igel et al. 1995), produce almost no effect on the surface tension of [3-1actoglobulin solution at short lifetimes, but decrease significantly the equilibrium surface tensions. However, no definite predictions can be made concerning the effect of ionic surfactants (like sodium alkyl sulphates) on dynamic surface tensions, because in this case the inter-ion interaction between surfactants and proteins can result in the formation of protein/surfactant complexes (Turro et al. 1995). For very small additions of ionic surfactants (100 times lower than the protein concentration) an increase in the surface tension of the mixture occurs, and for relatively high additions of the same surfactant the surface tension generally decreases, while in some concentration regions anomalous surface tension behaviour of the mixture was observed (Wiistneck et al. 1996b). For mixtures of surfactants such effects were predicted and observed by Fainerman & Miller (1997). The experimental and theoretical study of the adsorption behaviour of mixtures of the globular protein (HSA) and a non-ionic surfactant (decyl dimethyl phosphine oxide, CI0DMPO ) was carried out by Miller et al. (1998b). This particular system was chosen because it is a good model system for a theoretical analysis:

it can be described in the framework of known

theoretical models, and studies of the adsorption properties of such mixtures promote insight into the mechanisms goveming the variations of surface active characteristics of serum caused by various diseases. The adsorption of the proteins and their mixtures with surfactants was characterised by dynamic surface and interfacial tension measurements using the axisymmetric drop shape analysis (ADSA). The standard deviation of the ADSA method in these studies was 0.2-0.3 mN/m. The surface tension measurements of C10DMPO were performed using the tensiometers MPT1 (maximum bubble pressure method) and TEl (ring method), all manufactured by Lauda, Germany. The MPT1 device and measuring procedures are described in Chapter 2.

29

75 70 65

~ 6o ;~ 55 1 b

50 45 !

~

40-

T 0

~

5000

~

10000

15000

20000

t Is] Fig. 1.9. Dynamic surface tensions of HSA/C 10DMPO mixtures at various surfactant concentrations: 110 "9 (O), 110 -8 (A), 410 "s (A), 710 -8 (~3), 110 -7 ('r

210 -7 (*), 410 "7 (O), 710 "7 (11), 1.10 -6 (x)

mol/cm 3

75

ImVN

*

ii

65 xxX

55

Z

45

35

I' '

0

000

-

-

V'~.,IV

1

"

~

v

2

,..A-,

3

x X x

Oo

xX

9 00

0

v

4

5

t -I/2 [s -1/2] Fig.1.10. Dynamic surface tension of CIoDMPO solutions plotted as functions of t "~'z , vertically dotted line t = 100 s; concentrations: 110 "7 (O), 210 -7 (In), 5.10 -7 (A), 110 -6 (x), 210 "6 (0), 510 "6 (O) mol/cm 3

30 The dynamic surface tensions for HSA mixtures (concentration 10-7 mol/1) with C10DMPO for various surfactant concentrations are shown in Fig. 1.9. These have to be compared with the dynamic curves for pure HSA solutions (Figs. 1.5, 1.6), and pure C10DMPO solutions (Fig. 1.10), respectively. The dynamic curves for the surfactant are plotted in the coordinates c~ versus t l/z. The theory predicts that in these coordinates a linear dependence should exist at t > 100. This is clearly supported by experimental data. The intersection of the linear portion of the curves with the ordinate corresponds to the equilibrium surface tension. Note that for C~0DMPO solutions the time necessary for the equilibrium to be attained is rather short. The time value t = 100 s is marked in Fig. 1.10 by the dotted line. It is seen that the dynamic surface tensions for all the concentrations studied at this time moment differ from the equilibrium values by less than 1 mN/m. For HSA solutions the dynamics of surface tension decrease is rather different. Thus, in all the mixtures studied, preferential adsorption of CI0DMPO takes place first, followed by the adsorption of HSA. Therefore, the dynamic curves shown in Fig. 1.9 can be considered to consist of two sections: the first (t < 200 s) corresponding to the adsorption of C10DMPO, and the second (t > 200 s) - to the HSA adsorption. It is seen from Fig. 1.9, that for the concentrated (2-10 -4 mol/1 and higher) CIoDMDO solutions, the adsorption of HSA is almost absent, while for CIoDMPO concentrations c < 10.5 mol/1, only HSA is adsorbed from the solution. The equilibrium surface tension isotherms for CIoDMPO without and mixed with HSA are shown in Fig. 1.11. It is seen that for c > 10-4 mol/1, the two isotherms are almost indistinguishable. This also shows that the adsorption of HSA for the higher CIoDMPO concentrations is negligibly small. We conclude that the composition changes in the surface layer is rather sharp within a narrow range of C10DMDO concentration. This view is supported by the analysis of the curves of shear viscosity of mixed monolayers as shown in Fig. 1.12.

31

80

--

70-

i

E ;~60-

50-

40

........... -7

I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -6

-5

-4

I

-3

-2

1og c [mo 1/1] Fig. 1.11. Equilibrium surface tension isotherms for individual C10DMPO solutions ([2, solid line) and mixed C10DMPO/HSA solutions for the concentration 10 -7 mol/l (A), dotted line - equilibrium surface tension of pure HSA solution of c = 10-7 mol/l averaged over 6 measurements (~ = 57+ 1) mN/m.

100

-

80-

60-

Z K--

40

20

0 0

50

100

150

200

250

t [min]

Fig.l.12. Shear viscosity for surface layers of mixed solutions of HSA (c = 10 -7 mol/l) and C10DMPO at various surfactant concentrations" 10 -6 (m), 5.10 -6 ([3), 10 -5 ( 0 ) , 210 .5 (4"), 410 .5 (A), 710 .5 mol/l (A); the solid line corresponds to the surface shear viscosity of pure HSA, according to Miller et al. (1998b)

32 It is seen that for C10DMPO concentrations c _< 2.10 5 mol/1, the monolayer possesses a rather high viscosity, characteristic for pure HSA solutions. However, already at a concentration of 7.10 .5 mol/1, the shear viscosity decreases sharply almost to zero, which is characteristic of surfactant solutions. Therefore, both tensiometric and rheologic studies indicate that the compatibility of HSA and CIoDMPO in the mixed monolayer is very poor, in contrast, for example, to the mixtures of surface active homologues. For such mixtures no range of 'components antagonism' exists, that is, the addition of a second component always results in an extra decrease of surface tension for the mixture. This fact can be theoretically explained easily. It follows from the generalised Szyszkowski-Langmuir equation for the mixture of two components, 1 and 2, that RT In 1 + b2c2 ) AFI~2 = co 1 + b~cl

(1.44)

where AYI~2 is the extra decrease of surface tension for the solution of component 1, caused by the addition of component 2, e0 is the partial molar area of the surfactant, b the adsorption equilibrium constant. It is seen from Eq. (1.44) that, except for blC 1 >~b2c2, an addition of the second component leads to a substantial decrease of the surface tension of the mixed solution. Recalling Fig. 1.11, one sees that for CIoDMPO concentrations in the range c - (10 -5 - 10-4) mol/l, the surface tension of mixtures exceeds that of the individual HSA solution. (For CI0DMPO concentrations lower than 5.10 -7 mol/1, the surface tension of the mixtures was equal to that of the pure HSA solution). For concentrations in the range from c = 4.10 .5 mol/1 to 10 4 mol/l this surface tension excess amounts only to 1-1.5 mN/m. However, for c < 4-10 .5 mol/1, the surface tension of mixed solution exceeds that of the pure HSA solution by 3 to 4 mN/m. The theoretical model of Eq. (1.44) is unable to explain the increase of surface tension: an anomalous increase of the surface tension in the mixture of homologues, (that is, negative values of AFI~2) cannot follow from Eq. (1.44) by the addition of the second component. The hydrophobic interaction of C10DMPO hydrocarbon tails with HSA polypeptide chains can, in principle, lead to a hydrophilisation of the protein molecule. This effect, however, cannot be significant in our case, when only 10 molecules of C10DMPO (for c = 10-6 mol/1) exist in the

33 mixed solution per HSA molecule, which possesses 585 amino acid groups. This anomalous adsorption behaviour of a protein/surfactant mixture can be explained in the framework of equations of state (1.20) and protein adsorption isotherm (1.21) for a solution/fluid interface. Although 2 to 3 adsorption states may exist for HSA molecules in general, for surface pressures > 3 mN/m (c > 2.10 -8 mol/1) only one state persists. This state possesses minimal area per molecule O)min - - 4 0 nm 2. Therefore, in our case (c

= 1 0 "7

mol/1),

only one state has to be taken into consideration, resulting in an essential simplification of the model. Note that the adsorption of C10DMPO in absence of protein can be described quite well by the Szyszkowski-Langmuir equations. Thus, in a mixture of HSA (component 1) and C10DMPO (component 2), HSA exists in a single adsorption state.

VI = - RT [ln(1 - Fzmz)- ae,F2m~ ]

(1.45)

0) E

blC 1 =

FI (-1)1

(1 _ r~mz)~,/~ ~

(1.46)

F2m2

(1.47)

where Fz = F 1 + F 2. The average molar area of adsorbed components 1 and 2 can be expressed according to Eq. (1.6) by

mz =

Fl(O l + F 2 o ) 2

F~ + F 2

(1.48)

or

2

Flm~ + r2m2

(1.49)

(2) Z ~- rlfX)l -~-F20) 2

where the averaging in Eq. (1.48) and Eq. (1.49) was performed over the adsorptions and monolayer coverages, respectively. Equation (1.48) can be successfully used to describe mixtures of surfactants when the difference between the molar areas of the components does

34 not exceed one order of magnitude. Eq. (1.49) seems to be more appropriate for mixtures of a protein and a surfactant, where the % values differ by two orders of magnitude,. Note however, that for limiting cases, when a preferential adsorption of either of the two components takes place, the difference between the models of Eqs. (1.48) and (1.49) becomes negligible. For pure HSA solutions, the following values were found: col = 40 nm 2 (per molecule), b I = 2.1071/mol, and ael = 320. For pure C10DMPO solutions the corresponding values are co2 = 0.45 nm 2 (per molecule), and b 2 = 2.28.104 1/mol. The surface tension isotherm for CIoDMPO, calculated with these parameters from the Szyszkowski equation is shown in Fig. 1.11. The relation between the adsorptions of protein and surfactant can be derived from the adsorption isotherms of Eqs. (1.46) and (1.47):

:

F2co2

b,c,

(1 -

(1.5o)

b2c2

For a given ratio of the component concentrations in the solution bulk one can deduce from Eq. (1.50) that the portion of protein in the surface layer decreases sharply with the increase of the total adsorption

as coI ~ ~

The values calculated from Eqs. (1.45)-(1.49) for the

adsorption characteristics of HSA and C10DMPO as listed above, agree well with the experimental dependence shown in Fig. 1.11. This figure displays the anomalous increase of surface tension at low CIoDMPO concentration. Negligible adsorption of protein at high C~0DMPO concentration are predicted. For the limiting cases, one can derive simple analytical expressions for AFI. For low concentrations (adsorptions) of CIoDMPO, as col/coy~_=_1 and col/coz << 1, approximate relations follow from Eqs. (1.46) and (1.47)

F, coI = b ~ c , ( 1 - b 2 c 2 ) / 0

+ b,c~) and F2co 2 =b2c 2

(1.51)

Rewriting Eq. (1.44) for the case when no Ct0DMPO is added to the HSA solution, and subtracting this from the original equation, one obtains an expression for the jump of surface pressure of the mixture

35

1

/ . (blClo, ae 1+blCl/ t b c

Applying the parameters explained above into Eq. (1.52), one can see that the first term can be neglected as compared with the second term. It is known that, for protein solutions, the main contribution to the surface pressure is due to the inter-ion interaction, i.e., to the second term of Eq. (1.45). It follows from Eq. (1.52) that the value

AI'-II2 is negative

for low concentrations of

C10DMPO. Moreover, calculating the extreme value of the second term of Eq. (1.52) one sees that the maximum of the surface tension of a mixture takes place at b2c2 = 1, that is, for C10DMPO concentrations of approximately 4.10 .5 mol/1. It is seen from Fig. 1.11 that the actual maximum of

Al-[12 is

attained for lower C10DMPO concentration. This discrepancy can be

attributed to the approximate character of Eq. (1.52). For low or zero values of ael, the mixing of components 1 and 2, as it follows from eq. (1.52), cannot lead to an increase of the surface tension. Therefore, the anomalous surface tension behaviour for mixtures of HSA with C10DMPO most likely results from the large free charge of the protein molecule. This phenomenon can be explained rather simply from a physical point of view. The addition of C10DMPO results in an increase of the total surface layer coverage, which in turn leads to an increase in the first term of Eq. (1.52). However, it follows from the isotherm (1.51) that the addition of C10DMPO decreases the adsorption of HSA. This now results in a decrease of the second term of Eq. (1.45), which depends quadratically on the HSA adsorption. Therefore, a decrease of the surface pressure in the mixture takes place. In summary, the experimental results for mixtures of HSA and C10DMPO agree well with the theoretical model which accounts for large differences between the molar area of protein and surfactant, and for the existence of a significant unbounded charge of the protein molecule. From a practical point of view two results are important: 1)the prediction of an anomalous increase of surface tension by adding a small amounts of surfactant to HSA/DMPO mixtures; 2) the range of bulk concentrations for HSA and DMPO at which both components contribute equally to monolayer formation is very narrow. The dynamic surface tension for protein mixtures was studied by Suttisprasit et al. (1992) and Xu & Damodaran (1994). They found that mixing of proteins does not necessarily result in a decrease of surface tension. For example, the addition of a-lactalbumin and [3-1actoglobulin to BSA at constant total protein concentration leads to an increase of the dynamic surface tensions (Suttisprasit et al. 1992).

36 The results of model studies of dynamic surface tension performed on human biological liquids are still at their initial stage. The number of publications in this area has increased; however, they are dedicated mainly to low-concentrated protein solutions in the long lifetime range, while fast processes in concentrated solutions are of practical interest from a biological and medical point of view. There is still a considerable lack of general information concerning the dynamic adsorption behaviour of proteins at the solution/air and solution/liquid interface. This lack prevents a deep analysis of dynamic tensiograms of actual biological liquids.

1.5. Summary The basic theoretical models to describe the adsorption of surfactants and proteins and their mixtures at liquid interfaces are presented in a modem way, starting from the general Butler equation. As the result a set of equations is derived which allows to quantitatively understand the adsorption of protein and surfactants at a liquid interface. The description of mixtures is still in a qualitative state and requires further extended fundamental research. The adsorption of proteins and surfactants at interfaces is a time process. While the modelling of the adsorption dynamics of surfactants is on a high level, it is shown that there are only semi-quantitative models available to describe the process of protein adsorption. Some general ideas presented recently in literature are encouraging and represent a good starting point for future improvements of these theories. The key tool for the derivation of both equilibrium and dynamic theories is the principle of Braun- Le Chatelier applied to interfacial layers. Due to this principle each special adsorbed at an interface occupies a certain area. The molar area of the components and the composition of a mixed layer are controlled by the surface pressure, i.e. the change in surface tension due to adsorption. At the end of this chapter it is shown which parameters of a protein/surfactants adsorption layer can be measured in addition in order to better understand the formation process and the properties of these interfacial layers. In particular surface rheology provides important information about the structure of mixed protein/surfactant layers, as they are formed by biological liquids.

37 1.6. References

Atkinson, P.J., Dickinson, E., Home, D.S. and Richardson, R.M., ACS Symp. Ser., 602(1995)311 Benjamins, J., de Feijter, J.A., Evans, M.T.A., Graham, D.E. and Phillips, M.C., Disc. Faraday Soc., 69(1978)218 Borwankar, R.P. and Wasan, D.T., Chem. Eng. Sci., 43(1988)1323 Boury, F., Ivanova, Tz., Panaiotov, I. and Proust, J.E., Langmuir, 11(1995)599 Butler, J.A.V., Proc. Roy. Soc. Ser. A, 138(1932)348 Cheng, O., Li, D., Boruvka, L., Rotenberg, Y. and Neumann, A.W., Colloids Surfaces A, 43(1990)151 Clark, D.C., Husband, F., Wilde, P.J., Cornec, M., Miller, R., Kr~igel, J. and Wt~stneck, R., J. Chem. Soc. Faraday Trans., 91 (13)(1995) 1991 Davies, J.T., Proc. Roy. Soc., Ser. A, 208(1951)224 Davies, J.T., Proc. Roy. Soc., Ser. A, 245(1958)417; 419 de Feijter, J.A. and Benjamins, J., in "Food Emulsions and Foams" (E. Dickinson, Ed.), Special publication no.58, p.72. Royal Chem. Soc., London, 1987 de Feijter, J.A., Benjamins, J. and Veer, F.A., Biopolymers, 17(1978)1760 de Gennes, P.G., Adv. Colloid Interface Sci., 27(1987) 189 Dobrynin, A.V., Colby R.H. and Rubinstein, M., Macromolecules, 28(1995)1859 Douillard, R., Daoud, M., Lefebvre, J., Minier, C., Lecannu, G. and Coutret, J., J. Colloid Interface Sci., 163(1994)277 Douillard, R. and Lefebvre, J., J. Colloid Interface Sci., 139(1990)488. Dussaud, A., Han, G.B., Ter Minssian-Saraga, L. and Vignes-Adler, M., J. Colloid Interface Sci., 167(1994)247 Fainerman, V.B., Colloids Surf., 57(1991)249

38 Fainerman, V.B., Makievski, A.V. and Miller, R., Colloids Surf. A, 87(1994)61 Fainerman, V.B. and Miller, R., J. Colloid Interface Sci., 178(1996b) 168 Fainerman, V.B. and Miller, R., Langmuir, 12(1996a)6011 Fainerman, V.B. and Miller, R., Langmuir, 13(1997)409 Fainerman, V.B. and Miller, R. In "Proteins at Liquid Interfaces", in "Studies of Interface Science", D. M6bius and R. Miller (Eds.), Vol. 7, Elsevier, Amsterdam, 1998a, p. 51-102 Fainerman, V.B. and Miller, R., submitted to Langmuir, (1998b) Fainerman, V.B., Miller, R. and Wiistneck, R., J. Colloid Interface Sci., 183(1996a)26 Fainerman, V.B., Miller, R. and Wfistneck, R., J. Phys. Chem. B, 101 (1997)6479 Fainerman, V.B., Vollhardt, D., Melzer, V., J. Phys. Chem., 100(1996b) 15478. Flory, P.J., J. Chem. Phys., 10(1942)51 Flory, P.J., J. Chem. Phys., 9(1941)660 Franks, F., in: ,,Characterisation of Proteins", (F. Franks, ed.), Humana Press - Clifton, New Jersey, 1988, p.53 Ghosh, S. and Bull, H.B., Biochemistry, 2(1963)411 Gonzalez, G. and MacRitchie, F., J. Colloid Interface Sci., 32(1970)55 Graham, D.E. and Philips, M.C., J. Colloid Interface Sci.,70(1979a)403 Graham, D.E. and Phillips, M.C., J. Colloid Interface Sci., 70(1979b)415 Graham, D.E. and Phillips, M.C., J. Colloid Interface Sci., 70(1979c)427 Guzman, R.Z., Carbonel, R.G., Kilpatrick, P.K., J. Colloid Interface Sci., 114(1986)536. Hansen, F.K. and Myrvold, R., J. Colloid Interface Sci., 176(1995)408 Hermel, H. and Miller, R., Colloid Polym. Sci., 273(1995)387 Huggins, M.L., J. Phys. Chem., 46(1942) 151 Israelachvili, J., Langmuir, 10(1994)3774.

39

Joos, P. and Serrien, G., J. Colloid Interface Sci., 145(1991)291 Joos, P. and Serrien, G., J. Colloid Interface Sci.,127(1989)97 Joos, P., Biochim. Biophis. Acta, 375(1975)1 Kalischewski, K. and Schugerl, K., Colloid Polymer Sci., 257(1979)1099 Klein, J. and Luckham, P., Nature, 300(1982)429 Klein, J. and Luckham, P., Nature, 308(1984)836 Kragel, J., Wtistneck, R., Clark, D., Wilde, P. and Miller, R., Colloids Surfaces A, 98(1995)127 Lassen, Bo and Malmsten, M., J. Colloid Interface Sci., 179(1996)470 Leermakers, F.A.M., Atkinson, P.J., Dickinson, E., Home, D.S.,J Colloid Interface Sci., 178(1996)681. Lucassen-Reynders, E.H., Colloids Surf. A., 91 (1994)79 Lucassen-Reynders, E.H., J. Colloid Interface Sci., 41 (1972) 156 Lucassen-Reynders, E.H., J. Colloid Interface Sci., 85(1982) 178 Lucassen-Reynders, E.H., J. Phys. Chem., 70(1966) 1771 MacRitchie, F., Colloids Surfaces, 41 (1989)25 MacRitchie, F., Analytica Chimica Acta, 249(1991 )241 MacRitchie, F., J. Colloid Interface Sci., 61 (1977)223 Makievski, A.V., Fainerman, V.B., Bree, M., Wtistneck, R., Kr~igel, J. and Miller, R., J. Phys. Chem., 102(1998)417 Miller, R., Trends Polym. Sci., 2(1991)42 Miller, R., Policova, Z., Sedev, R. and Neumann, A.W., Colloids Surfaces A, 76(1993)179 Miller, R., Fainerman, V.B., WiJstneck, R., Kragel, J. and Trukhin D.V., Colloids Surfaces A, 131(1998a)225

40 Miller, R., Fainerman, V.B., Makievski A.V., Kr~igel, J. and Wtistneck, R., Colloids Surfaces A, (1998b), in press Murray, B.S. and Nelson, Ph.V., Langmuir, 12(1996)5973 Murray, B.S., Colloids Surfaces A, 125(1997)73 Paulsson, M., and Dejmek, P., J. Colloid Interface Sci., 150(1992)394 Peters, Th., Adv. Protein Chemistry, 17(1985) 161 Rotenberg, Y., Boruvka, L. and Neumann, A.W., J. Colloid Interface Sci., 37(1983)169 Ruckenstein, E., Bhakta, A., Langmuir, 10(1994)2694. Ruckenstein, E., Li, B., Langmuir, 11(1995)3510. Serrien, G., Geeraerts, G., Ghosh, L. and Joos, P., Colloids Surfaces, 68(1992)219 Suttisprasit, P., Krisdhasima, V. and McGuire, J., J. Colloid Interface Sci., 154(1992)316 Ter-Minassian-Saraga, L., J. Colloid Interface Sci., 80(1981)393 Tomberg, E. and Lundh, G., J. Colloid Interface Sci., 79(1981)76 Tripp, B.C., Magda, J.J. and Andrade, J.D., J. Colloid Interface Sci., 173(1995)16 Turro, N.J., Lei, X.-G., Ananthapadmanabhan, K.P. and Aronson, M., Langmuir, 11(1995)2525 Uraizee, F. and Narsimhan, G., J Colloid Interface Sci., 146(1991) 169. Ward, A.J.I. and Regan, L.H., J. Colloid Interface Sci., 78(1980)389 Wtistneck, R., Kr~igel, J., Miller, R., Fainerman, V.B., Wilde, P.J., Sarker, D.K. and Clark, D.C., Food Hydrocolloids, 10(1996a)395 Wtistneck, R., Kr~igel, J., Miller, R., Wilde, P.J. and Clark, D.C., Colloids Surfaces A, 114(1996b)255 Xu, S. and Damodaran, S., Langmuir, 10(1994)472

41

Chapter 2

Experimental technique and analysis of tensiograms The most frequently used experimental techniques to study adsorption phenomena are surface tension methods. Most of these methods give static surface tension characteristics. However, for biological systems dynamic surface tension characteristics are of utmost importance. As we are interested mainly in the dynamics of the adsorption processes of proteins and surfactants and their mixtures a dynamic method is required. This chapter gives an introduction into the application of the maximum bubble pressure technique and its practical use to determine dynamic surface tensions. This method is particularly suited to measure tensions in a broad time interval at surface lifetimes of the order of milliseconds up to about 100 seconds. The course of surface tension versus time dependencies are shown and procedures discussed which allow to determine dynamic surface tension characteristics which are of significant importance in medical applications.

2.1. Experimental methods A great variety of methods exist to measure surface tension, or. Among these methods one can distinguish between those based on direct measurements of capillary forces acting on curved liquid surfaces, those relying upon the analysis of the form of liquid meniscii, and those involving the measurement of the pressure difference between the two sides of a curved surface (Rusanov & Prokhorov 1996). In addition, the various methods differ from each other in the range of accessible interfacial lifetimes, in the rate and extent of surface deformation exerted by the measuring procedure, and in their precision and reproducibility of results. Many types of commercially available devices are produced, based on the du NoOy ring or Wilhelmy plate method. Their common deficiencies with respect to studies of biological liquids is the limited range of lifetimes measured (10 s and higher), and the demand for a rather large amount of liquid to be analysed (at least 20 ml), which is often inappropriate for clinical practice. The same problems are associated with the drop volume tensiometer TVT1 (Lauda,

42 Germany) which allows measurements for the lifetime range of 2 to 500 s and uses sample volume of 5 ml (Miller et al. 1992). The ADSA tensiometer (Kwok et al. 1995) based on the drop shape analysis using the Laplace equation of capillarity can provide the cy dependence within the lifetime range of 10 to 20000 s and using sample volumes less than 1 ml. However, the production of commercially available devices of this type, which we believe to be very promising for studying of biological liquids, is still at their initial stages. Tensiometers based on the maximum bubble pressure method (MBPM) meet the requirements imposed by medical applications most adequately. Different types of such devices are currently available: PBS (Electronetics Comp., USA), Sensadyn 5000 (Chem Dyne Research Corp., USA), BP1 (KriJss, Germany) and MPT2 (Lauda, Germany). The use of the maximum bubble pressure method (MBPM) for the measurements of surface tension was originally proposed by Simon (1851). The history of the method was reviewed by Mysels (1990), more brief descriptions can be found in the publications of Rusanov & Prokhorov (1996) and Miller et al. (1994). Simon (1851) had employed a single capillary; therefore the immersion depth of the capillary into the liquid was measured to take account for the hydrostatic pressure. This depth needs not be measured if the capillary is installed below the liquid level (Brown 1932) or if two capillaries (narrow and wide) are used, as proposed by Sugden (1922). The Sugden's idea was further developed by Jaeger (1917), Warren (1927), J

Pugachevich (1964), Belov (1981), Razouk & Walmsley (1974), Ross et al. (1992) and Lunkenheimer et al. (1982) leading to modifications of the method based on the measurements of the immersion depth difference for the capillaries with different diameters in the same liquid and for two identical capillaries in different liquids. One of the MBPM advantages is the possibility to study the surface tension in microvolumes of a liquid, which is particularly important in the investigations of biological liquids. Microvolume measuring cells developed for this purpose were described by Feldman et al. (1980) and Cuny & Wolf (1956). The main measured parameter in the MBPM is pressure. Modem devices, including all commercially available tensiometers, employ electrical pressure transducers. These pressure transducers have high sensitivity and low inertiality, which enable measurements of bubble formation frequency (Bendure 1971, Miller & Mayer 1984, Hua & Rosen 1988, Hirt et al. 1990

43 and Mysels 1986, 1989). The method of frequency registration is most accurate when the volume of the measuring system V s is relatively small with respect to the separating bubble volume V b. However, if the bubble formation frequency is high under such conditions, the error of maximum pressure measurements increases (Mysels 1989). Therefore other (independent) methods were proposed to measure the time interval between successive bubbles. These methods include stroboscopic (Austin et al. 1967 and Kloubek 1968, 1972a), photoelectric (Garrett & Ward 1989 and Kao et al. 1992), conductometric (Fainerman & Lylyk 1982) and acoustic (Fainerman et al. 1994a and Miller et al. 1997) techniques. Some devices employ also the measurements of gas (air) flow rate and bubble volume. Among the available MBPM devices the MPT2 tensiometer (Lauda, Germany) is the most technically advanced (Miller et al. 1994, 1997). This instrument is equipped with pressure and air (gas) flow rate transducers, and with two independent systems (acoustic and conductometric) for bubble formation frequency measurement. The advantages of MPT2 over other devices are: wide range of lifetimes measured (1 ms to 100 s), small volume of analysed sample (less than 1 ml), short time required to perform the analysis, completely automated measurement and computerised data processing. The MPT2 tensiometer enables one to obtain dynamic surface tension values with a reproducibility within 0.2 %. 2.2. The design of a maximum bubble pressure tensiometer

The MPT2 tensiometer (Lauda, Germany) is the most advanced tensiometer applying the maximum bubble pressure method. Such tensiometer is shown schematically in Fig. 2.1. The air coming from the microcompressor 1 is cleaned in a fine purification filter 2. Other gases (e.g. nitrogen) can be employed, in which case the gas reservoir is connected to the compressor via a reducer. The gas flows next into the throttling capillary 3 which is used to measure the air flow rate with the help of a differential electric pressure transducer 5. The excess pressure within the system is measured using the pressure sensor 6. The electric signals from the pressure transducer 6, air flow sensor 5, the electrodes (see fig. 2.2) and the microphone 7 are sent to the measuring-control block 8 connected to the computer 9 via a serial port. The electromagnetic valves 4 and compressor 1 are operated by the computer via the control block

44 8. The total air (gas) volume in the tensiometer MPT2 (the volume between the throttling capillary 3 and the measuring capillary (see fig.2.2), comprises of about 35 cm 3, of which approximately 30 cm 3 is the volume of an additional vessel with the built-in pressure sensor 6. For the volume of a separating bubble (3+4).10 -3 cm 3 and the measuring capillary radius of about 0.01 cm the maximum pressure drop in the system during the period of fast bubble growth does not exceed 0.5 %. The microcell used for medical and biological analysis is shown schematically in Fig. 2.2. The microcell comprises of the measuring capillary 1, the hollow containing the biological liquid studied 2, and the deflector (or the electrode which acts as deflector) of the bubbles 3; the cell is equipped by a thermostating water jacket. The immersion depth of the measuring capillary I into the liquid and the distance between the capillary and deflector-electrode 3 can be adjusted.

to measuring cell

Fig. 2.1. Schematic design of the MPT2 tensiometer from LAUDA. l-Pump

4-Electromagnetic valve

2-Filter

5-Differential

3-Throttling capillary

sensor 6- Pressure sensor

7-Microphone

pressure 8-Electronic control unit 9-Computer

45 from M PT2

.__f~ 1

---_3

Fig. 2.2. Special medical microcell (schematically); 1 - capillary, 2 - liquid to be investigated, 3 - deflector. The deflector-electrode and another electrode immersed into the liquid makes up the electrode system used for the measurement of bubble formation frequency. The second independent system, which monitors the bubble formation frequency in any type of a liquid, is the acoustic system which employs a high sensitive microphone 7 (see fig.2.1). When the bubble touches the electrode the resistance between the electrodes increases, while the collapse of the gas cavity at the moment of bubble separation creates a sound wave, which can be easily registered by the microphone. The calibration, measurement, and calculation procedures are completely automated. The operational program provides a number of measurement regimes. 2.3. Theory of the maximum bubble pressure method The bubble growth at the capillary tip can be divided into four main stages. The first stage starts the moment the bubble separates from the capillary tip. The meniscus curvature radius is approximately equal to the radius of the separating bubble r b (r b )) rc). At this time moment the next bubble begins to grow. During the time interval tll the meniscus curvature radius approaches the capillary radius, and the meniscus itself moves into the capillary by some depth h (forward meniscus motion), depending on the properties of the intemal surface of the capillary. In the second stage the meniscus driven by excess pressure in the system, moves during the time t12 to the end of the capillary (reverse meniscus motion). The third stage starts when the bubble radius decreases and becomes equal to the capillary radius (for narrow capillaries). It is determined by the air flow: the greater the air flow L, the shorter is this

46 'drawing-up' time t B. The time interval between the last bubble separation and the moment when r = r c is called the bubble lifetime t I where t I = tll + tl2 + t B (Dukhin et al. 1996, 1998). The last stage of the growing bubble evolution starts when the bubble has passed the hemispherical size. During this so-called deadtime interval t d between the moment of maximum pressure (r = re) and the separation of the bubble the bubble grows quickly and finally separates while the pressure in the growing bubble decreases. It was mentioned above that the amplitude of actual pressure oscillations in the system Ps does not exceed 0.5 %. The maximum pressure is achieved at the end of the lifetime period; at this moment the pressure in the bubble Pb differs only slightly from Ps. Throughout the deadtime and at the initial stage of the lifetime the pressure in the bubble decreases sharply. In summary, the time interval between the formation of two successive bubbles t b consists of the lifetime t I and the deadtime td; and the lifetime t~ can in general be subdivided into three further components. The surface tension cr is calculated from the values of maximum capillary pressure P and capillary radius r e using the Laplace equation: rcP cr=-~f

(2.1)

where f is a correction factor which accounts for the bubble non-sphericity. The capillary pressure P is expressed by the excess maximum pressure in the measuring system Ps, the hydrostatic liquid pressure PH = ApgH (Ap is the difference between the densities of liquid and gas, g the gravity and H the immersion depth of the capillary into the liquid), and the excess pressure Pd that arises between the measuring system and the bubble due to dynamic effects (aerodynamic resistance of the capillary, viscous and inertia effects in the liquid etc.). Therefore P = P~ - PH -- I'd

(2.2)

The correction factor f was calculated by Sugden (1924) and tabulated as a function of the ratio re/a, with a being the capillary constant and defined by

47

a =(2~/Apg) 1/2

(2.3)

Sugden's tables were later transformed into a polynomial form by a number of authors (Bendure 1971, Johnson & Lane 1974, Volkov & Volyak 1972 and Kisil' et al. 1981). Various corrections to the Laplace equation were analysed by Mysels (1990). For capillaries with radii r~ < 0.02 cm f is very close to unity. As narrow capillaries are used in the MBPM studies (re< 0.01 cm), this non-sphericity correction can be neglected; hence the formula for the calculation of surface tension can be expressed as

~ = ~rc- (Ps - P~ - Pd)

(2.4)

The deformation (expansion) of the bubble surface and the displacement of the meniscus into the liquid bulk during the lifetime can contribute to the excess pressure Pd. Keen & Blake (1996) have considered the effect of bulk and surface (dilatational)

viscosity on the growth

dynamics of the bubble on the capillary tip immersed into the liquid. It was shown, in particular, that the effect of dilatational viscosity k~ is significant for very large values of ks (k~/r~ bt > 1000, where g is the bulk viscosity of the liquid). According to the calculations performed by Kao et al. (1992), the excess pressure in the growing bubble arising from the bulk viscosity of the liquid can be expressed as ~tL Pd - ~r 3

(2.5)

where r is the current value of bubble radius, L the gas flow rate. At the final lifetime stage Eq. (2.5) transforms to within a numerical factor into the approximate expression which Fainerman at al. (1993) had obtained from the Stokes' law 3g P d - t~

(2.6)

This formula agrees qualitatively with the experiments performed for highly viscous liquids. The dilatational surface viscosity contribution to the P d value for the final lifetime stage in the MBP method was estimated by Kao et al. (1992) as

Pd -

ks rct~

(2.7)

48

It can be shown that the surface viscosity contribution to the Pd value in the MBPM can be neglected for the millisecond and sub-millisecond time range. The liquid inertia contribution to Ps was estimated by Dukhin et al. (1996). It was shown that while the inertial effects are most significant at the final lifetime stage for very wide and short

capillaries, P Ousually amounts to less than one percent with respect to P. A hydrodynamic theory for the MBPM was developed by Dukhin et al. (1996, 1998) and Koval'chuk et al. (1998a, 1998b). The analysis shows that the regime of the process which takes place in the capillary is determined by the dimensionless parameter K = nvrc2/81v, where v is the gas kinematic viscosity, v is the sound velocity and 1 is the capillary length. For K > 1 the excess pressure in the capillary P oscillates. For K < 1 the pressure in the capillary smoothens without any oscillations (aperiodic regime). The process regime is determined mainly by the geometric characteristics of the capillary: the shorter and wider the capillary, the higher is the K value. Therefore the pressure oscillation regime can exist in short capillaries, while for long capillaries the aperiodic pressure smoothening regime takes place because the oscillations are fading down rapidly. The important consequence of the oscillation process is a faster smoothening of the pressure throughout the capillary; therefore the value of PO decreases for K ~>1. Therefore one can reduce significantly the aerodynamic resistance contribution to the Pd value by a proper choice of the capillary geometry. Aerodynamic resistance could be further reduced by applying the theoretical results obtained by Koval'chuk (1998b). In this study the interrelation between the lifetime and deadtime was analysed on the basis of the fact that the gas velocity in the capillary at the end of the deadtime period acts as the initial condition for the lifetime period of the subsequent bubble, and vice versa. It was shown that for short wide capillaries (r E/1 > 10.4 cm) the excess pressure is reduced significantly due to high initial velocity of the gas. For capillaries for which the condition rc2/1 > 10.4 cm is satisfied, the value of Po does not exceed 0.5 % of P. Thus the hydrodynamic MBPM theory enables one to determine the conditions at which the aerodynamic component of the excess pressure Pd is minimised.

49 First theoretical calculations of the deadtime t d were performed with a Poiseuille approximation for the gas flow through the capillary by Fainerman (1979) 3

321q

o.

where r b is the separating bubble radius. The first term on the right hand side of Eq. (2.8) describes the gas expansion into an infinite space, while the second term corresponds to the capillary pressure in the growing bubble. The surface tension for a growing bubble cy* during the deadtime, which enters into the second term, is in fact unknown for surfactant solutions. The analysis performed by Fainerman (1990) had shown that for solutions the value of cy* in Eq. (2.8) is between the equilibrium value ooo and the dynamic value of cy for t = t l. Another important conclusion of this analysis is that a variation of cy* in the range o > ~* > cyoodoes not affect the t d value. This fact enables one to exclude o* from Eq. (2.8) by substituting cy instead. Thus in the Poiseuille approximation one obtains (Fainerman et al. 1994a) td=tb'~

1+~

where kp is the Poiseuille equation constant for the capillary not immersed into the liquid (L = kpP), L is the gas flow rate, P = Ps" P., and t b is the time interval between successive bubbles. A more rigorous deadtime theory was developed by Dukhin et al. (1996) and Koval'chuk et al. (1998b). These authors had shown that the corrections related to the nonstationarity of the gas flow through the capillary and to the effect introduced by the initial section of the capillary, do not exceed a few percent of the t d value calculated from Eq. (2.9). The hydrodynamic relaxation time lifetime:

th --

th

represents the sum of the first two components of the

tll + t12, that is, the sum of the times of forward and reverse meniscus motion. For

short capillary whose internal surface is hydrophobic, the liquid penetration depth h into the capillary is small, while for hydrophilic internal capillary surfaces h is of the order of the capillary radius. Therefore for hydrophilic capillaries the time interval th can contribute

50

significantly to tl. The values of tll and h for hydrophilic capillaries were first estimated by Dukhin et al. (1998). It was shown that for the aperiodic regime (K < 1) the value for h became 2- 3 % of the capillary length. Furthermore, the value for the forward meniscus motion time, tll, became 103s for long and narrow capillaries , and 10-Ss or less for short and wide capillaries ). It is to be noted that the value of h does not depend on the excess dynamic pressure in the system, P d = Ps -

PH - P ,

while the forward and reverse meniscus motion times depend strongly

on the ratio P/Pd" The larger the excess pressure, the lower is the meniscus hydrodynamic relaxation time. The value

PO

in turn depends on the capillary geometric characteristics and the

gas flow regime. For the aperiodic regime the value of t h is close to the lifetime in long narrow capillaries, while for short capillaries the inequality

t h << t I holds.

It means that short and wide

capillaries can be used for the MBPM in the range of milli- and submillisecond bubble lifetimes. The lifetime value is determined by subtracting the calculated deadtime value

td

from

t b.

The

deadtime value could be estimated using the method first proposed by Kloubek (1972a). This method takes advantage from the fact that during high frequency bubble formation the deadtime interval is equal to the time interval between successive bubbles. The transition from the regime with

to < tb

to the regime when

to = tb

is marked by a sharp pressure increase in the

measuring system which is evident when Ps is plotted against tb,. The

tb

value at the moment

when the pressure jump takes place is exactly equal to t d. This method, however, does not account for the t d dependence on t b. More precise is the combined experimental/theoretical procedure (Fainerman 1992, Miller et al. 1997) which is based on Eq. (2.9), where it is assumes that t a depends on t b and P. The dependencies of excess pressure in the measuring system on the gas flow rate L, for the capillaries of various length are shown in Fig. 2.3.

51 3500

--

3 0 0 0 --

,--., 2 5 0 0

--

o

m 9

9

.....II

~" 2 0 0 0

~A AA 9

9

~A D

1500

I

1000 0

b

0,05

0,1

I

I

0,15

0,2

L [cmVs] Fig. 2.3 Dependence o t pressure P in the measuring system on the air tlow rate L for a 0.2 % Triton X-100 solution, rc = 0.0084 cm, 1 = 6 cm (A), 3 cm (4,) and 1.5 cm (11)

All these curves possess the inflexion point with co-ordinates Pc and L c. The linear sections of the curves in the region L > L c are described by the Poiseuille equation and correspond to the jet regime of gas expansion from the capillary. For L < L c the gas flow results in a formation and separation of individual bubbles with t I > 0. However, in the critical point (L = Lc) the lifetime vanishes, therefore the time interval between successive bubbles becomes equal to the deadtime. Then, for constant t b one can combine two equations which follow from Eq. (2.9) for L = L c and L < L c to obtain

L~ t d = t b Lc P

(2.10)

It follows from Eq. (2.10) that

tl = t b - - t d =tb 1-- LcPj

(2.11)

Thus the procedure developed by Fainerman (1992) and Miller et al. (1997) allows the lifetime calculation using the parameters tb, P = Ps- PH and L determined from the experiment. The critical point co-ordinates on the P

vs

L dependence can be easily calculated by the algorithm

based on the Poiseuille equation. Equation (2.11) accounts for the dependence of t I on t b (or, more precisely, on P). The t d value usually increases with tb, because in this case the value of P

52 decreases (for solutions). Equation (2.10) was derived assuming a constant

tb

(or gb) value; this

can be achieved in the experiment if the distance between the capillary and the electrode (or by another body as deflector) is fixed, see Fig. 2.2. When the bubble expansion during the deadtime interval is unrestricted, which is the case for all known bubble tensiometers except the MPT2, the use of Eqs. (2.10) and (2.11) results in an erroneous value for h. When surfactant solutions are studied, it has to be considered that the surface of the bubble expands during the last (third) stage of the lifetime period. Various types of surface deformation are also used in other methods to measure surface tensions (drop volume, oscillating jet, dynamic capillary method etc.). To compare various methods, the results of surface tension measurements are usually represented in terms of the dependence on the so called effective adsorption time (Miller et al. 1994). The diffusional adsorption kinetics equation for the actual process, being expressed via the effective time, transforms into the equation for diffusion kinetics of adsorption taking place on a non-deformed plane surface, that is, the classic Ward & Tordai equation (1946)

('Dt) 1/2 F=2colv-7-)

(D] 1,2 t - 2 ~-

(2.12)

j'c(0,t-x)d('c 1/2) 0

where F is the dynamic adsorption value, D is the diffusion coefficient, c o and c(0, t) are the bulk and subsurface concentrations, respectively, and x is a dummy integration variable. If convection and surface deformation are considered for a radial symmetric drop or bubble growth under a constant flow rate, then the dynamic adsorption obeys the equation (Miller et al. 1994)

F : 2c 0 - ~ )

[ c(0'(7/3)'s3/7 0~ (x _ s),/2

s

(2.13)

where x =(3/7)t7 3, s is a dummy integration variable. It is easily seen that Eq. (2.13) can be transformed into Eq. (2.12) substituting 3 tef =~-t

(2.14)

53 where the numerical coefficient is defined by the surface deformation rule. For drops or bubbles growing under a constant flow rate it follows that (Joos & Rillaerts 1981)

O-

dlnA dt

-

2 3t

(2.15)

where A is the surface area. If the relative dilation rate 0 is described by a more general expression 0=oct -1

(2.16)

with c~ - const, then according to Joos & Rillaerts (1981) the effective time can be represented as

t ef -

t 2ct + 1"

(2.17)

It is seen that Eq. (2.14) is a special case of the more general expression (2.17) for m = 2/3. Calculations of the effective lifetime of the bubble surface during the whole stage of bubble lifetime were performed by Kloubek (1972a) and Makievski et al. (1994). For a hydrophilic capillary with the geometry corresponding to the condition K 2 > 10 or capillaries possessing a hydrophobic internal surface (in both cases the liquid does not penetrate into the capillary after bubble separation) the pressure within the bubble throughout the whole lifetime stage remains constant Pb = (2eye/re)COS% = const

(2.18)

where eye and q), are the current instantaneous values of surface tension and contact angle during the lifetime stage (0 < t < tl). Equation (2.18) is the basic expression for the calculation for the relative dilation rate of the bubble surface. The following expression was derived by Makievski et al. (1994)

where cy is the dynamic surface tension for x = tl. It is seen that the dependence of 0 on ~ and z is rather complicated and does not obey the simple relation (2.16). Assuming that the bubble

54 surface area increase during the lifetime stage is relatively small (in fact, the largest possible area variation is from nr 2 for x - 0 to 2nr 2 for x = t 0, the finite variations of bubble surface area were analysed to estimate 0. It was shown that to within a reasonable accuracy the relative dilation rate and effective time can be expressed by Eqs. (2.16) and (2.17). The following expression for the constant ot was derived by Makievski et al. (1994) 2sin% ot = ~ 2 + sin%

(2.20)

where % = arccos(o/%), o 0 the surface tension of the solvent. For o = o 0 the value of ot is equal to 0, that is, the bubble surface is virtually non-deformed. For surfactant solutions with cy/cy0 < 0.8 we have ot ~ 2/3; therefore, similar to the case of a growing drop, tef= (3/7)t. It is to be noted that Eq. (2.20) is valid for short and wide capillaries, when t h <
AP=Pa VB

(2.21)

Vs which corresponds to the surface tension decrease that equals to

Ao -

r~PaV~ 2Vs

.

(2.22)

Bubble separation became possible, however, if the bubble lifetime and the solution concentration are sufficient to reduce the surface tension by the value FI = cy0 c~0 is the solvent surface tension,

O'i_ 1

G'i. 1 - A(y,

where

the surface tension for the preceding bubble. As the

surface pressure H value increases with each successive bubble, so will the time interval

55 between the bubbles. For the MPT2 tensiometer the value of Acy is a few tenths of a mN/m. The efficiency of the method is increased with the increase of the measuring system volume. The formation of bubbles terminates when 1-I ~ G0 -cyoo. In the stopped flow regime the MBPM is capable of measuring the surface tension of the solutions for lifetime values of 50- 100 s and more. The results of dynamic surface tension measurements using the MBPM or other methods, e.g., Wilhelmy plate, dynamic capillary, inclined plate, strip, drop volume and oscillating jet methods, were compared for similar surfactant solutions by various authors , (Miller et al. (1994, 1997), Fainerman et al. (1994c), Makievski et al. (1994), Fainerman & Miller (1996), Kloubek (1972b), Van Hunsel & Joos (1987) and Geeraerts & Joos (1994)). The results between the different methods agreed very well, when the surface tension was presented as a function of the effective adsorption time. A satisfactory agreement between the results obtained by various methods was also found for diffusion coefficients or adsorption (desorption) rate constants that were calculated from the experimental data (Kragh 1964, Noskov 1996, Bleys & Joos 1985, Chang & Franses 1995, Li et al. 1994, Borwankar & Wasan 1986, Xua & Rosen 1991 and Joos et al. 1992).

2.4. Experimental technique The results of surface tension measurements using the MBPM for pure liquids and, especially, for solutions of surface active material, depend on the experimental conditions. Some problems arising in connection to the experimental technique were summarised by Mysels (1989, 1989, 1990). The measured surface tension value is affected by the capillary inclination angle and the intensity of the liquid intermixing in the measuring cell (Mysels & Stafford 1989) and the capillary immersion depth (Cuny & Wolf 1956), the diameter and wettability of the vessel containing the liquid under study (Huh & Striven 1969, Lane 1973, Bottomley 1974 and Campanini et al. 1976). For foaming solutions (usually mixtures) it makes no difference whether the surface tension measurements are started from minimal or maximal gas flow rates (Kloubek 1975 and Fainerman et al. 1987). Our own experience with the MBPM has shown that the reproducibility of the results depends significantly on the stability of the process of bubble formation and on the bubble volume. A stable bubble formation (without termination

56 and series formation) is promoted by a large value of the ratio Vb/V s, the increase of the capillary length, the quality of the capillary tip prepared by cutting or splitting and its hydrophilicity outside/hydrophobicity inside it. In addition, to enhance the separation and rise of the bubbles, the extemal diameter of the capillary tip should be somewhat smaller than the diameter of the separating bubble, while the immersion depth should exceed this diameter. A small inclination of the capillary (up to 10~ also improves the performance. And finally, the condition V b = const which means that the volume of separating bubbles is independent of the air flow rate and surface tension, is an extremely important prerequisite. This condition can be fulfilled by installing the electrode or any other sharp body opposite to the capillary tip, see Fig. 2.2. The distance between capillary and electrode should approximately be equal to the critical diameter of the separating bubble

dbc, which

can be defined from the balance of the

capillary and buoyancy forces: dbc = (12rccY/ Apg ) !,,3

(2.23)

To obtain correct values of surface tension of solutions, especially in the short time range, the values of the characteristic parameters of the tensiometer, in particular of Vs/V b and the bubble volume V b have to be chosen in an optimum way. The measured results are also affected strongly by the properties of the internal capillary surface and the capillary length 1, as mentioned above. The results of the studies concerning the influence of these parameters on the measured surface tension value and the recommendations in what regards the optimum choice of these parameters are presented in a paper by Lylyk et al. (1998). The increase of the ratio Vb/V s results in errors in measured surface tensions due to increased pressure oscillation amplitudes in the system. The increase of the separating bubble volume itself leads to an increased deadtime. These effects distort the results of surface tension measurements for solutions, restricting the MBPM applicability under these conditions to dilute solutions and large t I values. On the contrary, to study the surface tension of concentrated solutions in the milli- and submillisecond time range, one has to decrease t 0. To achieve this, one can either decrease Vb, or increase L c (employing shorter or wider capillaries, Fainerman & Miller 1995).

57 The properties of the tip of the capillaries and the internal surface employed in the MBPM vary. Completely hydrophobised glass capillaries (Austin et al. 1967, Hua & Rosen 1988, Miller et al. 1997 and Horozov et al. 1996), capillaries possessing a hydrophobised internal and hydrophilic external surface (Dushkin et al. 1991, Mysels 1986, Iliev & Dushkin 1992) and completely hydrophobic Teflon capillaries (Miller & Meyer 1984, Hirt et al. 1990, Hallowell & Hirt 1994) were used for measurements. The properties of the internal surface of the capillary affect the maximum rise height h of liquid in the capillary after bubble separation. However for short and wide capillaries (K ~ 10) the value of h is negligibly small. For the capillaries studied by Lylyk et al. (1998) (rc = 0.0084 cm, 1 = 1.5 cm) the value ofh determined by high-speed video filming was re to 2r c. The results of dynamic surface tension studies using capillaries with a hydrophobic intemal/hydrophilic external surfaces and entirely hydrophilic capillaries are compared by Lylyk et al. (1998). It was shown that for equal V b the values of cr measured using the hydrophilic capillary are higher than those obtained with a hydrophobic capillary, both for small and large bubble volumes. The increase of cr can be related to the expansion of the liquid surface during the reverse meniscus motion on the t~2 stage. The expansion of the surface decreases the adsorption, leading to the increase of cy for a given lifetime as compared to a non-deformed surface. In the MBPM narrow capillaries (re = 0.005 - 0.015 cm) are usually employed (Bendure 1971, Kloubek 1968, 1971, Woolfrey et al. 1986, Miller et al. 1997 and Horozov et al. 1996), which makes it unnecessary to introduce gravitational corrections. However the length of the capillaries is varied within a rather wide range, from a few millimetres to some centimetres. The effect of the capillary length and the wettability of the internal surface on the surface tension values was also studied by Lylyk et al. (1998) and both hydrophilic and hydrophobic capillaries were employed. The dynamic behaviour of the liquid meniscus within the hydrophobic long capillary is essentially the same as in the hydrophobic short capillary. However the pattern of the liquid flow within the hydrophilic long capillary is rather different. At the final stage of the bubble separation the liquid penetrates into the capillary from the rear side of the separating bubble and rises along the internal capillary surface in the form of an expanded drop. During this process both the volume and the surface of the drop are increasing rapidly until the whole cross-section of the capillary is filled by the liquid. The symmetric meniscus formed in this way subsequently rises to a significant height. The height of maximum

58 liquid rise h depends on the capillary length and the frequency of bubble formation: with the decrease of t b the value of h somewhat decreases. In long narrow capillaries the aperiodic gas flow regime takes place. This results in a prolongation of the meniscus hydrodynamic relaxation time, mainly during the reverse motion phase. The dependence of cy on t I for long hydrophobic capillaries agree satisfactorily with the standard dependence. For the hydrophilic capillaries however, the discrepancy between the results is rather strong. There is a sharp decrease of surface tension for t t --~ 0, while for large t i the values of cy exceed those characteristic for the standard capillary. A sharp decrease of cy at t I --~ 0 for long hydrophilic capillaries can be related to the process of dilation and subsequent fast compression of the liquid surface within the capillary. The fact that the slope of the cy dependence on t ! for a hydrophilic capillary is less pronounced, can be explained by the reverse meniscus motion. The duration of this reverse motion increases with the increase of l and t l, because the liquid penetrates deeper into the capillary. Therefore the longer the hydrophilic capillary, the greater is the part of the lifetime spent on the expansion of the liquid during the reverse meniscus motion. This expansion results in a slower decrease of c~ as compared to the hydrophobic capillary. Therefore the results of dynamic surface tension measurements using MBPM are essentially dependent on the characteristics of the capillaries employed. Reliable results can be expected for relatively short and wide capillaries possessing a hydrophobic intemal surface. The application of short hydrophilic capillaries with the same geometric characteristics (rE/1 > 5.10 .5 cm) does not lead to errors in determining the ~ value. The employment of hydrophilic long capillaries however leads to significant errors in the measured dynamic surface tensions. The relation between the volume of separating bubbles and the measuring system volume affects the accuracy of dynamic surface pressure measurements as well. A ratio of Vs/V b > 5000 is recommended. In addition, the volume of the separating bubble must not be too large, otherwise the increased

td

values would restrict the MBPM applicability to weakly

concentrated solutions and long times. The studies of biological liquids have revealed that capillary parameters and properties produce significant effects in the precision and reproducibility of the data obtained. Best results were obtained with glass capillaries of narrow sections between 7 to 10 m m , and possessing internal diameter of 0.25 to 0.2 mm. We found that these capillaries are most useful for studying

59 biological liquids, because the liquid cannot penetrate into the capillary irrespectively Of the properties of the internal surface.

2.5. Analysis of t e n s i o g r a m s

The results of tensiometric studies of biological liquid are surface tensions at different surface lifetimes (Fig. 2.4). Such tensiogram for serum usually shows a relatively weak decrease of cr at short lifetimes, followed by a rapid decrease at t > 0.1 s. The shape of the tensiograms for other biological liquids show a wide variety: a sharp decrease of ty at t < 0.01 s, the existence of linear sections or one or two extrema, almost no dynamic features throughout the whole measured time range, etc. It is therefore a rather complicated problem to make comparisons between curves. To determine which dynamic tensiogram parameters are most informative, the asymptotic equations of the diffusion controlled adsorption kinetics theory at liquid interfaces have been employed.

74 72

~176176176 ~176 o Oo ~

00%

70 ~

68

66 % 64 0,01

I

t

t

I

0,1

1

10

100

t I [s]

Fig. 2.4. Dynamic surface tension cr of a blood serum sample as a function of surface lifetime h.

The dependence of surface tension on surface lifetime is governed by adsorption/desorption processes of surface active components at the liquid interface. At the initial time moment

60 (t = 0) the surface layer contains no excess of these components, that is, the adsorption is zero, and the surface tension of the solution is equal to that of the solvent, cy0. For most biological liquids, cy0 is close to the surface tension of water D 70 to 74 mN/m. In general, the adsorption rate and the rate of surface tension decrease are determined by the diffusion of surface active molecules towards the surface, and by restructuring processes of the adsorbed molecules within the surface layer (see Chapter 1). The basic equation of the diffusion controlled adsorption kinetics theory was proposed by Ward & Tordai (1946, Eq. 2.12). However, its application is rather cumbersome, because the solution of the resulting integral equation (a Volterra type nonlinear equation) requires additional thermod3,namic and kinetic relations (see Chapter 1). Thus for multicomponent biological liquids one can hardly expect at present any success in the rigorous solution of diffusion kinetics problems. Instead we believe that using asymptotic equations of this adsorption kinetics theory (Van den Bogaert & Joos 1982, Fainerman et al. 1994b, Miller et al. 1994, Hansen 1964, Rillaert & Joos 1982 and Bleys & Joos 1985), provides a more simple, and at the same time a rather informative method of the analysis of dynamic tensiograms. For the case of extremely short times (t --~ 0) a simple relation follows from the general Ward & Tordai Eq. 2.12. For multicomponent solutions this relation can be written as (Fainerman et al. 1994b)

do

= -2RT t--~0

c~ i=l

71;

where the subscript 'i' refers to any i th of the n components. The derivative on the left hand side of this equation (~-0 = [do/dt 1/2 ],-~0)is the slope of o as a function of t 1/2. As the values of diffusion coefficients for different components are of the same order of magnitude, it follows from Eq. (2.24) that this slope is roughly proportional to the total concentration of surface active components of the mixture. The data presented in Fig. 2.4 are re-plotted in Fig. 2.5 in the cy versus tl/2 coordinates.

61 74 ~-

72t~ ~'70-

t~ 6 8 - -

0 0

0

O0

66-

0 O0

0 O O

64 0

1

I

r

2

3

teff 1

/2

/2 [S 1 ]

-

t

4

5

Fig. 2.5. Dynamic surface tension of blood serum sample as a function of teff 1/2. Characteristics of the linear part are: Cyo=72,7mN/m, ~,o= 2,8 mN m-1 s-1/2. It is seen that a linear part of the curves for t--~0 exists in this case. The intersection point of this line with the ordinate axis corresponds to the surface tension of water (in our example ~0=72,7 mN/m). In general, the value of % is determined mainly by the salt composition of the biological liquid. Thus, comparing the dynamic tensiogram slopes in the co-ordinates cy versus t 1/2 one can draw conclusions concerning the total concentration of surface active components in the studied sample. One more important relation, following from the Ward & Tordai theory, is the so-called JoosHansen equation, which is valid for the case of extremely large surface lifetimes. This equation, generalised to multicomponent system, can be represented as (Fainerman et al. 1994b):

•

dcy I RT Fi2 / n dt-1/2 t-.oo = 2 -~-]c~i i i = ,

(2.25)

where F i is the adsorption for the ith surface active component. Here the derivative on the left hand side ( )~ = [dcy/ dt -~/2 ]t_.oo) is taken with respect to (1/2, and is calculated in the limit t --~ oo (that is, t 1/2 --~ 0). As for most surface active components the ratio Fi/ci is constant (the so called Henry constant K i = Fi/ci), and the sum on the right hand side of Eq. (2.25) is an approximate expression for the total adsorption of all mixture components with reference to

62 their adsorption activity K i. Therefore, comparing the values of the derivative ~L=(do/dt-1/2)t _~oo for various samples of biologic liquids, one can deduce information on changes in the adsorptions. The experimental dependencies presented in the Figs. 2.4 and 2.5 are replotted in Fig. 2.6 in the coordinates of Eq. (2.25). It is seen that the dependence 0 versus t 1/2 in fact possesses a linear part at t -1/2 --~ 0 (t ~ oo). The intersection point of this linear part with the ordinate axis corresponds to the equilibrium surface tension Goo (i.e., reduced to infinite time t ~ oo). This characteristic is extremely important; it is seen that it can be rigorously obtained only from the extrapolation of a dynamic tensiogram in the co-ordinates G versus t -1/2.

74 7-

72 ~

OO

O

O

O

O

O

O

O

7O +

~

68! 66

,,

64 / 62 i 1

t

I

I

t

2

3

4

5

tef? 1/2 [S "1/2 ]

Fig. 2.6. Dynamic surface tension of a blood serum sample as a function o f teff"I/2, the characteristics of the linear part are: g~o=62,9mN/m, k =11,2 mN m-~ s ~/2. In addition

to

the

tensiographic

parameters

mentioned

above,

namely

00,

0o0--03,

Lo = -(dG/dtl/z)t - o, ~" = (dG/dtI/Z)t- oo, we have used also the dynamic surface tensions at two other points of the tensiogram: 0~ for t = 0.01 s, and 02 for t = 1 s. The values 0 and Lo are characteristic for solvent properties and adsorption processes in the short lifetime range, while the value of 02 is indicative of the properties and processes in the medium surface lifetime range. These processes are governed mostly by the presence of low- and medium-molecular

63 weight surfactants in the composition of biological liquids, while the values of cy3 and ~ are controlled by the properties of high-molecular weight fractions of albumins and other compounds. As the result of this chapter one can conclude that the maximum bubble pressure technique is uniquely suited for studies of biological liquids. The methodology is well elaborated, experimentally and theoretically, and provides reliable dynamic surface tension data in a time interval important for these liquids. It turns out that particularly the time range of milliseconds up to seconds is extremely sensitive to the composition of blood, urine and other medically relevant liquids. The characteristic values which can be extracted from the complete tensiogram are sensitive to changes in the liquid composition, and hence carry information useful for diagnostic and therapeutic matter. The subsequent chapters will demonstrate this fact systematically.

2.6.

Summary

The maximum bubble pressure tensiometry is a modern and reliable tool to accurately measure dynamic surface tensions. It is shown how the measured physical values, capillary pressure as a function of gas flow rate, are interpreted as dynamic surface tension in function of the effective surface lifetime. The method gives access to data even in a time interval down to less than one millisecond. The method is theoretically well founded and all phenomena observed under the wide variety of experimental conditions can be described adequately by hydrodynamic theories. Also for viscous liquids, such as serum, the measured data are quantitatively understood. The analysis of the dynamic surface tension curves provides a number of characteristic values which are of great importance for medical research. Particular plots are discussed which give easy access to these characteristic values. The subsequent chapters 3 to 8 will describe which of the defined characteristic values correlate with biochemical data and thus are relevant as diagnostic tool and for monitoring the progress of medical treatments.

64 2.7. References

Austin, M., Bright, B.B. and Simpson, E.A., J. Colloid Interface Sci., 23(1967)108 Belov, P.T., Zh. Fiz. Khim., 55(1981 )302 Bendure, R.L., J. Colloid Interface Sci., 35(1971)238 Bleys, G. and Joos, P., J. Phys. Chem., 89(1985)1027 Borwankar, R.P. and Wasan, D.T., Chem. Eng. Sci., 41 (1986) 199 Bottomley, G.A., Austr. J. Chem., 27(1974)2297 Brown, R.C., Philos. Mag., 13 (1932) 578 Campanini, R., Swanson, A. and Nicol, S.K., J. Chem. Soc. Faraday Trans. 1, 72(1976)2638 Chang, C.-H. and Franses, E.I., Colloids Surfaces A, 100(1995)1 Cuny, K.H. and Wolf, K.L., Ann. Phys. Leipzig, 17(1956)57 Dukhin, S.S., Fainerman, V.B. and Miller, R., Colloids Surfaces A, 114(1996)61 Dukhin, S.S., Mishchuk, N.A., Fainerman, V.B. and Miller, R., Colloids Surfaces A, 138(1998)51 Dushkin, C.D., Ivanov, I.B. and Kralchevsky, P.A., Colloid Surfaces,60(1991)235 Fainerman, V.B. and Lylyk, S.V., Kolloidn. Zh., 44(1982)598 Fainerman, V.B. and Miller, R., J. Colloid Interface Sci., 175(1995)118 Fainerman, V.B. and Miller, R., J. Colloid Interface Sci., 178(1996)168. Fainerman, V.B., Colloids Surfaces, 62(1992)333 Fainerman, V.B., Kolloidn. Zh., 41 (1979) 111 Fainerman, V.B., Kolloidn. Zh., 52(1990) 921 Fainerman, V.B., Lylyk, S.V. and Jamilova, V.D., Kolloidn. Zh., 49(1987)509 Fainerman, V.B., Miller, R. and Joos, P., Colloid Polymer Sci., 272(1994a)731 Fainerman, V.B., Makievski, A.V. and Miller, R., Colloids Surfaces A, 87(1994b)61.

65 Fainerman, V.B., Makievski, A.V. and Joos, P., Colloids Surfaces A., 90(1994c)213 Fainerman, V.B., Makievski, A.V. and Miller, R.,. Colloids Surfaces A, 75(1993)229 Fainerman, V.B., Zholob, S.A., Miller, R., Loglio, G. and Cini, R., Tenside-Detergents, 33(1996)452 Feldman, I.N., Malkova, I.V., Sokolovskij, V.I. and Zaturenskij, R.A., Zh. Prikl. Khim., 53(1980)1594 Garrett, P.R. and Ward, D.R., J. Colloid Interface Sci., 132(1989)475 Geeraerts, G. and Joos, P., Colloids Surfaces A., 90(1994) 149 Hallowell, C.P. and Hirt, D.E., J. Colloid Interfaces Sci., 168(1994)281 Hansen, R.S., J. Phys. Chem., 60(1964)637. Hirt, D.E., Prud'homme, R.K., Miller, B. and Rebenfeld, L., Colloids Surfaces, 44(1990)101 Horozov, T.S., Dushkin, C.D., Danov, K.D., Arnaudov, L.N., Velev, O.D., Mehreteab, A. and Broze, G., Colloids Surfaces A., 113(1996) 117 Hua, X.Y. and Rosen, M.J., J. Colloid Interface Sci., 141 (1991) 180 Hua, X.Y. and Rosen, M.J., J. Colloid Interface Sci.,124(1988) 652 Huh, C. and Scriven, E.L., J. Colloid Interface Sci., 30(1969)325 Iliev, Tz. H. and Dushkin, C.D., Colloid Polymer Sci., 270(1992)370 Jaeger, F.M., Z. Anorg.Chem., 101 (1917) 1 Johnson, C.H.J. and Lane, J.E., J. Colloid Interface Sci., 47(1974)117 Joos, P. and Rillaerts, E., J. Colloid Interface Sci., 79(1981)96 Joos, P. and Van Uffelen, M., J. Colloid Interface Sci., 171(1995)297 Joos, P., Fang, J.P. and Serrien, G., J. Colloid Interface Sci., 151 (1992) 144 Kao, R.L., Edwards, D.A., Wasan, D.T. and Chen, E., J. Colloid Interface Sci., 148(1992)247 Keen, G.S. and Blake, J.R., J. Colloid Interface Sci., 180(1996)625 Kisil', I.S., Mal'ko, A.G. and Dranchuk, M.M., Zh. Fiz. Khim.,55(1981)177

66 Kloubek, J., Colloid Polymer Sci., 253(1975)754

Kloubek, J., J. Colloid Interface Sci., 41 (1972a)7 Kloubek, J., J. Colloid Interface Sci., 41 (1972b) 17 Kloubek, J., Tenside, 5(1968) 317 Koval'chuk, V.I., Dukhin, S.S., Fainerman, V.B. and Miller, R., J. Colloid Interface Sci., 197(1998)383 Koval'chuk, V.I., Dukhin, S.S., Makievski, A.V., Fainerman, V.B. and Miller, R., J. Colloid Interface Sci., 198(1998) 191 Kragh, A.M., Trans. Faraday Soc., 60(1964)225 Kwok, D.Y., Hui, W., Lin, R. and Neumann, A.W., Langmuir, 11(1995)2669. Lane, J.E., J. Colloid Interface Sci., 42(1973)145 Li, B., Geeraerts, G. and Joos, P., Colloids Surfaces A, 88(1994)251 Lunkenheimer, K., Miller, R. and Becht, J., Colloid Polymer Sci., 260(1982)1145 Lylyk, S.V., Makievski, A.V., Koval'chuk, V.I., Schano, K.-H., Fainerman, V.B. and Miller, R., Colloids Surfaces A, 135(1998)27 Makievski, A.V., Fainerman, V.B. and Joos, P., J. Colloid Interface Sci., 166(1994)6 Miller, R., Fainerman, V.B., Schano, K.-H., Heyer, W., Hofmann, A. and Hartmann, R., Labor Praxis, N9(1994)65 Miller, R., Fainerman, V.B., Schano, K.-H., Hofmann, A. and Heyer, W., Tenside-Detergents, 34(1997)357 Miller, R., Hofmann, A., Schano, K.-H., Halbig, A. and Hartmann, R., Tenside-Detergents, 28(1992)435. Miller, R., Joos, P. and Fainerman, V.B., Adv. Colloid and Interface Sci., 49(1994)249 Miller, T.F. and Meyer, W.C., American Laboratory, February (1984) 91 Mysels, K.J. and Stafford, R.E., Colloids Surfaces, 36(1986)289

67 Mysels, K.J. and Stafford, R.E., Colloids Surfaces, 41 (1989)385 Mysels, K.J., Colloid Surfaces, 43 (1990) 241 Mysels, K.J., Langmuir, 2(1986)428 Mysels, K.J., Langmuir, 5(1989)442 Noskov, B.A., Adv. Colloid and Interface Sci., 69(1996)63 Pugachevich, P.P., Zh. Fiz. Khim., 38(1964) 758 Razouk, R. and Walmsley, D., J. Colloid Interface Sci., 47(1974)515 Rillaerts, E. and Joos, P., J. Phys. Chem., 86(1982)3471. Ross, J.L., Bruce, W.D. and Janna, W.S., Langmuir, 8(1992) 2644 Rusanov, A.I. and Prokhorov, V.A., Interfacial Tensiometry, in Studies of Interface Science, Vol.3, D. M6bius and R. Miller (Editors), Elsevier, Amsterdam, 1996 Simon, M., Ann. Chim. Phys. 32 (1851) 5 Sugden, S., J. Chem. Soc., 121 (1922) 858 Sugden, S., J. Chem. Soc.,125(1924) 27 Van den Bogaert, R. and Joos, P., J. Phys. Chem., 96(1982)3471 Van Hunsel, J. and Joos, P., Colloids Surfaces, 24(1987)139 Volkov, B.N. and Volyak, L.D., Zh. Fiz. Khim., 46(1972)598 Ward, A.F. and Tordai, L., J. Chem. Phys., 14(1946)453. Warren, E.L, Philos. Mag., 4(1927) 358 Woolfrey, S.G., Banzon, G.M. and Groves, M.J., J. Colloid Interface Sci., 112(1986)583

68

Chapter 3

Dynamic interfacial tensiometry of biological liquids obtained from healthy persons All biological liquids of the human organism contain surface active compounds, such as proteins, lipids, and molecules of other nature. These surfactants are characterised by a high adsorption activity at low bulk concentrations which significantly effects equilibrium interfacial properties and the kinetics of physicochemical processes taking place at interfaces (disperse systems of biological liquids, cell membranes). Surfactants may be synthesized endogenously by specific cells or enter the body exogenously through, e.g., the intestine, skin or lungs. Both types of surfactants may undergo various metabolic transformations. Some of the surfactants could be collected in blood others in urine samples. The fact that an appropriate theory was already elaborated, and advanced experimental techniques were available for tensiometric measurements (as discussed in the previous chapters) enabled us to perform systematic experiments with actual human biological liquids. This chapter will describe values of dynamic surface tension characteristics for healthy persons. We will show that dynamic surface tension depend on sex and age. In addition, surface tension characteristics various during pregnancy.

