Nutrition and biochemistry of phospholipids

Nutrition and Biochemistry of Phospholipids

Editors

Bernard F. Szuhaj Central Soya Co., Inc. Fort Wayne, Indiana

Willem van Nieuwenhuyzen Central Soya Specialty Products Aarhus, Denmark

PRESS Champaign, Illinois

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Nutrition and hiochemistry o f phospholipids I editors. Bemard F. Szuhaj. Wiltem van Nkuwcnhuyu-n. p. : cm. Include. bibliographical refemlCH and index. ISBN ]·893997-42·1 (alk. paper) I. Ph<»pholipids--Physiological effect [DNLM: I. Phospholipid •. 2. Biochemistry. 3. NutritlOln. QU 93 N976

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Preface The focus of the 8th International Congress was Nutrition and Biochemistry of Phospholipids. It was held September 7–10, 2002, at the University of Vienna in Vienna, Austria. The 8th Congress is a result of the International Lecithin and Phospholipid Society's scientific program to bring together leading scientists working in the area of phospholipids on a biennial basis if possible. The last ILPS Congress, organized and chaired by Drs. Szuhaj and Zeisel, respectively, was held in 1996 in Brussels, Belgium. This book is an extension of that Congress after six years of the efforts at Brussels. The venue in Austria at the University of Vienna was selected to bring together the invited speakers and an enthusiastic scientific group to be updated on the latest scientific information and technology for the nutrition and biochemistry of phospholipids. Coincidentally, the 3rd International Symposium on Soy Lecithin on “Lecithin and Health Care” was held April 13–14, 1984, in Vienna as well. Drs. Paltauf and Lekim were organizers of that program. As the Congress has expanded from Symposia to more scientific evaluations, we have gone from dietetic applications to biochemical, pharmaceutical, and analytical considerations of phospholipids. Also it included phospholipids characterization, metabolism, and novel biological applications to choline, phospholipids, health, and disease. Scientifically we are searching for the direction and conclusions for the application of phospholipids in human nutrition. Some of the papers presented show that we still have no clear solution to why and how and how much phospholipids make a difference in human nutrition. Nevertheless phospholipids are involved in many intrinsic applications within the cell. We know they are part of all major tissue and concentrated in vital organs that require neuronal interaction. This 8th Congress had four sessions covering phospholipids metabolism in brain function, choline and galactosphingolipids in health and disease, phospholipids in cardiovascular, liver, and muscle health, and finally, phospholipids in infant nutrition. This book, which contains these current research activities and updates, will stimulate the scientific community to continue working on phospholipids in biochemistry and nutrition. Understanding the mechanisms of phospholipids, including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol lipids, is important to general health and well being. Phospholipids are not miracle compounds but are heavily involved in most membrane processes and are important in overall health. The research activities on phospholipids bring together a small nucleus of interested scientists who are looking at opportunities for macronutrients and micronutrients in many health areas for the body. This particular Congress was divided into four separate sessions that expand the interest level and activities for phospholipids today and in the future. B.F. Szuhaj W. van Nieuwenhuyzen

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Acknowledgments The ILPS Congress Secretary and Chairman wish to thank and recognize several important areas as follows. First we would like to thank the ILPS Board for encouraging the organizers to complete this scientific challenge as part of the ILPS interests to promote scientific investigation of phospholipids. We also want to acknowledge the Scientific and Advisory committees and session chairpersons for their patience and efforts in carrying out their responsibilities. The location of the meeting at the University of Vienna was ideal for 100 plus participants. Without financial support from several companies this event would not be possible. Donations were provided by the following companies: American Lecithin Company, Archer Daniels Midland, Avanti Polar Lipids, Central Soya Company, Degussa BioActives, Riceland Foods, and Roland Arzneimittel. We also would like to recognize the contributions from the Austrian Medical Academy of Nutrition, AOCS Phospholipid Division, H&N Division and European Section, and the University of Vienna. We want to thank Mondial Congress for administration, registration, and social events for this Congress. Special thanks go to the ILPS President Willem van Nieuwenhuyzen for his extraordinary efforts in making this Congress happen in Europe and Carolyn Boone for keeping it all together for the ILPS Congress and these Proceedings. B.F. Szuhaj W. van Nieuwenhuyzen

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Contents

Chapter 1

Preface

Chapter 1 Phospholipid Transporters in the Brain Robert A. Schlegel, Margaret S. Halleck, and Patrick Williamson Chapter 2 Stimulation of Lipases and Phospholipases in Alzheimer Disease Akhlaq A. Farooqui, Wei-Yi Ong, and Lloyd A. Horrocks Chapter 3 Is There Evidence That Phospholipid Administration Is Beneficial for Your Brain? G. Pepeu Chapter 4 Phospholipid and Fatty Acid Metabolism in Schizophrenia and Depression M.S. Manku and D.F. Horrobin Chapter 5 Altered Prostaglandin Mediated Skin Flush in Schizophrenia—Implications for Early Psychosis Interventions Stefan Smesny, Timm Rosburg, Sven Riemann, and Heinrich Sauer Chapter 6 Nutritional Implications of Sphingolipids: Occurrence and Roles in Cell Regulation Alfred H. Merrill, Jr., Holly Symolon, Jeremy C. Allegood, Qiong Peng, Sarah Trotman-Pruett, and M. Cameron Sullards Chapter 7 Digestion and Absorption of Sphingolipids in Food Åke Nilsson, Erik Hertervig, and Rui-Dong Duan Chapter 8 Dietary Sphingolipids in the Prevention and Treatment of Colon Cancer Eva M. Schmelz Chapter 9 Compositional Analysis of Complex Mixtures of Sphingolipids by Liquid Chromatography–Tandem Mass Spectrometry M. Cameron Sullards, Elaine Wang, and Alfred H. Merrill, Jr. Chapter 10 Role of Dietary Gangliosides in Early Infancy Ricardo Rueda, Enrique Vázquez, and Angel Gil

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Chapter 11 The Benefits of Lecithin on Cardiovascular Disease Debra L. Miller Chapter 12 Is There a Rationale for Phospholipid Supplementation in Athletes? Fred Brouns Chapter 13 Effects of Phosphatidylcholine Intake on Liver Function and Liver Carcinogenesis David J. Canty Chapter 14 Polyunsaturated Phosphatidylcholine in Chronic Liver Disease—Past and Present K.-J. Gundermann and E.W. Scheele Chapter 15 Cyclic Phosphates Originating from Degradation of Phospholipids M. Shinitzky and A. Pelah Chapter 16 Effect of Two Diets in Children and Adolescents with Familial Hypercholesterolemia: Soy-Protein Diet Versus Low Saturated Fat Diet K. Widhalm and E. Reithofer Chapter 17 Essential Polyunsaturated Fatty Acids in Mothers and Their Neonates Gerard Hornstra Chapter 18 Liposomes in Nutrition B.C. Keller Chapter 19 Lower Incidence of Necrotizing Enterocolitis in Infants Fed a Pre-term Formula with Egg Phospholipids Susan E. Carlson, Michael B. Montalto, Debra L. Ponder, Susan H. Werkman, and Sheldon B. Korones. Chapter 20 Perinatal Supply and Metabolism of Long-Chain Polyunsaturated Fatty Acids Hans Demmelmair, Elvira Larque, and Berthold Koletzko Chapter 21 Phosphatidylcholine as Drug Substance and as Excipient— the Mechanism of Biological Activity Miklos Ghyczy and Mihály Boros

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Chapter 1

Phospholipid Transporters in the Brain Robert A. Schlegela, Margaret S. Hallecka, and Patrick Williamsonb aDepartment

of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania 16802 bDepartment

of Biology, Amherst College, Amherst, Massachusetts 01002

Organs in the body are specialized to perform the various functions required to sustain life. The cells from which these organs are assembled are highly specialized in turn, having elaborated upon some aspect of general cell function to acquire their specialized properties. The brain is assembled from cells that have elaborated upon general plasma membrane functions. Physically, the high surface/volume ratio of the elongated processes of axons and dendrites in neurons, and of folded myelin figures, ensures that membranes and their amphipathic constituents constitute a disproportionately large fraction of the molecular composition of brain. Functionally, the intra- and intercellular transmission of nerve impulses are the direct consequence of highly specialized membrane channels and active ion transport processes, as well as vesicle trafficking events at the plasma membrane. Adenosine triphosphate (ATP) hydrolysis provides the energetic input that establishes the ion gradients upon which nerve impulses depend by driving the Na,K-ATPase active transporter, as well as the plasma membrane Ca2+–ATPase. Both of these enzymes belong to a large family of ATP-dependent membrane transporters termed P-type ATPases. Recently, a new subfamily of these transporters has been characterized; these enzymes appear to catalyze the transbilayer transport of amphipathic ions, such as phospholipids. Such transporters might be expected to play a prominent role in brain function.

Transbilayer Phospholipid Asymmetry Virtually every cell in the body, including those in the brain, actively maintains an asymmetric distribution of phospholipids across the plasma membrane (1). By an energy-dependent process, aminophospholipids are translocated from the outer to the inner leaflet of the membrane bilayer. The resulting imbalance of phospholipid molecules in the two leaflets, biased toward the inner leaflet, is compensated by the passive diffusion of choline phospholipids from the inner to the outer leaflet. These processes set up a dynamic asymmetric steady state, with aminophospholipids concentrated in the inner leaflet and choline phospholipids in the outer leaflet (2). It has been estimated that the total energy requirements for phospholipid metabolism in

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the mammalian brain account for about 13% of total ATP consumption. The cost of maintaining plasma membrane lipid asymmetry at steady state is estimated to represent 60% of that amount (3). Such a high price for maintaining asymmetry suggests that an important function is served by the active transport of aminophospholipids. Indeed, display of one of the aminophospholipids, phosphatidylserine (PS), on the cell surface leads to recognition by macrophages (4). Failure to remove PS from the cell surface by inward transport results in phagocytosis and destruction of the cell. There are times, however, when it is desirable to mark cells for destruction. One of those instances is when cells undergo apoptosis or programmed cell death. As part of the apoptotic program, cells display PS on their surface; once engulfed by macrophages, the dying cells can be safely dismantled, thus avoiding inflammation around disintegrating corpses. Exposure of PS on the cell surface results in part from down regulation of aminophospholipid translocase activity. In addition, another activity is required because of the slow rate of passive diffusion of PS from the inner to the outer leaflet of the plasma membrane. Diffusion is accelerated by activation of another membrane protein, the scramblase, which permits all phospholipids to equilibrate across the bilayer in a matter of minutes. The combined inactivation of the translocase and activation of the scramblase ensures rapid exposure of PS and elimination of apoptotic cells in a timely fashion (2,5). One of the normal functions of apoptosis is the remodeling of tissues during embryogenesis. Indeed, apoptotic cells presenting PS are seen throughout mouse embryos, including both the central and peripheral nervous system (1). In fact, the development of the brain, like that of the immune system, depends on the overproduction of cells followed by selective removal of apoptotic cells that failed to establish functionality.

The Aminophospholipid Translocase Family of Proteins Although the aminophospholipid translocase was partially purified from erythrocytes and reconstituted in artificial lipid vesicles (6), this plasma membrane enzyme was never isolated from erythrocytes or any other cell type in sufficient quantity and purity to allow cloning the gene encoding the enzyme. However, aminophospholipid translocating activity is also present in the membrane of secretory vesicles, although the functional reason for its presence there is not known (7). When the gene encoding the vesicle enzyme was cloned, it defined a new subfamily (type IV) of mammalian P-type ATPases (8). The human genome contains 14 genes in the type IV P-type ATPase subfamily (2). The phylogenetic relationships between these genes compared to a Ca2+–ATPase (type IIA) are shown in Figure 1.1. Befitting its distinction as the first to be discovered, the gene encoding the vesicle enzyme was designated the 1a (ATP8A1) gene of the type IV subfamily. P-type ATPases include some of the most well-studied membrane transporters. Their name derives from the phospho-intermediate that is formed by transfer of the

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Fig. 1.1. Phylogeny of type IV P-type ATPases. The phylogenetic relationship among the 14 mammalian type IV P-type ATPases and the type IIa sarcoplasmic reticulum Ca2+–ATPase (SERCA2), expressed as a Neighbor-joining tree, using p-distance, complete deletion, and Felsenstein’s bootstrap test (24) (1,000 replications). Large Arabic numerals designate classes. The names of the genes adopted by the HUGO Nomenclature Committee or the Mouse Genome Database are in parentheses, followed by any common names: Ca2+ ATPase (ATP2A2; SERCA2); 1d (ATP8B2); 1m (ATP8B4); 1c (ATP8B1; FIC1); 1k (ATP8B3); 1a (Atp8a1; APLT); 1b (Atp8a2); 5c (ATP10c; pfatp); 5b (ATP10B); 5d (ATP10D); 6f (Atp11b); 6h (Atp11a); 6g (Atp11c); 2a (Atp9a); 2b (Atp9b).

gamma phosphate of ATP to an aspartate residue of the enzyme. The ability of solubilized recombinant enzyme to dephosphorylate in the presence of its substrate has confirmed that the 1a enzyme transports aminophospholipids (9), and it is widely assumed in the literature that all enzymes in the type IV subfamily transport aminophospholipids. This assumption is probably incorrect, and we have proposed that the subfamily transports a variety of amphipathic molecules.

Expression of Transporters in the Brain Several techniques have been used to examine where type IV transporters are expressed in mammalian organisms. Using Northern analysis, the expression of 7 different transporters was examined in eight different tissues of the mouse (10,11). None was expressed ubiquitously in all tissues, but five of the seven transporters

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were expressed in the brain. In situ hybridization is a more sensitive technique to examine expression and allows determination of the regions of a tissue, or even the individual cells, in which genes are expressed. This approach was used to examine the expression of the same seven transporter genes in mouse embryos and young mice (11). This study indicated that with the exception of the 1c gene, the other six genes were all expressed in the brain. No gene was expressed ubiquitously in all parts of the brain; rather, expression of a particular gene was restricted to several locations. No two genes exhibited the same pattern of expression, although multiple genes were expressed in some regions of the brain. Thus, the pattern of expression was complex and quite distinctive for each gene. Large sequencing projects have examined expressed genes by sequencing cDNA transcribed from mRNA isolated from various tissues. A compilation of the data from all tissues has been compiled in a searchable database (Gene Cards, http://bioinformatics.weizmann.ac.il/cards/), allowing the pattern of expression of given genes to be retrieved. This approach indicates that all 14 of the type IV Ptype ATPases are expressed at some level in the human brain. We have confirmed this result in mice using gene-specific primers and an immobilized murine brain cDNA library as template: 10/10 genes examined were expressed in brain. The greater sensitivity of the cDNA approaches (a single mRNA molecule may be sufficient to score as a positive) may explain the discrepancy with the results obtained by Northern and in situ analyses.

Transporters and Brain Disease The importance of the type IV transporters in human health and disease is readily apparent in conditions where normal expression of one of the genes is impaired. The most extensively investigated disorders are Summerskill syndrome and Byler disease. Both are hereditary disorders characterized by impaired bile flow, or cholestasis. Both are caused by mutation of the 1c (ATP8B1) gene of the type IV P-type ATPases (12), commensurate with its expression in the liver and intestine. Curiously, as indicated above, this enzyme is also expressed at low levels in the brain, although the functional significance of this expression is unclear. Mutations in a type IV transporter have also been implicated in disorders of the brain. Angelman Syndrome (AS), characterized by mental retardation, tremor, ataxia, abnormal gait, inappropriate laughter, and epilepsy, maps to the q11-13 region of human chromosome 15. This region is imprinted, or silent, in the paternally inherited chromosome, so that its deletion in the maternally inherited chromosome is responsible for 70% of known cases of AS; paternal inheritance of both chromosomes (disomy) is responsible for 2–3 % of cases. An additional 5% of AS cases are caused by imprinting defects, and 2% may be explained by gross chromosome rearrangements. Until recently, the UBE3A gene was the only gene known to be contained in the deleted region linked to AS. Yet only about 5% of cases appear to involve mutation of the UBE3A gene, which is required for long-

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term synaptic potentiation. Two recent reports, however, map the type IV 5c (ATP10C) transporter gene to the q11-13 region of chromosome 15, in close proximity to UBE3A (13,14). In mice, expression of the 5c gene is localized to the cerebellar granule cells, the hippocampus, cells adjacent to the corpus callosum, and the olfactory bulb mitral cells (11), which are the precise regions where Ube3a is maternally expressed (15). Using RT-PCR, maternal expression of the 5c gene has been shown to be absent from AS patients with 15q11-13 deletions. In addition, the gene is not expressed in an AS patient with an imprinting defect, suggesting that the AS-associated imprinting center also controls the expression of the 5c gene (14). As a result, the 5c gene is now a leading candidate for the gene whose impaired expression is responsible for AS. Interestingly, in mice, maternally inherited deletions of the 5c gene result in increased body fat and obesity (16). Since the obese phenotype is also a characteristic of a mouse AS model created by paternal disomy, and a subset of human AS patients are obese, some have speculated that the lack of 5c gene expression in certain brain tissues may also be related to obesity (14). Curiously, a recent report implies that overexpression of the15q11-13 region also has deleterious effects; maternal duplications of the region lead to an autistic phenotype, whereas paternal duplications of the region have no effect (13). Since mutations in the UBE3A gene are not seen in autistic individuals with a normal karyotype, it has been suggested that overexpression of the 5c gene might contribute to the autistic phenotype. Interestingly, the 5c gene is expressed at locations in the brain that have been indicated as being preferentially involved with autism (17).

Structural Analysis of Type IV P-Type ATPase Transporters Recently, new avenues for understanding the function of P-type ATPases have been opened by the availability of high resolution X-ray structures for a type II enzyme, the sarcoplasmic reticulum Ca2+–ATPase (SERCA2). The original structure reported by Toyoshima et al. (18) confirmed and illuminated previous biochemical and genetic studies and, in particular, provided a clear indication of the residues that bind the transported ions. Moreover, it became possible in the overall structure to visualize particular amino acids, such as those conserved in the P-type ATPase family and/or implicated in transport function by mutation studies. While a structure for a type IV P-type ATPase is not available, some insights into structure/function can be gained indirectly by taking advantage of the similarities between type II and type IV enzymes. Sequence analysis of the type IV subfamily not only indicated clear overall sequence similarity with type II transporters but also suggested that the Ca2+ transporters and the amphipath transporters share a similar transmembrane domain organization, with two pairs of membrane-spanning helices on the amino terminal side and three pairs of helices on the carboxyl terminal side of the largest cytoplasmic domain. These findings suggest the possibility that type IV enzymes might be threaded onto the structure of the Ca2+–ATPase to gener-

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ate a three-dimensional structural model of the former, with the goal of visualizing interesting regions of the protein sequence. The amino acid sequence of the Ca2+–ATPase (SERCA2) and the 1a protein were aligned using the CLUSTAL W program. Then, from viewing the structure of the Ca2+–ATPase, the alignment was refined manually. For example, substantial insertions in the 1a protein sequence that introduced amino acids into the middle of important structural domains were realigned to leave the basic structural elements intact. Similarly, deletions or insertions in transmembrane helices were adjusted to allow them to span the bilayer, as in the structural model, and in some cases were adjusted to align aromatic residues at the membrane interface. At later stages, the sequence was threaded onto the known Ca2+–ATPase structure (1EUL), and highly unfavorable sidechain clashes were identified using DeepView, the Swiss Protein Data Base (PDB) viewer, and the alignment further refined. Finally, prospective alignments were submitted to SWISS-MODEL (http://www.expasy.ch/swissmod/ SWISS-MODEL.html) for energy minimization. Alignments in which sidechain interactions could not be resolved were then further adjusted at the point in the sequence where the minimization failed to give an acceptable structure. The sequence alignment shown in Figure 1.2 gave rise to the successful homology model structure shown in Figure 1.3A. All transmembrane helices are clustered together to form transmembrane domain M, which is contained within the hydrophobic lipid bilayer. Above this domain in the figure are the three functional domains exposed in the aqueous compartment: the canonical DKTGTLT phosphorylation sequence of P-type ATPases is contained in the P domain, the N domain contains the ATP binding region, and the A domain is involved in activation. Once a model is generated, specific amino acids of interest may be identified and visualized. For example, conserved amino acids are of great interest, since they represent functionally important sites under constant surveillance by natural selection. Such residues were identified first by aligning the sequences of 46 type IV enzymes using CLUSTAL W. Residues in the structure were then shaded, according to the degree to which they were conserved at each position (Fig. 1.3A), using the ConSurf server (http://bioinfo.tau.ac.il/consurf/). For comparative purposes, a similar analysis was carried out on 46 homologs of the Ca2+–ATPase (Fig. 1.3B). In both cases, there is substantial conservation of the amino acids that comprise the helices in the interior of the transmembrane domain. The cytoplasmic domains are characterized by clusters of conserved sequences on the surfaces of the domains facing each other, with only a small degree of conservation of the residues on the faces of the domains facing outward. These conserved regions include the canonical phosphorylation sequence and nucleotide binding regions of P-type ATPases and constitute the interacting faces of the domains in the closed, E2, conformation of the P-type ATPases (19) that alternates in the catalytic cycle with the more open, E1, conformation shown in these enzyme models (18). Among the amino acid sequences conserved in the type IV subfamily are many that are conserved across the entire family. On the other hand, there are also conserved

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Fig. 1.2. Sequence alignment of the sarcoplasmic reticulum Ca2+–ATPase (SERCA2) and

the 1a (Atp8a1) transporter. Following alignment of the two sequences using CLUSTAL W, manual adjustments were made in the alignment as described in the text. Subfamily-specific consensus sequences A–J as well as those in P-type ATPase consensus sequences 1–4 (white letters on black background) are boxed. Transmembrane domains are underlined and the conserved aspartate characteristic of all P-type ATPases is marked with an arrow.

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A

B

Fig. 1.3. Structural models showing conserved residues in type IIA and type IV P-type

ATPases. A CLUSTAL W alignment of 46 type IV P-type ATPases or 46 type IIA P-type ATPases was used with the ConSurf server to shade either the space-filling structural model of the 1a protein generated by homology modeling using DeepView (see text) or (B) the known space-filling structural model of the SERCA2 Ca2+ transporter (1EUL). Two views are shown for each structure. Residues are shaded according to degree of conservation from most similar (black) to least similar (white). The three cytoplasmic domains, A (activation), P (phosphorylation), and N (nucleotide binding), as well as the M (transmembrane) domain, are indicated.

sequences that are specific to the subfamily or, in some cases, to specific classes within the subfamily. These subfamily-specific residues are scattered throughout the overall linear sequence, making it difficult to draw any conclusions about their functional implications. When these subfamily-specific sequences are visualized in the structure,

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however, an interesting pattern emerges. The first four of these conserved sequences (A–D, Fig. 1.2) include about 35 amino acids and are dispersed over the first 400 residues of the protein (10). As shown in Figure 1.4, these residues are not dispersed in the structure but rather are in immediate proximity and combine to form a single region in the protein. This region, which can be seen at the edges of the convex face on the right of the upper structure in Figure 1.3A, is not conserved in the corresponding location of the type II enzymes, which instead contains only a few isolated conserved amino acids. The crevasse formed on the surface of the type IV enzyme extends from the A cytoplasmic domain into the transmembrane region exposed to the lipid bilayer. The hydrophobicity of the residues in this crevasse is as expected—a mixture of polar and charged (largely cationic) residues in the cytoplasmic region and hydrophobic side chains exposed to the bilayer in the transmembrane region. The function of this structural feature cannot be determined with confidence until functional transport assays for type IV enzymes can be developed. Its location, however, suggests that it may form part of a pathway permitting exit or entrance of substrates into the protein interior during the transport cycle, a function consistent with the assignment of a similar role for a conserved aspartate and glutamates in this location in the Ca2+–ATPase (20). If so, the larger size of the feature in the type IV enzymes is consistent with a role in the transport of more bulky substrates, such as phospholipids and other amphipaths, in comparison with the simple metal ions transported by the Ca2+–ATPase.