3.1. Dynamic surface tension depend on sex and age All biological liquids of the human organism contain surface active compounds, such as proteins, lipids, and molecules of other nature. These surfactants are characterised by a high adsorption activity at low bulk concentrations which significantly effects equilibrium interfacial properties and the kinetics of physicochemical processes taking place at interfaces (disperse systems of biological liquids, cell membranes). Surfactants may be synthesised endogenously by specific cells or enter the body exogenously through, e.g., the intestine, skin or lungs. Both types of surfactants may undergo various metabolic transformations. Some of the surfactants could be collected in blood others in urine samples.

69 The fact that an appropriate theory was already elaborated, and advanced experimental techniques were available for tensiometric measurements (as discussed in the previous chapters) enabled us to perform systematic experiments with actual h u m a n biological liquids. Table 3.1 summarises averaged values o f dynamic surface tension parameters for serum and urine samples obtained from 80 healthy persons that were between 15 to 65 years old. However, the average data presented b e l o w do not discriminate between persons' sex and age. This will be considered later. Table 3.1. Normal values of dynamic surface tension parameters for serum and urine obtained from 80 healthy volunteers Parameter*

Biological liquid** Serum

Urine

70.0+0.41

71.5 + 0.33

67.7 + 0.35

69.3 + 0.21

60.0 + 0.44

61.8+0.36

X0 [ m N ' m l ' s "I/2]

4.5 + 0.74

4.9+0.65

)~ [ m N / m l . s 1/2]

12.6 + 0.54

13.5 + 0.47

0"1

[mN/m]

0"2 [mN/m]

0" 3

[mN/m]

* Cl = surface tension at t = 0.01 s, 0"2 surface tension at t = 1 s =-

0"3 = 0"~ derived obtained by extrapolation for t ~ oo )~ = ( d 0 " / d t l / Z ) t ~ oo.

9~o = -( d0./dtl/Z)t ~ o,

** Data are given as interval M+3m, with M characterising the average value of a parameter and

m2

the

distribution of this measured value (m2= ~;2/n), where e is the standard deviation, n is the number of volunteers. The interval M+3m corresponds to a probability of 0.9973 that the measured value occurs within the interval [M-3m, M+3m] and may serve as a normal value.

70 For our analysis of dynamic tensiograms obtained from biological liquids we used the following parameters (see last paragraph of Chapter 2 for a detailed explanation): (11 - surface tension at t = 0.01 s, (12 -- surface tension at t = 1 s (13 = (1ooderived obtained by extrapolation for t --~ oo = -(d(1/dtl/2)t -~ o, = (d(1/dtln)t -, oo. In the short time range the surface tensions of serum and urine is by few mN/m lower than that of pure water. The equilibrium surface tensions for both liquids is about 60 mN/m. We have performed an analysis of correlations between various parameters of dynamic surface tensions of biologic liquids taken from healthy persons. The purpose of this studies was twofold. First, high correlation coefficients between certain parameters can be regarded to as an indication for a link between these parameters as they are determined by the same constituents of biologic liquids, or by the same processes which take place therein. If the value of the correlation coefficient between two compared parameters is close to unity, it means that the choice of one of this parameters was inappropriate. On the contrary, low values of correlation coefficients can indicate that these parameters are independent of each other. Second, studies of the correlation between tensiographic parameters under 'standard' conditions, that is, for healthy persons, provides us with an extra tool for the analysis of pathologies, based at the difference of correlation coefficients for certain parameters of dynamic tensiograms. In fact, strong direct correlation (with coefficient r=0.8-0.9) was observed between some parameters of dynamic surface tension for serum and urine (see Fig. 3.1). As expected, a correlation exists in 'neighbouring' surface lifetime ranges ((11- (12, (12- (13). However, the slope L of serum tensiograms does not depend on the other surface tension parameters of this biological liquid, while the L value for urine exhibits a weak positive correlation with the dynamic surface tension crl at t = 0.01 s and (12 at t = 1 s, and a negative correlation with the equilibrium surface tension (13. Dynamic surface tensions of serum depend on its biochemical composition. There exists a strong dependence of (I2 of serum on the concentration of lipids, while ~3 strongly depends on the concentration of proteins (Kazakov

71 et al. 1996a). It can be argued that the excretion of these surfactants via kidneys can lead to the variations in the dynamic surface tension parameters of urine, that is, certain relationships can exist between surface tension parameters o f serum and urine. At the same time, a significant relationship between equilibrium surface tension t~3 and X for urine takes place only with dynamic surface tension parameter or1 of serum (Table 3.2).

a) serum _

0.8

-

0.6O

o r,.)

0.4-

9~

0.2 -

0

0

cj -0.2 -0.4 o1

~2

~3 b) urine

0.8

-

0.6

-

0.4o

o

O

0.2-

-o.2-0.4 -0.6 t~l

t~2

~3

Fig. 3.1. Correlations between surface tension parameters of biological liquids obtained from healthy persons, hatched - cq, black - or2,white - or3

72 Table 3.2. Correlation coefficients between particular surface tension parameters in serum and urine obtained from healthy persons

Serum Urine

0"1

0"2

0"3

0"1

+0.07

+0.23

+0.17

+0.38

0"2

-0.12

-0.13

-0.07

+0.26

0"3

-0.52

-0.36

~

+0.16

+0.53

+0.37

+0.14

-0.06

While surface active constituents of serum are well known and extensively studied, such constituents in urine are rather unknown. Proteinuria in healthy persons is unlikely; however, proteins are mostly responsible for the surface tension of urine even in a healthy person. Normally, slight proteinuria (100-150 mg per day) is attributed to the existence of functional kidney glomerular barriers characterised by selective permittivity with respect to plasma proteins. This permittivity depends on the size, electric charge and configuration of the protein molecules, and also on hydrodynamic factors and the intensity of re-absorption in the tubular apparatus. Among the proteins of unchanged urine, 40% are tissue proteins, secreted by the cells of tubules and mucous coating uropoietic organs, in particular, the Tamm-Horsfall mucoprotein, which possesses a high molecular mass (7000 kDa) and the ability for coagulation within the tubular lumen, forming the matrix of cylinders. Also traces of proteins from the secretion of sexual glands are present in healthy person's urine. All these proteins are likely to determine interfacial tensiometric parameters of urine. The osmolarity of urine probably can affect significantly its dynamic surface tension. Depending on the water/electrolyte balance of the organism, either osmotically concentrated, or hypotonic urine is secreted. The portion of plasmatic secreted proteins in this case is negligibly, and the excretion of osmotically active substances depends on the absolute and relative amounts of soluble electrolytes (sodium, potassium, ammonium). The amount of electrolytes in urine is determined by glomerular filtration, extent of tubular secretion, and reabsorption. These factors, quite naturally, can influence the parameters of dynamic surface tensions of urine.

73 The disbalance in the composition of protein and fat in blood can lead to a hemocoagulation even in healthy persons. This, in turn, can affect the parameters of interfacial tensiometry. For example, if a disbalance in lipid homeostasis exists, then an incorporation of free cholesterol into erythrocyte membranes can happen leading to a change in the cholesterol/phospholipid ratio, accompanied by a transfer of surface active phosphatidyl choline from cells to blood. This surfactant also affects the rheological characteristics of adsorbed layers, increasing its viscosity. Interactions of various metabolites with proteins lead to changes in the molecular structure of proteins, hence they determine changes in their physicochemical characteristics, e.g. viscosity and surface tension. It was mentioned in Chapter 2 that the surface viscosity can affect the results of dynamic interfacial tensiometry in the very short surface lifetime range; that is, surface tension cr~ to some extent reflects also the rheological characteristics of the surface layers. Due to the existence of hydrodynamic effects, a slight influence of the bulk viscosity of liquid also takes place. Table 3.3. Surface tension parameters of serum and urine for healthy persons with respect to sex Biological liquid

Serum

Urine

Parameter*

Male**

Female

or1, mN/m

69.2 + 0.50

70.8 + 0.59

cry, mN/m

67.1 + 0.39

68.3 + 0.54

~2, mN/m

59.3 + 0.18

61.3 • 0.65 w

Lo, m N ' m l ' s ''n

4.9 + 0.90

3.6 + 0.70

~, mN/ml.s 1/2

15.3 + 0.61

8.2 + 0.60 w

G1, mN/m

71.6 + 0.24

71.5 + 0.21

oh, mN/m

69.2 + 0.27

69.3 + 0.32

~3, mN/m

56.6 + 1.81

61.1 + 0.36 w

Lo, mN'm-l's -'n

5.5 + 0.70

5.2 + 0.65

~, mN/m-l.s '~

11.7+0.43

15.2 + 0.54 w

* see comments at table 3.1 **

Sex

see comments at table 3.1

w significantlydifferent between males and females

74 Differences in dynamic interfacial tensiometric parameters exist comparing sex and age (Table 3.3, Figs. 3.2., Fig. 3.3).

76 74

~

7o } 68 ~ 66 64 60 4

~

~

-2

-1

0

1

lg(tef) IS] Fig.3.2. Examples for serum tensiograms obtained from healthy persons of different age: solid thick-male 52 years, solid thin- male 27 years, dashed thick- female 37 years, thin dashed- female 26 years. Equilibrium surface tensions of serum and urine obtained from female are higher than male. The slope of the tensiograms ~. are also different comparing gender: it is higher in serum but lower in urine obtained from males. The relatively high g3 values for female serum can in part be attributed to lower contents of some proteins, lipids and hydrocarbon in their blood. In particular, female serum has lower physiological level of low density and very low density lipoproteins, and a number of ferments (creatine kinase, a-glutamyl transpeptidase, v-glutamate dehydrogenase etc., Kazakov et al. 1996a). In addition, sex related differences exist regarding variations of fractions of phospholipids, cholesterol, triglycerides, free fatty acids, polysaccharides

(galactose,

galactose

amine,

hexose,

fucose),

and

uric acid.

Furthermore, sex related differences exist in the occurrence of protein molecules that contain amino-acids possessing hydrophilic radicals (Grunenberg et al. 1996, Tamagur et al. 1992). One example for a sex related difference in protein concentration is fibronectin. Fibronectin is a high-molecular weight glycoprotein, comprised of two chains which have equal size, linked to each other by disulphide bridges, and contains up to 5% carbohydrates (mannose, galactose,

75 fucose, N-acetylglucosamine, sialic acids). The sources of circulating fibronectin are neutrophiles, macrophages, thrombocytes, fibroblasts, vascular endothelium, hepato- and nephrocytes.

Fibronectin

glycosaminoglycanes,

can be bound

actin,

immune

with

fibrinogen,

complexes

fibrin,

(containing

collagen,

gelatine,

immunoglobulin-G,

immunoglobulin-M, Clq- and C3b-components of complement) (Mosher & Williams 1978 and Baglin et al. 1987). Fibronectin participates in plasma coagulation leading to generation of a fibrin clot. Thus the concentration of fibronectin in serum is 35 to 40% lower than in plasma. The lower the amount of fibronectin in the blood, the higher is the level of circulating fibrinogen/fibrin complexes therein (Vasiliev et al. 1994). The concentration of fibronectin is directly correlated with the concentration of cholesterol, triglycerides and low density lipoproteins. It should be stressed that the content of fibronectin in blood serum for healthy males is much higher than for females (Vasilieva et al. 1991).

75

--

~

70

~

m

""

O.o

i,.,..,.i

65-

60

-2

-1

0

1

lg(tef) [S]

Fig.3.3. Examples for urine tensiograms obtained from healthy persons of different age: solid thick-male 27 years, solid thin - male 19 years, dashed thick- female 48 years, dashed thin- female 30 years. It was shown that an inverse correlation exists between equilibrium surface tension of blood serum for healthy people, and the concentration of such surfactants as cholesterol, triglycerides and fibronectin.

76 Differences between equilibrium surface tension values for persons of different gender may not only due to proteins but can result from different levels of eicosanoides (prostanoides, fatty oxyacids, leukotrienes), and non-lipid (palmitic and hyaluronic acid) or non-protein nitrous components (urea, creatinine, uric acid). Therefore, for males the differences in content and structure of protein and lipid components of urine result in a more pronounced decrease of surface tension. We have found correlations between particular interfacial tensiometric parameters that are sex related. For example, in serum and urine sampled from males the relation between 0"2 and 0"3 is stronger than in samples from females. However, in urine sampled from females equilibrium surface tension parameters depend on ~.. In general, serum and urine sampled from man or women usually show similar sex related correlation links between surface tension parameters. Some of these correlations are stronger than others. Strong correlations exist between X values of urine and 0"1 or 0"2 values of serum in male, see Table 3.4. In females, such correlations were generally not detected, but the relationship between the dynamic surface tension parameters for serum in the short and medium surface lifetime range, and the equilibrium surface tension for urine becomes stronger. For both sexes, the k values of serum are strongly correlated with the 0"1 value of urine. Table 3.4. Correlations between particular surface tension parameters of urine and serum sampled from healthy male or female.

Serum

Sex Urine Male

0"1

0"2

0"3

1"1"

0"1 0"2 0"3

1"1'1" Female

1"1" 1"1'

0"1 0"2 0"3

r162

t - positive correlation; $ - negative correlation; empty- no correlation r < 0.3; one symbol - r < 0.5; two symbols - r = 0.5-0.7; three symbols - r > 0.7

77 In blood of males the concentration of a number of proteins and lipids prevails. Therefore we speculated that a strong positive relation exists between the surface tension parameters of serum and urine in healthy men. However, such a relationship was not observed. The influence of such surfactants on the )~ values of urine (strong positive correlation with ~l and ~2) is difficult to explain. A rather weak, but opposite correlation of equilibrium blood surface tensions for males and females with the dynamic surface tension or1 at t = 0.01 s also remains unexplained. We conclude that dynamic tensiographic parameters depend on some particular, and possibly very complicated combinations of surfactants that are specific constituents of either blood or urine or may exist in both liquids but in different concentrations.. After describing gender related dependencies on dynamic tensiograms, we will now describe age related dependencies. With increasing age, a gradual growth of surface tension of blood, and a gradual decrease of surface tension of urine take place (Figs. 3.4 and 3.5), with most pronounced changes occurring in the very short time ranges.

171513 "

r,r

~

ll 9_

I

I

<20

I

25

I

I

30

I

I

35

I

I

40

I

I

45

I

I

50

t

t

55

I

I

60

I

I-I

>60

Age [years] Fig. 3.4. Values of ~, (mN.m-1.s 1/2) for serum (II~) and urine ([2) of healthy persons as a function of age.

The increase of

O" 1

values for serum during aging may be in part due to changes of the

biosynthesis and metabolism of proteins and lipids, that leds to changes in the level of surfactants in biological liquids. Examples are insulin and steroid and thyreoid hormones. The production and secretion of which gradually decrease during ageing. In addition, the response

78 of receptors with respect to arginine-vasopressin, adrenaline and thyroxin of target cells in kidney, liver, and the hypothalamo-hypophysiary system deteriorates during ageing (Kazakov et al. 1996a).

a) serum

75 70

m

~' 65

55! 50 +

'

~ t

<20

25

!

t

i -~

30

35

,

t

J

40

t

t

',

q-~

~

50

45

L

55

t

t

60

I

>60

Age [years] b)

urine

75 ~

7650 1 "

E~

--

--i

i

60 i 50

L i <20

! I 25

t 30

i

t 35

t

t 40

t

I 45

t

~ I 50

t 55

t

4

60

t

I -q

>60

Age [years]

Fig. 3.5. Surface tension parameters measured in serum and urine obtained from healthy persons as a function of age; (~')- crt, (11)- or2,(A)- or3parameters.

79 While the value of ~, for urine tensiograms increases over the first 40 years of life and then remains virtually constant, the ~, value of serum gradually decreases with age. The variations in the slope of tensiograms are related to ~l as was shown earlier. It supports our view that a positive correlation exists between these dynamic tensiographic parameters for different biological liquids. Specific features of dynamic surface tension parameters exist for serum and urine that are related to sex and age. These features are not reflected by the averaged data as presented in Table 3.1. Therefore, we present here a table that summarises normal values of dynamic interfacial tensiometric parameters with respect to sex and age. This table can be used by clinicians for practical purposes. Table 3.5 summarises our tensiographic data of serum and urine obtained from healthy persons of different sex and age. Surface tension parameters of the two biological liquids serum and urine are given as the range M+3m. Here M characterises the average value of a parameter and m 2 the dispersion of this measured value. The interval M+3m corresponds to a probability of 0.9973 that the measured value occurs within the interval [M3m, M+3m] and may serve as a normal value. The value for ~0 in the range M + 3m shows weak differentiation with respect to age and sex, and for both liquids. It is equal to 27 mN.ml.s v2. The normal values of dynamic surface tension parameters given in Table 3.5 were found in serum and urine that was obtained from healthy volunteers older than 15 years. However, dynamic tensiograms might be different when samples from children are analysed. Indeed, some differences were observed in the behaviour of dynamic tensiograms for children. 65 healthy children (boys and girls) of 2 months to 15 years old have been screened. The cyl value of serum from children is by 1.2 to 2 mN/m higher than for adults (see Table 3.3), while ~2 and cy3 of serum and urine differ from corresponding values for adults by less than 1 mN/m. It should be noted that the average tensiometric value L of serum for the two sexes in adults is virtually equal (12 mN.ml.s-l/2). In contrast, this value measured in samples from girls exceeds that of adult females by 50%, while for boys this value is by 20% lower than for adult males. Therefore, this parameter increases with males' age, and decreases with female's age. Fig. 3.6 which should be compared with the data presented in Fig. 3.4, summarises the values of this parameter for children and adults throughout the range of age. The ~ value of urine for children is by 20% lower than for adults of the same sex (see Table 3.3). While for adults a continuous

80 increase o f o l , ~2 and t~3 values for s e r u m takes place with age (see Fig. 3.5), no such d e p e n d e n c e w a s o b s e r v e d for children. Table 3.5. Normal values of dynamic surface tension parameters for serum and urine obtained from healthy volunteers Biological

Sex

Age

Surface tension parameter

Number of

liquid

volunteers

[mN/m] Serum

Males

Females

or2

or3

L

[mN/m]

[mN/m]

[mN.s 1/2.ml]

(~1

< 20

8

64-68

64-68

56-59

16-22

20-35

10

68-72

65-69

57-60

13-21

36-50

9

68-72

66-69

57-63

12-16

> 50

II

68-74

66-71

57-66

10-16

< 20

10

66'70

65-69

58-61

9-15

9

Urine

Males

._

20-35

11

68-74

66-70

59-62

8'14

36-50

10

70-74

67-70

59-65

5-10

> 50

11

70'76

67-72

59-68

4-10

< 20

8

72-73

69-71

61-63

7-12

20-35

10

70-72

68-70

59-62

10-15

36-50

9

70-72

68-70

57-60

10-15

> 50

11

70-73

67-71

56-61

10-16

_

"Females

< 20

10

72-73

69-71

65-68

11-16

20-35

11

70-72

68-70

64-66

14-19

36-50

10

70-72

68-70"

61-65

14-17

70-73

67-71

60-65

12-19

,.

> 50

11

o~ = surface tension at t = 0.01 s (~2 surface tension at t = 1 s or3 = crooderived obtained by extrapolation for t --~ oo L = (dc#dtV2)t ~ o~. Data are given as interval M+3m, with M characterising the average value of a parameter and m2 the dispersion of this measured value (m 2= t~2/n), where e is the standard deviation, n is the number of volunteers. The interval M+3m corresponds to a probability of 0.9973 that the measured value occurs within the interval [M-3m, M+3m] and may serve as a normal value. =

The values for ~, in s e r u m are rather high for children y o u n g e r than 1 year, decrease t o w a r d s the pubertal period, and then sharply increase, attaining its m a x i m u m in the age o f 16 to 20. S u b s e q u e n t l y a gradual decrease o f this p a r a m e t e r takes place. The ~,-values o f urine for juveniles

are

14 m N . m "l .s -1/2.

rather

stable

and

low

(10-12mN-ml.sl/2),

and

increase

with

age

to

81

1715-

I

13-

,<

9_

I

1

2

3

4

5

6

7

8

9

I 10

Age group

Fig. 3.6. Values of ~ for serum (white) and urine (hatched) for children and adults of various age 9age: 1 - less than 1 year, 2 - 1-5 years, 3 - 6-10 years, 4 - 11-15 years, 5 - 16-20 years, 6 - 21-30 years, 7 - 31-40 years, 8 - 41-50 years, 9 - 51-60 years, 10 - 60 years and more

In contrast to adults, the correlations between various values of surface tension of serum and urine for children typically do not exist. However, the tensiographic k values for these two biological liquids is much more dependent on the equilibrium surface tension of the same liquids ( r - 0 . 6 8 - 0 . 7 3 )

than for adults. Correlations between the parameters of different

biological liquids for children are also different to those characteristics for adults, see Tables 3.2 and 3.4. For example,'a dependence exists between ci, 13"2 and ~3 values of urine and the equilibrium blood surface tension, but the corresponding correlation coefficients are quite low, in the range of 0.33 to 0.35. The surface tensions of urine in the short time range is to some extent correlated with that of serum (r = 0.35 for boys and 0.43 for girls).

3.2. Dynamic surface tension of blood and amniotic liquid changes during pregnancy It is well-known that during normal gestation a variation in the surfactant spectrum in blood and amniotic liquid takes place (Radzinsky etal. 1993). Biochemical and hystochemical studies have shown that the placenta produces many proteins a great amount of which are "pregnancy-specific proteins". During the early period of gestation, the "requirements" of the

82 organ are satisfied at the expense of placental proteins, which support its growth, differentiation and normal existence. Starting from the 16th/17th week, and until the end of the gestation period, the produced proteins are exported to the organisms of mother and fetus to support the homeostasis of the fetoplacental complex. An extremely important feature of mature placenta is the production of secretory proteins, which are used for the building of enzymes, globulins and hormones which possess surface active properties. Therefore variations in the surfactant composition of blood and amniotic liquid should primarily affect the surface tensions of these biological liquids. In blood of pregnant women, a significant increase in the concentration of C-reactive proteins, ceruloplasmin

(ot-metalloseromucoid),

oxytocinase,

thermolabile

alkalic

phosphatase,

et-fetoprotein is observed. In the placenta, the synthesis of phospholipids (surfactants, which are the main structural components of the cell membrane, and model the functions of hormones and enzymes) and triglycerides occurs. Placental metabolism of fats plays an important role in determining the membrane characteristics and cell replication in immune reactions. The content of lipid fractions in maternal plasma is always higher than in the fetus. At the end of the gestation period the amount of free fatty acids and phospholipids, like lysophosphatidyl choline and sphingomyelin sharply increase in placental tissue while the contents of cholesterol and triglycerides remains higher in maternal blood (Radzinsky & Smal'ko 1987, Mclntosh et al. 1984, Moniz et al. 1985). It should be noted that lipids are synthesised by the placenta from maternal precursors, and the transport of fats is performed after their preliminary enzymatic splitting (Savelieva et al. 1991, Foster & Dos 1984). The increase in the amount of cholesterol, triglycerides and non-etherified fatty acids in the amniotic liquid, and the decrease of the phospholipid levels therein is considered to be a symptom of placental insufficiency, and is usually observed when complications of gestation occurs. The lipidemia level in the mother's organism strictly determines the mass of foetus and the so-called fatty status of infant (Komissarova et al. 1988, AI et al. 1995, 1996). It follows from the data of Kazakov et al. (1996b, 1997) that the increase of concentration of cholesterol, triglycerides and high density lipoproteins in blood takes place at the end of a normal gestation period. It should be noted that a continuous (almost linear) increase of very low density lipoproteins in serum during the gestation period is observed. In contrast the

83 amount of low density lipoproteins up to the 20th week of gestation decreases and attains a value characteristic to non-pregnant women in the 40th week of gestation. Very low density lipoproteins affect the concentration of cyclic nucleotides due to the inhibition of the phosphatidyl-inositol cycle. High levels of antibodies, low density lipoproteins and very low density lipoproteins is supposed to be one of the factors leading to abortion (Tuppla et al. 1995). The amount of low density lipoproteins in blood increases just in the 17th - 24th week of gestation. These contradictory results of studies might be explained by the diversity of studied populations with respect to age, genetic and environmental factors. A correlation exists between the level of low density lipoproteins and the age and number of previous pregnancies. In addition, levels of cholesterol, triglycerides and low density lipoproteins depend on the duration of gestation. While for non-pregnant women the relative amounts of low density lipoproteins, high density lipoproteins and very low density lipoproteins are 8:4:1, for the 20th week of gestation and the further period this relation becomes 2:1:1. The studies of possible relationships between the parturition course and state of infant with lipid exchange parameters are of special practical interest. An analysis has shown that the duration of the 1st period of parturition and the dimensions of the fetus are directly related with the concentration of cholesterol, triglycerides, and atherogenic low density lipoproteins and very low density lipoproteins in blood. High levels of atherogenic high density lipoproteins determine the status of the fetus, and, in combination with very low density lipoproteins, reflect the amount of amniotic liquid. It was shown that the increase of the cholesterol content in amniotic liquid indicates the decrease in the duration during the 3rd trimenon, and vice versa. It is quite clear that variations in the protein and lipid composition in the gestating organism should affect the surface tension of biological liquids which is supported by experimental data. The dynamic surface tension of serum and amniotic liquid obtained from 52 pregnant women was studied at various gestation periods. For 10 women, two and more analyses were performed. With increase of the gestation period, a gradual decrease in both dynamic and equilibrium surface tension of serum occurs, while a sharp increase of ~ is observed. It should be noted that up to the 20th week of gestation, the surface tension values in the short and medium time range are quite similar to those characteristic for the reference group of non-

84 pregnant w o m e n o f corresponding age. In the same period, however, the values of cy3 and already undergo significant changes, as depicted in Figs. 3.7, 3.8, 3.9 and 3.10.

75 70 65 E

604

i

501

45

i

0

i

i

5

10

1

I

i

i

15 20 25 30 Gestation period [weeks]

1

35

40

35

40

b)

2220r...-n

-~

18

-

16

-

J

J

E

Z 14E ,< 1 2 l0

J

J

t_...a

7 z~ 0

5

10

15

20

25

30

Gestation period [weeks]

Fig. 3.7. Surface tension characteristics of serum obtained from non-pregnant (0 weeks) and pregnant women as a function of gestation weeks; a) gives t~t - (+), a2 - (ll), a3 - (&), b) gives ~,. The correlation coefficients for interracial tensiometric parameters o f serum and amniotic liquid depend on the gestation period. For young non-pregnant women, a rather close direct correlation exists for serum between ~l and c~2 (correlation coefficients > 0.7),

(3" 1

and ~3 (>

0.5), t~2 and c~3 (> 0.7). During gestation, the surface tensions exhibit a strong interrelation with

85 ~. (correlation coefficients > 0.5). At the end of normal gestation, the correlation between surface tension values at t = 0.01 s and t = 1 s, 0.~ and O'2, disappear, while the correlation between equilibrium surface tension 0.3 and 0.1 reverses. This dynamics may indicate near parturition, thus being of certain practical interest. Generally, proceeding gestation, some correlation become insignificant, and other factors emerge. 75 o.......

70

o--.._. I1~

~ , 65

-o

60 t~ 55 50 45 lgr.

lgr.

2gr. Sertma

2gr.

Anmiotic liquid

Fig. 3.8. Surface tension characteristics of serum and amniotic liquid obtained from pregnant women with 20-21 weeks gestation (group 1) and 39-40 weeks gestation (group 2), (+) - oi, (11) - o2, (A) - o3 75

--

70 ~'65 N6o \

55

Qx

-

'X o~

50 -2

L

I

I

-1

0

1

lg(tef) Is]

Fig. 3.9. Tensiograms of serum obtained from pregnant women: solid line - 20 weeks, dashed line - 40 weeks of gestation.

86 This variation in the correlation between particular surface tension parameters of serum during the gestation period occurs also in amniotic fluid. Only c2 and c~3 remain strictly interrelated. This fact can be explained not solely by variations in the quantitative composition of surfactants. We rather believe that the state of the feto-placental complex is responsible for the production of qualitatively new surfactants. In addition, a deregulation in the excretion of electrolytes by kidneys of pregnant women appears. The level of contra-insular hormones (cortifan, placental lactogen) decreases during gestation promoting the increase of glycemia and the concentration of glucose in amniotic liquid (Solun 1992). This leads in part to a decrease in surface tension of amniotic fluid and to variations in the qualitative composition of proteins, thus affecting interrelations between tensiometric parameters. At the end of the gestation period, a significant decrease in the surface tension of serum and amniotic liquid at long times are observed, and these values become close to one another (of. Figs. 3.9, 3.10). This dependence is of particular interest, especially when considering that the level of serum proteins and lipids in serum is 10 to 23 times higher than in amniotic liquid. The amounts in the amniotic liquid is directly related to concentrations of albumins, al" and 7-globulins, high density lipoproteins and very low density lipoproteins in blood With increasing duration of pregnancy, the amount of proteins in serum becomes lower, but the level of lipids increases. Parameters of cholesterol and triglycerides in amniotic liquid are equal to each other, and in the gestation weeks 20-22, they are three times lower than in the weeks 39-40. There are strong links between the studied factors of fat exchange in both biological liquids, and the high density and low density lipoproteins in serum do not affect the amount of cholesterol and triglycerides in the amniotic liquid. With increasing duration of pregnancy, the transport of ot-fetoprotein through the placenta into mothers' blood increases, and the concentration of this glycoprotein (molecular mass 65 kDa) in serum attains a maximum in weeks 30-32 (cf. Fig. 3.11). At the same time, approximately from the 15th week, the level of a-fetoprotein in the amniotic liquid starts to decrease. The relative amounts of ct-fetoprotein in amniotic liquid and serum undergo variations during the

87 gestation period: 1000:1 in the first trimester, 100:1 in the second trimester, and 1 O: 1 in third trimester.

75

--

70 -

E65Z E b 60

II

I

I

I

|

|

|

I

I

I

IIII

I

I

I

I

I

I

I

I

I |

~

i~iii,

i~

ii~

,D

%

55 w~

50 -2

I

I

I

-1

0

1

lg(tef) [s]

Fig. 3.10. Tensiograms of amniotic liquid obtained from pregnant women: solid line-20 weeks, dashed line - 40 weeks gestation.

20-

v,a .t:=~

15

.~ 10 ~a 5

I

12

I

f

14

I

16

I

I

18

I

I

I

20

I

22

I

I

24

I

I

26

I

I

28

I

I '-I

I

30

32

I

I

34

I

1

36

I

I ~-]~

38

1

40

Gestation period [weeks]

Fig. 3.11. Variation in the concentration of fetoprotein ( , ) and estriol ([]) in mothers' serum during gestation.

88 The amount of amniotic liquid does not depend on the concentration of protein fractions therein, but exhibits a weak correlation with the level of triglycerides. The duration of pregnancy has a positive effect only on the amount of cholesterol and triglycerides in amniotic liquid. Only the level of txl-globulins in the amniotic liquid is interrelated with the concentrations of txl- and y-globulins in serum The influence of serum lipids on the amount of these substances in the amniotic liquid is more significant. Therefore, an interrelation exists between a number of parameters related to protein/fat composition of serum and amniotic liquid, in spite of significant differences in their concentrations. The comparison of interfacial tensiometric parameters for various biological liquids (Table 3.6) shows that surface tensions of serum at short surface lifetimes (0"1) for partus maturus correlates with the same parameter of amniotic liquid, while the values 0"2 and 0"3 for serum correlate with those for amniotic liquid. There is a negative dependence of ~, for serum on 0"1 for amniotic liquid. It can be thus concluded that a close interrelation exists between dynamic interfacial tensiometric parameters of the two biological liquids. For premature parturition many of these relations do not exist. Only the correlation coefficient between 0"2 for serum and 0"1 for amniotic liquid becomes more pronounced. Table 3.6. Correlations between particular surface tension parameters of serum and amniotic liquid sampled from women at various stages of gestation

Serum

Gestation

Amniotic

stage (weeks)

liquid

13"1

0"2

0"3

20-21

0"I

+0.60

+0.51

+0.25

+0.07

0"2

+0.27

+0.09

-0.21

+0.23

0"3

+0.14

+0.01

-0.18

+0.13

-0.18

-0.05

-0.11

+0.15

0-1

+0.33

-0.27

-0.24

-0.48

0-2

-0.27

+0.85

+0.67

-0.02

0-3

-0.10

+0.63

+0.43

+0.12

0.34

+0.08

+0.21

-0.04

39-40

89 Table 3.7. Correlations b e t w e e n some characteristics o f parturition and surface tension parameters o f serum and amniotic liquid obtained from pregnant w o m e n .

Parturition characteristic

Biological liquid Amniotic liquid

Serum 0.1

0.2

0.3

0.1

0.2

$

1'

Time of birth process

$$$

1'1'

1st period

$$$

t

3rd period Loss of blood

)~

1' $$

2nd period

0"3

$$ $$

1'

r

Amount of amniotic liquid Mass of fetus Growth of fetus APGAR I

1"1"1"

1'

r

APGAR II

1"1"

1"

$

1' - positive correlation; $ - negative correlation e m p t y - no correlation r<0.3; one s y m b o l - r < 0.5; two symbols - r = 0.5-0.7; three s y m b o l s - r > 0.7

The dependence of the parturition character on dynamic interfacial tensiometric parameters of amniotic liquid and serum have been analysed. Table 3.7 summarises our results. It can be noted that a significant dependence exists between the character of parturition and the surface tension level at t = 0.01 s for serum. In fact, this parameter reflects the duration of the first period, the extent of possible loss of blood and the state of the infant (Table 3.7). We believe that low surface tension values at short adsorption times (less than the M - 3m value for healthy non-pregnant women) are indicative of an unfavourable prognosis for the character of future parturition. This is also true, at least partly, with respect to high values of the equilibrium surface tension (exceeding M + 3m for healthy non-pregnant women). The decrease of 0.2 for serum and the increase of L can be regarded as implicit evidence of possible prolongation of the second and third period of parturition. In the organism of pregnant woman a specific hormonal system, the fetoplacental complex, is formed as the result of the mutual functioning of placenta and foetus. The main products of this complex are steroid, lactotrophic and somatotrophic hormones, among which placental lactogen,

90 estriol, oestradiol and progesterone are believed to be the most significant ones. The concentration of fetoplacental hormones in women's blood increases with the duration of gestation (for some hormones by hundreds times), corresponding to the increase of placenta and fetus mass (Fig. 3.11). The decrease in the level of serum placental lactogen, estriol, oestradiol and progesterone is indicative of the deterioration of the placenta function, and therefore, dangerous for the fetus. Placental hormones stimulate the growth of cells which perform the secretion of prolactin (molecular mass 20 kDa), whose contents in the female organism begins to increase already from the 8th week of gestation, and becomes still higher at time of parturition. One of the so-called ,,pregnancy hormones" is chorionic gonadotropin (molecular mass 38 kDa), which is synthesised in trophoblasts and syncytial cells of the placenta. During the first trimester this glycoprotein stimulates the production of estrogens which are necessary for the development of pregnancy. Then the decrease in the production of chorionic gonadotropin becomes less intensive and remains constant. The activity of estrogens is related to the concentration of (a2-glycoprotein), while progestins are related al-glycoprotein. Sex hormones retard the assimilation of glycine and the synthesis of proteins. In turn, insulin stimulates the protein metabolism in the placenta, while chorionic gonadotropin enhances placental glycogenesis and the production of proteins from carbohydrate and amino acid predecessors. Clearly, these extremely significant changes in protein and carbohydrate metabolism affect surface tension parameters of biological liquids in the organisms of pregnant women. A competition exists between sex hormones, aldosterone and glucocorticoids for the binding sites in kidney nephrons with respect to the control of sodium transport. Due to the enhanced adsorption of this inorganic ion, estriol and oestradiol lead to a retention of water in the organism irrespective of the activity of aldosterone. While the concentration of receptors of

17-13-oestradiol in the chromatin of kidney cell nuclei is low, and specific macromolecular complexes with oestradiol exist in the cytosol, the sites where the binding of estrogens in nephrons takes place are still unknown. Proximal tubules are the most probable place for the localisation of oestrogen receptors in kidney. Testosteron stimulates the activity of membranous sodium-potassium-ATPase, and progesterone decreases the reabsorption of sodium related to the competition of aldosterone receptors in kidney tubules. It is known that sodium ions in solution of low molecular surfactants can increase the dynamic surface tension at short surface lifetimes, which can in turn lead to the changes of interfacial tensiometric parameters observed for biological liquids of pregnant women.

91 Processes in the reproduction system are closely interrelated with the biological transport of steroid hormones. For estrogens and androgens, the corresponding compound is the steroidbinding globulin glycoprotein synthesised in the liver. The formation of this protein (molecular mass 120 kDa) is enhanced by oestradiol, while testosteron suppresses its formation. Steroidbinding globulin comprises N-glycoside-bound oligosaccharide chains and O-glycoside-bound carbohydrates, and has properties characteristic to biopolymers. It is believed that the main function of steroid-binding globulin is the protection of low-molecular hormones against the influence of blood factors and excessive excretion. Table 3.8. Correlations between concentration of various blood components and surface tension characteristics of serum obtained from pregnant women Surfacetension parameter

Blood component O'1

(Y2

(~3

$

Total protein Albumin c~l-globulin fraction ot2-globulin fraction 13-globulin fraction ?-globulin fraction Total cholesterol a-cholesterol $$

_Triglycerides

$$$

. High density lipoprotein fraction Low density lipoprotein fraction Very low density lipoprotein fraction Testosteron

$

$$

Estradiol

1"1"

Progesterone 9[3-chorionic gonadotropin

$

$

$

Placental lactogen 1" - p o s i t i v e c o r r e l a t i o n ; $ - n e g a t i v e c o r r e l a t i o n ; e m p t y -

n o c o r r e l a t i o n r < 0 . 3 " o n e s y m b o l - r < 0 . 5 ; .wo

s y m b o l s - r = 0 . 5 - 0 . 7 ; t h r e e s y m b o l s - r > 0.7

Up to 99% of circulating oestradiol and testosteron are bound to steroid-binding globulin, which are inactive in this state. Serum albumin also possesses some capability for binding with

92 steroids. However, under physiological conditions only negligible quantities of sex hormones form complexes with this protein. Steroid-binding globulin interacts mainly with androgens, and in spite of 20 times higher concentration of testosteron in males blood as compared with females low protein concentration entails that the amount of free hormone for males is 40 times higher. The concentration of steroid-binding globulin, while already prevailing in females' blood, significantly increases during gestation (Chajka & Matytsina 1994, Hadges et al. 1983). In the blood of gestating women, in addition to estrogens (oestradiol, estriol, estrone), significant amounts of androgens (testosteron, dihydrotestosteron) are also present. The decrease of testosteron during pregnancy, leads to a decrease in the contents of steroid-binding globulin. In practice, the enhanced production of steroid-binding globulin is indicative of increased contents of estrogens and a decreased level of androgens. Our data show that for pregnant women, the amount of serum testosteron are strongly related to ol and o2 of serum, while the amount of estrogens affect the equilibrium surface tension t~3 of biological liquids. The tensiographic parameter Ol in the short lifetime range depends on the level of chorionic gonadotrophine and placental lactogen (Table 3.8). These results are interesting not only from a purely scientific point of view, but may become important for obstetrical practice, enabling one to apply interfacial tensiometric measurements of female serum to estimate the hormonal status, and, therefore, to monitor the gestation process. During gestation the concentrations of triglycerides in serum and amniotic liquid are inverse correlated with the amount of estrogens. However, the dependence on cholesterol in the amniotic liquid is still more significant (Kazakov et al. 1996b, 1997). It should be stressed that the activity of serum progesterone strongly affects the composition of lipids in the studied liquids. This is especially true for the level of triglycerides in serum which, among other lipids, are most strongly affected by the hormonal status of gestating women, which is a significant fact for the clinical practice. In the framework of this discussion, the interrelation between lipids contained in amniotic liquid and the amount of sex hormones in serum is interesting. Keeping in mind that there is an equivalent dependence for serum fats, one can suppose that oestradiol and progesterone also produce similar effects on cholesterol and triglycerides in amniotic liquid. This hypothesis is further supported by correlations that exist between the lipids of various biological liquids, and by the similarity of chemical structure of sex hormones

93 to that of some lipids. The surface tensions of amniotic liquid significantly depend on the concentration of specific proteins and fats. The correlation coefficients at various periods of gestation are shown in Fig. 3.12. Such correlations do exist and exhibit correlation coefficients for certain substances of up to 0.83.

a) 0,6 0,5 o

0,4 0,3 0,2

.~

0,1

G

-0,1 0,2 -.-.

-0,3

1

2

3

4

5

6

7

8

b)

._

0,4

-

0,3

-

0,2

R

o

~, o

O

0,1

~

0

=

-0,1

"

~

-

1

-

-0,2

-

-0,3

-

-0,4

-

-0,5

-

-0,6

-

o

1

2

3

4

5

6

7

8

Fig. 3.12. Correlations between surface tension characteristics o f amniotic liquid (a) and biochemical components o f serum (b). Surface tension parameters are: ~ - hatched, or2- black, or3- white and Z, -grey. Biochemical components are: total p r o t e i n - 1 , albumin -2, Gtl-globulin fraction - 3, ct2-globulin fraction - 4, [3-globulins fraction - 5, y-globulin fraction - 6, total cholesterol - 7, triglycerides - 8.

94 It should be stressed that the dependence of surface tension on the protein-lipid composition is most important during the time of parturition. This is especially true for ~ and ~,. That is the level of particular proteins and lipids which determines the above dynamical tensiometric parameters of serum and amniotic liquid at the end of normal gestation. It enables one to use surface tension values for the estimation of the amount of surface active proteins and fats in biological liquids. For example, low surface tension values at t = 0.01 s and large X-values of serum are indicative of hypoproteinemia (hypoalbuminemia). For amniotic liquid, these tensiographic parameters have opposite effects. Up to the 20th week of gestation, the surface tension of serum is indicative mainly of the state of lipid exchange. From a practical point of view, interfacial tensiometric parameters of serum can be very useful for the estimation of the surfactant contents in amniotic liquid: ~ for serum is inversely related to the levels of protein and its specific fractions in amniotic liquid, while ~, is directly related. The equilibrium surface tension ~r3 is associated with the concentration of txl-globulins, while ~ exhibits a negative correlation with the contents of cholesterol and triglycerides. It is difficult to compare results obtained in the present studies with those published in the literature. The main reason is that there are no dynamic surface tension results for serum and urine of healthy persons in the literature. On the other hand, adsorption of serum albumin (the main surface-active component in the serum), and other serum components, have been widely investigated both at solid and liquid interfaces. The adsorption to solid surfaces is important for understanding the interaction between biological liquids with implant materials (Interaction of the Bloodwith Natural and Artificial Surfaces 1981). Influence of serum components, adsorbed at the liquid/gas interface was analysed in the context of serum proteins and the influence of lipids on the adsorption activity of lung surfactants (Pulmonary Surfactant: From Molecular Biology to Clinical Practice 1992, Surfactant Therapy for Lung Disease 1995, Pison et al. 1996, Manalo et al. 1996). Among all other biological liquids only the surface tensions of amniotic liquid was investigated systematically (Ruiz et al. 1989, Heytmanek et al. 1990, Moawad et al. 1991, Joura et al. 1995, Boda et al. 1997).

95 The composition of surfactants in amniotic liquid collected from pregnant women was studied with respect to the diagnostics of fetus lung maturity. The respiratory distress-syndrome, the primary cause of neonatal mortality, arises when the amount of lung surfactant is inappropriate. Ruiz et al. (1989) have studied the ability of some components of amniotic liquid to decrease the equilibrium surface tension. These studies have shown that fetus lung maturity can be easily estimated from surface tension measurements. Using the Wilhelmy method, Heytmanek et al. (1990) have measured surface tensions of amniotic liquid, and compared the results with the data of a gas chromatographic determination of dipalmitoyl phosphatidylcholine and lecithin choline in the liquid. They argued that the study of surface tensions enables one to estimate

the

total

effect

of

surfactant,

while

the

data

concerning

dipalmitoyl

phosphatidylcholine and lecithin choline provide information on the contents of the most important surfactants. Moawad et al. (1991) analyzed the capability of the drop volume method for measuring the surface tension of amniotic liquid with respect to predict respiratory distresssyndrome. Simultaneously the lecithin/ sphingomyelin index and phosphatidylglycerol concentration were determined. It was possible to predict the respiratory distress-syndrome for infants in 81.3% of all cases. In the studies performed by Joura et al. (1995) dedicated to the prognostics of fetus lung maturity, surface tension measurements of amniotic liquid (Wilhelmy method) were also combined with measurements of the lecithin/sphingomyelin coefficient. The results coincide in only 8.4% of all experiments, and the number of cases in which wrong positive information was obtained using biochemical methods was 4 times higher than that obtained from surface tensiometric studies. It can therefore be concluded that studies of surface tension of amniotic liquid allows reliable prognostics of fetus lung maturity. 60 amniotic liquid samples collected from pregnancies at different gestation times were studied by Boda et al. (1997). Measurements of surface tension of biological liquids were made by a pulsating capillary technique. A multiple regression analysis of the results, including other parameters (total protein contents, total lipid contents, phospholipid contents and micro-viscosity), indicated that this method may enhance the precision of the determination of gestation time.

96 Precise analysis of various samples proved that this technique gives well-reproducible results under the given standardised conditions. It can be concluded that surface tensions of biological liquids depend on the gestation time and surfactant contents in serum and amniotic liquid. The composition of these two biological liquids is strongly interrelated. Therefore, the dynamic interfacial tensiometry can be regarded as a useful tool for the prognostics of the gestation development and the state of the fetus. However, for final conclusions regarding relationships of physicochemical parameters of biological liquids with gestation complications, accompanying diseases and pathologic parturition far more experiments are to be done. New important results are necessary which would define the scope of informational criteria applicable for the efficient supervision of gestating and lying-in women. 3.3. Summary Dynamic surface tension characteristics of biological liquids depend on sex and age. In addition, duration of pregnancy influences values of surface tension, because surfactant contents in serum and amniotic liquid changes during pregnancy. The composition of these two biological liquids is strongly interrelated. Therefore, the dynamic interfacial tensiometry can be regarded as a useful tool for the prognostics of the gestation development and the state of the fetus. However, for final conclusions regarding relationships of physicochemical parameters of biological liquids with gestation complications, accompanying diseases and pathologic parturition far more experiments are to be done. New important results are necessary which would define the scope of informational criteria applicable for the efficient supervision of pregnant women. 3.4. References A1, M.D.M., Badarsmook, A. and Vanhouwelingen, A.C., J. Am. Coll. Nutrit., 15(1996)49. AI, M.D.M., Vanhouwelingen, A.C., Badartsmook, A. and Homstra, G., J. Nutrit., 125(1995)2822. Baglin, T.P., Simpson, A.W., Price, S.M. and Boughton, B.J., J. Clin. Pat., 40(1987)1468.

97 Boda, D., Eck E. and Boda, K., J. Perinat. Med., 25(1997)146. Chajka, V.K. and Matytsina, L.A., Arch. Clin. Exp. Med., 3(1994)67. Dang, C.V., Bell, W.R. and Shuman, M., J. Med., 87(1989)567. Foster, H.W. and Dos, S.K., Am. J. Obstet. Gynecol., 149(1984)670. Grunenberger, F., Lammi Keefe, C. J., Schlienger, J. L., Deslypere, J. P. and Hautvast, J.G., European Journal of Clinical Nutrition 50(1996)25. Heytmanek, G., Eppel, W., Lohninger, A. and Salzer, H., Z. Geburtshilfe Perinatol., 194(1990)65. Hodges, J.K, Eastman, S.A. and Jenkins, N., Journal of Endocrinology, 96(1983)443. Joura, E.A., Kainz, C. and Joura, E.M., Z. Geburtshilfe Neonatol., 199(1995)78. Kazakov, V.N., Sinyachenko, O.V., Fainerman, V.B., Barinov, E.F., Miller, R., Ermolaeva, M.V. and Sidorenko, I.A., Arch. Clin. Exp. Med., 5(1996a)3. Kazakov, V.N., Talalaenko, Yu.A. and Sinyachenko, O.V. Med. Soc. Probl. Semji, 2(1997)10. Kazakov, V.N., Talalaenko, Yu.A., Sinyachenko, O.V., Fainerman, V.B. and Miller, R., Med. Soc. Probl. Semji, 1(1996b)47. Komissarova, L.M., Burliev, V.A. and Golstian, A.A., Vopr. Ochr. Mat. Det., 5(1988)47. Manalo, E., Merritt, T.A., Kheiter, A., Amirkhanian, J. and Cochrane, C., Pediatr. Res., 39(1996)947. McIntosh, N., Rodeck, C.H. and Heath, R., Biology of the Neonate. 45(1984)218. Moawad, A.H., Ismail, M.A. and River, L.P., J. Reprod. Med., 36(1991)425. Moniz, C.F., Nicolaides, K.H., Bamforth, F.J. and Rodeck, C.H., Journal of Clinical Pathology, 38(1985)468. Mosher, D.F. and Williams, E.M., J. Lab. Clin. Med., 91 (1978)729. Pison, U., Herold, R. and Scht~rch, S., Colloid Surfaces A, 114(1996)165. Radzinsky, V.E., Kondratieva, E.N. and Milovanov, A.P., Pathology of Amniotic Medium, Kyiv, Zdorovja, 1993.

98 Radzinsky, V.E. and Smal'ko, P.Ya., Biochemistry of placental insufficiency, Kyiv, Naukova Dumka, 1987. Robertson, B. and Teusch, H.W. (Eds.), Surfactant Therapy for Lung Disease, Marcel Dekker Inc., New York, 1995. Robertson, B., Van Golde, L.M.G. and Batenburg, J.J. (Eds), Pulmonary Surfactant: From Molecular Biology to Clinical Practice, Elsevier, Amsterdam, 1992. Ruiz, B.J., Abbad, B.J. and Fabre, G.E., Clin. Chem., 35(1989) 800. Salzman, E.W. (Ed.), Interaction of the Blood with Natural and Artificial Surfaces, Marcel Dekker Inc., New York and Basel, 1981. Savelieva, G.M., Fedorova, M.V., Klimenko, P.A. and Siginova, L.G., Placental insufficiency, Moscow, Medicina, 1991. Solun, M.N., Ter. Arch., No.3(1992) 119. Tamugur, E., Ozer, M., Guener, G. and Djavani, M., Journal of Clinical Biochemistry & Nutrition 13(1992)63. Tuppla, M., Ailus, K., Palosuo, T. and Yeikorkala, O., Fertil. Steril., 64(1995)947. Vasiliev, S.A., Yefremov, E.E. and Savenko, T.A., Ter. Arch., 2(1994)63 Vasilieva, E.V., Mazveva, L.M., Golovanova, O.E. and Sura, V.V., Ter. Arch., 12(1991) 130

99

Chapter 4 Application of Surface Tensiometry in Nephrology The kidney is one of the most important structural and functional entities in the human organism. It maintains a constant volume and concentration of osmotically active substances in human liquids. It regulates ionic homeostasis, stabilises acid/base equilibrium, and excretes metabolic end products. It also takes part in the metabolism of proteins, lipids and hydrocarbons, and in the synthesis of hormones and other biologically active compounds (Borysov 1991, Cowly 1997, Forte et al. 1996, Clauser et al. 1996, Rosenthal et al. 1993). The entire variety of functions is related to processes of glomerular ultrafiltration (primary urine formation), and secretion and reabsorption processes through the collecting tubules (formation of final urine composition and elimination from the body), metabolic transformations and production of a number of substances, including surfactants. This chapter will describe some dynamic surface tension characteristics that could be found in biological samples obtained from patients with kidney diseases. When surfactants are released into the bloodstream of the kidney, the characteristics of the blood flow through the kidney vasal network changes. This could be demonstrated at the wave form of the rheonephrogram. In particular, the crest of systolic rheonephrogram wave becomes sharper with the dicrotic peak coming closer to the base of the curve. The shape of the catacrotie phase becomes more convex, indicating an imbalance in the venous deflux from the organ. This view is supported by the fact that the dicrote rises above the main wave with the formation of a systolic/diastolic plateau. Therefore, it can be argued that the release of surfactants into the bloodstream leads to significant variations in the kidney vasal system. Many kidney diseases causes variations in the protein and lipid composition in the blood resulting in changes of the rheologic properties such as the viscosity. This depends primarily on the concentration of fibrinogen, 3t-globulins and other high molecular proteins. The increase in viscosity results in an increased resistance to microcirculation through the kidney vessels. The deterioration of blood liquidity is also due to a water/salt imbalance. A variation in the rheological properties of a biological liquid is generally regarded as good indication of an

100 unfavourable development of kidney diseases (Ryabov et al. 1988, 1995, Gordge et al. 1988). The variation in the composition of a biological liquid affects not only their bulk characteristics, but also the surface rheological properties. Studies of the adsorption layer of urine of healthy subjects in a Langmuir trough indicate the existence of a hysteresis in monolayer expansion/compression cycles. Rheological characteristics of monolayers, including this hysteresis, exhibit variations for patients suffering from nephrotic pathology. These variations are especially significant when an excess of hydrogen ions in the media takes place. For kidney diseases violation of the protein, fat, hydrocarbon and electrolytic exchange significantly affects the surface tension of biological liquids. Thus, we believe that kidney malfunction can be considered as a natural clinical model for surface tensiometric studies of serum and urine.

4.1. Glomerulonephritis 4. I. 1 Variation in surface tensiometric parameters for various forms of glomerulonephritis Variations in the dynamic surface tension of serum are specific for various types of glomerulonephrites. The total number of patients in this study was 149 persons. For patients with acute glomerulonephritis (32 patients), lupus erythematodes associated glomerulonephritis (26 patients) and glomerulonephritis caused by hemorrhagic vasculitis (purpura SchrnleinGenoch) -16 patients the dynamic surface tensions of serum increase in all time ranges, while for chronic glomerulonephritis (86 patients) the values decrease in the short time range, and exhibits a slight increase in the long time range (cf. Fig. 4.1). Comparisons of serum and urine tensiometry for sick and healthy persons had to be matched. The values of surface tensiometric parameters for lupus glomerulonephritis were compared to those for healthy females because all patients screened were females. The mean age of patients who suffered from Genoch glomerulonephritis was 50 years, therefore the tensiographic parameters were compared with those measured for the group of healthy persons of the same age.

101

In comparative studies of serum tensiometry the increase in the dynamic surface tensions at t = 0.01 s and t = 1 s, ~ and o2, respectively, was rather insignificant as compared to the m e a n characteristics for all age groups. In contrast, a sharp increase in the slope of tensiographic curves o(t ~/2) = L was detected (cf. Fig. 4.2.). Only for acute glomerulonephritis was the value of ~, for serum unchanged. a) sertma _

4 321-

=

-2

-

-3

-

-4

-

AGN

CGN

LGN

GGN

b) urine _

320

-3-4

-

-5 -6

-

AGN

CGN

LGN

GGN

Fig. 4.1. Changes in surface tensiometric parameters measured in biological liquids obtained from patients with various forms of glomerulonephrites. Changes are given in % compared to sex and age corresponding healthy controls. AGN - acute glomerulonephritis, CGN-chronic glomerulonephritis, LGN-lupus glomerulonephritis, GGN - Genoch glomerulonephritis, hatched - Ol, black - or2, white - o3.

102 Besides comparisons of serum tensiometry for sick and healthy persons, comparisons of urine tensiometry were performed. Interesting data were obtained concerning the dynamic surface tension of urine (cf. Fig. 4.1.). An increase of crl was observed in all groups of patients, while an

increase

of

~2

was

characteristic

only

for

patients

suffering

from

Genoch

glomerulonephritis. In contrast, for chronic glomerulonephritis a decrease of cy2 takes place. The variations of equilibrium surface tension or3 for chronic glomerulonephritis and Genoch glomerulonephritis are opposite to each other: for the first group or3 decreased, while for the second group an increase was observed. No variations in ~. for urine were found for Genoch glomerulonephritis, unlike the behaviour characteristic for acute glomerulonephritis, chronic glomerulonephritis and lupus glomerulonephritis (cf. Fig. 4.2.). It is thus seen that various versions of glomerulonephritis are accompanied by variations in surface tensiometric parameters which are peculiar to a specific disease (cf. Table 4.1), and can be ascribed to various structural and functional kidney disturbances and hence leads to variations in the clinical manifestation of the disease. For example, the nephrotic syndrome often accompanied acute glomerulonephritis, while chronic glomerulonephritis was accompanied by a renal insufficiency (a terminal chronic renal insufficiency was diagnosed only in this group of screened patients). Serum

40 ~

~ ~

30 ~ 20 ~ 10 I 0

~

Urine

l/ II

I!

CGN

GGN

/!

i~ -lO -20

-30 ~

i

-40 l -50 AGN

LGN

AGN

CGN

LGN

GGN

Fig. 4.2. Changes in ~. values for patients with various forms of glomerulonephrites. Changes are given in % compared to corresponding healthy controls. AGN - acute glomerulonephritis, CGN-chronic glomerulonephritis, LGN - lupus glomerulonephritis, GGN - Genoch glomerulonephritis.

103 Differences of tensiograms were found for mesangiocapillary and mesangioproliferative states of glomerulonephrites. For mesangiocapillary glomerulonephritis the ~, value for serum is virtually equal to that characteristic of healthy persons, while for mesangioproliferative glomerulonephritis this value increases significantly. On the contrary, increase in values of 0-], 0-2 and 0"3 for urine was observed just for the group of patients with mesangiocapillary glomerulonephritis (cf. Fig. 4.3), which can be used to distinguish between these two morphologic versions of the disease. This fact is of considerable practical importance, because it makes a nephrobiopsy unnecessary for determining relevant pathogenetic treatment, and it predicts the development of pathologic processes.

Table 4.1. Differential diagnostic indicators of surface tension variation of biological liquids for various types of glomerulonephrites

Type of glomerulonephritis 0"1

0"2

+

+

Lupus

+

+

Genoch

+

+

Acute

Urine

Serum 13"1

0"3

0"2

0"3

+

+

t

Chronic +

+

+

+ statistically significant increase of parameter compared to normal, -statistically significant decrease of parameter compared to normal In fact, all known clinical-morphological differences between mesangioproliferative and mesangiocapillary glomerulonephritis are determined by a specific surfactant composition in blood and urine, which in turn leads to differences in the dynamic surface tension. An important point has to be emphasised: for both groups of screened patients the frequencies of the development of nephrotic syndrome and chronic renal insufficiency were virtually equal. The surface tension of urine (and, therefore, quantitative and qualitative surfactant composition and the composition of urine influencing the surface properties of surfactants) depends significantly on structural kidney imbalances which affect the glomerular filtration, selective tubular reabsorption and secretion of surfactants. It was shown that the surface tension of urine

104 depends directly on the proliferation extent of mesangial cells. In particular, the tensiographic parameters for the short surface lifetime were related to the increase of the mesangial matrix and glomerulus sclerosis-hyalinosis. The equilibrium surface tension o3 correlates with the beading of the basal capillary glomerular membrane, proliferation of Bowmans capsule epithelium or podocytes, and capsule sclerosis. The correlation features of ~, for urine are opposite similar to those of o3 (cf. Fig. 4.4.).

Serum

Urine

4~ 2

-2 -4 -6 ~ ol

o2

o3

ol

o2

o3

Fig. 4.3. Changes in surface tension parameters measured in biological liquids obtained from patients with various morphologic forms of chronic glomerulonephrites. Black-mesangioproliferative glomerulonephritis, whitemesangiocapillary glomerulonephritis. Changes are given in % compared to corresponding healthy controls.

From the presented data we conclude: (i)

The higher value mesangiocapillary

of surface tension in urine glomerulonephritis

compared

sampled to

from patients with mesangioproliferative

glomerulonephritis may be due to a more pronounced structural changes of the kidneys. (ii)

Selected parameters of dynamic surface tensions of urine indicate the extent to which the glomerular structure is affected.

(iii)

The extent to which the basal capillary glomerular membrane is affected determines the proteinuria level. Usually the equilibrium surface tension should decrease, when the protein concentration in urine increase. However, proteinuria increases o3.

105 (iv)

Kidney glomerulo sclerosis usually leads to a decreased surfactant filtration, which results in an increase of dynamic surface tension of urine at short times.

(v)

Increasing dynamic surface tensions and decreasing ~,-values for urine indicate an unfavourable development of chronic glomerulonephritis.

0.8 0.6 -1 0.4 ~9

0.2

O

0 -0.2

~-0.4 -0.6 -0.8 1

2

3

4

5

6

7

8

9

10

Fig. 4.4. Correlation coefficients between surface tension parameters of urine (hatched - 0-~,black -

0"2,

white -

0"3,

grey- ~.), obtained from patients with chronic glomerulonephritis, and various morphological kidney characterisation are given. 1 -increase in thickness of basal glomerular membrane, 2 - mesangial cells proliferation, 3- mesangial matrix increase, 4 - proliferation of capsule epithelium and podocytes, 5 capsula sclerosis, 6 - gyalinosis, glomerulosclerosis, 7 - degradation of tubular epithelium, 8 - increase in thickness of basal tubular membrane, 9- lymphohystiocitary infiltration of stroma, 10- stroma sclerosis; Glomerulonephritis can be accompanied by various complications. Tubulointerstitial damages in case of a chronic glomerulonephritis lead to a reduction in the osmotic concentration of urine, a suppression of ammonium and hydrogen ions excretion, and of the influx of various high-molecular proteins, which are secreted in tubules. The changes in the epithelium or basal tubular membrane do not affect dynamic surface tensions of urine, while the extent of lymphohystiocital infiltration and stroma sclerosis lead to variations in the dynamic surface

106 tensiometric parameters. Correlation links between the variations in tensiographic parameters and glomerular changes are of the same direction as glomerular changes themselves. The interrelation between various dynamic surface tension parameters of serum and urine obtained from patients with acute glomerulonephritis, chronic glomerulonephritis, lupus glomerulonephritis and Genoch glomerulonephritis is multivalent This is summarised in Table 4.2. Some interrelations typical for healthy individuals vanish, and some characteristics for particular diseases emerge. For example, the correlation between values of ~2 and cyl for urine becomes more evident, while the effect of gl of serum on the surface tensiometric parameters of urine becomes weaker (except for Genoch glomerulonephritis patients). In addition, the relationship of O'2 for serum with the dynamic surface tension parameters of urine emerges. It is interesting that for serum, in contrast to healthy persons and patients with other forms of glomerulonephritis, in hemorrhagic vasculitis we found a negative correlation between equilibrium surface tension and dynamic surface tension in the short and medium time range. This observation may be useful as an additional criterion for differential diagnostics between glomerulonephritis caused by hemorrhagic vasculitis as in purpura Sch6nlein-Henoch, chronic

glomerulonephritis

caused

by

other factors,

acute

glomerulonephritis,

and

glomerulonephritis in the cause of lupus erythematodes. The nephrotic syndrome often accompanied acute glomerulonephritis, while chronic glomerulonephritis was accompanied by renal insufficiency. Because the nephrotic syndrome changes protein and lipid metabolism a lot, and surface tensions of serum depend on the contents of proteins and lipids, it becomes necessary to perform additional analysis of screening results for patients suffering from the nephrotic syndrome. Proteinuria accompanies the nephrotic syndrome resulting in a decrease of the plasma colloid-osmotic pressure. This leads to several processes, a The transfer of liquid from within the vessels to the extra-vessel space is increased, decreasing the amount of circulating blood, and enhancing activity of the renin-angiotensin-aldosterone system and to an antidiuretic hormone secretion in case of hypovolemia, and to an increase in the reabsorption of sodium and water in kidney tubules. It is also supposed that the nephrotic syndrome is also accompanied by primary kidney defects of water and electrolyte excretion, because the hypervolemia is often developed in case of the nephrotic syndrome instead of hypovolemia.

107 The irregularity of the vascular permeability in case of the nephrotic syndrome produces relatively small effects on the variation in the blood amount and on the removal of sodium and water. Both the rate of albumin flux into extravascular space and the activity of kallicrein-kinin system increase, which can be regarded as implicit evidence of increased vascular permeability for such patients. The increase in albumin flux into the extravascular space enhances the transport through the lymphatic system returning proteins into the vascular network. This in turn leads again to an increased transcapillary oncotic gradient and, as the result, to an increase in the amount of the circulating blood volume. The pathogenesis of the nephrotic syndrome comprises of the mechanisms which are due to the disease itself, and also a number of non-specific processes, among which the disfunction of the lymph-draining activity of kidneys. The relation between the lymphatic kidney system and a violation of the protein metabolism is still obscure. The damage of glomerular filters leads to an increased burden laid upon the lymphatic system, both because of the reabsorption of increased amounts of protein, and because it is necessary to maintain the normal metabolism of the kidney tissue. Even a minimum proteinuria leads to an intensive functioning of the lymphatic system as the second link in the protein reabsorption process. The nephrotic syndrome leads to an overload of the tubular apparatus and interstitium by proteins, an intensification of the synthetic activity of kidneys. A necrosis of cells arises, and the collagenation increases (Shyshkin et al. 1989, Smoyer et al. 1998, Grone et al. 1988, Eddy et al. 1991). The excretion of sodium by kidneys is controlled by the variation in the rate of glomerular filtration and tubular reabsorption. The nephrotic syndrome, for which the main clinical symptom is the formation of oedema due to the retention of sodium and water, is a dynamic state, which develops through stages differing from each other in the type of sodium homeostasis: (i)

pronounced retention of sodium, characterised by an increase of oedema (positive sodium balance);

(ii)

stabilisation (while oedema are still present, the equilibrium between the consumption and excretion of sodium is establishes);

108 (iii)

the decrease of oedema accompanied by an excess of sodium excretion over its income (negative sodium balance).

Table 4.2. Correlations between various dynamic surface tension parameters of serum and urine. Serum and urine was obtained from patients with various types of glomerulonephrites and healthy persons

Blood

Screened groups Acute glomerulonephritis

Urine

0"1

0"1

t

0"2

t

0"2

0"3

(3"3

t

, .

Chronic glomerulonephritis

0"1 13'2 0.3

Lupus glomerulonephritis

0.1

t

tt

0.2

t

tt

0"3

t

0"1

t

0"2

ttt

0"3

t

_

Genoch glomerulonephritis

Healthy persons

tt

. _

_

ttt

. _

ttt

0"1

tt ttt

t

0.2 0.3

L

tt

t positive correlation; $ negative correlation; empty- lO correlation r<0.3" one arrow, r =0.3 to 0.5" two arrows, r - 0.5 to 0.7; three arrows, r > 0.7

The retention of sodium in the organism in case of the nephrotic syndrome is related to a decrease of the filtration load of the nephrone by sodium ions and to an enhanced reabsorption in the kidney tubules. It is known from model studies that for solutions of low-molecular surfactants the addition of sodium increases dynamic surface tensions in the short time range.

109 Similar phenomena possibly take place in the clinical practice, when biological liquids obtained from patients with nephrotic syndrome are studied. In patients who have nephrotic syndrome the dynamic surface tension parameters for serum are affected by increased albumin amount in the interstitial space and decreased colloid-osmotic pressure of plasma. The dynamic surface tension parameters for urine are affected by increased tubular reabsorption of sodium and water. There is no clear interrelation between the levels of proteinuria and albuminemia in cases of the nephrotic syndrome. One can find patients with extremely high proteinuria, but without any sharp decrease of albumin concentration in the serum; however, also cases are common where a rather low proteinuria level is accompanied by pronounced albuminemia with the formation of nephrotic syndrome. Clear correlations between the amount of daily loss of albumin through the kidneys, and albumin concentration in serum was observed only for chronic glomerulonephritis. The retention of liquid and the increase of the interstitial volume can be considered as one of the factors, which determine the decrease of albumin concentration in serum from patients with a nephrotic syndrome. The concentration of albumin within the vessels does not reflect the actual contents of this protein in the interstitial space. The total albumin pool size, however, can be calculated as the product of albumin concentration in serum by the volume of circulating blood and the ratio of the actual vascular pool. For patients with a hypervolemic type of the nephrotic syndrome, the albuminemia becomes evident at a lower decrease of the albumin contents within the vessels, which is considered to be the result of plasma decay. These data can to some extent explain the relation between the parameters of dynamic tensiometry of blood and urine, considered to reflect the dependence of the proteinemia level on proteinuria. It is not only the serum protein level, especially the concentration of albumin that affects dynamic surface tension, but also the molecular modification of proteins. Urea molecules, for example, possess the properties of dipoles and weak cations, and can therefore form rather stable complexes with other polar compounds like serum albumin. Albumin and other singlechain proteins, when introduced into an urea solution of physiological concentration (3 mmol/1) are able to bind equivalent quantities. In this case one urea molecule interacts with terminal

110 COOH groups of the polypeptide chain. The processing of albumin by strong urea solutions, the case that models a chronic renal insufficiency, leads to a reversible denaturation of the protein. Albumin molecules which had completely lost the secondary and ternary structure, and, therefore, its native properties, is capable for binding ca. 150 urea molecules (the mass of the complex depends on the concentration of both substances in blood). It follows from our studies and data presented in literature that the addition of urea to human serum albumin solutions in vitro results in a significant surface tension decrease. Therefore it can be expected also to take place in the organism of patients suffering from the nephrotic syndrome. The nephrotic syndrome is characterised by proteinuria, albuminemia and increased synthetic activity of the kidney. These characteristics are linked with increased serum concentrations for C-reactive

protein,

high-molecular

vitamin K - dependent

glycoprotein-C,

fibrinogen,

[3-thromboglobulin, etz-macroglobulin , et2-antiplasmin, and the coagulation factors II, V, VII, VIII and X (Cosio & Bakaletz 1986, Ryabov et al. 1989, Savitsky & Gordejev 1992, Kaysen 1993, Ota et al. 1992, Thiery et al. 1996). In addition to alteration of protein metabolism, the nephrotic syndrome is accompanied by alteration of lipid metabolism. The presence of lipid surfactants, namely phospholipids, cholesterol, triglyceride in the serum, while an inverse correlation exists for the level of albuminemia. Hyperlipemia is typical for the nephrotic syndrome and marked increase of cholesterol and very low density lipoproteins has been described (Ryabov et al. 1988, Hong et al. 1992, Faucher et al. 1993). In Figures 4.5, 4.6, 4.8 to 4.10 the tensiograms of biological liquids are presented for patients suffering from a nephrotic and uric syndrome during the formation of acute and chronic glomerulonephritis, respectively. It was found that the k-value is not only the acute glomerulonephritis are different depending on whether the nephrotic syndrome is present or not. This fact is important from a diagnostic and prognostic point of view. The value of ~ for serum for patients with a nephrotic syndrome exceeds that characteristic for healthy persons, while for the uric syndrome a decrease of k-values was found. Surface dynamic tensiometric parameters in these subgroups were rather similar, but the dynamic surface tensions of urine in the short time range were higher than those measured in the reference group for only the nephrotic syndrome. Thus, for patients with acute glomerulonephritis with a nephrotic syndrome an increase in X for serum and of crl for urine is common.

111

75

70

.....

.........

o

::::::::::::::::::::: 2:::

65

60

-2

1

t

t

-1

0

1

lg(tef) [S]

Fig. 4.5. Example for serum tensiograms obtained from patients with acute mesangiocapillary glomerulonephritis, one with additional nephrotic syndrome (male, age 32, thin line), one with additional uric syndrome (female, age 29, thick line); dotted curves correspond to average values for healthy subjects of

75

70

--

........

._

-::_:_.__._:_ .....

65

60

55

-2

-1

0

1

lg(tef) [s]

Fig. 4.6. Example for serum tensiograms obtained from patients with acute glomerulonephritis, one with additional nephrotic syndrome (male, age 48, thick line), one with additional uric syndrome (male, age 24, thin line); dotted curves correspond to average values for healthy subjects of corresponding age and sex.

112 6050403020=

10-

.~ >

0---10 -20 -30 -40 -50 AGN

CGN

AGN

Blood serum

CGN Urine

Fig. 4.7. Changes in ~ values of serum and urine obtained from patients with acute glomerulonephrites (AGN) and chronic glomerulonephrites (CGN) associated with nephrotic syndrome (black) and uric syndrome (white). Changes are given in % with respect to corresponding healthy controls.

75

70

+

..................... : --o ....

o. ~176

E

65

~

60 55 50

t

~

-2

-1

I 0

1

lg(tef ) Is]

Fig. 4.8. Example for urine tensiograms obtained from patients with acute glomerulonephritis, one with additional nephrotic syndrome (female, age 37, thick line), one with additional uric syndrome (female, age 32, thin line); dotted curves correspond to average values for healthy subjects of corresponding age and sex.

113

75

--

_

70-

"~ ....

~

-o. oo~

60

oo..

--

55

-

-2

t

I

I

-1

0 lg(tef) [s]

1

Fig. 4.9. Example for urine tensiograms obtained from patient with chronic glomerulonephritis, with additional lipoid nephrosis (female, age 22); dotted line corresponds to average values for healthy females of the same age.

75

--

70-

~65 o-,

60~

55

- - -

-2

I

-1

-

I

I

lg(tef) [s]0

1

Fig. 4.10. Example for urine tensiograms obtained from patients with chronic mesanglioproliferative glomerulonephritis, one with additional nephrotic syndrome (male, age 30, thin line), one with additional uric syndrome (male, age 28, thick line); dotted line corresponds to average values for healthy males of the same age. In the case of nephrotic syndrome a significant increase of ~, for serum and o l for urine is indicative o f patients suffering from chronic glomerulonephritis; here, however, a decrease of cr for urine at t = 1 s and t ~ oo is observed. The surface tensiometry parameters of urine for

114 the uric syndrome are virtually similar to those for healthy persons. We believe that if the nephrotic syndrome reflects the seriousness of kidney disease and is strongly indicative of the severity of pathologic processes, then the increase in k for serum and c l for urine can be regarded as evidence of a future recrudescence in the development of a chronic glomerulonephritis. While the nephrotic syndrome often accompanies acute glomerulonephritis, renal insufficiency accompanies

chronic

glomerulonephritis.

The development

of a renal

insufficiency

accompanying chronic glomerulonephritis results in a dynamic surface tension increase for serum at t = 0.01 s and t = 1 s (cf. Fig. 4.11.). It has to be stressed that significant changes in k are also important from a practical point of view, because differences between values of ~ , ~2 and k for patients with a non-affected kidney function and the parameters for a reference group is rather unreliable. Low values of ~. for urine can be cautiously regarded as a positive prognosis for the development of the disease in what regards the kidney function. Chronic renal insufficiency is accompanied by excessive urinal excretion of low molecular compounds like acetone, acetoacetate, acetylcarnityne, valine, glycine, lactate, as shown in Fig. 4.12. The influence of these metabolites on dynamic surface tensiometry parameters, in particular the ~, value for urine, is quite possible.

40 ~

3o -1 20- t 10-

-10 -20 I -30 l -40 cl

c2

~3

~.

crl

~2

~3

Fig. 4.11. Changes in surface tensiometric parameters measured in biological liquids obtained from patients with chronic glomerulonephritis with (black) and without (white) chronic renal insufficiency. Changes are given in % compared to corresponding healthy controls.

115

600 500 I .~" 400 ..o 300 200 100

A

AA

AC

AL

V

H

GL

D

C

L

T

Fig. 4.12. Increases of various low-molecular weight metabolites in urine for patients with chronic glomerulonephritis compared to normal. Black-without chronic renal insufficiency (CRI), white- 1st stage of CRI, hatched - 3rd stage of CRI. The metabolites are: A - acetone, AA - acetoacetate, AC acetyl cametine, AL - alanine, V - valine, H - hippurate, GL - glycine, D - dimethyl amine, C creatinine, L - lactate, T - trimethyl amine oxide

Arterial hypertension is one of the leading factors that degrade kidney structure and thus renal function in patients with chronic glomerulonephritis. Arterial hypertension was sometimes referred to as an indication of future chronic renal insufficiency. The increase in arterial hypertension is accompanied by increased urinary excretion of proteins, which are definitely related to the extent of morphological changes in kidneys. The characteristic feature of a glomerular

pathology

is the

10 to

30

times

increased

excretion

of

albumin

and

immunoglobulin-G. Arterial hypertension is accompanied by an increased excretion of acetyl-13-D-glucosaminopeptidase, the lysosome enzyme that is localised mainly in proximal tubular apparatus. This increase excretion of acetyl-13-D-glucosaminopeptidase into extracellular liquids can be explained by lysis of cells and exocytosis, which reflects the rather functional than structural stage of a kidney damage. A direct relationship between systolic blood pressure and the activity of acetyl-[3-D-glucosaminepeptidase in urine was found by Fomenko et al. (1992). This correlation can be ascribed to the dependence of lysosome cell metabolism on the intracapillary

116 pressure in the kidneys. In line with the enhanced arterial hypertension, an increase of albuminuria takes place, which of course affects variations in the dynamic surface tensions of urine.

In

kidney

diseases

Scherberich

et

al.

(1989)

observed

in

addition

to

acetyl-13-D-glucosaminepeptidase, the urinary excretion of other proteins of tubular (alanine aminopeptidase,

7-glutamiltranspeptidase,

alcalic

phosphatase)

and

serum

(albumin,

immunoglobulin-G, Otl-microglobulin) origin. The excretion of these proteins in patients with kidney disease was more pronounced than in healthy persons, and it increased further with the development of arterial hypertension, a fact that can be explained by an increase in the intraglomerular perfusion pressure. The presence of such proteinic surfactants in urine of patients suffering from glomerulonephrites with arterial hypertension,

and increased

concentration of these surfactants, can be regarded as one of the factors determining the dynamic surface tensiometry parameters of biological liquids. A weak ability of kidneys to concentrate urine is believed to be the early symptom of their degradation. The relative density of urine is related to surface tension parameters, because a decrease in its specific weight corresponds to smaller surfactant contents, caused by lower glomerular filtration and less intensive tubular secretion. It was shown that only the equilibrium surface tension is characterised by a definite direct dependence on the urine density. In fact, only a3 correlates with the contents of non-ionic low-molecular weight surfactants, which in vitro are mixed with albumin often lead to a decrease in g3. Therefore the level of albumin and low-molecular weight non-ionic surfactants in urine can affect both the relative density of this biological liquid and its dynamic surface tension parameters. The process of osmotic concentration and dilution of urine is based on different permittivities for water, electrolytes and urea. The initial singular mechanism, which is substantial for the whole system of osmotic concentration of urine, is related to the ionic pump activity performed by cells located in the ascendant part of the looped tubule of Henle. This effect is subsequently enhanced due to the difference in osmotic properties of intratubular liquids and the interstitial tissue at different levels of the renal medullar layer. The tubulointerstitial component in glomerulonephritis is just the damage of structures by which tubular transport processes are performed. These structures control electrolyte homeostasis. Changes of kidney tubules and stroma can be responsible for these damages. It

117 was shown by Ratner et al. (1991) that for patients with pronounced tubulointerstitial components the ability for a maximum osmotic concentration and excretion of ammonium and hydrogen ions is strongly reduced. As the osmotic concentration of urine still takes place, these morphologic variations are certainly absent. The damage of tubules and stroma in chronic glomerulonephritis correlate with parameters of urine equilibrium surface tension, which in turn depends on the osmolarity of biological liquid. In cases of glomerulonephritis with massive proteinuria the ability for osmotic concentration of urine is significantly reduced. A sharp increase of tubular loading by serum albumin, which is common to the nephrotic syndrome, leads to increased protein reabsorption within the kidney interstitium, irregularities of water and urea transport through the distal collecting tubules. The significance of changes in various surface tensiometric parameters of serum and urine as indicators for different forms of glomerulonephrites can be summarised as follows. Acute glomerulonephritis is characterised by increases of ol and

0"2

for blood and crl for urine, and a

decrease of ~, for urine. For chronic glomerulonephritis a decrease in equilibrium surface tension or3 of blood and urine, an increase of crl for urine and )~ for blood, and the decrease of 02 and )~ for urine are typical. Common features of lupus glomerulonephritis are the increase of all dynamic surface tensiometry parameters for blood serum and also the increase of Crl and decreased )~ values for urine. For Genoch glomerulonephritis the increase in Crl and ~2 for blood and all parameters of urine was observed. While the development of the disease is specific for each particular form (and systemic lupus erythematosus and hemorrhagic vasculitis are characterised not only by renal symptoms), their differential diagnostics is of practical interest only with respect to chronic glomerulonephritis, acute glomerulonephritis, lupus glomemlonephritis and Genoch glomerulonephritis. In view of this, criteria can be presented in a simpler way: 9 in acute glomerulonephritis only the ~i and

(~2

level of serum, and ~l of urine increase

9 in chronic glomerulonephritis one has to expect a decrease of equilibrium surface tension for blood and urine, and a decrease of cr2 for urine; 9 in lupus glomemlonephritis an increase of all dynamic tensiometry parameters of blood appear, 9 in Genoch glomerulonephritis the cr2 and ty3 values increase for urine.

118 Thus dynamic surface tension measurements of biological liquids enables one to perform differential diagnostics of various nosologic forms of glomerulonephrites, and to distinguish implicitly

between

mesangiocapillary

glomerulonephritis

and

mesangioproliferative

glomerulonephritis.

4.1.2. Influence of particular serum and urine components on dynamic surface tension To study the influence of particular components of a biological liquid on surface tension, a correlation analysis has been performed with respect to proteins, lipids and other surfactants. Significant interrelations between values of surface tensiometric parameters and contents of specific surfactants in serum was found; in some cases the correlation was expectedly negative, while in other cases positive correlation exists, as summarised in Table 4.3. In patients who have chronic glomerulonephritis the values of (3"1 and (52 are inversely proportional to the proteinurea level. In patients who have lupus glomerulonephritis there exists a strong direct proportionality of these parameters and the equilibrium surface tension, which on a first glance seems to be difficult to explain. In patients who have acute glomerulonephritis or Genoch glomerulonephritis such correlations were not found at all. Such diverse variations of correlation coefficients for patients with chronic glomerulonephritis and lupus glomerulonephritis can be explained by the fact that the concentration of specific proteins in blood serum is different. Acute glomerulonephritis and chronic glomerulonephritis, in contrast to lupus glomerulonephritis and Genoch glomerulonephritis, are characterised by a lower total concentration of proteins, albumins, [32-microglobulin,

C-reactive protein,

fibrinogen, transferrin, circulating immune complexes, cryoglobulins. Also some antibodies (autoantibodies) are absent in senma, which may be possibly regarded as factors leading to the diversity in variations of dynamic surface tensions and correlation links. It was shown by model studies (see Chapter 1) that, for example, the addition of other proteins to the solution of albumin results in either a decrease or increase in surface tension.

119 Table 4.3. Correlation coefficients between surface tension parameters measured in serum obtained from patients with various types of glomerulonephritis and serum components Serum component Acute Chronic Lupus Genoch glomerulonephritis ' ~;lomerulonephritis. glomerulonephritis glomerulonephritis 0"1

0"2

0"3

0"1

$

Total protein

i0"2

,0"3

,0-1

$

,0-2

1'1'

1'

0"3

(3"1

1'

Albumin otl-globulin fraction

i 0"2

/ ,0"3

i

11'

|

$$$

oh-globulin fraction

|

$ 1"

1" $

[3-globulin fraction

$

~/-globulin fraction

?

Immunoglobulin-G Immunoglobulin-A

$

$

$

$$

$

1'1'1' $$ $

Immunoglobulin-M 132-microglobulin

1,1, $

Fibrinogen Circulating immune

i

1'

complexes

$$

Total cholesterol

$$

$~

1"1' 1'1'

$ 1'1'

1"

1"

$

m-cholesterol

$

Triglicerides High density lipoprotein fraction

$

Low density lipoprotein fraction Very low density lipoprotein fraction

1'

1'1'

S : S

1' i~ |

|

Urea Creatinine

$

$

$?71'

$ $$

$ $$

Uric acid Oxypurinol Medium-size molecular compounds

$

positive correlation; $ negative correlation; empty - no correlation r<0.3; one arrow, r =0.3 to 0.5; two arrows, r = 0.5 to 0.7; three arrows, r > 0.7

?? 71'

1"

120 In acute glomerulonephritis, granular deposits of immune complexes and particular fractions of the complement in glomerule capillary walls, a decrease of the total complement contents and its C3-component in serum and an increase in the amount of circulating immune complexes were found. The damage of glomerular structures was related both to the deposition of circulating immune complexes in the kidneys, and to the formation of immune complexes in situ. While the concentration of serum immunoglobulin-M increases for all patients suffering

from chronic glomerulonephritis, the increase in concentration of immunoglobulin-G was observed only for the uric syndrome, and high levels of circulating immune complexes was characteristic only for patients with a nephrodc syndrome. Four factors are responsible for specific features of a glomerulonephrites pathogenesis: (i)

the localisation of immune complexes in glomerules;

(ii)

the type of immune complex damage;

(iii)

the character of reactions of the basal membrane and mesangium;

(iv)

the type of immune complex elimination.

The location of immune complexes in glomeruli depends on their size and subepithelial, subendothelial, intramembranous and mesangial deposition can take place. The size of immunoglobulin depends on parameters of antigen determinants and on the antigen-antibody binding strength. Agglutination of small immune complexes (with molecular masses of 300 to 500 kDa) can happen in the subendothelial space which explains the paradoxical fact that membranous glomerulonephritis is progressing when the serum contains no circulating immune complexes. The accumulation of medium size immune complexes (500-5000 kDa) takes place in the mesangium, leading to its hyperplasy (e.g., for mesangioproliferative glomerulonephritis). Deposits of immune complexes activate the complement system and release proteases from polymorphonuclear leukocytes which damage basal membranes of capillaries. No correlation between the level of circulating immune complexes in serum and the amount of their glomerular deposits exists, but the number of circulating immune complexes is directly linked to the activity of glomerulonephritis (Kolesnyk et al. 1992, Yagame et al. 1991, Miura et al. 1989).

121 Immune complexes stimulate the formation of superoxide radicals and other active forms of oxygen by kidney mesangial cells and tubular epithelium. This process is accompanied by a destabilisation of cellular membranes, a deterioration of the liquidity of the cytoplasmatic membrane of peripheral blood erythrocytes, and a decrease in the total contents of lipids and phosphatidylcholine. An increase in the circulating immune complex contents in serum for various clinicalmorphological forms of chronic glomerulonephritis was observed most frequently for patients with a nephrotic syndrome. No correlation dependence of the level of circulating immune complexes and daily proteinuria, total protein and serum cholesterol was found (Ryabov et al. 1989). For systemic lupus erythematosus an increase in the contents of circulating immune complexes, immunoglobulin, a2-macroglobulin, Ctl-antitrypsin, C-reactive protein, fibrinogen, fibronectin, low density lipoproteins, apolipoprotein-H in blood was observed. Immune complexes react not only with exogeneous high-molecular weight substances, but also with low-molecular weight compounds of endogenous nature. The theory for autoimmune regulation mechanism of chemical homeostasis was developed, based on the existence of antibodies with respect to ferments, mediators, hormones and their metabolites. The coupling ability of immunoglobulin to amino acids increases in line with the activity of systemic lupus erythematosus, while the concentration of immunoglobulin-G, A and M in serum varies only slightly. For lupus glomerulonephritis the immunoglobulins, especially in the combination immunoglobulin-G+immunoglobulin-M, exist as circulating immune complexes. The fact that for systemic lupus erythematosus high levels of antibodies to DNA and some other cell organelles and biopolymers were found may be explained as follows. There exists an immune interrelation between DNA and membrane phospholipids such as cardiolipin, phosphatidylglycine and phosphatidic acid. This interrelation is due to the location of chemical groups in DNA that is similar in phospholipids. It is to be noted that the affinity of cardiolipin to anti-DNA is stronger than that of DNA itself. The presence of anti-DNA in serum is one of the characteristic features of lupus glomerulonephritis. Therefore, if antibodies are actually directed not towards DNA, as is generally accepted, but to phospholipids of membranes, then

122 these antibodies damage the lipid membrane microsomal oxidation system in systemic lupus erythematosus, and explain the deposition of immunoglobulin in kidney basal membranes, which contain phospholipids, but not DNA. Links exist between the levels of anti-DNA and circulating immune complexes, which correlate with the development of lupus glomerulonephritis. The deposition of DNA-anti-DNA complexes in kidneys is regarded to be the primary factor for the development of lupus glomerulonephritis. The most pronounced clinical activity of systemic lupus erythematosus coincides with either high or unexpectedly low titres of antibodies with respect to native DNA, or with a significant decrease in the contents of serum proteins of the complement system (especially C 3- and C4-components ). The formation of complement-fixing DNA-anti-DNA

immune complexes in blood happens along with a suppression of antibodies with respect to native DNA. Figure 4.13 shows the tensiograms for serum obtained from patients with systemic lupus erythematosus with and without kidney damage.

80

75

~

70 ~ t~

~

.

_

................................

656055 50 -2

I

t

I

t

-1

0 lg(tef) IS]

1

2

Fig. 4.13. Example of serum tensiogramsobtained from patients with sub-acute form of systemic lupus erythematosis having 3rd activity degree. One with additional glomerulonephritisand uric syndrome (female, age 36, thick line), one without additional glomerulonephritis(female, age 34, thin line), dotted line correspondsto average values for healthy females of the same age.

123 During complement activation (this activation can take place either in the classic way, starting usually from binding of the Cl-component to immune complexes, or in the alternative way, which includes usually the deposition and covalent binding of Cab-components to a surface activated by carbons), the proteins of the system (more than 20 various types) undergo either proteolytic splitting or pronounced conformational changes, which vary the adsorption properties and, therefore, the dynamic surface tensions of serum. For some diseases, a significant increase in the dynamic surface tensions of serum (cf. Figs. 4.8, 4.13) and cerebrospinal liquid (cf. Chapter 7) was found in the short time range. At first sight, no explanation can be given for values of

O'1 =

73-75 mN/m, which exceed the surface

tension of pure water by 1-3 mN/m. However, similar anomalies in the short surface lifetime range were also found in a number of other studies (Ramachandran et al. 1982, Miller et al. 1993, WiJstneck et al. 1996) using various experimental methods for certain proteins ([3-casein, [3-1actoglobulin). To give a qualitative explanation for this apparent contradiction, the protein adsorption theory summarised in Chapter 1 can be used. It is seen from Fig. 1.2 that no decrease in the surface tension of protein solution takes place for a monolayer coverage lower than 10%. Note that in the calculations of surface pressure, only the contributions from the entropy of mixing and Coulomb interaction were taken into account. At the same time, 10% coverage of the monolayer by protein corresponds to a protein adsorption of ca. 0.3-0.5 mg/m 2, or (3-5). 10-6 mol/m 2 (per mole of amino acid groups). For adsorption layers of 1 nm thickness, this results in a value of 3-5 mol/1 for the concentration of ions within the surface layer. It is well known that in aqueous solutions of inorganic electrolytes at similar concemration increases the surface tension from 72-73 to 74-76 mN/m. It is proven that hemorrhagic vasculitis is a disease of immune complex nature, when aseptic inflammation develop in microvessels, leading to the destruction of their walls due to the damaging action of circulating low-molecular immune complexes and activated components of complement. The serum of such patients contains large amounts of immunoglobulin-A and circulating immune complexes containing immunoglobulin-A. Activation of the complement system leads to the formation of C5-C9 protein complexes, which afflicts the lipid bilayer of the cell membrane, resulting in an osmotic lysis of endothelial cells. This in turn leads to the

124

activation of the Hageman factor with the formation of fibrin degradation products in blood (Mazurin et al. 1996, Mammen et al. 1988, Fuhrer et al. 1990). For hemorrhagic vasculitis the increased contents of o~2- and )'-globulins, fibrinogen, C-reactive protein, circulating immune complexes, immunoglobulin-G, and cryoglobulins in serum leads to changes in blood viscosity. Variations in the amount of these components also influence the dynamic surface tension, as one can see in Fig. 4.14. An important feature of circulating immune complexes for Genoch glomerulonephritis is their ability of cryoprecipitation: in 50% of cases circulating immune complexes were found to be mixed with cryoglobulins with a composition identical to glomerular immune complexes of immunoglobulin-G and immunoglobulin-M. In 7% of studied Genoch glomerulonephritis cases, a M-gradient was found in serum, capable for exerting a significant influence on dynamic surface tensiometric parameters of this biological liquid.

7472 ~176176 ~

,_.70

.o, . . . . OOoo--,~ooooOOOOOoo "OO,,o Ooo

~ 68~

"~176

66-64-62 -2

~

I

t

I

I

-1

0 lg(tef) [S]

1

2

Fig. 4.14. Example of serum tensiograms obtained from patients with hemorrhagic vasculites, Genoch glomerulonephritis, CRI0. One with additional nephrotic syndrome (male, age 62, thick line), one with additional uric syndrome (male, age 50, thin line); dotted line corresponds to average values for healthy males of the same age.

125 Studies of the rheological properties of blood obtained from patients who have hemorrhagic vasculitis provide evidence that changes in aggregate composition take place due to the variation of circulating immune complexes, fibrinogen and other proteins. The presence of circulating immune complexes in the vascular bed leads to the activation of the coagulation system, resulting in further changes of rheological parameters. Recent fundamental investigations (experimental model studies of immunocomplex processes for animals, estimation of immunoglobulin, contents of complement and immune complexes in vessel wall for studies of biopsy material taken from patients) indicated the pathogenetic action of circulating immune complexes in the development of Genoch glomerulonephritis. The mean values of circulating immune complexes in cases of hemorrhagic vasculitis are almost 4 times higher than those characteristic for healthy persons, and were observed in all cases of this disease. A correlation between these values was also found, and both amount to the C-reactive protein in blood and y-globulinemia level. The presence of granular deposits containing immunoglobulin (mostly immunoglobulin-A) and Ca-components of the complement in capillary walls and kidney mesangium is indicative of the immunocomplex process in Genoch glomerulonephritis pathogenesis. Mesangial localisation of deposits enables one to presume that circulating immune complexes arising due to antibody excess are large. The contents of immunoglobulin and circulating immune complexes in serum depend on the particular form of glomerulonephritis and affects the dynamic surface tension. For patients with acute and chronic glomerulonephritis the inverse correlation exists between

0.1

and 0.2 and

the immunoglobulin concentration, while for lupus glomerulonephritis a pronounced positive correlation link exists between

O"2

and the immunoglobulin concentration. In this case the

equilibrium surface tension shows a pronounced negative correlation with the immunoglobulin concentration, while for Genoch glomerulonephritis this correlation was found to be unexpectedly positive. As one could expect, there is a negative correlation between the amount of circulating immune complexes and dynamic surface tension within groups of patients with acute and lupus glomerulonephritis. The fact that a relationship exists between parameters of the proteinogram and the X-value of dynamic tensiograms of blood serum is interesting with respect to differential diagnostics of

126 various nosologic forms of diseases. For example, the )~-value of blood for acute glomerulonephritis

correlates

with'

the

concentration

of

[3-globulins, for

chronic

glomerulonephritis with circulating immune complexes, for lupus glomerulonephritis with immunoglobulin, for Genoch glomerulonephritis with txl-globulins. One can assume that in case of systemic lupus erythematosus dynamic surface tensions of blood serum are largely dependent on the extent of immunoglobulinemia, which definitely reflects the pathogenesis of this particular disease (autoformation of antibodies, formation of circulating immune complexes). Currently the origin and biological role of low molecular [~2-microglobulin (with a molecular mass 11 kDa) are widely discussed. This protein, which is synthesised mainly by lymphocytes, participates in immune processes and its contents in blood remains approximately constant during the day, and depends only slightly on external factors. Due to its low molecular mass and negative charge, [32-microglobulin freely penetrates through the glomerular kidney filter, and subsequently becomes almost reabsorbed and catalysed by proximal tubular cells. There is evidence that for glomerulonephrites the contents of [~2-microglobulin in serum increases. The highest contents of [32-microglobulin in blood was found for patients suffering from chronic glomerulonephritis, while for lupus glomerulonephritis and Genoch glomerulonephritis this concentration was lower. For acute glomerulonephritis a definite correlation exists between [~2-microglobulinemia and the ~1 value of blood serum, while for chronic glomerulonephritis an inverse correlation of [32-microglobulinemia with the equilibrium surface tension cy3, and a direct correlation with k of blood is observed. For systemic lupus erythematosus and hemorrhagic vasculitis the contents of [32-microglobulin produces no significant effect on dynamic surface tensiometric parameters (cf. Table 4.3). The urinal excretion of [32-microglobulin for the acute form of chronic and lupus glomerulonephritis exceeds significantly that for healthy persons. The existence of a nephrotic syndrome and chronic renal insufficiency for these patients leads to even higher excretion of this protein. Chronic glomerulonephritis and lupus glomerulonephritis, independent of their morphologic forms, are frequently accompanied by pronounced tubulointerstitial components, which are exhibited by dystrophy and atrophy of the tubular epithelium, thickening and homogenisation of the tubular basal membranes, sclerosis and lymphohystiocital infiltration of

127 stroma. The damage in the proximal tubular apparatus can be one of the causes of improper reabsorption and catabolism of 132-microglobulin, leading to an increase in protein urinal excretion, resulting in an increased concentration in urine. In addition, immune imbalances specific to glomerulonephrites promote the development of hyper-132-microglobulinemia. The particular features of 132-microglobulin metabolism (free filtration through the basal membrane of kidney glomeruli and almost complete reabsorption by proximal tubular cells) constitute the physiological basis for the study of the tubular apparatus. For patients with a glomerulonephrite tubulointerstitial component, diffuse deposits of 132-microglobulin were found in the tubular epithelium cytoplasm and at basal membranes. To differentiate between glomerular and tubular proteinuria, simultaneous estimation of the 132-microglobulin contents in serum and urine can be suggested, because a glomeruli damage may not result in a variation of the 132-microglobulin contents. Some data support the view that the increase in albumin and 132-microglobulin excretion is specific to a pathologic process, which primarily extends to the glomerular or tubular apparatus of kidney. The isolated dystrophy of tubular epithelium, with other parenchyme structures being unchanged, leads to more pronounced increased 132-microglobulin excretion, which is indicative of the decrease in its readsorption by damaged tubular epithelium. Peritubular sclerosis and inflammative infiltration of interstitium result in a progressive increase in 132-microglobulin excretion, which is indicative of deterioration in tubular reabsorption of proteins. The fact that even severe damages of the kidney parenchyma does not increase significantly the 132-microglobulin excretion supports the view that this excretion is specific to tubulointerstitial changes. The so called adhesive proteins, i.e. proteins with similar structure and functionality (homologous proteins), in particular, fibrinogen and fibronectin, play a significant role in the system of regulation of kidney tissue damage and reparation. These proteins, together with some components of connective tissue, e.g. laminine, chondroitin sulfates, are responsible for the interaction of cells with one another and with the extracellular martix. The main biological function of fibrinogen is the formation of a fibrin network. Fibrinogen is one of the proteins specific to acute inflammation, and its concentration in blood is controlled by the contents of destruction products, which initiate the formation of fibrinogen by hepatocytes either directly or via the formation of a hepatostimulating factor by monocyte-

128 macrophages. The increase of fibrinogen in patients with the nephrotic syndrome is related to an increase in the fibrinogen synthesis by kidneys as response to proteinuria and subsequent hypoproteinemia. Deposits of fibrinogen can be detected in kidneys. For patients with lupus glomerulonephritis and Genoch glomerulonephritis sharp increases of the fibrinogen concentration in plasma is observed. In groups of patients with lupus glomerulonephritis and acute glomerulonephritis correlations between fibrinogen contents and surface dynamic tensiometry parameters were found. One of the most urgent problems in modern nephrology is the development of new screening tests, which can be used to perform differential diagnostics, to estimate activity of the disease, and to predict further development of a kidney disease. It is known from clinical experience that those indicators, which are either related specifically to a mechanism of development of a disease, or reflect various aspects of glomerulonephrites pathogenesis integrally, can be most useful for this purpose. This last group of indicators includes, in particular, fibronectin. Fibronectin is a common component of the mesangium. Its contents in kidneys increases for various glomerular disturbances, and it plays a key role in the formation of immune complexes in situ. For patients suffering from a nephrotic syndrome a moderate increase in the serum

fibronectin contents was observed. However, some data show a significant decrease in the fibronectin amount for pronounced proteinuria. Negative correlations exist between the fibronectin level and serum tensiographic parameters in the short and medium time range. These correlations were observed only for patients with lupus glomerulonephritis (Table 4.3). It follows, that two adhesive blood proteins, fibrinogen and fibronectin, produce opposite effects on the dynamic interface tensiometric parameters. For patients suffering from chronic glomerulonephrites no significant changes in fibronectin level were found. The extent of urinal excretion of fibronectin is defined by proteinuria. Therefore in cases of the nephrotic syndrome a most pronounced fibronectinuria is observed. The presence of fibronectin in urine can be explained by three possible causes. First, its filtration through damaged superpenetratable glomerular filters is possible. As fibronectin possesses a high molecular mass, it can be present in urine only for highly selective proteinuria, which accompanies acute inflammations of kidneys. Thus the highest level of proteinuria is observed for the nephrotic syndrome. Second, fibronectin can be produced

129 locally (in kidneys) due to the destruction of the glomerular capillary basal membrane, which contains fibronectin. Third, in cases of glomerulonephrites the activation of mesangial and endothelial cells in kidneys leads to the increase in the fibronectin synthesis. There is evidence that fibronectin participates in the reactions of cellular cytotoxicity, and is able to produce significant changes in the properties of immune complexes. The presence of fibronectin can affect both the formation of complexes in situ and the deposition process of circulating immune complexes in the kidney tissue. For example, for immunoglobulin-Aglomerulonephritis, immunoglobulin-A + fibronectin complexes were detected in serum of patients. There are data, which indicate that immunoglobulin deposition at the glomerular capillary basal membrane takes place due to the presence of specific parts in the fibronectin responsible for collagen binding. For patients suffering from lupus glomerulonephritis and Genoch glomerulonephritis strong negative correlations between the fibronectin level in urine and ~1. For chronic glomerulonephritis this correlation is weaker, while for the acute glomerulonephritis it becomes positive. Only [32-microglobulin is equally related with the surface tensions of urine for all types of glomerulonephritis. The value of (~1 for all nosologic forms of glomerulonephritis directly correlates with urea, creatinine, uric acid and oxypurinol (cf. Table 4.4). Cryoglobulins represent a heterogeneous group of immunoglobulins capable for anomalous precipitation or gel formation at temperatures lower than 37~

These pathological proteins

were detected for 46% of patients with systemic lupus erythematosus. Depending on their constituents, cryoglobulins can be divided into three types: type I corresponds only to monoclonal immunoglobulins of single classes (M, G, sometimes A) with one sort of light chains (• or ~); type II comprises monoclonal immunoglobulin bound to polyclonal immunoglobulin which belongs to other classes; type III includes only polyclonal immunoglobulins

in

various

combinations

(for

example,

immunoglobulin-G

+

immunoglobulin-M, immunoglobulin-G+ immunoglobulin-M+ immunoglobulin-A, etc.) The combination of immunoglobulin-M+ immunoglobulin-G is most common, while mixed cryoglobulins of type III represent true immune complexes where immunoglobulin-M frequently possessing the rheumatoid factor activity (Gordovskaya et al. 1990), act as an

130 antibody. The presence of cryoglobulins in blood are seen as typical serological markers of a wide range of autoimmune diseases, including systemic lupus erythematosus. For systemic lupus erythematosus definite relationships exist between the serum level of cryoglobulin and complement components, increased antibody titres to native DNA and the existence of lupus glomerulonephritis. Kidney damage by cryoglobulinemia is related to the derangement of microcirculation due to an increased blood viscosity. Table 4.4. Correlation coefficients between surface tension parameters measured in urine obtained from patients with various types of glomerulonephrites and urine components.

Acute

Chronic

glomerulonephritis

glomerulonephritis

glomerulonephritis glomerulonephritis

~1

~1

Urine component

Total protein

~2

~3

$$

$

Genoch

Lupus

~2

~3

~1

~2

$

$

$$

$$

~3

~1

~2

~3

$$

$

$

i

Albumin

' 1"?

'

$

$$

~2-macroglobulin -132-microglobulin

$$ ' $ $ $ +

' 1'

Fibrinonectin

?

Urea

?

Creatinine

?

$

$$ ' $ ,

$

$

$

$$

?

$

'$$$

$ '

$

$

$

6$ '

$

?71'

1'

?

1'

?

1"1"

?

1'

71"

?

???

?

Uric acid Oxypurinol

$$$

1"

$

~r

?

?

?

??

? positive correlation; $ negative correlation; empty - no correlation r<0.3; one arrow r =0.3 to 0.5; two arrows r = 0.5 to 0.7; three arrows r > 0.7

The contents of protein in serum cryoprecipitates for systemic lupus erythematosus increases in line with the severity of the disease. Fibronectin and imrn,unoglobulin-G, more rarely

131 immunoglobulin-M and immunoglobulin-A were found in cryoglobulins. For lupus glomerulonephritis with cryoglobulinemia, higher values of fibrinogen, cz2-globulins and C-reactive protein are characteristic, while hyper-7-globulinemia is more common for patients without cryoglobulins, which was explained by high levels of immunoglobulin-G (Vasilyev et al. 1994, Howard et al. 1991, Sikander et al. 1989, Adu et al. 1984, Phi et al. 1989). The association between the presence of antibodies in high titres and cryoglobulins leads to the supposition that B-cell hyperreactivity and monoclonal cryoglobulinemia can be regarded as parallel processes. Therefore, the decrease of antibodies for patients with cryoglobulins in blood can be regarded as evidence for switching from B-cell activation to monoclonal processes, and is an indication of a possible participation of cryoprecipitates in the pathogenesis of kidney damage for systemic lupus erythematosus. The data presented above enable us to expect that significant changes in dynamic interface tensiometric parameters will be obtained in the presence of cryoglobulins. However, only a trend in a decrease for or2, or3 and L was observed. As an inverse correlation exists between the level of cryoglobulinemia and the immunoglobulin-G contents, one could expect a remarkable surface tension decrease at medium surface lifetime, and a variation of the equilibrium surface tension. However, such effects were not found. Recently the role of lipids in pathogenesis and development of glomerulonephrites was intensively studied. Hyperlipoproteinemia can result in the damage of kidney endothelial cells and the deposition of lipids in mesangium stimulates the proliferation of mesangiocytes, and leads to tubulointerstitial pathologic processes caused by the precipitation of lipoproteins in tubules. Lipid inclusions in kidneys of patients suffering from a nephrotic syndrome are localised in the cytoplasm of podocytes, mesangiocytes, tubular epithelium and stroma cells. In some patients the deposition of lipids in the interstitium was so high that foam cells were formed. In morphological

studies

of kidney tissue

obtained

from

patients

with

chronic

glomerulonephritis and lupus glomerulonephritis, in 6% lipid deposits were found in glomeruli. Here subendothelial and mesangial localisation was more common than intramembranous localisation. No correlation between hyperlipidemia and the existence of glomerular lipid inclusions was found. The formation of low density lipoprotein deposits in glomeruli includes

132 the

following

stages:

1) binding

of

low

density

lipoproteins

with

polyanionic

glucosamynoglycanes; 2)neutralisation of glomerular negative charge; 3)an increase in the penetrability of glomerular filter; 4)penetration of lipoproteine molecules into mesangium; 5) binding of low density lipoproteins with mesangiocites; 6)excessive production of matrix substance. In addition to the deposits of low density lipoproteins and foam cells interstitium of patients with a nephrotic syndrome contains intracellular and intercellular lipid inclusions which cannot be identified as low density lipoproteins. These may be high density lipoproteins which are filtered in glomeruli, or prostaglandines synthesised in podocytes. For a completely developed nephrotic syndrome an increase in the contents of lipid surfactants (total cholesterol, triglycerides and phospholipids) in blood occurs. This is unambiguously related to the concentration of proteinic surfactants in blood serum. Simultaneously with decreased albumin, cholesterol and triglyceride concentrations in serum increase. High values of serum cholesterol were detected in all cases of hyperlipidemia. This fact, which causes certain difficulties in phenotyping, can be explained by the redistribution of cholesterol between various classes of lipoproteins. For the nephrotic syndrome the contents of cholesterol in very low density lipoproteins (types liB and IV) and in low density lipoproteins (type IIA) increases significantly. The dynamics of triglycerides can be more easily explained: their level corresponds to a certain type of hyperlipidemia. An unfavourable progress of the nephrotic syndrome is mostly accompanied by a type IV hyperlipidemia, while for moderate cases the type IIA is more common. The decrease of or-cholesterol was observed for type IV only, that is, when the triglyceride concentration in serum is highest, and the disease is more severe. For acute glomerulonephritis variations in lipid metabolism parameters are more pronounced, while they occur less frequently as is common for chronic glomerulonephritis. For patients with a nephrotic syndrome the qualitative distribution of lipoproteins is violated. While apolipoprotein-C2 is normally distributed in equal proportions between high density lipoproteins and very low density lipoproteines, for the nephrotic syndrome it is usually contained in the very low density lipoprotein fraction only. Other apoproteins in high density

133 lipoproteins are also subjected to variation: the contents of apolipoprotein-A is increased, the contents of apolipoprotein-C is decreased, and apolipoprotein-E is completely absent. The pathogenesis of the nephrotic hyperlipidemia is still unclear. The most common theory assumes that the hypoalbuminemia stimulates an increased synthesis of both proteins and lipoproteins in the kidney. Then the loss of albumin with urine takes place, while hyperlipoproteinemia continues to exist.

1510-

m

__.

r-----]

............ t.__-_l

105~ ...............

t

I

, ,

-15 I

IIA

IIB

III

IV

6040 =

20

][

][

T

-20

m

-40 I

IIA

IIB

III

IV

Fig. 4.15. Changes of surface tension measured in serum obtained from patients with chronic glomerulonephritis at various type of lipidemia (type I, IIA, IIB, III, IV). Changes are given in % with respect to corresponding values for healthy person. In the upper graph changes cyl- hatched, ~2 - black, and ~3 - white, in the lower graph for ~, are given. Type

I hyperlipidemia

was

detected

only for acute

glomerulonephritis

and

chronic

glomerulonephritis, while type IV was observed only for patients suffering from chronic

134 glomerulonephritis. For lupus glomerulonephritis type IV of lipid metabolism disorder was typical, while for Genoch glomerulonephritis this was of IIA type. For type I an increase of or2 and a decrease in the equilibrium surface tension was characteristic, for IIB a sharp increase of surface tension for all the surface lifetimes was observed with a decrease of L, while for type IV an increase of L results (cf. Fig. 4.15). It is to be noted that for type IIA and normal lipidemia the surface tensiometric parameters are virtually equal to those characteristic for healthy persons. These data are of certain interest for differential diagnosis of hyperlipidemia based on surface tensiometry of blood serum. On the other hand, they clearly demonstrate the influence of particular lipids on the dynamic surface tensions of blood. A correlation analysis of the effect produced by various lipids on surface tension parameters for serum yield that the most pronounced correlation exists for lupus glomerulonephritis, somewhat less pronounced for Genoch glomerulonephritis, and yet less marked for acute glomerulonephritis. For patients suffering from chronic glomerulonephritis the dependence of tensiographic parameters on the character of lipidemia was rather weak. Similarly to proteins, the effect on surface tension parameters caused by lipids for various nosologic types of glomerulonephritis was often diverse (cf. Table 4.3). The fatty acid composition of serum and cell membranes for patients with nephrotic syndrome is characterised by an increase in the contents of the oleic acid and a decrease of the linoleic acid concentration. The contents of cholesterol in erythrocyte membranes was significantly decreased. It was supposed by Clemens & Bursa-Zanetti (1989) that hyperlipidemia for the nephrotic syndrome can lead to qualitative and quantitative variations in membrane composition, which result in a deterioration of the cell antioxidant system function and a suppressing of the membrane lipid resistance to peroxide oxidation processes. In all groups of patients with glomerulonephrites a significant increase in the processes of peroxidic oxidation of lipids was observed, with an increase for lupus glomerulonephritis and Genoch glomerulonephritis taking place against the background of suppressed activity of antioxidant systems. Variations in peroxidic oxidation of lipids can create changes in dynamic surface tensions of blood serum and can increase these changes, if they already exist. Instabilities in the state of plasmatic membranes is related to the increase in the number of phospholipids, which possess pronounced surface active properties, leading to a "loosening"

135 and superpenetrability of membranes and to a deterioration of their integrity. This destabilisation of membranes is partially related to the stimulation of peroxidic oxidation of lipids, the substrate of which are unsaturated fatty acids (linoleic, linolenic, etc.) contained in phospholipids. Variations in contents and qualitative composition of phospholipids affect the blood rheology of patients suffering from glomerulonephrites. Nearly one half of the "essential" phospholipid fractions (phosphatidylcholine fraction, which contains significant amounts of poly-non-saturated fatty acids) is represented by the main ingredient 1, 2-dilinoleoylphosphatidylcholine glomerulonephritis

remission

usually phase

for

present the

in

small

isolated

quantities.

uric

In

syndrome

the only

lysophosphatidylcholine in serum is increased. During the exacerbation of the disease, increases in the total concentration of phospholipids and of the concentration of each fraction are observed, with most pronounced variations for patients suffering from a nephrotic syndrome. Serum phospholipids are contained mainly in the high density lipoprotein fractions. The increase in the contents of lysophosphatidylcholine and phosphatidylethanolamine is indicative of the vulnerability of high density lipoproteins with respect to Az-phospholipase, and their peroxidation by free radicals. Sphingomyelin possesses a specific stability with respect to peroxidic oxidation of lipids as it contains the lowest proportion of fatty acids and the phospholipases are unable to split acidic-amin bonds with fatty acid spresent in sphingomyelin. Thus, increase of the sphingomyelin contents can be regarded as defence reaction. An increased concentration in the total phospholipid and lysophosphatidylcholine contents in serum is directly related to the replenishment of the blood constituents due to membrane fragments (Neverov& Nikitina 1992). The destruction of cell membranes leads to tromboplastinemia, which affects the rheological properties of blood (Ryabov et al. 1995). The reactive products of oxygen, produced by blood cells and kidney mesangiocytes can lead to various damages of cells. The resulting peroxidic oxidation of lipids leads to changes in liquidity, ionic transport and fermentative activity of membranes. In addition to lipid oxidation, the reactive products of oxygen deactivates antioxidant enzymes. During pathologic processes, accompanied by intensified free-oxidant reactions and a break-off of the antioxidant system as defence against lipid peroxide oxidation, the phospholipid composition of membranes and their physical properties undergo changes. The decrease in the phosphatidylcholine contents in

136 membranes for chronic renal insufficiency is indicative of a lipoperoxidation activity. Under these conditions a decrease in the phosphatidylcholine contents is quite explainable, because phosphatidylcholine contains certain amounts of unsaturated fatty acids subjected to overoxidation processes. Increased amounts of phosphatidylcholine in blood serum can be the result of either the transfer of phosphatidylcholine from membrane into plasma, or of increased synthesis induced by hyperlipoperoxidation. It was shown by Grinshtein et. al. (1991) that patients with chronic renal insufficiency exhibit increased lipid peroxide oxidation. This is evident from the increase of dien conjugates and malonic dialdehyde amounts (in erythrocytes and blood serum). Studies of a-tocopherol support the idea that the activity of the antioxidant system for a chronic renal insufficiency is low. It was shown using the erythrocytal model that at the free radical oxidation level a destabilisation of membranes takes place accompanied by a deterioration of lipid bilayer liquidity, deformation of cell cytoplasmatic membranes, change in osmotic resistance, and decrease in level of total phospholipids and phosphatidylcholine. The state of lipid metabolism affects significantly the life prognosis of patients with chronic renal insufficiency. It was found by Rosental et al. (1989) that the lipid metabolism is damaged, namely the increase in total lipid concentration in blood serum due to increased amounts of cholesterol, triglyceride, low density lipoproteins and very low density lipoproteins. The trigliceride amount in serum was twice as much as that for the control group, while a deficiency of these lipids was detected in membrane erythrocytes. This redistribution of triglycerides is temporary, while its mechanism is related to a decreased rate of their elimination from plasma due a weaker lipoproteinlipase activity, which results from a decelerated kidney function. The depression of lipoproteinlipase is caused by low concentrations of apolipoprotein-C2, which acts as an enzyme activator, and by the accumulation of the inhibitor of this enzyme in blood. Low contents of triglicerides in erythrocyte membranes indicates that they are somewhat unstable. Processes generated during the interaction of protein exchange metabolites (e.g., urea) with proteins result in changes of the molecular structure of proteins, and consequently, their physico-chemical properties. In particular, variations in viscosity and surface tension of solutions of albumins and globulins were detected (Kazakov et al. 1995-1997). It was stated

137 earlier that urea is capable of forming rather strong complexes with other polar components, in particular blood serum albumins, and denaturate them under certain conditions. Blood serum albumins of patients suffering from chronic renal insufficiency are overloaded by endogenous urea, and its ability to isolate urea is significantly weaker than that characteristic for blood serum albumins of healthy persons. Qualitative variations in the protein composition of patients with chronic renal insufficiency significantly affect the surface tensions of blood serum. Imbalances in purine metabolism characterised by hyperuricemia, hyperuricosuria and a number of kidney disfunctions are indicative of unfavourable prognosis for most patients with chronic glomerulonephritis. Detailed morphologic study of tubulointerstitial changes for patients with high amounts of uric acid in blood showed that in almost all cases stroma sclerosis appeared, and atrophy of the tubular epithelium is more frequent than it usually occurs. In addition to the effect of microcrystallisation in kidneys and direct nephrotoxic action of uric acid, the developing changes in tubular and glomerular apparatuses enable one to assume the existence of immune mechanisms in the formation of hyperuricemial nephropathy (Sinyachenko et al. 1994-1997, Nickeleit et al. 1997). There is a weak dependence of the serum surface tension at short time on the uricemia level for patients suffering from Genoch glomerulonephritis. For lupus glomerulonephritis negative correlations are observed between the uricemia level and the values of ~ and ~2. In the group of patients suffering from chronic glomerulonephritis the average values of uric acid in blood were the highest, but no correlations with dynamic interface tensiometric parameters were found (Table 4.3). The development of a chronic renal insufficiency is accompanied by an increase in the contents of uremic toxines, among which the so-called medium-size molecules are of special interest. Each stage of chronic renal insufficiency is characterised by its specific contents of mediumsize molecular compounds in blood. Variations of this parameter can amount up to almost 200% (Rumiantsev et al. 1991), and highest values were detected for terminal renal insufficiency. The term "medium-size molecules" refers to substances of a molecular mass in the range of 0.5 to 5 kDa. The compounds with a molecular mass less than 1.5 kDa are usually regarded as low-

138 molecular weight. Thus the ranges of molecular mass for these two types of compounds partially overlap. Current classifications of meditun-size molecular compounds comprises more than 20 different groups. The chemical properties of these compounds are rather diverse: there are for example peptides, glycopeptides, aminosugars, polyamines, and multiatomic alcohols. Medium-size molecules include at least 30 peptides which possess well-defined chemical and biological activity, for example, vasopressin, oxytensin, neurotensin, angiotensin, glucagon, calcitonin, endorphins, and encefalins. These substances are common products of the vital activity of the organism, possess relatively low molecular mass and are efficiently removed from the bloodstream by kidneys in its normal function. 80 to 95% of medium-size molecules are split or deactivated in the proximal tubules of kidneys. During this process free amino acids are reabsorbed, and the residuals are removed by glomerular filtration. Concentrations of some substances are kept at a definite level, and represent biochemical constants of the organism. Under normal conditions an equilibrium exists between assimilation and excretion processes, but any disfunction is accompanied by enhanced catabolism, resulting in an increase of the amounts of these components. The deterioration of the elimination function affects the quantitative and qualitative composition of the spectrum of medium-size molecular compounds in blood serum. For patients with chronic renal insufficiency, the pool of medium-size molecules is constituted by polypeptides, oligosugars, derivatives of gluconic acids, polyamines and other substances (Gabrielyan et al. 1983, Kovalishyn 1987, Ringoir & De Smet 1988). For cases of uremia more than a twofold increase in the amount of components of a molecular mass of ca. 1 kDa is characteristic. Endogenous intoxication for chronic renal insufficiency can be caused both by the increase of the pool of medium-size molecules of peptide nature, and by non-proteinic substances. Chronic renal insufficiency is most common for patients with chronic glomerulonephritis. Therefore one can expect that correlations between the contents of medium-size molecular compounds in blood serum and the surface tension parameters exist, especially in this group of screened persons. However, such dependence was not found. The concentration of mediumsize molecules correlates with Cl and c2 for lupus glomerulonephritis and Genoch glomerulonephritis, with correlation coefficients equal to those obtained for correlations

139 between dynamic surface tension parameters and concentrations of uric acid and urea (Table 4.3). Studies on the selectivity of urine proteins and proteinuria are considered to be rather instructive in nephrologic diagnostics. There are various methods, which allow the differentiation between glomerular and tubular proteinuria. Traces of proteins and globulins are always present in healthy person's urine. However, for proteinuria the main protein is albumin. Tubular reabsorption of filtered proteins is incomplete (84 to 97%) and depends on their molecular mass. Direct relationships exist between albumin contents in blood and urine, thus hyperalbuminemia can lead to microalbuminuria. The reabsorption of high molecular proteins is more difficult than that of low molecular ones, and cationic proteins are reabsorbed better then anionic ones. Several hundreds of proteins can be detected in urine; however, only a few of them are identified, and a diagnostic function was ascribed to even less. Small molecules, for example [32-microglobulin (molecular mass 12 kDa), lysozyme (16kDa), retinol-binding protein (21 kDa) can penetrate through the basal glomerular membrane similarly to water. At the same time, not more than 0.1% of albumins contained in blood (with molecular mass 68 kDa) can pass through the glomerular filter. The negative charge at the surface of podocytes, which cover the basal glomerular membrane, is the main reason for the retention of albumin during the processes of filtration and formation of primary urine: as the charge of albumin molecules is also negative, it is rejected due to electrostatic repulsion, when its diameter is lower than the membrane pore size. A decrease of the negative charge leads to albuminuria and proteinuria. Immunoglobulins and plasma proteins of large molecular size can appear in urine only when the basal membrane is damaged (Titov & Tarasov 1988, Pasi et al. 1997). One of the types of microproteinuria is the fermenturia. Under physiologic conditions the sources of urine enzymes are blood plasma and epithelial cells of kidney tubules and the bladder. Only enzymes with molecular masses less than 70 kDa (e.g. amylase, pepsinogen, lipase) can be excreted from blood into urine in the glomerular filtration process, while enzymes possessing larger molecular masses (alanine aminopeptidase, lactate dehydrogenase etc.) cannot penetrate through the glomerular filter.

140 Selective reabsorption of enzymes is essentially a process where under normal conditions after glomerular filtration some of these enzymes are completely reabsorbed in the proximal tubules. Low-molecular lysozyme (molecular mass 16 kDa) and urokinase (53 kDa) can be mentioned as example: their urinal excretion can be regarded as the prediction of an affection of the nephron tubular part (Fomenko et al. 1991). When a pathology occurs leading to an increased penetrability of the glomerular basal membrane, the excretion of enzymes with molecular masses larger than 70 kDa becomes possible. The acetyl-13-D-glucosaminepeptidase indication in urine can serve as a rather sensitive test for the activity of chronic glomerulonephritis, and correlates well with albumin excretion. Urinal excretion of this enzyme becomes most pronounced for the mesangiocapillary form of chronic glomerulonephritis. The most reliable reason for an acetyl-13-D-glucosaminepeptidase excretion for glomerulonephrites can be the increased penetrability of the glomerular capillaries in the basal membrane (molecular mass of acetyl-13-D-glucosaminepeptidase is ca. 140 kDa), and also the damage of kidney parenchyma cells. Significant urinal excretion of alanine aminopeptidase, acidic phosphatase and 13-galactosidase can also be indicative of the damage of the glomerular apparatus. For patients suffering from acute glomerulonephritis the contents of alanine aminopeptidase is usually high. In glomerulonephritis, the detection of lysosomas with acid phosphatase in mesangial cells cytoplasm and in the mesangial matrix bulk can be regarded as evidence of a possible infection by enzymes both of mesangial matrix itself, and of some sections of the glomerular basal membrane to which hydrolases can diffuse. Acidic hydrolysis of the glomerular basal membrane can result in a partial destruction of its molecular framework accompanied by increased pore size and local increased penetrability of the glomerular filter with respect to macromolecules, including immunoglobulins. The increased transfer of protein into kidney tubular gaps caused by a damage of the glomeruli, and prolonged overload at all stages of protein absorption and catabolism in tubular cells leads to a disfunction of these transport systems, which plays a significant role in the development of tubulointerstitial damages. Increased load of the tubular apparatus by proteins, and

141 irregularities in the function of the lysosomic-vacuolar apparatus result in a progressing glomerulonephritis. The highest level of daily proteinuria was detected for acute glomerulonephritis. For lupus glomerulonephritis the amount of 132-microglobulin was highest. The contents of fibronectin was closely related to the total concentration of proteins in urine, and for acute glomerulonephritis this contents was higher than for chronic glomerulonephritis, lupus glomerulonephritis and Genoch glomerulonephritis. In contrast to blood serum, the correlation coefficients between protein contents in urine and its surface tension for patients with chronic glomerulonephritis, lupus glomerulonephritis and Genoch glomerulonephritis were negative. Only for patients with acute glomerulonephritis positive correlations were detected between the contents of proteins and Ol (cf. Table 4.4). Dynamic surface tensiogram parameters for urine were in fact determined by the amount of proteins. At the same time, a crucial effect remains unexplained: if the surface tension decreases of urine is in line with the extent of proteinuria, why then do the mean tensiometric parameters in particular groups exceed those characteristic for healthy persons, who do not suffer from any proteinuria? Paradoxically, the effect of proteinuria on blood surface tension is higher than that of the proteinemia level. For example, the correlation coefficients between albuminuria and particular parameters of serum surface tensiometry for chronic glomerulonephritis and lupus glomerulonephritis can be up to 0.8, and are positive for chronic glomerulonephritis, while negative for lupus glomerulonephritis (blood surface tension for systemic lupus erythematosus is negatively linked to proteinuria and fibronectinuria). In the screened group of patients suffering from chronic glomerulonephritis, only moderate correlations were found between urine fibronectin and

02

of blood, while for Genoch glomerulonephritis only the equilibrium

surface tensions for blood serum respond to the proteinic composition of urine. The violation of metabolism and excretion of proteins and lipids, most pronounced for the nephrotic syndrome, play a significant role in the increase of the amount of circulating plasmin inhibitors, in particular cz2-macroglobulin, the decrease of fibrinolysis activators, the degradation

of

plasma

fibrinolytic

activity.

We

believe

that

for

patients

with

142 glomerulonephrites, urinal excretion of those blood components, which determine its surface tension, can happen along with the excretion of proteins. It can be also supposed that a pronounced proteinuria can be related to variations in a qualitative composition of surfactants in the blood serum. Thus one can conclude that surface tension parameters of biological liquids for patients suffering from glomerulonephrites depend on a variety of factors, and the above analysis is by no means comprehensive. However, there are no doubts about the crucial effect of proteins and lipids contained in biological liquids, and also of many other compounds, irrespective of their surface active properties. Only first steps towards an understanding of this important and complicated problem are made, and its solution requires further detailed studies in future. 4.1.3. Effect of treatment on variations in surface tensiometric parameters

Patients with chronic glomerulonephritis were arbitrarily divided into subgroups of good or bad responder to medical treatment. These patients were not completely identical with respect to the duration of disease, form of chronic glomerulonephritis and extent of the therapy already performed. However, the data obtained in such a way are of some practical interest. It was found that high initial surface tension values of serum can be regarded as positive prognosis for a subsequent treatment, and vice versa. The dynamics of surface tensiometric parameters was analysed with respect to the application of glucocorticoid hormones for the treatment of glomerulonephritis. In 1.5 to 2 months from the beginning of the therapy with hormonal preparations, changes in surface tension of serum and urine were observed towards values characteristic for healthy subjects (cf. Fig. 4.16). The treatment of patients suffering from a nephrotic syndrome by glucocorticoids results in a decrease of the total and free cholesterol contents in serum, and an increase in concentration of high density lipoproteins (Aoki et al. 1993). It was supposed by Kuzemkova et al. (1989) that corticosteroid hormones directly affect the metabolism of lipids, either increasing the synthesis of high density lipoproteins in liver, or decreasing the intensity of their destruction by hepatolipase. A glucocorticoid treatment of patients leads to a chylomicronemia and to an increase in the concentration c f high density lipoproteins and very low density lipoproteins in blood.

143

a)senma 10_

F

r

-5-

-10

-

-15

-

AGN

CGN LGN Before treatment

GGN

AGN

CGN LGN GGN Atter treatment

b) urine 10-]

5-1 0 ..o

5 -10 -15 -20 AGN

CGN

LGN

Before treatment

GGN I AGN

CGN

LGN

GGN

Aider t r e a t m e n t

Fig. 4.16. Changes in surface tension parameters in serum and urine obtained from patients with glomerulonephrites before and after a glucocorticoid therapy. Changes are given in % compared to corresponding healthy controls. Hatched - ~ , black - g2, white - ~3,.

There is an inverse correlation between the contents of total cholesterol, triglycerides and low density lipoproteins, and the level of serum albumin. A reduction was observed in concentrations of cholesterol and low density lipoproteins in line with a decrease of

144 proteinuria. This fact led to the assumption that some irregularities take place in particular steps of the transformation of very low density lipoproteins into low density lipoproteins, or in the catabolism of medium density lipoproteins and low density lipoproteins in liver, caused by urinal excretion of serum albumin. Thus, variations in concentration, composition and structure of lipid and protein surfactants for patients with glomerulonephrites lead to changes in the dynamic surface tension parameters of biological liquids. The application of intra-vessel laser therapy enhances anti-oxidant protection processes (an increase of glutathione peroxydase, glutathione reductase, superoxyddismutase is observed) and makes the peroxidic oxidation of lipids weaker (decreasing the contents of dien conjugates and malonic dialdehyde). Intravenous laser irradiation of blood affects the hemorheologic and hemostatic properties, decreases the viscosity of plasma. Even a single application of the

laser therapeutic procedure leads to a decrease of ~ of blood

serum for patients with chronic glomerulonephritis with chronic renal insufficiency (cf. Fig. 4.17). Such single exposure as yet produces no effect on the concentration of peroxidic oxidation of lipid indicators and anti-oxidants in blood, and the contents of medium-size molecules and nitrous non-protein species also remains unchanged. It can be supposed that either surface active properties of the studied surfactants undergo some changes, or some new surfactants are formed. We believe that surface tensiometry can be regarded as a promising method for the estimation of the efficiency of intra-vessel laser therapy and the prognosis of its expected results, even at the very beginning of a performed treatment. It is generally believed that ultraviolet irradiation of blood leads to a more significant decrease of serum viscosity, than laser irradiation does. As many protein and lipid components affect both the liquidity and surface tensions of a biological liquid, one can expect that intra-vessel ultraviolet irradiation for patients suffering from chronic renal insufficiency will result in some changes in surface tension parameters. However, such changes in the surface tension dynamics were not found (cf. Fig. 4.18). Hemosorbtion, plasmapheresis and isolated ultrafiltration are so-called extracorporal methods of homeostasis correction. These are the methods for convection elimination of water and dissolved substances by the creation of an either positive hydrostatic pressure from the blood side, or a negative pressure from the external side of the semipermeable membrane. The

145 volume of liquid extracted during hemofiltration can amount to 20-801 (sometimes it exceeds the total volume of liquid contained in the organism). Partial replacement of water contained in the organism leads to the elimination of dissolved medium-size molecular compounds, urea and creatinine from patient's organism.

75

~

--

70 -

65

I -2

-1

lg(tef) [s]

I

I

0

1

Fig. 4.17. Example for serum tensiogram obtained from patient with chronic glomerulonephritis and terminal renal insufficiency (male, age 37) before laser irradiation of blood (thin line), and after (thick line).

72

E 70 Z

--

--

68--

66 -2

-1

lg(tef) [s]

0

1

Fig. 4.18. Example for serum tensiogram obtained from patient with chronic glomerulonephritis and terminal renal insufficiency (male, age 35) before ultraviolet irradiation of blood (thin line), and after (thick line).

146 For cases with the nephrotic syndrome a single application of the procedure does not result in any changes of electrolytes and concentration of non-proteinic products in blood. Osmolarity and viscosity of plasma also remain unchanged. However, the dynamic surface tension parameters are changed: for chronic glomerulonephritis the values of crl and or2 decrease, while for lupus glomerulonephritis these parameters increased. It is quite possible that after isolated ultrafiltration the values of 13" 1 and or2 for serum are determined by increasing the total contents of proteins, which induces these opposite changes of surface tension for patients suffering from chronic glomerulonephritis and lupus glomerulonephritis. Medium-size molecules, which exist in plasma and adsorb at the surface of blood cell elements, lead to a deterioration of the rheology of blood. During hemosorption the amounts of medium-size molecular compounds in blood decrease by 45%, a fact that directly correlates with the intensity of peroxidic oxidation of lipids (Kursakova et al. 1989). The decrease in the concentration of medium-size molecules during hemosorption therapy can be explained by both a direct elimination of these molecules from blood, and by a decelerated formation caused by an improved microcirculation, the influence exerted on the callicrein-kinin system and proteasic activity of plasma. A single application of the hemosorption procedure results in a ca. 40% decrease of the triglyceride concentration and 20% decrease in cholesterol and low density lipoproteins concentration in serum. The surface tension of blood serum also decreases. The correlation between blood serum surface tension and the concentration of lipids in blood is inversely related. Therefore it is necessary to take account of the variations in the contents of other (proteinous and lipid) surfactants and inorganic ions (Na+, K+, Mg 2+, Cl) in blood serum. The decrease in concentration of inorganic ions in low molecular surfactant solutions is usually accompanied by a decrease in surface tension in the short lifetime range (cf. Chapter 1). The elimination of urea from the organism during hemosorption can lead to either an increase or decrease of serum surface tension. This depends on the prevailing action of this nitrogenous product either on albumins (decreased protein denaturation) or on the composition of low molecular surfactants (changes in the structure of water). During hemosorption the urea

147 concentration in blood is decreased only slightly, therefore the suppositions presented above were analysed with reference to hemodialysis. Plasmapheresis is one of the extracorporal blood purification methods which is extensively used in nephrological practice. This method allows elimination of high- and low-molecular weight compounds from the organism which are excessively accumulated in blood of patients with chronic renal insufficiency (Kolesnyk et al. 1992, Bartges 1997). The application of this method leads to 40-60% decrease in the concentrations of cholesterol, triglycerides and low density lipoproteins, and to a decrease in the total contents of proteins, immunoglobulins, circulating immune complexes, amino acids, fibrinogen, sialic acids and to a decrease in blood viscosity. At the same time, the contents of albumins in serum increases, and the concentration of medium-size molecules remains unchanged (Dorofejev et al. 1991, Konovalov 1991, Fadul et al. 1997, Ciszewsi et al. 1993). 75

--

70,_., 65 Z

60-

i.....a

5550

~ o ,,.o

--

45-

I

-2

-1

lg(tef ) [S]

I

I

0

1

Fig. 4.19. Example for the changes of the of serum tensiograms obtained from the patient with chronic glomerulonephritis, terminal renal insufficiency (male, age 40), before hemosorption and plasmapheresis (solid line), and after (dotted line). Thickness of dotted curves corresponds to the number of therapeutic applications. Dynamic surface tension parameters were measured before and after plasmapheretic treatment of patients with chronic renal insufficiency (cf. Fig. 4.19). The blood circulation rate during plasmapheresis was 80-100 ml/min and the duration of a procedure was approximately 3 hours The plasma was removed at a rate of 40-60 ml/min (in all cases the exfusion was performed in

148

such a way that its level exceeded 1.5- 2 times the volume of circulating plasma). For the substitution of the plasma 5% and 10% albumin solution (in some cases lyophilised or fresh frozen plasma) and isotonic electrolyte solution were used. The contents of proteinic preparations was ca. 60% of the inserted volume. During the application of the plasmapheresis procedure a decrease of the dynamic surface tensions of blood serum was observed first in the short, and subsequently in the medium time range. These variations take place simultaneously with a decrease in the concentrations of lipids, fibrinogen, fibronectin and medium-size molecular compounds. The decrease of the dynamic surface tension was most significant for a combined therapy, when hemosorption and plasmapheretic treatment was applied in turns (cf. Fig. 4.19).

4 ._

3 2

TL

PL

CH

CHE

TG

FFA

LL

PS

PC

PE

Fig. 4.20. Effects of hemodialysis on the ratios of various lipid components in blood and the same component in erythrocyte membrane in patients with chronic glomerulonephritis. The various components are: TL - total lipids, PL - phospholipids, CH - cholesterol, CHE - cholesterol ethers, TG -triglycerides, FFA - free fatty acids, LL - lysolecithin, PS - phosphatidylserine, PC - phosphatidylcholine, PE phosphatidyl ethanol amine. The black columns represent the ratio before, the white ones after hemodialysis. The grey columns represent ratios for healthy controls. Among the extracorporal methods of treatment applied to patients suffering from chronic renal insufficiency the most important one is the chronic (programmed) hemodialysis. This method enables one to decrease significantly the concentration of medium-size molecular compounds, urea, creatinine, uric acid, some lipids, electrolytes and enzymes in blood. It is seen from

149 Fig. 4.20 that hemodialysis leads to a restoration of the interrelation of triglycerides in blood serum and erythrocyte membranes due to the suppression of lipidemia. Here the decrease of the contents of very low density lipoproteins takes place; for some patients also the amount of low density lipoproteins decreases. At the same time, this procedure of artificial extrarenal purification of blood involves a number of undesirable consequences regarding the lipid and proteinic metabolism, and hence the composition of surfactants in biological liquids. The application of hemodialysis is inevitably accompanied by a significant loss of amino acids and carnitine, (Alhomida 1997, Kim et al. 1998) and by an increase of the tissue resistance against insulin, which creates the conditions for an intensified protein catabolism (Shostka et al. 1990). A significant activation of plasma lipoprotein lipase entailed by the introduction of heparin during dialysis leads to an increased concentration of toxic free fatty acids. These toxic effects are increased by disproteinemia and the presence of deficient modified proteins. Lipid peroxide oxidation in erythrocytes becomes significantly more pronounced for patients treated by hemodialysis which is believed to be caused by an increased generation of free oxygen radicals by blood neutrophiles and monocytes (Balashova et al. 1992). When hemodialysis is performed, the amount of dien conjugates increases and the antioxidant protection is depressed. This was estimated from the contents of a-tocopherol, reduced glutathione, superoxide dismutase, catalase, glutation peroxidase (Matkovics et al. 1988, Stetsiuk et al. 1989, Trznadel et al. 1989, Biasioli et al. 1996, Zima et al. 1998). For patients with chronic renal insufficiency an accumulation in blood of various products which possess pro-oxidative properties is observed, for example malonic dialdehyde which is regarded as uremic toxine, and metal ions (aluminium, silicone). The hemodialysis promotes peroxidic oxidation of lipids in blood cells due to the enhancement of "oxygen boost" and the accumulation of toxic products of incomplete oxygen reduction (superoxide ion-radical, hydrogen peroxide). Changes in the peroxidic oxidation of lipids and damage of the antioxidant systems can affect the surface tension of blood serum, as was explained earlier. The dynamic behaviour of surface tensiometric parameters during hemodialysis for uremia caused by chronic glomerulonephritis is essentially different from that characteristic for uremia caused by polycystic renal diseases (cf. Figs. 4.21 and 4.22). Extracorporal purification of

150 blood for chronic glomerulonephritis leads to a decrease of crl followed by an increase to initial values, while a similar treatment applied in cases of a polycystic renal disease results in a continuous increase of surface tension: first the increase of the equilibrium surface tension or3 is observed, and then the increase of Ol and or2 is observed. It can be argued that these variations of surface tension parameters are caused by the elimination of surfactants like nitric nonprotein

substances

(urea, creatinine,

uric acid), medium-size molecular

compounds,

immunoglobulins, 132-microglobulin and lipids from patient's organism. The continuous decrease in concentrations of some surface active proteins and lipids in blood leads to increased surface tensions of serum.

75 q : _ _ _ -~_

~Q

71

--.

69 - ~ ,

"

~176

~63

~

61 I

"-\

59--

-.

57--

"-.