Fig. 1.4. Location of type IV-specific

sequences in the structural model of the 1a transporter. View of the space-filling structural model of the 1a transporter with residues comprising type IV-specific consensus regions A–D shaded in dark gray using Protein Explorer (http://molvis.sdsc.edu/protexpl/index.htm). Domains identified in the legend to Figure 1.3 are labeled with large, open letters.

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The mechanism of transport in the P-type ATPases requires linkage between large movements of the three great cytoplasmic domains and movements of the helices forming the transmembrane domain that traverses the bilayer. While the cytoplasmic domains must subsume similar functions of binding substrate and ATP across the whole family, the membrane helices must include residues that bind the tranported substrates, with the nature of these residues determining what substrate is transported by each enzyme. In the sarcoplasmic reticulum Ca2+– ATPase, two Ca2+ ions are transported in each round of the catalytic cycle, a finding reflected in the structure of the enzyme by the presence of two intramembrane Ca2+ binding sites, sites I and II in Figure 1.5A. As predicted from earlier studies (21–23), both of these sites include anionic carboxyl groups contributed by aspartates and glutamates that neutralize the charge of the Ca2+ ion. Interestingly, the

Fig. 1.5. Comparison of the Ca2+ binding sites of the SERCA2 Ca2+–ATPase with anal-

ogous sites in the1a transporter. (A) Amino acids in Ca2+ binding site I, contributed by amino acids in TM(transmembrane helix)5, TM6, and TM8, and binding site II, contributed by residues in TM4 and TM6 of the Ca2+ transporter. (B) Amino acids occupying the same positions in sites I and II in the 1a transporter. Hydrophobic (white), polar (gray), or charged (black) residues in sites I and II are shown as space-filling models. Traces of TM1 and TM10 are identified.

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plasma membrane Ca2+–ATPase (PMCA4), a quite different P-type ATPase than its relative from the cell interior (SERCA2), transports only a single Ca2+ ion at each turn of the transport cycle, and in this case, two of the three anionic side chains at site I have been replaced by uncharged residues (Table 1.1). The retention of the third may be related to a role for this residue in transferring the transported calcium ion into or out of the remaining binding site II. The congregation of the anionic side chains results in the production of highly charged binding sites in the interior of the transmembrane domain (Fig. 1.5A). The importance of the corresponding residues in the type IV enzymes is indicated by their high degree of conservation (Table 1.1), but the nature of the site is quite different (Fig. 1.5). Unlike the highly charged binding sites in the Ca2+–ATPase, the sites in the 1a protein, and thus in the subfamily in general, lack charged residues and are composed of a mixture of hydrophobic and polar uncharged amino acids (Fig. 1.5B). Such substrate binding sites are consistent with the transport of more bulky and complex amphipathic molecules, in contrast to the simple charged ions of the Ca2+–ATPase and its relatives.

Summary In addition to water-soluble metal ions, membranes in living systems must be traversed by amphipathic molecules, such as phospholipids. The brain, which is enriched in membrane constituents, contains a new type of ATP-dependent transTABLE 1.1 Comparison of Residues in Ca2+ Binding Sites to Those in Equivalent Sites in Amphipath Transporters Site I

Site II

Gene

TM5

TM6

TM8

TM4

TM

SERCA2 PMCA4 1a 1b 1c 1d 1k 1m 2a 2b 5b 5c 5d 6f 6g 6h

N,E N,A N,Y N,Y N,T N,T N,T N,T S,S G,S N,V N,V N,V N,I N,T N,I

T,D M,D F,T F,T Y,T Y,T Y,S Y,T Y,T Y,T F,T F,S F,T F,T F,T F,T

E Q C C N S T S L L L L L T T T

E E I I I I M I I I I I I V I V

N,D N,D N,T N,T N,T N,T N,S N,T S,T A,T N,T N,S N,T N,T N,T N,T

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porters that may be specific for such amphipathic molecules, and defects in at least one of the proteins may be responsible for a human brain disorder. Analysis of the structure of these transporters is consistent with their role in amphipath transport and identifies specific regions of the enzymes of potential functional importance. References 1. Van den Eijnde, S.M., Boshart, L., Reutelingsperger, C.P.M., De Zeeuw, C.I., and Vermeij- Keers, C. (1997) Phosphatidylserine Plasma Membrane Asymmetry in Vivo: A Pancellular Phenomenon Which Alters During Apoptosis, Cell Death Differ. 4, 311–316. 2. Williamson, P., and Schlegel, R.A. (2002) Transbilayer Phospholipid Movement and the Clearance of Apoptotic Cells, Biochim. Biophys. Acta, 1585, 53–63. 3. Purdon, A.D., and Rapoport, S.I. (1998) Energy Requirements for Two Aspects of Phospholipid Metabolism in Mammalian Brain, Biochem. J. 335, 313–318. 4. Schlegel, R.A., and Williamson, P. (2001) Phosphatidylserine, a Death Knell, Cell Death Differ. 8, 551–563. 5. Schlegel, R.A., Krahling, S., Christie, A.J., and Williamson, P. (1998) in Choline, Phospholipids, Health, and Disease, Zeisel, S.H., and Szuhaj, B.F., AOCS Press, Champaign, IL, pp. 57–68. 6. Auland, M.E., Roufogalis, B.D., Devaux, P.F., and Zachowski, A. (1994) Reconstitution of ATP-Dependent Aminophospholipid Translocation in Proteoliposomes, Proc. Natl. Acad. Sci. USA 91, 10938–10942. 7. Williamson, P., and Schlegel, R.A. (1994) Back and Forth: The Regulation and Function of Transbilayer Phospholipid Movement in Eukaryotic Cells, Mol. Memb. Biol. 11, 199–216. 8. Tang, X., Halleck, M.S., Schlegel, R.A., and Williamson, P. (1996) A Subfamily of PType ATPases with Aminophospholipid Transporting Activity, Science 272, 1495–1497. 9. Ding, J., Wu, Z., Crider, B. P., Ma, Y., Li, X., Slaughter, C., Gong, L., and Xie. X-S. (2000) Identification and Functional Expression of Four Isoforms of ATPase II, the Putative Aminophospholipid Translocase: Effect of Isoform Variation on the ATPase Activity and Phospholipid Specificity, J. Biol. Chem. 275, 23378–23386. 10. Halleck, M. S., Pradhan, D., Blackman, C., Berkes, C., Williamson, P., and Schlegel, R.A. (1998) Multiple Members of a Third Subfamily of P-type ATPases Identified by Genomic Sequences and ESTs, Genome Res. 8, 354–361. 11. Halleck, M.S., Lawler, Jr., J.F., Blackshaw, S., Gao, L., Nagarajan, P., Hacker, C., Pyle, S., Nakanishi, Y., Ando, H., Weinstock, D., Williamson, P., and Schlegel, R.A.. (1999) Differential Expression of Putative Transbilayer Amphipath Transporters, Physiol. Genomics 1, 139–150. 12. Bull, L.N., van Eijk, M.J., Pawlikowska, L., DeYoung, J.A., Juijn, J.A., Liao, M., Klomp, L.W., Lomri, N., Berger, R., Scharschmidt, B.F., Knisely, A.S., Houwen, R.H., and Freimer, N.B. (1998) A Gene Encoding a P-type ATPase Mutated in Two Forms of Hereditary Cholestasis, Nature Genet. 18, 219–224. 13. Herzing, B.K., Kim, S.-J., Cook, Jr., E.H., and Ledbetter, D.H. (2001) The Human Aminophospholipid-Transporting ATPase Gene ATP10C Maps Adjacent to UBE3A and Exhibits Similar Imprinted Expression, Am. J. Hum. Genet. 68, 1501–1505. 14. Meguro, M., Kashiwagi, A., Mitsuya, K., Nakao, M., Kondo, I., Saitoh, S., and Oshimura, M. (2001) A Novel Maternally Expressed Gene, ATP10C, Encodes a

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Putative Aminophospholipid Translocase Associated with Angelman Syndrome, Nature Genetics 28, 19–20. Albrecht, U., Sutcliffe, J.S., Cattanach, B.B., Beechey, C.V., Armstrong, D., Eichele, G., and Beaudet, A.L. (1997) Imprinted Expression of the Murine Angelman Syndrome Gene, Ube3a, in Hippocampal and Purkinje Neurons, Nature Genetics 17, 75–78. Dhar, M., Webb, L.S., Smith, L., Hauser, L., Johnson, D., and West, D.B. (2000) A Novel ATPase on Mouse Chromosome 7 is a Candidate Gene for Increased Body Fat, Physiol. Genomics 4, 93–100. Lord, C., Cook, E.H., Lenenthal, B.L., and Amaral, D.G. (2000) Autism Spectrum Disorders, Neuron 28, 355–363. Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Crystal Structure of the Calcium Pump of Sarcoplasmic Reticulum at 2.6Å Resolution, Nature 405, 647–655. Toyoshima, C., and Nomura, H. (2002) Structural Changes in the Calcium Pump Accompanying the Dissociation of Calcium, Nature 418, 605–611. Lee, A.G., and East, M. (2001) What the Structure of a Calcium Pump Tells Us about Its Mechanism, Biochem. J. 356, 665–683. Clarke, D.M., Loo, T.W., Inesi, G., and MacLennan, D.H. (1989) Location of High Affinity Ca2+–Binding Sites within the Predicted Transmembrane of the Sarcoplasmic Reticulum Ca2+–ATPase, Nature 339, 476–478. Andersen, J.P.(1995) Dissection of the Functional Domains of the Sarcoplasmic Reticulum Ca2+–ATPase by Site-Directed Mutagenesis, Biosci. Rep. 15, 243–261. Zhang, Z.S., Lewis, D., Strock, C., and Inesi, G. (2000) Detailed Characterization of the Cooperative Mechanism of Ca2+ Binding and Catalytic Activation in the Ca2+ Transport (SERCA) ATPase, Biochemistry 39, 8758–8767. Felsenstein, J. (1985) Confidence Limits on Phylogenies: An Approach Using the Bootstrap, Evolution 39, 783–791.

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Chapter 2

Stimulation of Lipases and Phospholipases in Alzheimer Disease Akhlaq A. Farooquia, Wei-Yi Ongb, and Lloyd A. Horrocksa aDepartment

of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio, 43210 bDepartment of Anatomy, National University of Singapore, Singapore, 119260

Stimulation of glutamate receptors is involved in the release of arachidonic acid through degradation of phospholipids in neural membranes. A direct mechanism of arachidonic acid release involves the activation of phospholipase A2 and an indirect mechanism utilizes the phospholipase C/diacylglycerol lipase pathway. In kainic acid-induced neurotoxicity, the stimulation of kainic acid receptors results in the activation of phospholipase A2 and a rapid release of arachidonic acid from neural membrane phospholipids. A decrease in reduced glutathione immunoreactivity and an increase in 4-hydroxynonenal immunoreactivity accompany degeneration of neurons. This indicates that these neurons are under oxidative stress. Neurodegeneration is prevented and the immunoreactivities of cPLA2 and 4hydroxynonenal are decreased in hippocampal slice cultures when the application of kainic acid is accompanied with treatment with quinacrine and arachidonyltrifluoromethylketone. Treatment of neuron-enriched cultures with glutamate or Nmethyl-D-aspartate (NMDA) produced activation of the activities of diacylglycerol lipase and monoacylglycerol lipase. Dextrorphan and MK-801 blocked this activation. Marked elevation in activities of cytosolic phospholipase A2, diacylglycerol lipase and monoacylglycerol lipase was observed in Alzheimer disease (AD) brain compared to age-matched control human brain. Our studies suggest that the over-stimulation of glutamate receptors initiates a cascade of neurochemical events, including stimulation of lipases and phospholipases A2, enhancement of glycerophospholipid turnover, alterations in membrane permeability and fluidity, excessive calcium ion entry, reduction in ATP, accumulation of lipid peroxides including 4-HNE, and proteolysis. All these processes are closely associated with cell death in AD.

Introduction Alzheimer disease (AD) is a progressive neurodegenerative disease characterized by massive neuronal death associated with histopathological hallmarks such as extracellular deposition of amyloid fibrils and intracellular accumulation of neu-

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rofibrillary tangles (1). The main constituent of the amyloid fibrils is β-amyloid (Aβ), which is produced by the degradation of the amyloid precursor protein (APP). The cleavage of APP at the extracellular N-terminus and the intramembrane C-terminus of the Aβ domain liberates potentially amyloidogenic or neurotoxic Aβ peptide (1). Membrane phospholipids and their degradation products are increasingly becoming associated with the synthesis and secretion of APP. Second messengers generated by glutamate-induced stimulation of phospholipases A2, C, and D, such as arachidonic acid, diacylglycerol, inositol 1,4,5-trisphosphate, and eicosanoids, increase APP secretion (2–5). Additionally, levels of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), and especially plasmalogens (PlsEtn and PlsCho) are decreased in brain samples from AD patients (6–13), leading to destabilization of the neuronal plasma membrane. Hydrolysis of neuronal membrane phospholipids exposes the abnormal cleavage site in the APP transmembrane region, thereby generating amyloidogenic fragments (14). Thus alterations in neural membrane composition along with abnormal ion homeostasis and redox status may influence the pathogenesis of AD (15). The hydrolysis of phospholipids is accompanied by an increase in phospholipid degradation products, such as phosphodiesters (PDE) and phosphomonoesters (PME) (16,17). Changes in the composition of brain membrane phospholipids and in high-energy phosphate metabolism seem to occur before any clinical manifestation of AD (18). This suggests that changes in neural membrane composition may constitute a major pathogenic molecular mechanism of abnormal signal transduction in AD (19,20). Glutamate is a major excitatory neurotransmitter in the central nervous system. It is known to stimulate phospholipid hydrolysis and arachidonic acid release by direct and indirect enzymic mechanisms (19,20). The direct mechanism involves the stimulation of phospholipase A2 resulting in generation of arachidonic acid and lysophospholipids (21–23). The indirect mechanism of arachidonic acid release requires the activation of phospholipase C, followed by diacylglycerol lipase (24). Although the relative contributions of these mechanisms to the release of arachidonic acid and degradation of phospholipids are still obscure, the importance of the direct deacylation of phospholipids by phospholipase A2 and the action of diacylglycerol lipase preceded by phospholipase C has been clearly established (15). An early study from our laboratory showed a marked increase in activities of diacylglycerol and monoacylglycerol lipases in a small number of patients with AD (25). Others have reported elevated cPLA2 immunoreactivity in the brains of AD patients (26,27). The purpose of this commentary is to describe the effect of glutamate and its analogs on activities of phospholipase A2 and diacylglycerol and monoacylglycerol lipases in potential animal and cell culture models of neurodegeneration and to discuss the implications of these neurochemical findings for the pathogenesis of AD. We also present new results on activities of diacylglycerol lipase and phospholipase A2 in different regions of normal human brain and brains from AD patients.

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Effect of Kainic Acid on Brain Cytosolic Phospholipase A2 (cPLA2) Activity

pmol/min/mg protein

Treatment of neuron-enriched cultures with kainic acid and other glutamate analogs results in the stimulation of cPLA2 activity in a dose- and time-dependent manner (Fig. 2.1). A kainate/AMPA antagonist, 6-cyano-7-nitroquinoxaline (CNQX), blocks this increase in cPLA2, suggesting that the stimulation of cPLA2 is a receptor-mediated process (Table 2.1). Quinacrine, a nonspecific PLA 2inhibitor, also prevents the kainic acid-induced stimulation in cPLA2 activity (Table 2.1). This is supported by our studies on cPLA2 immunoreactivity in rats injected with kainic acid (28,29). The systemic administration of kainic acid into adult rats markedly increases cPLA2 immunoreactivity in neurons at one and three days after injection. The increased cPLA2 immunoreactivity in degenerating pyramidal neurons in the CA1 area of the hippocampus at one day after kainic acid injection (Fig. 2.2A, B) suggests that this enzyme may be involved in neurodegeneration (29). Electron microscopic studies after three days of kainic acid injection confirmed that cPLA2 immunoreactivity was present in degenerating neurons with indistinct nuclear and cell outlines but was absent from viable glial cells with distinct nuclear outlines (28). The cPLA2 immunoreactivity is also increased in astrocytes at 1, 2, 4, and 11 weeks after kainic acid injection (28). The increase in cPLA2 immunoreactivity in astrocytes, even after 11 weeks, suggests that this increase may be associated with astrogliosis that occurs in brain tissue after injury. A similar increase in cPLA2 immunoreactivity occurs in astrocytes in AD (26,27). It remains an open question whether the increased cPLA2 immunoreactivity in

µM) KA (µ Fig. 2.1. Effects of kainic acid on cPLA2 activity of neuron-enriched cultures from rat

cerebral cortex. Enzymic activity was determined by a procedure described earlier (34). Results are the mean ± SEM for three different cultures.

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TABLE 2.1 Effects of CNQX and Quinacrine on cPLA2 Activity in Kainate-Treated NeuronEnriched Cultures from Rat Cerebral Cortex Treatment Control Kainate (50 µM) Kainate (50 µM) + CNQX (20 µM) Kainate (50 µM) + Quinacrine (50 µM) Kainate (50 µM) + AACOCF3 (20 µM) aResults

cPLA2 activitya (pmol/min/mg protein) 5.2 ± 1.4 34.3 ± 4.2 7.5 ± 2.3 8.5 ± 1.9 6.5 ± 1.7

are the mean ± SEM for three different cultures.

reactive astroglia is involved in neurodegeneration or whether the enhanced cPLA2 immunoreactivity in astrocytes and microglia provides second messengers for the survival of sick neurons. It is interesting to note that the increased immunoreactivity of cPLA2 in degenerating neurons at one and three days after kainate injection is associated with decreased levels of the reduced glutathione (30) and increased immunoreactivity of 4-hydroxynonenal (4-HNE). This indicates that degenerating neurons are under oxidative stress (Fig. 2.2C, D) (29,31). 4-HNE is a peroxidized product of arachidonic acid. It impairs the activities of key metabolic enzymes, including Na+, K+–ATPase, glucose 6-phosphate dehydrogenase, and several protein phosphatases and kinases (32,33). The impairment of Na+, K+–ATPase by 4-HNE can result in the depolarization of neuronal membranes leading to the opening of NMDA receptor channels and additional Ca2+–influx. This can make neurons vulnerable to NMDA receptor excitotoxicity (19,20). The generation of 4-HNE not only disrupts ion homeostasis but also disrupts transmembrane signaling pathways. 4-HNE also impairs glucose and glutamate transporters in astrocytes (33). When hippocampal slice cultures were treated with kainate alone or kainate and quinacrine together, significant differences in cPLA2 immunoreactivity were observed (29). At one week post-kainate application, significantly less cPLA2 immunoreactivity was observed in slices treated with quinacrine. This indicates that these inhibitors protect the hippocampal culture slices from neural injury induced by kainic acid. Similarly, when kainate-treated slice cultures were treated with arachidonyl trifluoromethyl ketone, there was considerable protection against neurodegeneration in hippocampal slice cultures, again suggesting that cPLA2 is associated with neurodegeneration (Fig. 2.2E, F) (29). At one week post-kainate application of quinacrine or AACOCF3, there was a significant decrease in 4-HNE immunoreactivity compared to kainate alone, indicating that these inhibitors block the formation of 4-HNE by inhibiting cPLA2 (29). The molecular mechanism by which cPLA2 inhibitors reduced kainic acid neurotoxicity is not fully understood. However, it is possible that cPLA2 inhibitors modulate kainic acid receptors by controlling the levels of eicosanoids. Eicosanoids have been reported to be

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Fig. 2.2. Sections (A–D) and organotypic slice cultures (E–F) of the hippocampus that were immunostained with affinity-purified goat polyclonal antibody to cPLA2 (A, B) or mouse monoclonal antibody to HNE (C–F). The cPLA2 and HNE antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and a kind gift from Dr. Georg Waeg, respectively. (A) A hippocampal CA1 field from a control rat that received an intracerebroventricular injection of PBS one day earlier. There is very light staining of pyramidal neurons (arrows) in these sections. (B) A hippocampal CA1 field from a rat that had received an intracerebroventricular injection of kainate (1 µL of a 1 mg/mL kainate solution) one day earlier. An increase in cPLA2 immunoreactivity is observed in the cell bodies of pyramidal neurons (arrows) and the neuropil (asterisk). (C) A hippocampal CA1 field from a control rat that had received an intracerebroventricular injection of PBS one day earlier (same animal as in A). The pyramidal neuronal cell bodies are very lightly or not stained for HNE (arrows). (D) A hippocampal CA1 field from a rat that had received an intracerebroventricular injection of kainate one day earlier (same animal as in B). An increase in HNE immunoreactivity is observed in pyramidal neurons (arrows) and the neuropil (asterisk). (E) Dense HNE immunoreactivity is present in pyramidal neurons (arrows) in hippocampal slices that were treated with kainate one week earlier. (F) In contrast, significantly less HNE staining is observed in the pyramidal layer (arrows) in slices that were treated with kainate and AACOCF3 (a specific inhibitor of cPLA2), indicating that cPLA2 plays a role in HNE formation after kainate treatment. The quantitative data are given in a previous publication (29). Scale: 100 µm.

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involved in the regulation of hippocampal glutamate receptors. cPLA2 inhibitors can also modulate the expression of cytokines, nuclear factor kappa B (NFκB), AP-1, and intracellular adhesion molecules (34,35). All these molecules are associated with the induction and maintenance of inflammatory processes after neuronal injury. Kainate injections into the right lateral ventricle of rat brain result in significant increases in cPLA2 mRNA in the hippocampus. The levels of cPLA2 mRNA remained significantly above the control level at one week after kainate injection. The kainate-induced increase in cPLA2 mRNA levels was abolished when rats were treated with quinacrine. This suggests that in addition to its well-known effect on the inhibition of cPLA2 activity, quinacrine also inhibits the expression of cPLA2 mRNA (35). Thus our studies on kainate-induced neurotoxicity indicate that the over-stimulation of kainate receptor results in activation of cPLA2. This stimulation alters the composition of neural membrane glycerophospholipids, raises arachidonic acid levels, and produces changes in membrane fluidity and permeability. This leads to Ca2+-influx, production of lipid peroxides including 4-HNE, and loss of glutathione. Thus the consequences of altered glycerophospholipid metabolism in kainate-induced neurotoxicity can include cell death.