55

J -2

-1,5

-1

i

i

I

-0,5 lg(tef) [s]

0

0,5

I

Fig. 4.21. Example for the changes of serum tensiograms obtained from patient with chronic glomerulonephritis and terminal renal insufficiency (male, age 29) before hemodialysis (solid line), and during hemodialysis with 2 hours intervals (dotted lines). Thickness of lines corresponds to hemodialysis time Significant changes in dynamic tensiometry parameters occur after kidney transplantation. Sharp increases in surface tensions are observed already the next day; subsequently the values of dynamic tensions gradually approach the levels characteristic of healthy persons (cf. Fig. 4.23). It is evident that rapid removal of surfactants out of the organism determines the dynamics of surface tensions. Thus it seems obvious that in future dynamic surface tensiometry

151

of biological liquids can become a highly informative indicator for the development of a transplant rejection crisis. 8 0

- -

75

70-

60 55 o

50

t -2

-1

lg(tef ) Is]

0

1

Fig. 4.22. Example for the changes of serum tensiograms obtained from patient with kidney polycystosis and terminal renal insufficiency (male, age 42). Solid line - before hemodialysis, dotted lines - with 2 hours intervals during hemodialysis, thickness of lines corresponds to hemodialysis time

75 73 71

_-+-.-

~ . .

~ __

w

m

~

~

~

-....,..

"~ .

- .......

69

E 67

__

65

__

E) 63

o~

61 59 57 55

I -2

-1,5

I -1 lg(tef ) [s]

F

I

-0,5

0

Fig. 4.23. Example Ibr the changes of serum tensiograms obtained from patients with chronic glomerulonephrites and terminal renal insufficiency (male, age 50). Solid line - before kidney transplantation, thin dotted line - one day after transplantation, thick dotted line - one week after transplantation

152

4.2. Chronic pyelonephritis Chronic pyelonephritis is the most common renal disease., It is often accompanied by an imbalance in all metabolic activities, irregularities of blood biochemical homeostasis, and the presence of a number of inorganic and organic surface active substances in the urine. In primary pyelonephritis (and also secondary forms caused by urolithiasis) C-reactive protein, some globulins, sialic acids, mucoproteins, ceruloplasmin and haptoglobin levels in blood increases, while the contents of albumin and transferrin (siderophilin) decreases (Gavrylov 1987). For patients suffering from calculous pyelonephritis, pronounced irregularities of the hemostatic system are fotmd, displayed as hyperfibrinogenemia, thrombinemia, decrease of plasma heparin cofactor activity and XII-dependent fibrinolysis, leading to variations of rheological properties of blood (Neymark & Mazyrko 1986). In chronic pyelonephritis the increase of concentrations of malate dehydrogenase, leucine aminopeptidase, 7-glutamil-trans-peptidase, lactate dehydrogenase (lactate dehydrogenase isoenzymes LDG 1 and LDGs), lysozyme (muramidase) and 13-glucuronidase in urine, correlates with total

proteinuria

level.

The

clinical

relevance

of low molecular

lysozyme,

~l-microglobulin, 132-microglobulin and ribonuclease contents in serum and urine was considered by Jung et al. (1989). Under normal conditions these proteins filtered through the glomerular basal membrane undergo subsequent reabsorption and catabolism in the kidney tubular apparatus, thus excreted into the urine in minor quantities only. The lesion of tubules in chronic pyelonephritis leads to a decrease in albumin reabsorption, which results in an increased proteinuria. As mentioned above, variations in the surfactant composition of biological liquids can affect the surface tension parameters. Dynamic surface tensiometry was performed with serum and urine sampled from patients with primary pyelonephritis (58 patients) and secondary pyelonephritis caused by urolithiasis (54 patients) (cf. Fig. 4.24). In serum samples the following results were obtained. For males with primary pyelonephritis a decrease in 03 was found, while for females a decrease of both c2 and c3 was accompanied by a significant (almost twofold) increase of ~.-values in the serum tensiograms. Urolithiasis in females also leads to an increase of ~.-values of serum, while in males this parameter decreases.

153 In general, the surface tensiometry parameters of serum in male patients can be used to differentiate between primary and secondary pyelonephritis, caused by urolithiasis. While the former leads to a decrease of equilibrium surface tensions, the urolithiasis is characterised by decreased ~.-values. a) serum 120 100 80-

6040tD

20I "-t-~

' ~[____j , m

L---J ' m

-20 t~l

~2

~3

~,

PPN

UL

b) u ~ e 30 20 10 =

0

-10 -20 -30 -40 crl

or2 PPN

or3

~

I

~1

or2

cr3 UL

Fig.4.24. Changes in surface tensiometric parameters of biological liquids obtained from patients with primary pyelonephritis (PPN) and urolithiasis causing secondary pyelonephritis (UL), depending on patients' sex. Changes are given in % from corresponding values for healthy persons. Males - black, females - white.

154 In urine samples the following results were obtained. The ~.-values obtained from dynamic tensiograms of urine sampled from female patients decrease significantly, regardless of the form of pyelonephritis, while for males this parameter increases. Decreased o2-values were observed for females suffering from primary pyelonephritis, and for male patients with urolithiasis causing pyelonephritis. Thus it can be concluded that surface tension parameters of biological liquids in the cases of chronic pyelonephritis should be considered with regard to patients' sex. Fig. 4.25 shows serum tensiograms for patients with chronic pyelonephritis. 75 70 E65 ~176

t~60

55I 50 -2

-1

0 lg(tef), [s]

J

I

1

2

Fig. 4.25. Example of serum tensiogram obtained from patients with chronic pyelonephritis. One is male, age 44, without CRI (thin line), the other is female, age 30, with CRI1 (thick line). The sex and age corresponding tensiograms for healthy persons are given in dotted lines. We performed correlation analysis between serum and urine data. The value of Ol for urine correlates with Ol and o3 for serum. In turn, ol of serum correlates with 02 and o3 from serum, while equilibrium surface tension correlates with 02. Similar correlation links were found also for urine. Urolithiasis causing pyelonephritis leads to more pronounced interrelations between surface tensiographic parameters of urine and serum (here only 02 of urine exhibits a weak relation with 01 of serum), while for primary pyelonephritis a correlation between surface tensions for the two biological liquids was found at short times (t = 0.01 s) only. The ~. value for urine correlates directly with the duration of chronic pyelonephritis, while other surface tensiometric parameters do not depend on patient's age or disease duration. Quite

155 opposite to our expectations, no surface tension dependence on the arterial blood pressure was found. For patients with chronic pyelonephritis, the renal hemodynamics is characterised by a decrease in both effective bloodstream and glomerular filtration rate, the increase in the resistance of renal vessels, which in turn is related to the extent of arterial hypertension and with the mass of functioning parenchyma. Arising irregularities in the renal circulation of blood lead to a water-osmotic imbalance, while a developing hypovolemia leads to an increased blood viscosity, which changes of the rheological properties of blood serum (Spector et al. 1987). For patients suffering from urolithiasis, the possible effect of concrement composition on surface tension of urine was estimated (spontaneous discharge of calculus was studied for patients who have undergone lithotripsy, pyelolithotomy and ureterolithotomy). The chemical origin of a calculus is usually related to uric or oxalic acid, with a composition comprised of [(Ca3PO4)2], [CaC2Oa.H20], [CaC204.2H20], [MgNHaPO4.6H20], and ammonium urate

[(NH4CsH3N403)2]. The processes of urolithiasis are caused by filling tubular over its stability limit, the presence of respective inhibitors, and the formation of crystallisation activators. The relation between the albumin contents in urine and the concentration of cells in interstitial space appears to be important. Significant correlations were found between the accumulation of macrophagocytes in the interstitium and proteinuria parameters. A decrease in proteinuria is accompanied by less extensive damages of tubular cells. The formation of microcrystals in kidney stroma reflects the breakdown of adaptation reactions, which in turn can be caused by the disfunction of humoral regulation, the irregularity of intercellular interactions, and a degeneration of the physiological response of nephron and collecting tube cells to biologically active substances (hormones, mediators). The maintenance of the acid/base balance within the organism is determined, in addition to other factors, by the productivity of excretion of hydrogen ions via the kidney, which controls the necessary level of plasma bicarbonate concentration. The mechanism of urine kidney acidification is especially interesting, because changes in this lead to a decrease in the pH value of cortical and medullar substance in the interstitial space, resulting in a decreased salt

156 solubility leading to salt precipitation. The formation of urate and oxalate microcrystals in kidneys interstice represents an important pathogenetic factor in the development of urolytic nephropathy.

5OOO

~ o

4500 t 4000 3500 3000 2500 2000 1500 1ooo

500 ! 0 UA

X

HX

AL

Fig. 4.26. Solubilityof various purines, mg/l, in urine pH=5 (black), urine pH-7 (white), and blood (grey). UA uric acid, X - xantine, HX - hypoxantine,AI - alanine. Black- urine pH=5, white- urine pH=7, greyblood. One of the final products of the degradation of puric compounds is 2, 6, 8-trioxypurine, which exists predominantly in oxyform. The solubility of this form in water is extremely low. The shift of the tautomeric equilibrium towards well-soluble oxyforms is controlled by the polarity, ionic strength of the medium, the association type, the stability of formed complexes, etc. The decrease of the pH of tubular liquid in a nephrone from 7.4 in the initial part of proximal tubule to 4.5 - 5.0 in the collecting tubes leads to a shift of HUrAUr+H § equilibrium towards the nondissociated, and therefore poorly soluble uric acid. For pH values of 5.0 and 6.5 of urine its solubility limit is 100 mg/1 and 1200 mg/1, respectively. The data concerning the solubility of basic purines in biological liquids are presented in Fig. 4.26. Increased biosynthesis of oxalic acid salts for pyelonephritis is always accompanied by hyperoxaluria (Ebisuno et al. 1986). This phenomenon is in most cases secondary, while it was proven that local synthesis of oxalates in kidneys can take place due to the destruction of membrane phospholipids. This destruction can be the result of kidney ischemia, activation of

157 endogenous phospholipases, or the effect of membrane-toxic compounds (Neiko & Del'va 1991). Hyperoxaluria accompanies the primary pyelonephritis and urolithiasis (Koide et al. 1985, Cill & Rose 1986 and Mitwalli 1988). Oxalic acid is formed as the final product of the metabolism of purines, ascorbic acid, glycine, serine, hydroxyproline, tryptophane etc. (Balche 1983, Swartz et al. 1984 and Ono 1986). For such patients the glucose load leads to a further increase of oxaluria. The daily amount of oxalate excretion for healthy subjects is 20 to 50 mg (Pendse et al. 1985 and Mitwalli 1988), while for patients with urolithiasis the excretion rate is much higher (Norman et al. 1984 and Jaeger et al. 1985).

Table 4.5. Correlationsbetween particular surface tension parameters of serum and urine obtained from patients with chronic pyelonephritis and the amount of the concentration of different components measured in the same liquid.

Component

urine

serum 13"I

0"2

0-3

0-3"1

0"2

0-3

Urea Creatinine Oxypurinol Uric acid Oxalic acid

$

$ $$$

$$

$

$$

$$

1"positive correlation, $ negative correlation, empty - no correlationr<0.3; one arrow, r =0.3 to 0.5; two arrows, r -- 0.5 to 0.7; three arrows, r > 0.7 The in vitro variations of the pH for serum albumin solution towards increased acidity leads to a pronounced decrease of 0-1, 0-2 and 0-3 of blood serum. Note that the main urine proteins for patients with calculus pyelonephritis are also albumins, and the pH of this biological liquid usually decreases (especially for uratic urolithiasis). One should expect a decrease in surface tensiometric parameters of urine for urolithiasis. These suppositions, based on model studies of simple systems, were not supported, however, in clinical practice.

158 It is seen from Table 4.5 that both uric acid and oxalic acid possess rather pronounced surface active properties, resulting in a decrease of surface tensions, especially in the short time range. Note that surface tensiometric parameters for urine exhibit some (negative) correlation with albuminuria

level,

while

for serum these

parameters

depend

on the

contents

of

132-microglobulin, immunoglobulins, cholesterol, a-cholesterol and the high density lipoprotein fraction. One can argue that there is a significant effect of uric acid and oxalic acid on the surface tension of biological liquid. The level of uric acid in blood is closely related to surface tension parameters also for urine: here similarly, an inverse dependence exists with correlation coefficients ranging between -0.47 at t ~ oo arid -0.78 at t = 0.01 s. In contrast to glomerulonephritis, chronic pyelonephritis exhibit no correlation between tensiographic parameters and the contents of urea and creatinine in serum. Thus significant changes in water and albumin structure (leading to surface tension variation) are expected and the concentration of urea should be higher than that detected for pyelonephritis. It was mentioned above that the presence of inorganic ions in solutions of low molecular weight surfactants, although increasing the surface tension in the short surface lifetime range, can either increase or decrease surface tension at medium and large lifetimes. Human biological liquids also contain low molecular surfactants. However, their amount and quantitative relations to proteins composition and mixed model solutions can differ essentially. In addition, no in vitro studies have as yet been performed in what regards various high molecular surfactants contained in serum; it is known however that the concentration of these surfactants often increases or decreases for chronic pyelonephritis and other renal diseases. While it seems impossible to extrapolate available surface tensiometry data of such model solutions onto actual patients, we believe that studies of correlation links between the contents of inorganic ions in a biological liquid and surface tension parameters would be rather instructive (see Table 4.6). The following preliminary conclusions can be drawn on existing correlations: (1) the presence of sodium, potassium and calcium in serum leads to increased values of (Yl and 0"2; (2) chlorine produces the most significant effect on surface tensiometric parameters; (3) the effect of sodium, chlorine and magnesium on surface tension parameters of serum is opposite to that for urine; (4) surface tensions of urine are independent of the calciuria level.

159 This last fact is especially interesting for patients suffering from urolithiasis, because the development of this disease depends closely on the state of calcium metabolism, in particular, on the calcium contents in urine. Calcium-containing concrements are formed due to distal tubular acidosis and hypercalcemia, often caused by hyperparathyriodism. An inverse dependence between the level of parathyroidin in blood serum and calcium urinal excretion exists, and a direct correlation between calciuria and parathyreoidhormoneuria. High concentrations of calcium in urine is a risk factor, which predisposes for the formation of not only calcium, but also urate and oxalate concrements. The hyperuricosuria can also play a significant pathogenic role in the formation of concretions of various composition, even in absence of hypercalciuria. Theories which explain the formation mechanism of calcium calculus caused by hyperuricuria consider uric acid as the heterogeneous centre for calcium salts crystallisation. Table 4.6 Correlationsbetween particular surface tension parameters of serum and urine obtained from patients with chronic pyelonephritis and the amount of the concentration of inorganic iones measured in the same liquid.

Inorganic ion

urine

serum ol

or2

Sodium

1"

1"

Potassium

1"

1'

Chlorine

1' $

1' $

Phosphor

$

or3

crl

o2

$

$

1'

1"

or3

$

Total calcium Ionised calcium

Magnesium

$$$

1'

1"1'

$

1' positive correlation, $ negative correlation, empty - no correlation r<0.3; one arrow, r =0.3 to 0.5; two arrows, r -- 0.5 to 0.7; three arrows, r > 0.7; - - - not studied Tubular affections, caused by hypercalcemia and explicit action of parathyroid hormone, can play a certain role in the pathology of the lithogenesis. These factors suppress the cell mitochondrial activity, resulting in an accumulation of mucoproteins in cells. This in turn leads

160 to a destruction of the epithelium, and to the excretion of proteinic compounds, which form the organic matrix of the concrement. The salts begin to adsorb at this matrix, thus forming spherolithes, which grow and form microlithes and subsequently transform into macrolytic nuclei in the calycle-pelvis system. There is an interrelation between the testosteroneuria level and the progress of urolithiasis. The indicators of androgens in urine for urolithiasis are significantly lower than those characteristic of healthy persons. In this case a positive correlation exists between the testosteron concentration and the activity of uricase, which controls the contents of uromucoid which in turn stimulates the formation of crystals. For patients with chronic pyelonephritis the contents of testosteron, estradiol, progesterone and other hormones in serum was compared with the surface tension parameters. It is seen from Fig. 4.27 that estradiol (for males only), insulin and thyroxine do not affect the surface tensions of serum. 1

-

0.8

E

0.6

i

0.4

tj

0

t

-0.2 ~

-0.4 -0.6 !

~

-0.8 -1 Tm

Tf

Om

Of

Pm

Pf

I

C

TIN

T3

T4

TG

Fig. 4.27. Correlations between surface tension characteristics of serum and biochemical components of serum. Surface tension parameters are Crl (hatched), t~2(black), c3 (white), ~. (grey). Biochemical components are testosteron (males) - Tm, testosteron (females) - Tf, oestradiol (males) - Om, oestradiol (females) - Of, progesterone (males) - Pm, progesterone (females) - Pf, insulin - I, cortifan - C, thyrotropic hormone TTH, triiodothyronine - T3, thyroxine - T4, thyroglobulin - TG.

For males the levels of testosteron and thyroglobulin positively correlate with the surface tension of serum, while for progesterone and thyrotropic hormone this correlation is negative.

161 The parameters of progesteronemia exhibit opposite dependencies on equilibrium surface tension and ~, values of serum for patients of different sexes. Surface tensions in the short and medium surface lifetime range are determined by the concentrations of testosteron and progesterone (for males), cortifan and thyroglobulin; the equilibrium surface tensions by the contents of thyrotropic hormone and thyroglobulin, testosteron (for males) and progesterone (for females); the values of ~ by the concentrations of testosteron and estradiol (for females), progesterone (independently of patient's sex), triiodthyronine and thyroglobulin. It can be presumed that this dimorphism of surface tensiometric parameters with respect to patients sex is mainly due to the results of correlation studies presented above. Insulin, hydrocortisone, sex hormones and thyroid hormones are involved in the metabolism of surface active albumins, lipids and carbohydrates, which affect the surface tension of a biological liquid. Correlation links between surface tension parameters and various hormones are by no means limited to the dependencies outlined above. In addition, one has to take into account a great variety of combinations of surface active compounds, which can multivariably affect the tensiographic parameters, e.g. the presence of inorganic compounds in biological liquid, etc. 30 -

Serum

Urine

2010-

=

0

~

-10 -

Uf//

'

-20 -30 -40 ~1

~2

~3

~,

~1

~2

~3

~,

Fig. 4.28. Changes in surface tension parameters measured in biological liquids obtained from patients with chronic pyelonephritis with either preserved (white) or decreased (black) renal function. Changes are given in % compared to corresponding healthy controls.

162

a) serum

8~1 60 40-

/

20-

o

"--"-'

I

'----------'

I

--L._...J

I

' mB----J ' ~___J ' -20 0"1

0"2

0"3

0"1

~,

0"2

PPN

0"3

%

UL

b) urine

-5 d J t

-10 o

-15 -20 J -25 j al

~2 PPN

a3

~,

~1

c~2

~3

)~

UL

Fig. 4.29. Changes in surface tensiometric parameters measured in of biological liquids obtained from patients with primary pyelonephritis and secondary pyelonephritis caused by urolithiasis who have single (white) or both (black) kidneys. Changes are given in % compared to corresponding healthy controls.

For male patients the contents of testosteron in senun is inversely related to 0.1 and 0"2 of urine, while for females this relation is direct. There are positive correlations between the progesteronemia level and 0"1 and 0"2 ( f o r males) and 0"3 for females, respectively. The

163 equilibrium surface tension of urine for females is to some extent determined by the contents of estradiol in the blood. Urine tensiografic parameters at short times exhibit positive correlations with the contents of insulin, thyroglobulin, [32-microglobulin , high density lipoproteins and a-cholesterol, and negative correlations with the levels of cortifan, thyrotropic hormone, ot2-globulins, circulating immune complexes, immunoglobulins G, A, M, and uric acid. The deterioration of the kidney function for chronic pyelonephritis is accompanied by a significant increase of ~. for urine (cf. Fig. 4.28). For urolithiasis with chronic renal insufficiency, in addition, blood serum displays a decrease in

0"2

and

0"3, and

an increase in ~,.

We believe that this dynamics of surface tensiometry parameters can be indicative of a negative prognosis for the development of the disease. Dynamic surface tension data for patients with primary pyelonephritis and secondary pyelonephritis caused by urolithiasis, who have a single or both kidneys (including the cases of one functioning kidney) are of some interest (cf. Fig. 4.29). In these cases a nephrectomy was performed because of a hydronephrosis caused by additiohal vessels and urolithiasis. A sharp increase of ~, for serum was observed for patients with a single kidney; this increase was more pronounced for primary pyelonephritis patients. At the same time L for urine also displayed an increasing trend, approaching values characteristic of healthy persons. In general, for chronic pyelonephritis the increase in ~. for biological liquids during the observation was indicative of unfavourable developments of the disease.

4.3. Diabetic nephropathy The effect on kidneys due to microangiopathy is one of the most serious epiphenomena, which mainly determines the prognosis of Diabetes mellitus (Zatzet 1986). Diabetes mellitus is often accompanied by chronic pyelonephritis, which can make the diagnosis of true diabetic nephropathy

very

complex.

Various

glomerulonephritis

versions

(mesangiocapillary,

membraneous, or extracapillary) (Shyshkin et al. 1989, Maueret et al. 1992, Yoshikama et al. 1990, Chihara et al. 1986) can develop, which are usually combined with diabetic nephropathy, bolstering the progressive diabetic lesion of kidneys, which finally results in chronic renal insufficiency. The incipience of the glomerulonephritis accompanying diabetes mellitus is

164 attributed to the immunogeneity of some components of the collagen (response to endogenous insulin), and to the fact that mesangial cells are unable to perform the phagocytosis of substances which attain the glomerular filter. Even at the early stages of the disease, microscopic studies show that morphological changes take place in kidney glomeruli and tubules. The sponginess of the glomerular basal membrane increases its permittivity with respect to albumin. At initial stages this process is completely compensated by the reabsorbance of proximal tubular nephrocytes; this is implicitly confirmed by the fact that hyaline degeneration of the tubular epithelium is observed. The further genesis of non-specific structural degradation of kidneys essentially depends on the increase in the renal blood flow and hyperfiltration. Both these factors leads to increased penetration of plasma albumin through the basal glomerular membrane. This results in an albuminuria and the deposition of blood proteins within the mesangium, stimulating the mesangial proliferation. Increased amounts of glucose, galactose, hydroxylysine, collagen, some ferments in glomerular basal membrane were also observed (Abbakumova et al. 1985, Berg et al. 1997, Osterby 1993). The hyperfiltration is believed to be one of the leading factors determining progressive diabetic nephropathy (Muchin et al. 1990, Shestakova et al. 1991, O'Bryan et al 1997, Buckalew 1994). For most patients characterised by high levels of glomerular filtration, all nephrones are working with maximum efficiency, which results in a rapid burn-out and subsequent progressive deterioration of the glomerular filtration. The progress of hyperfiltration for diabetes mellitus is characterised by a dilatation of afferent arterioles, with an unchanged tonus of efferent vessels of glomeruli. This results in a sharp increase in the gradient of the intraglomerular hydrostatic pressure. This in turn contributes to the deterioration of basal membrane permittivity, leading to proteinuria and the deposition of proteins in the mesangial matrix. Microalbuminuria is believed to be the only reliable criterion for the diagnosis of diabetic nephropathy in its subclinical stage. Variations in the composition of many low-, medium-, and high-molecular surface active compounds were observed in the blood of patients with diabetes mellitus and in the contents of soluble complexes of monomeric fibrin and its degradation products (Belitskaya et al. 1991, Carmassi et al. 1992, Velikov et al. 1991, Van Wersch et al. 1990, Lapchinskaya 1991, Simuda et al. 1997). Villebrand's factor and 13-thromboglobulin (Sokolov et al. 1991, Bath et al. 1996,

165 Trovati et al. 1992) are increased along with the plasma viscosity (Zhumatova 1991, Zimmermann et al. 1996). The development of diabetic nephropathy is accompanied by an increase in the total concentrations of cholesterol and the low density lipoprotein fraction in serum (Balabolkin et al. 1996, Morishita et al. 1995, Mancini et al. 1988).

a) serum 0.8 0.6 0.4 o

0.2~

0

-0.2-0.4 -0.6 ol

o2 o3 Serum

;L

ol

o2

X

o3 Urine

b) urine 0.8 ~9 0.6 0.4 0 0.2 o -o.2 o

0.4 -0.6

-0.8 ol

o2 Bbod

o3

X

ol

o2

o3

X

Urine

Fig. 4.30. Correlations between particular surface tension parametersof biological liquids obtained from patients with diabetic glomerulosclerosis.Surface tension parameters are o~ (hatched), o2 (black), o3 (white) and X(grey).

166 It was already noted above that dynamic surface tensiometric parameters in the medium surface lifetime range are controlled mainly by the contents of low- and medium-molecular surfactants in the studied liquids, while the values of equilibrium surface tension are determined mainly by the presence of high-molecular weight compounds. The values of o2 (for type I of the disease) and 0.3 for serum are decreased in case of diabetes mellitus with kidney affection. In addition, for patients with type I diabetes mellitus, the value of ~, for serum is sharply increased, which can possibly be used for differential diagnosis. The values of ol for serum correlate with 0.2, while 0.3"2correlates with 0.3 (see Fig. 4.30). The number of patients with diabetic nephropathy used in this study was 32. A direct correlation was observed between surface tensiographic parameters in short and medium time range, and ~, values of serum. However, the value of equilibrium surface tension, quite expectedly, displays a negative correlation with ~. The above relations indicate that these tensiographic characteristics are affected by the presence of the same compounds in serum. The value of urine 0.3 correlates with serum 0"2, while 0.2 and 0.3 values for urine correlate with the parameter ~, of the serum. The parameter ~, depends on the patients age (negative correlation) and arterial blood pressure level (positive correlation).

Urine

Senlnl

4t 2

II

0-

~'~

-2

~

-6

"[1

-8

_10 j 0.1

0.2

0.3

0.1

0.2

0.3

Fig. 4.31. Changes in surface tension parameters of biological liquids obtained from patients with diabetic glomerulosclerosis with either preserved (white columns) or decreased (black columns) renal function. Changes are given in % compared to corresponding healthy controls.

167 While for the uric syndrome with unaffected kidney function the values of 0"2 do not differ from those characteristic for healthy persons, the development of a nephrotic syndrome and chronic renal insufficiency is accompanied by a decrease of 0-2 and 0-3 (see Figs. 4.31 and 4.32), which can serve as an additional prognostic criterion for the development of diabetic nephropathy.

Serum

_

Urine

_

o•

0

- - -

-2-4 -6 -8-

0-1

0-'2

0-3

0-1

0-2

0-3

Fig. 4.32. Changes of surface tension parameters of biological liquids obtained from patients with diabetic glomerulosclerosis with uric (white columns)and nephrotic (black columns) syndrome. Changes are given in % comparedto correspondinghealthy controls. For patients who suffer form chronic renal insufficiency, even irrespective of diabetic nephropathy, hyperglycemia and decreased tolerances with respect to glucose (so-called uremic pseudodiabetis) were often observed. For healthy persons the level of renal metabolism of insulin contained in arterial blood is up to 40%; on the contrary, this hormone undergoes virtually no destruction in kidneys of patients with chronic renal insufficiency, especially in the terminal stage. In addition, a potassium deficiency arises, leading to the decay of metabolic processes in the cells and a reduced insulin secretion. Acidosis, the low activity of the somatotrophic hormone, enhanced gluconeogenesis from alanine and hyperglucagonemia, depressed peripheral utilisation of glucose due to special peptides (possessing molecular mass ca. 1.2 kDa) occurs in the blood of patients suffering from chronic renal insufficiency are the

168 factors which lead to the development of a hyperglycemia. The imbalances of the carbohydrate exchange for chronic renal insufficiency related to diabetic nephropathy are characterised by some specific features, because for patients with diabetes mellitus, the defects in glucose metabolism arise when the renal function is still unaffected. It is this fact which enables to discriminate between the variation of blood surface tension for patients with chronic renal insufficiency due to the diabetic nephropathy, and a chronic renal insufficiency due to another renal diseases. There exists a close relation between carbohydrate and lipid exchange. For chronic renal insufficiency the continuous excess of insulin in blood stimulates the production of fatty acids (from glucose metabolites) and triglycerides (due to their delayed elimination from the circulating flow and enhanced synthesis in the liver). The deterioration of the qualitative and quantitative composition of carbohydrates, proteins and lipids affects the dynamic surface tensions of blood serum. Table 4.7 illustrates the correlations which exist between surface tensiometric parameters and various compounds present in blood serum of subjects with diabetic nephropathy. The presence of glucose promotes the increase of blood surface tension at short and medium surface lifetimes. A similar effect is also characteristic for calcium and, especially, for sodium. The arising electrolytic imbalance and the extension of the hyperglycemia which accompanies the chronic renal insufficiency for patients suffering from diabetes mellitus, can possibly lead to a decrease in ~2 and ~3 values of serum. The excretion of sodium by kidneys is controlled via variations in the rate of glomerular filtration and tubular reabsorption of electrolyte. Here the hormonal systems of the organism (cortifan, aldosterone, renin, hypertensin, catecholamines, prostaglandins,) play an essential role (Kutyrina et al. 1991, Kemppainen et al. 1997, Blaine 1990, Bemheim et al. 1986). Tubular reabsorption of sodium is affected by physical factors, such as the redistribution of renal blood flow. The balance of water and sodium in the organism depends on the consistent performance of the regulation systems; otherwise the water/electrolyte equilibrium is violated, which can lead to surface changes of blood serum.

169 Table 4.7. Correlations between the concentration of various blood components and surface tension characteristics of serum obtained from patients with diabetic nephropathy

Blood component Glucose Total protein Albumin Otl-globulin fraction ~z2-globulin fraction 13-globulin fraction 7-globulin fraction Immunoglobulin-G Immunoglobulin-A Immunoglobulin-M Circulating immune complexes Total cholesterol a-cholesterol Triglycerides High density lipoprotein fraction Low density lipoprotein fraction Very low density lipoprotein fraction a2-macroglobulin 132-microglobulin Fibronectin Urea Creatinine Uric acid Oxypurinol Sodium Chlorine Potassium Total calcium Ionised calcium Magnesium Phosphorus

Surface tension parameter (3"1

(Y2

1'

1'1'

$$

t~3

1'

1' 1"1' $ 1"1'

$$

$$

$ 1'1'

1'1' $$

1'1" $

1'1' 1'1' 1"1'

1'1' 1"1'

1'1' 1'1'1'

1" positive correlation, $ negative correlation, empty - no correlation; one arrow, r < 0.5; two arrows, r - 0.5 to 0.7; three arrows, r > 0.7

1"1" $$ 1'1"

$

1'1'

1'1" 1'1'1' 1"1" 1"1" 1"

170 Proteins and lipids, which are strong surface active components, can decrease the surface tension of serum significantly. In our experiments a decrease in surface tension of serum with increased concentrations of these compounds was observed only for short and medium surface lifetimes (for t = 0.01 s and t = 1 s), while their effect on the equilibrium surface tension was rather unexpected. Alterations of the physicochemical properties of proteins for patients with diabetic nephropathy can be hypothetically ascribed to the glycosilation process, that is, the non-fermental attachment of glucose to the amino groups of proteins. The glycosilation is a non-specific reaction, which involves proteins of serum, basal membranes, erythrocyte membranes etc. The flexibility of blood cell membranes depends on the existence of a lipid bilayer and proteins, capable of being glycosilated. Violations of the carbohydrate exchange initiate disturbances in the kidney microvessels (Galenok & Chanykina 1991). Urea, creatinine, uric acid and other low molecular compounds affect the structure of proteins, making the amino groups less accessible for linking with glucose; this leads to violations in the protein glycosilation processes (Lebedeva 1996, Barbagallo et al. 1993). We believe that this fact can explain the decrease of equilibrium surface tensions of blood for patients suffering from diabetic nephropathy with chronic renal insufficiency (g3 is negatively correlated to the nitrous non-proteinic constituents of blood serum). For diabetic nephropathy, pronounced dependencies exist between surface tensiometric parameters and the amounts of immunoglobulins and circulating immune complexes present in blood. It is known that serum of patients suffering from diabetes mellitus of type I (insulindependent) and II (insulin-independent) exhibits an increase in the immunoglobulin level. Usually the concentration of immunoglobulins-G becomes higher. It was argued, however (Saltykov et al.

1990) that a concentration

increase

of immunoglobulins-A

and

immunoglobulins-M also takes place. Our data show that hyperimmunoglobulinemia results in a concentration

increase

for all

classes

of such proteins,

where

the

increased

immunoglobulin-G level is most pronounced. However, their concentration becomes significantly lower with the development of a nephrotic syndrome. Patients with diabetic nephropathy exhibit large amounts of circulating immune complexes able to fix the C3-component of the complement and to incorporate insulin. Close correlations were found between the existence of a renal microangiopathy and the presence of circulating immune complexes containing immunoglobulins-A1 and immunoglobulins-A2.

171 The proteinuria is the most early symptom of kidney affection for diabetes mellitus, while structural lesions of the kidney tissue arises much earlier as proteinuria becomes recognisable. The earliest preclinical signs of diabetic nephropathy are the microalbinuria (the excretion of albumins below the threshold of common methods used for the detection of proteinuria, i.e. 30-300 ~tg per day) and high urinal concentrations of the specific ferments localised in the epithelium of kidney proximal tubules. While the mechanisms of glomerular and tubular disturbances for diabetes mellitus are different, a direct correlation between microalbuminuria and the activity of acetyl-[3-D-glucosamine peptidase and alanineamine peptidase in urine exists (Jung et al. 1989), suggesting that a high excretion of these enzymes is a marker of diabetic nephropathy. The damage of the tubular epithelium leads to urinal excretion of ferments specific to the membranes of proximal tubule brush border (alkaline phosphatase, ,/-glutamyl transferase), cytoplasm (LDG) and lysosomic ~-glucuronidase (Dedov et al. 1989). The enhanced urinal excretion of low molecular [32-microglobulins indicates a change in the tubular reabsorption process. Therefore, the increase in urinal excretion of surfactants, such as albumins, ferments and [32-microglobulins, can lead to varied dynamic surface tensions even at a preclinical stage of diabetic nephropathy. For pronounced glomerulosclerosis, the dynamic surface tensiometry parameters of urine will exhibit most appreciable variations. For diabetes mellitus, activated T-lymphocytes become capable of producing a factor which enhances the penetrability of vessels and the synthesis of endoglycosidase - the ferment for the degradation of heparane sulphate of protein glycanes of the subendothelial matrix and basal glomerular membrane. Heparane sulphate determines the negative charge of the glomerule filtration barrier, and acts as a physiological anticoagulant. Therefore, a lack of negative charge of the glomerular filter, a decreased local anticoagulant potential, and extended penetrability of the vessels (caused by products of activated lymphocytes) promote the enhanced excretion of negatively charged albumins (Salozhin et al. 1991). An interrelation exists between albuminuria and the concentration of lipoproteins in blood serum (Muchin et al. 1990, Schnack et al. 1994). Each diabetes mellitus patient without any clinical-laboratory symptoms of kidney damage displays a change in the glomerular filtration, while for one patient out of five an affection of the proximal tubules was found. In 75% of all these cases a hidden proteinuria was found by

172 special tests. The presence of proteins with an electrophoretic mobility characteristic to prealbumin, albumin and post-albumin in urine was found for 6% of patients suffering from diabetes mellitus of type I, while for type II this portion increased to 41%. For transferrin and haptoglobulin these percentages are 38% and 59%, respectively. The presence of uromucoids in the region of immunoglobulins and tx2-microglobulin was detected for 57% of patients with diabetes mellitus type I (insulin-independent) only. The increase of urinal excretion of surfactants filtered and secreted by kidneys for diabetes mellitus type I leads to decreased o2 values. The pronounced proteinuria for patients with nephrotic syndrome leads to strongly decreased surface tensions of urine (cf. Fig. 4.32). Hampered reabsorption processes in kidney tubules for chronic renal insufficiency result in more pronounced shifts of the dynamic tensiograms at medium surface lifetimes (cf. Fig. 4.31). The values of 0.1 for urine correlate directly with the parameters 0.2 and 0.3 of this biological liquid. The correlation between the parameters in the medium and large time range is still more significant. There is a strong negative dependence of the ~ values for urine on the equilibrium surface tension. Thus, all the correlations just mentioned indicate that the variation of dynamic tensiometry parameters for this type of the disease are caused by single reason. Interrelations between the tensiographic parameters of different biological liquids also exist. In particular, the ~. value of serum for diabetic nephropathy is related to 0.2 and 0.3 of urine, and 0.3 of urine correlates with the parameter 0.2 of serum (cf. Fig. 4.30b). The dynamic surface tensiometry parameters for urine exhibit inverse correlations with the duration of diabetes mellitus, but do not depend on the glucosuria level. However, there are weak negative dependencies of 0.1 and 0.3 for urine on the glucose concentration in blood (Fig. 4.33). A correlation between the extent of the albuminuria and the 0.a-value for serum is observed as well. The level of albuminemia should determine the values of equilibrium surface tension for serum; therefore for patients with diabetic nephropathy, the concentrations of albumin in various biological liquids will affect significantly the value of c3 in serum. In this connection, both the direct correlation between the albumin concentration in serum and equilibrium surface tension of serum, and the inverse correlation between the concentration of albumin in urine and 0"3 of

serum remains unclear. First, increased concentrations of albumin cause a surface tension

fall of the model solutions; second, the albumin contents in blood decreases with increased

173

proteinuria. Hence the two biological liquids exhibit opposite dependencies with respect to the albumin concentrations. These anomalies of surface tension are possibly related to the conformations of proteins caused by the interaction with low molecular nitrous compounds, and a variation in their adsorption properties caused by the glycosilation processes. Senna

Urine

0.8 0.6

-

0.4o

O

0.2-

I

-0.2-0.4 -0.6 al

a2

a3

k

al

a2

a3

L

Fig. 4.33. Correlation coefficients between surface tension parameters measured in biological liquids obtained from patients with diabetic glomerulosclerosis and the level of glycemia.

0,2

-

0,1 ~

0

~

-0,1 -

O

0,2

=

0,3

7/.,_-s $22

:::" "":" .... :" ..... "::" ............ : ::": """ ":":": :"" ..... ""' ..... : :" ::::::: : ......

0,4

.............

L!.;.

"~

-05

7_72 S7_,,ii 7"",.........i $2 .. ,,.;TZ ,',"..'.'?Z 2"Z"S ZZ2 ?7.,221 ,SZ

75.S 77,_,S

-~

-0,6 -

~ i -0,7 -0,8 -

P

A

F

MG

Fig. 4.34. Correlation coefficients between surface tension parameters measured in urine obtained from patients with diabetic glomerulosclerosis and the concentration of proteins in urine. Surface tension parameters are o~ - hatched, tr2- black, a3- white and ~ -grey. Measured proteins are albumin - A, fibronectin- F, 132-microglobulin - MG, total protein- P.

174 There exists quite expectedly a negative correlation between urine tensiographic parameters and the concentrations of various proteins (cf. Fig. 4.34). This is primarily true for the parameters of surface tensiograms in the short time range. Both fibronectin and 132-microglobulin affect the Z, value of urine. As chronic pyelonephritis often accompanies diabetes mellitus in general and diabetic nephropathy in particular, the results obtained by surface tensiometry of biological liquids for female patients possessing this pathology have been analysed carefully in that respect. It was mentioned above that for female patients suffering from primary pyelonephritis, decreases in o2 and

03

for serum, o2 and ~, for urine, and increased values of ~, for serum were observed.

One should naturally expect equivalent variations of surface tension parameters for diabetic nephropathy accompanied by chronic pyelonephritis. On the contrary, it was found that this combination results in an increase (not decrease) of o2 of urine and of the equilibrium surface tensions for both the biological liquids (cf. Fig. 4.35). The only tensiographic parameter whose increase was reliably stated for chronic pyelonephritis, is ~ for serum.

Serum

4q 24 0-2

-

-4

-

Urine

-6 -8 10 -12 i -14 j 16 18 ol

o2

o3

ol

o2

o3

Fig. 4.35. Changes in surface tension parameters measured in biological liquids obtained from patients with diabetic nephropathy. Black columns- without accompanyingpyelonephritis, white columns- with accompanyingpyelonephritis. Changes are given in % comparedto correspondinghealthy controls.

175 To summarise, the diabetic nephropathy combined with pyelonephritis results in quite unusual variations of the dynamic surface tensiometric parameters, characteristic neither for primary pyelonephritis, nor for isolated diabetic glomerulosclerosis. Therefore, a future task in these studies should be to compare the characteristics of surface tensions between the patients who do not suffer from the renal syndrome and possess quite similar parameters of the renal function, similar arterial blood pressure, disease duration, etc. It would be interesting to study the surface tension of biological liquids for patients suffering from diabetes mellitus causing no renal lesions, as compared with pyelonephritis not accompanied by diabetic nephropathy. At the present stage it can be argued that for the cases of diabetic nephropathy, the dynamic surface tensiometric parameters can be regarded as rather informative criteria for differential diagnosis, which enable one to predict further developments of the pathologic process. We believe that dynamic surface tension studies of serum and urine will become a reliable auxiliary method for monitoring various treatments. 4.4. Other renal diseases

Other renal disease that were studied using dynamic surface tension analysis of biological fluids include podagric nephropathy (40 patients), kidney amyloidosis (21 patients), kidney sarcoidosis (19), hypertension disease accompanied by nephrosclerosis (18), myelomic nephropathy (18). All these diseases either are kidney specific or affect kidneys secondarily. Among other renal diseases, the amyloidosis is remarkable for the hyperproduction and the decrease in the catabolism of free light chains, and the structural components of immunoglobulins (Resnikov et al. 1996) having surface activity. For the primary variant of this disease, the amyloid fibrillae consist of polypeptide fragments of normal serum proteins and light chains of immtmoglobulins or their fragments. For the secondary variant, the amyloid protein differs in its amino acid sequence, and is represented by the protein AA (molecular mass ca. 9 kDa), which forms from the serum albumin SAA. For the hereditary form, the amyloid fibrillae were identified as the prealbumins (with a molecular mass in the interval of 8 kDa to 40 kDa). The P-component of any amyloid deposition corresponds to the blood serum glycoprotein (molecular mass 23 kDa), and contains large levels of glutaminic and aspartic acids, glycine, leucine, and, in a lower extent tryptophane.

176 The protein SAA is synthesised by hepatocytes, and becomes present in blood in significant amounts as a result of prolonged antigenic stimulation. In the blood circulation, this protein is closely related to the high density lipoprotein fraction, forming complexes with them (with molecular masses around 180 kDa). For all types of the disease, the amyloid fibrillae are of pronounced polyanionic character, and therefore bind other proteins (Bannikova 1987). The specific composition of blood proteins for kidney amyloidosis should affect the state of dynamic surface tensions of biological liquids - not only of serum, but also of urine, because pathologic proteins are filtrated in the kidney glomeruli. Surface tensiometry of biological liquids has been performed for patients with secondary kidney amyloidosis caused by rheumatoid arthritis (the diagnosis was confirmed by biopsy). Decreased surface tension of serum in the short and medium surface lifetime range, and a decrease in the ~. values were observed (cf. Fig. 4.36).

75

....

70

.o

.............. ......

~.............

~ 1 7 6O~176176 176 ~176176 ~ ~ o~176 ~ ~ o~ ~ ~ ~176

t_.__a

~ ~ ~176 ~176

[

60

55

t

-2

. . . .

-1

t

b

t

0 lg(tef) [S]

1

2

Fig. 4.36. Example of serum tensiogramobtained from patient (female, age 58) with rheumatoid arthritis accompanied by secondary kidney amyloidosis,nephrotic syndrome,chronic renal insufficiencyof 2nd stage, dotted line correspond to average value for healthy females of the same age. The urine of such patients is characterised by high

(Yl

and

03

values, with a significant decrease

in the ~, values. It should be stressed that for all patients screened, the nephrotic syndrome and normal renal ftmctions were diagnosed.

177 When morphologic studies of the nephrobioptates are not available, clinicians encounter severe difficulties in the differential diagnosis of kidney amyloidosis and chronic glomerulonephritis. It should be recalled in this connection that the nephrotic syndrome for chronic glomerulonephritis is characterised by a significant increase in ~, for serum and decreased ~2 and ~3 values of urine, while for acute glomerulonephritis an increase in L of serum and the Crl values of urine is observed. Therefore the results of dynamic surface tensiometry for biological liquids enables one to distinguish rather reliably between glomerulonephrites and kidney amyloidosis at the nephrotic stage of the pathologic process, which is of a significant practical value. One approach to the study of the intensity of cellular receptors metabolism is the determination of R-protein concentrations in biological liquids. These are products of the catabolic decomposition of external parts separated from the cells. The serum R-proteins are capable of binding ligands of catalytic activity similar to that of superoxyde dismutases, and can circulate either in a free state, or in complexes formed with immunoglobulin-G. For kidney amyloidosis, the level of R-proteins in blood increases as the result of the peculiar (~desquamation>> of cells; this leads to a distortion of their response to various stimuli (Kozlovskaya et al. 1992). The concentration of R-proteins, rather expectedly, displays an inverse correlation with surface tension parameters of serum in the short and medium adsorption time range. For patients suffering from kidney amyloidosis, the contents of fibronectin in serum is somewhat higher than that characteristic of chronic glomerulonephritis. The level of fibronectinuria increases sharply, especially for patients with a nephrotic syndrome. The urinal excretion of this high molecular plasmatic glycoprotein is enhanced due to the increase in the penetrability of the vessels, the glomerular filtration of the glycoprotein, and the intensified metabolism of glomerular capillary basal membranes (Karryjeva et al. 1992, Westermark et al. 1991). The dynamic surface tensions of urine in the short time range exhibits direct correlations with the concentration of fibronectin in urine for kidney amyloidosis accompanied by the nephrotic syndrome. The monoclonal free light chains take their part in the pathogenesis, not for the amyloidosis only, but also for the myelomic disease. They possess a nephrotoxic effect, and determine the prognosis of the disease evolution. Low molecular free light chains are easily filtered and

178 almost completely reabsorbed in the kidneys. However, when the number of functioning nephrones decreases and, therefore, the glomerular filtration becomes less intensive, then these free light chains cannot arrive in the region of their catabolism. This results in a decrease of their excretion, and an increase of their contents in blood. A reliable marker for highly differentiated B-cell tumours, one example of which is the multiplex myeloma, is the production of monoclonal immunoglobulins and/or free light chains -

Bens Jones protein. While the diagnostics capability for serum monoclonal immunoglobulins

is by no means absolute, the presence of free light chains in urine can be regarded as evidence for the tumoral nature of a process. In spite on the fact that K-chains are much more frequently incorporated into immunoglobulins-G, there are Z,-chains which prevail in free light chains (because they are less capable of binding to immunoglobulins-G). The same difference exists between the polyclonal and monoclonal free light chains: in monoclonal free light chains the occurrence of K-chains is twice as high as that of ~-chains. In the pre-clinical stage of myelomic nephropathy, moderate effects of nephrothelium albuminous degeneration can be detected, while no changes in glomeruli and interstice are as yet present. Then a pronounced granular hyaline and hydropic degeneration develops, along with a moderate atrophy of tubular epithelium and eosinophilic cylinders in their lumina. An ectasia of stroma takes place with separate focuses of sclerosis. Further stages of the development of myelomic disease are characterised by the thickening of the glomerular capillary basal membranes, an increase of the mesangium, and an extensive process of glomeruli elimination, controlled by the periglomerular (or, less frequently, capillary collapse) sclerotic mechanism. Finally, the nephrocalcinosis becomes evident (Sidorova et al. 1988, Ivanyi 1993, Mundy 1990). One could expect similarities between the parameters of surface tension of serum for myelomic disease and those for kidney amyloidosis. However, the multiplex myeloma is characterised by decreased surface tensiometric parameters of seruna in the medium and long time range, with a significant increase of ~,. It has to be noted that the myelomic disease is accompanied by hyperproteinemia and more pronounced ~-globulinemia, relatively low contents of cholesterol and triglycerides in blood, even for cases of myelomic nephropathy with nephrotic syndrome. Typical for such patients is the increase of crl and X values of serum. The development of amyloidosis for myelomic disease leads to an increase in the or3 and ~ values of urine.

179 The dynamic surface tensiometric parameters in the medium surface lifetime range (t = 1 s) directly correlate with the total concentration of proteins and the level of immunoglobulins M and G in blood. The parameter 0-1 for urine depends inversely on total proteinuria (not albuminuria solely), while

0"3

inversely depends on the fibronectinuria.

While the hypercalciemia, hyperuricemia and high blood viscosity contribute significantly to the development of nephropathy for multiplex myeloma, the main role is believed to be played by the renal excretion of anomalous immunoglobulins (proteinuria of repletion) whose accumulation in the interstice, glomerular and tubular basal membrane leads to a damage of the nephrothelium and locking of tubular lumen. The plasmapheretic treatment of such patients makes it possible to remove large masses of pathologic proteins, to decrease plasma viscosity and the oncotic pressure, to improve the rheologic properties of blood and the performance of microcirculation processes in kidneys (Abdulkadyrov et al. 1991, Abdulkadyrov and Bessemeltsev 1992, Reinhart et al. 1992). Reciprocal variations of blood macroproteins and albumins, rather moderate for the plasmapheresis,

become

more

significant

for

the

cytapheresis

and,

especially,

plasmacytapheresis. At the beginning of the second stage the level of macroproteins somewhat exceeds its initial value, while the concentrations of albumins and globulins remain lower than their initial values. As the relative contents of proteinic fractions in blood is rather inertial, the level of macroproteins can be regarded as merely the general indicator of the treatment efficiency. Repeated procedures lead to the increase in the total amount of eliminated proteins and macroproteins. One can presume that at least two mechanisms for the compensation of the blood proteinic system exist in respect to a plasmapheresis treatment, a fast and a slow mechanism, respectively. The fast mechanism becomes active at the commencement of the procedure, and controls efficiently the proteinemia parameters during tens of minutes, while the slow mechanism remains effective after many hours. The fast mechanism is supported preferentially due to the penetrability, deposition and re-deposition of proteins and liquid constituents of blood. The crucial role in the performance of the second mechanism is played by processes of biosynthesis of plasma proteins.

180 Variations in the total concentration of proteins, and the decrease of the albumin/globulin factor for the myelomic disease, lead to the increase of the plasma colloid-osmotic pressure. The plasmapheresis process leads to the elimination of globulin fractions of the proteins, to a decrease of the levels of proteinemia, circulating immune complexes, and blood viscosity. The rheological properties are improved due to the normalisation of the plasma colloid-osmotic pressure (osmolarity). The application of a treatment makes it possible to eliminate the paraproteins, to improve the coagulation state of the blood and the drainage function of the tissue.

410 . . 390 ~

m m

370

m

--

m

m

m

350 O .~

330-

_~

310

0

~ 9 r~

O""""<~~"~------~'~

290

"~

~

O

270 250

I

before

0

I

1

I

5

I

7

1

10

I

12

I

15

Time after plasmaferesis [days]

Fig. 4.37. Dynamicsof serum and urine osmolarityduring a plasmapheresistreatment of patients with myelomic nephropathy; + - serum osmolarity,~- urine osmolarity Immediately after a single seance of plasmapheresis, the colloid-osmotic pressure of blood and urine decreases significantly, which affects the surface tensions of the biological liquids. Subsequently the osmolarity of urine increases somewhat, while the osmolarity of blood remains unchanged (cf. Fig. 4.37). The application of plasmapheresis results in larger values of c2 and cr3 of serum, and smaller ~,values, in line with the variation in osmolarity. In fact, the surface tension of serum after a plasmapheretic treatment approaches a value characteristic for healthy persons. This dynamics

181 of surface tensiometry during the treatment process can be explained by the elimination of a number of albumin surfactants, including pathological proteins, from the organism of the patient with multiplex myeloma. At the same time, the surface tension parameters for urine remain virtually the same. Therefore, surface tensiometry of serum performed before and after plasmapheretic seance can be regarded as a rather promising method for the control of the efficiency of this treatment. Significant variations in surface tension values of urine were found for patients suffering from sarcoidosis without any changes in the clinical-laboratory characteristics which are related to the kidneys. A significant decrease of ~1, ~2, cy3 and )~ values for urine is observed, while the surface tensiographic parameters of blood remain constant. Sarcoidosis is one of the system diseases, the morphologic substrate of which is the necrotizing granulomatosis which sometimes affects the kidneys. The clinical-morphological versions of sarcoidosis are glomerulonephritis, interstitial and "calcic" nephritis. Both hypercalciemia and hypercalciuria can result in an urolithiasis. Glomerulonephritis which accompanies the sarcoidosis can persist in mesangioproliferative, mesangiocapillary and membranoproliferative forms, while immunofluorescence microscopy detects deposits of immunoglobulins G, A, M, and the complement at the basal glomerular membrane and in the mesangium. The amyloidosis can be referred to as the form which rarely affects the kidneys (Dobin & Kalinichev 1991, Kornev & Potapova 1991, Casella et al. 1993). The development of glomerulonephritis in the case of sarcoidosis produces no additional changes in the surface tension parameters of urine. Therefore dynamic surface tensiometry of urine can serve as a diagnostic method to detect latent nephropathy accompanied with sarcoidosis. This hypothesis can be verified in future, by a comparison of the morphological pattern of "intact" with the surface tension parameters of urine. However, it can turn out that such a dependence does not exist, because the detected variations in urine tensiograms can be caused by some extrarenal factors. To finish this chapter, some results are presented to illustrate the multivarious character of dynamic tensiograms for renal diseases. Figure 4.38 shows the ratio of serum tensiografic parameters to the corresponding parameters of urine for various renal diseases.

182

A 1.1

1

13 0.9

0.8

,

HC

HM

HF

AGN CGN L ( ~

PPN

UL

HD

KA

MN

DN

PN

KS

l-IF

AGN CGN LGN C_K3N PPN

UL

HD

KA

MN

DN

PN

KS

~

4 3.5 3 2.5 2 ,,< 1.5

0"5/ 0

i

HC

l

HM

Fig. 4.38. Ratios of surface tension parameters measured in serum and urine that was obtained from healthy individuals and patients with various renal diseases; HC - healthy controls (general group), HM - healthy males, HF - healthy females, AGN - acute glomerulonephritis, CGN - chronic glomerulonephritis, LGN - lupus glomerulonephritis, GGN - Genoch glomerulonephritis, PPN - primary pyelonephritis, UL - urolithiasis, HD hypertension disease accompanied by nephrosclerosis, KA - kidney amyloidosis, MN - myelomic nephropathy, DN - diabetic nephropathy, PN - podagric nephropathy, KS - kidney sarcoidosis. In graph A the serum/urine ratios of a~ (black) and a3 (white) are given. In graph B the serum/urine ratios of ~. are presented.

183 It is seen that for some diseases a decrease of this ratio is obtained, while for other diseases an increase

is

observed.

glomerulonephritis

this

For

example,

ratio

for

increases

lupus in

the

glomerulonephritis short

time

and

range,

sarcoidosis while

for

arteriolonephrosclerosis and kidney amyloidosis this ratio decreases. A similar behaviour is characteristic for the equilibrium surface tensions. The ratio of the ~-values usually increases: the most pronounced increase is observed for patients with arteriolonephrosclerosis, while for myelomic nephropathy the ~ values of serum tensiograms is obviously lower than those for urine. As gout is a disease prevailed in males, it can be argued that a decrease of the serum to urine ratio of the parameters should result, while the surface tension parameters themselves depend on the type of the nephropathy. This will be considered in the following chapter.

4.5. Summary In summary, dynamic tensiograms of serum and urine sampled from patients with various kidney diseases have a multivarious character. Figure4.38 shows the ratio of serum tensiographic parameters to the corresponding parameters of urine for various renal diseases. We have shown that for some diseases a decrease of this ratio is obtained, while for other diseases an increase is observed. For example, for lupus glomerulonephritis and sarcoidosis glomerulonephritis

this

ratio

increases

in

the

short

time

range,

while

for

arteriolonephrosclerosis and kidney amyloidosis this ratio decreases. A similar behaviour is characteristic for the equilibrium surface tensions. The ratio of the )~-values usually increases: the most pronounced increase is observed for patients with arteriolonephrosclerosis, while for myelomic nephropathy the )~ values of serum tensiograms is obviously lower than those for urine. As gout is a disease prevailed in males, it can be argued that a decrease of the serum to urine ratio of the parameters should result, while the surface tension parameters themselves depend on the type of the nephropathy.

4.6. References Abbakumova, M.V., Dedov, I.I., Dreval', A.V., Muchin, N.A., Ter. Arch., 6(1985)147. Abdulkadyrov, K.M., Bessemeltsev, S.S., Liubimova, N.Yu., Ter. Arch., 7(1991) 122.

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185 Cill, H.S., Rose, G.A., Urol. Int., 41 (1986)393. Ciszewski, T.S., Ralston, S., Acteson, D., Wasi, S., Strong, S.J., Transfusion Medicine. 3(1993)59. Clauser, E., Curnow, K.M., Davies, E., Conchon, S., Teutsch, B., Vianello, B., Monnot, C., Corvol, P., European Journal of Endocrinology, 134(1996)403. Clemens, M.R., Bursa-Zanetti, Z., Nephron, 53(1989)325. Cosio, F.G., Bakaletz, A.P., J. Lab. Clin. Med, 107(1986)453. Cowley, A.W. Jr., American Journal of Physiology, 273(1997) 1. Dedov, I.I., Muchin, N.A., Paltsev, M.A., Ter. Arch., 12(1989)73. Dobin, V.L., Kalinichev, G.A., Ter. Arch., 6(1991) 140. Dorofejev, S.B., Tov, N.L., Movchan, E.A., Ter. Arch., 10(1991)119. Ebisuno, S., Morimoto, S., Yoshida, T., Acta Urol. Jap., 32(1986)1773. Eddy, A.A., McCulloch, L., Liu, E., Adams, J., American Journal of Pathology. 138(1991)1111. Fadul, J.E., Linde, T., Sandhagen, B., Wikstrom, B., Danielson, B.G., Journal of Clinical Apheresis, 12(1997)183. Faucher, C., Doucet, C., Baumelou, A., Chapman, J., Jacobs, C., Thillet, J., American Journal of Kidney Diseases, 22(1993)808. Fomenko, G.V., Arabidse, G.G., Titov, V.N., Ter. arch., 6(1991)142. Fomenko, G.V., Lipitskaya, I.Ya., Tuminen, T., Ter. arch., 4(1992)30. Forte, L.R., Fan, X., Hamra, F.K., American Joumal of Kidney Diseases, 28(1996)296. Fuhrer, G., Gallimore, M.J., Heller, W., Hoffmeister, H.E., Blut, 61 (1990)258. Gabrielyan, N.I., Levitsky, E.R., Scherbaneva, O.I., Ter. arch, 6(1983)76. Galenok, V.A., Chanykina, I.V., Ter. Arch., 12(1991)113. Gavrylov, V.N., Ter. Arch., 10(1987)125.

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191

Chapter 5

Surface tensiometry in rheumatology In this chapter we will discuss dynamic surface tensiometry as a tool for differential diagnosis of connective tissue disorders and for monitoring purposes of therapeutic interventions.

5.1. Pathogenesis of rheumatic diseases Degradation of connective tissue which occurs with rheumatic diseases is caused by the disturbance in the biosynthesis of macromolecules, in particular, various types of collagens, proteoglycans, elastin, and structural glycoproteins. Collagen and elastin undergo a degradation, which results from the decreased activity of lysonoxydase and production of tropoelastin, and enhanced elastolytic processes.

The turn-over of connective tissue

metabolism takes place from the ordinary exocrine to endocrine route. Elastase is responsible for the control of metabolic processes in the connective tissue. One important class of components in connective tissue are the glycosaminoglycanes, which largely determine the flexibility, strength and elasticity of articular (arthroidal) cartilage, the course of ossification, calcification and fibrillogenesis processes. Disintegration of collagen and its destruction is manifested by the increase of the amount of oxyproline, which is virtually contained in collagen fibres only.

121086

4_

- - -

-f

0 Healthy persons

I

1

II

--1

III

Activity degree

Fig. 5.1. Levels of oxyproline and glycosaminoglycanesin serum obtained from patients with rheumatoid arthritis and healthy persons, hatched - free oxyproline, black - protein associated oxyproline, white glycosaminoglycanes

192 For rheumatoid arthritis (cf. Fig. 5.1) and other rheumatological diseases, significant increases in the elastolytic activity and the contents of glycosaminoglycanes and oxyproline in blood were observed (Mikunis et al. 1985, Jensen et al. 1989, 1991). During the development of rheumatoid arthritis the proteolytic ferments play a significant role (due to their universal ability to impact the cartilage and other articular structures). Proteolysis leads to the formation of biologically active substances which influence the course of inflammatory processes, participate in the production of the rheumatoid factor and cause degradation of the connective tissue. The highest proteolytic activity is characteristic for elastase, cathepsin D and trypsin-like protamin. The level of proteolytic ferments in blood increases with the concentration of cz2-globulins, and does not depend on the level of seromucoids, C-reactive protein and ,/-globulins (Rudenko 1987). One of the factors of the pathogenesis of inflammatory rheumatic diseases is the change of the theological properties of blood, related to the immune disbalance (mainly with the enhanced synthesis of immunoglobulin and circulating immune complexes). Intermediate immune complexes, isolated for such patients suffering from the hyperviscous syndrome, are by their configuration cyclic dimers, and are polymers possessing a sedimentation constant of 6.6S-19S. It has to be noted that increased viscosity was found for every other patient with rheumatic disease (Gundmundsson et al. 1993), but the correlation analysis of the theological (plasma viscosity) and immunological parameters (contents of immunoglobulin-G and circulating immune complexes) reveals strong correlations only for systemic lupus erythematosus and rheumatoid arthritis (Loskutova et al. 1989). For patients suffering from rheumatoid arthritis, pronounced variations of visco-elastic properties of blood are caused by the presence of intermediate immune complexes possessing sedimentation constants of 10S18S, or their mixtures with 22S complexes in serum. For rheumatoid arthritis, the extensive presence of immune complexes with a sedimentation constant of 22S is characteristic determining the viscosity of blood. It was reported by Jara et al. (1989) that the hyperviscous syndrome for rheumatoid arthritis and systemic lupus erythematosus arises in connection with the presence of intermediate circulating immune complexes which contain antibodies to DNA and the

rheumatoid

factor,

aggregated

immunoglobulin-G

and

small

amounts

of

immunoglobulin-A and immunoglobulin-M. The increased viscosity of blood is determined by the molecular composition and the configuration of proteins (Balabanova et al. 1990, Harreby et al. 1987, Silberman et al. 1986).

193 A direct relation was found between high viscosity and the inflammatory activity of rheumatoid arthritis. A more pronounced increase in the parameters of plasma viscosity was found for patients with systemic vasculitis and digital arteritis. For such cases the most significant decrease of the flow properties of the blood, and therefore, the existence of most pronounced haemorheological variations in the vessels of various diameter are observed. A phenomenon common for rheumatic diseases is the increase in the contents of degradation products of fibrin and fibrinogen in the blood. These products, which are various substances formed during the fermentative hydrolysis of fibrinogen and fibrin, can exist as high molecular compounds or molecular fragments of monomeric fibrin or fibrinogen. They can make bonds with fibronectin and can be found in the cryoprecipitates prepared from serum (Schepotin et al. 1989). Up to 25 kinds of dysfibrinogenemia are known and develop as the result of improper polymerisation processes. (Dang et al. 1989). An increase in the concentration of serum fibrinogen is found when the total and euglobulin fibrinolitic activity of the blood and plasminogen activators are decreased, and the fibrin stabilising factor increased (Yerov 1986, Canesi et al 1980). For patients suffering from rheumatoid arthritis, a significant increase in the contents of fibronectin-fibrin complexes in serum is found. There is an inverse correlation between the amount of plasma fibronectin and circulating fibronectin complexes, and a direct correlation between these complexes and the concentrations of sialic acid and fibrinogen (Vasil'ev et al. 1994). The fibronectin in blood serum was found only for patients characterised by a moderate or high activity of the inflammation process. The quantitative and qualitative variations of proteins in blood, quite naturally, produce their effect on the state of surface tension in rheumatic diseases. The total number of patients with rheumatic disease was 322 of which 33 had rheumatism, 45 systemic lupus erythematosus, 20 sclerodermia systemica, 22 heamorrhagic vasculites, 43 rheumatoid arthritis, 12 Bechterew's disease, 29 Reiter's disease, 23 psoriatic artropathica, 46 gout, 49 osteoarthrosis. The shifts of surface tensiograms observed for patients with systemic lupus erythematosus and hemorrhagic vasculitis were already discussed in the previous chapter. It is seen from Table 5.1 and Figs. 5.2 and 5.3 that the peculiarities in the dynamic surface tensions and the slope of the curves are observed for all rheumatologic diseases, with particular features for each disease. This fact is very important from a differential diagnosis point of view.

194

80

6o I 40=~'

20-

9~

0

o

-20 -

--UI

/:

I

BD

RD

II

/

-40 -60 -80 R

SLE

SS

HV

RA

PA

G

OA

Fig. 5.2. Variation of ~.-values in serum (black) and urine (white) obtained from patients with rheumatic diseases. The changes are given in % compared to corresponding healthy controls. R - rheumatism, S L E systemic lupus erythematosus, SS - sclerodermia systemica, HV - heamorrhagic vasculites, RA rheumatoid arthritis, BD-Bechterew's disease, RD-Reiter's disease, PA - psoriatic artropathy, G gout, OA - osteoarthrosis; black - blood serum, white - urine Table 5.1. Differential diagnostic indicators of surface tension variation of biological liquids for rheumatic diseases.

Diseases

Surface tension parameter Sertma 0"1

0"2

Urine

0"3

~

0"1

0"2

G3

4-

Rheumatism Systemic lupus erythematosus

+

Sclerodermia systematica

+

Haemorrhagic vasculitis

+

R h e u m a t o i d arthritis

+

44-

4-

B e c h t e r e w ' s disease Reiter' s disease

+

Psoriatic arthropathy Gout Osteoarthrosis "+" - statistically significant increase of parameter compared to normal; "-" - statistically significant decrease of parameter compared to normal.

195

serHm

-1 -2

-

R

SLE

SS

HV

RA

BD

RD

PA

G

OA

b) urine 1412lOm-,,-,i

8-

= 6:~ 4

4

~[--] -2

-

-4

-

R

SLE

SS

HV

RA

BD

RD

PA

G

OA

Fig. 5.3. Changes of surface tension parameters measured in biological liquids obtained from patients with rheumatic diseases. Changes are given in % compared to corresponding healthy controls. R- rheumatism, SLE - systemic lupus erythematosus, SS - sclerodermia systematica, HV - haemorrhagic vasculitis, RA - rheumatoid arthritis, BD-Bechterew's disease, R D - Reiter's disease, PA - psoriatic arthropathy, G - gout, OA - osteoarthrosis; hatched - gl, black - g2, white - ~3

5.2. Systemic lupus erythematosus W e studied 45 patients with systemic lupus erythematosus and compared dynamic tensiograms o f serum and urine with data obtained for normal patients. Systemic lupus erythematosus is

196 characterised by increased surface tensions of serum in the short time range, and by a decrease of o2 and ~ for urine. These deviations which exceed the range M + 3m as determined for healthy persons, were detected in 62.7%, 57.3% and 71.6% of all cases, respectively. It should be noted that differences in the variations of dynamic surface tensiograms exist with respect to sex (cf. Fig. 5.4). Senma

40

Urine

30 -I 20 .,o

10

-10 -20 -30 ol

02

o3

~,

ol

o2

03

k

Fig. 5.4. Changes in surface tension parameters measured in biological liquids obtained from male patients (black columns) and female patients (white columns) with systemic lupus erythematosus. Changes are given in % compared to corresponding healthy controls.

For example, for male patients the increase in o~ is accompanied by an increase in o2 and decrease in ~, of serum, while for females a significant increase in ~ is observed but the other surface tension parameters of serum remain unchanged. For males an increase in the equilibrium surface tension for urine was observed. On the contrary, for female patients suffering from systemic lupus erythematosus, a decrease of o2 and ~, for urine was found. Thus, similarly to healthy persons, certain specifications of dynamic surface tension parameters with respect to sex were observed. However, these differences for patients with systemic lupus erythematosus are quite non-similar to those characteristics for healthy persons. We believe that these ambiguous inconsistencies in surface tension parameters of biological liquids with respect to patients' sex can be explained by a number of specific features characteristic to the clinical progress of the disease. First, the stage of the disease was different in male and female. All screened males had acute or sub-acute forms of systemic lupus

197 erythematosus, while 54% of all females had the chronic form. Secondly, accompanying diseases were different in male and female. In the male group a statistically reliable prevalence of myositis, erythema of face, pneumonitis, hepatomegalia (with a specificity of 74%, 60%, 74% and 74%, respectively) was detected, while for females capillaritis of fingers, Raynaud syndrome, endocarditis, pericarditis, and cerebral vasculitis (with a the specificity of 100%, 100%, 63%, 100%, 100%) were found. In addition, the specificity of the development of the nephrotic syndrome for females was 62% (for males was 38%). It should be stressed that the deterioration of kidney function was 49% in females and only 14% in males. And finally, all screened male patients were 20 to 25 years old, while 10% of females were younger, and 15% older. No differences were found between sex groups in regard of leukopenia, anaemia, thrombocytopenia, hyper-7-globulinemia, occurrence of lupus cells in blood, anti-nuclear factor and antibodies to native DNA. Thus, the specific features and stage of the disease can be responsible for differences in the variations of dynamic surface tensiometric parameters for patients of opposite sex. Correlations between particular parameters of the surface tension of serum and urine for systemic lupus erythematosus are much lower than for healthy persons. For healthy subjects (cf. Fig. 3.1) high correlations exist between ~1 and

0"2,

and between

13"2 and

~3 values for both

serum and urine. Almost no relation exists between surface tension values and the slope of the tensiographic curve L for serum taken from healthy persons. At the same time, L for serum of patients with systemic lupus erythematosus exhibit negative correlation with ~ ,

CY2

and cy3

values (correlation coefficients r =-0.51, -0.50 and -0.56, respectively), while L values for urine correlate inversely with the equilibrium surface tension (r =-0.69). For systemic lupus erythematosus, virtually no interrelations were observed between L and the surface tension values. This behaviour is also non-similar to that characteristic for healthy persons, for which significant correlations were found (cf. Table 3,4). The tensiogram slope of serum and urine is the parameter, which is mostly affected by the character of the development of systemic lupus erythematosus. For the acute form of this disease, the decrease in L for all biological liquids is common. This usually takes place in combination with the increase in surface tension of urine in the short time range. In most cases the sub-acute systemic lupus erythematosus is accompanied by high values of the cyz-values for serum.

198 As the amount of surface active and surface inactive compounds in biological liquids depends on the activity of systemic lupus erythematosus, one can expect that variations in the dynamic surface tension parameters exist for patients suffering from a disease in its mild, moderate and high activity forms. In fact, the high activity form is characterised by a reliable increase in the surface tension of serum and a decrease of 0"2 and 0-3 for urine. The moderate and mild forms of the disease are characterised by decreased K values for urine (26% and 40%, respectively). The dependence of dynamic surface tensions of biological liquids on the clinical symptoms of the disease in patients with systemic lupus erythematosus are of special interest, as our results indicate. It was shown that myocarditis, endocarditis, pneumonitis, hepatomegalia and lymphadenopathy do not affect the surface tensiograms of serum. The development of capillaritis and myositis, however, is accompanied by a significant increase of 0-I for serum. In patients with Raynaud syndrome accompanying SLE 0-2 and 0-3in serum was decreased. In patients with accompanying serositis these parameters increase, and for capillaritis, erythema and Raynaud syndrome an increase of ~, takes place. Low values of L can correspond to the presence of cerebral vasculitis. Alopecia correlates with the total activity of systemic lupus erythematosus and is characterised by increased values of 0-1 and 0-2, and relatively low values of ~, for serum. These results are summarised in Fig. 5.5. It should be noted that if no kidney manifestation occurs, then an increase of equilibrium surface tension of serum and a corresponding decrease of ~, is usually observed, while the development of glomerulonephritis generally results in quite opposite changes of surface tensiograms. Obviously, this fact is especially important, because the prognosis of SLE is usually determined by kidney manifestation, the presence of lupus glomerulonephritis. Urine sampled from patients with lupus glomerulonephritis had decreased 0-2 value and significant decreased K values . In fact, the value of urine tensiographic L value is indicates the development of kidney manifestation of SLE, because the absence of nephritis is characterised by a very high value for ~, in urine. The fibrinolytic activity of urine from patients with lupus glomerulonephritis varies from zero to very high values. The activity of plasminogen activator in urine is high, while the level of tissue type antiplasminogen is normal. Kidney vascular endothelium possesses a rather high potential of tissue plasminogen activator, which can be regarded as a defence-adaptive reaction of kidney fibrinolytic system with respect to the local action of thrombogenic factors (Polyantseva et al. 1995, Shibata et al. 1993, Sakakibara et al. 1987).

199 In rapidly progressing lupus glomerulonephritis, the average value of plasminogen activator in

urine is lower than in healthy persons 9 Zero activator activity of urine is often observed. We believe that the variations in the fibrinolytic activity of urine can affect the dynamic surface tensiometric parameters of this biological liquid in patients with kidney pathology 9 a) serum 1086

..q

il

10

11

4

m

2 o ~

r

-2 -4

-

-6

-

-8

-

-10 1

2

3

4

5

6

7

8

9

12

13

14

15

b) urine 8 6 4 2

J

-4 -6 -8

-

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Fig. 5.5. Variations of dynamic surface tension parameters of biological liquids obtained from patients with systemic lupus erythematosus with various clinical symptoms of the disease. The various symptoms are: 1- arthrous syndrome; 2-muscular syndrome; 3-alopecia; 4-affection of the skin; 5-finger capillaritis; 6 - Raynaud syndrome; 7 - lymphadenopathy; 8 - nephritis; 9 - myocarditis; 10 - endocarditis; 11 - serosites; 12 - pneumonitis; 13 - hepatomegalia; 14 - splenomegaly; 15 - cerebral vasculitis. We compared ol, (hatched), (~2 (black) and 03 (white) mean-values measured for the group of SLE -patients with one of the symptom to the remaining SLE-patients without this symptom.

200 An increase of 0"1 in urine was detected in patients with capillaritis, cerebral vasculitis, alopecia, pneumonitis and splenomegaly. Low 0"2 values were observed when no skin manifestation, muscular syndrome, alopecia, myocarditis or pneumonitis took place, while low 0"3 values

were characteristic for patients showing no Raynaud syndrome, cerebral vasculitis,

myocarditis or pneumonitis. When the nephrotic syndrome occurs, we found the following characteristics for serum and urine tensiograms. Tensiograms of serum had a decreased 0"2 and 0"3 values. Tensiograms of urine had decreased 0.~, 02 and ~, values. If kidney function deteriorates we found decreased values of 0.1, 0.2 and 0.3 in serum and decreased values of 0.2, 0.3 and ~ in urine (cf. Fig. 5.6). Serum

5~

Urine

= 15 l ~9 -20 -25 q -30 t -35

L

-40 0.1

0.2

0.3

~

cl

0.2

0.3

Fig. 5.6. Variations of dynamic surface tension parameters of serum and urine for patients suffering from lupus glomerulonephritis with non-deteriorated (black) and deteriorated (white) kidney function (+% deviations from parameters for healthy persons). These results are of major interest for the prognosis of the disease and the control of the efficiency of therapy. While for kidney manifestation of SLE changes of the dynamic tensiograms of serum were observed, no such changes were found for fever and skin manifestation of SLE., However, fever and skin manifestation are the clinical symptoms that often reflect the progress of systemic lupus erythematosus and always correlate with the activity of this disease. It should be noted in this regard that interleukin-1 plays a certain role in the regulation of the pathologic processes for systemic lupus erythematosus. Interleukin-1

201 exists in form of imerleukin-1 ot and imerleukin-ll3 (main form). This cytokine is produced in various cells of the organism, and determines the extent of an inflammatory response (Gabay et al. 1997). The amount of interleukin-1 [3 in the serum of patients suffering from active systemic lupus erythematosus increased, and correlates with the presence of fever and skin manifestation (Ketlinsky et al. 1993). Probably the absence of differences in dynamic surface tensions of serum for such patients can be explained to some extent by similar levels of interleukin-113. The detection of C-reactive protein in serum can be primarily regarded as a sign of the acute phase response (Lacki et al. 1997, Hilliquin 1995). This compound is the classical acute-phase protein whose synthesis takes place in response to fever and tissue damage. By its structure, this protein belongs to the pentraxine family of proteins, and consists of 5 identical nonglycosilated polypeptide subunits with a molecular mass of ca. 23 kDa, which form a cyclic disk-like pentameric structure via non-covalent bonding (Nasonov et al. 1997a, Kilpatrick et al. 1991). Under normal conditions, only trace quantities of C-reactive protein are detected in serum, and for 90% of healthy persons its concentration does not exceed 3 mg/ktl (Calvin et al. 1988). However, in case of inflammation, its concentration can increase 100 and more times, doubling each 6 hours after activation of its synthesis. The increase in the concentration of C-reactive protein can be observed even 4-6 hours after tissue damage, with a maximum content reached within 24-72 hours. The synthesis and secretion of C-reactive protein is controlled by interleukin-1 and interleukin-6 anti-inflammatory cytoxines (Hirano et al. 1990, Horwitz & Jacob 1994). In systemic lupus erythematosus and other rheumatic diseases, a close correlation was observed between the concentration of C-reactive protein and the activity of the inflammatory processes (Medvedev et al. 1996). In addition, there is a relation between the hyperproduction of C-reactive protein and anti-phospholipid antibodies (Klukvina et al. 1997). It is shown in Table 5.2 that the

serum concentration of C-reactive protein for screened

patients correlates with the surface tension parameters of serum at short and medium lifetimes. Similar correlations were found also with fibronectin. While the synthesis of C-reactive protein takes place in the liver, no significant differences in dynamic surface tensions between patients with or without hepatomegalia were found. The closest correlation links between surface tension parameters of serum were shown to exist with respect to the concentration of immunoglobulin. It should be noted that a similar correlation was observed between surface tensiographic parameters and the level of binding

202 ability of immunoglobulins with amino acids (proline, oxyproline, arginine, glutaminic acid, lysine). Some comments should be made in this regard. Because no other protein than collagen contains proline and oxyproline (these substances are specific labels of collagen and its degradation products), the increased binding capability of immunoglobulin to these amino acids and increased concentrations of these amino acids in blood specifically reflect the extent and intensity of destructive processes in the connective tissue. Table 5.2. Correlation coefficients between surface tension parameters measured in serum obtained from patients with systemic lupus erythematosus and serum components

Blood component

tensiometric parameters t~2

Total protein

,

Albumin oil-globulin fraction

t~3

1" $

$

1"

1'

.

a2-globulin fraction fl-globulin fraction y-globulin fraction 132-micr~176

~

$ i

1"1"1"

$

Immunoglobulin-G

~$

$

$

tt

Immunoglobulin-A

$$$ $$$

$$$ $

$$ $

ttt

$

tt

t

$$

Immtmoglobulin-M Circulating immune complexes C-reactive protein Fibrinot~en

tt $

Fibronectin

tl'

tl"

t

Urea Creatinine Uric acid

tt

Oxypurinol 1" positive correlation; $ negative correlation; empty - no correlation r<0.3" one symbol - r = 0.3 to 0.5" two symbols - r = 0.5-0.7; three symbols - r > 0.7

Comparing the parameters which reflect an increase in the concentration of serum immunoglobulins G, A and M with increased binding ability to amino acids we have shown

203 that in patients with grave manifestation of systemic lupus erythematosus, the amount of these immunoglobulins had increased approximately 2.3, 2.1 and 1.3 times, respectively, as compared to normal concentrations. The corresponding increase in their binding ability with amino acids was increased 3.4-5.7, 2.7-4.7 and 2.4-2.6 times (Zamulko et al. 1991). In patients with moderate severity of systemic lupus erythematosus no changes in the concentrations of these immunoglobulins were found as compared with mild SLE. The binding ability of immunoglobulin with amino acids increases with severity of the disease. The most significant increase was found for the binding ability of immunoglobulin-G with arginine (13.5 times), glutaminic acid (7.8 times) and lysine (5.3 times) (Amosova et al. 1994). The data presented above provide an argument that the dynamic surface tensions of serum are necessary integral parameters for the estimation of the activity of the pathologic processes of systemic lupus erythematosus. The dependence of equilibrium surface tension of serum on the glucose content should be mentioned, and is demonstrated in Table 5.3. This relation is important in patients suffering from systemic lupus erythematosus with latent diabetes mellitus, especially for its version induced by prolonged application of glucocorticoid hormones. Table 5.3 also illustrates the correlations between surface tension parameters of serum and the concentration of particular lipids. It should be mentioned that the effect of studied lipid surfactants on the parameters of dynamic surface tensiograms for systemic lupus erythematosus is less pronounced than for other diseases; however, in the pathogenesis of systemic lupus erythematosus deteriorations in the lipid metabolism are always accompanied by a hyperproduction of antibodies directed against phospholipids (a-phospholipids, Popkova et al. 1998, Lipp et al. 1998, Marquerie et al. 1997), which possess a pronounced surface activity, a-phospholipids represent a heterogeneous population of autoantibodies which react with antigenic determinants formed during the interaction between phospholipids and phospholipid-binding serum proteins (Nasonov et al. 1995). The production of t~ -phospholipids is associated with the development of the antiphospholipid syndrome, whose main signs are the venous and arterial thromboses of the vessels of any diameter and localisation. The diagnostics of the anti-phospholipid syndrome is based upon the determination of antibodies to negatively charged membranous phospholipids

204 bound to proteinic phospholipids (132-glycoprotein-I, annexin-V, protothrombin etc.), and the detection of the effects of 'lupus anticoagulant'. One of the possible mediators of vascular lesion for systemic lupus erythematosus is the lipoprotein (a) (Borba et al. 1994), whose structure resembles that of low density lipoproteins, but involves apolipoprotein (a) [apo (a)] bound to apo-B-100 by disulphide bonds. Apolipoprotein (a) possesses a structural homology with plasminogen. In vitro apolipoprotein (a) inhibits the activation of plasminogen, competes with it and tissue plasminogen activators for the binding with fibrin, suppresses the absorption and destruction of fibrinogen by mononuclear cells (Ezratty et al. 1993), modulates the fibrinolysis and plasminogen activator inhibition by vascular epithelium cells (Ettinger et al. 1991).

Table 5.3 Correlation coefficients between surface tension parameters measured in serum obtained from patients with systemic lupus erythematosus and serum components Serum component

Surface tension parameter (yl

t~2

(Y3 1'1'

Glucose Total cholesterol a-cholesterol Triglycerides Phospholipids

$$

High density lipoprotein fraction Low density lipoprotein fraction Very low density lipoprotein fraction ....

1"positive correlation; $ negative correlation; empty - no correlation r<0.3; one symbol - r = 0.3 to 0.5; two symbols - r - 0.5-0.7; three symbols - r > 0.7

$

205 For systemic lupus erythematosus, an increase of lipoprotein (a) concentration in blood was observed, which can be related to a hyperproduction of t~ -phospholipids. A correlation was detected between the increase in concentrations of lipoprotein (a) and hypoalbuminemia, while a treatment with glucocorticoid hormones results in a decreased concentration of lipoprotein (a) (Aoki & Kawai, 1993). For the nephrotic syndrome, a negative correlation was detected between the concentrations of lipoprotein (a) and albumin, and a direct relationship of lipoprotein (a) on the total serum cholesterol and the extent of proteinuria (Fujita et al. 1992). For patients with systemic lupus erythematosus with a-phospholipids detected in blood, an 'atherogeneous' profile of lipids was observed, resulting in a decrease of cholesterol, high density lipoproteins and apo-A-1. Momot et al. (1997) have shown that a consistent and significant decrease in the coagulation activity of phospholipid membranes takes place for 'lupus anti-coagulant'. The application of discrete plasmapheresis leads to an increase in the activity of phospholipid membranes, explained by the removal of 'membrane phospholipids - - a-phospholipid' complexes from the blood of such patients. The instability of the state of erythrocyte membranes, caused by an increased number of lysoforms of phospholipids and a change in the ratio of phospholipids fractions, leads to a 'sponginess' of the membranes, increases their penetrability and, finally, leads to their disintegration. In addition, the destabilisation of the cell membranes is related to a stimulation of the peroxide oxidation of lipids, whose substrates are the unsaturated fatty acids as constituents of phospholipids. The variation in the level and qualitative composition of blood phospholipids can affect its surface tension parameters. For patients with an isolated uric syndrome an increase in the phosphatidylcholine level in plasma was observed, and when arterial hypertension was also present an increase of sphingomyelin was also detected. During the acute phase of the disease, an increase in the total concentrations of phospholipids and all their fractions in blood appeared, with maximum variations found for patients with nephrotic syndrome (Ryabov et al. 1995). Blood phospholipids usually exist in the high density lipoprotein fraction. The increase in the levels of lysophosphatidylcholine and phosphatidylethanolamine indicates the action of phospholipase Aa and their peroxidation by free radicals on the high density lipoprotein fraction. The increase in the contents of sphingomyelin, which possesses enhanced stability

206 with respect to peroxide oxidation of lipids, can be regarded as a defence reaction of the organism. For systemic lupus erythematosus, ot-phospholipids do not react with anionic phospholipids themselves, but with complex epitopes formed during the interaction between phospholipids and 132-glycoprotein-I. This protein possesses a natural anti-coagulant activity and acts as co-factor of the interaction between (z-phospholipids and anionic phospholipids. The 132-glycoprotein-I is structurally homologous to apolipoprotein H. The surface structure formed by the low density lipoprotein fraction, which comprises apolipoprotein-B and the monolayer consisting of phospholipids and cholesterol, resembles the epitope formed during the interaction between phospholipids and 132-glycoprotein-I to some extent (Nasonov et al. 1997b). Thus changes of the lipid composition in blood of patients suffering from systemic lupus erythematosus is typical and is especially related to lipids of pronounced surface activity. The contents of phospholipids clearly determine the surface tension parameters. A weaker correlation exists between surface tensiometric parameters and particular lipids (lipoproteins) which can be possibly explained by the 'evening-out' of other substances, for example electrolytes, the concentration of which is also subjected to changes during the progress of systemic lupus erythematosus. It is seen from Table 5.4 that the surface tension parameters also depend on the contents of potassium, calcium, phosphorus and magnesium. This fact can possibly be especially significant for patients taking glucocorticoid hormones, and for chronic renal insufficiency related to lupus glomerulonephritis. Table 5.4 Correlation coefficients between surface tension parameters measured in serum obtained from patients with systemic lupus erythematosus and serum electrolytes.

Electrolyte CY2

CY3

?t

tt

?tt

$$$

??

t

??

$

CYl

Sodium Potassium

$ .....

Total calcium

-

_

Ionized calcium _

Chlorine Phosphorus Magnesium

.

$ "

-

t

t

-

tt

tt

"

t?

$$

t?

,1,$ J

1' positive correlation; $ negative correlation; empty - no correlation r<0.3; one symbol - r = 0.3 to 0.5" two symbols - r = 0.5-0.7" three symbols - r > 0.7

207 Table 5.5 Correlation coefficients between surface tension parameters measured in urine obtained from patients with systemic lupus erythematosusand urine components Urine component O1

O2

O3

L

$

$ $$

1"

Total protein 132-microglobulin

$$

Fibronectin

$$$

$

Urea

?

?

Creatinine

1"?

?

Oxypurinol

?

Uric acid

$$

$

Relative density 1" - positive correlation; ,1,- negative correlation; empty- no correlation r<0.3; one symbol - r 0.3 to 0.5; two symbols - r = 0.5-0.7; three symbols - r > 0.7

To some extent the factors which determine the dynamic surface tension of urine for patients with lupus glomerulonephritis have been discussed earlier. Table 5.5 summarises the correlations, which exist between surface tensiometric parameters of urine and the level of proteins and non-protein nitrous products. The concentration of these compounds affects primarily the values of surface tension in the short time range. Equilibrium surface tensions depend mostly on the concentration of 132-microglobulin. It is interesting that the relative density of urine, closely related to the amount of urea, produces no effect on the behaviour of dynamic surface tensiograms. It should be recalled that the surface tension parameters for serum depend significantly on the concentration of immunoglobulin-A. In turn, the amount of serum immunoglobulin-A for lupus glomerulonephritis correlates with the level of haematuria (Shvetsov & Kozlovskaya, 1996). It should be mentioned in this connection that the extent of haematuria does not affect the state of dynamic surface tension for these patients. 5.3 Rheumatism

Rheumatism is characterised by increased dynamic surface tensions for serum (Fig. 5.7) and decreased values of o2 and L for urine. A feature of the disease which is characteristic for

208 women is an increase of k for serum combined with a decrease of ol for urine. It should be stressed that in the male group of patients no change in the dynamic surface tension parameters for urine was observed. The most pronounced increase of o~ for serum is typical for patients from the oldest age groups, and the lowest equilibrium surface tensions of urine were found for young patients. In general, the ol values of serum exceeding M + 3m were detected for 72%, and the o3 values of urine lower than M - 3m for 55% of screened patients. For rheumatism, the dynamic surface tension parameters of urine exhibit weak correlations with those parameters for serum. Only relations exist between equilibrium surface tension of urine and surface tension parameters for serum at medium and large times. The most significant deviations of surface tensiometric parameters of biological liquids as compared with corresponding values for healthy individuals were detected for the duration of the disease of 10-20 years. The increase in the activity of rheumatism results in an increase of cri and decrease of cr3 of serum and lowered 02 and 03 values for urine (cf. Fig. 5.8). The activity of the pathologic process affects also the ~. values for the two liquids (Fig. 5.9): the most significant increase of ~, for serum is observed for the high activity degree (Am), while for urine exhibits a continuous decrease when passing from AI to

AIIl.

75

7O

65 ~

60

. . . . -2

t -1

-

J

I

lg(tef) [s] 0

1

....

Fig. 5.7. Examples for serum tensiograms obtained from patients with rheumatism, with rheumatic carditis, 1 st activity degree; thin line - female, age 50; thick line- female, age 50, dotted lines correspond to average values for healthy females of corresponding age.

209 For patients with r h e u m a t i s m and aortal valve defects, the

(3" 1

value for serum significantly

exceeds the c o r r e s p o n d i n g values for mitral defects. A similar d e p e n d e n c e was found also for 13'3 o f urine.

Serum

Urine

80-

706o 50 40

I

4

0

I

I

II

III

0

I

II

III

AcfMty degree

Fig. 5.8. Dynamic surface tension (or) of serum and urine obtained from patients with rheumatism of various degrees of activity indicated as 0, I, II, III. Grey - Crl, black - or2, white - c3.

Serum

Urine

302010-

0

I

I

J

I

1

-10-20 -30 -40 -50 0

I

II

III

0

I

II

III

AcfMty degree Fig. 5.9. Changes in ~ - values measured in serum and urine obtained from patients with rheumatism of various degrees of activity indicated as 0, I, II, III. Changes are given in % compared to corresponding healthy controls.

210 The results obtained for patients with cardial arrhythmia, as compared with data for persons showing not such arrhythmias, have been quite unexpected. If ventricular or supraventricular extrasystoles occur, then the observed values of cry, or2 and or3 for serum are significantly lower than corresponding values for other patients, while ~, for serum is increased. If auricular fibrillation or flutter takes place, then the equilibrium surface tension sharply decreases. Therefore, decreased surface tensions at long times (~3) can be used as a prognostic criterion of the deterioration of the rhythm for patients with rheumatism and heart manifestations of the disease. The surface tension data at various stages of circulatory insufficiency are of special interest with respect to the diagnostics of the disease and monitoring of the treatment (Fig. 5.10). Increases of surface tensiographic parameters have been observed from the 1st stage to stage IIA, and subsequently a decrease at liB and, especially, at stage IIId. At this point, we cannot present any explanation of this fact; however, there are variations in the composition of surfactants and surface inactive compounds in the blood of patients with rheumatic heart disease with various degrees of circulatory insufficiency. Senma

Urine

7570~' 65t~ 6 0 55-

1

50

. 0

. I

. IIA

.

.

.

.

.

.

IIB III 0 I IIA Stage of cardiac insut~iency

IIB

III

Fig. 5.10. Dynamic surface tension (a) of serum and urine obtained from patients with rheumatism of various degrees of circulatory insufficiency indicated as 0, I, llA, liB, III. Grey - ~, black - a2, white - ~3. It should be stressed that increased surface tension values for serum and urine were observed during the treatment of patients suffering from heart insufficiency using various combinations

211 of angiotensin-converting ferment, calcium antagonists, selective 13-adrenoblockers, nitrates, heart glycosides and diuretics (cf. Fig. 5.11). Correlation links have been analysed between the dynamic surface tension of serum and urine for

patients

with

rheumatic

heart

diseases

and

echocardiographic

and

Doppler

echocardiographic parameters (cf. Table 5.6). Surface tensiograms are significantly affected by the size of the right ventricle, and, therefore, by the pressure in the lung circulation. The aortic pressure (in fact, in the systemic circulation) correlates with surface tension parameters of serum. Surface tension values in the short surface lifetime range correlate with the final systolic size of the left ventricle and interventricular septum thickness, while the equilibrium surface tension correlates with the decrease in the left ventricle size. Final systolic volume and size of the left ventricle correlate with ~. of urine. These data can possibly provide an explanation of some variations of surface tensiograms for patients with mitral and aortal valve diseases. It can be argued that the haemodynamic derangements result in a disproportion of surfactants and the presence of new surface active and surface inactive substances in the blood. Serum

75 q

Urine

70 65 t~ 60 55 50

I

I

~1

c~2

c~3

I

I

~1

~2

~3

Fig. 5.11. Dynamic surface tension characteristics of serum and urine obtained from patients with rheumatism during treatment (black - before, white - after), and for healthy persons (grey). Surface tension parameters for serum at medium and long times exhibit inverse correlations with the level of average arterial blood pressure (r =-0.33 and r = -0.46, respectively), and total peripheral vascular resistance (r =-0.36 and r=-0.45, respectively). One can presume that

212 variations in the arterial blood pressure and total peripheral vascular resistance, which are capable of affecting the renal bloodstream and the intensity of glomerular filtration, could also affect to some extent the dynamic surface tension of urine. Indeed, it is seen from Fig. 5.12 that close negative correlations exist between

surface tensiographic parameters and

the

haemodynamic parameters indicated above, with correlation coefficients of about -0.7. Table 5.6 Correlation coefficients between surface tension parameters measured in serum and urine obtained from patients with rheumatismand various echocardiographicparameters. Echocardiographic parameter

Biological liquid serum (~1

i Urine o2

(~3

~

C~l

!(~2

t~3 t

Left ventricle final diastolic size tt

Left ventricle final systolic size

t

$

tt

Right ventricle final diastolic size

t

Right ventricle final systolic size

tt $$

Interventricular septum thickness

t

$

t

tt

$

t

t

t

t

Left ventricle posterior wall thickness Left

ventricle

anterior/posterior

size

t

shortening fraction Stroke volume Ejection fraction

$

t tt

Right ventricle size t

tt

Aortic valve opening magnitude

$

$

Aortic pressure

$

$$

Left auricle size

$

t

t

t

t

$

t

ttt

ttt

$$

$

$

Aorta diameter

$$

tt

$

Mitral valve pressure gradient Aortic bloodstream rate Mitral valve bloodstream rate

t

$

Pulmonary artery bloodstream rate 1' - positive correlation; $ - negative correlation; empty - no correlation r<0.3; one symbol - r = 0.3 to 0.5" two symbols - r = 0.5 to 0.7; three symbols - r > 0.7

213

Urine

Serum

-0.1

-

-0.2

r0 -0.3 r,..) =

r

-0.4

-

-0.5 -

o -0.6 r,.) -0.7 -0.8

-

o'1

o'2

o'3

cyl

r

~3

Fig. 5.12. Correlation coefficients between parameters of dynamic surface tension and the level of average arterial blood pressure (black) and total peripheral vascular resistance (white). Surface tension parameters were measured in serum and urine obtained from patients with rheumatism.

With reference to certain specific features of dynamic surface tensiograms for patients exhibiting a different activity of the rheumatic process, we have compared the surface tension parameters of serum with some characteristic values reflecting the activity degree of the disease.

It was

found

that high

correlations exist between

(5" 1

and

the

level of

antistreptolysine-O ( r - 0 . 7 5 ) , and between O'2 and seromucoid, sialic and diphenyl amine (r=0.49, 0.49 and 0.48, respectively). Correlations with these and other parameters are summarised in Table 5.7. It is seen that for sermn the

O" 1

value directly correlates with the concentration of

immunoglobulin, while for (Y2 this correlation is inverse. The equilibrium surface tension essentially depends on circulating immune complexes. In general, it can be concluded that the state of dynamical surface tension for patients with rheumatism depends on the concentration of many proteins, lipids and other substances in blood. For example, the total calcium concentration significantly affects

(5"1,

while (5"2 depends on the concentration of ionised

calcium, and (Y3 on the chlorine concentration.

214 Table 5.7. Correlation coefficients b e t w e e n surface tension p a r a m e t e r s m e a s u r e d in s e r u m obtained from patients with r h e u m a t i s m and s e r u m c o m p o n e n t s

Blood component o'1

Total protein Albumin c~-globulin fraction ot2-globulin fraction

(Y2

~3

$

$

1"1"

$$ $

13-globulin fraction ]~-globulin fraction 132-microglobulin Immunoglobulin-G Immunoglobulin-A Immunoglobulin-M C-reactive protein Circulating immune complexes Fibrinogen Fibronectin Urea Creatinine Uric acid Oxypurinol Total cholesterol et-cholesterol Triglycerides Phospholipids High density lipoprotein fraction Low density lipoprotein fraction Very low density lipoprotein fraction Potassium Sodium Total calcium Ionised calcium , Chlorine Glucose I

!

!

1'1"1' 1"1' 1'1"1'

$$$ $$$ $$

I

I

$ $ $$ $ 1"1"1'

!

t t1'1" l"t ttl"

1'1'1"

1"1'1'

1"1'

1"

1'1" 1'1'

1"1' $$$ $ ,

$$

$ $$$ i$$ $,1,

1" - positive correlation; $ - n e g a t i v e correlation; e m p t y - no correlation r<0.3; one s y m b o l - r = 0.3 to 0.5" two symbols - r = 0.5 to 0.7; three symbols - r > 0.7

$$ $$$

1"1"1" 1"1"

215 5.4. Sclerodermia systematica Possibly the most significant surface tension variations in serum are characteristic of patients suffering from sclerodermia systematica (20 female patients were screened). We have found that the parameters of surface tension were increased, see Fig. 5.13.

80

--

75 E

70

65

_

60

~ .~ .~176

1

-2

-1

.~ .~

I

I

0

1

lg(tef) [s] Fig. 5.13. Examples of serum tensiograms for patients with sclerodermia systematica. Thin line - female, age 37, 2"d degrees of activity, thick line - female, age 41, 3rd degrees of activity; dotted curves correspond to average values for healthy females of corresponding age For patients with sclerodermia systematica an increased excretion of glycosaminoglycanes (carbohydrate component of proteoglycans) was observed. These compounds are important constituents of the connective tissue. It was noted by Fusailov et al. (1984) that the more serious the illness is, the higher the contents of glycosaminoglycanes in serum. When fibrosis and sclerosis processes become more pronounced, the urinal concentration of orcine increases, while the carbazoluria level decreases. These processes take place due to the significant excretion of uronic acids (due to the prevalence of the polymers which contain iduronic acids, in particular, dermatan sulphates). Iduronic acids have an increased affinity to collagen, and therefore participate efficiently in the stabilisation of collagen fibres. In contrast to glycosaminoglycanes, the glycoproteins in blood (a2-globulins, fibrinogen, seromucoid, ceruloplasmin etc.) not always reflect the true activity of pathological processes for sclerodermia systematica.

216 It was shown that a direct correlation exists between 02 of serum and the concentration of immunoglobulins, [32-microglobulin and fibronectin, and between or1 of urine and the contents of glycosaminoglycanes. Possibly the enhanced excretion of proteoglycans affects the surface tensiograms in the short time range for sclerodermia systematica patients. In addition, the crl values of urine partly correlate with the contents of glycosaminoglycanes for patients with rheumatoid arthritis and osteoarthrosis. At the same time, the dynamic surface tensiometric parameters of urine can reflect morphological changes of the kidneys. Biopsies of kidneys show a vessel pathology, with preferential lesion of interlobular arteries and glomerular capillaries. In some cases pronounced changes of the microcirculatory bed are observed, accompanied by a destruction of endotheliocytes up to the "denudation" of glomerular capillary loops, the symptoms of functional activity of the endothelium and the increase of the thickness of basal membranes (Buchmann et al. 1974). One of the ultrastructure defects is the reticulation of the endotheliocyte cytoplasm, which correlates with renal functions (Anikina et al. 1986). The dynamic surface tension parameters of urine have been compared for patients who do not suffer from kidney lesion with those characteristics for sclerodermic glomerulonephritis. It was found that the tensiograms in the medium and large surface lifetime ranges are directly correlated with the presence of a kidney pathology for patients with sclerodermia systematica, while an inverse correlation exists with the total amount of protein, fibronectin and 132-microglobulin in urine. Glomerulonephritis is also accompanied by increased equilibrium surface tension and ~. values of serum. These features of surface tensions for patients suffering from sclerodermia systematica accompanied by kidney pathologies are of significant practical interest for a diagnosis of sclerodermic nephropathy and for monitoring the treatment of these patients. 5.5. Rheumatoid arthritis

If biologically active compounds (hormone, neurotransmitter, transport protein, ferment) act as an antigen during a rheumatic disease, a wide range of biochemical variations arise in the organism, caused by antibodies (Azarenok & Generalov 1990, Misaki et al. 1994, Corper et al. 1997, yon Mikecz et al. 1994). For patients with rheumatoid arthritis, antibodies for the hydrolysis of hyaluronic acid are produced. It is known that, after binding to antigen, the

217 antibodies change there conformation and form immune complexes. These complexes are capable of stimulating the superoxide dismutase activity. The presence of superoxide dismutase enhance inflammation and introduce a disbalance in the surfactant composition of serum and in the interstitium. For rheumatoid arthritis, the presence of various antibodies in serum is characteristic, along with an increase of circulating immune complexes that activates the complement system leading to concentration increases of C3, C3a, C3bi, C3dg components of the system (Taylor 1990). The proteins of the complement system, during its activation, acquire new surface active properties, which eventually leads to variations in surface tensions of serum. The 100 to 1000 times increase in the contents of amyloid acute phase SAA-protein is similar to C-reactive protein. Note that the concentration of other acute phase proteins (o~-antitrypsin, ttl-antichemotrypsin, fibrinogen, haptoglobin, oq-acidic glycoprotein) increase only by a factor of 2 to 4 (Bannikova 1987). The tensiographic parameters for serum in the short and medium time range for rheumatoid arthritis exceed significantly those characteristic of healthy persons. This behaviour of 0-~ (> M + 3m) was found for more than 3/4 of patients, and for 0-2 for almost 2/3 of the patients (cf. Table 5.8). It should be noted that for healthy persons, a strong dependence exists between 0-~ and 0-2 of serum and urine. In addition, 0-3 values of serum correlate with ~rl and 0-2, and the value of 0"3 of urine with 0-2 and ~,. For rheumatoid arthritis, the dependence of ~, on 0-~ and 0-2 for serum, and of ~, on 0-3 for urine becomes evident. The value of ~, for synovial fluid is in fact entirely determined by its values of 0-1 and 0-3 (cf. Fig. 5.14). All the parameters of dynamic surface tensiometry for synovial fluid exhibit direct correlations with surface tensions at t = 0.01 s and t ~ ~, and inverse correlations with L of serum, as one can see in Table 5.9. It was already noted above that the mean surface tension values of serum for patients suffering from rheumatoid arthritis exceed those found for healthy persons, while the values of ~, are lower. However, this is only the case for patients with severe rheumatoid arthritis. If the activity of the diseaseis low, a decrease of 0-1, 0-2 and 0-3 are observed, while the L value remains unchanged. It is worth noting that ~, decreases in line with an enhanced activity of

218 rheumatoid arthritis. This fact is quite significant for monitoring the efficiency of a specific treatment. There is only a weak dependence of dynamic surface tensiometric parameters on the stage of the disease, as demonstrated in Fig. 5.15 (Kazakov et al. 1996). Table 5.8. Differences between values for various surface tension parameters measured in serum and urine from rheumatoid arthritis patients and corresponding controls. Differences are given as the frequency (in %) of values for patients that lie above M + 3m or below M - 3m for controls.

Biological liquid

Serum

Urine

Parameter deviation, %

Surface tension parameter

>M+3m

<M-3m

0"1

76.7

18.6

0"2

62.8

14.0

0"3

67.4

9.3

7.0

53.5

0"1

41.9

16.3

0"2

51.6

27.9

0"3

53.5

37.2

9.3

74.4

Table 5.9. Correlation coefficients between surface tension parameters measured in biological liquids that were obtained from patients with rheumatoid arthritis.

Biological liquid

Urine

Synovial fluid

Surface

tension

Serum

parameter

0"1

0"2

0-3

0"1

+0.06

+0.12

+0.17

+0.03

0"2

-0.03

+0.16

+0.15

+0.17

0"3

+0.02

+0.29

+0.26

+0.29

+0.01

-0.22

-0.24

-0.20

0"1

+0.73

-0.25

+0.62

-0.70

0"2

+0.85

+0.02

+0.81

-0.88

0"3

+0.77

-0.18

+0.69

-0.73

+0.46

+0.59

+0.54

-0.61

219

a) serum 0.9

-

0.8 .~ 0.7 -:--:-..-:.:-

~ o

o

C

-:-:-:-:-:-:-.. ....... :-.... .... :..:...:.-. ". .:.'.:.' .: .2 2 " : ' : ..... ..-. :-...:-:.-. ". .:.2. .2. 2. " : 2 2 ~:..-.-...: ..:..-.-..: ~-.-.-.-.:-. -. .: .-.:.-.:.- : - - : - : .".:.' .:.' .: .' . ' 2 " : ' : -:-:-:-:-:-:-: ....... ..-...-.- ..-. .: .-.:.:.:.T. : ..-...-.-..: :-.......... -...... .... :..:........ ............ ........ -. .. . . . . ..-. ...........-. .. . . . . ..-.

0.6 0.50.4-

0.3 oo

:. :. .- .:.-.-.:. -. :. .- .f : :-.. .....

0.2-

"-.222222 ...... -.

:....-.......

0.1

iiiiiiiii ?:-55555:

I

I

o'1

o2

0"3

b) synovial fluid _

0.8 0.6 0.4 0~

o

0.20 -0.2 -0.4-

o

-0.6 -0.8 -1crl

or2

o3

Fig. 5.14. Correlations between various dynamic surface tension parameters of biological liquids obtained from patients with rheumatoid arthritis. Hatched - o 1, black -o2, white - or3, grey - X.

Of some interest are the data concerning the variations in the tensiograms of serum for patients with the seropositive version of the disease, when the rheumatoid factor in the serum leads to a sharp increase of the surface tension (cf. Figs. 5.16 and 5.17). One can presume therefore that the concentration of immunoglobulins, which contributes to the formation of the rheumatoid factor, can affect the surface tension of serum for rheumatoid arthritis.

220

Activity degree

Stage

10

~

I

JI

I

,,'.,, ~,.~

"~ -20 -25 t -30 t -35 -40 ~

I

II

III

I

II

III

IV

Fig. 5.15. Changes in surface tension parameters measured in serum obtained from patients with rheumatoid arthritis and various activity degrees indicated as I, II, III and various stages of the disease indicated as I, II, III, IV. Changes are given in % compared to corresponding healthy controls. Hatched - a~, black - a2, white - a3, grey - ~,.

25201510-

t_____a

5

..o

0 -~

;>

! !

-5 -10 -15 -20 -25 al

a2

a3

~,

Fig. 5.16. Changes in surface tension parameters measured in serum obtained from patients with rheumatoid arthritis for various serologic activities of the disease; black - seronegative, white - seropositive. Changes are given in % compared to corresponding healthy controls.

221

75

-............

_....

o. . . . . . . . . . . . . . .

7 0 - -

~ .............

65

~ ~ ~

E 60 Z ~-~55 50 45 40 -2

I

I

I

-1

0

1

lg(tef) Is]

Fig. 5.17.Examples for serum tensiograms obtained from patients with rheumatoid arthritis, one with seropositive version (female, age 69, thin line), one with seronegative version (female, age52, thick line); dotted curves correspond to average values for healthy females of corresponding age. In some cases, common laboratory methods fail to detect the rheumatoid factor. First of all, for 1/5 of all patients, the immunoglobulins-G are blocked by the immunoglobulin-M-rheumatoid factor; secondly, the immunoglobulin-G-rheumatoid factor, which cannot be registered by known agglutination tests, can be present instead of the immunoglobulin-M-rheumatoid factor, recognised as "seronegative" cases (Aho 1986). It is quite possible that more precise randomisation of the patients with respect to serologic versions of the disease (we have used the Waaler-Rose and latex-test) could affect the mean surface tension parameters of serum. We believe, however, that the variations detected would in any case display at least pronounced trends. It should be noted that the dynamic surface tension of serum increases with the duration of rheumatoid arthritis (cf. Fig. 5.18), becoming apparent only after 7 years with a twofold increase of the parameters after about 15 years. We believe this phenomenon can be explained either by the formation of some new substances in blood, or by the fact that substances already existing can acquire unusual surface active or surface inactive npon~pxt~cy (e.g. due to the effect of medication).

222

1210~

o .~

8._o

64-

21 1

1

3

1

5

I

7

I

9

I

11

I

13

15

Duration [years]

Fig. 5.18. Changes of surface tension parameters measured in serum obtained from patients with rheumatoid arthritis as a function of the disease duration. Changes are given in % compared to corresponding healthy controls. (~) - ol, (m) _ o2, (A) - o3. It was already mentioned in the previous chapter that variations in surface tensiograms of urine appear during the development of a secondary kidney amyloidosis for patients suffering from rheumatoid arthritis. At the same time it should be noted that even if no clinical-laboratory symptoms of any nephropathy exist, then the ~, value of urine decreases. These deviations from characteristic values of healthy subjects were found in 3/4 of all cases of rheumatoid arthritis studied (cf. Table 5.8). It is well known that kidney lesion can result from rheumatoid arthritis. In addition to the formation of amyloidosis, glomerulonephritis and interstitial nephritis often occur, the development of which can be latent, without apparent symptoms. Morphological studies show the thickening and doubling of the tubular basal membrane, albuminous degeneration and focal atrophy

of

the

epithelium

of

convoluted

tubules,

stroma

oedema

and

sclerosis,

lymphohistiocytic infiltration, hyalinosis of arterioles, segmental ectasis and sclerosis of glomerular mesangium, focal proliferation of mesangial cells, sometimes accompanied by the thickening of the capillary basal glomerular membrane, external capsules, and sclerosis of vascular ansae (Krel' et al. 1990). The detection of mesangioproliferative glomerulonephritis

223 using kidney biopsy is possible even if the urine analysis shows no changes, and the symptoms of the uric syndrome do not necessarily correspond to the extent of kidney pathology. While the equilibrium surface tension and L of serum for patients who do not suffer from cardiac or hepatic pathology are virtually equal to the reference values of respective characteristic parameters in healthy persons, the existence of cardiac or hepatic pathology is accompanied by an increase of 0-3 and a decrease of L. Note that the cardiac degradation does not result in any changes of 0-1 and 0"2 for serum, while the hepatic pathology leads to an increase of these surface tensiographic parameters. Table 5.10 demonstrates the correlations between surface tension parameters of serum and concentrations of some proteins and lipids. The results obtained in these studies are quite complex, therefore some additional comments are to be made. In fact, a direct correlation has been expected between the dynamic surface tension and the contents of these surfactants in blood, because the surface tensiographic parameters for serum often exhibit an increase when the activity of the pathologic process increases. In turn, the activity of rheumatoid arthritis positively correlates with the levels of immunoglobulins and 132-microglobulin. It is quite possible that the state of surface tension is determined also by other constituents of serum (including those of non-protein and non-lipid nature). The answer to this question is as yet unknown,

The synovial fluid is the most available indicative medium regarding the character of the articulatory lesion. It should be kept in mind that 0-1 and ~, for synovial fluid are lower than those of blood. At the same time, synovial fluid of patients with rheumatoid arthritis possess large concentrations of immunoglobulins-G, immunoglobulins-M and immune complexes which contain large quantities of immunoglobulins G, M and A (Shine et al. 1991). The activity of acidic phosphatase exceeds 8 times that characteristic for patients suffering from posttraumatic arthritis, while the acetyl-13-D-glucosaminepeptidase activity is three times as high as that for posttraumatic arthritis, and two times higher than that for patients suffering from Reiter's disease. The level of 132-microglobulin is twice as high as the contents of this protein in blood, and correlates with the total amount of protein in synovial fluid. During the phagocytosis of immune complexes by neutrophiles and mononuclear phagocytes, lysosomic ferments are released to the extracellular medium (Henderson & Pettinher 1985).

224 Table 5.10 Correlation coefficients between surface tension parameters measured in serum obtained from patients with rheumatoid arthritis and serum components.

Serum component 0"1

0"2

0'3

$$

$$

$$

$$$

$

$ $

$$

$$

$$$

$$

Total protein Albumin ot~-globulin fraction a2-globulin fraction 13-globulin fraction T-globulin fraction Immunoglobulin-G Immunoglobulin-A

$$

$$

Immunoglobulin-M 132-microglobulin Fibrinogen Fibronectin

i"

Circulating immune complexes Total cholesterol

$

or-cholesterol Triglycerides High density lipoprotein fraction Low density lipoprotein fraction Very low density lipoprotein fraction

$

$

1" p o s i t i v e c o r r e l a t i o n ; ,1, n e g a t i v e c o r r e l a t i o n ; e m p t y - no c o r r e l a t i o n r<0.3; o n e s y m b o l - r = 0.3 to 0.5; t w o s y m b o l s - r = 0.5 to 0.7; three s y m b o l s - r > 0.7

Synovial fluid of patients with rheumatoid arthritis contains large immunoglobulin-G complexes, which can react with the immunoglobulin-M-rheumatoid factor forming very large, stable and insoluble intercross-reacting compounds. Also specific antibodies directed against immunoglobulins are found, which cannot be regarded as rheumatoid factors, because they

225 react with other regions of the immunoglobulin-G molecule or with immunoglobulins belonging to other classes. Immunoglobulin-G-rheumatoid factor is most often found in biological liquids that contain many

immunoglobulin-G-immune

complexes.

Even

at

low

concentrations

of

the

immunoglobulin-G-rheumatoid factor, immunoglobulins-G dimerise due to self-association. The significant level of immunoglobulins-G in blood results in an excess of antigen and prevents from the formation of increased size of immune complexes, therefore these complexes are small and usually incapable of binding to complement. There is a significant amount of immunoglobulins-G-rheumatoid factor in the joint cavity, which reacts with normal immunoglobulins-G with the formation of immune complexes that undergo partial aggregation. The attachment of immunoglobulins-G-rheumatoid factor and complement to such megacomplexes leads to a further increase of their size. The molecules of normal immunoglobulin-G and immunoglobulin-G-rheumatoid factor possess two antigen determinants in the region of the Fc-fragment which bind the immunoglobulins-G-rheumatoid factor. This leads to a rotation and steric blocking of the second determinant of unchanged immunoglobulin-G, distorting the position of the Fab-fragment. Thus the excess of normal immunoglobulins-G prevents a further polymerisation of immunoglobulin-G-rheumatoid factor. Their dimers cannot block the second determinant; therefore large polymers are formed (Munthe & Egeland 1984). Such remarkable differences in the composition of immune complexes in blood and synovial fluid is responsible for the diversity in the variations of surface tension between these biological liquids. Morphological studies of the synovial membrane, even for a prolonged course of seronegative rheumatoid arthritis, do not display the classic appearance of rheumatoid synovitis. The proliferative reaction of synovial cell is observed with the formation of multilayer strata, the hypertrophy of the endothelium of venulae and capillaries of microcirculatory bed with a deterioration of the penetrability of vessels and the deposition of fibrinous masses. No antibodies are locally synthesised or lymphoid-plasmacellar follicles formed (Ivanova et al. 1986, Busso et al. 1998). One of the most significant mechanism, which cause a distortion in the metabolism of the bone/cartilage matrix in rheumatoid arthritis is the pathology of the metabolism of tissue

226 (cartilage) proteoglycans. Both the concentration and the qualitative composition of proteoglycans and glycosaminoglycanes affect the morphological and functional state of the connective tissue. Certain dependencies were observed in the variation of the contents and qualitative composition of glycosaminoglycanes in synovial fluid for various degrees of synovitis. A direct correlation between the gravity of a local inflammation process and the amount of sulphated glycosaminoglycanes exists, and an inverse correlation with nonsulphated (by hyaluronic acid) glycosaminoglycanes (Astachova et al. 1989, Silbermann et al. 1990, Bensouyad et al. 1990). Hyaluronic acid, which is the main glycosaminoglycane among those constituting the synovial fluid, determines its viscosity. For articular diseases the viscosity of synovial fluid decreases, which can be explained by the depolymerisation of hyaluronic acid or the formation of lowpolymeric hyaluronates due to demage of the synthesis process. This depolymerisation is caused by the action of a number of lysosomic ferments (e.g., [3-glucuronidase) and peroxide radicals. The increased contents of chondroitynsulphates in synovial fluid correlates with the gravity of the articular inflammation, and is determined by a destruction of tissue (cartilage) proteoglycans. These molecules possess a unique property, the ability to form large aggregates, comprised of proteoglycane sub-units, hyaluronic acid and connecting proteins. This protein stabilises the proteoglycane-hyaluronic complex, preventing its dissociation. The chondrocytes which produce glycosaminoglycanes and are influenced by lysosomic ferments, perform the synthesis of anomalous proteoglycans, incapable of aggregation; therefore these chondrocytes are more vulnerable to the action of hydrolases. The discussion above can also explain differences in surface tension between blood serum and synovial fluid for patients with rheumatoid arthritis. Rheumatoid arthritis is accompanied by a decreased activity of the fibrinolytic system in synovial fluid. Increased amounts of fibrin degradation products were found, which correlate with the concentration of coagulation factor XI and XII. The deficiency in the amount of plasminogen activators is caused by a decrease in their synthesis by the articulation synovial membrane. At the same time, the concentration of plasminogen activation inhibitors is increased, which suppresses the fibrinolysis. Eventually, the contents of fibrinogen and fibrin

227 in synovial fluid increases. The deposition of fibrin at the synovial membrane hampers the exchange between the articulation liquid and the cartilage (Murav'iev et al. 1989, Clemmensen et al. 1983). For rheumatoid arthritis an increase of the fibronectin concentration in synovial fluid was observed (Vasil'eva et al. 1991, Scott et al. 1981). The analysis of the composition of polyethylene glycolic precipitates in articulation liquid has shown that fibronectin as their integral constituent, can be related to immunoglobulins, rheumatoid factor and the components of the complement. There are some differences between fibronectin molecules in synovial fluid and those in blood serum, therefore the contents of proteins in the immune complexes of these biological liquids is different. For patients suffering from rheumatoid arthritis, a degradation of fibronectin molecules take place and complexes with other proteins are formed. It should be stressed that for arthritis with other etiology, the contents of fibronectin in synovial fluid virtually does not change. For healthy subjects fibronectin is the only protein, which has a concentration in the articular liquid similar to that in serum, while the amount of other synovial proteins (which originate from blood) is significantly lower. This indicates an important contribution of local synthesis of fibronectin by synovial cells and/or cells which are present in the synovial cavities. The variations in the amount of synovial fibronectin are accompanied by a development of its physicochemical inhomogeneity. The pathological forms of the protein and the derivatives of fibronectin can differ significantly from the native forms in their bonding with gelatine, which is the product of collagen denaturation (Abdullin et al. 1988). A direct correlation link exists between the gelatine-bonding ability of fibronectin in synovial fluid and the gravity of the rheumatoid arthritis. The malfunction of the fibrinolytic system Of synovial fluid for patients with rheumatoid arthritis controls in many ways the dynamic surface tensiometric parameters. In the short surface lifetime the surface tensions of serum exhibit some correlations with the concentrations of fibrinogen and fibronectin (cf. Table 5.10). For synovial fluid, this dependence becomes more pronounced, and a correlation to the value of tensiographic quantity ~. becomes apparent, having however negative correlation coefficients. Therefore it can be argued that other factors are responsible for the differences in surface tension of serum and synovial fluid.

228 5.6. Reiter's disease

Reiter's disease is characterised by an increase of and an increase in cl and

(Y3

13'1 and

decrease of ~, for serum tensiograms,

for urine. Surface tensiographic parameters of synovial fluid are

significantly lower than corresponding serum parameters, see Fig. 5.19. Such finding could be explained by the differences in the contents of surface active and inactive substances in these biological liquids.

In particular,

synovial fluid contains lower amounts

of albumin,

immunoglobulins-G and immunoglobulins-M, txl-antitrypsin , glycosaminoglycanes, C-reactive protein and electrolytes, and higher concentrations of immunoglobulins-A, antithrombin III, ceruloplasmin, 132-microglobulin , fibronectin, alkaline phosphatase and lactate dehydrogenase.

80 T

E 70 t3

65-

60 -2

-1

lg(tef) [s]

0

1

Fig. 5.19. Examples of tensiograms of various biological liquids obtained from a male patients, age 35, with reactive urinogenous chlamydial arthritis (Reiter disease ). Thick curve - serum, thin curve - synovial fluid, dotted curves correspond to average values for healthy males of corresponding age (serum) and for control group (synovial fluid).

Serum R-proteins are proteins of non-immunoglobulin nature which possess the catabolic activity with respect to peroxide oxidation reactions. They are present in the circulating blood both in the free form and as complexes formed with immunoglobulins-G, whose properties correspond to the naturally formed antibodies with various specificity (agglutinants or homoreactants.

Such complexes

reaches

up to 5% of the total

amount

of serum

immunoglobulins. For Reiter's disease the free fraction of R-proteins is reduced due to the

229 transition of these proteins into a form bound to immunoglobulins-G. This process is accompanied by a significant increase in the catalytic activity of R-proteins with respect to the peroxide oxidation reactions, a fact important for its destructive activity for arthritis (Salihbayeva et al. 1989). Note that the surface tension parameters of serum at t = 1 s and t --~ oo for Reiter's disease inversely correlates with the contents of serum immunoglobulins-G, and directly with the concentration of R-proteins. 5.7. Psoriasis

During the development of psoriatic arthropathy (PA), an improper microcirculation determined by rheological properties of blood in the articulatory synovia plays a certain role. For most of the patients with microcirculatory derangements, heavy hemorheological disbalances were found with an increase in plasma viscosity causing the hemorheological syndrome (Korotayeva et al. 1991, Wolf et al. 1981). The hemorheological syndrome develops via activation of fibrocytes located in the synovial membrane by such cytokines as interleukin1 that is released by the macrophagic-phagocytic system. Chronic inflammation of microvessels of the articulatory membrane is accompanied by hyperfibrinogenemia, degrading fibrinogen into the fragments D and E, which in turn stimulates the release of interleukin-1 by synovia macrophages. This haematological stress-syndrome is manifested by an increase in blood viscosity. In all cases of psoriatic arthropathy, the viscosity increases due to the increase in the concentration of high molecular proteins, mainly of fibrinogen. For psoriasis, the total concentrations of lipids, triglycerides, cholesterol, and phospholipids were increased, along with the variation in the contents of neutral fats and lipoproteins (Seishima et al. 1994, Imamura et al. 1990). The most significant changes of the lipid profile were observed for large-plaque type and pronounced exudative components of eruptions (Panasiuk 1988). For one-third of all patients a hyperuricemia develops due to the enhanced maceration of the epithelial cells and the output of nucleic acids. In addition, the purine exchange is deranged, which is related to the deficiencies of lipid metabolism (Sinyachenko & Barinov 1994). In spite of these significant changes in protein and lipid metabolisms, the dynamic surface tension parameters of serum for psoriatic arthropathy only slightly differ from those characteristic for healthy persons. There are, however, certain negative correlations between

230 and lipoprotein fractions of various densities, and also between ~ and the total contents of cholesterol in blood. 75

70

oo

65 I

~

I

~o ,,o

60

i

-2

-1

-

lg(tef) [s]

t 84

t

0

1

Fig. 5.20. Example for tensiograms of urine obtained from a male patient, age 37, with psoriasis, psoriatic arthropathy and nephropathy (male, age 37). Dotted curve correspond to average values for healthy males of corresponding age. It was rather unexpectedly found that significant variations of surface tension parameters in urine were found for patients with this disease (increase of t~2 and (r3, and increase of ~, see Fig. 5.20). It is quite possible that the genesis of surface tension variations is local (renal), in spite of ostensibly "intact" kidneys for psoriatic arthropathy found in the course of clinical, laboratory and instrumental studies. The alterations in kidneys during psoriatic arthropathy are manifested by segmental proliferation of mesangial cells, the increase of mesangial matrix volume, dystrophic changes in the epithelium of convoluted tubules, and interstitial oedema. For serious cases of this disease, the extent of damage suffered by glomerular and tubulostromal component increases, correlating with the character of articular syndrome (Shlopov & Shevchenko 1993). Most observations show the typical picture of mesangioproliferative glomerulonephritis. In some glomerules a symphysis of peripheral capillary loops with Bowman's capsule takes place, a focal proliferation of the epithelium of the external layer, with a possible subsequent sclerosis. T-helper-inductor lymphocytes prevail in the periglomerular cellular infiltration. The

231 tubulointerstitial component is characterised by the presence of protein masses in the tubular lumina, focal proliferation of the tubular epithelium, lymphoid infiltrates in the stroma with the presence of macrophages of haematogenic origin. The role of the antigen-antibody complexes in the development of psoriatic nephropathy deserves special attention, because the mechanisms responsible for glomerular filtration promote the retention and deposition of circulating immune complexes in kidneys. This effect is related to the existence of the receptors for the C3-component of the complement and F3-fragment of immunoglobulin-G at the epithelial and mesangial cells. Immune complexes are deposited in kidney glomerules due to the presence of antigens, similar to those in the glomerular basal membrane. All these factors lead to the angiopathy at the microcirculatory level, the increase in the contents of immunoglobulins (in particular, immunoglobulin-A), and to the activation of the complement system (Panasiuk et al. 1990, Imai et al. 1995, Yamamoto et al. 1994). 5.8. Gout

Among the dynamic surface tension parameters of serum sampled from patients with gout, only the ~ values decreased compared to healthy controls, despite the fact that this disease involves major irregularities in the composition of serum proteins (immunoglobulins, circulating immune complexes, ferments, hormones), lipids, non-albumin-type nitrous compounds, electrolytes and other surface active substances. Hyperuricemia is the most demonstrative evidence of gout. It was already mentioned that uric acid could affect surface tension parameters of a biological liquid. Similarly to other products of purine metabolism, uric acid correlates with the contents of sex hormones and gonadotrophic hormones in blood. For gout, the concentrations of these hormones undergo significant changes. In turn, the contents of proteins in blood depend essentially on androgens, estrogens and progestins. The metabolism of purines is closely related to the metabolism of fats. In 74% of gout cases a hyperlipoidemia was detected (types IIB and IV predominate), demonstrated by increased total concentrations of lipids, cholesterol, triglycerides and the low density lipoprotein fraction. The hyperlipoidemia is caused by an enhanced production of cholesterol-rich apolipoproteins and

232 triglyceride-rich very low density lipoproteins, and also by a decreased rate of degradation of very low density lipoproteins due to a suppressed activity of lipoprotein lipase, hepatic triglyceride lipase, letinin cholesterol transferase. Sytematic studies of various clinical forms of nephropathy complicating gout enabled us to differentiate between 3 types of this disease, which provide the most comprehensive description of the developments of the disease and its prognosis: urolithic, latent and proteinuric gout. The characteristic features of damages of the main kidney structures for various types of nephropathy are roughly the same, although some distinctions exist. The urolithic type is more often accompanied by damages of tubules, the proteinuritic type by damages of glomerules, while for the latent type damages of stroma dominate. Only in the urolithic type of nephropathy small kidney stones from calcium salts were found. Plasmocytic infiltration of kidney stroma occurs in urolithic and proteinuritic types of nephropathy. Plasmatic infiltration of the vessels occurs in the proteinuritic and latent types. The most significant extent of damages to kidney structures was found for patients suffering from the proteinuritic type of the disease. The lipid profile for the proteinuritic version of gouty nephropathy is characterised by hypercholesterolemia and hypertriglyceridemia, an increase of the cholesterol contents in the low density lipoprotein and very low density lipoprotein fraction, and a decrease of the amount of high density lipoproteins in serum. At the same time, increased concentrations of apolipoprotein-B, apolipoprotein-C and apolipoprotein-E, and lowered concentrations of apolipoprotein-A 1 and apolipoprotein-A 2 appeared. The development of chronic renal insufficiency leads to an increased production of low density lipoproteins and a slowdown of their degradation. It follows from our data that for gout (especially for female patients) the concentrations of calcium, either ionised, total or corrected with respect to albumin, in the peripheral blood is decreased, while its clearance is significantly increased. This effect is accompanied by an increase in the concentrations of parathyroid hormone, calcitriol and calcitonin. For the chronic form of gouty arthritis the levels of parathyroidin and calcitriolum are twice as high as those characteristic for the intermittent form. The most significant variations in parathyroid hormones were observed for patients with urolithiasis, cases where the most pronounced

233 disturbance of calcium homeostasis was observed. The indications of calcitonin in blood for the urolithic and latent types of nephropathy are the same, and do not differ significantly from those characteristic for healthy persons. On the contrary, for the proteinuretic version of kidney pathology the level of this hormone even increases significantly.

0 . 6

-

0.50.4E

0.3-

o

0.20.1 -

.~

0

m

::

-~ -0 1

iD~:~il I

o -0.2 k9 -0.3 -0.4 -0.5 TCa

ICa

PH

CTr

CT

Fig. 5.21. Correlations between the parameters of calcium and purine metabolisms for patients suffering from gout. Parameters of calcium metabolism are: TCa - total calcium; ICa - ionised calcium; PH - parathyroid hormone; CTr - calcitriol; CT- calcitonin. Parameters of purine metabolism are: hatched - uric acid, black - oxypurinol, white - xanthine oxydase, grey - adenosine desaminase

A direct correlation was found between the concentration of ionised calcium and the parameters of oxypurinolemia, and also a trend in an inverse correlation of total calcium in blood with oxypurinol, of calcitonin with oxypurinol, uric acid and adenosine desaminase (Fig. 5.21). High parathyroid hormone concentrations lead to a decreased tubular reabsorption of phosphates, resulting in a hypophosphatemia with subsequent compensatory mobilisation of calcium from the skeleton, accompanied by the development of hypercalciemia and hypercalciuria. It can be thus concluded that the qualitative and quantitative compositions of both surface active and surface inactive substances in blood for gout depend on the concentration of various hormones that are sometimes associated specifically with different forms of nephropathy. In this connection, a comparison study has been performed of dynamic surface tensiographic

234 parameters of serum obtained from patients with different versions of kidney lesion. For the proteinuric type, the decrease of k becomes yet more significant, while other surface tension parameters remain virtually constant. It should be noted that intermittent gouty arthritis is accompanied by the trend to decreased equilibrium surface tension of serum, while for the chronic version an increase was observed. The ~ values of serum tensiograms have opposite direction, and the values for the chronic form of the articular syndrome is two times lower than those characteristic for the intermittent form. It can be supposed therefore that the increase in the equilibrium surface tension and the decrease of L of blood for patients with gout can be regarded as evidence for the transformation of intermitting arthritis into the chronic form. Nephropathy is one of the most common visceral symptoms of gout. In fact, the disease is always associated not only with articular lesions, but also with renal lesions. Disturbances of renal functions in special examinations can be found even when no obvious clinical symptoms of nephropathy exist. Morphological changes in kidneys were detected by optical spectroscopy for all gout patients. The lesion of glomerules and stroma occurred in 100% of cases, tubular lesion in 88%, and lesions of vessels were found in 73% of cases. Most typical variations in glomerules are characterised by focal thickening of basal tubular membranes, an increase of mesangial matrix therein, and by focal (or, less common, diffuse) proliferation of mesangial cells, which are more or less pronounced. Glomerular sclerosis and hyalinosis can often be found. These changes, in 80% of all cases, resemble focal mesangioproliferative glomerulonephritis, while for the other 20% a similarity with mesangiocapillary glomerulonephritis was found. At

the

basal

membranes

of glomerular

capillaries

a

subendothelial

fixation

of

immunoglobulins-M and C3-component of the complement prevail. For this form of kidney lesions, a small increase of the mesangial matrix is typical. Weak proliferative reactions of mesangial cells can be explained by the minimum nephrotoxical ability of immunoglobulins-M as compared to other immunoglobulins. The glomerular depositions of immunoglobulins-G and complement are seldom detected, accompanied by a yet pronounced increase of the mesangial matrix and the creation of synechiae in the Bowman's capsule cavity. The occurrence of fibrosing mesangial cells for this type of gouty nephropathy is caused by the

235 stimulating influence of immunoglobulins-G on mesangiocytes, and by the hypoxia of tissue which takes place due to violations of the glomerular bloodstream. Electron microscopy studies show a proliferation and pronounced swelling of endothelium in some capillary loops, a proliferation and activation of mesangial cells with the tendency towards an interposition of their processes between the endothelium and basal membrane of the capillary loop. In many podocytes an expansion of cisterns of the granulated endoplasmic network is observed, accompanied by the loss of ribosomes. In uric space separated cellular organelles, fragments of the membrane and cellular cytoplasm can be found. In addition, podocytes contain a number of ribosomes and polysomes, and a hyperplasia of granulated endoplasmic network occurs, which is indicative of the increase in their metabolic activity (in particular that responsible for the synthesis of albumins). At some sections of the capillary loops the fusion of small podocytes, and the destruction of cells is seen.

7472 ,---, 7 0 -

{

68

~ 66 64 6260---2

~176

f

-1.5

-t

-

-1

t

-0.5

t-

--%-

f

0

0.5

1

--I

----q

1.5

2

lg(tef) IS] Fig. 5.22. Examples for urine tensiograms obtained from patients with gout and chronic arthritis, one with additional urolithic type of nephropathy (male, age 58, thick line), one with additional latent type of nephropathy (male, age 44, thin line); dotted curve correspond to average values for healthy males of corresponding age.

Dystrophy, atrophy and desquamation of the epithelium in tubules are determined, the expansion of lumen, hyalinosis and a thickening of the basal membrane, albuminous cylinders, leukocytes, small kidney stones from calcium salts and deposits of uric acid crystals are observed. The sclerosis of interstice is immanent to gouty nephropathy. The lesion of stroma

236 also demonstrates itself in the infiltration by lymphocytes, histiocytes, plasmocytes with giant cells. These effects are accompanied by great changes in vessels (sclerosis, proliferation of endothelium, lumen arctation, mioelastofibrosis, plasmatic steep, mucoid and fibrinoid swelling.) For gout, crl for urine increases. However, for the urolithic type strong incresing surface tension values of urine in all studied surface lifetime ranges result (Fig. 5.22). The latent type of nephropathy is accompanied by significant increases of the tensiographic parameter ~,. For the urolithic type no changes of ~, is obtained. For urolithiasis combined with gout, normal or2 values and slightly increased equilibrium surface tension were observed. 5.9. Osteoarthrosis Dynamic surface tension of urine sampled from patients with osteoarthrosis is quite similar to that characteristic for gout, while morphological changes in kidneys are absent. We believe that the increase in crl, cr2 and ~r3 values can be explained by the enhanced urinal excretion of glycosaminoglycanes, whose levels correlate with surface tensiometric parameters. It can be argued that the development of pathological processes in articulations for osteoarthrosis are characterised by disturbances in the metabolism of proteoglycans, which are the carbohydratecontaining polymers which play an important role in the construction of the connective tissue. The progressive dismetabolism of this tissue leads to a sharp decrease of the proteoglycans contents in the synovial media, while glycosaminoglycanes are the products of their degradation. High levels of glycosaminoglycanes in urine are related to the superactivity of [3-glucuronidase in blood (Astachova et al. 1987). In general, the destruction of the articular cartilage is determined essentially by the activation of hydrolytic ferments, which participate in the degradation of the main substances of the connective tissue: lysosome hydrolases and neutral proteinases, which include 13-glucuronidase (Gardner 1994). No dependence exists between clinical-roentgenological peculiarities of osteoarthrosis and glycosaminoglycaneuria, which can be explained by the heterogeneity of glycosaminoglycanes, represented by low-molecular forms, and also by non-dialysing high-molecular forms (Kosjagin et al. 1988). At further stages of the pathologic process, the dynamic surface tensions of urine increase. We can therefore

237 conclude that the surface tensiometry can be regarded as a better indicator of the progress of osteoarthrosis, than the contents of glycosaminoglycanes in urine. For patients suffering from osteoarthrosis, the synovial fluid contains extremely low amounts of immunoglobulins and 132-microglobulins, no fibronectin and c~2-macroglobulin, and low concentrations of lysosomic enzymes. At the same time, crystals of calcium pyrophosphate and hydroxyapatite are often present. We presume that these calcium salts contribute to the variation of surface tension parameters of synovial fluid during osteoarthrosis. This hypothesis is based upon the following findings: -

calcium ions in aqueous solutions of low molecular surfactants can increase the surface tension in the short surface lifetime range only;

-

low levels of albumin in synovial fluid only slightly affect the quantitative surface tension parameters;

-

calcium salts, which possess negative charges, promote the activation of factor XII (Hageman factor) in articulation cavity; this factor, in turn, activates kinins, low molecular mediators of inflammation, whose formation takes place via thrombin and plasmin, through the action of prekallikrein and high molecular kininogen;

-

the absorption of hydroxyapatite and calcium pyrophosphate by articulation cartilage chondrocytes leads to a significant release of neutral protease, collagenase (which is extremely active with respect to type I and II collagens) and adenosine triphosphate pyrophosphohydrolase; crystals of calcium salts lead to the increase of proteoglycans and mucin in the synovial fluid.

5.10. Effect of glucocorticoid therapy and plasmapheresis

The data concerning the effect of a glucocorticoid therapy on the state of surface tension for patients with glomerulonephrites are presented in chapter 4. It was mentioned that there is a trend towards a normalisation of dynamic tensiographic parameters during glucocorticoid therapy. Figure 5.23 shows variations of surface tension parameters of serum before and after administration of hormones to patients with rheumatoid arthritis, systemic lupus erythematosus and sclerodermia systematica. It can be concluded that glucocorticoid therapy increases surface tensions of serum, especially for sclerodermia systematica. For the cases of rheumatoid arthritis

238 and sclerodermia systematica, this is true mostly for (Y3, while for systemic lupus erythematosus the important parameter is el. For patients with rheumatoid arthritis the hormonal treatment leads to a normalisation of cl and or2, while for sclerodermia systematica the normalised parameters are g2 and cr3. For systemic lupus erythematosus (patients who do not suffer from lupus glomerulonephritis were also incorporated in the screened group) increased surface tensions of serum were observed. Although clinical-laboratory effects have been obtained, increased deviations of the parameters from normal values characteristic of healthy individuals occurs. The above is true also for the equilibrium surface tension of patients suffering from rheumatoid arthritis, and for g l values with respect to patients with sclerodermia systematica. The reactivity of the organism for rheumatoid arthritis is characterised by the contents of serum macroproteins, which promote the deterioration of the rheological properties of blood, and a more rapid decrease of the compensatory potential of vascular tension. The application of plasmapheresis leads to a decrease in the levels of immune complexes and immune active proteins. During the medication, an unambiguous correspondence was found between the parameters characteristic to macroproteinemia, and the concentrations of immunoglobulins-M and circulating immune complexes (but not with concentrations of immunoglobulins-G, fibrinogen and C-reactive protein). RA

10-]

SLE

SS

-10 -15 ~1

~2

~3

cl

~2

~3

~1

c2

~3

Fig. 5.23. Changes in surface tension parameters measured in serum obtained from patients with rheumatic diseases before (black columns) and after glucocorticoid therapy (white columns). Changes are given in % compared to corresponding healthy controls. RA - rheumatoid arthritis, SLE - systemic lupus erythematosus, SS - sclerodermiasystematica.