Effects of Glutamate on Diacylglycerol Lipase and Monoacylglycerol Lipase Activities in Neuron-Enriched Cultures The indirect mechanism of arachidonic acid release involves the action of phospholipase C on phospholipids results in generation of diacylglycerols. In brain tissue, diacylglycerols are either phosphorylated to phosphatidic acids by a diacylglycerol kinase or hydrolyzed to monoacylglycerols and arachidonic acid by diacylglycerol lipase. The monoacylglycerol is then hydrolyzed to glycerol and free fatty acid by a monoacylglycerol lipase (24). Glutamate acts on neuron-enriched cultures to produce a concentration and time-dependent stimulation of diacylglycerol and monoacylglycerol lipase activities. Thus exposure to 50-µM glutamate for 15 min produces a threefold increase in the specific activities of diacylglycerol and monoacylglycerol lipases (Table 2.2). Higher glutamate concentrations result in lower specific activities of these enzymes. However, even at 100-µm glutamate, diacylglycerol and monoacylglycerol lipase activities were higher than the basal activities present in the control cultures. The time dependence of the glutamate effect on diacylglycerol and monoacylglycerol lipases is shown here. The activities of diacylglycerol and monoacylglycerol lipases were unchanged during the initial 4 min of exposure to glutamate. A twofold stimulation of enzymic activities was observed at 6 min with maximal stimulation of lipase activities between 12 and 15 min. At longer times, the enzymic activities were decreased (36).

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TABLE 2.2 Effects of Glutamate and NMDA on Diacylglycerol and Monoacylglycerol Lipase Activities in Neuron-Enriched Cultures from Rat Cerebral Cortexa Excitatory amino acid (µM) Glutamate Control 10 20 50 75 100 NMDA Control 10 20 50 75 100 Control Glutamate (50 µM) Dextrorphan (50 µM) + Glutamate (50 µM) Control NMDA (50 µM) NMDA (50 µM) + MK-801

Diacylglycerol lipase

Monoacylglycerol lipase

5.20 ± 0.54 6.85 ± 0.62 12.57 ± 0.73 17.59 ± 1.2 12.63 ± 0.97 10.59 ± 0.57

12.6 ± 1.6 16.3 ± 1.4 25.8 ± 1.8 32.8 ± 2.8 29.3 ± 2.4 21.6 ± 2.7

5.05 ± 0.63 6.82 ± 0.57 15.73 ± 0.48 20.79 ± 1.60 14.73 ± 0.82 13.27 ± 0.92 5.27 ± 0.73 18.93 ± 1.3 6.27 ± 0.82

11.9 ± 1.8 13.2 ± 2.0 23.1 ± 2.3 29.8 ± 2.5 26.3 ± 2.4 24.3 ± 2.1 11.6 ± 1.7 28.7 ± 2.8 15.7 ± 1.8

5.32 ± 0.63 18.32 ± 1.30 5.39 ± 0.82

12.1 ± 1.9 27.3 ± 2.3 11.9 ± 1.9

aEnzymic

activities were determined by procedures described earlier (36). Specific activity was expressed as nmol/min/mg protein. Results are the means ± SEM for three different cultures.

Effect of NMDA on Diacylglycerol and Monoacylglycerol Lipases of Neuron-Enriched Cultures Like glutamate, NMDA also stimulates diacylglycerol and monoacylglycerol lipase activities in a dose- and time-dependent manner. Thus a 15-min treatment with NMDA (50 µM) causes fivefold and threefold increases in the specific activities of diacylglycerol and monoacylglycerol lipases (Table 2.2). Maximal stimulation was again achieved between 12 and 15 min. As with glutamate, enzymic activities are decreased at longer times of exposure to NMDA. Dextrorphan (75 µM) had no effect on diacylglycerol and monoacylglycerol lipase activities of neuron-enriched cultures. This glutamate receptor antagonist prevented the stimulation of diacylglycerol lipase and monoacylglycerol lipases by glutamate. Similarly MK801 (150 µM) had no effect on diacylglycerol and monoacylglycerol lipase activities, but it blocked the stimulation of lipases by NMDA or glutamate (36). Thus our studies suggest that the stimulation of diacylglycerol and monoacylglycerol lipases by glutamate or NMDA plays an important role in the release of arachidonic acid from neural membrane phospholipids.

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Neurochemical Implications of Elevated Lipase and Phospholipase A2 Activities in Neural Cell Injury Induced by Glutamate and Its Analogs Activation of lipases and cPLA2 by glutamate and its analogs in animal and cell culture models of neurodegeneration may be harmful to neurons in many ways. • It results in the loss of essential phospholipids with the accumulation of arachidonic acid and lysophospholipids that may have a detergentlike effect on neuronal membranes. • Free fatty acids in general and arachidonic acid in particular can uncouple oxidative phosphorylation resulting in mitochondrial dysfunction and reduction in ATP production. Arachidonic acid can also produce changes in membrane permeability by regulating ion-channels. It also inhibits glutamate uptake. • Platelet activating factor, which is formed by acetylation of lysophospholipids, can produce activation of leukocytes and inflammatory reactions at the endothelial cell surface. • The accumulation of arachidonic acid can trigger an uncontrolled “arachidonic acid cascade”. This can result in a marked increase in eicosanoids and set the stage for increased production of free radicals and 4-HNE, which may cause oxidative damage to membrane proteins. • The polyunsaturated fatty acid-induced oxidative stress is accompanied by an increase in AP1 and NFκB activity (37). Hydrogen peroxide or hydroxyl radicals, which are generated due to polyunsaturated free fatty acid breakdown, may increase the DNA binding activity of AP1 transcription factor. Superoxide anions and hydrogen peroxide also activate NFκB. The activation of these transcription factors is involved in regulation of specific genes associated with neural cell injury. Another consequence of increased polyunsaturated free fatty acid induced oxidative stress is the activation and inactivation of redox-sensitive proteins (38). The transcription factor NFκB is known to respond to changes in the redox state of the cytoplasm. It translocates in response to NMDA- or glutamate-induced cellular stress (39). • Finally, an uncontrolled sustained increase in Ca2+–influx through increased phospholipid degradation may lead to the stimulation of many enzymes that are involved in lipolysis and proteolysis (40). Thus glutamate receptor-mediated elevations in activities of lipases and cPLA2 produces changes in the composition of neural membrane phospholipids, permeability, fluidity, and membrane bioenergetics. Phospholipid degradation by lipases and cPLA2 may lead to alterations in activities of transporters and functional failure of excitable neuronal membranes (20). cPLA2 activation has also been reported to affect the processing of APP (41) and induction of cyclooxygenase. The most compelling evidence for the involvement of cPLA2 in neuronal injury comes from

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reports indicating that mice with a targeted deletion of the gene encoding cPLA2 show reduced infarct size following cerebral ischemia (42) and resistance to MPTP-induced neurotoxicity (43).

Activities of Lipases and Phospholipase A2 in AD

Activity (nmol/min/mg protein)

Two plasma membrane fractions were prepared from different regions of human brain from normal human subjects and from patients with AD (25). One plasma membrane fraction was from synaptosomes (SPM) and the other was from glial cells and neuronal cell bodies (PM) (25). The main objective of our study was to determine the activities of lipases and cPLA2 in SPM and PM fractions obtained from different regions of both normal and AD brain. So far we have assayed autopsy samples from 20 normal human brains and 65 AD brains. All subjects were above 60 years of age. All AD subjects were diagnosed as moderately advanced or advanced AD on the basis of neurological and neuropathological criteria proposed by the National Institute of Health. The time between death and removal of the brain varied from 1 to 4 h. The specific activities of diacylglycerol lipase in nucleus basalis were four- to sixfold higher in SPM fractions from AD patients than in the corresponding fraction from normal subjects (Fig. 2.3). SPM from the hippocampal region of AD

Brain region Fig. 2.3. Specific activities of diacylglycerol lipase in synaptosomal plasma membrane

(SPM) fractions from different regions of normal and AD autopsy brains. Enzymic activity was determined as described earlier (15). NB, nucleus basalis, CN, caudate nucleus, FC, frontal cortex, PC, parietal cortex, OC, occipital cortex, HC, hippocampus, and CC, corpus callosum.

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Activity (nmol/min/mg protein)

subjects also show consistently higher activities of diacylglycerol lipase than do those from control human brains. The specific activities of diacylglycerol lipase in nucleus basalis were also four to six times higher in PM fractions from AD subjects than in PM from normal human brains (Fig. 2.4). Also, the PM fractions prepared from the hippocampal region of AD subjects show consistently higher activities of diacylglycerol lipase. Similarly, the specific activities of monoacylglycerol lipase were four to six times higher in SPM and PM fractions from AD brain than in SPM and PM fractions from normal human brain in the nucleus basalis. The specific activities of monoacylglycerol lipase were consistently higher in the hippocampal region of AD subjects than in normal human subjects (25). The cytosolic fraction obtained from the nucleus basalis and hippocampal regions showed four- to fivefold higher specific activity of cPLA2 than the corresponding fraction from normal human brain (Fig. 2.5). The determination of the kinetic parameters of diacylglycerol lipase, monoacylglycerol lipase, and cytosolic

Brain region Fig. 2.4. Specific activities of diacylglycerol lipase in plasma membrane (PM) fractions from different regions of normal and AD autopsy brains. Enzymic activity was determined as described earlier (25). NB, nucleus basalis; CN, caudate nucleus; FC, frontal cortex; PC, parietal cortex; OC, occipital cortex; HC, hippocampus; and CC, corpus callosum.

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Activity (nmol/min/mg protein)

Brain region Fig. 2.5. Specific activities of cPLA2 in cytosolic fractions from different regions of normal and AD autopsy brains. Enzymic activity was determined as described earlier (34). NB, nucleus basalis; CN, caudate nucleus; FC, frontal cortex; PC, parietal cortex; OC, occipital cortex; HC, hippocampus; and CC, corpus callosum.

phospholipase A2 in membrane and cytosolic fractions of nucleus basalis, hippocampus, and frontal cortex indicated that the Km values of these enzymes in AD brain were similar to those in the corresponding preparation from normal human brain. Only Vmax values of these enzymes were markedly increased in AD brain as compared to normal human brain. The exact cause of the increased activities of lipases and phospholipases A2 is not fully understood. However, there are several possibilities. Aβ, which accumulates in AD, has been reported to activate cPLA2 activity in vitro (44) and in LAN-2 cells (45). Thus the formation of Aβ is expected to accelerate neural membrane breakdown through the activation of lipases and phospholipase A2. It is also suggested that PDE and PME formation by cPLA2 favors the aggregation of Aβ and therefore may be associated with the pathogenesis of AD (46). The second possibility is that the activation of astrocytes and microglia in AD may result in the expression of cytokines. These are known to stimulate cPLAβ activity (47,48) and the influx of calcium ion. This is known to promote the activation and translocation of cPLAβ from cytosol to neural membranes resulting in their breakdown and abnormal signal transduction in AD. Thus the increase in activities of lipases and phospholipases may be due to alterations in levels of Aβ, calcium ion, and cytokines. It remains to be seen whether the increases in activities of lipases and phospholipase A2 correlate with the number of senile plaques or neurofibrillary tangles (16,49). At this stage of the research, we do not know whether changes in lipases and phospholipase A2 are the cause or the consequence of neurodegenera-

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tive process or whether changes in these lipolytic enzymes are primary or secondary (20). Furthermore, we do not know whether changes in lipases and phospholipase A2 are specific for AD or whether other neurodegenerative diseases also show similar increases in these lipolytic enzymes. Our studies on ischemic injury in rat brain and traumatic injury in rat spinal cord indicate that these enzymes are also stimulated in brain and spinal cord tissue after ischemic and traumatic insults (19,20). This suggests that glutamate-induced abnormal phospholipid metabolism may be a common mechanism involved in acute neural trauma and neurodegenerative diseases. Acute neural trauma is accompanied by a rapid release of glutamate and a sustained increase in calcium influx (19,20). In contrast in chronic neurodegenerative diseases such as AD, there is not an excessive release of glutamate. However, the number of NMDA and other glutamate receptors is decreased in neocortex and hippocampal regions compared to age-matched controls (50). This results in alterations in calcium homeostasis and breakdown of neuronal cytoarchitecture (19,20). In ischemia and spinal cord trauma, the glutamate-induced neurodegeneration may be rapid (in days) because of the sudden lack of oxygen and a quick drop in ATP and alterations in ion homeostasis. In neurodegenerative diseases in which oxygen and nutrients and ATP are available to the nerve cell and ion homeostasis is maintained to a limited extent, neural cells may take a longer time period (years) to die. AD is a complex and multifactorial disease that involves not only abnormal APP metabolism, elevations in lipases and phospholipase A2 activities, and increased peroxidative damage but also depressed energy metabolism and aberrant calcium homeostasis. All these processes are closely associated with neural cell injury.

Summary The following are conclusions from our studies. • cPLA2 and diacylglycerol lipase hydrolyze phospholipids and diacylglycerols in neural membranes. • Systemic administration of kainic acid into adult rats increases cPLA2 activity and immunoreactivity in brain tissue. Quinacrine, AACOCF3, and CNQX block this stimulation, indicating that the stimulation of cPLA3 is a receptormediated process. • Glutamate or NMDA in neuron-enriched cultures produce a three- to fivefold stimulation of diacylglycerol and monoacylglycerol lipase activities. This stimulation can be blocked with dextrorphan or MK-801, indicating that the stimulation of lipases is also a receptor-mediated process. • The activities of cPLA2 and diacylglycerol and monoacylglycerol lipases in brain tissue from AD subjects are markedly higher in nucleus basalis and hippocampal regions than in the control human brain regions indicating the enhancement of neural membrane phospholipids metabolism in AD.

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• We propose that the over-stimulation of glutamate receptors initiates a cascade of neurochemical events, including stimulation of lipases and phospholipases A2, enhancement of glycerophospholipid turnover, alterations in membrane permeability and fluidity, excessive calcium ion entry, reduction in ATP, accumulation of lipid peroxides including 4-HNE, and proteolysis. All of these processes are closely associated with cell death in AD. Acknowledgment We thank Siraj A. Farooqui for providing figures.

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45. Kanfer, J.N., Sorrentino, G., and Sitar, D.S. (1998) Phospholipases As Mediators of Amyloid Beta Peptide Neurotoxicity: An Early Event Contributing to Neurodegeneration Characteristic of Alzheimer’s Disease, Neurosci. Lett. 257, 93–96. 46. Klunk, W.E., Xu, C.J., McClure, R.J., Panchalingam, K., Stanley, J.A., and Pettegrew, J.W. (1997) Aggregation of B-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer’s Disease Brain, J. Neurochem. 69, 266–272. 47. Stella, N., Estelles, A., Siciliano, J., Tencé, M., Desagher, S., Piomelli, D., Glowinski, J., and Prèmont, J. (1997) Interleukin-1 Enhances the ATP-Evoked Release of Arachidonic Acid from Mouse Astrocytes, J. Neurosci. 17, 2939–2946. 48. Jayadev, S., Hayter, H.L., Andrieu, N., Gamard, C.J., Liu, B., Balu, R., Hayakawa, M., Ito, F., and Hannun, Y.A. (1997) Phospholipase A2 Is Necessary for Tumor Necrosis Factor αInduced Ceramide Generation in L929 Cells, J. Biol. Chem. 272, 17196–17203. 49. Pettegrew, J.W. (1989) Molecular Insights into Alzheimer Disease, Ann. N. Y. Acad. Sci. 568, 5–28. 50. Geddes, J.W., Ulas, J., Brunner, L.C., Choe, W., and Cotman, C.W. (1992) Hippocampal Excitatory Amino Acid Receptors in Elderly, Normal Individuals and Those with Alzheimer’s Disease: Non-N-Methyl-D-Aspartate Receptors, Neuroscience 50, 23–34.

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Chapter 3

Is There Evidence That Phospholipid Administration Is Beneficial for Your Brain? G. Pepeu Department of Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy

Introduction Phospholipids can be administered for therapeutic purposes in the form of lecithin, which is a mixture composed mostly of phosphatidylcholine (PtdCho), followed by phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (1) or purified compounds. The structures of the main phospholipids are shown in Figure 3.1. Figure 3.2 illustrates schematically the pathways leading to the synthesis of PtdCho, PtdEtn, PtdSer, and PtdIns starting from dihydroxyacetone phosphate (DHAP), which derives from glycolysis, through the formation of the common precursor phosphatidic acid (2). Lecithin, which contains approximately 20% PtdCho, 12% PtdEtn, and 1.5% PtdSer, with slight differences according to the source (3), has been used for therapeutic purposes, both by injection and oral administration, for a long time. For instance, in Medicamenta (4), an Italian dictionary of medicines published in 1933, lecithin was indicated for many pathological conditions, including tuberculosis, diabetes, “psychosis of organic origin,” and depression and for facilitating recovery after infectious diseases. Many pharmaceutical companies were marketing lecithin for oral and parenteral administration, and bromine lecithin and iodine lecithin were also available. However, even at that time the question was asked whether it was really useful to administer 0.1–0.5 g per day of a substance of which 1.7 g could easily be consumed by simply eating an egg. Seventy years later, lecithin is still mentioned in the tenth edition of Goodman and Gilman’s The Pharmacological Basis of Therapeutics (5) as a doubtful therapy for Alzheimer disease. The Martindale (6) lists seven proprietary preparations of lecithin and about 40 multi-ingredient preparations containing lecithin, and PtdCho is an important component of the solutions for parenteral nutrition. Moreover, lecithin is largely available as a “nutraceutical” in health and food stores. Is there a rational basis for this success, and what evidence is there of lecithin’s therapeutic usefulness? I believe that the first reason for considering lecithin a drug lies in a remote, almost instinctive, human belief that is at the origin of the so-called “physiological agent” therapy. According to this belief, to eat the heart or liver of your enemy gave you strength and courage. No wonder that, ages later, when phosphorus was detected in the brain, it was believed that its administration could be useful for improving brain functions. Shortly thereafter, the dis-

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Y

Lipid

Abv.

H

Phosphatidate

PtdOH

+ CH2–CH–NH3

Phosphatidylethanolamine

PtdEtn

+ CH2–CH–N(CH3)3

Phosphatidylcholine

PtdCho

Phosphatidylserine

PtdSer

NH3+ |

CH2–C–COO– |

H PLA1

↓ PLA2 CH –O–X 2 ↓

|

R′–C–O–CH O || | || O CH2–O—P—O–Y

↑ O– ↑ PLC PLD |

X = a long chain hydrocarbon (C16, C18) PL = Phospholipase

Fig. 3.1. The chemical structures of the principal phospholipids. From Seigel et al. (52), modified.

covery of phospholipids in all tissues, including the brain, by Tudichum lead to their use in the variety of diseases mentioned above. According to Sollman (7) “the unfounded assumption that these compounds (lecithin and other Phospho-lipins and Nucleins, Glycerophosphates and Hypophosphites) would be more easily assimilated by the tissues and particularly by the brain, gave rise to their employment as tonics and “brain food” in all sort of depressed conditions. . . .” Fifty years later, the rationale became different and had two prongs. According to Growdon (8), it is based upon (i) the observation that pharmacological amounts of PtdCho provide an exogenous source of choline that enhances the biosynthesis of the neurotransmitter acetylcholine (ACh) and (ii) the fact that PtdCho is an integral part of membrane phospholipids. This leads to two overall postulates for PtdCho administration: to correct phospholipids abnormalities and to stabilize neuronal membranes

Effects of Lecithin Administration Lecithin is mostly formed of PtdCho, and indeed in many publications the word lecithin indicates PtdCho. PtdCho is the main exogenous source of choline.

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Glycolysis DHAP = dihydroxyacetone phosphate;

DHAP 1 DHAP

Gro-3P = sn-glycerol-3-phosphate; PtdOH = phosphatidate;

Gro-3P 3

DAG = diacylglycerol;

AcylCoA

SAM = S-adenosylmethionine;

1-acyl-Gro-3P 6

cer = ceramide; SM = sphingomyelin

AcylCoA

P1 7

PtdOH

DAG CDPEtn

SM

CDPCho 9

8 CMP

CMP

20 Cer

10 PtdEtn SAM SAM SAM CO2

Ser 12

11 Etn

PtdSer Fig. 3.2. The pathways leading to the synthesis of PtdCho, PtdEtn, PtdSer, and PtdIns.

From Siegel et al. (52), modified.

Therefore, as already mentioned, it has been assumed that the administration of pharmacological amounts of PtdCho may provide choline for enhancing the biosynthesis of ACh (8), restoring the cholinergic function impaired by several pathological conditions and by aging. The rationale for administering PtdCho to patients with neurological and psychiatric disorders is based upon this assumption. The pathological conditions in which a cholinergic deficit, caused by loss of cholinergic neurons in the nucleus basalis, has been demonstrated include Alzheimer disease, Parkinson’s disease, postalcoholic Korsakoff’s disease, and dementia pugilistica, just to mention some of the most common conditions (9). A decrease in the number of forebrain cholinergic neurons, associated with a reduced density of the cortical cholinergic network (10) and a decrease in ACh release (11) has also been observed in

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man during normal aging (12). These findings are similar to what has been repeatedly described in aging animals (13). The presence of a cholinergic deficit, demonstrated by a significant reduction in cholinergic markers, has been also described in vascular dementia (14). Much evidence demonstrates a pivotal role of the cholinergic system in learning and memory (15), and therefore the impairment of the cholinergic system is considered responsible for at least part of the cognitive deficits that characterize the above mentioned pathological conditions and, to a lesser extent, aging. On this basis, lecithin therapy was used for Alzheimer disease and subjective memory complaints and is popularly used for improving memory. Choline derives from lecithin through the hydrolysis of PtdCholine by phospholipases (16). The administration of lecithin as a source of choline presents advantages over the administration of choline because formation of trimethylamine in the gut is avoided. There is some concern about the possible toxicity of trimethylamine, which is also the cause of the smell of rotten fish that accompanies choline treatment (17). The administration of lecithin increases plasma choline concentration more effectively than choline itself (18). According to Pomara et al. (19), lecithin administration for 7 days at daily doses ranging from 4 to 20 g leads to a dose dependent increase in plasma and red blood cell choline levels. In some patients a fourfold increase was found, but individual differences were very large. Rabin et al. (20) observed a pronounced and persistent threefold increase in serum choline two days after a diet supplementation with 100 g of lecithin. It has been shown that an increase in plasma choline results in an increase in choline-containing compounds in human brain measured by magnetic resonance spectroscopy (21). The effectiveness of lecithin therapy has been tested in many clinical trials. Unfortunately, they were usually carried out on small groups of patients, many were uncontrolled, and contrasting results have been obtained. A comprehensive and critical review of the best trials was published by Higgins and Flicker (22). The summary of the results and the conclusions are as follows: “Twelve randomized trials have been identified involving patients with Alzheimer’s disease (265 patients), Parkinsonian dementia (21 patients) and subjective memory problems (90 patients). No trials reported any clear benefit of lecithin for Alzheimer’s disease or Parkinsonian dementia. Few trials contributed data to meta-analysis. . . . A dramatic result in favour of lecithin was obtained in a trial of subjects with subjective memory problems” (23). According to the reviewers “evidence from randomized trials does not support the use of lecithin in the treatment of patients with dementia. A moderate effect cannot be ruled out, but results from small trials to date do not indicate priority for a large randomized trial.” This critical analysis may provide a justification for the use of lecithin in “subjective memory complaints,” a clinical picture that can probably be assimilated to the “minimal cognitive impairment” and could evolve in Alzheimer disease (24). “Subjective memory complaints” is actually the condition for which lecithin is now largely used outside any medical prescription.

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The limited therapeutic efficacy of lecithin administration in cognitive impairment finds an explanation in the mechanisms involved in the regulation of choline uptake and ACh synthesis. In the brain, the physiological choline extracellular levels of about 4–6 µM are maintained through the blood-brain barrier uptake and the release of choline from phospholipids (25). According to Klein et al. (26), the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet-induced changes of the net uptake and release of choline. Presumably because of these elaborated regulatory mechanisms, contrasting results were obtained by studying the effect of dietary increase in brain choline on brain ACh levels, as reviewed by Haubrich et al. (27). A number of authors reported an increase in ACh synthesis and levels after choline administration, but other investigators have been unable to confirm these findings. Among them, Pedata et al. (28) found no increase in striatal and cortical steady state levels of ACh after acute administration of large doses of choline, and Wecker (29) observed that after choline supplementation, even if choline efflux from the brain tissue was enhanced, ACh synthesis was unaffected. According to Löffelholz (25), under normal conditions, the high affinity choline uptake (HACU), through which the cholinergic neurons obtain their choline supply, is the rate-limiting step for the synthesis of ACh. Choline concentration is above the Km of HACU and is not rate limiting. However, stimulationinduced activation of HACU leads to a decrease in the synaptic choline level, which then may become rate-limiting for ACh synthesis. In this case, the reduction of ACh tissue content can be prevented by exogenous choline and lecithin (30). Therefore, it is plausible that stimulated but not basal ACh release could be enhanced by lecithin administration. Cohen et al. (31) demonstrated that after administration of choline the increase in brain cytosolic choline-containing compounds, measured with proton magnetic resonance spectroscopy, is significantly smaller in aging than in young subjects. Therefore it may be assumed that in Alzheimer disease, and to a smaller extent in aging, the extra choline made available by lecithin supplementation in the diet is not utilized, due to a reduction in the cholinergic network (10) and brain metabolism (32). However, even if the benefits of lecithin supplementation as a source of choline in normal or pathological brain aging are still matter of debate, recent work (33) demonstrating that choline plays a critical role in brain development, although carried out only in the rat, needs to be considered when answering the question whether phospholipid administration is beneficial for the brain. It has been shown (34) that manipulation of dietary choline levels during gestation results in enduring neurobehavioral changes in the offspring that last into adulthood. Prenatal choline supplementation tends to promote excitatory synaptic efficacy in the hippocampus, while prenatal choline deficiency diminishes it. It has also been shown (35) that pre- and postnatal choline supplementation reduces seizure-induced memory impairment. The mechanism through which choline exerts these neurotrophic and neuroprotective actions has not yet been clarified, and there is no evidence supporting an involvement of the cholinergic system (35).

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Since lecithin is the best dietary source of choline, the potential benefits of lecithin supplementation in maternal and neonatal diets should be investigated.

Effects of Phosphatidylserine Administration Phosphatidylserine (PtdSer) is the second component of lecithin, which has been and still is the object of much investigation and many therapeutic trials. Whereas PtdCho supplies the building blocks for the cell membrane and is the source of choline, PtdSer has a fundamental role in many physiological and pathological processes. Dr. Schlegel (Chapter 1 of this book) has described some of the biochemical properties of this phospholipid, which is found in the inner leaflet of plasma membrane of virtually every cell in the body (36). PtdSer externalization is a crucial event in the recognition and phagocytosis of apoptotic cells and in platelet activation (37). Moreover, PtdSer is the most effective acidic phospholipid in activating calcium-activated phospholipid-dependent protein kinase (38). Administered to animals, PtdSer exerts a number of pharmacological effects that have been reviewed by Pepeu et al. (39). PtdSer given parenterally to aging rats improved the acquisition and retention of passive avoidance responses (40). The onset of the effect required 3–7 days of administration (41,42) and the effective doses range between 15 and 50 mg/Kg. The cognition-enhancing properties in aging rats were recently confirmed by Blokland et al. (43). The effects of PtdSer from bovine cortex, chicken eggs, and soybeans were compared and egg-PtdSer was inactive. Suzuki et al. (44) demonstrated that oral administration transphosphatidylated PtdSer obtained from soybean lecithin was able to restore spatial memory and escape latency in aging rats. The behavioral improvement in aging animals has been attributed to the recovery of the brain cholinergic function. PtdSer administration, but not PtdCho, restored ACh release from cortical slices in aging rats (41). This effect was confirmed in vivo by Casamenti et al. (45) studying by microdialysis the PtdSer effect on ACh release from the cerebral cortex in aging rats. Recently, Suzuki et al. (44) demonstrated an increase in ACh release in synaptosomes isolated from old rats treated with PtdSer. The mechanism through which PtdSer restores ACh release in aging rats is not known, although it has been shown in old rats that PtdSer is able to increase the availability of endogenous choline for ACh synthesis (42) to restore Na+, K+-ATPase activity (44) and 45Ca2+ uptake into K+ depolarized cortical synaptosomes (46). An enhancement of central cholinergic activity by PtdSer is also demonstrated by the observation that it antagonizes scopolamine-induced amnesia in both rats (47) and mice (48). However, it has been recently shown that PtdSer also reverses reserpineinduced amnesia, which depends on catecholamine depletion (49), a finding that adds to the multiplicity of PtdSer pharmacological actions reviewed by Pepeu et al. (39). Since it has been shown that PtdSer is able to improve many age-associated behavioral and neurochemical alterations in rodents and rabbits, this phospholipid was tested in man with the aim to improve age-associated cognitive decline, including the

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memory impairment characterizing Alzheimer disease. Seven controlled trials were carried out in the years from 1987 to 1994, the largest by Amaducci et al. (50) on 142 subjects meeting the NINCDS-ADRDA criteria for probable Alzheimer disease. The trials were reviewed by Pepeu et al. (39). It may be concluded that the controlled clinical trials carried out on patients with probable Alzheimer disease, age-associated memory impairment (AAMI), and “mental deterioration” indicate that prolonged (2–6 months) treatment with PtdSer, at doses of 200–300 mg per day per os, was followed by a consistent, albeit slight, improvement in subgroups of patients demonstrated by neuropsychological and instrumental tests. To what extent the improvement could translate into significant advantages in daily life needs to be established. Recently, an open trial of plant-source derived PdtSer on a small group of subjects with AAMI was carried out (51). The results obtained after 6 weeks of treatment (300 mg per day p.o.) were encouraging and the effect was still present after 12 weeks. However, this trial, like the preceeding ones, awaits double-blind controlled verifications on larger subject numbers.

Conclusions The question asked in the title of this review was “Is there evidence that phospholipid administration is beneficial for your brain?” The answer, based on the existing large number of excellent preclinical investigations and the much smaller number of clinical studies carried out on PtdCho (lecithin) and PtdSer, may be only: “perhaps, it cannot be excluded.” For both PtdCho and PtdSer, slight to moderate positive therapeutic effects, with practically no adverse effects, have been reported. For a definite, final response, large multicentric controlled trials with well-defined groups of subjects of different age and clear endpoints are still needed. References 1. Merck Index, XIII edn., (2001) Merck & Co., Inc., Whitehouse Station, N.J. 2. Agranoff, B.W., Benjamins, J.A., and Hajra, A.K. (1999) Lipids, in Basic Neurochemistry, Sixth Edn., Siegel, G. J. Lippincott-Raven, Philadelphia, pp. 47–67. 3. Herslöf, B., Olsson, U., and Tingvall, P. (1990) Characterization of Lecithins and Phospholipids by HPLC with Light Scattering Detection, in Phospholipids, Biochemical, Pharmaceutical and Analytical Considerations, Hanin, I., and Pepeu, G., Plenum Press, New York, pp. 295–298. 4. Medicamenta, IV edn., (1933) Cooperativa Farmaceutica, Milano, pp. 2122–2126. 5. Goodman and Gilman’s The Pharmacological Basis of Therapeutics, X edn. (2001) Hardman, J.G., and Limbird, L.E, McGraw-Hill, New York, p. 561. 6. Martindale The Complete Drug Reference, 32nd edn. (2002) Sweetman S.C., Pharmaceutical Press, London. 7. Sollman, T. (1932) A Manual of Pharmacology, Saunders, Philadelphia, pp. 850–851. 8. Growdon, J.H. (1987) Use of Phosphatidylcholine in Brain Diseases: An Overview, in Lecithin. Technological, Biological and Therapeutic Aspects, Hanin, I., and Ansell G.B., Plenum Press, New York, pp. 121–127.

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9. Bigl, V., Arendt, T., and Biesold, D. (1990) The Nucleus Basalis of Maynert during Aging and in Dementing Neuropsychiatric Disorders, in Brain Cholinergic Systems, Steriade, M., and Biesold, U., Oxford University Press, Oxford. 10. Geula, C., and Mesulam, M.M., (1989) Cortical Cholinergic Fibers in Aging and Alzheimerís Disease: A Morphometric Study, Neuroscience 33, 469–476. 11. Feuerstein, T.J., Lehman, J., Sauermann, W., Van Velthoven, V., and Jackisch, (1992) The Autoinhibitory Feedback Control of Acetylcholine Release in Human Neocortex Tissue, Brain Res. 572, 64–71. 12. de Lacalle, S., Iraizos, I., and Gonzalo, I.M., (1991) Differential Changes in Cell Size and Numbering Topographic Subdivision of Human Basal Nucleus in Normal Aging, Neuroscience 43, 445–456. 13. Pepeu, G., and Giovannelli, L. (1994) The Central Cholinergic System During Aging, Progr. Brain Res. 100, 67–71. 14. Tohgi, H., Abe, T., Kimura, M., Saheki, M., and Takahashi, S. (1996) Cerebrospinal Fluid Acetylcholine and Choline in Vascular Dementia of Binswanger and Multiple Small Infarct Types as Compared with Alzheimer-Type Dementia, J. Neural. Transm. 103, 1211–1120. 15. Everitt, B.L., and Robbins, T.W. (1997) Central Cholinergic Systems and Cognition, Ann. Rev. Psychol. 48, 649–684. 16. Löffelholz, K. (1990) Receptor Linked Hydrolysis of Choline Phospholipids: The Role of Phosholipase D in a Putative Mechanism of Signal Transduction, in Current Aspects of the Neurosciences, Osborne, N.N. MacMillan Press, New York, pp. 49–76. 17. Eckernäs, S.-A., and Aquilonius, S.-M. (1979) Use of Choline in Five Patients with Huntington’ Disease, in Nutrition and the Brain, Barbeau, A., Growdon, J.H., and Wurtman, R.J. Raven Press, New York, pp. 325–330. 18. Wurtman, R.J., Hirsch, M.J., and Growdon, J.H. (1977) Lecithin Consumption Raises Serum Free-Choline Levels, Lancet 2, 68–69. 19. Pomara, N., Goodnick, P.J., Brinkman, S.D., Domino, E., and Gershon, S. (1982) A DoseResponse Study of Lecithin in the Treatment of Alzheimer’s Disease, in Alzheimer’s Disease: A Report of Progress in Research, Corkin, S., Davis, K.L., Growdon, J.H., Usdin, E., and Wurtman, R.J., Raven Press, New York, pp. 379–383. 20. Rabin, P.L., Gooch, B.R., Teschan, P.E., Schmidt, D.E., Island, D.P., and Rabin, D. (1983) Effects of Dietary Lecithin on Hormonal and Neurobehavioral Profiles in Normal Subjects, J. Clin. Psychiatry 44, 136–138. 21. Stoll, A.L., Renshaw, P.F., De Micheli, E., Wurtman R., Srinivisan, S.P., and Cohen, B.M. (1995) Choline Injection Increases the Resonance of Choline Containing Compounds in Human Brain: An in Vivo Proton Magnetic Resonance Study, Biol. Psychiatry 37, 170–174. 22. Higgins, J.P.T., and Flicker, L. (2002) Lecithin for Dementia and Cognitive Impairment (Cochrane Review), in The Cochrane Library, Issue 2, 2002, Oxford, Update Sofware. 23. Panijel, M. (1986) Therapeutische Wirksamkeit von Lecithin bei Gedachtnis-und Konzentrationsstorungen, Therapiewoche 36, 5029–5034. 24. Grön, G., Bittner, D., Schmitz, B., Wunderlich, A.P., and Riepe, M.W. (2002) Subjective Memory Complaints: Objective Neural Markers in Patients with Alzheimer’s Disease and Major Depressive Disorder, Ann. Neurol. 51, 491–498 25. Löffelholz, K. (1998) Brain Choline Has a Typical Precursor Profile, J. Physiol (Paris) 92, 235–239.

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26. Klein, J., Köppen, A., and Löffelholz, K. (1991) Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation, J. Neurochem. 57, 370–375. 27. Haubrich, D.R., Gerber, N.H., and Pflueger, A.B (1978) Choline Availability and the Synthesis of Acetylcholine, in Nutrition and the Brain, Barbeau, A., Growdon, J.H., and Wurtman, R.J., Raven Press, New York, pp. 57–71. 28. Pedata, F., Wieraszko, A., and Pepeu, G. (1977) Effect of Choline, Phosphorylcholine and Dimethylaminoethanol on Brain Acetylcholine Level in the Rat, Pharmacol. Res. Comm. 9, 755–761. 29. Wecker, L. (1990) Dietary Choline: A Limiting Factor for the Synthesis of Acetylcholine by the Brain, in Advances in Neurology, Vol. 51: Alzheimer’s Disease, Wurtman, R.J., Corkin, S., Growdon, J.H., and Ritter-Walker, E., Raven Press, New York, pp. 139–145. 30. Trommer, B.A., Schmidt, D.E., and Wecker, L. (1982) Exogenous Choline Enhances the Synthesis of Acetylcholine Only Under Conditions of Increased Cholinergic Neuronal Activity, J. Neurochem. 3, 1704–1709. 31. Cohen, B,M., Renshaw, P.F., Stoll, A.L., Wurtman, R.J., Yurgelun-Todd, B., and Babb, S. (1995) Decreased Brain Choline Uptake in Older Adult, JAMA 274, 902–907. 32. Rossor, M.N., Tyrrel, P.J., and Frackowiak, R.S.J. (1990) Patterns of Cerebral Metabolism in Degenerative Dementia, in Imaging, Cerebral Topography, and Alzheimer’s Dementia, Rapoport, S.I., Petit, H., Leys, D., and Christen, Y., Springer Verlag, Berlin, pp. 121–128. 33. Blusztajn, J.K. (1998) Choline, a Vital Amine, Science 281, 794–795. 34. Montoya, D.A., White, A.M., Williams, C.L., Blusztajn, J.K., Meck, W.H., and Swartzwelder, H.S. (2000) Prenatal Choline Exposure Alters Hippocampal Responsiveness to Cholinergic Stimulation in Adulthood, Dev. Brain Res. 30, 25–32. 35. Holmes, G.L., Yang, Y., Liu, Z., Cermak, J.M., Sarkisian, M.R., Stafstrom, C.E., Neill, J.C., and Blusztajn, J.K. (2002) Seizure-Induced Memory Impairment is Reduced by Choline Supplementation Before or After Status Epilepticus, Epilepsy Res. 48, 3–13. 36. Schlegel, R.A., and Williamson, P. (2001) Phosphatidylserine, a Death Knell, Cell Death Diff. 8, 551–563. 37. Bevers, E.M., Comfurius, E.P., and Zwaal, R.F.A. (1983) Changes in Membranes Phospholipid Distribution During Platelet Activation, Biochim. Biophys. Acta 736, 57–62. 38. Kaibuchi, K., Takay, Y., and Nishizuka, Y. (1981) Cooperative Roles of Various Membrane Phospholipids in the Activation of Calcium-Activated, PhospholipidsDependent Protein Kinase, J. Biol. Chem. 256, 7146–7149. 39. Pepeu, G., Marconcini Pepeu, I., and Amaducci, L. (1996) A Review of Phosphatidylserine Pharmacological and Clinical Effects. Is Phosphatidylserine a Drug for the Aging Brain? Pharmacol. Res. 33, 73–80. 40. Corwin, J., Dean, R.L., Bartus, R.T., Rotrosen, J., and Watkins, D.L. (1985) Behavioral Effects of Phosphatidylserine in the Aged Fisher 344 Rat: Amelioration of Passive Avoidance Deficit Without Changes in Psychomotor Task Performance, Neurobiol. Aging 7, 11–15. 41. Pedata, F., Giovannelli, L., Spignoli, G., Giovannini, M.G., and Pepeu, G. (1985) Phosphatidylserine Increases Acetylcholine Release from Cortical Slices in Aged Rats, Neurobiol. Aging 6, 337–339. 42. Vannucchi, M.G., Casamenti, F., and Pepeu, G. (1990) Decrease of Acetylcholine Release from Cortical Slices in Aged Rats: Investigations into Its Reversal by Phosphatidylserine, J. Neurochem. 55, 819–825.

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43. Blokland, A., Honig, W., Brouns, F., and Jolles, J. (1999) Cognition-Enhancing Properties of Subchronic Phosphatidylserine (PS) Treatment in Middle-Aged Rats: Comparison of Bovine Cortex PS with Egg PS and Soybean PS, Nutrition 15, 778–783. 44. Suzuki, S., Yamatoya, H., Sakai, M., Kataoka, A., Furushiro, M., and Kudo, S. (2001) Oral Adminstration of Soybean Lecithin Transphosphatidylated Phosphatidylserine Improves Memory Impairment in Aged Rats, J. Nutrition 131, 2951–2956. 45. Casamenti, F., Scali, C., and Pepeu, G. (1991) Phosphatidylserine Reverses the AgeDependent Decrease in Cortical Acetylcholine Release: A Microdialysis Study, Eur. J. Pharmacol. 194, 11–16. 46. Pepeu G., Giovannelli, L., Giovannini, M.G., and Pedata, F. (1986) Effect of Phosphatidylserine on Cortical Acetylcholine Release and Calcium Uptake in Adult and Aging Rats, in Phospholipid Research and the Nervous System. Biochemical and Molecular Pharmacology, Horrocks, L.A., Freysz, L., Toffano, G., Liviana Press, Padova, pp. 265–271. 47. Zanotti, A., Valzelli, L., and Toffano, G. (1986) Reversal of Scopolamine-Induced Amnesia by Phosphatidylserine in Rats, Psychopharmacol. (Berlin) 90, 274–275. 48. Claro, F.T., Silva, R.H., and Frussa-Filho, R. (1999) Bovine Brain Phosphatidylserine Attenuates Scopolamine-Induced Amnesia, Physiol. Behav. 67, 551–554. 49. Alves, C.S.D., Andreatini, R., da Cunha, C., Tufik, S., and Vital, M.A. (2000) Phosphatidylserine Reverses Reserpine-Induced Amnesia, Eur. J. Pharmacol. 404, 161–167. 50. Amaducci, L., and SMID Group (1988) Phosphatidylserine in the Treatment of Alzheimer’s Disease. Results of a Multicentric Study, Psychopharmacol. Bull. 24, 130–134. 51. Schreiber, S., Kampf-Sherf, O., Gorfine, M., Kelly, D., Oppenhaim, Y., and Lerer, B. (2000) An Open Trial of Plant-Source Derived Phosphatidylserine for Treatment of Age-Related Cognitive Decline, Isr. J. Psychiatry Relat. Sci. 37, 302–307. 52. Basic Neurochemistry (1999) VI edn., Siegel, G.J., Agranoff, B.W., Albers, R.W., Fisher, S.K., and Uhler, M.D. Lippincot-Raven, Philadelphia, pp. 47–67.

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Chapter 4

Phospholipid and Fatty Acid Metabolism in Schizophrenia and Depression M.S. Manku, and D.F. Horrobin Laxdale Ltd., Kings Park House, Laurelhill Business Park, Stirling FK7 9JQ, Scotland

Introduction Phospholipids and closely related compounds make up roughly 60% of the brain by weight (1). Dendrites and synapses are particularly rich in phospholipids, and neurotransmitters in nerve endings are wrapped in phospholipid vesicles. The brain is highly metabolically active, consuming around 20% of the human body’s energy even though it is only about 2% of the bodyís weight. The mitochondria that generate most of that energy consist of enzymes tightly arranged within a phospholipid structure. Calcium ions, which are central to many neuronal activation processes, are contained within mitochondria and the phospholipid endoplasmic reticulum. The receptors, ion channels, ATPases, and many other proteins that are central to brain function are for the most part embedded in or attached to phospholipid membranes. Signal transduction processes that follow the occupation of receptors by neurotransmitters almost invariably involve the activation of one or more phospholipases from the A2, C, or D groups. Release of fatty acids, diacylglycerols, lyso-phospholipids, and phosphatidic acid as a result of phospholipase activity generate compounds that can influence many aspects of neuronal function from the genome to ion channels. Given the central role that phospholipids and their metabolites play in brain function, it would not be surprising if abnormalities in phospholipid metabolism were to play some role in psychiatric disorders, such as depression and schizophrenia. What is surprising is how little attention has been paid to this concept within the psychiatric research community. However, that situation is now changing rapidly and many research groups around the world are investigating phospholipid metabolism in psychiatric disorders. Most of the progress has been made in depression and in schizophrenia (1).