239 The application of plasmapheresis to patients suffering from rheumatoid arthritis leads to higher surface tensions of serum. This effect is caused by the excretion of some proteinic surfactants from the organism, while the contents of macroproteins is decreased along with an increase of the parameters indicative of relative albuminemia. The requirement to balance a protein deficiency is one of the problems, which arise in connection with a plasmapheretic treatment. In this regard, the main attention is paid to the development of specific methods aimed at the removal of those components from the plasma which are considered to play a key role in the pathogenesis of rheumatoid arthritis, while the plasma being returned back to the patient. During a selective plasmapheresis, fibronectin, complement components, cryoglobulins, circulating immune complexes and cryofibrinogen are removed. As there are no significant changes in the levels of albumins, the total protein contents at the end of a series of plasmapheretic procedures is similar to its initial value. The comparison of surface tensiometric parameters measured before and after plasmapheresis once again shows that pathologic proteins affect the surface tension of serum for rheumatoid arthritis. The elimination of pathological proteins from blood during plasmapheresis is clearly accompanied by the return of surface tensiometric parameters to values characteristic for healthy persons. Kidney lesions accompany many rheumatic diseases. Comparisons of the ratio of surface tensions for serum and urine at t = 0.01 s and t ~ oo have been performed for patients with such lesions. It was found that diseases of the connective tissue (systemic lupus erythematosus, sclerodermia systematica, rheumatoid arthritis) are characterised by significant increases of these parameters,

while in metabolic

and degenerative

articulation diseases

(gout,

osteoarthrosis) these values were decreased, see Fig. 5.24. Low equilibrium surface tension ratios were found also for non-specific aortal arthritis and seronegative arterites. In contrast, the serum to urine ratio for ~, significantly prevails for non-specific aortal arthritis, two or more times exceeding the corresponding values in other groups of patients, see Fig. 5.24. To summarise, we believe that the study of dynamic surface tension of biological liquids as applied to rheumatology is of significant practical interest, due to its capability of providing a differential diagnosis and monitoring of the efficiency of therapy. With regard to the above analysis of correlations between interface tensiographic parameters and the contents of surfactants it is now possible to indicate some surfactants which affect the surface tension state

240

o f serum, urine and synovial fluid. In turn, the d y n a m i c surface t e n s i o m e t r y o f biological liquids for rheumatic diseases is capable o f p r o v i d i n g rapid and rather accurate reflection o f the total c o m p o s i t i o n o f surfactants, including pathological proteins and other c o m p o u n d s formed and a c c u m u l a t e d during the d e v e l o p m e n t o f the disease. A

1.1

1.05 1 ~, 0.95 0.9 0.85 0.8 HC

HM

HF

*

R

SLE

HM

HF

*

R

SLE

SS

SS

HV NAA RA

BD

RD

PA

G

RD

PA

G

OA

OA

3 2.5 2

,<

1.5

1 0.5 0 HC

HV NAA RA

BD

Fig. 5.24.. Ratios of surface tension parameters measured in serum and urine that was obtained from healthy individuals and patients with various rheumatic diseases; HC - healthy controls (general group), HM healthy males, HF- healthy females; R - rheumatism, SLE - systemic lupus erythematosus, SS - sclerodermia systematica, HV - heamorrhagic vasculites, NAA - non-specific aortal arthritis, RA - rheumatoid arthritis, BD- Bechterew's disease, RD - Reiter's disease, PA - psoriatic arthropathy, G - gout, OA - osteoarthrosis. In graph A the serum/urine ratios of cr~ (black) and cr3 (white) are given. In graph B the serum/urine ratios of k are presented.

241

5.11. Summary

To summarize, we believe that analysis of dynamic surface tension of biological liquids obtained from patients with connective tissue disorders are of significant practical interest, due to its capability of providing a differential diagnosis and monitoring of the efficiency of therapy. With regard to the above analysis of correlations between interface tensiographic parameters and the contents of surfactants it is now possible to indicate some surfactants which affect the surface tension state of serum, urine and synovial fluid. In turn, the dynamic surface tensiometry of biological liquids for rheumatic diseases is capable of providing rapid and rather accurate reflection of the total composition of surfactants, including pathological proteins and other compounds formed and accumulated during the development of the disease. 5.12. References

Abdullin, A.R., Litvinov, R.I., Zinkovich, O.D. and Salichov, I.G., Ter. Arch. (Therapeutic Archive), 4(1988)89 Aho, K., Ter. Arch., 7(1986)9. Amosova, E.N., Fedorich, V.N., Zamulko, A.A., Vrach. Delo, 2(1994)20 Anikina, N.V., Guseva, N.G. and Mach, E.S., Ter. Arch., 8(1986)62 Aoki, K., Kawai, S., Intern. Med., 32(1993)382. Astachova, T.A., Volkova, Z.I., Trofimova, T.M. and Mul'diyarov, P.Ya., Ter. Arch., 4(1987)62. Astachova, T.A., Volkova, Z.I., Trofimova, T.M. and Mul'diyarov, P.Ya., Ter. Arch., 5(1989)55. Azarenok, K.S. and Generalov, I.I., Ter. Arch., 5(1990)149. Balabanova, R.M., Loskutova, T.T., Saikovskaya, T.V., Revmatologija (Rheumatology), 1(1990)36 Bannikova, E.M., Ter. Arch., 8(1987) 146 Bensouyad, A., Hollander, A.P., Dularay, B., Bedwell, A.E., Cooper, R.A., Hutton, C.W., Dieppe, P.A., Elson, C.J., Annals of the Rheumatic Diseases, 49(1990)301. Borba, E.F., Santos, R.D., Bonfa, E., J. Rheumat., 21 (1994)220. Buchmann, C., Zoller, H., Gutmann, W., Medizinische Welt, 25(1974)174.

242 Busso, N., Peclat, V., Van Ness, K., Kolodziesczyk, E., Degen, J., Bugge, T., So, A., Journal of Clinical Investigation, 102(1998)41. Calvin, J., Neale, G., Potherby, K.J., Price, C.P., Ann. Clin. Biochem., 25(1988)60. Clemmensen, I., Holund, B., Andersen, R.B., Arthritis & Rheumatism, 26(1983)479. Corper, A.L., Sohi, M.K., Bonagura, V.R., Steinitz, M., Jefferis, R., Feinstein, A., Nature Structural Biology, 4(1997)374. Dang, C.V., Bell, W.R. and Shuman M., J. Med., 87(1989)567. Ettinger, O.R., Hajjar, D.P., Hajjar, K.A., J. Biol. Chem., 226(1991)2459. Ezratty, A.M., Simon, D.I., Fless, G., Scanu, A.M., Clin. Res., 41(1993)183. Fujita, T., Saito, E., Ohi, H., Nephron., 61 (19920122. Fusailov, O.R., Astachova, T.A. and Guseva, N.G., Ter. Arch., 5(1984)45. Gabay, C., Cakir, N., Moral, F., Roux-Lombard, P., Meyer, O., Dayer, J.M., Vischer, T., Yazici, H., Gueme, P.A., Journal of Rheumatology, 24(1997)303. Gardner, D.L., Journal of Anatomy, 184 (1994)465. Gudmundsson, M., Bjelle, A., British Journal of Rheumatology. 32(1993)774. Sakakibara, K., Nagase, M., Takada, Y., Takada, A., Thrombosis Research, 45(1987)403. Harreby, M.,. Danneskiold-Samsoe, B., Kjer, J., Lauritzen, M., Annals of the Rheumatic Diseases, 46(1987)601. Henderson, B. and Pettinher, R., Semin. Arthr. Rheum., 15(1985)1 Hilliquin, P., Cellular & Molecular Biology, 41 (1995)993. Hills, B.A. and Butler, B.D., Ann. Rheum. Dis., 43(1984)641 Hirano, T., Abira, S., Taga, T., Immunol. Today., 11(1990)443. Horwitz, D.A., Jacob, C.O., Springer Sem. Immunopath., 16(1994)181. Imai, H., Kodama, T., Ishino, T., Yasuda, T., Miura, AB., Asakura, K., Nishimura, Clinical Nephrology, 44(1995)64. Imamura, T., Takata, I., Tominaga, K., Yamamoto, T., Asagami, C., Japanese Journal of Dermatology. 100(1990)1023. Ivanova, M.M., Akimova, T.F., Tzurko, V.V. and Venikova, M.S., Ter. Arch., 7(1986)14 Jara, L.J., Capin, N.R. and Avalle, C., J. Rheumatol., 16(1989)225 Jensen, H.S., Mogensen, H.H., Kjaersgaard, E., Scandinavian Journal of Rheumatology. 18(1989)297.

243 Jensen, H.S., Tvede, N., Diamant, M., Mogensen, H.H., Hansen, M.B., Thomsen, B.S., Pedersen, P.B., Bendtzen, K., Scandinavian Journal of P&eumatology, 20(1991)83. Kazakov, V.N., Fainerman, V.B. and Sinyachenko, O.V., Advanced treatment of rheumatic diseases, Donetsk, (1996)25. Ketlinsky, S.A., Alekseeva, T.G., Perumov, N.D., Simbirtsev, A.S., Ter. Arch., 12(1993)51. Kilpatrick, J.M., Volanakis, J.E., Immunologic Research, 10(1991)43. Klukvina, N.G., Baranov, A.A., Aleksandrova, E.N., Nasonov, E.L., Clin. Med., 8(1997)24 Korotayeva, T.V., Firsov, N.N. and Mach, E.S.,., Ter. Arch., 5(1991)78 Kosjagin, D.V. and Karyakina, E.V., Revmatologija, 4(1988)52 Krel', O.V., Varshavskij, V.A. and Kanevskaya, M.Z., Ter. Arch., 6(1990)104 Lacki, J.K., Leszczynski, P., Kelemen, J., Muller, W., Mackiewicz, S.H., Journal of Medicine, 28(1997)99. Lipp, E., von Felten, A., Sax, H., Muller, D., Berchtold, P., European Journal of Haematology, 60(1998)283. Loskutova, T.G., Koreshkov, G.G. and Nasonov, E.L., Ter. Arch., 5(1989)51 Marguerie, C., Bunn, C.C., Black, C.M., So, A.K., Walport, M.J., QJM, 90(1997)347. Medvedev, A.N., Korshunov, N.I., Nasonov, E.L., Clin. Rheumatol., 1(1966) 12 Mikunis, R.I., Dubovoj, A.F., Kozlova, T.V., Ter. Arch., 8(1985)100 Misaki, Y., Pruijn, G.J., van der Kemp, AW., van Venrooij, W.J., Journal of Biological Chemistry, 269(1994)4240. Momot, A.P., Barkagan, Z.S., Mamayev, A.N., Byshevsky, K.M., Clin. Labor. Diagnost., 8(1997)20. Movshev, B.E., Markova, M.L., Kalinin N.N., Revmatologija, 1(1988)9 Munthe, E. and Egeland, T., Ter. Arch., 5(1984) 10 Murav'iev, Yu.V., Loskutova, T.T. and Anikina, N.V., Ter. Arch., 12(1989)106 Nasonov, E.L., Baranov, A.A., Shilkina, N.P., Alekberova, Z.S., Vascular pathology for antiphospholipid syndrome, "Medicina", Yaroslavl', 1995. Nasonov, E.L., Chichasova, N.V., Baranov, A.A., Clin. Med., 7(1997b)26 Nasonov, E.L., Popkova, T.V., Efremov, E.E., Clin. Med., 9(1997a)49 Panasiuk, N.N., Muchin, N.A. and Varshavskij, V.A., Ter. Arch., 6(1990)99 Panasiuk, N.N., Ter. Arch., 6(1988) 130 Polyantseva, L.G., Bobkova, I.N., Tareeva, I.E., Ter. Arch., 5(1995)17. Popkova, T.V., Pokrovsky, S.N., Alekberova, Z.S., Clin. Med., 8(1998)21

244 Rudenko, V.G., Revmatologija (Rheumatology), 2(1987)27 Ryabov, S.I., Kulikova, A.I., Mitrofanova, O.V., Ter. Arch., 2(1995)51. Salihbayeva, U.S., Kozlovskaya, L.V. and Zaitseva, L.I., Ter. Arch., 11(1989) 134 Schepotin, B.M., Ena, Ya.M. and Bodaretskij, G.M., Revmatologija, 2(1989)64 Scott, D.L., Wainwright, A.C., Walton, K.W., Williamson, N., Annals of the Rheumatic Diseases, 40(1981) 142. Seishima, M., Seishima, M., Mori, S., Noma, A., British Journal of Dermatology. 130(1994)738. Shibata, T., Sumie, A., Ishii, T., Tomo, T., Magari, Y., Sato, J., Yasumori, R., Nasu, M., Japanese Journal of Nephrology, 35(1993)687. Canesi, B.A., Banff, F., Rossi, A.F., Sinigaglia, L., Scandinavian Journal of Rheumatology, 9(1980)266. Shine, B., Bourne, J.T., Begum Baig, F., Dacre, J., Doyle, D.V., Annals of the Rheumatic Diseases, 50(1991)32. Shlopov, V.G. and Shevchenko, T.I., Arch. Clin. Exp. Med. 2(1993)136. Shvetsov, M.Yu., Kozlovskaya, N.L., Ter. Arch., 1(1996)57. Silberman, S., Holmes, E.W., Miller, B.J., Messmore, HL.Jr., Barr, W.G., Annals of Clinical & Laboratory Science. 16(1986)26. Silverman, B., Cawston, T.E., Page Thomas, D.P., Dingle, J.T., Hazleman, B.L., British Journal of Rheumatology, 29(1990)340. Sinyachenko, O.V. and Barinov, E.F. Gout, Donetsk, Med. Univ., 1994. Taylor, R.P., Ter. Arch., 5(1990)27 Vasil'ev, S.A., Yefremov, E.E. and Savenko, T.A., Ter. Arch., 2(1994)63 Vasil'eva, E.V., Mazneva, L.M., Golovanova, O.Ye. and Sura, V.V., Ter. Arch., 12(1991)130. von Mikecz, A., Hemmerich, P., Neu, E., Peter, H.H., Krawinkel, U., Zeitschrift ftir Arztliche Fortbildung, 88(1994)501. Wolf, R., Machtey, I., Feuerman, E.J., Creter, D., Acta Dermato-Venereologica, 61(1981)153. Yamamoto, M., Yorioka, T., Kawada, M., Nishimura, K., Kumon, Y., Yasuoka, N., Suehiro, T., Hashimoto, K., Japanese Journal of Nephrology, 36(1994)779. Yerov, N.K., Revmatologija, 2(1986)26 Zamulko, A.A., Fedorich, V.N., Amosova E.N., Vrach. Delo, 7(1991)147

245

Chapter 6

Surface tensiometry in pulmonology This chapter will deal with dynamic surfactometry in patients with respiratory diseases. The role of pulmonary surfactant for lung physiology is known for almost fifty years. Pulmonary surfactant can be regarded as stable complex compounds of lipoprotein origin, whose main surface active component is dipalmitoyl phosphatidyl choline (DPPC).

6.1. Pathogenesis of respiratory diseases The presence of surfactant in the alveoli leads to a decrease of the surface tensions which prevents the alveoli from collapsing, ensures the protection of walls from exudation, plays a defensive role against infiltration of micro-organisms and noxious substances, promotes the dissolution of oxygen during the diffusion into capillaries, and improves the circulation of blood (Pylishchev 1985, Pilipchouk et al. 1993, Wright 1997, Hills 1990). The activity of lung surfactant is higher than that characteristic of other surfactant present in the organism; they are responsible for the strong decrease of surface tension at the water/air interface close to zero, which ensures the extensibility and elasticity of the lung tissue. The in vivo studies of surface active properties of lung surfactant using the captive bubble

method were introduced quite recently. For an implicit investigation of the lung surfactant system and lung tissue homogenate, broncho-alveolar liquid and expired air condensate are used. Lung surfactant systems include extracellular material (surfactant) and cells which participate in its metabolism. The synthesis of lung surfactant happens within type I! epithelial pneumonocytes which are located in alveoli. In these type II cells, from which the secretion of lung surfactant is performed via exocytosis, the surfactant itself is stored within lamellar bodies (Taganovich 1996). In addition to lipids, specific proteins, surfactant protein SP-A, SP-B, SP-C and SP-D essentially constitutes pulmonary surfactant. The proteins are synthesised in the endoplasmic reticulum of the type II cells and accumulated within lamellar bodies. These proteins enhance

246 the adsorption of phospholipids in to the surface active film that floats on the liquid covering alveoli. The contents of proteins in lung surfactant is usually less than 20%, while the main lipid component is phosphatidylcholine. The most significant neutral lipid is presumably the cholesterol which constitutes some 10% of the total lung surfactant mass. Phospholipids contain relatively high amounts of phosphatidylglycerol (3-12%), while the dipalmitoyl molecular form is an essential constituent of phosphatidylcholine is the most abundant phospholipid molecule (54-87%) (Tanaka & Takei 1983, Shelley et al. 1984, Khan et al. 1985) The main constituent of lung surfactant proteins is the protein which possess molecular masses between 28 and 36 kDa, generally known as SP-A, and the multimeric protein with a molecular mass of 39 kDa, SP-D (Persson et al. 1990, Weawer & Whitsett 1991, Johannson et al. 1994). They acquire a negative charge in neutral media, and possess isoleucine at the aminoacyl end of their polypeptide chains. Enzymatic cleavage of SP-A with neuraminidase and a subsequent isoelectric focusing has shown that the different molecular masses of SP-A are due to posttranslational modification (Kuroki et al. 1988). Proteins with a molecular mass less than 10 kDa constitute a relatively small portion of the lung surfactant apoprotein components (ca. 1%) (Yu & Possmayer 1986). These components are called SP-B and SP-C. The synthesis of lung surfactant protein components is controlled by glucocorticoid hormones, estrogens, androgens, insulin and vasopressin (Liley et al. 1989, Wright & Hawgood 1989, Wirtz & Schmidt 1992). Intracellular processing of the primary translation product of the SP-A gene(s) results into a hydrophobic signal peptide, and the mature SP-A. SP-A can freely interact with lipids and accelerates the process of aggregation of fatty acid vesicles. The presence of this protein is necessary for the formation of tubular myelin, increases the absorption of liposomes, and inhibits the secretion of lung surfactant by type II cells. These effects require calcium ions. Surfactant proteins B and C also undergo a intracellular processing, resulting in the two mature proteins with molecular masses of 7 kDa and 5 kDa, respectively. Both proteins are necessary for the actualisation of surface active properties of dipalmitoyl phosphatidyl choline (Possmayer 1988).

247 Alteration of the lung surfactant system caused by pulmonary diseases result in a decrease of alveolar stability, wrong distribution of air flow between alveoli, a deterioration of gas exchange, and of the microcirculation, which is considered to be one of the important significant mechanisms leading to an aggravation of ventilation (Silber 1986, Taskar et al. 1997, Petty 1990, Mason 1987). The comparison of lung ventilation and gas exchange function with surface tension parameters of expired air condensate measured by the du Notiy ring method, and the contents of lipids in expired air condensate that may reflect the state of lung surfactant, was performed by Geltser et al. (1990), see Fig. 6.1.

0,8

-

0,6

-

/ft.

0,4o

O

0,20 -0,2 -0,4 -0,6 -0,8 -1CH

CHE

TG

DG

FFA

ST

Fig. 6.1. Correlation coefficients between lipid composition in expired air condensate and equilibrium surface tension and lung ventilation parameters. The lipid components are: C H - cholesterol; C H E - cholesterol ethers; TG - triglycerides; DG - diglycerides; FFA - free fatty acids; ST - surface tension; lung ventilation parameters are: hatched horizontal - vital lung capacity, black - forced expiratory forced volume, hatched - breathing volume; white - inspiratory reserve volume, light grey - maximum pulmonary ventilation, dark grey- partial oxygen pressure.

An inverse correlation exists between surface tensions of expired air condensate and the parameters of vital lung capacity, forced expiration volume, breathing capacity and inspiratory reserve volume. The same inverse correlation exists between respiration parameters and the contents of cholesterol, cholesterol ethers and free fatty acids in expired air condensate. However, the levels of triglycerides and diglycerides in expired air condensate directly correlate with lung ventilation function. The contents of particular lipid fractions in expired air condensate (cholesterol, cholesterol ethers, phospholipids and free fatty acids) of patients

248 suffering from acute pneumonia was similar to that of healthy persons in 39 %, while in 6 1 % increased concentrations of particular lipid fraction was found. In each fourth patient increased surface tensions were measured in expired air condensate. The lipid profile of expired air condensate, and thus of lung surfactant, undergoes changes with the age of healthy persons, as documented in Fig. 6.2. 800 700 - 600 L_____., ~ 500

"~ 4 0 0 300 O

200 100

I

0 <30

30-40

41-50

51-60

>60

Age [years] Fig. 6.2. Lipid composition of expired air condensate (mg/l) obtained from healthy subjects as a function of age, columns: l - total lipids, 2 - phospholipids, 3 - cholesterol, 4 - cholesterol ethers, 5 - triglycerides, 6 - free fatty acids, 7 - monodiacylglycerides. The main variation of lipid composition in expired air condensate during ageing is found in the level of fat, which is decreased in older healthy subjects resulting in an increase of surface tension of expired air condensate. Here only monodiacylglycerides and cholesterol do not comply with this trend: the first compound does not depend on age, and its concentration in expired air condensate remains relatively stable, while the contents of the second component increases in the older groups. The detected relations between the ventilation and gas exchange derangements, on one hand, and the metabolic variations on the other hand, reflect certain interrelations between the studied phenomena. It was argued that the increase in surface tension of expired air condensate together with the high contents of phospholipids and some fractions of neutral fats, indicate severity of lung surfactant alteration. High surface tension values of expirations cannot be

249 regarded as a contradiction while the contents of lipid surfactants shows a maximum. Pathological processes in the lungs increases some phospholipid fractions with high and decreases others with low surface activity (Ado et al. 1986, Silber 1986, Yuldashev et al. 1987, Bernhard 1997). The negative correlations that exist between surface tension of expired air condensate or some lipid content (cholesterol, cholesterol ethers, free fatty acids), and the pO 2 in the blood, could be related to the effect of lung surfactant on gas exchange. This effect is altered and characterises most respiratory diseases. The positive correlations that exist between the concentrations of diglycerides and triglycerides, respectively, and ventilation parameters and the PO2, show that the suppression of lipolytic processes in the lung surfactant system influences lung volume and bronchial permeability. This can be explained by a decrease in the sensitivity of the lung tissue and an improvement in the course of metabolic processes (e.g., due to the reduction in bronchiconstrictogenic effects of phospholipid lysoforms and arachidonic acid metabolites in lung surfactant). It was mentioned above that for surface tension studies either native biological liquids (lung tissue homogenate, bronchi-alveolar liquid, expired air condensate), or their surface active fraction can be used. The isolated fractions provide the 'purest' (mainly lipid-like) substances of the lung surfactant system. There exist two main approaches to the estimation of surface properties of lungs: the explicit approach is based on direct measurements of surface tension; the implicit approach relies on the variation in levels of main structural and functional components of the lung surfactant measured by biochemical analysis (Bestouzheva 1995). For the explicit evaluation of lung surfactant the Wilhelmy balance was used. This method showed that expired air condensate has a notable surface activity, and the results do not depend significantly on the amount of liquid used. The presence of lung surfactant in expired air condensate leads to lower surface tensions as compared with the effect produced by the presence of surfactant in broncho-alveolar lavage liquid. As expired air condensate contains very low, almost trace-like quantities of surfactant new methods are needed for the study of lung surfactant.

250 For the implicit evaluation of the lung surfactant system, usual biochemical approaches can be applied. These include methods for the determination of fat metabolism parameters using the integral test with Sudan dye, or the determination of separate lipid components using thin-layer chromatography. These are the fractions of neutral lipids, and not phospholipids, which are more accessible for the analysis. However, lipid components possess different adsorbtion behaviour with respect to dyes and, therefore, different intensities for equal lipid concentrations can be ambiguous. On the contrary, the initial total amount of phospholipids on the chromatogram of neutral lipids can include non-phospholipid fractions capable of increasing the density of an initial spot, resulting in an overestimation of the contents of total phospholipids. An important property of lipid surfactant is their capability for micellar solubilisation (dissolution), in particular, with respect to the Sudan dye. The colorimetric method for determining lung surfactant is based on this property. Here the measured value is the solubilisation ability of fats in expired air condensate: the higher the solubility, the higher is the surface activity of a lung surfactant (Khasina et al. 1989). The draw-backs of such methods is that rather large amounts of expired air condensate samples are required, and that it is specific to lipid components of the lung surfactant only. Surface active characteristics of expired air condensate monolayers spread onto a physiological salt solution were studied in a Langmuir trough (Kurik et al. 1987, Geltser et al. 1990). Prior to the measurements the condensate was mixed with isopropyl alcohol in a ratio 1:1 (thus ensuring a homogeneous distribution of expired air condensate monolayer), and then spread dropwise on the surface of the physiological subphase. Such spread layers decrease the surface tension from 72 mN/m down to 60 mN/m. There are other approaches to quantitatively estimate the contents of lipid fractions in expired air condensate. In particular, after a separation in thin layer chromatography, cholesterol and cholesterol ethers can be determined by the Liebermann-Burchardt method, while for basic phospholipids (lecithin, sphingomyelin, cephalin) the Morrison method can be used (Sidorenko et al. 1980, Franzini et al. 1990). The methods used in the studies of expired air condensate (Bestouzheva 1995) were those generally applied to blood serum and other biological liquids. It was found that these methods are by no means capable of quantitatively determining all

251 surfactant. A further extension of the range of detected physicochemical properties of expired air condensate and "missing" products of the metabolism, and basic reactions of analytical chemistry should be employed to provide new prospects for the theoretical disentanglement of mechanisms involved in the regulatory-excretory function of lungs. For patients suffering from chronic bronchitis, the total contents of lipids and cholesterol ethers in expired air condensate decreases in line with the extent of respiratory insufficiency. This dynamics of lipid composition is less characteristic of other components of lung surfactant, as it becomes visible from Fig. 6.3 800 700 -

~

600500 ,: :7 ..... ....

..::.,-

400-

~'i:'.i -':7: ...... i'i!:i .::::: ...... :S: .... ..:.:: .,. : ,,,., .,.

300O

200-

..... .... .::.:.: .: ,,: .,:::,

.

100 -

i'i ......i:i ...... .... ..... ......

_

[

TL

PL

CH

[

CHE

I

TG

I

FFA

MDG

Fig. 6.3. Lipid composition o f expired air condensate (mg/l) obtained from patients with chronic bronchitis and various extent o f respiratory insufficiency, and for healthy persons. TL - total lipids; PL - phospholipids; CH - cholesterol; CHE - cholesterol ethers; TG - triglycerides; FFA - free fatty acids; MDG - monodiacyl glycerides; hatched - no respiratory insufficiency, b l a c k - 1st degree of respiratory insufficiency, white 2 "ddegree o f respiratory insufficiency, grey - healthy persons.

It is quite obvious that an enhanced deterioration of respiratory functions affects the surface tension of expired air condensate, whose parameters are controlled by the level of lipid surfactant in the alveolar-bronchial lining layer. The expired air condensate is produced in part by filtration from the pulmonary circulation through the alveolar-capillary membrane and may be modified by various endogenous and exogenous mechanisms. Exercise reduces of the permeability at the blood/air interface. Furthermore exercise is characterised by complicated interrelations between the variations in plasma volume, the loss

252 of electrolytes and perspiration: the volume of plasma decreases, and the contents of potassium increased due to an adrenergic stimulation and the electrolytic disbalance characteristic of the acidosis. Exercise promotes alterations of the membranes, which results in an activation of peroxide oxidation of lipids, intensifies the expiratory loss of moisture (via the interrelation between the transport of water and protein), and the hampering of selective transfer of soluble substrates onto the alveoli surfaces, with a subsequent reduction of the amount of these substrates in the condensate. The activity of pulmonary surfactant affects expired air condensate, because the permeability of the alveolar capillary membrane and tile fluid balance between blood, the alveolar space and finally the expired air condensate is modified by pulmonary surfactant. The alveolar lining layer and the film of surfactant floating on this layer facilitates the transfer of water molecules from liquid to gaseous media, thus performing a drainage of the lung and preventing hyperhydration. The data which demonstrate a relationship between the age-specific dynamics of surface tension for expired air condensate, and the excretion of medium-size molecules, illustrating the blocking role played by pulmonary surfactant. The ability of lungs to excrete moisture plays a major role in the respiration physiology. A proper relative humidity of expired air is required to maintain the normal work of mucociliar clearance, while an excessive saturation by water vapour is observed for the increased permeability of capillaries of the pulmonary circulation, and is believed to be the precursor of the illness (Protsiuk & Pikas 1996). To understand the mechanisms involved in the transport function of the alveolar lining layer, the basic reactions used for the determination of biochemical parameters in expired air condensate need to be analysed. The composition of moisture expired by healthy persons depends on sex and age. For females, the amount of expired lipid peroxides, ceruloplasmin and malonic acid is significantly lower than for males, while the total contents of lipids, total contents of phospholipids, the contents of acylglycerides, free fatty acids, ammonia and pyruvate kinase are higher. The levels of these surfactant, and also of triacylglycerides, lactic and pyruvic acids decrease with the age, while an increase of surface tensions of expired air condensate, and the concentration of lipid peroxides are observed (Bestouzheva 1995). The parameters of lung surfactant in expired air condensate correlate with those in blood. The correlations are positive with respect to total lipids, cholesterol ethers and the products of

253 peroxide oxidation of lipids, and the correlation is negative for total phospholipids. A surface tension analysis of expired air condensate for healthy subjects shows the existence of direct correlations with the parameters of cholesterol, and an inverse correlation with total lipids, phospholipids and ammonia. Expired air condensate reflects adequately the biochemical composition of the liquid layer that covers bronchi of different generations and alveoli. For bronchial asthma, the level of calcium and magnesium in this biological liquid decreases. The endobronchial deficiency of bivalent cations for bronchial asthma can arise due to a hampered transport through the alveolocapillary barrier, and also because of an enhanced consumption by lung tissue (Fedoseyev et al. 1992). The former supposition can be supported by the fact that for patients with bronchial asthma, the expirate volume is 71% lower than for healthy persons (Yakovleva 1990, Antczak et al. 1997). The increase in the activity of ion transport through the mucous that coat epithelium of respiratory tracts due to the contact of this coat with an allergen was experimentally proven (Robinson et al. 1989, Sestini et al. 1990). The detection of particular phospholipids is sometimes impossible even in a large volume of expired air condensate; therefore it is often preferred to study lung broncho-alveolar lavage fluids sampled by using techniques. In addition, the analysis of expired air condensate allows conclusions about catabolic products of native, pulmonary surfactant. Nevertheless, these considerations cannot be regarded as detractions from the merits of using expired air condensate as a valuable and easily accessible material for of diagnostic and prognostic purposes in a number of lung diseases. One classic method for collecting of pulmonary surfactant is the broncho-alveolar lavage technique. Lung washing sampled using the technique could be further fractionated to isolate and purify the most surface active components in lung (Wright 1991; Bernhard & v o n der Hardt 1995). Histochemical analysis detects the existence of a hypophase at the alveoli surface, covered by a film that reassembles artificial phospholipid membranes. Three main components of lung surfactant in the bronchi-alveolar liquid (phospholipids, cholesterol and protein) were studied by Doyle et al. (1994). Close direct correlations exist between different parameters; moreover, fast changes in the relative proportions of surfactant were found to be caused by the respiratory load.

254 The utilisation of broncho-alveolar lavage in clinical practice enabled to perform extensive studies of the factors which provide local protection against various lung diseases. The protein to albumin ratio in bronchi-alveolar lavage liquid is significantly higher than in blood. Especially large values of the excretion coefficient were found for immunoglobulins-A (1 for monomer, and 20 for polymer). No selective influx of immunoglobulins-G into the bronchialveolar liquid happens under normal conditions, while for patients with lung diseases (cancer, tuberculosis, sarcoidosis, pneumosclerosis) an increased local synthesis of albumin takes place. For healthy subjects the concentration of immunoglobulins-M and ct2-macroglobulin in bronchi-alveolar liquid is lower than in blood, which supports the view that the principal mechanism for the influx of serum proteins into the bronchi-alveolar liquid is transudation, whose intensity is inversely proportional to the molecular mass of the proteins. For lung pathologies, the level of immunoglobulins-M and tx2-macroglobulin in bronchi-alveolar liquid increases 70 times. This fact is attributed to structural changes of the alveoli capillaries, the increase in diffusion of these macromolecules out of the blood, and/or the increase of the local synthesis and trans-epithelial transport (Titanian et al. 1987, Roche 1992, Out et al. 1991, Nagy et al. 1986). Eicosanoids, i.e. arachidonic acid metabolites, play an important role in the pathogenesis of chronic obstructive lung disease. Eicosanoids lead to the development of hyper- and dyscrinism, and also takes part in the formation of interstitial oedema and pulmonary hypertension. One of the most important mechanisms responsible for the hypersensitivity of bronchi exposed to various influences is the increase in the secretion of bronchi-constriction and inflammation mediators (leukotrienes, prostaglandins, thromboxane A2). Studies of bronchi-alveolar liquid for catarrhal forms of the bronchitis during the exacerbation period had shown the increased contents of leukotriene B4 for patients suffering from chronic obstructive bronchitis, as compared with chronic non-obstructive bronchitis (Efimov et al. 1990). A displacement is observed of the prostacyclin/thromboxane

equilibrium towards the

thromboxane A 2, which leads to strong bronchi-obturation effects. A decrease in the prostacyclin/thromboxane ratio happens mainly due to the increase in the thromboxanesynthetasous activity, while the prostacyclin concentration in the bronchi-alveolar liquid does not vary significantly.

255 Changes of the bronchial mucous lead to an increase in the amount of bronchial secretion and to variations of the rheological properties (viscosity, elasticity, adhesivity) of the secretion. This results in a deterioration of the efficiency of lung clearance, and consequently to the obturation of the respiratory tract. The adhesive properties of the sputum can be estimated from measurements of the normal force required to detach two glass plates covered with the sputum. The value of adhesive contact strength is proportional to the force of preliminary pressure exerted by the cover glass on the material studied. It was shown that the strength of adhesive contact is directly proportional to the time of pre-compression, and inversely proportional to the acceleration, i.e. the rate of increase of the force which destroys the adhesive contact (Punin et al., 1991). Good correlations were found between the adhesive properties of sputum and the contents of water fractions therein (direct correlation for bound water, and inverse correlation for free water). The high adhesion of sputum with high contents of bound water promotes the development of mucociliar insufficiency. The amount of bound water, or the hydration degree, has significant effects on the functional state of macromolecules, and governs their biophysical properties. Free water interacts with the electrolytes of the tissue, thus contributing to the physiological processes (Farashchuk 1989, Winters et al. 1997, Folkesson et al. 1994, Teubner et al. 1983). A reaction of the normal bronchial tree to unfavourable external factors is almost equal to an increase in the amount of glycoproteins and serum transudate into mucous membranes. However, changes in the type of glycoproteins take place and the amount of silated and sulphated groups, and their contents outgoing the gains of serum transudate increases, while the contents of the transudate becomes higher with the addition of infectious agents. Inflammatory processes are a common pathogenic pathway in many chronic non-specific lung diseases; these processes are accompanied by significant disturbances of the lipid metabolism in the organism, which lead to pathochemical variations in the lung tissue. The main role is believed to be played by structural components of cellular and sub-cellular membranes and the level of their functional activity. Lungs are capable of capture and zymolysis of the incoming blood lipids, by incorporating them into the surfactant. Hypoxia and bacterial infection of the

256 lung lead to deterioration in the synthesis of phospholipids and cholesterol, which in turn results in a decrease of their contents in blood. The qualitative composition of blood phospholipids present with chronic non-specific lung diseases is similar to that characteristic of healthy persons; these phospholipids are represented by phosphatidyl choline, sphingomyelin, lysophosphatidyl choline, phosphatidyl ethanol amine and phosphatidyl inositol. The relative amount of phospholipids in lung surfactant sharply increases together with the total lipid contents. Changes in the relative quantities of phospholipid fractions also take place: at low contents of phosphatidyl choline the concentration of sphingomyelin increases. This can be ascribed to the pathogenetic factors, because phosphatidyl choline inhibits collagen formation, while phosphatidyl choline participates in the destructions of the inflamed foci. Similar changes in the phospholipid metabolism are responsible for the deterioration of the permeability of capillary walls and cellular membranes, and contribute to structural changes, which is one of the factors which lead to a diffusive lipoidosis of lung tissue and cause the progressive development of the disease. The decrease in the phosphatidyl choline level can be explained by its weak synthesis in the lung (Ivanov et al. 1989). Lipoproteins are responsible for the transport of fatty acids in the bloodstream, which possess individual physico-chemical properties. Complex lipids are formed through the reaction of etherification of acids with alcohols. Pronounced steric differences between saturated and polyunsaturated fatty acids determine their structurisation of various complex lipids. Triacyl glycerides are the form by which the transport of saturated fatty acids is arranged (Titov 1996). Phosphatidyl choline, phosphatidyl serine, phosphatidyl inozytol, and phosphatidyl ethanol amine represent the functional series of complex lipids which have various degrees of saturation of the headgroups. The saturated and poly-unsaturated fatty acids, even in the form of complex lipids, transport various apoproteins in the bloodstream. There are different ways for the transport of f2-9, f2-6 and f2-3 families of polyene fatty acids in the bloodstream. Unsaturated f]-9 acids are first arranged by enterocytes into triacyl glycerides, then released from apo-B48 and apo-Bl00 lipoproteins during the lipolysis, and finally enter the cells in association with albumin. Polyene acids f2-6 are passively transported by high density lipoproteins, via the interaction of a-spiral chains of apo-Al with hydrophobic

257 clusters of the plasmatic membranes of the cells, and re-etherification of fatty acids from phospholipids of high density lipoproteins into membrane phospholipids. The transport of f2-3 polyene acids into cells is accomplished by an active receptor-mediated mechanism. The apolipoprotein-B100 ligand-receptor interaction is the main stage of the active transport of etherised cholesterol of poly-unsaturated fatty acids into the cells. In human blood, bacterial lipo-polysaccharides do not exist in a free state, but are associated with high density lipoproteins and low density lipoproteins. A lipo-polysaccharide molecule, being incorporated into lipoprotein particles, retains its ability for interaction with lipopolysaccharide cell receptors, and, in addition, acquires the capability for binding via apo-B/E-receptors. Lipo-polysaccharides, when existing in complexes with lipoproteins, introduce

modifications

into

the

lipid

metabolism.

The

properties

of

lipo-

polysaccharide/lipoprotein complexes are determined by its structure. The lipo-polysaccharide molecule interacts with the surface of lipoprotein particles through its segment, so called lipid A, with the hydrocarbon chains pointing towards the liquid phase. Lipid-A incorporates two D-glucosamine molecules, which are linked together by a 13(l_6)-glycoside bond, contain two phosphate groups and are etherised by six saturated fatty acids. When lipo-polysaccharides are incorporated into low density lipoproteins,

the proteinic composition of lipo-

polysaccharide/low density lipoprotein complexes (with dominating apo-B100) remains virtually unchanged, while variations in the contents of phospholipids (decrease of phosphatidylcholine concentration versus increase of phosphatidyl-ethanolamine portion) and neutral lipids (decrease of etherised cholesterol and increase of free fatty acids) contents take place (Viktorov et al. 1989). The lipo-polysaccharide/low density lipoprotein complex incorporates, on an average, 9 to 10 lipo-polysaccharide molecules per lipoprotein particle. The lipo-polysaccharide is incorporated into the lipid monolayer of low density lipoproteins, with six hydrophobic hydrocarbon chains of lipid-A immersed into this monolayer. Approximately one-half of the low density lipoprotein surface is occupied by the sub-units of one apo-B100 molecule. Phospholipids cover about 2/3 of that portion of the low density lipoprotein surface which is free from apo-B. The remaining 1/3 is occupied by molecules of free cholesterol and free fatty acids. As no phase segregation of lipids takes place, the surface concentration of free cholesterol in the

258 phospholipid monolayer is ca. 30%. Free cholesterol molecules do not affect the conformation of N+(CH3)3 groups at the surface of the particle, and interact directly with apo-B, because they are capable of binding to proteins. The total amount of phospholipids in lipo-polysaccharide/low density lipoprotein complexes is decreased, mainly due to a decrease in phosphatidyl choline. With the incorporation of lipopolysaccharide molecules, the packing density of lipids within the monolayer increases, and, as the size of the polar head of phosphatidyl choline molecules is quite large, some phosphatidyl choline molecules are ousted from the lipo-polysaccharide/low density lipoprotein complex. In addition, the incorporation of lipo-polysaccharides into the surface layer of low density lipoproteins affects the orientation of some fragments of apo-B, because their neighbourhood becomes more hydrophilic. Therefore, vacancies are created between the fatty acid chains of lipo-polysaccharide molecule, which can be filled by molecules of free cholesterol; this can explain the binding of lipo-polysaccharide with particular lipoproteins which possess higher amounts of free cholesterol. To summarise, the existence of a microbial flora in the respiratory system of patients with inflammatory lung diseases can affect the lipid composition of lung surfactant components significantly. This leads to variations in the surface tension of expired air condensate and bronchi-alveolar liquid. Certain interrelations exist between the state of the lung surfactant system and the composition of surface active substances in blood. Bacterial infections of the upper and/or lower respiratory tract produce marked changes in dynamic surface tensiograms of lung lavage and serum. 6.2. Bronchitis

The number of patients with bronchitis in this study was 56. 26 had chronic obstructive lung disease, 30 had non obstructive lung disease. The dynamic surface tensiograms of serum for chronic non-obstructive bronchitis is shown in Fig. 6.4. In general, increased surface tension parameters of serum in the short surface lifetime range, and a decrease of these values in the medium and long surface lifetime ranges, was observed for chronic non obstructive bronchitis. It should be pointed out, that these variations in surface tension parameters of serum are characteristic only for the acute phase of the disease, while

259 during remission the changes of surface tension are either less pronounced, or the parameters of surface tensiometry do not differ from those measured for healthy persons.

75

--

70-

65I......d

o~ OOo

60-

55

I -2

-1

0 lg(tef) Is I

1

2

Fig. 6.4. Example for serum tensiogram obtained from male patient, age 17, with chronic non-obstructive bronchitis and respiratory insufficiency I; dotted curve correspond to average values for healthy males of same age. For chronic obstructive bronchitis, both qualitative and quantitative changes in lung surfactant are more pronounced than for chronic non-obstructive bronchitis; therefore we can expect to find more significant variations also in surface tension parameters of serum. However, the mean values of 0-2 for serum remain within the normal range, while the values of 0-1 and 0"3 do not differ significantly from the reference values measured for healthy persons. This implies that an additional analysis of dynamic surface tensiograms of serum for patients with chronic obstructive bronchitis should be performed. With increasing age, the contents of total lipids, phospholipids, cholesterol ethers, triglycerides and free fatty acids in lung surfactant decreases with increasing total amount of cholesterol. At the same time, an increase in the concentrations of cholesterol, triglycerides, low density lipoproteins and very low density lipoproteins in serum takes place. These features of the lipid composition of blood lead to larger values of 0-2 and 0-3 for serum, as one can see in Fig. 6.5.

260

75

70 + ...........................

;~ 6 5 + oo . o . ~

~ 60 i 55

I

-2

-1

t

r

i

0

1

2

mg(t~f) [s] Fig. 6.5. Examples for serum tensiograms obtained from patients with chronic obstructive bronchitis and respiratory insufficiency II. Thick line - male, age 61; thin line - male, age 47; dotted lines correspond to average value for healthy males of the same age. It is to be noted that similar changes occur also for healthy persons. To demonstrate the influence of the age factor on the character of dynamic surface tensiograms, a group of patients of the same sex was chosen, with the same duration of the disease and severity of respiratory insufficiency. It was found that the duration of the disease also affects the surface tension parameters of blood serum; in particular, studies of patients with similar age and sex, but different time scales of chronic obstructive bronchitis lead to increases in the values of ~2 and o3 (cf. Fig. 6.6). No reliable correlations were found between surface tension parameters and the level of lipids in serum, therefore we conclude that changes found are related to other factors, e.g., to the pathomorphism of the disease caused by treatments performed earlier. In addition, chronic bronchitis leads to increases in the amount of immunoglobulins, C3r complement,

Cl-inactivator

and

C3-activator,

tx2-macroglobulin,

txl-antitrypsin

of the and

(zi-antichimotrypsin in blood serum (Lutoshkin et al. 1991, Prinsen et al. 1989, Dasgupta et al. 1986, Plusa et al. 1985), while for prolonged diseases a further significant accumulation of these proteins in the peripheral blood takes place, which affects the dynamic surface tensions.

261

74

--

71-

--o .... ....

;~

~176

-~176

68-

65

-o

62 -2

oo

oo

Ooo

I

I

I

I

-1

0 lg(tef) [s]

1

2

Fig. 6.6. Examples for serum tensiograms obtained from patients with chronic obstructive bronchitis and respiratory insufficiency II. Patients are males, age 61; thickness of lines is proportional to the duration of the disease; dotted line correspond to average values for healthy males of the same age.

10

0

-5 .~ 9

-10

-

r

-15 -20 -25 -30 ~1

~2

~3

)~

Fig. 6.7. Changes in surface tension parameters measured in serum obtained from patients with chronic obstructive bronchitis with various severity of respiratory insufficiency. Changes are given in % compared to corresponding healthy controls. Hatched - 1st degree of respiratory insufficiency, black - 2 nd degree, w h i t e - 3 rd degree.

262 The deterioration of respiratory functions for patients suffering from chronic obstructive bronchitis is combined with changes of the equilibrium surface tension and ~ of serum tensiograms, as demonstrated in Figs. 6.7 and 6.8. While for the 1st degree of respiratory insufficiency the equilibrium surface tension values are quite the same as those found in the reference group of healthy persons, the 2 nd degree and, especially, the 3 rd degree of respiratory insufficiency is accompanied with significant decreases of this parameter.

75 -7 70

--

~.,~65! t~ 60 55 50 ~ -1

t

~

-0.5

0

lg(tef) [S]

i

I

i

0.5

1

1.5

Fig. 6.8. Examples for serum tensiograms obtained from patients with chronic obstructive bronchitis. Patients are males, age 50 and higher; thickness of curves is proportional to the respiratory insufficiency degree, dotted line correspond to average values for healthy males of same age. The variations in ~, are still more pronounced. The decrease of the equilibrium surface tension of serum which accompanies the deterioration in the performance of the bronchi-alveolar system can be ascribed to the accumulation of some surfactants in blood; among other factors, this can be explained by changes of the lung surfactant. This supposition is based on the existence of an inverse correlation between the equilibrium surface tension of serum and expired air condensate for the respiratory insufficiency caused by chronic obstructive bronchitis. The decrease of equilibrium surface tension and ~, values of serum during the dynamic observations of such patients can be regarded as indications of an unfavourable prognosis for the development of the disease.

263 Figure 6.9 shows the correlations between some particular parameters of surface tension of serum for healthy subjects and patients suffering from chronic obstructive bronchitis (a group of screened males was taken as an example). While for healthy patients a direct dependence of ~, on tensiographic parameters in the short and medium time range exists, chronic obstructive bronchitis is characterised by strong negative correlations of L with the values of 0-1, 0-2 and 0-3; a similar dependence is observed for 0"3.

a) healthy males 0.9

.,o 0

c,.)

0

-

0.80.70.60.5 0.40.3 0.2 0.1

I

i, ,,,, ,,

i : ,.,

' :,i . . . .

: , ,: . . . . ,,. , : ,

, : ,,

0

- - -

-0.1

-

-0.2

0-1

0-2

0-3

b) chronic obstructive bronchitis _

0.8

-

0.6.~, 0.4o 0.2r,.) = 0 ~-0.2 -0.4 ~ 0 o -0.6

-0.8 -1 0-1

~2

o'3

Fig. 6.9. Correlations between various surface tension parameters in serum obtained from healthy males (upper graph) and patients with chronic obstructive bronchitis (lower graph), hatched -cr~, black - or2,white - o3, grey- ~,.

264 6.3. Bronchial asthma and other pulmonary diseases

We studied 19 patients with bronchial asthma, 7 patients with acute pneumonia, 9 patients with pulmonary abscess, 26 with chronic obstructive bronchitis, 30 with non obstructive bronchitis, 10 with idiopathic lung fibrosis. In bronchial asthma, the surface tension of blood decreases in the short surface lifetime range, while the value of L increases (in contrast to chronic obstructive bronchitis, where increased 0-~ and decreased ~, were found), see Table 6.1 and Fig. 6.10. Table 6.1. Differential diagnostic indicators of surface tension variation of serum for various lung diseases

Disease 0-1

0"2

0"3

Chronic non-obstructive bronchitis Chronic obstructive bronchitis Bronchial asthma Acute pneumonia Pulmonary abscess Idiopathic lung fibrosis + statistically significant increase of parameter compared to normal, -statistically significant decrease of parameter compared to normal

Such particular features of dynamic interface tensiograms are very important for the differential diagnosis of bronchial asthma and chronic obstructive bronchitis. Unfortunately, no difference was found between the dynamic surface tension parameters of serum for the infection-dependent form of bronchial asthma as compared to the atopic form, while certain direct correlations were found between equilibrium surface tensions and the severity of the disease. During the remission of the pathological process (especially for atopic bronchial asthma), the surface tension parameters of serum in fact return to their normal values, which is very important for monitoring the efficiency of a healing process.

265 The variations in the dynamic surface tensiometric parameters of serum for acute pneumonia and pulmonary abscess, see Fig. 6.11, are roughly the same, they are characterised by increased values of crl and decreased values of)~.

75

--

70~

w,,

?" 6 5 -

~

60-

t3

555045-1

I

I

-0.5

0

....

tg(t~f) [s]

i-

- - t

0.5

1

1.5

Fig. 6.10. Example for serum tensiogram obtained from patient (male, age 61) with infection-dependent bronchial asthma, dotted line correspond to average values for healthy males of corresponding age.

75

--

72~ ' 69

~ 66

~

~ 1 7 6O o o o

63 60 -2

~176 o

I

I

I

-1

0

1

........ - - - I 2

lg(te0 [S] Fig. 6.11. Example for serum tensiogram obtained from patient (male, age 61) with pulmonary abscess, dotted line correspond to average values for healthy males of corresponding age.

266 The surface tension variations in the short time range are visible even when the disease cannot be detected by X-ray methods. As the surface tension parameters of serum correlate with those of expired air condensate, it can be argued that only the return of surface tension parameters of serum can be indicative of a complete recovery. The shape of surface tensiograms of serum for patients suffering from idiopathic lung fibrosis, is rather similar to that characteristic of chronic obstructive bronchitis (see Table 6.1), however, the decrease in ~, becomes more significant. At first one expects that for these diseases pronounced differences should exist between the surface tension values of serum, rather than morphologically and pathogenically. However this is not the case, because variations in the surfactant composition of blood serum for both these diseases are quite similar. Qualitative and quantitative differences in the composition of lung surfactant for these pathologies are rather significant, which explains the differences in the composition of expired air condensate. The decrease in the level of phospholipids, cholesterol ethers and free fatty acids in expired air condensate for idiopathic lung fibrosis is much more pronounced than for chronic obstructive bronchitis, which leads to the sharp increase in the equilibrium surface tensions. The variations of surface tension parameters of serum for patients with various pathologies of the lung are summarised in Table 6.1 and Fig. 6.12. 40 -~

,

I

i

30 20 10-

-10 -20 -30 -40 CNB

COB

BA

AP

PA

IFA

Fig. 6.12. Changes in surface tension parameters measured in serum obtained from patients with various lung diseases. Changes are given in % compared to corresponding healthy controls. CNB- chronic nonobstructive bronchitis, COB-chronic obstructive bronchitis; BA-bronchial asthma; AP-acute pneumonia; PA - pulmonary abscess; IFA - idiopathic fibrosing alveolitis; hatched -gl, black - c2, white c3, grey - ~,.

267 Among all diseases, bronchial asthma is characterised by the lowest values of O'1 and (Y3, and increased )~-values. The unusual state of surface tensions for serum during the exacerbation of bronchial asthma is caused in part by peculiar features of the enzyme composition in serum. It is known that lysosomic enzymes are the inflammation mediators in bronchopulmonary diseases. These enzymes are soluble hydrolases, and they are most efficient in acidic medium. The characteristic feature of lysosomic enzymes is their capability of sedimentation (the formation of bonds with particles and creation of a non-penetrable membrane barrier which restricts the access to lysosomic enzymes, their heterogeneity and complex composition, they comprise cathepsins, nucleases, glucosidases, esterases, phosphatases, lipases, sulfatases, etc.), enabling the lysis of virtually any biopolymer to low-molecular products (Yakushin & Stroyev 1989). For chronic non-specific lung diseases, the highest activity in the lung tissue fraction is exhibited by acid phosphatase, 13-galactosidase, [3-N-acetylglucosaminidase,

DNA-ase,

RNA-ase. While the severity extent of inflammatory diseases of the lungs is not accompanied by a dependence on the acid phosphatase level, the concentration of enzymes in blood of patients suffering from bronchial asthma increases in line with the severity of the disease and reflects the degree of allergic reactions. Our data indicate that the presence of acid phosphatase in serum either significantly affects its surface tension, or acts as a marker of the changes in some other surfactants (of protein or lipid nature). For patients suffering from bronchial asthma an inverse correlation of the acid phosphatase concentration in blood with surface tension parameters in the short time range and tensiographic )~ values obtained with serum was detected. For chronic non-specific lung diseases, the fermental activity of acid phosphatase in bronchial contents is many times higher than in blood; this indicates the importance of lysosome structure variations just in the cells of the bronchopulmonary apparatus which releases this enzyme to the bronchial secretum. The concentration of acid phosphatase in the bronchialveolar liquid depends on the type of the inflammatory process which happens in the bronchial tree. The electronic microscopy of bronchi epithelium biopsies taken from patients suffering from chronic non-obstructive bronchitis and chronic obstructive bronchitis shows an increase of the total number of lysosomes with high contents of acid phosphatase for hypersecretoryresorptive types of this disease, and a decrease in the number of primary and secondary

268 lysosomes for atrophic and hyperplastic versions which are characterised by the planocellular metaplasia of the bronchial epithelium.

a) 10 1 0-]

-10 9

-2o

-

-30 -40 -50 61

o2

o3

%,

b) 4030 20 . .O , .,..,

10-

-10

ol

62

o3

~,

Fig. 6.13. Changes in serum surface tension parameters in patients with chronic obstructive bronchitis during treatment, a) Differences of surface tension parameters are given in % compared to healthy people (black - before treatment, white - atter treatment), b) Differences of surface tension parameters before and after treatment. For patients suffering from chronic non-specific lung diseases, the peroxide oxidation products of lipids affect the membrane-receptor system of the cell, and activate citodestructive processes. Studies of the lipid metabolism and peroxidation process showed that a sharp increase in the contents of hydroperoxides, malonic dialdehyde, medium mass molecules and other products of peroxide oxidation of lipids in blood takes place, with suppressed anti-

269 oxidant protection leading to a decreased level of superoxide dismutase (Silvestrov et al. 1991, Kurosawa et al. 1991, Sanz et al. 1997, Venge 1994). Such variations are mostly characteristic of patients suffering from chronic obstructive bronchitis. While high concentrations of the peroxide oxidation products of lipids in blood affect the surface tension value of serum (these data were obtained by the authors for some renal and articulation diseases), no clear dependence of dynamic tensiographic parameters of serum on the characteristics of peroxide oxidation of lipids for chronic non-specific lung diseases was found. Peroxidation of lipids is a general finding in patients with chronic obstructive lung disease. They are characterised by a deficiency of the anti-oxidant system. Treatment of patients suffering from chronic obstructive bronchitis therefore include the administration of vitamin E (a-tocopherol). In addition to this basic treatment patients received either broncholytics or broncholytics plus antibiotics. The combined therapy using the anti-oxidant (vitamin E) plus broncholytics or broncholytics and antibiotics restores the form of the serum tensiograms by increasing k - values (Fig. 6.13). is accompanied by a decreased equilibrium surface tension of serum. In patients who received antibiotics higher surface tensions in the short time range were found. Thus the dynamic surface tensiometry can be used for monitoring the treatment of chronic obstructive bronchitis. 6.4. Pneumoconiosis and other lung diseases caused by inhaled dust The changes in the lung surfactant system play a significant role in the development of dust induced lung diseases (Velichkovskij 1994, Schengrund et al. 1995, Lesur et al. 1994, Richards et al. 1984). The influence of various damaging factors (coal, silica, asbestos etc.) enhances the synthesis of lung surfactant and its secretion onto the alveolar surface, resulting in a decrease in surface tension of lung tissue homogenate, bronchi-alveolar lavage liquid and expired air condensate. The maximum amount of lung surfactant in the alveoli was observed during the periods of intensified lung parenchyma proliferation; this fact supports the theory of the protective role of lung surfactant in the formation of pneumosclerosis. The decrease in the production of phospholipids promotes the damage of the alveolar epithelium and fatty infiltration of lung tissue.

270 Compensatory-adaptive reactions of the lung surfactant system characteristic of dust exposed persons without initial lung diseases (healthy colliers) demonstrates an increase in the phospholipid amounts in expired air condensate and a decrease in the surface tension of expired air condensate. Initial forms of dust lung pathologies are observed with the increase in the surface tension of broncho-alveolar lavage liquid, accompanied by developing ventilatory and hemodynamic disturbances (Korzh & Fainerman 1997). A continuous inhalation of silica particles leads to the development of an alveolar lipoproteinosis with increased production of phosphatidylcholine, which can be regarded as a defence reaction against dust inhalation, and thus prevents epithelial cells from damage. At later stages of pneumoconiosis, smaller amounts of phospholipids are found in the lung surfactant. The surface tensions of bronchi-alveolar lavage liquid and expired air condensate, taken even from those colliers showing no roentgenologic signs of dust lung pathology are decreased. However, with the development of anthracosis and/or silicosis the surface tensions of these liquids increase due to lowered contents of phosphatidylcholine in the lung surfactant. The deficiency of phospholipids results in the formation of cholesterol deposits in the colliers' lung tissue, with maximum contents in the bronchi-alveolar liquid and lung tissue homogenate. The excess of phospholipids for patients suffering from chronic dust bronchitis leads to a hypercholesterolemia and increased excretion of cholesterol from lungs via the lavage liquid. Animal studies in experimental pneumoconiosis using Wistar rats revealed the following results. A compulsory inhalation of dust containing 29% of free silicon dioxide (SiO2) was performed for 2 hours 5 times a week over a period of 8 months. In these experiments, the first group of experimental animals were kept at 26-28~ 38-40~

while the second group were kept at

Every month a number of animals were taken out of the experiment under an ether

anaesthesia, and lung tissue homogenate was prepared (100 mg per 1 g of physiologic salt solution) and centrifuged (15 rain at 1500 rpm). In addition to surface tension measurements of the supematant liquid by using the MPT2 tensiometer, studies were also performed on the contents of lipids, peroxide oxidation of lipids and antioxidants.

271

a) temperature 26-28~

8o 1 60 40 i i i

20

"~

0 i

~ -20 -40 -60 ] -80 1

2

3

4

5

6

Time [month]

7

8

9

10

7

8

9

10

b) temperature 38-40~

120

1

I

100 80-

60= 40~, 9 20 0-

t____._a

-20 t -40 -60 1

2

3

4

5

6

Time [month]

Fig. 6.14. Changes of dry lung mass (black) and oxyproline contents in dry lungs (white) compared to healthy controls. Animals received compulsory inhalation (for details see text) and were kept at 26~176 (a) and at 38-40~ (b).

272

a) temperature 26-28~

10

9

8 \

6 I

4

..

v

\

I

~

2

\

t

m..

-.

// _.

o t

X

i

I

..

-I~

_

l

--B

//

/

l

-2 j 1

2

3

4

5 T~

6 [month]

7

8

9

10

b) t e n , e r a s e 38-40~

4

. " -' ~" : - 7 . . r

----~

/=

~ - ~

0

-

~--

,

~

~

/

~-~--.-

i,

'~

t

~

,

-2 1

2

3

4

5

6

7

8

9

10

Time [month]

Fig. 6.15. Changes in surface tension parameters of lung tissue homogenates of experimental animals after compulsory inhalation in dependence of time and temperature. Changes are given in % compared to corresponding healthy controls. + - 0.~,I - 0.2, Ai, - 0 3 The heated microclimate, while promoting a retardation of the development of the dust pathology in the early evolution period (due to the activation of adrenal cortex), subsequently enhances the fibrous degeneration of lungs. It has to be noted that the extent of pneumoconiosis

273 cases for colliers increases with the increase of ambient temperature; this fact can be explained by an increased amount of dust transferred to lungs due to more frequent and deep inhalation under heat. The combined action of dust and heat also leads to more pronounced pathological effects for experimental animals. After one month from the beginning of dust inhalation, the mass of lungs for "hot" rats was higher than for those kept under comfortable conditions. The adaptation reactions which develop under these conditions promote the increase of stresslimiting factors (glucocorticoid hormones etc.), which results in the decrease of the collagen amount in lungs (estimated from oxyproline concentration). After 6 months of the experiment, the mass of lungs decreases and becomes approximately equal between the two groups of rats, while the oxyproline dynamics shows opposite trends (Fig. 6.14).

Serum

Urine

I

1-

0.8

.~ o c,.) =

O

0.6 0.4 0.20---0.2 -0.4-0.6 -

l

l

l

-0.8 -It~l

c~2

t~3

t~l

~2

c~3

Fig. 6.16. Correlation coefficients between surface tension parameters of lung tissue homogenates and dry lung mass (black) or oxyproline contents in dry lungs (white) after silica inhalation in rats. With the increase of the experiment duration, the mass of lungs for animals subjected to dust aspiration at 26-28~

again increases, which can be regarded as a symptom of fibrous

degradation. At this time, the level of oxyproline increases for the two groups of animals. Subsequently a stabilisation of the pathological process was observed, and signs of recovery, if the damaging factors were abated. It has to be noted that in the first group of animals the mass of lungs correlates directly with the oxyproline contents, while for the second group an inverse relation is observed.

274 The dynamics of surface tension parameters for lung tissue homogenates during the experiment almost closely correspond to the changes in lung mass; this is especially true for the surface tensions at medium and long times (Fig. 6.15). At the same time, the contents of oxyproline was related more closely to values of 0"1, with a negative correlation for both groups of rats (cf. Fig. 6.16). For the experimental animals the surface tension parameters of lung tissue homogenate in the short and medium time range are characterised by significant interrelations, and in fact, determine the ~, value, which is inversely correlated with the equilibrium surface tension (cf. Fig. 6.17).

0.8 0.6 -

9~

0.4

O 0.2 =

__1__~~ _

1

0---0.2

O -0.4 -0.6 -0.8 -1 0"1

0"2

0"3

~,

Fig. 6.17. Correlation coefficients between various surface tension parameters measured in lung tissue homogenates obtained from animals with experimental pneumoconiosis. Hatched
0.1

and

0"3

values and the equilibrium surface tension

is closer for healthy rats. The combined action of dust and hot microclimate leads to an increase of the total amount of lipids, cholesterol and phospholipids in lung tissue homogenate already even one month from the beginning of the experiment, attaining its maximum after 6 months. After two more months, the total level of lipids decreases in respect to cholesterol, with a further increase in the contents of phospholipids. For animals subjected to a dust inhalation at 26-28~

the increase

275 of phospholipids in the lung tissue homogenate was observed after 8 months only. Therefore, the combined influence of the two damaging factors (dust and high ambient temperature) leads to earlier and more pronounced changes in the lung surfactant system. Moreover, a positive relationship between the level of phospholipids and oxyproline was observed in the second group only. Table 6.2 demonstrates the correlations between surface tension parameters of lung tissue homogenates for experimental animals, and the lipid contents. The surface tensiographic parameters depend mostly on the concentration of cholesterol; the values of Crl and or2 are related to phospholipids, and tyl depends on the contents of total lipids. Table 6.2. Correlation coefficients between surface tension parameters measured in lung tissue homogenates obtained from animals with experimental pneumoconiosis and serum components

Serum component _ l

CYl

Total lipids

~2

0"3

1'1'

Cholesterol Phospholipids

.........1'1' ....

Dien conjugate $

Mal0nic dialdehyde Cataiase

r162162

Superoxide dismutase Glutation peroxidase

1"

t positive correlation; ,!, negative correlation; empty - no correlation r<0.3" one symbol - r = 0.3 to 0.5; two symbols - r = 0.5 to 0.7; three symbols - r > 0.7

Rather unexpectedly, we obtained negative correlation coefficients between surface tension parameters and cholesterol, while for the correlation between surface tension parameters and total amounts of lipids or phospholipids these values turn out to be positive. It should be remembered that the levels of all the three lipid surfactant in lung tissue homogenate are mutually dependent; in addition, for animals with pneumoconiosis the phospholipids are surface-inactive substances. It is quite possible that dynamic surface tensiograms, which

276 comprehensively reflect the complex qualitative and quantitative changes in the lung surfactant system, can depend on the presence of other surfactant. In addition, phospholipids and other surfactant can acquire specific properties when influenced by damaging factors such as silica dust. This hypothesis is supported by the data obtained from studies of intact animals, where an inverse correlation was found between the amount of phospholipids in lung tissue homogenate, and surface tension values. From a hysto-morphologic and functional point of view, lungs are a giant membrane. Therefore, membrane-dependent processes are extensively studied, in particular, peroxide oxidation of lipids and the lung anti-oxidant system. Peroxide oxidation of lipids is accompanied by a decelerated function of lung 13-adrenergic structures, which leads to a deterioration of the bronchial penetrability. Enhanced peroxidation processes affect the metabolism related to the exchange of arachidonic acid. Hypoxia leads to the oxidation of lipids by free radicals in the lung tissue, and increased amounts of arachidonic, oleic and linolenic acid due to the activation by phospholipase A. Therefore, the parameters of peroxide oxidation of lipids in expired air condensate can be used as rather simple and quite objective measures to evaluate damages of the lung surfactant system (Chyshiktuiev et al. 1990, Haddad et al. 1997, Hallman et al. 1996). This can be explained by the fact that the lungs are the only organ which continuously and directly interacts via the lung surfactant system with air which contains oxygen and other initiators of peroxide oxidation. However, in some studies doubts have been expressed about the possibility of an unambiguous determination of the products of peroxide oxidation of lipids in expired air condensate (Aleksandrov et al. 1992). The properties of lung surfactant are determined by its specific lipid composition. In lung surfactant the processes of peroxide oxidation of lipids are induced by contact with ozone, nitrogen oxide and other reactive constituents of air. Forced respiration with resistance in the aspiration phase is accompanied by loss of mono-unsaturated phospholipids. Not only aerodynamic factors, but also the process of peroxide oxidation of lipids controls the rate of synthesis, secretion and degradation of lung surfactant (Verbolovich et al. 1985, Haddad et al. 1993). A possible formation of substances which can react with thiobarbituric acid was investigated using refined lung surfactant for either spontaneous peroxidation (incubation in the media

277 containing no initiators of peroxide oxidation of lipids) or peroxidation induced by ascorbate and nicotinamide dinucleotide phosphate (NADP). The rates of NADP-dependent processes are essentially higher than those dependent on ascorbate. With respect to the level of spontaneous peroxide oxidation, human lung surfactant are less stable than those of experimental animals. The intensity of spontaneous and induced peroxide oxidations of lipids in the surfactant and microsomal fraction of lungs are comparable to each other; however, the resistance of lung surfactant against peroxidation is somewhat higher as compared with membranes of the endoplasmatic reticulum (this can possibly be explained by higher amounts of non-saturated fatty acids). The biological role of lung surfactant oxidation by free radicals is the promotion of the physiological degradation of polyenol phospholipids. Under pathological conditions these processes can cause a major destruction of lung surfactant (Gusev & Danilovskaya 1987, Kolodub et al. 1993, Rtistow et al. 1993). The presence of superoxide dismutase, glutathione peroxidase and glutathione reductase was detected in lung tissue homogenates; these substances were interrelated with each other. Active processes of fermentative peroxide oxidation of lipids should correspond to a more efficient system of enzyme antioxidants. This is especially important for lungs, because the antioxidant activity of the extract from lungs is significantly lower than that from other organs (liver, kidney). Even at the very beginning of a compulsory inhalation of dust by experimental animals, variations in the processes of peroxide oxidation of lipids in lung surfactant were observed, with an activation at high ambient temperatures, and a suppression under more comfortable conditions. Subsequently the processes of peroxide oxidation of lipids were activated also for the first group of animals (comfortable ambient conditions); the amount of dien conjugates in lung tissue homogenate attains its maximum after 6 months, and malonic dialdehyde after 8 months start of experiments. The contents of dien conjugates does not affect the parameters of dynamic surface tensiograms for lung tissue homogenate, whereas the level of malonic dialdehyde exhibits weak positive correlations with the parameter ~1 (Table 6.2). The values of tyl positively correlate with the total contents of lipids and phospholipids in lung tissue homogenate, while no statistically

278 reliable data can be presented for a correlation between the concentrations of lipids and malonic dialdehyde. To investigate the regulation mechanisms of peroxide oxidation of lipids in experimental animals, the state of the lung antioxidant system was studied. After one month from the beginning of the compulsory inhalation of dust, a decrease in the coefficients of anti-oxidation activity was observed. For the first group of rats after 6 months this effect leads to an additional mobilisation of endogenous antioxidants and a stabilisation of processes of peroxide oxidation of lipids. For the second group of experimental animals, a similar dynamics was observed at later times, because in this case a more intensive generation of active oxygen forms are present, thus requiring greater amount of antioxidants for their neutralisation. The activity of superoxide dismutase at the beginning of the experiment decreases sharply, which can be explained by the intensive production of superoxide anion-radicals (O 2-) by phagocytes of the lung tissue. The amount of hydrogen peroxide (H202) thus produced in rats subjected to dust inhalation at 26-28~

is much lower, and therefore the hydrogen peroxide

becomes more accessible to the action of glutathione peroxidase. The activity of catalase in lung tissue homogenate of the first group of animals remains unchanged; this can be explained by the fact that catalase is "not involved" in the neutralisation of hydrogen peroxide. In the second group the level of glutathione peroxidase is quite similar to that in the control group of animals (intact rats), while an unambiguous trend towards the increase of the amount of catalase was observed. This fact indicates high concentration of H202 in the lung tissue. The activity of the ferments during the experiment was found to be flexuous, which reflects the existence of different phases in the development of the adaptation response of the rat organism to the action of damaging factors. Note that oscillations of superoxide dismutase were observed for the first animal group only. After six months from the beginning of the compulsory inhalation of dust, the level of this enzyme in the lung tissue homogenate attained its maximum, while the activity of glutathione peroxidase in this period was lowest. There exists a pronounced negative correlation between the contents of superoxide dismutase and glutathione peroxidase in lung tissue homogenate, and the values of or2, see Table 6.2. It is interesting that the value of ~, strongly and negatively depends on c2, while no correlation between ~ and superoxide dismutase or glutathione peroxidase was found. In fact, surface

279 active properties of catalase and superoxide dismutase are significant in the time ranges t - 0.01 s and t -- 1 s, respectively. Therefore, the existence of pronounced correlations between the amount of enzymes in lung tissue homogenate and dynamic surface tension parameters indicate that anti-oxidant enzymes, acting as the markers of the activity of other systems (e.g., peroxide oxidation of lipids), can be supposed to determine implicitly the dynamic surface tensions of lung tissue homogenate. Treatment methods of bronchopulmonary diseases (including those of dust aetiology) using corrections of the lung surfactant system become increasingly popular. Prescriptions of essential phospholipids and substances which enhance the synthesis of lung surfactant are believed to be helpful in this regard. With respect to the state of the processes of peroxide oxidation of lipids and anti-oxidant activity in lung tissue homogenate, the observed effects in animals suffering from experimental pneumoconiosis using the following drugs for the correction of deviations seem to be rather interesting: lipocerebrin (as the source of exogenous phospholipids), choline chloride (the donator of methyl groups and participant in the synthesis of lung surfactant phospholipids, in particular, of the phosphatidyl choline fraction), N-metoxybenzoyl- 1-(indolyl-3)- 1,2-dihydroisoquinoline (Mb) and di-3-5-methylpyrazolyl-4selenide (Mp). Due to its high hydrogen electron donating ability, Mb inhibits the peroxide oxidation of lipids, while Mp possesses a wide range of biological activity, including anti-oxidation. The increase in the level of phospholipids in lung surfactant is both a symptom of the development of fibrous degradation in the lung tissue, and a protective response of the lung surfactant system to dust particles entering the alveolar space. The amount of collagen in lungs increases in this case which is also characteristic of rats which were given, Mp. Continued application of lipocerebrin produces favourable effects on the lung surfactant system, leading to a reduction in the surface tension of lung tissue homogenate. Exogenous essential phospholipids cause a decrease of surface tension at t - 0.01 s and t - 1 s. The administration of choline chloride is accompanied by a decrease of the equilibrium surface tension of lung tissue homogenate. The impact of choline chloride on the lung surfactant system depends on the time of its first administration, and on the duration of the course: for example, the administration of choline chloride during the period of compulsory inhalation of dust leads to an increased concentration of surfactant and enhances their adsorption, while the same

280 medication used after this compulsory inhalation period results in an inverse, non-favourable effect.

a) 15 10

F

5 0

-10 -15 I 2

_

II

III

IV

V

VI

b)

I

1.8 -4! 1.6 4 1.4

1.2 i

'< 0 . 8 1 0.6 0.4 0.2 l 0 --

I

II

III

IV

V

II VI

Fig. 6.18. Treatment effects on surface tension parameters measured in lung tissue homogenates obtained from animals after 3 month of silica inhalation. I -no treatment; II -treated with choline chloride during the compulsory inhalation period only; III- continuously treated with choline chloride; IV- treated with lipocerebrin; V- treated with N-metoxy benzoyl-l-(indolyl-3)-l,2-dihydroisoquinoline; VI- treated with di-3-(5)-metylpyrazolyl-4-selenide. The upper graph gives differences to healthy control in % for o~ (hatched), o2 (black) and ~3 (white). The lower graph gives k ratios between silica inhaled and healthy animals. The application of Mb decreases the adsorption, i.e. leads to an increase in the equilibrium surface tension. Mp decreases the level of those surfactants which determine the parameters of

281 surface dynamic tensiograms in the short- and medium surface lifetime range, thus increasing the values of cyl and (3"2 (of. Fig. 6.18). It follows that the negative effect produced by Mb and Mp on the state of lung surfactant which results in an increase of the surface tension of lung tissue homogenate prevents these antioxidants for therapeutic purposes. Here choline chloride and, especially, lipocerebrin should be preferred. To summarise, the above discussion was restricted to studies of dynamic surface tensions of serum for various pathologies of the respiratory organs, and lung tissue homogenate for animals with experimental pneumoconiosis. In future studies it is planned to apply bubble pressure tensiometry to determine parameters of dynamic surface tensiograms of bronchialveolar liquid, expired air condensate, sputum and pleural liquid for patients suffering from infectious inflammatory and autoimmune diseases of the bronchopulmonary system, bronchial asthma, lung tumours, pneumoconiosis of various geneses etc. It should be especially interesting to determine which surface active and inactive substances affect surface tension parameters of biological liquids, and to estimate trends in the changes of surface tensiograms which take place in the course of various treatment methods. In this regard, comparative studies of dynamic surface tensions of expired air condensate for practically healthy persons of various age and living in various environmental conditions (in particular, in ecologically unfavourable regions) can be considered as an initial step. Dynamic surface tensiometry of expired air condensate has the potential of providing new information concerning the state of the bronchopulmonary system. 6.5. Summary To summarise, the above discussion was restricted to studies of dynamic surface tensions of serum for various pathologies of the respiratory organs, and lung tissue homogenate for animals with experimental pneumoconiosis. In future studies it is planned to apply bubble pressure tensiometry to determine parameters of dynamic surface tensiograms of bronchialveolar liquid, expired air condensate, sputum and pleural liquid for patients suffering from pneumonia and autoimmune diseases of the bronchopulmonary system, from bronchial asthma, lung tumours, and pneumoconiosis of various types. It is of special interest to determine which surface active and inactive substances affect surface tension parameters of biological liquids,

282 and to estimate trends in the changes of surface tensiograms which take place in the course of various treatment methods. In this regard, comparative studies of dynamic surface tensions of expired air condensate obtained from healthy persons will clarify the influence of age and environmental conditions (in particular, in ecologically unfavourable regions) on the pulmonary surfactant system. Dynamic surface tensiometry of expired air condensate has the potential of providing new information concerning the state of the bronchopulmonary system using a non-invasive sampling technique. 6.6. References

Aleksandrov, O.V., Dobrinina, O.V., Sevrunova, O.A., Ther. Arch. (Therapeutic Archive), 10(1992)105. Antczak, A., Nowak, D., Shariati, B., Krol, M., Piasecka, G., Kurmanowska, Z., European Respiratory Journal, 10(1997) 1235. Bernhard, W., Haagsman, H.P., Tschernig, T., Poets, C.F., Postle, A.D., van Eijk, American Journal of Respiratory Cell & Molecular Biology, 17(1997)41. Bernhard, W., vonder Hardt, H., Appl. Cardiopulmon. Pathophysiol., 5(1995)6. Bestuzheva, S.V., Clin. Labor. Diagnost., 3(1995)32. Chyshiktuiev, B.S., Ivanov, V.N., Solovieva, N.V., Zhiz, M.S., Laboratornoe delo (Laboratory work), 5(1990) 18. Dasgupta, D.J., Singhal, S.K., Goyal, A., Singh, A., Mohil, M., Sooch, N., Gupta, Journal of the Association of Physicians of India, 34(1986)503. Doyle, R., Barz, N.A., Orgeig, S., Amer. J. Breath. Reanimatol., 149(1994) 1619. Efimov, V.V., Blazhko, V.I., Voiekova, L.S., Ther. Arch., 4(1990)94. Fedoseyev, G.B., Emelyanov, A.V., Goncharova, V.A., Ther. Arch., 12(1992)58. Franzini, C., Luraschi, P., Journal of Clinical Chemistry & Clinical Biochemistry, 28(1990)913. Geltser, B.I., Khasina, M.A., Sobina, A.I., Ther. Arch., 12(1990)20.