Depression Depression is one of the most common of all psychiatric disorders, affecting anywhere from a low of around 2% to a high of around 20% of the population at any one time. There are many subvarieties of depression, but the main ones are major

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depressive disorder (MDD), depression in bipolar disorder, and depression associated with pregnancy and lactation. In all these conditions there are sensations of despair, hopelessness, lassitude, sleep problems, and guilt feelings, often accompanied by irrational anxiety. Usually these feelings are unrelated to any obvious external situation, or they may be an exaggerated and prolonged response to something that would cause a relatively minor upset in most people. In MDD the condition may show relatively little fluctuation, whereas in bipolar depression there may be cycles of depression and normality or oscillations between depression and abnormal feelings of excitement, euphoria, and high energy (technically known as hypomania or mania). It is now increasingly recognized that many women become depressed during pregnancy, which can cause particular distress because they may feel that this should be a particularly happy time and hence be unwilling to admit their difficulties. Post-partum depression can be profound. Three main lines of evidence suggest that fatty acids and phospholipids play major roles in depression. There are biochemical changes in plasma and red cells, epidemiological data relating diet to depression, and clinical trials that have demonstrated that particular fatty acids may alleviate depression. Biochemistry The first evidence relating lipids to depression was collected around 25 years ago, when it was demonstrated that standard antidepressant drugs, among other things, were prostaglandin antagonists (2,3). This raised the possibility that depression might be associated with over-production of prostaglandins from arachidonic acid (AA). Another piece of evidence pointing in the same direction was that depressed patients bruise easily, a phenomenon that may also be related to platelet activation, commonly associated with eicosanoid over-production. These findings were then followed by several studies in the 1980s and 1990s that showed that depressed patients had elevated levels of thromboxane B2 and various 2 series prostaglandins in plasma, cerebrospinal fluid, and saliva (4–6). Finally, in the 1990s studies from Australia, Japan, Europe, and North America showed that either in plasma or in red cells, depressed patients consistently showed an elevated ratio of AA to eicosapentaenoic acid (EPA) (7–10). Docosahexaenoic acid (DHA) levels were also low in some studies, but this finding was less consistent than that for EPA. Thus, overall the biochemistry is remarkably consistent. There is over-production of 2 series eicosanoids, AA levels are relatively or absolutely high, and EPA levels are relatively or absolutely low. Moreover, antidepressants are either prostaglandin antagonists or inhibitors of prostaglandin synthesis, although these facts are not widely known in either the lipid or the psychiatric communities. Epidemiology One of the puzzles of depression is why there are such large country-by-country variations in its prevalence. Many explanations have been proposed, but none is as

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convincing as the relationship between depression prevalence on the one hand and fish or seafood consumption on the other. Countries whose population have a high intake of fish or seafood show low prevalence of depression and vice versa (11). The relationship is very strong and holds true for major depression, for bipolar depression, and for post-partum depression. There is something in fish or seafood that seems to protect against depression. However, country epidemiological studies must be interpreted with caution. They are notorious for giving misleading information that has explanations other than the one initially proposed. So epidemiologists have learned to mistrust countryby-country associations that are not also supported by individual-by-individual associations within country studies. But here too the association stands. In both prospective and cross-sectional studies, individuals who regularly eat fish and seafood are at much lower risk of depression than are individuals who rarely eat these foods (12). So the evidence for the protective effect of fish and seafood is now strong. Of course, these foods contain many ingredients and the weakness of epidemiological studies is that it is very difficult to tease these out. However, fish and seafood may contain substantial amounts of EPA and DHA, and it is generally felt that it is these components that most likely explain the protection against depression. Clinical Trials Ultimately biochemical and clinical evidence can only be suggestive. Proof that a particular biochemical abnormality is the cause of depression can be obtained only by correcting that abnormality and seeing what happens. Already clinical trials have both provided support for the picture generated by the biochemistry and epidemiology and also demonstrated how shaky reasoning based only on these sciences can be. Most people in the field predicted that DHA would be the key fatty acid. This was primarily based on the fact that it is found in very large amounts in the human brain (roughly 6–7% by weight), whereas EPA is found in only tiny amounts, although it is found in vascular endothelial cells, which play important roles in modulating brain function. However, trials that compared DHA with placebo in depressed patients showed no beneficial effects at all (13). Trials of DHA in attention deficit hyperactivity disorder in children have proved equally negative (14). The situation with EPA is dramatically different. One trial looked at an EPA/DHA mix (15), whereas six (two as yet unpublished) looked at purified EPA alone (16–18, Frangou et al. unpublished data). All seven have shown that EPA is effective in relieving depression. This is dramatic because less than half of all trials studying standard drugs, such as fluoxetine (Prozac), paroxetine (Paxil), or sertraline (Zoloft), have shown any beneficial effect. The first published study in major depression was a report of case in which EPA relieved depression in a young man who had been seriously suicidal and who had failed to respond to any other form

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of treatment (16). The other six studies were all randomized, double-blind, and placebo-controlled, three involved major depression in patients who were resistant to standard therapies, two involved bipolar depression, also not responding well, and one involved depression in borderline personality disorder, which is notorious for failing to respond to standard drugs. These studies therefore leave little doubt that ethyl-EPA is a major new treatment for various types of depressive disorder. These studies also raise two important issues that are relevant not just to psychiatric disorders but to studies of omega-3 fatty acids and lipid preparations in general. These are the issues of purity and of dose. In other areas of pharmacology it has in general been learned that the properties of a drug can be defined only when it is administered in pure form. If it is given in the form of a mix of closely related compounds, some of which may have similar (agonist) actions, some of which may be neutral but may simply dilute the molecule of interest, and some of which may actively antagonist it, then it is impossible to tease out with any confidence what actually is happening. The second issue that has wide relevance is that of dose. Very few biologically active compounds have actions only at a single site (19). Certainly EPA and related fatty acids have multiple specific binding sites in the cell. Just a few of them in the case of EPA are peroxisome-proliferator activated receptors (PPAR), which can regulate many aspects of lipid metabolism and multiple genes; ion channels; various enzymes; binding proteins; and transport proteins (19). The ligand binding characteristics at each site are different, and different concentrations are required. Thus, as the dose of a fatty acid, such as EPA, is increased, binding to different sites of activity will kick in leading to highly complex dose/response relationships. Some of these mechanisms will have negative feedback characteristics. As a result it is common for an action that is present at low concentrations to be reversed at high concentrations. Moreover, the behavior of a biological system may be quite different if a fatty acid is administered in pure form as compared to the situation after it is administered as a mix with other compounds (19). One of the real tragedies in the lipid field is that a very large part of the biological and clinical work has been wasted because attention was not paid to these two simple principles. The fatty acids have been administered as complex mixtures with little consistency between one experiment and another. Scientists have behaved as though, for example, EPA will have the same effects irrespective of what form it is in and irrespective of what other fatty acids are present. This is highly unlikely to be true. The second error has been to ignore the issue of dose and simply assume that it is almost irrelevant, more will always be better and what happens at one dose will be qualitatively the same as what happens at another dose, the only difference being quantitative. Neither proposition is true, which has led to total confusion. For example, no one seems to have appreciated that in the cardiovascular field, most human studies that have used relatively low doses have produced desirable clinical effects, whereas most studies that have used higher doses have completely failed to

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find positive outcomes. The reasons have not been critically discussed or thought through, and as a result the field is in a state of considerable confusion (20). The potential value of a pure single compound and of careful dose-response studies is well illustrated by a study in patients with treatment-unresponsive depression that compared placebo with 1 g, 2 g, and 4 g doses of ethyl-EPA (18). The 1 g dose proved much more effective than any of the others, and as a result future studies, which have all been positive, were conducted at that dose. Had a dose-response study not been done initially it would have been easy to follow a “more is better” strategy, to choose the wrong dose, and to conclude that EPA was ineffective. This is a principle that is applicable to all other indications. Lipids as therapeutic agents will become respected and effective only when consistent results are achieved by using pure compounds and doses chosen on the basis of careful dose-response studies.

Schizophrenia In schizophrenia, similar sorts of evidence have accumulated for a lipid abnormality. This is quite different from the abnormality in depression but has also led to novel approaches to treatment (1,21). Schizophrenia is a very serious psychiatric illness that, contrary to popular belief, does not mean a “split mind” or a “multiple personality.” Rather, it indicates a disintegration of the personality. Symptoms can be very complex and differ substantially from person to person. They usually begin somewhere between the ages of 15 and 35, often a little earlier in men. In some patients they may begin even earlier or later. Complete recovery, at least in developed countries, is relatively rare. Symptoms often begin with withdrawal from friends and family and a loss of normal emotional responses (normal “affect”). Patients often have auditory hallucinations (hearing voices) that may sometimes give insistent instructions. They may become paranoid and feel that other people are constantly talking about them and thinking about them. A good impression of the experiences of some people was given in the film about John Nash, “A Beautiful Mind.” Although there are many drugs that have actions in schizophrenic patients, they are not very effective. Using standard rating scales, the extents of improvement are only of the order of 15–17%. The new drugs are no better than the first anti-schizophrenic drugs, which were introduced fifty years ago, although the side effects are somewhat different (22). There is therefore a great need for new understanding of the disease and new approaches to treatment. Epidemiology The epidemiology of schizophrenia is quite different from that of depression. There is almost no difference between countries and peoples in the lifetime prevalence of the illness. In all countries the lifetime risk of schizophrenia is somewhere between 0.7 and 1%. There is also no impact of diet on this lifetime risk.

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There is, however, an impact of diet on the lifetime severity of the disease. In some countries, notably the industrialized west, the lifetime outcome is all too often depressingly serious. Relatively few patients recover and most are ill to some degree for most of their lives after their first episode. In contrast, in less industrialized countries and in Japan the lifetime outcome in many patients is less serious. There may be multiple episodes of psychosis but these episodes are often interspersed with long periods of relative abnormality. Danish investigators first noted that the most adverse outcomes were in countries that had a relatively high intake of foods rich in saturated fats and a relatively low intake of foods rich in unsaturated fats (23). Their calculations have been checked by others and confirmed in various ways. Consistently, a high ratio of saturated to unsaturated fats in the diet is associated with poor outcome but does not influence whether or not someone becomes schizophrenic. Clinical Observations Although the main features of schizophrenia relate to mental function, schizophrenia is a whole body disease with a number of features that suggest that the phospholipid/fatty acid/eicosanoid system may be involved (21,24,25). • Schizophrenic patients are often highly resistant to pain, for example, not experiencing the usual symptoms when they break a bone or develop appendicitis. • Schizophrenic patients develop arthritis and other inflammatory disorders less frequently than expected. • Schizophrenic patients show a reduced cutaneous flushing response to the oral or topical application of niacin (nicotinic acid). • Schizophrenic patients may show a remarkable but transient improvement in their mental state when they develop a febrile illness. • Schizophrenic patients may have an increased susceptibility to diabetes with an increased risk of abdominal obesity. Since AA conversion to prostaglandins (PG) is important in pain, in inflammation, and in the response to niacin, these observations point to some impairment of AA/PG metabolism. Since AA and PG are released during fever, the improvement of mental state during fever raises the possibility that the presumed PG deficit may be playing a causative role in the mental disturbances. Since PPAR are involved in the control of visceral obesity and diabetes, the clinical observations in this field raise the possibility that abnormalities of PPAR may be involved. Biochemical Observations Three groups of biochemical observations have been made in schizophrenic patients (1). They all point to an abnormality of phospholipid metabolism as playing a central role.

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1. Phospholipase A2 (PLA2). There are consistent reports of elevated phospholipase A2 activity in plasma, red cells, and brains of schizophrenic patients. These are most consistent with respect to cytosolic PLA2, although a calciumindependent PLA2 may also be involved. 2. Blood and brain fatty acid levels. Plasma fatty acid levels are normal or near normal in schizophrenia. However, the fatty acid composition of red cell membranes is consistently abnormal. Red cell AA levels are low, and this abnormality may also be accompanied by low EPA and DHA levels. Red cell linoleic and alpha-linolenic acid levels are normal. The specific abnormality of long-chain highly polyunsaturated fatty acids in membranes suggests that there is a problem of incorporation of these fatty acids into membrane phospholipids. Consistent with this are reports of increased concentrations of lysophospholipids and of reduced rates of incorporation of labelled AA into platelet phospholipids. 3. Oxidative metabolites of lipids. There are consistent reports of increased levels of malondialdehyde and thiobarbituric acid, which are reactive substances in schizophrenia. Schizophrenia is now recognized to be a disease that has a strong genetic component but that cannot be explained by any single gene. Almost certainly several genes must be simultaneously present for the illness to occur. The observations to date suggest that there are at least three and possibly more abnormalities in lipid metabolism. These are increased loss of AA and other highly unsaturated fatty acid (HUFA) from membranes because of increased PLA2 activity coupled with a reduced rate of incorporation. As a result there is a reduced pool of AA available for signal transduction processes, which could explain the impaired pain responses and reduced niacin flushing and risk of inflammatory disorders. When, during fever, the mechanisms governing AA release are overactivated, enough may be released to allow relatively normal signal transduction. It is not yet clear whether the third abnormality, increased lipid peroxidation, is simply a consequence of the increased levels of free HUFA or whether it may constitute a third primary abnormality. Treatment with Lipids Unlike the situation with depression, it is unlikely that any single lipid manipulation will produce very substantial improvements in schizophrenia. It is more likely that more than one approach will be helpful. The epidemiological studies suggest that the outcome may be most likely to improve if there is a substantial reduction in intake of saturated fats, coupled with an increase in the intake of unsaturated fats. Which fatty acids are relevant can be established only by clinical trials. Such trials may influence not only the psychotic process itself but also the associated condition of tardive dyskinesia (TD). TD is a syndrome of distressing and uncontrollable movement disorders. It occurs to a limited degree even in unmedicated

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schizophrenic patients, but is frequently made very much worse by the standard drugs that are used in schizophrenia. Initial trials involved the administration of EPA/DHA mixes, which gave modestly positive results. Then trials compared partially purified EPA with partially purified DHA. As with depression, the expectation was that DHA would be more effective. This turned out not to be the case: DHA was without effect, or even slightly worsened symptoms, whereas EPA produced clearly beneficial results (26). Some patients could be treated by EPA without any requirement for standard drugs. Since these standard drugs all produce severe side effects that is a major step forward. Finally, once ultra-pure ethyl-EPA became available, a careful dose response study became possible. This compared placebo with 1 g, 2 g, or 4 g per day of ethyl-EPA (27). This time the best outcome was in the 2 g/d group with the 4 g/d group being of no benefit. The biochemical analyses of red cell fatty acids in the dose ranging study provided a rational basis for understanding (20,27). Red cell AA levels in schizophrenia tend to be low. Two grams per day of ethyl-EPA produced a clear elevation of red cell AA, whereas 4 g/d produced a clear depression of levels (see Fig. 4.1). When clinical change was correlated with red cell fatty acid level, by far the strongest correlation was with red cell AA. An increase in AA was related positively to clinical improvement. The explanation for the dose-response effect may be that at doses of up to 2 g/d the dominant effect of EPA is inhibition of PLA2 leading to a conservation of AA in membrane phospholipids and a partial correction of the abnormality seen in schizophrenia. At 4 g/d, in contrast, the predominant effect becomes displacement of AA by EPA, leading to a further fall in AA that is certainly not beneficial and may have adverse consequences. Once again, careful dose-response studies with pure compounds lead to progress.

Percentage change (%)

Percentage changes from baseline of 4 fatty acids in red cell PL membranes in patients on Neuroleptics.

AA EPA DPA n3 DHA

Placebo

1 g/d

2 g/d

4 g/d

Fig. 1. Dose response effect of ethyl eicosapentaenoate (E-EPA). Red cell membrane phospholipid (PL) fatty acids.

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Conclusions The findings to date in depression and schizophrenia suggest that detailed exploration of phospholipid, fatty acid, and eicosanoid metabolism may lead to many new insights in these and other psychiatric and neurological disorders. These new insights may lead to new and better therapeutic approaches as has already happened in depression and schizophrenia. Referenceses 1. Peet, M., Glen, I., and Horrobin, D.F. (Eds.) (1999) Phospholipid Spectrum Disorder in Psychiatry, Marius Press, Carnforth, UK. 2. Horrobin, D.F. (1977) The Roles of Prostaglandins and Prolactin in Depression, Mania, and Schizophrenia, Postgrad. Med. J. 53 Suppl 4, 160–165. 3. Horrobin, D.F., Manku, M.S., and Mtabaji, J.P. (1977) A New Mechanism of Tricyclic Antidepressant Action. Blockade of Prostaglandin-Dependent Calcium Movements, Postgrad. Med. J. 53 Suppl 4, 19–23. 4. Lieb, J., Karmali R., and Horrobin, D.F. (1983) Elevated Levels of Prostaglandin E2 and Thromboxane B2 in Depression, Prostaglandins Leukot. Med. 10, 361–367. 5. Linnoila, M., Whorton, A.R., Rubinow, D.R., Cowdry, R.W., Ninan, P.T., and Waters, R.N. (1983) CSF Prostaglandin Levels in Depressed and Schizophrenic Patients, Arch. Gen. Psychiatry 40, 405–406. 6. Ohishi, K., Ueno, R., Nishino, S., Sakai, T., and Hayaishi, O. (1988) Increased Level of Salivary Prostaglandins in Patients with Major Depression, Biol. Psychiatry 23, 326–334. 7. Adams, P.B., Lawson, S., Sanigorski, A., and Sinclair, A.J. (1996) Arachidonic Acid to Eicosapentaenoic Acid Ratio in Blood Correlates Positively with Clinical Symptoms of Depression, Lipids 31, S157–S161. 8. Maes, M., Smith, R., Christophe, A., Cosyns, P., Desnyder, R., and Meltzer, H. (1996) Fatty Acid Composition in Major Depression: Decreased Omega 3 Fractions in Cholesteryl Esters and Increased C20:4 Omega 6/C20: 5 Omega 3 Ratio in Cholesteryl Esters and Phosopholipids, J. Affective Disord. 38, 35–46. 9. Seko, C. (1997) Relationship Between Fatty Acid Composition in Blood and Depressive Symptoms in the Elderly, Jpn. J. Hyg. 52, 539–42. 10. Edwards, R., Peet, M., Shay, J., and Horrobin, D. (1998) Omega-3 Polyunsaturated Fatty Acid Levels in the Diet and in Red Blood Cell Membranes of Depressed Patients, J. Affect. Disord. 48, 149–155. 11. Hibbeln, J.R. (1998) Fish Consumption and Major Depression, Lancet 351, 1213. 12. Tanskanen, A., Hibbeln, J.R., Hintikka, J., Haatainen, K., Honkalampi, K., and Viinamaki, H. (2001) Fish Consumption, Depression, and Suicidality in a General Population, Arch. Gen. Psychiatry 58, 512–513. 13. Marangell, L.B., Zboyan, H.A., Cress, K.K., Vogelson, L., Puryear, L.J., Jensen, C., and Arterburn, L. (2000) A Double Blind, Placebo-Controlled Study of Docosahexaenoic Acid in the Treatment of Depression, Inform 11, S78. 14. Voigt, R.G., Llorente, A.M., Jensen, C.L., Fraley, J.K., Berretta, M.C., and Heird W.C. (2001) A Randomized, Double-Blind, Placebo-Controlled Trial of Docosahexaenoic Acid Supplementation in Children with Attention-Deficit/Hyperactivity Disorder, J. Pediatr. 139, 189–196.

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15. Stoll, A.L., Severus, W.E., Freeman, M.P., Rueter, S., Zboyan, H.A., Diamond, E., and Cress, K.K. (1999) Omega 3 Fatty Acids in Bipolar Disorder—A Preliminary DoubleBlind, Placebo-Controlled Trial, Arch. Gen. Psychiatry 56, 407–412. 16. Puri, B.K., Counsell, S.J., Richardson, A.J., and Horrobin D.F. (2002) Eicosapentaenoic Acid in Treatment-Resistant Depression, Arch Gen Psychiatry 59, 91–92. 17. Nemets, B., Stahl, Z., and Belmaker, R.H. (2002) Omega-3 Fatty Acid Treatment of Depressive Breakthrough During Unipolar Maintenance, Am. J. Psychiatry 159, 477–479. 18. Peet, M., and Horrobin, D.F. (2002) A Dose-Ranging Study of the Effects of EthylEicosapentaenoate in Patients with Ongoing Depression Despite Apparently Adequate Treatment with Standard Drugs, Arch. Gen. Psychiatry 59, 913–919. 19. Horrobin, D.F. (2002) A New Category Of Psychotropic Drugs: Bioactive Lipids As Exemplified By Ethyl Eicosapentaenoate (E-E), in Progress in Drug Research, Zucker, E., Berghauser Verlag, Basel. 20. Horrobin, D.F., Jenkins, K., Bennett, C.N., and Christie, W.W. Eicosapentaenoic Acid and Arachidonic Acid: Collaboration and Not Antagonism Is the Key to Biological Understanding, Prostaglandins Leukot. Essent. Fatty Acids 66, 83–90. 21. Horrobin, D.F. (1998) The Membrane Phospholipid Hypothesis as a Biochemical Basis for the Neurodevelopmental Concept of Schizophrenia, Schizophr. Res. 30, 193–208. 22. Khan, A., Khan, S.R., Leventhal, R.M., and Brown, W.A. (2001) Symptom Reduction and Suicide Risk Among Patients Treated with Placebo in Antipsychotic Clinical Trials: An Analysis of the Food and Drug Administration Database, Am. J. Psychiatry 158, 1449–1454. 23. Christensen, O., and Christensen, E. (1988) Fat Consumption and Schizophrenia, Acta Psychiatr. Scand. 78, 587–591. 24. Horrobin, D.F. (1977) Schizophrenia As a Prostaglandin Deficiency Disease, Lancet 1, 936–937. 25. Horrobin, D. (2001) The Madness of Adam and Eve, Bantam Press, London. 26. Peet, M., Brind, J., Ramchand, C.N., Shah, S., and Vankar, G.K. (2001) Two DoubleBlind Placebo-Controlled Pilot Studies of Eicosapentaenoic Acid in the Treatment of Schizophrenia, Schizophr. Res. 49, 243–251. 27. Peet, M., and Horrobin, D.F. (2002) EPA Multicentre Study Group. A Dose-Ranging Exploratory Study of the Effects of Ethyl-Eicosapentaenoate in Patients with Persistent Schizophrenic Symptoms, J. Psychiatric Res. 36, 7–18.

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Chapter 5

Altered Prostaglandin Mediated Skin Flush in Schizophrenia–Implications for Early Psychosis Interventions Stefan Smesny, Timm Rosburg, Sven Riemann, and Heinrich Sauer Department of Psychiatry, Friedrich-Schiller-University of Jena, Philosophenweg 3, D-07740 Jena, Germany

Introduction Phospholipids are essential components of biological membranes and are supposed to be altered in schizophrenia (1). Metabolites of lipid degradation represent precursors for the synthesis of potent cellular second messengers, e.g., prostaglandins (PG) (2–4). Therefore, alterations of PG-mediated processes give information about disturbances of membrane lipid degradation pathways. One PG-mediated process with clinical implications is the erythema and oedema response after systemic or topical exposure to niacin (vitamin B3). When Murrell and Taylor (5) performed their first niacin tests in 1959, the underlying biochemical mechanisms of the skin reaction were quite unclear. Now we have much more insight into the physiological background of the skin reaction. Investigations by Morrow (6) suggested that prostaglandin D2 (PGD2) released by skin macrophages (7) may be the striking mediator of the skin response. PGD2 triggers an increase of the intracellular cAMP production causing in turn a vasodilatation of the superficial capillary microvessels of the skin (8). There is rising evidence for an indirect linkage between the niacin skin reaction and alterations of cerebral phospholipids in schizophrenia patients (9,10). Highly unsaturated fatty acid (e.g., arachidonic acid) depletion of cell membranes (11,12) caused by oxidative damage (13,14) or deregulated phospholipase A2 activities (15,16) may lead to deficient prostaglandin precursor availability and, respectively, to a deficiency of PG-formation. Given the fact that alterations of lipid breakdown processes occur not only in nervous tissue but potentially in all tissues of the body, niacin skin tests have the potential to indicate cerebral phospholipid alterations. In several first generation studies using 200 mg oral niacin about 25–50% of the schizophrenic patients failed to flush compared to depressive patients or healthy controls (12,17,18). Patients suffering from bipolar psychosis exhibited a normal or exaggerated skin flushing (18). In a multicenter study of schizophrenic patients with predominantly negative symptoms the clinical accompaniments of the niacin

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response were investigated (12). Patients failing to flush with niacin showed significantly reduced levels of arachidonic and docosahexanoic acids. Conversion from nonflushing to flushing during 6 months of fatty acid supplementation was predicted by an increase in arachidonic acid levels in red blood cell membranes, suggesting an association between niacin sensitivity and the availability of prostaglandin precursors. Furthermore the normalization of diminished niacin flush reaction after clinical improvement of schizophrenic symptoms to evening primrose oil supplementation was reported in a single case report (19). In contrast to those promising results, attempts to quantify the flush reaction by measuring skin temperature changes or changes in blood flow were not successful (17,20,21). Oral niacin stimulation tests are time consuming and can only be properly performed on fasting patients. Considerable side effects may be quite unsettling. As such an approach is difficult to use in wide clinical studies, Pauline Ward and coworkers (22) developed a topical variant of the niacin test. Results of different groups showed diminished niacin sensitivity in up to 50% of schizophrenia patients using the topical variant of stimulation and a descriptive four-point rating scale (22–29). Early studies on psychiatric controls also showed a slightly diminished skin flush in bipolar (22) and depressive (29) patients. With regard to psychopathology, results in comparably small samples of schizophrenic patients are suggestive for an association of negative symptomatology and impaired niacin response (26,30–32) . The fact that topical niacin test studies corroborate previous results after oral niacin exposure is incentive to perform further studies in this field. However, some limitations of the current methodical standard have to be taken into account. To date, niacin sensitivity has been assessed on the basis of descriptive rating scales, which raises the problem of possible user bias and the need for objective measures. Apart from one study (29), all investigations have been performed on unmatched samples, a possible confounding effect of age and gender has yet to be proved. Because of the small sample sizes the influence of psychopathology on the niacin response and the specificity of the impaired skin reaction have to be further explored. So far the target group of most studies was multiple episode schizophrenia patients at different stages of the disease. Therefore, the interpretation of diminished niacin sensitivity as state or trait-related phenomenon and, not least, information about its causative pathophysiological background is still limited (33). Nevertheless, we regard the niacin skin test as a promising tool for schizophrenia research. The notion that schizophrenia comprises a group of disorders with a similar clinical picture but different endophenotype (34) emphasizes the need for markers to select more homogeneous subgroups. Biological markers, such as niacin sensitivity, may be able to define such endophenotypes with possible relevance for the implementation of alternative treatment options [e.g., (ω-3)-longchain fatty acid supplementation, for review see (1)]. Given the fact that lipid metabolism is altered not only at the time of first acute illness (35–37) but also at a

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pre-psychotic state (38), differentiation of patients with regard to a possible phospholipid alteration would enable attempts of new early intervention strategies. Overall, the availability of an easy applicable clinical test that is able to identify subjects with similar biochemical impairments would have major implications not only for diagnosis and treatment of a group of schizophrenia patients but also as a possible indicator for prodromal patients or people at risk to develop psychosis who would benefit from more specific preventive treatment options. The aim of this study was to characterize the niacin sensitivity on the basis of objective measures in an early state of the disease. Therefore, we applied a new optical spectroscopic niacin skin test technique to a group of first acute episode patients and age and gender matched healthy controls.