283 Gusev, V.A., Danilovskaya, E.V., Voprosy Medicinskoi Kchimii (The Questions Of Medical Chemistry), 5(1987)9. Haddad, I.Y., Ischiropoulos, H., Holm, B.A., Beckman, J.S., Baker, J.R., Matalon, S., American Journal of Physiology, 265( 1993)555. Haddad, I.Y., Nieves-Cruz, B., Matalon, S., Journal of Applied Physiology, 83(1997)1545. Hallman, M., Bry, K., Seminars in Perinatology, 20(1996)173. Hills, B.A., British Journal of Anaesthesia, 65(1990) 13. Ivanov, E.M., Novogorodzeva, T.P., Endakova, E.A., Svetaschev, V.I., Ther. Arch., 3(1989)94. Johalmson, J., Curstedt, T., Robertson, B., Eur. Respir. J., 7(1994)372. Khan, A.Q., Sikpi, M.O., Das, S.K., Lipids, 20(1985)7. Kolodub, F.A., Cleiner, A.I., Makotchenko, V.M., Medicina Truda i Prom. Ecol., 1112(1993)31. Korzh, E.V., Fainerman, V.B., Vrach. Delo, 2(1997)54. Kuroki, Y., Mason, R.J., Voelker, D.R., J. Biol. Chem., 263(1988)3388. Kurosawa, M., Kobayashi, H., Kobayashi, S., Nakano, M., Allergy: European Journal of Allergy & Clinical Immunology, 46(1991) 173. Lesur, O.J., Mancini, N.M., Humbert, J.C., Chabot, F., Polu, J.M., Chest, 106(1994)407. Liley, H.G., White, R.T., Warr, R.G., J. Clin. Invest., 83(1989)1191. Lutoshkin, S.F., Makarovski, V.V., Silvestrov, V.P., Vrachebnoe Dielo (Medical Work), 9(1991)47. Mason, R.J., European Journal of Respiratory Diseases - Supplement, 153(1986)229. Nagy, B., Marodi, L., Jezerniczky, J., Karmazsin, L., Acta Paediatrica Hungarica, 27(1986)205. Out, T.A., van de Graaf, E.A., van den Berg, N.J., Jansen, H.M., Scandinavian Journal of Immunology, 33(1991)719. Persson, A., Chang, D., Crouch, E., J. Biol. Chem., 265(1990)5755.

284 Petty, T.L., Disease-A-Month, 36(1990)1. Pilipchuk, N.S., Prokchorovich, I.V., Procyuk, R.G., Pilipchuk, V.N., Ukr. Pulmon. J., 1(1993)63. Plusa, T., Tchorzewski, H., Allergie und Immunologie, 31(1985) 169. Possmayer, F.A., Amer. Rev. Resp. Dis., 138(1988)990. Prinsen, J.H., Schweisfurth, H., Rasche, B., Breuer, J., Clinical Physiology & Biochemistry, 7(1989)198. Procyuk, R.G., Pikas, O.B., Vrach. Dielo, 7(1996)37. Punin, A.A., Diakov, M.Y., Faracshyuk, N.F., Ther. Arch., 3(1991)37. Pylishchev, V.V., Problemy Tuberculosa (The Problems Of Tuberculosis), 10(1985)18. Richards, R.J., Curtis, C.G., Environmental Health Perspectives, 55(1984)393. Robinson, N.P., Kyle, H., Webber, S.E., Widdicombe, J.G., J. Appl. Physiol., 66(1989)2129. Roche, W.R., Journal of Clinical Pathology, 45(1992)46. Roche, W.R., Journal of Clinical Pathology, 45(1992)46-8. Rtistow, B., Haupt, R., Stevens, P.A., Kunze, D., American Journal of Physiology, 265(1993)133. Sanz, M.L., Parra, A., Prieto, I., Dieguez, I., Oehling, A.K., Allergy: European Journal of Allergy & Clinical Immunology, 52(1997)417. Schengrund, C.L., Chi, X., Sabol, J., Griffith, J.W., Lung, 173(1995)197. Sestini, P., Bienenstock, G., Crowe, E., Amer. Rev. Resp. Dis., 141(1990)393. Shelley, S.A., Paciga, J.E., Batis, J.M., Lipids, 19(1984)857. Sidorenko, G.I., Sborovsky, E.I., Levina, D.I., Ther. Arch., 3(1980)65. Silvestrov, V.P., Nikitin, A.V., Chesnokova, I.V., Ther. Arch., 12(1991)7. Taganovich, A.D., Pulmonology, 2(1996)45. Tanaka, Y., Takei, T., Chem. Pharm. Bull., 131(1983)4091.

285 Taskar, V., John, J., Evander, E., Robertson, B., Jonson, B., American Journal of Respiratory & Critical Care Medicine, 155(1997)313. Titanian, A.S., Selickaya, R.P., Kim, A.Ch., Ther. Arch., 12(1987)48. Titiov, V.N., Ukr. Cardiol. J., 3(1996)66. Velichkovskij, B.T., Medicina Truda i Prom. Ecol., 5(1994)3. Venge, P., Allergy Proceedings, 15(1994)139. Verbolovich, V.P., Petrenko, E.P., Podgomyi, Yu.K., Voprosy Medicinskoi Kchimii, 5(1985)65. Viktorov, A.V., Gladkaya, E.M., Yurkiv, V.A., Biokchimia, 54(1989)549. Weaver, T.E., Whitsett, A., Biochem. J., 273(1991)249. Wirtz, H., Schmidt, M., Clin. Investing., 70(1992)3. Wright, J.R., Ann. Rev. Physiol., 53(1991)395. Wright, J.R., Hawgood, S., Clin. Chest. Med., 10(1989)83. Wright, J.R., Physiological Reviews, 77(1997)931. Yakovleva, O.A., Ther. Arch., 3(1990)102. Yakovleva, O.A., Thertyshnaya, E.V., Ther. Arch., 12(1991)11. Yakushin, S.S., Stroyev, E.A., Ther. Arch., 12(1989)130. Yu, S.-H., Possmayer, F., Biochem. J., 236(1986)85.

286

Chapter 7

Surface tensiometry in neurology In modem neurology, cerebrospinal fluid and blood are widely used as diagnostic material. Cerebrospinal fluid protects the brain from physical injuries, maintains a stable intracranial and osmotic pressure in brain tissue, participates in metabolic processes, neurohumoral and neuroendocrinal regulation, and reacts with the compensatory-protective mechanisms during central nervous system diseases. Any pathological processes in the central nervous system are accompanied by changes of cerebrospinal fluid. The changes in composition and properties of surfactants contained therein and its impact on the dynamic surface tension characteristics will be described in this chapter

7.1. Tensiogram parameters for diseases of the nervous system The results obtained in comprehensive studies of dynamic surface tension of cerebrospinal fluid and serum obtained from patients suffering from various diseases of the nervous system are summarised in Table 7.1. In the control group were included patients without damage of the nervous system. Table 7.1. Differential diagnostic indicators of surface tension variation of biological liquids for various types of nervous system diseases

Nervous system disease type Liquor

Serum

ol

02

03

Z.

13"1

Spondylogenic

0"3

+

+

+

+

+

+

+

+

+

Infection Vascular

0"2

+ +

Neoplasm Trauma

+

+

+

" + " - statistically significant increase of parameter compared to normal; "-"-statistically significant decrease of parameter compared to normal

287 All patients were subdivided into the following groups: 1st group: 49 patients with infection (encephalitis, meningoencephalitis, arachnoiditis, myelitis, polyneuritis and slow infections - encephalomyelitis and multiple sclerosis); 2 nd group: 27 patients suffering from vascular brain diseases (discirculatory encephalopathy and acute brain circulatory disturbances); 3rd group: 41 patients with spondylogenic diseases (spinal osteochondrosis, radiculitis, vertebrogeneous myelopathy, spondylolisthesis); 4th group: 36 patients with neoplasms of the nervous system (accusticus neurinoma, meningioma, tumours of cerebellum, fourth ventricle, posterior cranial fossa, trunk and spinal cord), 5th group: 38 patients with traumatic brain damages (contusions of various degree of severity). The control group consisted of patients with diseases involving no damage of the nervous system

(vascular

dystonia,

Harris

facial

sympathalgy, porencephalic

cyst,

residual

encephalopathy with liquor-vascular discirculation). Averaged values of dynamic tensiographic parameters of cerebrospinal fluid for the control group (16 patients) were:

0"1 -"

71.7 mN/m, 0"2 = 66.6 mN/m,

0"3 ---- 60.4

mN/m and ~. = 7.6 mN.ml.s 1/2.

For infectious, tumour and trauma, the general feature is a decrease of blood serum. A decrease of

0"3

0"2

and

0"3

values for

of serum is also observed for spondylogenic diseases, while 0"2

tends to increase (Fig. 7.1). Some increase of all surface tension characteristics of serum was found for patients suffering from vascular brain diseases. It is to be noted that only a pathological process in the spinal column results in a significant increase of the ~ value for serum (Figs. 7.2 and 7.3). While infection lead to the decrease of all serum surface tension parameters, vascular diseases are accompanied by their increase (however, statistically unreliable). Examples of dynamic serum tensiograms for patients with infection are shown in Figs. 7.4 - 7.8.

288

a) serum 4 1

7

J 0 i.

.

.

.

.

.

.

.

-6 -8 ~

-10 1

2

3

4

5

b) cerebrospinal fluid 12 10

6

f I

2 O~ -2 A -41

2

3

4

5

Fig. 7.1. Changes in surface tension parameters measured in biological liquids obtained from patients with nervous system diseases. Changes are given in % compared to corresponding healthy controls. 1 - infection; 2 - vascular diseases; 3 -spondylogenic diseases; 4 -neoplasm; 5 -trauma; hatched -o~, black - 02, white - 03.

289

251

Blood

Liquor

20

-~

15

10

1

2

3

4

5

6

7

1

2

3

4

5

6

Fig. 7.2. )~ values of serum and liquor tensiograms obtained from patients with various nervous system diseases and from control groups. 1-infection; 2 - vascular diseases; 3-spondylogenic diseases; 4-neoplasm; 5 -trauma; 6 - diseases involving no affections of nervous system; 7 - healthy persons.

Serum

140 l

Liquor

120 100 80 60 40 20 0 -20 J

1

2

3

4

5

1

2

3

4

5

Fig. 7.3. Changes in ~, values of serum and liquor tensiograms obtained from patients with various nervous system diseases. Changes are given in % compared to healthy controls. 1-infection; 2- vascular diseases; 3 -spondylogenic diseases; 4 -neoplasm; 5 -trauma

290

75

--

7~ ....................................................................

~,65

60 ~55 50-45 -+

i

-2

-1

1

-

t

0

1

lg(te f) [S] Fig. 7.4. Example for serum tensiogram obtained from patient (female, age 29) with sub-acute polyradiculoneuritis (female, age 29); dotted line correspond to average values for healthy females of corresponding age

7 5 -~

70 -~ ......

~ 1 7 .6.

~.

~17~ 6 .....

o . . . . . . . o-o..

65--

~176176 oo,,. ~176176 ~176176176176176

;~ 60 -

t

50

45

t .

-2

-1

.

.

.

t

t

1

0

1

2

lg(tef ) [S] Fig. 7.5. Example for serum tensiogram obtained from patient (male, age 27) with slow encephalomyelitis; dotted line correspond to average values for healthy males of corresponding age

291

7472 70

~

66~176 o

64-

_

o

oo

oo 00

62-

6~176

60-

I

-2

-1

1

I

0

1

lg(tef) [S]

Fig. 7.6. Example for serum tensiogram obtained from patient (male, age 35) with spinal osteochondrosis and disk prolapse at L5-S 1" dotted line correspond to average values for healthy males of corresponding age

75

-...........

o.. ....

o~176

70~176

o

~65

~ 1 7 6 1 7 6~ 1 7 6 1 7 6

6055-2

-1

0 lg(tef) [S]

1

2

Fig. 7.7. Example for serum tensiogram obtained from patient (female, age 69) with spinal osteochondrosis and vertebrogeneous myelopathy; dotted line correspond to average values for healthy females of corresponding age

292

75-70

- ...................................................................

65 .

.

.

.

.

.

.

.

.

.

.

.

~'60 ;~55 § 50 45 40

+ -2

-1

lg(t~f) [s]

+

......... ' .........

0

1

Fig. 7.8.. Examples for serum tensiograms obtained from two patients with trauma. One with open penetrating severe craniocerebral trauma, haematoma of left frontal, parietal and temporal region (male, age 56, thick curve), one with closed craniocerebral trauma, brain contusion, sub-arachnoidal haemorrhage (male, age 47, thin curve); dotted line correspond to average values for healthy males of corresponding age Our results that infectious disease lead to decreased surface tension parameter are supported by studies of other physicochemical and biochemical characteristics of serum from such patients. For example, plasma viscosity increased during infection (Pokrovskij et al. 1989). Infection lead to increased levels of amino acid and circulating immune complexes in blood, while the concentration of serotonin decreased. (Laurent & Schott 1986, Luca & Hategan 1986). One of the most important finding in a patients with slow damage of the nervous system is the decrease in the contents of unsaturated fatty acids in blood. The most significant changes are observed for multiple sclerosis, where a reduction of the ratio of linolic to arachidonic acid in the high density lipoprotein and complex ethers fraction of cholesterol is observed (Navarro & Segura 1988). For a vascular pathology, the level of serum orosomucoid was increased (Vrethem et al. 1987). After cerebral damage, an increase of total cholesterol and the low density lipoprotein fraction in blood takes place, while the concentrations of the high density and very low density lipoprotein fractions remain virtually constant. At the initial stage of the disease, a tendency to hypotriglyceridemia was observed (Mendez et al. 1987). It is quite

293 probable that these (and other) substances can affect the values of surface tensiographic parameters of serum. For patients suffering from infectious and vascular diseases, close interrelations exist between various parameters of serum surface tension. For tumours and traumas (4 th and 5th groups) a correlation between ~2 and or3 exists, while for spondylogenic diseases (3 rd group) a correlation between Cyl and ~2 is obtained. Discirculatory encephalopathies and acute brain circulatory disturbances (2 nd group) are characterised by large negative correlations of all surface tension parameters with the k value of serum. For the 1st (infection) and 3 rd group an analogous dependence was observed with respect to o3, for the 5th group - with respect to orl, and for neoplasm all correlation coefficients were positive. For patients with various diseases of the nervous system, different correlations between dynamic surface tension parameters of serum and cerebrospinal fluid were observed as demonstrated in Table 7.2.

72

--

70-~'68

~

--

66 -" ~ 1 7 6 1 7 ~Oo 6 OOoo '*o o oo

64--

Oo Oo o o ~

62--

"~ o - ~ . .

60---2

-1.5

-1

-0.5

0

0.5

1

1.5

2

tg(tef) [s] Fig. 7.9. Example for cerebrospinal fluid tensiogram obtained from male patient, age 34 with slow multiple encephalomyelitis; dotted line correspond to average values for control group

294 Table 7.2. Correlations between various dynamic surface tension parameters o f serum and cerebrospinal fluid obtained from patients with various types of nervous system diseases

Nervous system disease

Liquor

Sertm~

type Infection

0"1

0"2

0"3

t?

?t

tl"

??

???

tl"t

o3

??

??

tt

ol

$

i

$

$$$

1"1"1"

??? ??t $$

l i

??? "l'?t

ttl'

$$

1"t1'

$$$

tit

t

t**

1"

02

$$$

~

$$$

$$

o3

$$$

$$

$$$

ol 02

Vascular

o2 0"3 Spondylogenic

i

,

,

$$

1"1'1' Neoplasm

1"1"1"

ol 0"2 o3

Trauma

ol

$$$

r162

02 o3 $$

$

1" positive correlation; $ negative correlation; empty - no correlation r<0.3; one symbol - r = 0.3 to 0.5; two symbols - r = 0.5 to 0.7; three symbols - r > 0.7

For example, infection and spondylogenic diseases showed a direct dependence of

0-1, 0"2

and

o3 values of liquor on 0"~ of serum, while for vascular diseases this dependence is inverse. For infection and vascular diseases, positive correlations exists between surface tension parameters of the cerebrospinal fluid and serum in the medium and long time ranges, while for vertebrogeneous diseases negative correlations were found. For patients with nervous system neoplasm and brain traumas such correlations were not found. There exists an interrelation

295 between the composition of surfactant in the cerebrospinal fluid and in serum, which can explain the observed correlations between dynamic surface tension parameters of these biological liquids.

72

--

7068

--

~66 t~

64 ~ "" "~ "~ "~ ~

oo O o o o o o o

o oo oo o . ~ 1 7 6

62-60-

t

-2

t

-1

I

0 lg(tef)

1 [sl

Fig. 7.10. Example for cerebrospinal fluid tensiogram obtained from male patient, age 46 with acute meningoencephalitis; dotted line correspond to average values for control group

76

--

72 --

.

.

~

Y

"-..... 64 ~ 1 7 6~1 7~6 Oo o~ oo o ...~ o o ~ 1 7.o6 . O . o o ,

60 -2

I

I

-1

0

I

[s]

1

Fig. 7.11. Example for cerebrospinal fluid tensiogram obtained from male patient, age 61 with cerebral atherosclerosis, discirculatory encephalopathy; dotted line correspond to average values for control group

296

78 ~75 + ~,72

1

;~ 69

"

~ 66• 63 ~i

60

"~ - ..~176

'.

~

-2

-1

t

t

0

1

tg(t O Is] Fig. 7.12. Example for cerebrospinal fluid tensiogram obtained from female patient, age 55 with spinal osteochondrosis, C2-C3 spinal cord pressure; dotted line correspond to average values for control group

7 2

--....

. ......

~

70 ~

"'-

~

~ ~ ~

;~ 66 ~

.. ,

~

-~

1 64 +

"'..

o

-... ~176

62

....

60 t

!

t

-2

-1

0

--~176

t

1

[s] Fig. 7.13. Example for cerebrospinal fluid tensiogram obtained from male patient, age 57 with cervical myelopathy; dotted line correspond to average values for control group Variations in the surface tension parameters of cerebrospinal fluid for screened patients are shown above in Figs. 7.1 and 7.3. Figures 7.9 to 7.13 illustrate cerebrospinal fluid tensiograms for patients with various neurological diseases. For all types of neurological diseases except

297 trauma some of the surface tension parameters increase: (Y2 increased in the 1 st, 2 nd and 3 rd groups, or2 - in the 2 nd and 3 rd groups, (Y3 " in the 1st and 2 nd groups. The increase of ~ values for liquor was characteristic for all studied groups; however, the most significant increase was observed for patients with vascular brain pathologies. L o w surface tension values at t = 0.01 s and t ~ oo might be significant for intracranial neoplasm.

7.2. I n f l u e n c e o f the c a u s e o f i n f l a m m a t i o n on s u r f a c e tension c h a r a c t e r i s t i c s

An additional analysis of dynamic surface tensiograms of biological liquids obtained from patients with inflammation of the central nervous system conceming different causes have been performed. It was found that polyradiculoneuritis and, to a lower extent, multiple sclerosis and myelitis, are accompanied by a decreased surface tension parameters of serum, while for meningoencephalitis an increase was usually observed. Myelitis leads to larger values of ~3, while for arachnoiditis ~3 decreases. It should be noted that the most significant changes were usually observed for the equilibrium surface tensions of serum, as one can see in Fig. 7.14.

151

Serum

Liquor

10

0 -5I

-10 1

2

3

4

5

6

3

6

Fig. 7.14. Changes in surface tension parameters measured in serum and liquor obtained from patients with nervous system inflammation of different causes. Changes are given in % compared to controls. 1 - multiple sclerosis; 2 - polyradiculoneuritis; 3 - myelitis; 4 - encephalomyelitis; 5 - meningoencephalitis; 6 - arachnoiditis; hatched - gl, black - or2,white - ~3.

298 The parameter ~ 1 of cerebrospinal fluid is rather stable and shows only a minor dependence on the cause of inflammation of the central nervous system. For multiple sclerosis, small decreases of ~ 1 result, while for patients with polyradiculoneuritis, a minor increase was observed. There is a significant increase in the dynamic surface tensions in the medium and long time ranges, with a maximum deviation from the reference group observed for arachnoiditis. It can be argued therefore that dynamic surface tensiometry of cerebrospinal liquid should be included in the scope of differential diagnostic tests for infection of the nervous system. For myelitis, lower L values for the two liquids are common, while in cases of multiple sclerosis and arachnoiditis L decreases for liquor only. In contrast, for polyradiculoneuritis and meningoencephalitis the increase in L values for cerebrospinal fluid is usually remarkable, as shown in Fig. 7.15. The ratio of L-values serum/liquor for multiple sclerosis is increased, while for polyradiculoneuritis, myelitis, encephalomyelitis and meningoencephalitis this ratio drops as compared to the control group. These data can be employed as an additional differential diagnostic criterion in practical neurology.

7.3. Role of patients age and duration of a disease

Two major factors influence dynamic surface tension parameters in biological liquids: age and duration of a disease. The age of patients is a factor which should be considered in the analysis of surface tensiometric parameters, because the surfactant composition of serum and cerebrospinal fluid changes with aging. All observed patients with vertebral disorders were older than 30 years, while 33.4% of screened patients suffering from tumours were below 30. In this regard, a correlation analysis of surface tension parameters for biological liquids have been performed with respect to the age in all five groups of screened neurological patients (Fig. 7.16). The most significant dependence among correlations found was that between L for serum and the age of screened patients for vascular diseases (direct correlation) and for spondylogenic diseases (inverse correlation). For other diseases no significant correlation between patients' age and tensiographic parameters was registered.

299

a) 25

20

-'~

15

~

10

m

I 5

i

..

'

I

1

~

I

2

1

1

3

L

4

I

5

L

6

C

b) 4 3.5 3 .~

2.5

~ ,<

2 1.5

1 0.5 0

- -

1

]

I-

2

3

~

r

4

I ~

-

-

5

T

.

.

.

.

6

T.

.

.

i .

.

I

C

Fig. 7.15. Influence of nervous system inflammation of different causes on k - values. 1- multiple sclerosis; 2 - polyradiculoneuritis; 3 - myelitis; 4 - encephalomyelitis; 5 - meningoencephalitis; 6 - arachnoiditis; C - control group. The upper graph gives Z, values in mN~n siX2for serum (black) and liquor (white). The lower graph gives ~, ratios between serum and liquor.

300

0.8

a) serum

v

0.6 -~ ~-9 o

rj

0.4 -1 1

o.2 0

..o -0.2 -0.4o -0.6 -; -0.8 -1-

J

U

U

b) liquor 0.6

!

0.4 1 (D O

0.2 ] 0 -0.2

o

t

-0.4 j -0.6

1

2

3

4

5

Fig. 7.16. Correlations between surface tension characteristics of serum and liquor obtained from patients with various type o f nervous system diseases and patients' age. Surface tension parameters are: hatched - cry, black - o2, white - cr3, grey - ~,. Nervous system diseases are: 1-infection; 2 - vascular diseases; 3 -spondylogenic diseases; 4 -neoplasm; 5 - trauma

301 The duration of a disease also affects the parameters of dynamic surface tensiograms. The results of corresponding correlation analysis are shown in Fig. 7.17.

a) serum 0.8

-

0.6

-

0.4-

0.2I

o

~

-0.2 0.4 0.6 -0.8 -1-

1

2

3

4

3

4

b) liquor 0.8 0.6 0.4 c)

..o

0.2 0~ -0.2

o

L) -0.4 -0.6 -0.8

1

2

5

Fig. 7.17. Correlations between surface tension characteristics of serum and liquor obtained from patients with various type of nervous system diseases and the duration of disease. Surface tension parameters are: hatched - a~, black - a2, white - a3, grey - ~. Nervous system diseases are: 1-infection; 2 - vascular diseases; 3 -spondylogenic diseases; 4-neoplasm; 5 - trauma

302

a) s e n m a

0.8 J

0.6 I .~ 0 . 4 0.2 0

s=

O+

~--

i ~-0.4

~

-0.6 j -0.8 ~

b) liquor 0.6 0.4

:

~0.2

g

o

.

.

.

.

.

-o.21 -0.4 -0.6 -

1

2

3

4

5

Fig. 7.18. Correlations between surface tension characteristics of serum and liquor obtained from patients with various type of nervous system diseases and the affection severity of nervous system. Surface tension parameters are: hatched - ol, black - o2, white - o3, grey - ~,. Nervous system diseases are: 1 -infection; 2 - vascular diseases; 3 -spondylogenic diseases; 4 -neoplasm; 5 - trauma Concerning the duration of a disease the most pronounced correlations were found for vascular pathologies of the brain. For spondylogenic diseases the dependence of surface tension of

303 cerebrospinal fluid on the duration of the pathological process is almost identical to that found for vascular pathologies, while the correlation between the duration of the disease and surface tensions of serum is the opposite. It seems interesting to estimate the possibility of prognosis of the severity of a disease using the data obtained from the dynamic surface tensiometry of biological liquids. Corresponding correlations are illustrated in Fig. 7.18. The factors that predict a bad prognosis are: -

for infection -~ low serum values of ~l and )~

-

for vascular diseases -~ high serum values of )~ for serum and high ~

values of

cerebrospinal fluid accompanied by small )~ values of liquor and low equilibrium surface tensions of both liquids; -

for spondylogenic diseases -~ increase ~ and ~2 values of serum;

-

for neoplasm --, decrease of ~l and increase of ~3 values for serum;

-

for trauma ~ increase of ~2 and ~3 of serum with a decrease of ~2 values for liquor.

These data are important from a practical point of view, because the application as tensiometric analysis of biological liquids might be helpful for monitoring the course of treatment of neurological disorders.

7.4. Correlation between surface tension parameters and amount of proteins and other components Cerebrospinal fluid is composed of water (98-99%), organic (proteins, amino acids, carbohydrates,

urea,

glycoproteids

and

lipoproteids),

and

inorganic

(electrolytes

and

microelements) substances. The amount of proteins in cerebrospinal fluid is usually 200 to 400 times lower than in serum. In addition, two other fractions, prealbumin and 0-fraction (intermediate between ]3- and y-globulins) are present. The proportion of prealbumin in liquor sampled from different sites is different. The fraction of prealbumin in ventricular liquor is 13-20% with respect to total protein, while this amount is 7-13% in liquor sampled from major cistern, and 4-7% in the lumbar liquor. In some cases this fraction cannot be detected in the cerebrospinal liquid, because it can be masked by albumins, and appears to be entirely absent when the amount of protein is large.

304 The protein composition of normal cerebrospinal fluid is essentially constant. For pathologies which result in a malfunction of liquor circulation and hampering of venous deflux (e.g. intracranial tumours), pronounced typical variations in both qualitative and quantitative composition of proteins in cerebrospinal liquid are noticed. It should be stressed that dysproteinrachia was observed also in cases where the total concentration of proteins in the cerebrospinal fluid did not exceed the normal values. The increase in the total concentration of proteins in cerebrospinal fluid is accompanied by a simultaneous increase in the amount of transferrin, cholesterol, some enzymes, zinc, copper etc. (Kapaki et al. 1989). In the cerebrospinal fluid both high-molecular (molecular mass higher than 10 kDa) and lowmolecular (molecular mass lower than 1.5 kDa) fractions are present. The high-molecular fraction

contains

various

proteins

(albumins,

pre-albumins,

y-globulins,

transferrin,

ceruloplasmin etc.), while the low-molecular fraction composed of carbohydrates and vasoactive peptides

(vasopressin,

serotonin,

catecholamines,

acetylcholine,

prostaglandins,

thromboxane, histamine, y-hydroxybutyric acid). The most abundant proteins of cerebrospinal liquid, i.e., albumin, prealbumin, transferrin and tx2-macroglobulin, constitute ca. 90% of the total protein pool in liquor. They participate in the binding and transport of biologically active substances of both endogenous and exogenous nature. The concentration of other proteins (e.g. immunoglobulin) is essentially lower. The formation of cerebrospinal fluid proteins takes place mainly in the ventricles of the brain; therefore variations in the composition of ventricular liquid indicate that special mechanisms of the liquor production are altered. In addition, inflammation processes leads to variations in the protein quantity in the cerebrospinal liquid. However, the absolute quantity of proteins shows virtually no correlation with the severity of a disease (Gebesh et al. 1988). Hyperproteinrachia is related to an enhanced permittivity of the blood-brain barrier, a slower removal of proteins from the liquor due to a decreased circulation, or an increase in the extrachorioidal (o.k) formation of liquor (Tsvetanova 1986). The total amount of protein is closely correlated to the amount of albumin in cerebrospinal liquid, and the albumin and prealbumin contents. The origin of the components of ctl-globulins, such as orosomucoid, al-antitrypsin and txl-lipoproteids, is mainly plasmatic.

305 For various neurological diseases, variations in liquor proteins can result from a transudation (increased influx from the plasma), and enhanced local formation. While the albumin level is indicates transudation processes which prevail, the enhanced local synthesis can be determined from the level of immunoglobulins. In cerebrospinal liquid, immunoglobulin-A (molecular mass 160 kDa) exists mainly in the monomeric and (more rarely) dimeric form; its molecule is built of two heavy chains (ct) and two light chains (K and k). It contains two subclasses (immunoglobulin-A 1 and immunoglobulin-A2), and activates the complement system through the altemative pathway by cleaving the C3-component. The secretory component of immunoglobulin-A is a glycoprotein, which imparts the ability to the protein to resist proteolytic enzymes. Immunoglobulin-G (molecular mass 160 kDa) is the main carrier of specific antibodies and enters the cerebrospinal fluid from the blood compartment, but intrathecal synthesis takes place in pathological states. This molecule also comprises of two heavy chains and two light chains, and the processing by papain leads to the formation of Fab and Fc fragments. There exist four subclasses of immunoglobulins-G, which differ in their ability of complement fixation. Immunoglobulin-M is comprised of 5 monomers with a high amount of hydrocarbons, and cannot penetrate through the blood-brain barrier easily due to its large dimension (molecular mass 900 kDa). A decreased amount of immunoglobulins in cerebrospinal fluid results either from a primary or secondary deficiency in the formation of antibodies; however, for most cases of nervous system diseases, there is a hyperimmunoglobulinrachia take places, with the normal levels of immunoglobulins in the blood. Hyperimmunoglobulinrachia in liquor could be related to a specific immunoglobulin class or to poly-, mono-, or olygoclonal types of immunoglobulins within a class. For example, in 90% of patients with multiple sclerosis, an increase in the production of immunoglobulins-G in brain was observed, while for acute and sub-acute inflammation processes in the central nervous system, the synthesis of this protein was found to increase in 70-80% of all cases (Safar et al. 1986). It was shown by Rao & Boker (1987) that high levels of immunoglobulins-G and albumins exists in the liquor for spondylogenic diseases of the nervous system, which correlate with the concentration of the same proteins in blood. Hyperimmunoglobulinrachia was observed also for patients with tumours of the central nervous system (Hrazdira et al. 1987). Correlations between surface tension parameters of

306 cerebrospinal fluid and the amount of various proteins therein are presented in Table 7.3 and Table 7.4. Table 7.3. Correlations between surface tension parameters measured in cerebrospinal fluid obtained from patients with various types of nervous diseases and protein components of liquor. Protein component

Disease Infection

Vascular

Spondylogenic

Neoplasm

Trauma

i ~1

, ~2

~3

~1

~2

~3

~1

i

Total protein

,1,

Albumin

~2

~3

~1

~2

~3

~!

~2

G3

!

$

$$

a~-globulin fraction

S J,

1"1"

$,1, ,l,,l,,[, 1'

$

1"1"1" i ,1,

1' !

,l,,I,,I,,

,l,,l, ,l,,l,,l,

1" !

a2-globulin fraction I

1"

1'

~-globulin fraction y-globulin fraction

,1,

]

lmmunoglobulin-G

$,1,

,l,,[,

[mmunoglobulin-A

,l,

,1,

lmmunoglobulin-M

,l,

a2-macroglobulin

,l,

132-microglobulin

i

,l,

,l, 1"

$

Fibrinogen

1"

,l,

,l,,l,

i

,l,,[,

,1,,1 i 1"

Transferrin

$

,1,

1" 1"1"'I"

,1,

$$

I

C-reactive protein .....

I ;

$$

,I, . . . . . .

1" positive correlation; ,1, negative correlation; empty - no correlation r < 0.3; one symbol - r = 0.3 to 0.5; two symbols - r = 0.5 to 0.7; three symbols - r > 0.7

307 Table 7.4. Correlation coefficients between K- value of cerebrospinal fluid tensiograms obtained from patients with various types of nervous diseases and protein components of liquor. Protein component Infection

Vascular

Spondylo-

Neoplasm

Trauma

genic Total protein Albumin Ctl-globulin fraction cz2-globulin fraction [3-globulin fraction ),-globulin fraction Immunoglobulin-G Immunoglobulin-A Immunoglobulin-M

1"1"

ct2-macroglobulin 132-microglobulin Fibrinogen Transferrin

1"1"

C-reactive protein 1'

positive correlation; $ negative correlation; empty - no correlation r

< 0.3;

one symbol - r - 0.3 to 0.5; two symbols - r = 0.5 to 0.7 No interrelation between the values can be detected for patients with craniocerebral traumas. The level of total protein shows a very close correlation with crl for vascular and spondylogenic diseases of the nervous system; here the correlation in the 2 no group is negative, while in the 3 rd group a positive correlation exists. The equilibrium surface tensions inversely depend on the

308 total concentration of proteins in the cerebrospinal fluid for patients with spondylogenic diseases of the nervous system and brain neoplasms. Surface tensions of liquor in the medium time range show moderate correlations with parameters of total protein and albumin: for patients with vascular encephalopathy and acute derangements of blood circulation in the brain a positive correlation was observed, while for other groups of screened patients this correlation was negative. The amount of proteins in the albumin and pre-albumin fraction in cerebrospinal fluid shows a negative correlation with ~2 and ~3 values for infection, and cl for vascular diseases of the nervous system. For tumours and trauma, the dependence of surface tension parameters on the extent of proteins level in cerebrospinal fluid is somewhat less pronounced. One can see from Tables 7.3 and 7.4 that the same substances are characterised by opposite signs of the correlation coefficients either for different pathologies of the nervous system, or for different surface lifetimes. In addition, in a number of cases, some surface active substances (e.g., albumin) show no correlation at all with dynamic surface tension parameters of the liquor. It has to be stressed once more that cerebrospinal liquor is an extremely complicated biological liquid comprising a great variety of proteins, lipids and other compounds. The composition of liquor undergoes quantitative and qualitative variations during any pathology of the nervous system, and therefore the results obtained in dynamic surface tension studies of liquor in some cases can disagree with the results of in vitro studies performed with model solutions. At this point, some general conclusions could be made, which are interesting not only from a theoretical point of view, but have practical consequences (for the estimation of the amount of particular proteins, the determination of the activity of pathological processes and the stage of development of a disease, the control of treatments etc.). In particular, it can be concluded that for infection of the nervous system, surface tension parameters of the cerebrospinal fluid correlate primarily with the amount of albumins and immunoglobulins-G in the liquor, for vascular diseases a correlation with albumins and fibrinogen exists; for spondylogenic diseases there is a correlation with immunoglobulin-M and transferrin; for brain tumours a correlation was found with the extent of [32-microglobulinemia, while the ~, values of liquor exhibit the most pronounced correlation with the protein level for spondylogenic diseases. Of some interest are the studies of brain-specific compounds in the cerebrospinal liquid, in particular, the dipeptide homocarnosine (7-aminobutyril-l-histidine), which acts as an inhibitory mediator in the central nervous system. The presence of homocarnosine in the liquor

309 or the change in their concentration can be used for diagnostics, estimation of the severity and the dynamics of a neurological disease. The study of such compounds is especially relevant for the cerebrospinal liquid, enabling one to make conclusions about the metabolism of the nervous tissue. The metabolism of homocamosine in brain is altered for a number of central nervous system diseases; this results in a sharp increase of the concentration of this compound in liquor and its occurrence in blood (Bondarenko & Makletsova 1992). In severe cases of the diseases of the central nervous system, morphological alterations of the blood-brain barrier occur. This affects the barrier-transport function of microvessels, and leads to an enhanced penetration of proteins (Barshtein et al. 1989). The "breakdown" of the bloodbrain barrier, and the influx of uncontrolled amounts of proteins into the perivascular and pericellular space results in disturbances of the colloidal-osmotic equilibrium, aggravating changes in the brain tissue (Yarosh 1991). For differential diagnostics of neurological diseases, the oxidation potential of cerebrospinal fluid has been studied, which depends on the level of proteins in the liquor. The level of proteins in the liquor reflect the permittivity of the blood-brain barrier. The substances which determine the oxidation potential are oxyreductases, protein detritus, carbohydrates and toxic metabolites (Zaitsev 1994). Erythrocytes which enter the liquor spaces undergo alterations, and the released haemoglobin, affected by the ferments of the meninges endothelial system, transforms into bilirubin, the concentration of which in the cerebrospinal fluid determines the extent of xanthochromia, which is extremely significant for the diagnostics of subarachnoidal haemorrhage, craniocerebral trauma and malignant tumours of the central nervous system. Cerebrospinal fluid contains encephalines, endorphins, hormones of the hypophysis and hypothalamus, insulin, cortifan etc. Their amounts depend on biological circadian rhythms of the organism, physical activity, and derangements in the circulation of cerebrospinal liquor. Modern biochemical methods enable scientists to estimate the quantitative level of particular fractions of lipids, such as cholesterol and itsethers, free fatty acids, kephalin, lecithin, sphingomyelin, cerebrosides, sulphatides, gangliosides, lipoproteids in cerebrospinal liquid,

310 and the activity of a number of ferments (creatine phosphokinase, lactate dehydrogenase, adenylate cyclase, aldolase, isocitric dehydrogenase, [3-glucuronidase, amylase etc.). The total phospholipids constitute approximately one half of the total lipid contents, even exceeding the concentration of cholesterol (with etherified fraction amounting to about 1/3, and free fraction about 1/3 of total cholesterol). The relative amounts of lysolecithin, sphingomyelin, kephalin and lecithin are 1:3:3:8. Pathological derangements of the fat metabolism are displayed either by variations in the concentration of all or particular fractions, or by changes in the relative concentration of the fractions for the normal total amount of lipids. Hyperlipidrachia can result from the influx of fats from brain tissue, lysis of cells of the cerebrospinal fluid and plasma. It was found that for patients suffering from vascular diseases of the nervous system, direct correlations exist between the values of cy2 and

0"3

and the cholesterol level in cerebrospinal

liquid, while ~, of the liquor inversely correlates with the extent of cholesterol level in liquor. The penetration of enzymes from blood to liquor depends on the functional state of the bloodbrain barrier, as well as on physicochemical properties of the ferment molecules. One source which delivers ferments into the cerebrospinal fluid is the brain, where the activity of some glycolytic ferments is significantly higher than in other tissues. The release of ferments contained in the cytoplasm (aldolase, creatine kinase etc.) happens more easily than for enzymes contained in lysosomes (acid phosphatase, [3-glucuronidase) and cell membranes (alkaline phosphatase, ATPase, adenylate cyclase). Serious damage of cells releases mitochondrial ferments (malate- and isocitrate dehydrogenase) into the cerebrospinal liquid. It is seen from Table 7.5 that correlations exist between surface tension parameters of cerebrospinal fluid with the level of some ferments in the liquor. Some conclusions can be made: (1) dynamic

surface

tensions

depend

significantly on the

concentration

of lactate

dehydrogenase and alkaline phosphatase in cerebrospinal liquid; (2) the same enzymes produce different effects on surface tension parameters for different surface lifetimes; (3) in spondylogenic diseases and neoplasm, the enzymatic composition of liquor has little effects on surface tension parameters;

311

(4) in trauma, surface tensions of cerebrospinal fluid positively correlates with the level of enzymes in liquor; (5) the correlation of tensiographic parameters with the concentration of muramidase (lysozyme) and malate dehydrogenase exists only in infection diseases, while for neoplasms these parameters correlate with choline esterase, and in trauma with acid phosphatase and leucine aminopeptidase; (6) dynamic surface tensions do not correlate with the amount of aldolase and amylase in cerebrospinal liquid

Table 7.5. Correlation coefficients between surface tension parameters of cerebrospinal fluid obtained from patients with various types of nervous diseases and ferments concentration of liquor. Ferment Infection ~1

~2

Vascular ~3

~1

~2

Spondylogenic ~3

~1

~2

~3

Neoplasm ~1

U2

Trauma ~3

~1

~2

~3

Aldolase Amylase

1"

Acid phosphatase

$$

Lactate

1"

1"1'

dehydrogenase Leucine aminopeptidase Muramidase

$

$

Malate

$

$

dehydrogenase Choline esterase Alkaline

$ ,~

$,~

,1,$,~

1"

$

$

phosphatase

1" positive correlation; $ negative correlation; empty - no correlation r < 0.3; one symbol - r = 0.3 to 0.5; two symbols - r - 0.5 to 0.7; three symbols - r > 0.7

The concentration of amino acids in cerebrospinal fluid is about 1/3 of their concentration in blood. The influx of amino acids into the liquor occurs from the central nervous system and

312 plasma. Their total quantity is 0.8-0.9 mmol, with a glutamine amount of 70 to 75% of this quantity. There exists a positive correlation between the total level of proteins in cerebrospinal liquid, and the amount of glutamine and alanine. The most abundant carbohydrates in cerebrospinal fluid are glucose, galactose, fructose, mannose, ramnose and ribose. The concentration of glucose is determined by the activity of the transport through the blood-brain barrier, and the rate of metabolism in the brain tissue. When the aerobic glycolysis, which is typical for the brain, transforms into the anaerobic mode, and the rate of the glycolysis becomes slower, then of glucose is released into the extracellular space, and subsequently into the cerebr'ospinal fluid. The high level of glucose in liquor becomes most pronounced during the sleep, which can be explained by retarded circulation of the blood, hypoxia and a decrease in the total brain metabolism. It was mentioned before (see Chapter 1) that the addition of glucose to solutions of lowmolecular surfactants in vitro results in a decrease of surface tension in the entire surface lifetime range. Table 7.6 summarises correlations between the parameters of dynamic surface tensiograms for cerebrospinal liquid, and the contents of glucose.

Table 7.6. Correlation coefficients between surface tension parameters o f cerebrospinal fluid obtained from patients with various types o f nervous diseases and glucose level in liquor.

Nervous system disease type t3"1

0"2

0"3

tt

Infection Vascular Spondylogenic Neoplasm

1'1"

1'1"1"

Trauma ,

1" - positive correlation; $ - negative correlation; empty - no correlation r < 0.3; one symbol - r = 0.3 to 0.5; two symbols - r = 0.5 to 0.7; three symbols - r > 0.7

It is seen that a certain discrepancy exists between the data obtained by in vivo studies, and the results of in vitro model measurements.

313 How could this discrepancy be explained? The cerebrospinal fluid is composed of a great variety of high-, medium- and low-molecular weight compounds, which in different combinations and in presence of glucose can produce various influences on dynamic surface tensions. Changes in the correlations of surface tensiometric parameters for patients suffering from different diseases can be explained by peculiar features of the qualitative and quantitative composition of surfactants and surface inactive substances in the liquor. In addition, proteins of cerebrospinal fluid could be glycosylated, which imply a dependence on the glucose level in the liquor, and the ability to undergo structural changes. Therefore, their surface active properties can vary. These questions have been discussed in more detail in Chapter 4. Under normal conditions, the concentration of inorganic compounds in cerebrospinal fluid is constant, and almost independent of their concentration in blood. Measurable quantities of sodium, chlorine, potassium, calcium, magnesium, boron, aluminium are present, with traces of tin, iodine, chromium, manganese, strontium and barium. The transfer of ions from plasma to cerebrospinal fluid happens by active transport and passive penetration mechanisms. Passive transfer is arranged via the extracellular shunting path (through intercellular links and lateral intracellular spaces). The motion of sodium, chlorine, potassium and calcium is unidirectional from blood to liquor. The concentration of chlorine and potassium in cerebrospinal fluid is similar to that in blood, while it is higher for sodium and magnesium, and lower for calcium. Almost no correlations were found between dynamic surface tension parameters of cerebrospinal fluid and concentration of particular electrolytes; only for tumour diseases a dependence of dynamic surface tensions of cerebrospinal fluid (at t = 1 s and t ~ oo ranges) on the level of chlorine exist.

7.5. Role of tensiometry in therapy, diagnosis and prognosis Among the various types of infection of the nervous system, meningitis is the most common. Figure 7.19 summarises the results of dynamic surface tensiometry of cerebrospinal fluid for patients with purulent and serous meningitis.

314

353025 -~

=20 ~

I I

"~15 1 105-

I

!

IIII

0~-cl

~2

r

k

Fig. 7.19. Surface tension changes of liquor obtained from patients with meningitis Black- purulent meningitis, white - serous meningitis. Changes are given in % compared to controls.

107 I

i 0 ~

..... ~

-

! I

~-10~ ~9

-2o 4 -30

_40 ~' -50 j r

~2

c3

Fig. 7.20. Variations of antibiotic treatment on surface tension parameters measured in liquor obtained from patients with purulent meningitis. Changes between initial and subsequent measurements are given in %.

Obvious is the increase of surface tension in the medium and long time ranges, and in the value of liquor. The variations in c2 and (~3 are more pronounced for purulent meningitis, while the increase in ~, becomes more apparent for serous meningitis.

315 During the process of antibiotic therapy, changes in dynamic surface tension parameters of cerebrospinal fluid for patients suffering from meningitis were observed, as shown in Fig. 7.20. Table 7.7. Correlations between surface tension parameters measured in liquor and liquor components for patients with purulent and serous meningitis. Liquor component

Serous meningitis

Purulent meningitis Cyl

cy2

cy3

~.

Protein

$

1"

$$$

Glucose

$

r

1'

$$

1"1'

r

Chlorides Lymphocytes

r

Neutrophiles

$$r

1"

Cyl

r

c~2

cy3

~.

r162

1"1"1"

$$

r162

1"1'1"

1"

1"

1" - p o s i t i v e c o r r e l a t i o n ; $ - n e g a t i v e c o r r e l a t i o n ; e m p t y - no c o r r e l a t i o n r < 0.3; o n e s y m b o l - r - 0.3 to 0.5; t w o s y m b o l s - r - 0.5 to 0.7; t h r e e s y m b o l s - r > 0.7

Most pronounced is the significant decrease of ~; during the treatment it approaches the characteristic value for the reference group of patients. The extent of ~. decrease can possibly be considered as one of the criteria of an efficient treatment, keeping in mind also the correlation between the value of X and the severity of the disease. As surface tensions depend on the composition of the liquor, we have analysed the correlations which exist between the concentrations of protein, glucose or chlorides, and dynamic surface tension parameters (cf. Table 7.7). It was found that for purulent and serous meningitis the correlations between similar values are often opposite to each other. For example, the extent of proteins level in cerebrospinal fluid for serous meningitis displays a pronounced positive correlation with ~ value ( r - +0.86), while for purulent meningitis this correlation is negative (r= -0.71). In addition, correlations of surface tensions with the cellular composition of liquor were analysed. Liquor cells do not influence the surface tension values directly; however, they affect the level of ferments and other synthesised substances, by which the surface tensions of liquor

316 can be influenced. It was found that the value of

(Yl

is closely related to the neutrophilosis of

cerebrospinal fluid for patients suffering from purulent meningitis (negative dependence), while the cy2 value for serous meningitis correlates with the lymphocytosis parameters (positive dependence). As the quantity of neutrophiles and lymphocytes in liquor is essentially characteristic for various types of meningitis, the application of surface tensiometry enables one to access implicitly the cellular composition of the liquor and therefore to perform a differential diagnostics of meningitis. The composition of cerebrospinal fluid has its peculiar features depending on the disease. For infection, increased concentrations of ceruioplasmin, fibrinogen, immune complexes, C-reactive protein, cholesterol, interleukin-6, lactate, acid phosphatase, isoferments - lactate dehydrogenase and kininase-II (hypertensin-I), glutamine, methionine, phenyl alanine, histidine, 7-aminobutyric acid, calcium were found, whereas for high density lipoproteids, C3and C4- components of complement the concentrations decreased (Dequette & Charest 1986, Dougherty & Jones 1986, Laurent & Schott 1986, Luca & Hategan 1986, Pitkanen et al. 1986 and Hashim 1995). The studies of liquor are believed to be important for the diagnostic of multiple sclerosis (gradual infection of the nervous system), as these can be used to estimate the intrathecal humoral immune response, which is the main constituent of the pathogenesis of the disease (Sch~idlich 1990). The specific form of leucine aminopeptidase exists in the cerebrospinal fluid of patients with multiple sclerosis, having physicochemical properties (molecular mass, optimum pH value, substrate specificity, electropheretical mobility) different from other isoforms of the ferment, also present in the liquor (Chochlov et al. 1987). The activity of [3-glucuronidase, neutral and acid proteinases in liquor was measured by Halonen et al. (1987). While changes in the concentration of lysosome hydrolases in cerebrospinal fluid are unspecific for multiple sclerosis, distinctive features of this disease are the increase in the concentration of neutral proteinase and a decrease in the concentration of acidic proteinase. The acute phase of multiple sclerosis is characterised by increased amounts of Oil-acidic glycoprotein (Tsukamoto et al. 1986) and circulating immune complexes, which comprise immunoglobulins-G, immunoglobulins-M, complement and [32-microglobulin (Procaccia et al. 1988). Both the level

317 of 132-microglobulin and the decrease in the index of C9-component of the complement show a direct dependence on the activity extent of the pathological process (Compston et al. 1986, Bjerrum et al. 1988). In 57 to 96% of all multiple sclerosis cases, the oligoclonal fractions of immunoglobulins, represented mainly by immunoglobulin-G with prevailing light n-type chains, were found in cerebrospinal liquor. For 46-78% of all cases, total hyper-7-globulinemia had developed (Ganes et al. 1986, Safar et al. 1986). With a decreased quantity of synthesised immunoglobulin-G, the oligoclonal spectrotype of immunoglobulin-G becomes more complex. Prolonged and progressive multiple sclerosis is accompanied by low amounts of immunoglobulins-G in the liquor, with a number of anomalous oligoclonal bands characterising the permittivity of the blood-brain barrier for albumins (Livrea et al. 1987). The data reported by Grucker et al. (1989) indicate an increase of the immunoglobulin-G index for 3% of healthy persons, 27% of patients suffering from central nervous system pathology, and 82% of patients with multiple sclerosis, with an increase in local

synthesis of

immunoglobulins-G found for 3%, 55% and 81% of all cases, respectively. The corresponding proportions for oligoclonal immunoglobulin-G are 0%, 79% and 17%, while the increase in the local production of immunoglobulin-M was found for 5%, 58% and 30% of all cases, respectively. With respect to the frequency of immune disorders, the presence of free light chains in cerebrospinal fluid for multiple sclerosis is second as compared to the presence of immunoglobulin-G oligoclonal bands, see (Bracco et al. 1987). This is possibly the reason why the correlation dependence of surface tension parameters for liquor on the level of immunoglobulin-G, immunoglobulin-A and C-reactive protein was found only for patients suffering from infectious pathologies. Amarenco et al. (1987) have studied the level of immunoglobulin-G and C3-component of the complement in cerebrospinal ~fluid for the Guillain-Barr6 syndrome (acute primary idiopathic polyradiculoneuritis). Local synthesis of immunoglobulin-G in the central nervous system for the stabilised paralysis T2 phase was higher than that in the progressive paralysis T~ phase and in the motion recovery phase T3. No correlation was found between immunoglobulin-G and the characteristics of albuminemia. The decreased amount of the complement C3-component was observed, especially in T2 phase. The higher the production of immunoglobulin-G in the cerebrospinal fluid was, the more severe were the clinical symptoms of the Guillain-Barr6

318 syndrome. No correlations were detected between the extent of transudation (total concentration of proteins and albumins in cerebrospinal liquor), and the severity of pathological processes. The studies performed by Papen & Warecka (1989) had shown that only for 10% of patients with Guillain-Barr6 polyradiculoneuritis, a synthesis of immunoglobulins in cerebrospinal fluid took place, and 67% of such patients exhibit altered blood-brain barrier functions. This course of the disease is characterised by significant (tens times) increases in C3a- and Csa-components of the complement, activated via the proteolytic splitting of C3- and Cs-components. It is worth noting that for vascular brain diseases, no changes of the complement system took place (Hartung et al. 1987). For vascular diseases of the brain, increased concentrations of alkaline phosphatase, lactate dehydrogenase, ATPase, ~/-glutamiltranspeptidase, creatine kinase, aspartate transaminase, adenylate kinase, serotonin, orosomucoid, alanine, potassium and etheric fraction of cholesterol is usual, with normal values of immunoglobulin-M and decreased concentrations of choline (c.f. Buttner et al. 1986, Nappi et al. 1986, Popova & Todorov 1986, Vrethem et al. 1987, Akimov et al. 1990, Manyam et al. 1990). It should be recalled that high inverse correlations between the amount of alkaline phosphatase and lactate dehydrogenase, and surface tension parameters of cerebrospinal fluid in the short time range was detected for vascular pathology only. Wester et al. (1987) had studied the level of monoamine metabolites and the activity of cholinesterase in the liquor of patients with brain blood circulation disorders. In 1/3 of all cases, an increase in the concentrations of 3-metoxy tyramine, homovanillin acid and 5-hydroxy indoleacetic acid was observed along with an increase in the level of spinocerebral fluid ferments (acetylcholinesterase and butyrylcholinesterase). The amount of serotonin and 3-metoxy-4-hydroxyphenyl glycol remains constant. A detailed analysis of surface tensiometric parameters for biological liquids with respect to brain and spinal turnouts is presented in Chapter 8; here it should be noted only that for oncologic diseases increases in the concentrations of 132-microglobulin, 13-globulins, fibropectin, astroprotein, and uric, lactic, pyruvic and fatty acids are observed with simultaneous hypopotassirachia and hypochlorinerachia. For patients suffering from neoplasm these amounts of 132-microglobulin and chlorine determine the surface tension parameters of liquor in the medium and long time ranges (inverse and direct correlations, respectively). The

319 concentration of creatine kinase in liquor becomes higher, and a correlation exists between the severity of the neurological symptoms and the amount of this ferment in cerebrospinal fluid (Matias-Guiu et al. 1986). The increase in the activity of ~,-enolase in liquor for brain tumours (especially for astrocytoma) indicates that a degradation of neurones occurs. It should be stressed that for slow infections, vascular diseases of the nervous system and craniocerebral traumas, the cerebrospinal fluid contains normal amounts of this ferment (Van den Doel et al. 1988). For 77% of all patients with nervous system neoplasm, an increase of the concentration of immunoglobulins in cerebrospinal fluid takes place (in 54% of cases - immunoglobulin-G, in 9% - immunoglobulin-M), while for 20% of patients, derangements in the synthesis of immunoglobulin-G were found (see Rao & Boker 1987). Increases in the index of immunoglobulin-M, oligoclonal immunoglobulin-G, 132-microglobulin and albumins in liquor were observed (Ernerudh et al. 1987). Antigenic heterogeneity of neoplasm, various localisation, the intensity of tumour growth lead to the formation of a great variety of antibodies and various rates of their influx into the cerebrospinal fluid (Hrazdira et al. 1987). Traumatic diseases of the central nervous system is the main source of primary damages of meninges, intermeningeal spaces and brain tissue. Irrespective of the trauma mechanism, a displacement of the brain takes place, which is inevitably accompanied by dynamic redistribution of liquor in the subarachnoidal space and ventricular system. Traumas of the central nervous system are often accompanied by epidural, subdural or subarachnoidal haemorrhages. Subarachnoidal haemorrhage is accompanied by the occurrence of blood in liquor; therefore the processes of blood coagulation, formation of clots and their lysis (at various stages of the disease) lead to the presence of a number of additional surfactants in the cerebrospinal fluid, which can affect surface tension parameters. It should be stressed that only the concentration of fibrinogen, which affects the coagulation ability of blood, correlates with cy2and ~3 parameters of liquor in cases of traumatic brain damage. The diagnostic significance of the determination of creatine kinase in cerebrospinal fluid for craniocerebral traumas is well known (Moshkin 1989). This ferment is contained in cells of brain tissue (astrocytes, dendrites, axons of neurone body), and during the first day after the

32O trauma its amount in the cerebrospinal liquor is increased. We have determined the activity of creatine kinase and its isoferments by adding 1 mg of the stabiliser ditiotreitol per 0.1 ml of biological liquid. The final concentration of magnesium in the reaction mixture was increased to 15 mmol/1, and the concentration of Trilon-B to 3 mmol/1. If the stabiliser was absent, then for brain damages reliable correlations do not exist between surface tensiometric parameters of cerebrospinal fluid and the concentration of creatine kinase. The addition of ditiotreitol results in a more frequent detection of isoferment BB with activity higher than 5 E/I, and in these cases a direct correlation exists between ferment concentrations and surface tensions of cerebrospinal fluid at t = 1 s and t ~ oo. Therefore, the application of dynamic surface tensiometry of cerebrospinal liquor may be considered as a reliable method for the estimation of the severity extent of traumatic brain damages. It should be noted that the concentration of fibrinogen and creatine kinase in cerebrospinal fluid is higher for open craniocerebral trauma, and correlates with the extent of brain injury. It is seen from Fig. 7.21 that the shape of tensiograms also depends on the character of the trauma. Moreover, direct correlations exist between the parameters of equilibrium surface tension for liquor and the amount of fibrinogen. 75 ~ 72 ' . . . . . . . . . . . .

o.o~

~

oo

~

~" 69 -~ t~ 66 ' 63 T I

~176

60 i . . . . . . . . . . . . . . . . . § . . . . . . . . . . . . . . -2

-1

+-. . . . . . . . . . . . . . . . . . . 4-. . . . . . . . . . . . . . .

q

0

2

Nt~f/[s]

1

Fig. 7.21. Examples for cerebrospinal fluid tensiograms obtained from patients with craniocerebral traumas and severe brain injuries. Thick line - open trauma (girl, age 5), thin line - closed trauma (boy, age 8); dotted line correspond to average values for control group.

321 7.6. Summary

In summary, dynamic surface tensiometry of serum and cerebrospinal fluid is useful for diagnostic and prognostic purposes and may have scoring potential to describe the severity of a given disease. We believe that further studies of surface phenomena in biological liquids taken from patients with neurological diseases should be extended into the following three areas. (a) The determination of unambiguous surface tensiometric parameters of biological liquids with respect to specific infection, vascular, spondilogenic, neoplasm and trauma related diseases should include patients sex and age; (b) The detection of surface active and surface inactive compounds which affect the state of surface tension of biological liquids, should include experimental in vitro studies employing the modelling of the composition of cerebrospinal liquor; (c) Estimation of the dynamic properties of surface tensiograms of biological liquids for neurological diseases should be given with respect to treatment and prognosis. 7.7. References

Akimov, G.A., Barsukov, C.F., Kurbatov, O.I., J. Nevrol. Psichiatr., 7(1990)3. Amarenco, P., Sauron, B., Schuller, E., J. Neurol. Sci., 80(1987)129. Barshtein, Yu.A., Yarosh, O.A., Persidsky, Yu.V., Vrach. Delo, 10(1989) 118. Bjerrum, O.W., Bach, F.W., Zeeberg, I., Acta Neurol. Scand., 78(1988)72. Bondarenko, T.I., Makletsova, M.G., Labor. Delo, 4(1992)12. Bracco, F., Gallo, P., Menna, R., J. Neurot., 234(1987)303. Buttner, T., Hornig, C.R., Busse, O., Dorndorf, W., J. Neurol., 233(1986)297. Chochlov, A.P., Baskaeva, T.S., Ckhrustaliova, N.A., Vopr. Med. Kchimii, 2(1987)58. Compston, D.A.S., Morgan, B.P., Oleesky, D., Neurology, 36(1986)1503. Dequette, P., Charest, L., Neurology, 36(1986)727.

322 Dougherty, J.M., Jones, J., Ann. Emerg. Med., 15(1986)317. Emerudh, J., Olsson, T., Berlin, G., von Schenck, H., Arch. Neurol., 44(1987)915. Ganes, T., Brautaset, N.J., Nyberg-Hansen, R., Vandvik, B., Acta Neurol. Scand., 73(1986)472. Gebesh, V.V., Muravskaya, L.V., Kononenko, V.V., Vrach. Delo, 10(1988)115 Grucker, M., Rumbach, L., Kiesmann, M., Sem. Hop., 65(1989)1253. Halonen, T., Kilpelainen, H., Pitkanen, A., Riekkinen, P.J., J. Neurol. Sci., 79(1987)267. Hartung, H.P., Schwenke, C., Bitter-Suermann, D., Toyka, K.V., Neurology, 37(1987)1006. Hashim, I.A., J. Ann. Clin. Biochemistry, 32(1995)289. Hrazdira, C.L., Hrazdirova, V., Polcakova, M., Ces. Neurol. Neurochir., 50(1987)238. Kapaki, E., Sogditsa, J., Papageorgiou, C., Acta Neurol. Scand., 79(1989)373. Laurent, B., Schott, B., Acta Neurol. Scand., 73(1986)477. Livrea, P., Simone, I.L., Trojano, M., Rev. Neurol., 57(1987)189. Luca, N., Hategan, D., Neurol. Psychiatr. Rev. Rom. Med., 24(1986)153. Manyam, B.V., Giacobini, E., Colliver, J.A., Ann. Neurol., 27(1990)683. Matias-Guiu, J., Martinez-Vazquez, J., Ruibal, A., Acta Neurol. Scand., 73(1986)461. Mendez, I., Hachinski, V., Wolfe, B., Neurology, 37(1987)507. Moshkin, A.V., Labor. Delo., 9(1989)48. Navarro, X., Segura, R., Acta Neurol. Scand., 78(1988)152. Nappi, G., Facchinetti, F., Bono, G., J. Neurol. Neurosurg. Psychiatry., 49(1986)17. Papen, R., Warecka, K., Psychiatr. Neurol. Med. Psychol., 41 (1989)334. Pitkanen, A.S., Halonen, T.O., Kilpelainen, H.O., Riekkinen, P.J., J. Nurol. Scand., 74(1986)45. Pokrovsky, V.I., Radsivill, V.I., Smysgova, A.V., Ther. Arch., 5(1989)130.

323 Popova, M., Todorov, V., Nevrol. Psikshiatr. Nevrokchir., 4(1986)6. Procaccia, S., Lanzanova, D., Caputo, D., Acta Neurol. Scand., 77(1988)373. Rao, M.L., Boker, D.-K., Europ. Neurol., 26(1987)241. Safar, J., Vymazal, J., Tichy, J., Ces. Neurol. Neurochir., 49(1986)382. Sch~idlich, H.J., Fortsch. Neurol. Psychiatr., 58(1990)247. Tsukamoto, T., Seki, H., Takase, S., J. Neurol. Sci., 75(1986)353. Tsvetanova, E.M., Liqvorologiya, Zdoroviya, Kiev, 1986. Van den Doel, E.M.H., Rijksen, G., Staal, G.E.J., Rev. Neurol., 144(1988)452. Vrethem, M., Ohman, S., von Schenck, H., Acta Neurol. Scand., 75(1987)328. Wester, P., Puu, G., Reiz, S., Acta Neurol. Scand., 76(1987)473. Yarosh, O.O., Vrach. Dielo, No 12(1991)58. Zaitsev, I.A., Arch. Clin. Exper. Med., 3(1994)215.

324

Chapter 8

Interfacial tensiometry in oncology Neoplasms are associated with compositional changes of blood. These variations change dynamic surface tension parameters tremendously. Therefore, dynamic interfacial tensiometry has potential concerning the diagnosis of certain types of tumours and monitoring of treatment.

8.1.