Methods Niacin Skin Test Protocol Niacin was applied simultaneously in three dilutions (0.1 M, 0.01 M, 0.001 M) of 50 µL each (approximately one drop of a standard eye-dropper) to the skin at the inner side of the forearm using chambered plaster for epicutaneous testing (Hermal, Reinek, Germany). After 90 s of niacin skin exposure the plaster was removed. Skin reaction was quantified before stimulation and subsequently at 3min intervals up to 15 min, starting 90 s after the removal of the niacin patches. Circadian fluctuations of the natural skin color were taken into account by constant measurement times. Due to photoinstability, the test solutions were freshly prepared each measurement day. Optical Reflection Spectroscopy (ORS) ORS enables the objective assessment of color changes. As the skin color is determined mainly by the content of oxygenated blood, the oxyhemoglobin (HbO2) absorption double peak at 542 nm and 577 nm has been shown to be useful for the assessment of skin color changes (39). A handheld reflection spectrometer (Dr. Lange, Berlin, Germany, spectral range 400 nm to 700 nm, area of measurement ∅ 5 mm) was used for the quantification of the skin flush, whereby each measurement was repeated three times (within 10 s) and then averaged. Spectroscopic data were processed automatically using a proprietary developed MATLAB-based software. In consideration of the individual basic skin color of each person, difference spectra were created subtracting the prestimulation reflection intensities (measured also three times) from the test intensities. In a second step two Gaussian curves were fitted to the HbO2-absorption double peak. As the area under the resulting sum curve is proportional to the HbO2-content of the superficial capillaries of the tested skin area, it was taken as a measure of the current skin redness (measured in arbitrary units, AU). A more detailed description of the data processing is given by Smesny et al., 2001 (40).

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Subjects This technique was applied to a group of 37 first episode psychosis patients fulfilling DSM IV criteria for schizophreniform psychosis or schizophrenia (25 m, 12 f; age: mean 23.9 ± 5.2, range 18–43 years) from the inpatient unit of the Department of Psychiatry of the University of Jena. The mean duration of the acute psychotic episode measured as period of time between the day of admission and the day of skin testing was 19 days (min. 1 day, max. 74 days). Diagnoses were made by two independent experienced psychiatrists (SS and HS) and confirmed by a structured clinical interview (SCID IV). Apart from five patients without medication, all were on stable mainly atypical antipsychotic medication (atypical 30, typical 2; risperidone: n = 16, mean dose 4.5 mg/d, range 2–6 mg/d; olanzapine: n = 9, mean dose 15 mg/d, range 5–20 mg/d; quetiapine: n = 2, 300 mg/d and 600 mg/d; clozapine: n = 2, 200 mg/d and 275 mg/d; others: n = 3). Patients were compared to 37 age- and gender-matched healthy volunteers recruited from staff of the Department of Psychiatry of the University of Jena (25 m, 12 f; age: mean 24.3 ± 4.7, range 18–42 years). Current mental status and personal or family history of any mental disorder was assessed by unstructured interviews. Volunteers with a personal or family history of mental disorders were excluded. Present or history of alcohol and drug abuse was assessed in each participant (nicotine use: patients 30, controls 16; cannabis use: patients 17, controls 7). There was as expected a heavier nicotine and cannabis consumption in the patient group. Subjects with colored or sun-tanned skin, any current or history of skin disorders (eczema, atopical dermatitis, psoriasis) or recent treatment with nonsteroidal anti-inflammatory drugs (e.g., aspirin) were excluded from the study. The study was approved by the Research and Ethic Committee of FriedrichSchiller-University of Jena. All subjects gave written informed consent to participate in the study. Data Analysis The data were statistically analyzed by repeated measure analysis of variance (ANOVA) with time (3, 6, 9, 12, and 15 min) and concentration (0.1 M, 0.01 M and 0.001 M) as within-subject factors and gender and group (patients vs. controls) as between-subject factors. Where necessary, a Greenhouse-Geisser correction was performed. Post-hoc comparison of single values was conducted between groups using an unpaired t-test.

Results Data of all participants was successfully obtained. Repeated measure ANOVA revealed strong effects of the factors time (F(4,280) = 145.894, P < 0.001) and con-

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Mean value ± SE in (a.u.)

centration (F(2,140) = 69.692, P < 0.001) and a significant time*concentration interaction (F(8,560) = 7.975, P < 0.001). As shown in Figure 5.1, clear differences of skin flush were noticed between the different niacin concentrations (0.1 M, 0.01 M, and 0.001 M) and the different measurement intervals (3, 6, 9, 12, and 15 min). Whereas the skin redness continuously increased over time at the lowest niacin concentration, it reached a plateau at the middle concentration and was already decreased after 6 min at the highest concentration. Repeated measure ANOVA of all spectroscopic data revealed a significant group effect between patients and matched healthy controls (F(1,70) = 4.778, P = 0.032) and a group*concentration interaction at trend level (F(2,70) = 2.490, P = 0.095). ANOVA separately performed for each niacin concentration revealed only at the lowest concentration (0.001 M) a highly significant group effect (F(1.70) = 9.644, P = 0.003) and a significant time*group interaction (F(4,70) = 4.974, P = 0.006). Patients exhibited a diminished niacin sensitivity as compared to matched controls, significant at 6, 9, 12, and 15 min after niacin exposure at the lowest niacin concentration (Fig. 5.2).

Time interval (min) Fig. 5.1. Time course of the flush response within 15 min averaged over the total population (patients and controls, n = 74) ± SE shown for each niacin concentrations.

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Mean value ± SE in (a.u.)

Time (min)

*t-test, P < 0.05

Fig. 5.2. Group separated mean flush response ± SE at the lowest niacin concentra-

tion (0.001 M) for each measurement interval. Significant group differences were obtained at 6, 9, 12, and 15 min after niacin exposure, t-test: P < 0.05.

The power of skin reaction was not correlated to the duration of acute psychotic symptoms. Smoking had no observable effect on the skin redness. Interestingly, repeated measure ANOVA of all data (n = 74) revealed a significant effect of the factor gender (F(1,70) = 4.322, P < 0.041) (Fig. 5.3). Significant interactions could not be detected between concentration and gender (F(2,140) = 0.350, n.s.), between time and gender (F(4,280) = 0.787, n.s.), or between group and gender (F(1,70) = 0.05, n.s.). Therefore, it can be concluded that the deficient niacin sensitivity in patients did not depend on gender differences.

Discussion An objective ORS-based niacin skin test variant was applied to a group of 37 medicated first episode schizophrenia patients and a group of healthy controls matched for age and gender. Comparison of niacin sensitivity revealed highly significant impairment of skin reaction in the patient group, most pronounced at the lowest niacin concentration. To our knowledge, gender-specific differences in the niacin sensitivity have not yet been studied on the basis of objective measures. In both patients and controls of our population, females exhibited a quicker and more intense erythema than males, again primarily measurable at the lowest niacin concentration.

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Mean sum value ± SE in (a.u.) Fig. 5.3. Gender separated mean flush response ± SE of all subjects (patients and controls, n = 74) for each concentration summed up over time (15 min). Females exhibited stronger niacin response as compared to males, t-test: +P < 0.1, *P < 0.05.

Group effects between patients and controls were most pronounced at the lowest niacin concentration. Looking at the time course of erythema and oedema response within 15 min after topical niacin exposure, it becomes obvious that the reaction starts with purely erythema followed by oedema. Preliminary investigations on healthy controls revealed a diminishing influence of oedema on erythema (40). Thus, the longer the latency after niacin stimulation and the higher the niacin concentration, the likelier the skin erythema is influenced by oedema. As the strong oedema response of healthy control subjects to higher niacin concentrations diminishes the skin erythema measurable by ORS, only the lowest niacin concentration revealed clear group effects. Our spectroscopic results corroborate a diminished niacin sensitivity in schizophrenia patients, obtained on the basis of descriptive ratings (22,26,28,29,41,42). Previous studies were mainly performed on multi-episode or mixed patient populations, whereas in this study exclusively first episode patients were investigated. Given the fact that altered lipid metabolism is an immanent phenomenon of a schizophrenia subtype, which is expressed throughout the body (43), we might have characterized the same subgroup of patients that has been described by various investigators using 31P-MR-spectroscopy (35–37). From a clinical point of view, our results suggest that an easy applicable skin test can be used to identify patients with metabolic impairment at an early stage of disorder. At this stage

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diminished niacin sensitivity is far from being diagnostic for schizophrenia. But as altered lipid degradation pathways were described years prior to first psychotic symptoms (38) and in unaffected relatives of schizophrenia patients (44), it seems reasonable to value impaired niacin sensitivity as a possible clinical sign of a metabolic risk to develop psychosis. Only five patients were free of medication, therefore a possible influence of antipsychotic drugs on niacin response might be discussed. In previous investigations both medicated (22) and unmedicated (28) patients displayed an impairment of niacin skin reaction. As niacin sensitivity was equally impaired in treated and untreated subgroups of different study populations (12,29) it might not be influenced by neuroleptic medication. The focus of further ORS-based niacin skin test investigations could be the assessment of specificity and sensitivity of altered niacin sensitivity in larger populations of different mental disorders. Analysis of the discriminative power of certain test parameters (e.g., latency of skin reaction, steepness of erythema response, saturation value of skin redness, or measures of single intervals or concentrations) in large samples would enable the definition of a normal range of niacin sensitivity and possible cut-off values for certain mental disorders. Prospective skin test studies on prodromal patients or genetic risk populations would be necessary to assess the predictive values for transition to psychosis and for the treatment response to certain antipsychotics or alternative treatment options. Acknowledgments Dr. Stefan Smesny is supported by the German Research Foundation (DFG), grant Sm 68/1-1. The authors would like to acknowledge the assistance of Mrs. Kati Baur and Mrs. Nicole Rudolph. Furthermore, the authors would like to thank the staff of the Department of Psychiatry of the University of Jena for their extensive support in participating in this study as volunteers.

References 1. Fenton, W.S., Hibbeln, J., and Knable, M. (2000) Essential Fatty Acids, Lipid Membrane Abnormalities, and the Diagnosis and Treatment of Schizophrenia, Biol. Psychiatry 47, 8–21. 2. Nunez, E.A. (1997) Fatty Acids Involved in Signal Cross-Talk Between Cell Membrane and Nucleus, Prostaglandins Leukot. Essent. Fatty Acids 57, 429–434. 3. Sumida, C., Graber, R., and Nunez, E. (1993) Role of Fatty Acids in Signal Transduction: Modulators and Messengers, Prostaglandins Leukot. Essent. Fatty Acids 48, 117–122. 4. van Kammen, D.P., Yao, J.K., and Goetz, K. (1989) Polyunsaturated Fatty Acids, Prostaglandins, and Schizophrenia, Ann. N. Y. Acad. Sci. 559, 411–423. 5. Murrell, T., and Taylor, W. (1959) The Cutaneous Reaction to Niacinic Acid (Niacin)Furfuryl, Arch. Dermat. 79, 545–552. 6. Morrow, J.D., Awad, J.A., Oates, J.A., and Roberts, L.J.d. (1992) Identification of Skin as a Major Site of Prostaglandin D2 Release Following Oral Administration of Niacin in Humans, J. Invest. Dermatol. 98, 812–815.

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7. Urade, Y., Ujihara, M., Horiguchi, Y., Ikai, K., and Hayaishi, O. (1989) The Major Source of Endogenous Prostaglandin D2 Production Is Likely Antigen-Presenting Cells. Localization of Glutathione-Requiring Prostaglandin D Synthetase in Histiocytes, Dendritic, and Kupffer Cells in Various Rat Tissues, J. Immunol. 143, 2982–2989. 8. Andersson, R.G., Aberg, G., Brattsand, R., Ericsson, E., and Lundholm, L. (1977) Studies on the Mechanism of Flush Induced by Nicotinic Acid, Acta Pharmacol. Toxicol. 41, 1–10. 9. Stanley, J.A., Pettegrew, J.W., and Keshavan, M.S. (2000) Magnetic Resonance Spectroscopy in Schizophrenia: Methodological Issues and Findings—Part I, Biol. Psychiatry 48, 357–368. 10. Keshavan, M.S., Stanley, J.A., and Pettegrew, J.W. (2000) Magnetic Resonance Spectroscopy in Schizophrenia: Methodological Issues and Findings—Part II, Biol. Psychiatry 48, 369–380. 11. Peet, M., Laugharne, J., Rangarajan, N., Horrobin, D., and Reynolds, G. (1995) Depleted Red Cell Membrane Essential Fatty Acids in Drug-Treated Schizophrenic Patients, J. Psychiatr. Res. 29, 227–232. 12. Glen, A.I., Cooper, S.J., Rybakowski, J., Vaddadi, K., Brayshaw, N., and Horrobin, D.F. (1996) Membrane Fatty Acids, Niacin Flushing and Clinical Parameters, Prostaglandins Leukot. Essent. Fatty Acids 55, 9–15. 13. Mahadik, S.P., and Mukherjee, S. (1996) Free Radical Pathology and Antioxidant Defense in Schizophrenia: A Review, Schizophr. Res. 19, 1–17. 14. Huang, Y., Liu, D., and Sun, S. (2000) Mechanism of Free Radicals on the Molecular Fluidity and Chemical Structure of the Red Cell Membrane Damage, Clin. Hemorheol. Microcirc. 23, 287–290. 15. Gattaz, W.F., Hubner, C.V., Nevalainen, T.J., Thuren, T., and Kinnunen, P.K. (1990) Increased Serum Phospholipase A2 Activity in Schizophrenia: A Replication Study, Biol. Psychiatry 28, 495–501. 16. Gattaz, W.F., Schmitt, A., and Maras, A. (1995) Increased Platelet Phospholipase A2 Activity in Schizophrenia, Schizophr. Res. 16, 1–6. 17. Rybakowski, J., and Weterle, R. (1991) Niacin Test in Schizophrenia and Affective Illness, Biol. Psychiatry 29, 834–836. 18. Hudson, C.J., Lin, A., Cogan, S., Cashman, F., and Warsh, J.J. (1997) The Niacin Challenge Test: Clinical Manifestation of Altered Transmembrane Signal Transduction in Schizophrenia? Biol. Psychiatry 41, 507–513. 19. Horrobin, D.F. (1980) Schizophrenia: A Biochemical Disorder? Biomedicine 32, 54–55. 20. Fiedler, P., Wolkin, A., and Rotrosen, J. (1986) Niacin-Induced Flush as a Measure of Prostaglandin Activity in Alcoholics and Schizophrenics, Biol. Psychiatry 21, 1347–1350. 21. Wilkin, J.K., Wilkin, O., Kapp, R., Donachie, R., Chernosky, M.E., and Buckner, J. (1982) Aspirin Blocks Nicotinic Acid-Induced Flushing, Clin. Pharmacol. Ther. 31, 478–482. 22. Ward, P.E., Sutherland, J., Glen, E.M., and Glen, A.I. (1998) Niacin Skin Flush in Schizophrenia: A Preliminary Report, Schizophr. Res. 29, 269–274. 23. Ward, P., and Glen, I. (2001) Niacin Skin Response and Phospholipids in Psychiatry, World J. of Biol. Psychiatry 2, 396S. 24. Ward, P.E., Glen, A.I.M., Glen, A.C.A., Macdonald, P.C., Boyle, R.M., and Horrobin, D.F. (2000) Phospholipids, Niacin Skin Test and Type IV cPLA2 in Schizophrenia, Schizophr. Res. 41, 244.

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25. Ward, P., Maclean, R., and Roberts, S. (2001) Neuroleptics and the Niacin Skin Response, World J. of Biol. Psychiatry 2, 313S. 26. Das, I., Easton, T., Richardson, A.J., and Hirsch, S.R. (1999) Niacin Skin Test in a Small Group of Schizophrenic Patients, Schizophr. Res. 36, 307. 27. Easton, T., Richardson, A.J., Hall, A.D., Kidane, L., Das, I., and Puri, B.K. (1999) The Niacin Flush Test as an Index of Fatty Acid Deficiency in Schizophrenia, Schizophr. Res. 36, 307. 28. Shah, S.H., Vankar, G.K., Peet, M., and Ramchand, C.N. (2000) Unmedicated Schizophrenic Patients Have a Reduced Skin Flush in Response to Topical Niacin, Schizophr. Res. 43, 163–164. 29. Tavares Jr., H., Yacubian, J., Elkis, H., and Gattaz, W. (2001) Niacin Test in Schizophrenia and in Major Depression, World J. of Biol. Psychiatry 2, 138S. 30. Puri, B.K., Richardson, A.J., and Easton, T. (2000) Association of Niacin Flush Response with Schizophrenia Symptoms, Schizophr. Res. 41, 251. 31. Berger, G.B., Smesny, S., Yuen, H.P., Riemann, S., and McGorry, P.D. (2002) The Topical Niacin Flush Test in Early Psychosis, Schizophr. Res. 53, S38. 32. Berger, G., and McGorry, P. (2001) The Topical Niacin Flush Test—A New Assessment Scale, World J. of Biol. Psychiatry 2, 134S–135S. 33. Ward, P.E. (2000) Potential Diagnostic Aids for Abnormal Fatty Acid Metabolism in a Range of Neurodevelopmental Disorders, Prostaglandins Leukot. Essent. Fatty Acids 63, 65–68. 34. Garver, D.L., Holcomb, J.A., and Christensen, J.D. (2000) Heterogeneity of Response to Antipsychotics from Multiple Disorders in the Schizophrenia Spectrum, J. Clin. Psychiatry 61, 964–972. 35. Pettegrew, J.W., Keshavan, M.S., Panchalingam, K., Strychor, S., Kaplan, D.B., Tretta, M.G., and Allen, M. (1991) Alterations in Brain High-Energy Phosphate and Membrane Phospholipid Metabolism in First-Episode, Drug-Naive Schizophrenics. A Pilot Study of the Dorsal Prefrontal Cortex by in Vivo Phosphorus 31 Nuclear Magnetic Resonance Spectroscopy, Arch. Gen. Psychiatry 48, 563–568. 36. Fukuzako, H., Fukuzako, T., Hashiguchi, T., Kodama, S., Takigawa, M., and Fujimoto, T. (1999) Changes in Levels of Phosphorus Metabolites in Temporal Lobes of DrugNaive Schizophrenic Patients, Am. J. Psychiatry 156, 1205–1208. 37. Stanley, J.A., Williamson, P.C., Drost, D.J., Carr, T.J., Rylett, R.J., Malla, A., and Thompson, R.T. (1995) An in Vivo Study of the Prefrontal Cortex of Schizophrenic Patients at Different Stages of Illness via Phosphorus Magnetic Resonance Spectroscopy, Arch. Gen. Psychiatry 52, 399–406. 38. Keshavan, M.S., Pettegrew, J.W., Panchalingam, K.S., Kaplan, D., and Bozik, E. (1991) Phosphorus 31 Magnetic Resonance Spectroscopy Detects Altered Brain Metabolism Before Onset of Schizophrenia, Arch. Gen. Psychiatry 48, 1112–1113. 39. Frank, K.H., Kessler, M., Appelbaum, K., and D¸mmler, W. (1989) The Erlangen Micro-Lightguide Spectrophotometer EMPHP I, Phys. Med. Biol. 34, 1883–1900. 40. Smesny, S., Riemann, S., Riehemann, S., Bellemann, M.E., and Sauer, H. (2001) Quantitative Measurement of Induced Skin Reddening Using Optical Reflection Spectroscopy-Methodology and Clinical Application, Biomed. Tech. 46, 280–286. 41. Puri, B.K., Easton, T., Das, I., Kidane, L., and Richardson, A.J. (2001) The Niacin Skin Flush Test in Schizophrenia: A Replication Study, Int. J. Clin. Pract. 55, 368–370.

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42. Easton, T., Richardson, A.D., Hall, A.D., Kidane, L., Das, I., and Puri, K. (1999) The Niacin Flush Test as an Index of Fatty Acid Deficiency in Schizophrenia, Schizophr. Res. 36, 36. 43. Horrobin, D.F. (1996) Schizophrenia as a Membrane Lipid Disorder Which is Expressed Throughout the Body, Prostaglandins Leukot. Essent. Fatty. Acids 55, 3–7. 44. Waldo, M.C. (1999) Co-Distribution of Sensory Gating and Impaired Niacin Flush Response in the Parents of Schizophrenics, Schizophr. Res. 40, 49–53.

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Chapter 6

Nutritional Implications of Sphingolipids: Occurrence and Roles in Cell Regulation Alfred H. Merrill, Jr.a, Holly Symolona,b, Jeremy C. Allegooda,c, Qiong Penga, Sarah Trotman-Pruettd, and M. Cameron Sullardsa,c aSchool

of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332

bDivision cSchool

of Biological and Biomedical Sciences, Emory University, Atlanta, Georgia 30322

of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332

dDepartment

of Chemistry, Emory University, Atlanta, Georgia 30322

Introduction The sphingoid base backbone(s) by which all sphingolipids are categorized was first described by Johann L.W. Thudichum in A Treatise on the Chemical Constitution of Brain (1884) (1) as having “. . . an alkaloidal nature, and to which, in commemoration of the many enigmas which it has presented to the inquirer, I have given the name of Sphingosin.” Thudichum’s discovery was rejected by some of the famous scientists of his day and only acknowledged about 20 years later and by subsequent appreciative generations (2). This initial unpleasantness did not prevent Thudichum from making numerous additional contributions to science, including one of the earliest books on the preparation of food from a systematic, scientific perspective (3). In the preface to the latter treatise, Thudichum comments that: “Cookery has attained its present development by a long process of experimental empiricism, at which all mankind has labored from very early days of its existence. Such being its origin, the only means for its transmission and perpetuation was apprenticeship. . . . In the present treatise it has been attempted to produce . . . a system of general rules as will enable those who thoroughly master them to perform the principal culinary operations without reference to the frequently unintelligible records of the details of mere empiricism.” Then, he asserts that: “Physiologic deduction proves that perfect cookery is the greatest economy, and that no cookery is rational which does not attain the utmost theoretically possible effect, namely, the production of the highest physiological force. . . . It is believed and hoped that the medical profession will find in this work many materials to assist them in dietetic disquisitions, and in the synthesis of rules to give into the hands of patients or their providers. Such advice is often asked for, and is always well received; and this experience has been one of the motives for the composition of this treatise.” These observations are equally relevant today, especially in light of the findings—almost one century after Thudichum’s death—that there are many links between sphingolipids and nutrition (4), some of which will be summarized in this review and by other authors in this symposium.

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Structures and Occurrence of Sphingolipids Because they are usually described in less detail in most reviews on lipid structure, some background information on the structures of sphingolipids is warranted. There is a systematic nomenclature for sphingolipids (5,6); however, some of the “official” names for sphingolipids are not used commonly in the research literature, so this review will use the names most likely to be encountered by the reader. The sphingolipids have a common backbone referred to as a “long-chain” or “sphingoid” base. The major sphingoid base of mammals is “sphingosine” (the name that is recommended by the IUPAC is (E)-sphing-4-enine or (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol), which is shown in Figure 6.1. The next most prevalent sphingoid bases of mammals are sphinganine (dihydrosphingosine) and 4-hydroxysphinganine (also called phytosphingosine, or (2S,3S,4R)-2-aminooctadecane-1,3,4-triol). For most mammalian sphingolipids, the sphingoid base backbone is composed of a linear alkyl chain of 18 carbons; however, exceptions are encountered, such as a C20homolog that is found in brain gangliosides (the proportion of which increases with age) (7) and shorter and odd-carbon number sphingoid bases in dairy products (8). Greater variation is seen in other organisms (plants, insects, etc.) (9) that vary with respect to (i) alkyl chain lengths from 14 to 22 carbon atoms, (ii) different degrees of saturation at carbons 4 and 5, (iii) a hydroxyl group at positions 4 or 6, (iv) double bonds at other sites in the alkyl chain, and (v) branching (methyl groups) at the ω-1 (iso), ω-2 (anteiso), or other positions. Sphingoid bases are abbreviated by citing (in order of appearance in the abbreviations) number of hydroxyl groups (d and t for diand tri-hydroxy, respectively), chain length, and number of double bonds; therefore, sphingosine is d18:1, sphinganine is d18:0, and phytosphingosine is t18:0.

Fig. 6.1. General structures of sphingolipids. Complex sphingolipids are elaborations of long-chain (sphingoid) bases by the addition of long-chain fatty acids in amide linkage (to form ceramides) and polar head groups. Sphingoid bases are abbreviated by citing (in order of appearance in the abbreviation) the number of hydroxyl groups (d and t for di- and tri-hydroxy, respectively), chain length, and number of double bonds (with the double bond position indicated by a superscript). Two common sphingolipids of food are shown: sphingomyelin and glucosylceramide (GlcCer).

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The majority of the sphingoid bases in cells are N-acylated with long-chain fatty acids to produce ceramides(s) followed by addition of a headgroup on the hydroxyl at carbon 1 (Fig. 6.1). The fatty acids of ceramide vary in chain length (14 to 30 carbon atoms), degree of unsaturation (but are mostly saturated), and presence or absence of a hydroxyl group on the α- or ω-carbon atom. More complex sphingolipids are often grouped based on the headgroups into the phosphosphingolipids (sphingomyelins) and glycosphingolipids (cerebrosides, gangliosides, sulfatides, etc.); however, these categories are not mutually exclusive (for example, yeast has ceramide phosphorylinositols—some of which is covalently attached to protein) (10). Glycosphingolipids are classified into broad types on the basis of carbohydrate composition. Neutral glycosphingolipids contain uncharged sugars, such as glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and fucose (Fuc). Acidic glycosphingolipids contain ionized functional groups: sulfate (sulfatoglycosphingolipids), phosphate, or charged sugar residues, such as sialic acid (N-acetylneuraminic acid) in gangliosides or glucuronic acid in some plant glycosphingolipids. Gangliosides are the prevalent acidic glycosphingolipids of most mammalian tissues, and are often described by the “Svennerholm” nomenclature (6) that is based on the number of sialic acid residues (e.g., GM1 refers to a monosialo-ganglioside) and the relative order in which the members of that ganglioside series migrate on a thin-layer chromatoplate (for example, GM3 > GM2 > GM1). Cells contain a number of other sphingolipids that may be present in small amounts but are highly bioactive, such as sphingosine 1-phosphate, lyso-sphingolipids (sphingosylphosphocholine, 1-O-glucosylsphingosine, and others), Nmethylated sphingoid bases, and ceramide phosphate.

Cell Signaling via Sphingolipids Sphingolipids are members of a signaling paradigm shown in Figure 6.2, wherein receptor activation by agonists such as tumor necrosis factor-α and platelet derived growth factor—as well as numerous stresses, such as irradiation—induces sphingomyelin turnover to elevate ceramide and/or sphingosine and/or sphingosine 1-phosphate (5,11,12). Agonists and stress also produce these and related bioactive species via perturbation of de novo sphingolipid biosynthesis (13). The downstream signaling pathways that are activated or inhibited by these mediators include protein kinases, phosphoprotein phosphatases, ion transporters, and others that control cell growth, differentiation, and survival (including apoptosis). Because ceramide and sphingosine 1phosphate often have opposing signaling functions (e.g., induction versus inhibition of apoptosis or inhibition versus stimulation of growth), Sarah Spiegel has proposed that cells utilize a ceramide/sphingosine 1-phosphate “rheostat” in deciding between growth arrest/apoptosis versus proliferation/survival (14). Sphingosine 1-phosphate can be released from cells and serve as an agonist for some of the S1P family receptors (12,14); hence, this compound serves as both a first and second messenger.

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Fig. 6.2. Depiction of cellular functions of sphingolipids. The exploded diagram high-

lights the predominantly extracellular orientation of sphingolipids in the plasma membrane (shaded grey), interactions of sphingomyelin (SM) with cholesterol (shaded black), and the aggregation of many of the sphingolipids and cholesterol in microdomains such as “rafts” and caveolae. Also shown are interactions between gangliosides (such as GM3) with cell receptors as well as extracellular proteins (matrix proteins, bacterial, and viral binding proteins). Also shown is cell signaling via turnover of sphingomyelin in response to agonists (left) or formation of bioactive compounds via de novo biosynthesis (right). One signaling product, sphingosine 1-phosphate, is secreted by cells to act as an extracellular agonist.

Cells, therefore, have the capacity to modulate downstream responses to the type of agonist (15), the state of the agonist (for example, the degree of oxidation of low-density lipoproteins) (16), and the agonist concentration (17) and—one presumes—the capacity to coordinate actions of multiple agonists by changing the relative activities of these steps of sphingolipid metabolism and the bioactive species that are formed. The recent development of mass spectrometric methods that allow measurement of all of these species (18) should facilitate the decoding of this complex pathway.

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Nutritional Implications of the Biophysical and Signaling Roles of Sphingolipids The described chemical properties and biological activities of sphingolipids have numerous implications for nutrition (4)—many of which have emerged in just the last few years. Starting with their biophysical properties, the presence of sphingomyelin in cholesterol-containing foods suppresses cholesterol absorption, presumably through interactions in the digestive tract (19,20) analogous to those that occur in membranes and micelles (21,22). In addition, a number of dietary factors (casein, pectin, oleic acid, and inter alia) (23–25) alter the amounts and distribution of sphingomyelin in circulating lipoproteins, and the fatty acid composition of sphingomyelins can be affected by the types of dietary fat (26–28). Thus, sphingolipids should heretofore be given attention both as components of the diet that can influence the utilization of other dietary lipids and as something that can be affected by the other lipids in the diet. Complex sphingolipids are also attachment sites for bacteria, bacterial toxins, and viruses (reviewed in reference 4). There is considerable interest in using dietary sphingolipids to “trap” harmful gut bacteria (i.e., by competing for endogenous sphingolipids that the organisms would use to invade the intestine), to favor the growth of beneficial microflora, and to modulate the intestinal immune system (29). A number of organisms are toxic because they interfere with sphingolipid metabolism to produce highly bioactive products, such as ceramide phosphate, produced by sphingomyelinase D, which is the toxic factor in the venom of the brown recluse spider (30); sphingosylphosphorylcholine (lyso-sphingomyelin) in atopic dermatitis (31); and sphinganine, which accumulates in blood and tissues when animals consume fumonisins, a family of mycotoxins sometimes found in corn (32). Fumonisins are an interesting prototype of how nature can utilize an existing cell death pathway (i.e., the signaling of cell death via sphingoid bases) to achieve its end—in this case to kill plants, which also use sphingolipids for cell signaling. Fumonisins are a family of mycotoxins produced by Fusarium verticillioides (formerly F. moniliforme), which are common fungal contaminants of corn and some other grains (33). As much as 59% of the corn and corn-based products worldwide have been estimated to be contaminated with variable amounts of fumonisin subspecies (33). Fumonisins cause an at-first-glance puzzling range of diseases: liver and kidney toxicity and carcinogenicity, pulmonary edema, immunosuppression (and sometimes immunostimulation), neurotoxicity, and probably others (including neural tube defects) (34). Most or all of the toxicities resulting from exposure to these compounds can be explained by their ability to alter sphingolipid metabolism by inhibiting ceramide synthase (32,35), perhaps combined with activation of cytokine signaling (36). The precursors for de novo sphingolipid biosynthesis are serine and palmitoylCoA (4,5); hence, one might envision how elevations of these common biochemicals—both as nutrients and as metabolites of other nutrient precursors—might also

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elevate flux through this pathway and have deleterious effects on cells. This was first demonstrated using cells with a genetic defect in mitochondrial uptake of palmitoyl-CoA (37) and has subsequently been found in several other cases (4,38) leading to formulation of the hypothesis that aberrant formation of sphingolipids may be a major mechanism for “lipotoxicity” (38). Furthermore, this pathway may mediate the pathophysiology induced by some nutrient deficiencies either via ceramide accumulation (39) or the inability to produce needed complex sphingolipids (40–42). Clearly, much remains to be learned about the relationship between dietary sphingolipids and their physiologic effects, as well as how other dietary factors affect sphingolipid metabolism and biochemical functions. A recent cross-sectional survey (43) found significant differences in serum sphingosine among strata of age, menstruation status, serum cholesterol, carotenoids, retinol, tocopherols, and fresh and dried vegetable and fresh fruit consumption. Using multivariate linear regression with stepwise selection, the significant predictors for serum sphingosine included total tocopherols, age, serum selenium, and retinol. That ceramides and sphingoid bases are growth suppressive and inducers of apoptosis makes this a potentially dangerous pathway (4), but also might make it a useful one for some diseases—namely, cancer. Since abnormal control of growth and apoptosis are hallmarks of cancer, our laboratory has explored the feasibility of using dietary sphingolipids (in amounts comparable to those found in foods, such as dairy products or soybean) (4) to control cancer (44). These studies initially showed that feeding sphingolipids to mice that had been administered a colon carcinogen suppressed the early markers (increased proliferation and appearance of aberrant colonic crypt foci) and late stages (numbers of adenocarcinomas) of colon carcinogenesis (44). This has recently been confirmed (45). Subsequent studies (46) of colon tumorigenesis using mice with a mutation of a gene that is also frequently abnormal in human colon cancer (truncation of APC protein) have shown that feeding sphingolipids not only reduces the tumor number but also the abnormal cytosolic and nuclear accumulation of βcatenin; hence, this dietary intervention is effectively “normalizing” or “reversing” the abnormal phenotype of this gene defect and, in so doing, inhibiting tumorigenesis. Other work (47) has shown that dietary fiber elevates endogenous intestinal sphingolipids, which raises the possibility that sphingolipids may act directly as components of the diet and as mediators of the effects of other foods that have been associated with reduction of colon cancer risk.

Summary Sphingolipids are food components with a high degree of structural diversity but all sharing a common feature, a sphingoid base backbone. They are involved in membrane structure as well as many cell regulatory pathways; however, little is known about how modulation of sphingolipid amounts and type by diet affects (patho)physiology. One of the known links between diet and health via sphingolipids is the disruption of sphingolipid metabolism by fumonisins (mycotoxins

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found in corn), which results in a broad spectrum of disease. Dietary sphingolipids, however, appear to be beneficial because they suppress colon carcinogenesis. Much remains to be learned about the nutritional aspects of sphingolipids. Acknowledgments The authors are grateful to the numerous colleagues who helped with the work described in this review as well as to the NIH for funding for our basic research (GM46368) on sphingolipid metabolism and function and translation to studies of cancer (NCI-U19-CA87525).

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the Mitogenesis Induced by Oxidized LDL in Smooth Muscle Cells via Activation of Sphingomyelinase, Ceramidase, and Sphingosine Kinase, J. Biol. Chem. 274, 21533– 21538. Nikolova-Karakashian, M., Morgan, E.T., Alexander, C., Liotta, D.C., and Merrill, A.H. Jr. (1997) Bimodal Regulation of Ceramidase by Interleukin-1β: Implications for the Regulation of Cytochrome P450 2C11 (CYP2C11), J. Biol. Chem. 272, 18718– 18724. Sullards, M.C., and Merrill, A.H. Jr. (2001) Analysis of Sphingosine 1-Phosphate, Ceramides, and Other Bioactive Sphingolipids by High-Performance Liquid Chromatography-Tandem Mass Spectrometry, Sci. STKE 67, PL1. Nyberg, L., Duan, R., and Nilsson, A. (2000) A Mutual Inhibitory Effect on Absorption of Sphingomyelin and Cholesterol, J. Nutr. Biochem. 11, 244–249. Eckhardt, E.R., Wang, D.Q., Donovan, J.M., and Carey, M.C. (2002) Dietary Sphingomyelin Suppresses Intestinal Cholesterol Absorption by Decreasing Thermodynamic Activity of Cholesterol Monomers, Gastroenterology 122, 948–956. London, E., and Brown, D.A. (2000) Insolubility of Lipids in Triton X-100: Physical Origin and Relationship to Sphingolipid/Cholesterol Membrane Domains (Rafts), Biochim. Biophys. Acta 1508, 182–195. Cremesti, A.E., Goni, F.M., and Kolesnick, R. (2002) Role of Sphingomyelinase and Ceramide in Modulating Rafts: Do Biophysical Properties Determine Biologic Outcome? FEBS Lett. 531, 47–53. Bladergroen, B.A., Beynen, A.C., and Geelen, M.J. (1999) Dietary Pectin Lowers Sphingomyelin Concentration in VLDL and Raises Hepatic Sphingomyelinase Activity in Rats, J. Nutr. 129, 628–633. Geelen, M.J., van Hoorn, D., and Beynen, A.C. (1999) Consumption of Casein Instead of Soybean Protein Produces a Transient Rise in the Concentration of Sphingomyelin in VLDL in Rats, J. Nutr. 129, 2119–2122. Geelen, M.J., and Beynen, A.C. (2000) Consumption of Olive Oil Has Opposite Effects on Plasma Total Cholesterol and Sphingomyelin Concentrations in Rats, Br. J. Nutr. 83, 541–547. Gerasimova, E.N., Levachev, M.M., Perova, N.V., Nikitin, Iu.P., and Ozerova, I.N. (1986) Characteristics of Fatty Acid Composition of Phosphatidylcholines and Sphingomyelins of Low-Density Lipoproteins in the Plasma of Native Inhabitants of Chukotka, Vopr. Med. Khim. 32, 66–72. Bettger, W.J., and Blackadar, C.B. (1997) Dietary Very Long Chain Fatty Acids Directly Influence the Ratio of Tetracosenoic (24:1) to Tetracosanoic (24:0) Acids of Sphingomyelin in Rat Liver, Lipids 32, 51–55. Cook, C., Barnett, J., Coupland, K., and Sargent, J. (1998) Effects of Feeding Lunaria Oil Rich in Nervonic and Erucic Acids on the Fatty Acid Compositions of Sphingomyelins from Erythrocytes, Liver, and Brain of the Quaking Mouse Mutant, Lipids 33, 993–1000. Vazquez, E., Gil, A., and Rueda, R. (2001) Dietary Gangliosides Positively Modulate the Percentages of Th1 and Th2 Lymphocyte Subsets in Small Intestine of Mice at Weaning, Biofactors 15, 1–9. Merchant, M.L., Hinton, J.F., and Geren, C.R. (1998) Sphingomyelinase D Activity of Brown Recluse Spider (Loxosceles reclusa) Venom as Studied by 31P-NMR: Effects on the Time-Course of Sphingomyelin Hydrolysis, Toxicon. 36, 537–545.

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31. Okamoto, R., Arikawa, J., Ishibashi, M., Kawashima, M., Takagi, Y., and Imokawa, G. (2003) Sphingosylphosphorylcholine is Upregulated in the Stratum Corneum of Patients with Atopic Dermatitis, J. Lipid Res. 44, 93–102. 32. Merrill, A.H. Jr., Sullards, M.C., Wang, E., Voss, K.A., and Riley, R.T. (2001) Sphingolipid Metabolism: Roles in Signal Transduction and Disruption by Fumonisins. Environ. Health Perspect. 109 Suppl. 2, 283–289. 33. Marasas, W.F.O. (2001) Discovery and Occurrence of the Fumonisins: A Historical Perspective, Environ. Health Perspect. 109 Suppl. 2, 239–243. 34. Sadler, T.W., Merrill, A.H., Stevens, V.L., Sullards, M.C., Wang, E., and Wang, P. (2002) Prevention of Fumonisin B1-Induced Neural Tube Defects by Folic Acid, Teratology 66, 169–176. 35. Desai, K., Sullards, M.C., Allegood, J., Wang, E., Schmelz, E.M., Hartl, M., Humpf, HU., Liotta, D.C., Peng, Q., and Merrill, A.H. Jr. (2003) Fumonisins and Fumonisin Analogs as Inhibitors of Ceramide Synthase and Inducers of Apoptosis, Biochem. Biophys. Acta (in press). 36. Bhandari, N., Brown, C.C., and Sharma, R.P. (2002) Fumonisin B1-Induced Localized Activation of Cytokine Network in Mouse Liver, Food Chem. Toxicol. 40, 1483–1491. 37. Paumen, M.B., Ishida, Y., Muramatsu, M., Yamamoto, M., and Honjo, T. (1997) Inhibition of Carnitine Palmitoyltransferase I Augments Sphingolipid Synthesis and Palmitate-Induced Apoptosis, J. Biol. Chem. 272, 3324–3329. 38. Unger, R.H. (2002) Lipotoxic Diseases, Annu. Rev. Med. 53, 319–336. 39. Yen, C.L., Mar, M.H., Craciunescu, C.N., Edwards, L.J., and Zeisel, S.H. (2002) Deficiency in Methionine, Tryptophan, Isoleucine, or Choline Induces Apoptosis in Cultured Cells, J. Nutr. 132, 1840–1847. 40. Krigman, M.R., and Hogan, E.L. (1976) Undernutrition in the Developing Rat: Effect upon Myelination, Brain Res. 107, 239–255. 41. Vaswani, K.K. (1985) Effect of Neonatal Thiamine and Vitamin A Deficiency on Rat Brain Gangliosides, Life Sci. 37, 1107–1115. 42. Sundaram, K.S., Fan, J.H., Engelke, J.A., Foley, A.L., Suttie, J.W., and Lev, M. (1996) Vitamin K Status Influences Brain Sulfatide Metabolism in Young Mice and Rats, J. Nutr. 126, 2746–2751. 43. Abnet, C.C., Borkowf, C.B., Qiao, Y.L., Albert, P.S., Wang, E., Merrill, A.H. Jr., Mark, S.D., Dong, Z.W., Taylor, P.R., and Dawsey, S.M. (2001) A Cross-Sectional Study of Human Serum Sphingolipids, Diet and Physiologic Parameters, J. Nutr. 131, 2748–2752. 44. Merrill, A.H. Jr., and Schmelz, E-M., (2000) Sphingolipids: Mechanism-Based Inhibitors of Carcinogenesis Produced by Animals, Plants, and Other Organisms, in Handbook of Nutraceuticals and Functional Foods, Wildman, R.E.C., CRC Press, Boca Raton, Chapter 20. 45. Berra, B., Colombo, I., Sottocornola, E., and Giacosa, A. (2002) Dietary Sphingolipids in Colorectal Cancer Prevention, Eur. J. Cancer Prev. 11, 193–197. 46. Schmelz, E.M., Roberts, P.C., Kustin, E.M., Lemonnier, L.A., Sullards, M.C., Dillehay, D.L., and Merrill, A.H. Jr. (2001) Modulation of Intracellular β-Catenin Localization and Intestinal Tumorigenesis in Vivo and in Vitro by Sphingolipids, Cancer Res. 61, 6723– 6729. 47. Jian, Y.H., Lupton, J.R., Chang, W.C., Jolly, C.A., Aukema, H.M., and Chapkin, R.S. (1996) Dietary Fat and Fiber Differentially Alter Intracellular Second Messengers During Tumor Development in Rat Colon, Carcinogensis 17, 1227–1233.

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Chapter 7

Digestion and Absorption of Sphingolipids in Food Åke Nilsson, Erik Hertervig, and Rui-Dong Duan Department of Medicine, University Hospital of Lund, S-22185 Lund, Sweden

Background Meat, milk, and fish are important sources of sphingolipids in the diet. Whereas sphingomyelin (SM) is primarily an animal product, the sphingolipids in plants are mainly cerebrosides (mono- and oligohexosylceramides) with glucose, galactose, mannose, and inositol. Vegetable material contains small amounts of glycosphingolipids, with the exception of soybeans, which contain as much as 2410 µmol/kg of total sphingolipids (1). The membranes surrounding the milk fat globule is formed from the secreting plasma membrane of the mammary gland and contains about equal amounts of phosphatidylcholine and SM. Of the polar milk lipids 20–40% are SM and small amounts are cerebroside and gangliosides. In human milk the content of SM is 160–210 nmol/mL. A newborn baby ingests 400–500 mL and a 4-month-old baby 550–1000 mL milk per day. Suckling babies thus ingest 80–200 mg SM per day plus some glycosphingolipids. A review of the literature on sphingolipid contents of different foods show that the per capita consumption in the United States is on the order of 15–180 mmol per year (115–140 g), or 0.3–0.4 g per day (1). The dietary sphingolipids are mixed with an endogenous pool originating from bile and from sloughed mucosal cells. The amount of SM secreted in bile varies with species; the sheep bile contains a particularly high proportion (2,3). Even if SM accounts for as little as about 1% of the phospholipids in human bile, this still corresponds to 50–100 mg per day. The brush borders of mucosal cells in the gastrointestinal tract contain high levels of glycosphingolipids and SM; the glycosphingolipid/total phospholipid ratio is about one. Substantial amounts from sloughed mucosal cells must therefore enter lumen during the normal mucosal turnover, although the exact amount is difficult to estimate. The mucosal sphingolipids differ with regard to sphingosine bases and fatty acid composition (4). The glucocerebrosides contain a large proportion of hydroxylated fatty acids, and the bases are both saturated and unsaturated and contain 18–20 carbons. Bioactive sphingolipid metabolites may thus be formed from both endogenous and exogenous sphingolipids, and the amounts vary with dietary factors. An important feature is that there are no pancreatic sphingolipidases that hydrolyze the dietary sphingolipids rapidly, but the digestion is slow and depends on mucosal enzymes.