Pathogenesisof oncologicai disease

Patients with cancer of various organs have increased concentrations of 7-globulins, circulating immune complexes, [32-microglobulin, Otl-antitrypsin, haptoglobin, ferritin, orosomucoid, (z2glycoprotein, T-globulin, C-reactive protein and polyamines in blood (Guy etal. 1981, Mattison et al. 1981, Berdinskikh et al. 1987). An intensive synthesis of surface active fibronectin is performed by epithelial tumour cells, which leads to a hyperfibronectinemia (Titov & Sanfirova 1984). Amyloid P-component-glycoprotein is produced by hepatocytes. This glycoprotein is increased in serum obtained from patients with malignant tumours (Bannikova 1987). During oncological diseases, significant decreases in the level of vitamin-K-dependent glycoprotein C (molecular mass 62 kDa) in serum was observed (Ryabov et al. 1989). It was shown by Baskies et al. (1980) that direct correlations exist between the extensiveness of tumoural processes and the concentration of haptoglobin, orosomucoid and ot-antitrypsin in blood, while there are inverse correlations with the concentration of albumin, pre-albumin and ct2NS-glycoprotein. The ability of hepatoma to synthesise hepatic embryonic protein, ct-fetoprotein, was discovered thirty years ago. This fact strongly promoted the searches for new proteins, which arise during the development of neoplasms. The detection of oncofetal antigens can be regarded in some cases as an indication of tumoural development at its early stage, because the presence of these antigens depends on the degree of differentiation of tumour cells and the damage of intercellular links, and does not depend on the extend of the tumour. The characteristic feature of epithelial tumours is the increase of carbohydrate antigen 19.9 and carcinoembryonic antigens

(G[~I~ ~2-,

ct2H-, [~-, ~/1-, ,/2-fetoprotein, sulpho-glycoprotein) in

325 blood. The characteristic feature of tumours of the ovary is increased amounts of carbohydrate antigen 125, sialyl-SSEA, tissue polypeptide antigen, acid glycoprotein IAP and ferritin. The comedocarcinoma is accompanied by high levels of mucin-like glycoprotein and carbohydrate antigens 15.3 and 549. In serum obtained from patients with lung cancer, the concentrations of neurone-specific enolase and mucin-like glycoprotein are increased. Malignant tumours that arise from tissue that normally does not produce any hormones, often start hormone production. This secretion of hormones is often called ectopic secretion. It should be kept in mind in this regard that, under normal conditions the production of hormones happens not only in the endocrine glands, but also in the cells of so-called APUD (Amino Precursor Uptake and Decarboxylation) system. This (diffuse neuroendocrinal) APUD system consists of a complex of hormone-synthesising cells, specialised in the secretion of more than 35 various hormones and amines. It consists of neurosecretory cells of the brain, lungs, gastrointestinal tract, anterior lobe of the hypophysis, epiphysis, substantia medullaris of adrenal gland, C-cells of thyroid gland and D-cells of pancreatic gland. Therefore, the ectopic secretion is performed by cells which are neither endocrine cells, nor APUD system cells, and for which the production of hormones is not inherent. Table 8.1 summarises data concerning the ectopic production of hormones for malignant neoplasm of various localisation. Table 8.1. Ectopicallysecreted hormonesand localisationof tumours Hormone

Tumour localisation

Parathyroid hormone

Lung, mammary gland, kidney

Thyreotrophic hormone

Lung, mammary gland, chorionepithelioma

Gonadotrophic hormone

Lung, kidney

Somatotrophic hormone

Lung, stomach, uterus

Somatomammotrophin

Lung, intestine, mammary gland, uterus, thyroid

Somatoliberin

Lung, pancreas

Prolactin

Lung, kidney

Chorionic gonadotrophin

Lung, ovary, testicle, mammary gland, kidney, adrenal gland, liver, stomach, pancreas, intestine

326 The extracts of malignant neoplasm of lung, oesophagus and mammary gland contain much higher amounts of adrenocorticotrophic hormone and related peptides (13-1ipotropin, 13-endorphin, a-melano-stimulating hormone, enkephalins) compared to normal tissue. The synthesis of adrenocorticotrophic hormone and chorionic gonadotropin is common for all malignant tumours. Malignant neoplasm of the mammary gland is accompanied by a variations in the concentration of gonadotropic hormones in blood with an increased follicle-stimulating hormone level. The extent of such a hormonal imbalance depends on the stage of the disease and the size of the tumour. An inverse correlation was found between the level of the follicle-stimulating hormone, and the amount of prolactin, which possesses certain anti-gonadotropic properties (Agranat et al., 1991). Increased levels of calcitonin in blood was detected for the vast majority of patients suffering from lung cancer, pancreas carcinoma and struma maligna. The ectopic secretion of parathyroid hormone, together with osteoclast-stimulating factors and prostaglandin E2, results in a hypercalcemia for 10% of patients with malignant neoplasm. This fact was supported by tensiometric studies which have shown, that for lung cancer direct correlations exist between serum surface tensions in the short surface lifetime range, and the concentration of total and ionised calcium. However, no interrelation was detected between interfacial tensiographic parameters and the level of calcium-regulating hormones in blood. The somatotrophic hormone plays an important role in the regulation of carbohydrate and lipid metabolisms which is significantly disbalanced during tumourous processes (Dilman 1983). The increase in the amount of somatotrophic hormone in blood, and stimulation of lipolysis and ketogenesis during fasting indicates that somatotrophic hormone is contra-insular and acts as glucose-conserving agent. The hypersecretion of somatotrophic hormone in the organism of patients suffering from tumour increases the catabolic action of glucagon and corticosterone. The anabolic effect of somatotrophic hormone is hampered due to the decrease in the molar ratio insulin/somatotrophic hormone. The hypoglycaemic stress acts as a factor which initiates an increased secretion of the somatotrophic hormone. In addition, for patients suffering from oncological diseases somatotrophic hormone complements and enhances the biological effects produced by many other hormones (Shelepov et al. 1987).

327 Both the decrease in oxygen consumption by the tissue and the increase of lactate released into the blood directly correlates with the enhanced discharge of glucose (Bennegard et al. 1982, Edstrom et al. 1983). Thyroid hormones increase the rate of oxyhaemoglobin dissociation, which intensifies the transport of oxygen in tissue. The hypofunction of thyroid leads to a deterioration of this process, resulting in a tissue hypoxia. Usually a decreased glucose level in blood is observed for extended tumoural processes. Possible sources of hypoglycaemia are the ectopic production of somatostatin, somatomedin, proinsulin and insulin, the formation of insulinase inhibitors, retardation of glycogenesis in the liver, an increase in the glucose consumption by the tumour, intensification of glycolysis due to the suppression of lipolysis and the production of tryptophane (Dedov et al. 1988). Changes in the glucose concentration of a biological liquid can affect its surface tension, as it was demonstrated in Chapter 1. In fact, it was shown that increasing glucose levels in blood of patients with cancer is accompanied by increased surface tensions of serum in the short and medium time range. Interfacial tensiometric parameters for t ~ oo correlate negatively (however, less pronouncedly) with the glycemia level. The hormones secreted by any gland affect (directly or indirectly) other endocrine organs. Therefore, any imbalance of the endocrine equilibrium caused by tumours extends more or less to other endocrine secretion glands. Whatever preferential action is characteristic of the particular hormone (morphogenetic, metabolic or neurotropic), all the effects are based on the influence of hormones on the ferment system. Hormones can be involved in fermentative reactions as specific activators or inhibitors, which may result in a imbalance of the enzyme ~set)) of reacting cells. The main process in the invasion of malignant cells is the lysis of the extracellular matrix which acts as a barrier preventing the migration of invading cells. This lysis is performed by various ferments produced by the tissue. Proteinases are considered as the main enzymes (plasminogen activators, collagenase, cathepsins, proteoglycane-degrading ferments, elastase, gelatinase, etc.). A significant increase in the level of proteinases for tumours was observed (Geshelin et al. 1989, Varbanets 1990). The extracellular matrix is comprised of basal membranes and interstitial connective tissue. The main proteins of basal membranes are collagen type IV, laminine, and proteoglycans,

328 while the main proteins of the interstitial connective tissue are proteoglycans, fibronectin and elastin. In the cells of malignant phenotype, a high activity of plasminogen activators is observed, which transform plasminogen into plasmin. Plasmin splits laminine and fibronectin, and activates the latent forms of pro-collagenases. The activity of lysosome proteinases correlates with the rate of proteins metabolism in the tissue; therefore an increased activity in transformed cells can reflect the intensification of the intracellular metabolism (Sologub et al. 1992). Cathepsin B degrades collagen type I, laminine and proteoglycanes, and activates latent pro-collagenases of the connective tissue (De Bruin et al. 1988). The level of cathepsin H in brain tumours increases (Chernaya & Reva 1989). A similar increase of cathepsin D was observed in hepatoma cells by Maguchi et al. (1988). Cathepsin D intensively splits various proteins, including basal membrane proteoglycanes (Briozzo et al. 1988). In tumour cells and blood serum obtained from patients with malignant neoplasm, type IV collagenase is increased, which splits type IV collagen of the basal membranes (Kimura et al. 1990). The activity of this ferment correlates with the metastatic potential of tumours (Tryggvason et al. 1987). The components of the extracellular matrix are split by stromelysine - a ferment which is formed by connective tissue cells. Its primary structure is somewhat similar to that of collagenases, however, in contrast to these, stromelysine intensively degrades proteoglycanes and fibronectin, while it does not effect laminine, elastin and collagens. Elastase splits proteoglycanes, fibronectin, elastin and collagens of type III and IV. In the cells of carcinoma of the stomach, high molecular metalloproteinase (molecular mass ca. 1000 kDa) exists, which efficiently splits albumins, laminines, fibronectin, casein, haemoglobin and collagen type IV. The activity of this ferment remains unchanged in the presence of inhibitors of serine and cysteine proteinases (Tsuda et al. 1988). 8.2.

Serum tensiograms for different tumour localisations

The compositional changes of blood due to neoplasm depend on the localisation, size and histologic structure of the tumour. The total number of patients with malignant tumours studied was 165. Most changes in blood composition were observed for carcinoma of the stomach, lung and liver. At the same time, surface tension parameters of serum obtained from patients with carcinoma of the stomach (26 patients) show almost no differences in values of the

329

reference group (healthy persons). Lung malignant neoplasm (17 patients) was characterised by increased surface tensions in the short time range, while for liver malignant neoplasm (23 patients) equilibrium surface tension decreased, as shown in Fig. 8.1.

a) c~l, ~2, ~3 10-

I

_

J

0 ~ 9

-5

>

-10 -15 -20 St

Lu

Li

Ge

Mg

Br

Sc

Mg

Br

Sc

b)~, 100 80 60 ,-..,

40

~

2o

~9 .~. >

//

/

o

-20 -40 -60 -80 -100 St

Lu

Li

Ge

Fig. 8.1. Changes of surface tension parameters in serum obtained from patients with tumours of different location. Changes are given in % to corresponding healthy controls. St- stomach tumours, Lu- lung tumours, Li- liver tumours, Ge- genitals tumours, Mg- mammary gland tumours, Br- brain tumours, Sc - spinal cord tumours. The upper graph gives changes for crl - hatched, ~2 - black, cr3 - white. The lower graph gives changes for )~.

330 It should be mentioned that metastases in the liver following carcinoma of the stomach or lungs lead to a decrease in the equilibrium surface tension and an increase of ~ values of serum. In such cases, the interfacial tensiometric parameters approach those characteristic for primary hepatoma. These data let us conclude that decreases in equilibrium surface tensions of serum for patients with carcinoma of the stomach or lungs indicates metastatic spreading of the tissue into the liver, and represents evidence of the involvement of the liver in the formation of additional surfactants which can affect the dynamic surface tensions of biological liquids. For tumours of the female reproductive organs (42 patients) the lowest equilibrium surface tensions of blood serum was detected, and these changes do not depend on metastatic spreading into the liver. For neoplasm of genitals, the ~, values are increased up to values higher than those characteristic of patients suffering from primary carcinoma of the liver. It should be noted that the parameters of interfacial tensiograms virtually do not depend on the particular localisation of a tumour. No significant deviations from the normal amounts of the total protein in blood, its fractions, of cholesterol, triglycerides and lipoproteids of various density were detected; however, an increase in the level of some ectopically secreted hormones (in particular, chorionic gonadotropin and somatotrophic hormone), which can probably determine the serum surface tensions (either directly or indirectly via other surfactants) takes place. For malignant neoplasm of female reproductive organs, a significant increase of the [32-microglobulin in serum was observed. However, its concentration does not exceed a value characteristic for stomach or lung tumours, that is, for cases when either no change in the ~, values takes place, or these values become lower. In addition, no correlations exist between surface tension parameters and [32-microglobulinemia. For tumours of the mammary gland (21 patients) virtually no changes in averaged surface tension parameters of serum were detected. In some observations the dependence of equilibrium surface tension on the stage of pathological process was found, and correlations with some parameters of peroxide oxidation of lipids were detected which partly determine the composition of serum surfactant. This question will be discussed in more detail below. For brain tumours (26 patients) the values of gl and g2 decrease. The variations of these parameters for spinal cord neoplasm (10 patients) are still more pronounced, and the ~ value also becomes lower. For patients suffering from carcinoma of the stomach, liver and genitals,

331 inverse correlation exists between 9~ and equilibrium surface tension, while for spinal cord tumours this correlation is direct. For tumoural processes in the brain the behaviour of surface tensions of serum depends on the tumour location (Fig. 8.2). Serum

Liquor

1510-

o

0

2qu

.

-10 -15 2

3

4

5

6

1

2

3

4

5

6

Fig. 8.2. Changes surface tension parameters measured in serum and liquor obtained from patients with nervous system neoplsasms. Changes are given in % compared to corresponding controls. 1-accusticus neurinoma, 2- meningioma, 3- cerebellum turnout, 4- IV ventricle tumour, 5- posterior cranial fossa tumour, 6 - spinal cord tumour; hatched - 61, black -62, white - 63. For example, accusticus neurinoma is accompanied by low cl and c3 values of serum, while for turnouts of posterior cranial fossa these values are significantly increased. Equilibrium surface tensions for patients with meningioma becomes increased, while turnouts of cerebellum and ventricle IV lead to a decrease of this parameter. The localisation of pathological processes in the brain affects significantly the parameters of dynamic interfacial tensiograms not only for serum, but also for cerebrospinal liquid, as it is demonstrated in Figs. 8.3 to 8.5. Neoplasm of posterior cranial fossa are characterised by low 0.1 and 0.2 values with normal O"3 value of the liquor (cf. Fig. 8.2 and 8.3). Figure 8.4 shows the tensiogram of cerebrospinal liquid for patients suffering from ventricle IV turnouts. It is seen that the value of 0.3 is significantly lower, and the slope is higher, as compared with the reference curve.

332

a)

35 30 ,...,25 20

z15 ~1o 5

1

2

3

4

5

6

C

b) 300

I

200

~_~ 150 4 = r

!

"~ 100 t~

'

9

0 1 ~

--

-50

i

-100 J 1

2

3

4

5

6

Fig. 8.3. Z, values of biological liquids tensiograms obtained from patients with nervous system neoplasms.. 1 - accusticus neurinoma, 2 - meningioma, 3 - cerebellum tumour, 4 - IV ventricle tumour, 5 - posterior cranial fossa tumour, 6 - spinal cord tumour; C - control group. The upper graph gives ~, values in mN/m s ~/2, for serum (black) and liquor (white). The lower graph gives Z, deviation in % compared to corresponding controls.

W e have analysed the ratio of ~, values b e t w e e n serum and cerebrospinal liquid for brain tumours. W e found that this value remains normal only for tumours located in the posterior cranial fossa, see Fig. 8.6. For other types o f tumours, more or less p r o n o u n c e d decrease o f this p a r a m e t e r takes place.

333

7472,---, 70 68 66 64 6260 -1.5

-0.5

0.5 lg(tef) [s]

1.5

Fig. 8.4. Example for cerebrospinal liquid tensiogram obtained from girl, age 5 years with brain ventricle IV tumour; dotted curve correspond to average values for the control group

74 72 70 68

66 64 62 60

....

-

I

-1.5

-0.5

t

0.5 lg(tef) [s]

~176

1

1.5

Fig. 8.5. Example for cerebrospinal liquid tensiogram obtained from girl, age 3 years with cerebellum tumour; dotted curve correspond to average values for the control group

Table 8.2 summarises the variations in dynamic interracial tensiograms of biological liquids for patients with brain neoplasm.

334 Table 8.2. Differential diagnostic indicators of surface tension variation of biological liquids for different brain

neoplasm types Neoplasm Type Liquor

Serum (3"1

o2

o3

~,

a~

c:

or3

Accusticus neurinoma

-t-

Meningioma

+

+

Cerebellum tumour

+

+

iV ventricle tumour

~, -t+

+

Posteriorcranial fossa tumour

+

"+" - statistically significant increase of parameter compared to control; " - " - statistically significant decrease of parameter compared to normal

2.5

~9

2i 1.5-~ 1

0.5 l 0 --

// 1

2

3

4

5

6

C

Fig. 8.6. Ratio of serum to liquor tensiographic X values for nervous system tumours and control group; 1 accusticus neurinoma, 2 - meningioma, 3 - cerebellum tumour, 4 - IV ventricle tumour, 5 - posterior cranial fossa tumour, 6 - spinal cord tumour, C - control group.

It is seen that each kind of tumour has its specific features, which is important from a practical point of view. The differences in the dynamic surface tensions are caused by different compositions of surfactant in tumours and, in addition, can be determined by the age of

335 patients and the duration of the disease. For example, tumours of cerebellum, ventricle IV and posterior cranial fossa were characteristic primarily for children, whose liquor contains even for healthy children levels of ~,-globulins and [32-microglobulin lower than for adults, while the concentrations of amino acids are higher. In addition, the values of cy2, or3 and Z, inversely correlate with the duration of the disease, so that this factor should be considered in the analysis of interfacial tensiograms along with the patient's age.

8.3.

Correlation between surface tensions and biological liquid's composition

Dynamic surface tension parameters do not only correlate with turnout location but also with biological liquid composition. The presented data show that the ~2 value of liquor correlates directly with the total concentration of immunoglobulins, while ~1 depends inversely on the concentration of [32-microglobulin. The concentration of immunoglobulins-A in the cerebrospinal liquid taken from patients with benign neoplasm of posterior cranial fossa is usually higher than for malignant processes. For ca. 60% of adult patients with such a malignant turnout, hyper-immunoglobulin-A-emia was detected, while for children this number was three times lower. Similar data can be presented concerning the amount of immunoglobulins-G. The levels of immunoglobulins-M in liquor for benign and malignant tumours of posterior cranial fossa are roughly the same, and for children these levels are significantly higher than for adults (Rudenko et al. 1992). The protein composition of the cerebrospinal liquid for brain tumours is characterised by a significant increase in the amount of transferrin and high molecular ~2-macroglobulins (Vasilieva et al. 1995). No correlation was found between the dynamic surface tension parameters and the concentrations of transferrin and ~2-macroglobulin in cerebrospinal liquid. For patients with tumours of posterior cranial fossa, the concentration of transferrin in blood correlates positively with equilibrium surface tensions of serum. It was shown by Lampl et al. (1990) that the concentration of lactate dehydrogenase in cerebrospinal liquid increases even in the early stage of brain turnout processes. The dependence of interracial tensiometric parameters of biological liquids on the concentration of lactate dehydrogenase in liquor has been analysed and no reliable correlation was found between these parameters.

336 For

astrocytomas,

olygodendrogliomas,

glioblastomas

and

medulloblastomas

a

normoproteinrachia was observed, while for patients with accusticus neurinoma and corpus callosum neoplasm, a significantly increased protein concentration in cerebrospinal liquid was observed (Tsvetanova 1986). Inverse correlations were detected between the parameters crl and or3 of liquor, and the level of proteinemia. Therefore a more detailed analysis of dynamic interfacial tensiograms for various morphological versions of central nervous system tumours has been performed. Serum 20

Liquor

i

15 ~,

lO!

5 ~

oo!

'

U'

-5

-10 J -15 J Eb

Ar

As

Eb

Sert~

Ar

As

Liquor

250 200 1 r

150 t 100 ~

sol

~I-

-50

//

-100

Eb

Ar

As

Eb

Ar

As

Fig. 8.7. Changes of surface tension parameters measured in serum and liquor obtained from patients with various type of brain neoplasms. Changes are given in % compared to corresponding controls. Eb - ependymoblastoma, Ar- angioreticuloma, As - astrocytoma; The upper graph gives changes for ~ hatched, a2 - black, o3 - white. The lower graph gives changes for ~..

337 For astrocytomas, virtually no changes in surface tension parameters of serum were observed; for angioreticulomas 0.1 and 0.2 parameters for serum increased, while for ependymoblastomas these parameters decreased (cf. Fig. 8.7). In addition, ependymoblastomas are characterised by high equilibrium surface tensions and low ~ values for blood serum. The dynamic surface tension parameters of cerebrospinal liquid are also rather different: for ependymoblastomas a decrease of 0"2 takes place, for angioreticulomas a decrease of 0"2 and increase of 0"1 and 0"3 was observed, while for astrocytomas a significant increase of surface tensions in the short time range and a decrease of the equilibrium surface tension was detected. The 9~value for liquor for angioreticulomas was significantly lower, while for astrocytomas it was higher than the normal values. These data are of significant practical importance, enabling one to predict the morphological type of tumoural processes before a surgical treatment. Investigations were carried out to characterise the features of dynamic interracial tensiograms of biological liquids characteristic to patients with primary and metastatic neoplasm of the spinal cord. It was found that primary tumours of the spinal cord lead to a sharp increase of X for liquor, while for metastatic spreading into the spinal cord a decrease of ~ for serum takes place, as one can see in Fig. 8.8 and Fig. 8.9. These data are of certain practical importance for the differential diagnostics of pathological process in the spinal cord. Serum

Liquor

80 60 40 =

(D

20 0

-20 -40 -60 J 0.1

0.2

0.3

X

0.1

0.2

0.3

Fig. 8.8. Changes in surface tension parameters measured in serum and liquor obtained from patients with primary (black columns) and metastatic (white columns) various spinal cord neoplasms. Changes are given in % compared to corresponding controls.

338

75 70 65

.........

E

60 t~

55

45 -2

t

t

t

t

I

t

I

-1.5

-1

-0.5

0 lg(tef) [s]

0.5

1

1.5

Fig. 8.9. Example for serum tensiogram obtained from female, age 64 with tumour metastases into spinal cord; dotted curve correspond to average values for healthy females of corresponding age.

0.6 i

0.4 i

i

0.2

i

0 ~

-0.2

b ~)

-0.4

-0.6

i

-0.8 I

II

III

IV

V

VI

VII

VIII

IX

X

XI

XII

XIII

XIV

Fig. 8.10. Correlation coefficient between cerebrospinal liquid surface tension parameters and protein contents therein for patients with nervous system tumours. I - total protein, II - albumin, III - Oil-globulin fraction, IV - Ctx-globulin fraction, V - 13-globulin fraction, VI - ),-globulin fraction, VII - immunoglobulin-G, VIII - immunoglobulin-A, IX - immunoglobulin-M, X - ct2-macroglobulin, XI - 132-microglobulin, XII - fibrinogen, XII - transferrin, XIV - C-reactive protein; hatched - al, black -or2, white - cr3.

339 The variation in the composition of serum proteins is uncommon for neural system tumours. The disproteinemia in such cases, if any, is rather low. The correlation between interfacial tensiometric parameters and the concentration of serum proteins has been analysed and the results presented in Fig. 8.10. It can be concluded that concentration of proteins affects mainly the equilibrium surface tension values, and that a maximum correlation appears between equilibrium surface tension and the total concentrations of protein, albumins, 3,-globulins and [32-microglobulin. In the cells of human tumours (carcinoma of ovary, mammary gland, larynx) an albumin-like antigen (ALA, molecular mass ca. 68 kDa) was found (Bobrova et al. 1993). While the albumin contents in blood serum of patients suffering from oncological diseases, as measured using traditional methods, was usually found to be decreased (Lewis et al. 1991), the results obtained by immunoassay indicate an increase in the albumin concentration. The increase in the albuminemia level correlates with certain types of cells and the stage of the disease (Andersenn & Christensen 1991). Possibly for patients with malignant neoplasm, with respect to human albumin mouse antiserum detects an increased contents of some other antigens, which possesses crossed immune reactivity with albumins. With respect to some properties (thermoinstability, molecular mass, distribution in patients' serum) ALA is similar to cysteine proteinase (molecular mass 68 kDa), which acts as pro-coagulant, initiating blood coagulation (Gordon et al. 1990). The level of ferments in serum for patients with malignant tumours is much higher than for healthy persons, and corresponds directly to the stage of the disease. Qualitative and quantitative variations of serum albumin influence the surface tension parameters of blood exhibiting the most pronounced effects for patients suffering from hepatoma. The concentration of albumins inversely correlates with dynamic surface tensions in the short and medium time range, while there is a direct correlation with )~. During the development

of

the

nephrotic

syndrome

caused

by

paraneoplastic

membranous

glomerulonephritis accompanied by a significant decrease in the level of serum albumin, a decrease of cyl and

(5"2 values

was observed. This is illustrated by Fig. 8.11, showing the serum

tensiogram for female patients with carcinoma of the stomach, with a serum albumin concentration of 16.8 g/1.

340

72T "'~176176

.....

~ ~ 1 7 6 1 7 6 1 7 6 1. .7. 6. .

69 ~

............ ~ 1 7 6~ o

,

~

63

~

OOo

""

60 i -2

-1

0 lg(tef)[s]

t

t

1

2

Fig. 8.11. Example for serum tensiogramobtained from female patient, age 68 with carcinoma of the stomach, paraneoplastic membranous glomerulonephritis, nephrotic syndrome, chronic renal insufficiency 1st stage; dotted curve correspondto average values for healthy females of corresponding age. We believe that the variation in the surface tension of serum for malignant neoplasm is determined not only by the quantitative composition of albumins, but also by other factors: (a) variations in the qualitative composition of proteins caused by specific features of the metabolism related to the development of the nephrotic syndrome (cf. Chapter 4); and (b) the occurrence of hyperlipidemia (for patients suffering from carcinoma of the stomach, lungs and large intestine this is supported by a correlation between O'1 of blood serum and the amount of cholesterol). During oncological diseases serum albumin, which is highly surface active, undergoes pronounced conformational changes, caused by the increased load of ligands (Tolkacheva et al. 1991). In particular, extended binding of surface active poly-unsaturated fatty acids and products of peroxide oxidation of lipids by modified albumin occurs. For patients with malignant neoplasm, the total amount of lipids bound by albumin significantly exceeds that characteristic of healthy persons. This also depends on the location of the tumour and the extent to which the affected organ is involved in the metabolism of albumin ligands. The same fatty acids are present both in the albumin fraction of oncological patients and in albumin fraction of healthy persons; however, relative amounts of particular kinds of these compounds are essentially different, as one can see in Fig. 8.12.

341

250 200 1

il!ili

,.._150 = 100 ~9 50 0

--

-

-

.

.

.

.

.

-50 100 St

Pc

Li

EL

RP

Fig. 8.12. Changes of the amount of fatty acids bound to albumin in serum obtained from patients with malignant tumours of various localisations. Changes are given in % to values for healthy controls. St- stomach, Pc - pancreas, Li - liver carcinoma; EL - extraliver bile ducts, RP - retroperitoneal space; hatched-oleic acid, black-polyunsaturated fatty acids, white- c06-type polyunsaturated fatty acids, grey - 0~3-typepolyunsaturated fatty acids. Irrespectively of the location of the tumour, an increase is observed in the partial portion of 033 fatty acids which act as an important regulator of metabolic processes. The variations of the 0~6 series are less pronounced. However, for patients with neoplasm in the extrahepatic biliary tracts and retroperitoneal space, a significant reduction of the sum of basic fatty acids was observed. For patients suffering from oncological diseases, the increase in the concentration of oleic acid in the albumin fraction takes place (except for the cases when the tumour is localised in the extrahepatic biliary tracts). Both the oleic acid and poly-unsaturated fatty acids produce similar effects on some stages of the exchange process. As for oncological diseases the amount of poly-unsaturated fatty acids becomes significantly lower, it can be hypothesised that a substitution increase in the concentration of oleic acid happens in these cases. One of the important mechanisms for the support of homeostasis of the organism is the peroxide oxidation of lipids, which regulates the structure and function of biological membranes and their accessibility to various influences (Burlakova & Palmina 1982). The system of peroxide oxidation of lipids consists of activated metabolites of oxygen (superoxide anion-radicals, hydroxyl radicals, hydrogen peroxides, etc.), non-ferment and ferment antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase etc., cf.

342 Proter 1986). The formation and development of malignant neoplasm leads to changes in the intensity and character of peroxide oxidation of lipids in organs and tissue (Spector & Burns 1987, Kogarko et al. 1991, Korman et al. 1995, Gurevich et al. 1993, Gordon & Weizman 1993 and Casado et al. 1995), which reflects the mechanisms of the organism's reaction to emerging neoplasm (Potapov & Korman 1997; Korman et al. 1995). An integral parameter which enables one to estimate the intensity of peroxide oxidation of lipids is the total extent of unsaturation of fatty acidic components of lipids: the lower the number of double bonds, the more extensive is the oxidation by free radicals (Korman & Potapov 1994). The existence of tumours results in an attenuation of peroxide oxidation of lipids to an extent determined by the spreading of the process; for generalised tumoural processes this is accompanied by an increased proportion of unsaturated fats. Peroxide oxidation of lipids is a physiologic process required to support homeostasis and normal operations of the organism. The activation of the peroxide oxidation of lipids can be considered as a reaction towards the elimination of nosopoietic factors or as an adaptation to them, regulated by the system of anti-oxidant protection. In the initial period of the development of a tumour, a 'switching on' of the anti-tumoural protection mechanisms occurs, including the intensification of peroxide oxidation of lipids. However, at the same time an excessive oxidation by free radicals also becomes one of the pathogenic factors leading to a further development of pathological processes.

100

P

80 ~ i

6o ~ 40 -i ._o 20

o

0 4-

-20 f -40 " ,,

-60 J I

II

III

IV

Stage o f t u m o u r process Fig. 8.13. Levels of malonic dialdehyde and superoxide dismutase in serum obtained from patients with lung carcinoma (+% deviations from the parameters characteristic for healthy persons), black-malonic dialdehyde, white- superoxide dismutase

343 Figure 8.13 illustrates the level of malonic dialdehyde and superoxide dismutase in serum obtained from patients suffering from lung carcinoma at various stages. With the development of pathological processes, increased amounts of anti-oxidant ferments appear, which may be considered as the compensatory response to the peroxidation mechanisms. Simultaneously the surface tensions of serum are decreased (cf. Fig. 8.14).

MD

0.6

SD

0.4 -~ 9

0.2

o r..)

0

~

-0.2

o -0.4 -0.6 -0.8 I

II

III

IV

I

II

III

IV

Stage ofttmaour process

Fig. 8.14. Correlation coefficients between surface tension parameters and peroxide oxidation of lipids in serum obtained from patients with lung carcinoma of different stages. M D - m a l o n i c dialdehyde, SD - superoxide dismutase; hatched - a~, black -a2, white - a

The surface tension values in the short time range correlate inversely with the concentration of malonic dialdehyde, and directly with the concentration of superoxide dismutase. It appears that a2 for serum does not depend on the state of the peroxide oxidation system of lipids. -._

In kidneys malignant neoplasm, a strong intensification of the peroxide oxidation of lipids happen and result in an increased amount of malonic dialdehyde formed by both the ascorbatedependent and nicotinamide-adenine-dinucleotide-phosphate (NADP) -dependent mechanisms (Gorozhanskaya et al. 1995). One of the reasons for an enhanced peroxide oxidation of lipids is the low activity of protective ferments which destroy peroxides or prevents their formation (for example, the activity of superoxide dismutase is suppressed to a value two times lower than

344 that characteristic of normal conditions). It is generally believed that the formation of tumours is related to the decrease in the amount of superoxide dismutase in cells and even its complete disappearance. In addition, low catalase activity was found in tumours, caused by the suppression of the synthesis of ferments and the inactivation by formed hydroperoxides. One of the most important factors which regulate the peroxide oxidation of lipids is believed to be the amount of bioantioxidants in blood, which is significantly decreased during the progress of tumoural processes (Gorozhanskaya et al. 1989, Morozkina 1989). This is accompanied by either a decrease or an increase of a-tocopherol and retinol in tumours (Ostrowsky et al. 1989, Nikiforova et al. 1993). The amount of products of peroxide oxidation of lipids in serum albumin for patients suffering from oncological diseases is significantly higher than that for healthy people. However, when the tumoural process is localised in the liver or pancreas, increased concentrations of these products are accompanied by slight decreases in the amount of arachidonic acid, which is the main substrate of the peroxidation process. For carcinoma of the stomach, even an increase in the arachidonic acid amount was observed. In this case the activated formation of peroxides happens at the expense of other polyunsaturated fatty acids. The enhanced ability of serum albumin to bind peroxide oxidation products of lipids is considered to be a demonstration of the albumin antioxidant functions (Tolkacheva et al. 1995). In the albumins of patients with oncological diseases, essential decreases in the number of spiral structures and tyrosiles capable of perturbations was observed. The most significant variations in the structure of proteins were detected for liver tumours, which can be explained by a high contents of ligands. Therefore, for oncological diseases of various localisation, a pronounced structural change of serum albumins along with a decrease in the concentrations were observed. Large amounts of lipids, products of peroxide oxidation of lipids and polyunsaturated fatty acids of the

0) 6

family

in albumin is indicative of a disturbance of metabolic processes in the patient's organism, and also of large capabilities of the albumins to transport lipid-related ligands. Variations in the qualitative and quantitative composition of proteins and fats in serum affect its surface tension characteristics. The growth of tumours requires increased amounts of fats (Denisov et al. 1992). During the cultivation of cancer cells in the medium, depleted with respect to lipids, a significant decrease

345 in the concentration of cholesterol and phospholipids in cytomembranes was observed, while the ratio of saturated to unsaturated fatty acids amount had increased. The increase in the concentration of free fatty acids in blood serum and cell membranes is accompanied by the enhanced consumption of carnitine and the precursors of its synthesis, which is indicative of the increase in the utilisation of fats by the tumour. Unsaturated fatty acids possess a selective anti-tumoural activity, which can be blocked by antioxidants. The absorption of arachidonic and eicosapentaic acids by cancer cells decreases, while the absorption of linoleic acid remains unchanged. The excessive influx of fatty acids from cell membranes leads to growing concentrations in blood. High amounts of stearic and oleic acids in blood serum were observed for the carcinoma of the large intestine, while a higher concentration of arachidonic acid was detected for patients suffering from osteoblastoma, and higher levels of linoleic and a-linoleic acids was common for female patients suffering from breast cancer. Eicosanoids are metabolites of highly polyunsaturated fatty acids. These are prostaglandins, prostacyclins, thromboxanes, leucotrienes, various hydroxy- and hydroperoxy derivatives of fatty acids. The main sources for eicosanoids in the organism are believed to be linoleic, cx-linolenic and arachidonic (formed from linoleic) acids. The precursors of eicosanoids are contained in phospholipids and other complex lipids, which are surface active and determine the surface tensions of many biological liquids. It was shown in experiments that polyunsaturated fatty acids inhibit the synthesis of

prostaglandins E 2 and Fza in tumour cells, and these effects depend on the concentration of the acid and the particular acid species. The ranking of the acids is in the following order: with respect to prostaglandin inhibition: docosahexaenic > dihomo-y-linolenic > eicosapentaenic > a-linolenic > linoleic; with respect to a concentration decrease: dihomo-~,-linolenic > eicosapentaenic > docosahexaenic > c~-linolenic > linoleic. During the evolution of tumoural processes, significant disturbances take place in the eicosanoids metabolism and substantial changes were observed in the level of prostaglandins E 2, D 2, FI~, F2~ and thromboxane B 2 (Chiabrando etal. 1985, 1987). We believe that the

346 studies on the ferment activity in respect to the synthesis and catabolism of prostaglandins are very important. For example, the production of prostaglandin E2 in tumours is enhanced by a factor of 65 as compared to normal tissue, while for phospholipase A 2, which acts as catalyst for the release of arachidonic acid from phospholipids contained in the membranes, an eightfold increase was observed (Calo et al. 1984). It remains unclear whether during the development of a tumour the increase in the concentration of prostaglandins in blood reflects local or general disturbances of their metabolism (Kudriavtsev 1988). In many cases the removal of a tumour leads to a significant decrease in the level of prostaglandins E 2 and" E2~ for patients' serum. This effect, however, was observed only for certain localisations of pathological processes, and does not affect the amount of prostaglandin Fl,~ and thromboxane B 2 (Nigam et al. 1985). It was argued by Chaimoff et al. (1985) that high concentrations of prostaglandin E 2 in serum cannot be explained by its synthesis in tumour tissue. It was shown by Yoshino (1980) that for malignant tumours the level of low density lipoproteids in blood is lowered significantly, accompanied by higher amounts of fats and proteins. For female patients the type of lipidemia is related to the localisation of a tumour (uterus, mammary gland, intestine, stomach, cf. Kovalenko & Berstein 1993). Carcinoma of mammary gland is accompanied by increased concentrations of triacylglycerol, cholesterol and phospholipids, which exceed the corresponding values for patients with fibroadenoma and fibrous-cystic mastopathy (Araki et al. 1980). It should be noted that for the benign neoplasm of mammary gland, particular variations in the phospholipid contents were detected: increase of sphingomyelin with complete absence of lysophosphatidyl choline for patients with fibroadenoma, and an increase in the concentration of phosphatidyl ethanol amine together with decreased phosphatidyl choline contents for individuals with fibrous-cystic mastopathy. At the initial stage of a malignant growth lysophosphatidyl choline is also absent, while higher concentrations of serum sphingomyelin are found. A decrease in the concentration of phosphatidyl choline, and an increase in the amount of phosphatidyl ethanol amine is observed. The further development of a neoplastic process is characterised by a still more significant increase in the amount of sphingomyelin in serum (for patients with tumours < 4 cm in diameter) and phosphatidyl ethanol amine (for tumour diameters > 5 cm). For patients with

347 malignant tumours exceeding 6 cm in diameter, a suppressed anti-oxidant activity of blood lipids was observed. The application of radiotherapy results in a normalisation of the state of the anti-oxidant system and of the phospholipid levels: these characteristics become closer to those common for patients at early stages of tumoural processes (Kalnov & Palmina 1991). The irradiation leads to decreased concentrations of prostaglandins E 2 and F2~ in serum. These changes in the state of eicosanoids are accompanied by a reduction of peroxide oxidation of lipids and the activation of the anti-oxidant system (Shinjiro et al. 1989, Bilynskij et al. 1992). 8.4.

Influence of radiation therapy on dynamic surface tensions

For female patients with tumours of the reproductive organs, the application of a combined (remote and intracavitary) radiation therapy results in a decrease of the concentrations of malonic dialdehyde and dien conjugates in serum, while the amount of (z-tocopherol becomes higher. Such variations in the peroxide oxidation system of lipids correlate with the equilibrium surface tension. It is interesting that these variations are accompanied by a decrease in the interfacial tensiographic parameters of urine at t-~oo, see Fig. 8.15. At the same time, a pronounced decrease in X values of serum and urine results (Fig. 8.16). Therefore, an inverse correlation exists between equilibrium surface tension and the ~, values of serum, while for urine tensiograms these parameters are inversely related to each other. Serum tensiograms for patients suffering from uterus body, or neck carcinoma, which have been treated by remote radiation therapy are presented in Figs. 8.17 and 8.18. It should be noted that for healthy females no correlation exists between the values of ~3 and of different biological liquids, cf. Chapter 3. In this regard, the following suppositions can be made: 1) during radiotherapy for patients with reproductive organs malignant neoplasm, a decrease of surfactants (including the pathological ones) in blood happens, which leads to increased equilibrium surface tension values; 2) enhanced urinal excretion of some portion of blood surfactants leads to a decrease in the equilibrium surface tensions.

348

Serum

62 -t !

Urine

6o I

58 ~ ~" 56~ ~ 54 ~ 525048 ,~-

,~

~

,t

~

II

- - T

III

.

.

.

.

H

T - - ~

- - T - - ~

I

l

II

Serum

-

-

III

1

- -

H

Urine

. . . . . .

-2-4 -

9

-8-

"~ -10 -12 t 14 -16 I

II

III

I

II

III

Fig. 8.15. Influence of radiation therapy on equilibrium surface tension in serum and urine obtained from patients with genital tumours and healthy controls. Upper graph gives the equilibrium surface tension for patients and healthy controls in mN/m. Lower graph gives changes in % compared to healthy controls. I - before the treatment, II- after remote radiation therapy (22-26 Gy) with intracavitary radiotherapy (20 Gy), III- after remote radiation therapy (46-48 Gy) with intracavitary radiotherapy (40-50 Gy), H- healthy females

349

Senun

Urine

1, t 16

14 12lO,<

64_

_ _ _

1

~

1

~

II

1

III

~

~ - - T - - ~ - - T

T

H

I

1

II

Serum

III

H

Urine

120 100 80r"--'!

604020 0

1

- - -

1

I

I

-2O -40 I

II

III

I

II

III

Fig. 8.16. Changes in ~, values of serum and urine tensiograms obtained from patients with genital tumours during radiotherapy course. Upper graph gives the ~, values in mN/m-s ~ patients and healthy controls. Lower graph gives changes in % compared to healthy controls.

I- before the treatment, II- after remote

radiation therapy (22-26 Gy) with intracavitary radiotherapy (20 Gy), Ill - after remote radiation therapy (46-48 Gy) with intracavitary radiotherapy (40-50 Gy), H - healthy females

350

75 T 73 ~

................

.~176 ~176176 ~176176176

69 -~ E

~176176176176176176176176176 Oo

67--

~176176176176176 O4o~

~

Z 65 -E t~ 6 3 - 61

~

.......

~

59

57-55-~ -2

I

~

t

t

-1

0 lg(tef ) [ s ]

1

2

Fig. 8.17. Examples of serum tensiograms obtained from patient with body uterus carcinoma during the radiation therapy. Thick line - before treatment, thin line - after treatment, dotted line correspond to average values for healthy controls.

75T i. . . . . .....

70

.~

I

65+

E Z 60 E i

55-~

50 ~45 ....... -1

+----! -0.5

0

0.5

1

~

t

1.5

2

lg(tef ) [ S]

Fig. 8.18. Examples of serum tensiograms obtained from patient with neck uterus carcinoma during the radiation therapy. Thick line - before treatment, thin line - after treatment, dotted line correspond to average values for healthy controls.

Among other factors which affect the surface tension of urine for patients suffering from malignant neoplasm, free light chains of immunoglobulins, which possess a pronounced surface activity, play a certain role. The enhanced synthesis of these low molecular

351 compounds, which takes place in addition with tumour processes, leads to their enhanced urinal excretion (Reznikov et al. 1996). It was mentioned in Chapter 1 that non-ionic low molecular surfactants, mixed with serum albumin, can increase significantly the in vitro value of the equilibrium surface tension of a solution. One can suppose that the application of a radiotherapy suppresses the synthesis of free light chains of immunoglobulins; this, in turn, returns the surface tension of serum to its previous values. At this stage, no definite explanation can be given for the changes observed in the tensiograms of biological liquids during a radiation therapy treatment. The results of the studies of the effects of a radiotherapeutic treatment on surface tensions of biological liquids for patients with tumours of the reproductive organs can be generalised in as much as that similar mechanisms take place also for the radiotherapy of malignant neoplasm localised in other organs. It should be noted once again that for patients with tumours the development of hypo- and disproteinemia happens due to the lowered amounts of albumins and increased globulin fractions. Also increased levels of serum mucoids (approximately by 1/3) and free sialic acids (by 2/3) are observed. These variations during a radiotherapy can be different: for some patients the concentrations of these substances remain constant, while for other patients these values either increase or decrease. Usually the dynamics of seromucoids and free sialic acids directly correlate with the concentration of oxyproline in blood, while an inverse correlation exists with the amount of fucose. The application of X-ray treatment decreases the activity of alkaline phosphatase and arylesterase in blood, and increases the amount of cholinesterase, nitrous non-protein products and glucose (Ulianenko et al. 1991). We have found that for blood serum an inverse correlation exists between the amount of oxyproline and equilibrium surface tensions, while for urine a positive correlation exists. The surface tension of serum exhibits a negative correlation with the fibronectin concentration, and a positive correlation with the extent of fibrinogenemia. The surface tensions of serum in the medium and long surface lifetime range is directly related to the concentrations of uric acid and urea. It is quite possible that the enhanced excretion of non-protein nitrous products by kidneys is one of the reasons for the decrease of the equilibrium surface tension of urine during a radiotherapeutic treatment.

352 The malignant process is accompanied by an imbalance in the functional state of the hemostatic system; this leads to the enhanced capability of the intravascular thrombogenesis, which increases with the growth of a tumour. It is known that cancer cells possess high coagulation and adhesive activity, excrete the coagulation factor and contain large amounts of pro-coagulative and fibrin-stabilising factors. The presence of tumours leads to changes in the functional state of the coagulative and anti-coagulative systems and a decrease in antithrombogeneous properties of vascular walls (Baluda & Chnychev 1987). Tumoural cells, due to their pronounced pro-coagulative and adhesive activity, aggregate thrombocytes at their surface and become abducted by a fibrin layer, which results in the organisation of an oncogeneous embolus. For patients with metastases, large amounts of fibrin degradation products (D and E fragments) were detected accompanied by high otl-antitrypsin contents. No increase in the concentrations of antiprotease inhibitors and c~2-macroglobulin was found. It was shown by Groop et al. (1980) that the action of immune complexes on granulocytes leads to the release of elastase, which performs the digestion of fibrinogen and fibrin, with the formation of fibrin degradation products. Tumoural hypoxia is an unfavourable factor, which restricts the applicability of radiotherapy. For female patients suffering from malignant neoplasm, local irradiations lead to a disorder in the functional state of the hemostatic system, and results in an increase in the capability of the blood to form intravascular clots. The imbalance of blood proteins, which participate in these processes, can be one of the reasons for the changes in surface tension of biological liquids during a radiotherapeutic treatment. During the growth of a tumour and a radiation therapy, the fibrinolysis processes are also activated. With small total amounts of antithrombins, a decrease in the amount of free heparin is detected; however, the net fibrinolytic activity becomes increased at the expense of the nonfermentative fibrinolysis. It was shown that irradiations lead to a relative decrease in some fractions of esterase, alkaline phosphatase, amylase and lactate dehydrogenase; however, no correlation was found between the enzymemia level and surface tension parameters of biological liquids. Thus, radiotherapy affects the values of serum and urine surface tension parameters via changes in the qualitative and quantitative composition of surfactants contained in these

353 biological liquids; these effects are caused not only by a positive (curative) influence of X-ray irradiation, but also by secondary factors. 8.5.

Effects of surgical treatment

The results obtained from a screening of patients with tumours performed before and after an surgical treatment are rather interesting. Here the group of patients with nervous system neoplasm will be considered as examples. It is seen from Fig. 8.19 that the excision of brain tumour leads to a normalisation of the surface tension of the cerebrospinal liquid. 40

3O 25 =

20

;~

15

m

10

O

5 0

7--~----

---

-5 cyl

cy2

~3

Fig. 8.19. Changes in cerebrospinal liquid surface tension parameters before (black) and after (white) surgical treatment for patients suffering from brain tumours. Changes are given in % comparing to controls. Here the positive dynamics of the parameter 13"2 is especially demonstrative for patients with accusticus neurinoma and ventricle IV tumours, while for or3 the most representative patients in this sense are those with meningioma and cerebellum tumours. In these cases, decreased ~, values were observed, and the average values of this parameter after an operation were virtually the same as those in the control group. Only for patients with tumours of posterior cranial fossa this positive dynamics of interfacial tensiograms following an surgical treatment was not so pronounced; however, for these patients deviations of the initial parameters were also rather insignificant. Nevertheless, for this group of patients, the trend to a normalisation of dynamic surface tensions was also observed, and the equilibrium surface tension had attained its initial values.

354 The

excision

o f spinal

cord

tumours

is also

characterised

by

a subsequent

trend

to

n o r m a l i s a t i o n o f the d y n a m i c interfacial p a r a m e t e r s for b o t h b l o o d and c e r e b r o s p i n a l liquid, as one can see in Figs. 8.19, 8.20 and 8.21.

75 T f

70 -~--... 65 ;~

.. 60

"~

55+ i

50 -2

-1

0 lg(tef) [S]

i

1

1

2

Fig. 8.20. Examples of serum tensiograms obtained from male patient, age 31, with spinal cord tumour. Thick line - before surgical treatment, thin line - after surgical treatment; dotted line correspond to average values for healthy males of corresponding age.

7 5

--

70-E Z E 65 + ~

... ,.,,.

60

--2

~ -1

-

~ 0 lg(tef) [S]

.-

o

1

Fig. 8.21. Examples of serum tensiograms obtained from male patient, age 60 with tumour metastasis into epidural space of spinal cord. Thick line- before surgical treatment, thin line - after surgical treatment; dotted line correspond to average values for healthy males of corresponding age.

355 In particular, significant increases of surface tension values for blood serum were observed, while for liquor these parameters decreased. While studies of surface tension of biological liquids at later stages of the observations exist, the analysis of the dynamic tensiographic parameters with respect to the efficiency of treatments, a correlation analysis of the variables with the level of proteins, lipids and other surfactants are still to be done, even at a stage one can apply the method of dynamic interfacial tensiometry for monitoring of curing and diagnostics. Table 8.3 summarises the data concerning the variations of interfacial tensiograms of serum obtained from patients with various localisation of tumour. Table 8.3. Differential diagnostic indicators of serum surface tension variation for various localisation of turnouts

Tumour localisation (3"1

0"2

0"3

Stomach Lung Liver

+

Genitals

+

Mammary gland Brain Spinal cord "+" - statistically significant increase of parameter compared to normal; "-" - statistically significant decrease of parameter compared to normal

8.6. Summary Changes in the composition of surface active substances in the blood and other biological fluids of patients with neoplasms are complex and include hormonal and fermental disturbances, variations in the eicosanoid and peroxide oxidation of lipid systems, peculiar features of the electrolyte and immune imbalance. Chemotherapy and/or radiation treatment, duration of the disease, patient's sex and age also contribute to dynamic surface tension

356 chharacteristics of the various biological liquids that could be sampled from patients with neoplasms. For the analysis and interpretation of surface tension parameters all these factors must be considered. At the present stage, however, no comprehensive analysis and interpretation is available. Nevertheless, the presented approach based on dynamic interracial tensiometry of biological liquids can be regarded to as a promising, quite reliable and simple diagnostic method, magistral to all kinds of patients. 8.7. References

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358 Mattison, P., Cove, D.H., Walsh, L., Brit. J. Cancer, 43(1981)542. Morozkina, T.S., Energeticheski obmen i pitanie pri zlokachestvennikh novoobrazovaniyakh, Belarus, Minsk, 1989. Nigam, S., Becker, R., Rosendahl, U., Prostaglandins, 29(1985)513. Nikiforova, N.V., Karpatovsky, V.I., Chumakov, A.M., Bull. Eksper. Biol., 3(1993)290. Ostrowsky, J., Jarosz, D., Butruk, E., Neoplasma, 36(1989)353. Potapov, S.L., Korman, D.B., Bull. Eksper. Biol., 113(1994)631. Proter, N.A., Accounts Chem. Res., 19(1986)262. Ryabov, G.A., Pasechnik, I.N., Azizov, Yu.M., Ther. Arch., 7(1989) 151. Reznikov, Yu.P., Kamaeva, O.I., Pimenova, N.S., Ther. Arch., 1(1996)52. Rudenko, V.A., Lisyanyi, I.I., Skobsky, E.I., Clin. Kchir., 12(1992)36. Shelepov, V.P., Pasha-zade, G.R., Chekulaev, V.A., Shapot, V.S., Eksper. Oncol., 6(1987)62. Shinjiro, O., Keiki, O., Shinjiro, M., Clin. Chim. Acta, 182(1989)209. Sologub, L.I., Pashkovskaya, I.S., Antonyak, G.L., Eksper. Oncol., 6(1992)7. Spector, A.A., Bums, C.P., Cancer Res., 47(1987)4529. Titov, V.N., Sanphirova, V.M., Ther. Arch., 7(1984) 147. Tolkacheva, N.V., Levochev, M.M., Medvedev, F.A., Vopr. Oncol., 37(1991)293. Tolkacheva, N.V., Borisenko, S.N., Kulakova., S.N., Vopr. Oncol., 41 (1995)29. Tsuda, M., Yamagishi, Y., Katsunuma, T., FEBS Lett., 232(1988) 140. Tsvetanova, E.M., Liqvorologiya, Zdoroviya, Kiev, 1986. Tryggvason, K., Hoyhtya, M., Salo, T., Biochim. Biophys. Acta, 907(1987)191. Ulianenko, S.E., Surinov, B.P., Karpova, N.A., Med. Radiol., 5(1991)29. Varbanets, V.F., Vopr. Med. Kchimii., 2(1990)33. Vasilieva, T.G., Bersnev, V.P., Valberg, A.Yu., Kchachtriyan, V.A., Vopr. Neirokchir., 1(1995)21. Yoshino, M., Jap. J. Clin. Oncol., 10(1980)229.

359 List of Symbols

A

surface area

a

intermolecular interaction constant

(

a = 2~/Apg

)1/2

capillary constant

ael = z 2F/c0z(8~RTcz) 89 electrostatic constant b

parameter of the adsorption isotherm

C

bulk concentration of surfactant or protein

D

diffusion coefficient

E

energy

F

Faraday constant

f

correction factor

fi

activity coefficient of component i

g

gravity constant

H

immersion depth of the capillary into the liquid

H

enthalpy

Ki = (x~/x~lx._.o

distribution coefficients of state or component i at infinite dilution

k

kinetic rate constant

L

gas flow rate

1

capillary length

M

molecular weight

M

average value in statistical calculations

m

aggregation number of 2D-aggregates

m2= r

distribution of values measured for a number n of persons

Ni

number of moles of the ith state or component i

n

number of experiments or measured values in statistical calculations

P

pressure

Pb

pressure in the bubble

P~

pressure in the measuring system

R

gas law constant

r

radius

360 radius of a separating bubble capillary radius time tb

bubble time

td

deadtime

t~ff

effective surface age

th

hydrodynamic relaxation time

t~

bubble lifetime

T

absolute temperature

xi

=

N i / EN i

molar fraction of component i

Vb

bubble volume

Vs

volume of the measuring system sound velocity number of unbound unit charges measure of the surface activity of a protein configuration adsorption rate constant

A9

difference between the densities of liquid and gas standard deviation of measured values in statistical calculations surface concentration

Fc

critical adsorption of protein aggregation in the surface layer

Fi

adsorptions of component dielectric permittivity of the medium

X0 = -(d~/dt 1/2)t ~ 0 initial slope of surface tension curve X = (dcy/dt-1/2)t __~

final slope of surface tension curve liquid bulk viscosity

v

gas kinematic viscosity

ei = F~e~

relative surface coverage standard chemical potential of component i

l - [ = 0"0 - O"

surface pressure

361 surface tension 0"0

surface tension for t--->O

0"1

surface tension for t = 0.01 s

0"2

surface tension for t = 1 s

0"3

surface tension for t--->oo

0)i

partial molar area of component i

q0 = zFw/2RT

dimensionless electric potential electric potential

ae = (eRT/F2cz) v2

Debye length

Subscripts and superscripts c

critical value

max

maximum value of a parameter

s

surface or interface phase

Z

average

List of abbreviations for diseases and substances A

acetone

AA

acetoacetate

AC

acetyl carnetine

GL

glycine axisymmetric drop shape analysis

ADSA AGN

acute glomerulonephritis

AL

alanine

BD

Bechterew's disease

BSA

bovine serum albumin

362 C

creatinine

CGN chronic glomerulonephritis CH

cholesterol

CHE cholesterol ethers CRI

chronic renal insufficiency

C 10DMPO

decyl dimethyl phosphine oxide

D

dimethyl amine

DEL

electric double layer dipalmitoyl phosphatidyl choline

DMPC

DPPC dipalmitoyl phosphatidyl choline DN

diabetic nephropathy

FFA

free fatty acids

G

gout

GGN Genoch glomerulonephritis GHV glomerulonephritis accompanying hemorrhagic vasculitis H

hippurate

HD

hypertension disease accompanied by nephrosclerosis

HSA human serum albumin HV

haemorrhagic vasculitis

HX

hypoxantine

I

insulin

IgG

immunoglobulin G

IgM

immunoglobulin M

KA

kidney amyloidosis

363 KS

kidney sarcoidosis

L

lactate

LDG lactate dehydrogenase LGN lupus glomerulonephritis LL

lysolecithin

MBPM

maximum bubble pressure method

MCG mesangiocapillary glomerulonephritis MDG monodiacyl glycerides MN

myelomic nephropathy

MPG mesangioproliferative glomerulonephritis MPT 2 maximum bubble pressure tensiometer OA

osteoarthrosis

Of

oestradiol (females)

Om

oestradiol (males)

PA

psoriatic arthropathy

PC

phosphatidylcholine

PE

phosphatidyl ethanol amine

Pf

progesterone (females)

PL

phospholipids

Pm

progesterone (males)

PN

podagric nephropathy

PPN

primary pyelonephritis

PS

phosphatidylserine

R

rheumatism

364 RA

rheumatoid arthritis

RD

Reiter' s disease

SLE

systemic lupus erythematosus

SPN

secondary pyelonephritis

SS

sclerodermia systematica

T

trimethyl amine oxide

TEl

ring tensiometer method

Tf

testosteron (females)

Tm

testosteron (males)

TG

triglicerides

TL

total lipids

T3

triiodothyronine

T4

thyroxine

TTH

thyrotropic hormone

TWEEN 20

oxyethylated surfactant

UA

uric acid

UL

uro|ithiasis

V

valine

X

xantine

365

Subject index ~t-fetoprotein 82, 86, 324

adhesive proteins 127

c~-phospholipids 203

adrenocorticotrophic hormone 326

~t-lactalbumin 36

ADSA 40

ot-tocopherol 136, 149

adsorption isotherm 14

oq-antichimotrypsin 260

adsorption kinetics 17

c~-antitrypsin 228,260 ctl-globulins 86 a2-antiplasmin 110 c~2-macroglobulin 110, 237, 254, 260, 335

adsorption layer thickness 13, 27 adsorption ofpolyelectrolytes 1 adsorption of surfactants 1 adsorption rate constant 23, 54 aerodynamic resistance 45

13-casein 21 13-galactosidase 140 13-glucuronidase 236 132-glycoprotein-I 205 132-microglobulin 126, 175,216, 223,228, 237

age, effect of 259 alanine aminopeptidase 139, 140, 175 albumin 86, 107, 109, 137, 139, 143, 155, 167, 228, 339 albumin-like antigen 339 aldosterone 90

13-1actoglobulin 21, 36

alkaline phosphatase 228

13-thromboglobulin 11O, 168

alopecia 198

7-globulins 86

alveolar lipoproteinosis 271

7-glutamate dehydrogenase 74

alveolar membrane permeability 254

7-glutamyl transpeptidase 74

amino acids 323

abortion 83

ammonia 253

acetyl-13-D-glucosamine peptidase 115, 140,

amniotic liquid 82, 86, 95

175

amylase 139

acid phosphatase 140, 269

amyloid P-component-glycoprotein 324

acute glomerulonephritis 100, 117, 120

amyloidosis 179

acylglycerides 252

androgens 92

366 angioreticulomas 337

Bowman's capsule 230, 234

antibody 118, 121, 217

brain tumours 330, 353

anti-coagulant activity 205

bronchial asthma 253,264

antigen 216

bronchial secretion 255

antigen-antibody complexes 231

Bronchitis 259

antioxidant system 134, 135, 268, 276, 340

BSA 21, 28, 36

anti-phospholipid syndrome, 203

bubble deadtime 45, 50

apolipoproteid-C 133

bubble formation 54

apolipoproteid-E 133

bubble formation frequency 42

apolipoprotein (a) 203

bubble life time 44, 50

apolipoprotein-A 133

bubble pressure tensiometer 42

apolipoprotein-B 232

buoyancy forces 55

apolipoprotein-C 232

calcitonin 232, 326

apoprotein B 246

calcium 233, 237, 253

apoprotein C 246

calculous pyelonephritis 155

apoprotein D 246

capillaritis 198

APUD system 325

capillary constant 45

arachidonic acid 303

capillary pressure 45

arterial blood pressure 211

carbohydrates in cerebrospinal fluid 323

Arterial hypertension 115

carcinoembryonic antigens 324

astrocytoma 336

carcinoma of the stomach 328

asymptotic equations 59

cardial arrhythmia 209

bacterial lipo-polysaccharides 257

catalase 149, 279

basal membrane 139, 327

cathepsins 328

B-cell 131

cellular composition of liquor 326

Bechterew's disease 194

cerebrospinal fluid 297, 314

Bens Jones protein 182

ceruloplasmin 82, 155,228, 252

binding ability 202

cholesterol 74, 82, 86, 132, 136, 143, 146,

blood-brain barrier 320, 321

168, 246, 253,274, 303

367 choline chloride 279

denaturation 26

chondroitynsulphates 226

Diabetes mellitus 167

chorionic gonadotropin 90

Diabetic nephropathy 167

chronic bronchitis 251

dien conjugates 136, 149, 277

chronic glomerulonephritis 102, 105, 117,

diffusion kinetics 51

118

diffusional adsorption 50

chronic non-obstructive bronchitis 258

dipalmitoyl phosphatidyl choline 95,245

chronic obstructive bronchitis 259, 264

disbalance in lipid 73

Chronic pyelonephritis 155

dividing surface 5

Chronic renal insufficiency 114, 136, 137,

DNA 121

149, 166, 171 circulating immune complexes 123, 124, 125, 174, 192, 217, 239 circulatory insufficiency 210 coagulation factors 110

drop volume tensiometer 40 dynamic surface tension 42 dynamic tensiogram parameters 58 dynamic tensiographic parameters of cerebrospinal fluid 298

coagulation system 125,352

effective adsorption time 50

collagen 191,327

eicosanoides 76, 254

colloid-osmotic pressure 109, 183

elastase 191

complement system 122, 217, 231,260

elastin 191

configurations of protein molecules 3

electric double layer model 7

conformation distribution of adsorbed

electrical pressure transducer 41

molecules 21

electrolyte homeostasis 116

contra-insular hormones 86

ellipsometry 27

C-reactive protein 82, 110, 124, 155, 201,

endocarditis 198

228 creatine kinase 74, 331 creatinine 161 cryofibrinogen 239 cryoglobulin 124, 129

ependymoblastoma 337 equation of state 9 equilibrium surface tension 60 essential phospholipids 279 estradiol 163

368 estriol 90

glycoproteids 215

evolution of tumoural processes 344

glycoproteins 256

experimental pneumoconiosis 270

glycosaminoglycanes 191, 215,226, 235

expired air condensate 246, 250, 251,267

gonadotropic hormones 325

fatty acid 134, 172, 255, 339

gout 194, 231

fermenturia 139

gouty arthritis 234

fetoplacental complex 89

gouty nephropathy 232, 234

fetus plasma 82

growing bubble 44

fibrin 193,226

Guillain-Barr6 syndrome 328

fibrinogen 110, 124, 127, 193,226, 330

haematuria 207

fibrinolytic system 226

Hageman factor 124, 237

fibroadenoma 345

haptoglobin 155

fibrocytes 229

healthy persons 69, 252

fibronectin 74, 128, 141, 181, 216, 227, 228,

healthy persons, surface tension of urine 77

239,323

heamorrhagic vasculites 194

fibronectinuria 181

hemodialysis 148

fibrous degradation 272

hemorheological syndrome 229

fibrous-cystic mastopathy 345

hemorrhagic vasculitis 123

free fatty acids 74, 251

hemosorbtion 144

Genoch glomerulonephritis 117

hepatoma 324

gestation period 83

hepatomegalia 198

glioblastoma 335

high density lipoprotein 82, 86, 135, 142,

glomerular basal membrane 140, 167

205,301

glucocorticoid hormones 142

homocamosine 319

glucocorticoid therapy 237

hormone production of tumours 324

glucose 86, 203,322, 325

HSA 21, 27

glutathione 149

dynamic surface tension of 22

glutathione peroxidase 149, 277

hyaluronic acid 216, 226

glycolytic ferments 320

hydrodynamic relaxation 56

369 hydrodynamic theory 46

inorganic electrolytes 27, 146, 161

hydrogen peroxide 278

insulin 77, 171

hydrophilic capillary 52

interleukin- 1 200

hydrophilisation of a protein molecule 33

intravessel laser therapy 144

hydrophobic capillary 52

invasion of malignant cells 327

hydrophobised glass capillaries 55

ionic surfactants 29

hydrostatic pressure 41

kidney transplantation 150

hyperfiltration 168

kidney, general functions 99

hyperlipidemia 110, 132

lactate dehydrogenase 139, 228, 335

hyperlipoproteinemia 131

lamellar bodies 245

hyperparathyriodism 162

Langmuir trough 27

hyperuricemia 231

Laplace equation 45

hyperuricemial nephropathy 137

lecithin / sphingomyelin index 95

hypovolemia 106

lecithin choline 95

immune complexes 120, 123

leucine aminopeptidase 327

immunoglobulin 121, 174, 201, 216, 221,

Liebermann-Burchardt method 250

237, 260, 327 immunoglobulin-A 123, 125,228, 255, 317, 336

linolic acid 303 lipase 139 lipid 110, 131,203,247, 250, 259, 274, 321,

immunoglobulin-G 120, 124, 182, 223,228,

346

254, 316, 328

lipid

rheumatoid factor 225

lipid peroxide oxidation 136

immunoglobulin-M 120, 223,228, 254, 316

lipocerebrin 279

inertia effects 45

lipoprotein (a) 203

infectious brain diseases 298, 319

liver malignant neoplasm 328

inflammation mediators 254, 267

long time approximation 60

inflammatory processes 255

low density lipoproteids 74, 83, 131,136,

inorganic compounds in cerebrospinal fluid 324

143, 146, 168, 303,346 lung lavage 254

370 lung malignant neoplasm 329

microalbinuria 174

lung surfactants 245,249, 270

microangiopathy 167

lung tissue homogenates 274

microcirculatory bed 216

lupus glomerulonephritis 100, 122, 198

microvessels 123

lymphadenopathy 198

microvolume measuring cell 41

lysophosphatidyl choline 135,346

millisecond time range 46

lysosome hydrolases 327

mitochondrial ferments 321

lysosomic enzymes 237, 267

mixtures of protein and surfactant 29

lysozyme 21, 140

mucoproteins 155

magnesium 253

multiple sclerosis 303,308, 327

malonic acid 252

myelitis 308

malonic dialdehyde 136, 268, 277, 343

myelomic disease 181

maternal plasma 82

myelomic nephropathy 182

maximum bubble pressure method 29, 40,

myocarditis 198

41

myositis 198

theory 44

nephrectomy 167

medical microcell 43

nephrotic syndrome 106, 132, 197, 200, 204

medium mass molecules 137, 146, 268

nitrous components 76

medulloblastoma 336

non-ionic surfactants 28

membrane phospholipids 121

olygodendroglioma 336

meningitis 324

oncogeneous embolus 352

mesangial cell 230

operative treatment of tumours 353

mesangiocapillary glomerulonephritis 103,

orosomucoid 303

118 mesangioproliferative glomerulonephritis 103, 118, 222, 230

osmolarity of urine 72 osmotic concentration 116 osteoarthrosis 194, 236

metastatic neoplasm of the spinal cord 337

oxalic acid salts 160

methylpyrazolyl 279

oxidation potential of cerebrospinal fluid

metoxybenzoyl 279

320

371 oxyproline 273

prealbumins 314

oxytocinase 82

pressure oscillation 47

parameters of dynamic surface tension 70

primary pyelonephritis 155

parathyroid hormone 163,232

progesterone 163

P-component 179

prostaglandins 345

penetration depth 48

protein adsorption theory 123

pepsinogen 139

protein S A A 179

peroxide oxidation of lipids 134, 205,268,

proteinases 327

276, 341

proteins 314

phosphatidyl choline 135, 205, 256

proteins at liquid interfaces 1

phosphatidylethanolamine 135

proteinuria 72, 106, 109, 117, 139, 141, 174

phospholipid fractions 74

proteoglycans 226

phospholipids 82, 132, 134, 203,252, 256,

proteolytic ferments 192

274,321

psoriasis 229

placenta 81

psoriatic arthropathy 229

placental hormones 90

psoriatic nephropathy 231

plasma viscosity 229

purine metabolism 137

plasmapheresis 144, 147, 183,237

purines 231

plasminogen 198, 203,226

pyruvate kinase 253

plasminogen

radioactivity method 27

pneumoconiosis 271

radiotherapy 348

pneumonitis 198

Raynaud syndrome 198

pneumonocytes 245

reconformation rate 18

podocytes 139

Reiter's disease 194

Poiseuille approximation 47, 48

Reiter's disease 228

polyelectrolytes 6

renal hemodynamics 158

polyradiculoneuritis 308

renin-angiotensin-aldosterone system 106

polysaccharides 74

respiratory distress-syndrome 95

poly-unsaturated fatty 341

respiratory insufficiency 262

372 rheological properties of blood 125, 135, 155, 158

SP-B 245 SP-C 245

rheonephrogram 99

SP-D 245

rheumatism 194, 207

sphingomyelin 82, 135, 205, 256, 346

rheumatoid arthritis 192, 194, 216

spondylogeneous diseases 298

rheumatoid factor 219

steroid hormones 77

ribonuclease 21

steroid-binding globulin 91

R-protein 181,228

stopped flowregime 53

sarcoidosis 185

stromelysine 328

sclerodermia systematica 194, 215

submillisecond bubble lifetime 49

sclerodermic glomerulonephritis 216

superoxide dismutase 149, 217, 268, 278,

secondary kidney amyloidosis 222

343

secondary pyelonephritis 156

surface activity of a protein molecule 8

secondary structure of protein molecules 28

surface aggregation of the proteins 15

selective permittivity 72

surface pressure 11

selective reabsorption 140

surface tensiometry in nephrology 99

seronegative rheumatoid arthritis 225

surface tension

serum albumins 344

during gestation 81

serum mucoids 351

effect of age 77

serum tensiograms for tumours 328

for female 74

serum transudate 255

for male 74

shear viscosity 32

isotherm 31

of mixed monolayers 32

of serum, correlation with amniotic

short time approximation 59

liquid 84

sialic acid 155

of healthy children 79

sodium 107, 172

surfactant monolayers 1

transport of 90

surfactant protein A 246

somatotrophic hormone 325

surfactant proteins B 246

SP-A 245

surfactant proteins C 246

373 synovial fluid 223,237

ultraviolet irradiation 144

synovial membrane 225

unsaturated fatty acids 303,345

systemic lupus erythematosus 121, 130, 194,

unsaturated fatty acids

195

urea 109, 136, 161

Szyszkowski-Langmuir equation 33

uric acid 74, 137

Tamm-Horsfall mucoprotein 72

urine 103, 141,236

Teflon capillaries 55

urokinase 140

thyreoid hormones 77

urolithiasis 158, 185

transferrin 155, 335

vascular brain diseases 298, 329

transudation of proteins 316

vaso-active peptides 315

traumatic brain damage 330

very low density lipoproteids 74, 86, 132,

traumatic brain damages 298

136, 142, 303

treatment of bronchopulmonary diseases 279

Villebrand's factor 168

triglyceride 146

viscosity 46, 73,124, 158, 192

triglycerides 74, 82, 86, 92, 132, 136, 143, 172 trioxypurine 159

of blood 99 vitamin K - dependent glycoprotein-C 110, 324

tubulointerstitial damage 105

vitamin E 268

tumoural hypoxia 352

Waaler-Rose test 221

tumours

wettability of the capillary surface 56

of nervous system 298, 329

Wilhelmy balance 249

of female reproductive organs 330

Wilhelmy plate 27

of the mammary gland 330

xanthochromia 319

ultrafiltration 144

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