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The Course of SM Absorption Whereas the bulk of the main lipids in the diet, i.e., about 100 g of triglycerides and 2–3 g glycerophospholipids, are hydrolyzed rapidly by pancreatic enzymes in the upper gastrointestinal tract, the digestion of sphingolipids follow a different course. The digestion is slower, extended over the whole gut, and catalyzed by mucosal rather than pancreatic enzymes. Early studies showed that when 14C-stearoyl- or 3-3H-sphingosine-labeled synthetic SM was fed to lymphatic duct cannulated rats, little or no intact SM was transported by the chyle (5). The appearance of radiolabeled ceramide and other sphingolipids in chyle was also low. With 3H-labelling at position three of the sphingosine portion, little radioactivity was recovered in chyle when free 3H-sphingosine, 3H-ceramide, or 3H-SM was given orally. With dihydrosphingosine labeled at position 11–12 as substrate, the recovery of radioactivity in chyle was high, mainly in triglyceride (Table 7.1). The explanation was that the sphingosine bases had been converted to palmitic acid in the mucosa and incorporated into the chylomicron triglyceride. When 14C-stearoyl-labeled SM was given, 30–40% of the radioactivity was recovered in chyle, primarily in triglycerides and glycerophospholipids (Table 7.2). Data thus indicated a partial absorption, and some intact radioactive SM and ceramide were also recovered in feces. Also in experiments with 3-3H-sphingosine-labeled SM about 30% of the radioactivity was recovered in feces, mainly as ceramide. More recently, Schmeltz et al. (6) fed or injected 3H-dihydrosphingosinelabeled SM into closed loops from various levels of the gastrointestinal tract. The data obtained indicated that the digestion and absorption were extended over the whole length of the intestine, and they also found some reincorporation of the labeled sphingosine into ceramide and glycosphingolipids of the mucosa. In another study, Nyberg et al. (7) fed various mass amounts, up to 25 mg, of radiolabeled SM orally to rats, and the digestion at various levels of the small intestine was examined. Analysis of tissues after 2–4 h demonstrated that the uptake and hydrolTABLE 7.1 Distribution of Radioactivity in the Lymph Lipids After the Ingestion of Fatty Acid-Labeled Sphingomyelin and Ceramidea

Fed substrate [14C]-stearoyl-SM (n = 6) [3H]-palmitoylSphingosine (n = 4) aRats

Distribution of Radioactivity (%)

Recovery of radioactivity in lymph lipids (%)

Glycerides

Ceramide

SM

35.7–59.9 M = 43.9

90.8–94.8 M = 92.7

0–1 —

0–1 —

31.1–50.3 M = 39.3

91.8–95.5 M = 93.6

0–0.5 —

0–0.6 —

were fed 5 mg of substrate and the lymph collected for 24 h. M = mean.

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PC 5.5–7.4 M = 6.8 4.0–8.0 M = 6.0

TABLE 7.2 Distribution of Radioactivity in the Lipids of Abdominal Lymph After Feeding Sphingosine-Labeled Substratea Distribution of Radioactivity (%)

Substrate

Recovery in lymph lipids (%)

SM

Ceramide

Glycerides

PC

[3H]-SM (n = 6)

4.0–9.7 M = 7.9

0.4–2.6 M = 1.9

28.5–33.0 M = 30.9

57.5–62.5 M = 60.7

4.7–9.0 M = 6.5

[3-3H]-sphingosine (n = 8)

3.6–9.5 M = 7.1

2.1–9.6 M = 4.9

15.9–35.7 M = 27.1

53.4–69.8 M = 63.1

3.7–8.8 M = 4.9

46.9–78.5 M = 62.0

0.2–1.5 M = 0.8

1.8–4.0 M = 2.8

85.9–92.5 M = 90.2

4.1–11.6 M = 6.2

[11,12-3H2]dihydrosphingosine (n = 4)

aRats were fed 5 mg labeled substrate in 1 mL milk-protein-triolein emulsion and lymph was collected for 24 h. M = mean.

ysis of SM were largest in the middle and lower small intestine, where the levels of alkaline sphingomyelinase (alk-SMase) is highest (8). The data thus support a role of this enzyme in digestion. The capacity to digest SM seems, however, to be limited; when 25 mg was fed, substantial amounts of undigested SM were found in the intestinal content of the small intestine after 2–4 h and in feces collected over 24 h. In studies on patients who had an ileostomy after a colectomy due to severe colitis, liquid meals containing 250 mg of milk SM were fed and the ileostomy content was collected over 8 h and analyzed. The meal significantly increased the output of ceramide and undigested SM in ileostomy content. The conclusion was that dietary SM increases exposure of the distal small intestine and colon to sphingolipid metabolites also in humans.

Digestion and Absorption of Cerebroside The course of absorption of radioactive 3H-sphingosine-labeled cerebroside was similar to that of SM (9) (Table 7.3). No cerebrosidase activity could be demonTABLE 7. 3 Distribution of Radioactivity of the Lymph Lipids After Feeding Palmitoyl-Labeled Glucosyl-Ceramidea Distribution of Radioactivity (%) Recovery (%)

Glycerides

Lecithin

Cerebroside

Ceramide

20.2–41.5 M = 29.5

92.2–96.0 M = 93.8

4.0–7.8 M = 6.2

0–traces —

0–traces —

aRats

were fed 1 (mole [9,10-3H2]-pamitoyl glucosyl ceramide in 1 mL milk-protein-triolein emulsion. Lymph was collected for 24 h.

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strated in rat pancreatic juice, whereas an enzyme activity with a slightly acid pH optimum in the intestinal wall catalyzed the degradation to ceramide, which was then metabolized as described above. Also, the cerebroside absorption was incomplete; some intact cerebroside and ceramide appeared in feces. Interestingly Brady, as long ago as 1965, purified a glycosyl- and galactosyl-ceramide cleaving enzyme from rat small intestine that had a neutral pH optimum (10). Leese and Semenza concluded that this enzyme was probably identical to the lactase-phlorizine hydrolase, an enzyme that is located in the brush border and that is known to occur in highest levels during the suckling period (11). Later studies by Kobayashi and Suzuki confirmed the ability of this enzyme to hydrolyze glycosylceramides, and the action has been ascribed to a separate active site compared to lactase hydrolysis (12,13). Both glycosyl and lactosylceramide of the milk fat globule were physiological substrates for the enzyme.

Metabolism of the Sphingoid Bases The studies conducted in lymphatic duct cannulated rats demonstrated that the sphingosine base was metabolized to the fatty acids in the mucosal cell, which were then incorporated mainly into chyle triglyceride. In accordance with studies on liver tissue, mucosal homogenate from guinea pig small intestine was able to convert 3H-dihydrosphingosine to palmitic acid with palmitaldehyde as an intermediate (14). The reaction was stimulated by pyridoxin, with both liver and intestine as enzyme sources, supporting the formation of an intermediary Schiff base. Later studies demonstrated that sphingoid bases are degraded in two steps. They are first phosphorylated at the 1-hydroxyl group and then cleaved to a long-chain aldehyde and ethanolamine phosphate. The first step is ATP-dependent and is catalyzed by sphingosine kinases, which cause the formation of sphingosine-1-phosphate. The second step, i.e., the formation of an aldehyde and ethanolamine phosphate, is catalyzed by sphingosine-1-phosphate lyase. The palmitaldehyde formed is then oxidized to palmitic acid. Early studies demonstrated high levels of sphingosinemetabolizing enzymes in guinea pig small intestinal mucosa (14). Later studies compared lyase acitivities of different tissues and found the highest levels in intestinal mucosa, two- to threefold higher than in liver expressed per mg protein (15). The capacity of the intestinal mucosa to catalyze phosphorylation of sphingoid bases was also found to be high (16). Palmitaldehyde may also be reduced to hexadecanol and specifically utilized to form ester bonds of plasmalogens.

Enzymes Hydrolyzing Dietary SM Early studies attemptying to find pancreatic enzymes hydrolyzing sphingolipids were unsuccessful. Pancreatic tissue contains acid and neutral Mg2+-dependent SMase like other tissues, but in rat and human pancreatic juice we were able to find only small amounts of acid SMase acitivity. Chen et al. reported SMase activ-

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ity in human pancreatic juice obtained during endoscopic cannulation of bile ducts (17). The authors remarked, however, that samples with highest activity were bile colored, and we believe that the enzyme activity was due to the presence of SMase of biliary origin (see later). In 1969 Nilsson reported an enzyme activity in rat and pig small intestinal mucosa and human duodenal content that hydrolyzes SM to ceramide and phosphocholine (18). The enzyme had an alkaline pH optimum around 9, and subcellular fractionation suggested an enrichment in brush border preparations. The enzyme was also present in human duodenal contents but not in pancreatic juice. It was therefore postulated that the enzyme was released from the gut mucosa. The enzyme has not been further studied until recently, when Duan et al. examined the longitudinal distribution of the enzyme in the gastrointestinal tract of the rat (8). The enzyme was not present in stomach or upper duodenum but increased more distally to reach the maximum in the middle and lower part of the small intestine. The enzyme was also demonstrated in colonic mucosa but was not of bacterial origin since a similar longitudinal distribution could be demonstrated in germfree mice. The presence of the enzyme in meconium of newborns further supports its tissue rather than bacterial origin. The enzyme was found in intestinal tissue from rats, mice, humans, pig, and hamster but not in the guinea pig, which had higher intestinal acid SMase than the other species (19). Nyberg et al. found an alk-SMase activity with similar properties as the gut enzyme in bile of humans but not in bile of other species, including rat, pig, sheep, and cow (20) Similar activities were found in both bacteria-positive and bacterianegative bile samples. It is thus likely that secretion in bile contributes to the alkSMase activity of human duodenal contents and possibly to the mucosal enzyme activity. The bile alk-SMase has also been purified to electrophoretic homogeneity (21). Recently the alk-SMase was purified from rat intestinal mucosa (22) and from human intestinal content (Duan et al., unpublished). A luminal bile salt eluate from rat intestine was used as starting material, and the enzyme was purified by a combination of acetone precipitation, DEAE-Sepharose-, High Q anion exchange-, Phenyl Sepharose hydrophobic, and gel chromatography. The molecular mass of the enzyme of rat and human are 58 and 60 kDa, respectively. In contrast to the neutral SMase found in several other tissues, the enzyme was strongly inhibited by Triton X 100 and did not require magnesium ions. The enzyme is bile salt dependent, and the activity is particularly increased by taurocholate and taurochenodeoxycholate, two primary bile salts. The enzyme is extremely resistant to pancreatic proteolytic enzymes. The antibodies against both rat and human intestinal alk-SMase were developed and Western blots show only positive expression of the enzyme in the intestine. Whether the alk-SMase is synthesized by the mucosal epithelial cell is a question that has not earlier been clarified. Recent immunohistochemical studies in our laboratory showed that the enzyme is localized at the surface of microvillar mem-

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brane, and positive labels were also found in Golgi structures and endosomes, indicating that the enzyme is synthesized in the mucosal cells and successively transferred via the Golgi apparatus to the apical membrane (Duan et al., unpublished).

Sphingolipid Digestion During Suckling Since SM is an important polar lipid in milk, an important question is how the suckling neonate metabolizes milk SM. Nyberg et al. (23) found an acid SMase in human and cows milk, which is probably identical to the acid lysosomal SMase. The possibility that this enzyme may act at acid pH in the stomach was, however, not supported by analyzing gastric content of suckling infants. No ceramide was formed in the stomach, but only intact SM was found. Duodenal content and ileal samples obtained during surgery for malformations contained, however, alkaline SMase activity. Furthermore, meconium from both term and preterm newborn infants contained substantial levels of alkaline SMase. Lillienau et al. (unpublished data) examined alkaline SMase levels of the intestine of rat fetuses and newborn rats and found that the enzyme is expressed on day 22 after conception, i.e., just before delivery. The conclusion is that alk-SMase is likely to be the most important enzyme in the initial step of SM digestion in the newborn.

Hydrolysis of Ceramide Studies by Nilsson in 1969 (18) indicated that ceramide was not rapidly absorbed intact but was hydrolyzed to sphingosine and fatty acid before absorption. No enzyme hydrolyzing the ceramide could be demonstrated in pancreatic juice in incubations with taurodeoxycholate as detergent. Such enzyme activity was, however, demonstrated in human duodenal contents and in the mucosal wall of rat and pig small intestine. This enzyme activity had neutral to slightly alkaline pH optimum and catalyzed also the reversed reaction with a more acid pH optimum. Like the alk-SMase, the ceramidase activity was enriched in brush border fractions. Nyberg et al. (22) conducted experiments with pure (23) bile salt stimulated lipase (BSSL) from human milk and from pancreas and found that these enzymes had ceramidase activity in the presence of taurocholate. Earlier, Hui et al. (24) reported that this enzyme had lipoamidase acitivity with artificial substrates. Since BSSL is strictly dependent on cholate for its action, the earlier negative report may be due to the use of taurodeoxycholate. Recent studies characterized the longitudinal distribution of the bile salt dependent mucosal ceramidase and clearly demonstrated different distribution patterns between BSSL and intestinal neutral ceramidase (25). The neutral intestinal ceramidase could be separated from BSSL on high quaternary aminoethyl anion (HQ) chromatography, and Western blot using specific antiserum toward BSSL did not identify BSSL in the lower and middle parts of the gut, which had high ceramidase acitivty. In contrast BSSL levels were highest in the duodenum (26). The occurrence of a

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mucosal ceramidase in BSSL gene knockout mice further confirms the existence of this enzyme (27). Interestingly, the hydrolysis of ceramide by BSSL could be linked to an effect on secretion and size distribution of intestinal lipoproteins (27).

Factors Affecting Sphingolipid Absorption Due to the strong interaction between SM and sterols the question has been raised whether the compounds may exert a mutual effect on absorption. Feeding sonicated mixture of SM and either cholesterol or sitosterol and estimating the effects on cholesterol absorption, Nyberg et al. (28) found that cholesterol was poorly absorbed from cholesterol/SM mixture in a ratio of 1:1. A degree of absorption as low as 9% was seen. Both cholesterol and plant sterols were found to inhibit the hydrolysis of SM and increase exposure of colon to ceramide and intact SM. Physicochemical studies showed that incorporation of cholesterol in SM-phosphatidylcholine vesicles has profound effects on detergent-induced phase transitions. Using caco-2 cells and in vivo studies with the fecal dual isotope method in mice, Eckhardt et al. found that milk SM and dipalmitoyl-phosphatidylcholine reduced the cholesterol uptake (29). Moschetta et al. (30) found that inclusion of SM in phosphatidylcholine vesicles enhanced the bile salt-induced transition to micelles, but addition of cholesterol to the SM-containing vesicles strongly counteracted this transition. The conclusion is that the interaction between sterols and SM can be exploited both to inhibit cholesterol absorption and to increase the exposure of colon to ceramide. In vitro studies with purified alk-SMase revealed that presence of other glycerophospholipids in mixed bile salt micelles with radioactive SM inhibited SM hydrolysis (31). Also, neutral glycerides and sterols in mixed substrates, but not free fatty acids, inhibited SM hydrolysis. Intraluminal factors may thus strongly influence the course of SM digestion and thereby the exposure of colon to sphingolipid metabolites (32).

Alk-SMase and Cancer The occurrence of alk-SMase in colon mucosa initiated studies in which the enzyme levels were compared in tumors and surrounding mucosa. The findings were that the level was 50% lower in tumor tissue (33) . In mucosa of patients with familial adenomatous polyposis the level was about 10% that of normal (34). Additional support that the enzyme may influence cell growth was obtained in cell culture studies in which the enzyme was found to inhibit proliferation of HT 29 cells but not of the small intestinal epithelial cell line IEC6 (35). Since the risk of colon cancer is known to be influenced by hereditary as well as environmental factors, including diet, we examined whether dietary factors or drugs known to protect colonic mucosa against colon cancer also influence levels of alkaline SMase. Ursodeoxycholic acid, a bile acid known to counteract chemically induced colon cancer, increased alk-SMase and caused a parallell increase in

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caspase 3, a key executive enzyme in apoptosis (36). Feeding rat or mouse a high-fat diet for 3 to 4 weeks significantly reduced alk-SMase levels in the colonic mucosa by more than 50% (37). The levels of SMase may also be changed by the diet containing a high amount of fiber. Psyllium, a type of water soluble fiber, significantly increased alk-SMase in the colon and partly reversed the effect of high fat on alk-SMase levels. Water insoluble fiber, such as cellulose, only slightly increased neutral SMase activity but had no effect on alk-SMase activity. The altered activity of alk-SMase induced by psyllium and fat is a consequence of changed expression of the enzyme as Western blot showed the enzyme protein level is significantly increased by psyllium and decreased by a high fat diet (Cheng et al. unpublished data). Several pieces of evidence thus point to a possible link between alk-SMase and colon cancer. The enzyme may act on both exogenous and endogenous SM and the products linked to antiproliferative signaling mechanisms.

Future Perspectives Although the main features of the sphingolipid digestion have been characterized, further information about the enzymes involved is crucial. The cloning and characterization of the factors that regulate expression of alk-SMase and intestinal ceramidase are key issues. Factors regulating the intracellular level of sphingolipid metabolites, particularly in humans, need to be determined and the signaling pathways mediating the effects of the sphingolipid metabolites clarified. Furthermore intraluminal and mucosal factors influencing the rate of digestion and thereby the exposure of the intestine to the metabolites need to be further investigated. References 1. Vesper, H., Schmelz, E.M., Nikolova-Karakashian, M.N., Dillehay, D.L., Lynch, D.V., and Merrill, A.H., Jr. (1999) Sphingolipids in Food and the Emerging Importance of Sphingolipids to Nutrition, J. Nutr. 129, 1239–1250. 2. Alvaro, D., Cantafora, A., Attili, A.F., Ginanni Corradini, S., De Luca, C., Minervini, G., Di Biase, A., and Angelico, M. (1986) Relationships Between Bile Salts Hydrophilicity and Phospholipid Composition in Bile of Various Animal Species, Comp. Biochem. Physiol. 83, 551–554. 3. Moschetta, A., Van Berge-Henegouwen, G.P., Portincasa, P., Palasciano, G., Groen, A.K., and Van Erpecum, K.J. (2000) Sphingomyelin Exhibits Greatly Enhanced Protection Compared with Egg Yolk Phosphatidylcholine Against Detergent Bile Salts, J. Lipid Res. 41, 916–924. 4. Bouhours, J.F., and Guignard, H. (1979) Free Ceramide, Sphingomyelin, and Glucosylceramide of Isolated Rat Intestinal Cells, J. Lipid Res. 20, 879–907. 5. Nilsson, Å. (1968) Metabolism of Sphingomyelin in the Intestinal Tract of the Rat, Biochim. Biophys. Acta 164, 575–584. 6. Schmelz, E.M., Crall, K.J., Larocque, R., Dillehay, D.L., and Merrill, A.H., Jr. (1994) Uptake and Metabolism of Sphingolipids in Isolated Intestinal Loops of Mice, J. Nutr. 124, 702–712.

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7. Nyberg, L., Nilsson, Å., Lundgren, P., and Duan, R.D. (1997) Localisation and Capacity of Spingomyelin Digestion in the Rat Intestinal Tract, J. Nutr. Biochem. 8, 112–118. 8. Duan, R.D., Nyberg, L., and Nilsson, Å. (1995) Alkaline Sphingomyelinase Activity in Rat Gastrointestinal Tract: Distribution and Characteristics, Biochem. Biophys. Acta 1259, 49–55. 9. Nilsson, Å. (1969) Metabolism of Cerebroside in the Intestinal Tract of the Rat, Biochim. Biophys. Acta 187, 113–121. 10. Brady, R.O., Gal, A.E., Kanfer, J.N., and Bradley, R.M. (1965) The Metabolism of Glucocerebrosides. 3. Purification and Properties of a Glucosyl- and GalactosylceramideCleaving Enzyme from Rat Intestinal Tissue, J. Biol. Chem. 240, 3766–3770. 11. Leese, H.J., and Semenza, G. (1973) On the Identity Between the Small Intestinal Enzymes Phlorizin Hydrolase and Glycosylceramidase, J. Biol. Chem. 248, 8170–8173. 12. Kobayashi, T., and Suzuki, K. (1981) The Glycosylceramidase in the Murine Intestine. Purification and Substrate Specificity, J. Biol. Chem. 256, 7768–7773. 13. Kobayashi, T., and Suzuki, K. (1981) A Taurodeoxycholate-Activated Galactosylceramidase in the Murine Intestine, J. Biol. Chem. 256, 1133–1137. 14. Nilsson, Å. (1970) Conversion of Dihydrosphingosine to Palmitaldehyde and Palmitic Acid with Cell-Free Preparations of Guinea Pig Intestinal Mucosa, Acta Chem. Scandinavica 24, 598–604. 15. Van Veldhoven, P.P., and Mannaerts, G.P. (1993) Sphingosine-Phosphate Lyase, Adv. Lipid Res. 26, 69–98. 16. Gijsbers, S., Van der Hoeven, G., and Van Veldhoven, P.P. (2001) Subcellular Study of Sphingoid Base Phosphorylation in Rat Tissues: Evidence for Multiple Sphingosine Kinases, Biochim. Biophys. Acta 1532, 37–50. 17. Chen, H., Born, E., Mathur, S.N., Johlin, F.C., Jr., and Field, F.J. (1992) Sphingomyelin Content of Intestinal Cell Membranes Regulates Cholesterol Absorption. Evidence for Pancreatic and Intestinal Cell Sphingomyelinase Activity, Biochem. J. 286, 771–777. 18. Nilsson, Å. (1969) The Presence of Spingomyelin- and Ceramide-Cleaving Enzymes in the Small Intestinal Tract, Biochim. Biophys. Acta 176, 339–347. 19. Duan, R.D., Hertevig, E., Nyberg, L., Tauge, T., Sternby, B., Lillienau, J., Farooqi, A., and Nillson, Å. (1996) Distribution of Alkaline Sphingomyelinase Activity in Human Beings and Animals. Tissue and Species Differences, Dig. Dis. Sci. 41, 1801–1806. 20. Nyberg, L., Duan, R.D., Axelson, J., and Nilsson, Å. (1996) Identification of an Alkaline Sphingomyelinase Activity in Human Bile, Biochim. Biophys. Acta 1300, 42–48. 21. Duan, R.D., and Nilsson, Å. (1997) Purification of a Newly Identified Alkaline Sphingomyelinase in Human Bile and Effects of Bile Salts and Phosphatidylcholine on Enzyme Activity, Hepatology 26, 823–830. 22. Cheng, Y., Nilsson, Å., Tomquist, E., and Duan, R.D. (2002) Purification, Characterization, and Expression of Rat Intestinal Alkaline Sphingomyelinase, J. Lipid Res. 43, 316–324. 23. Nyberg, L., Farooqi, A., Blackberg, L., Duan, R.D., Nilsson, Å., and Hernell, O. (1998) Digestion of Ceramide by Human Milk Bile Salt-Stimulated Lipase, J. Pediatr. Gastroenterol Nutr. 27, 560–567. 24. Hui, D.Y., Hayakawa, K., and Oizumi, J. (1993) Lipoamidase Activity in Normal and Mutagenized Pancreatic Cholesterol Esterase (Bile Salt-Stimulated Lipase), Biochem. J. 291, 65–69.

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25. Lundgren, P., Nilsson, Å., and Duan, R.D. (2001) Distribution and Properties of Neutral Ceramidase Activity in Rat Intestinal Tract, Dig. Dis. Sci. 46, 765–772. 26. Duan, R.D., Cheng, Y., Yang, L., Ohlsson, L., and Nilsson, Å (2001) Evidence for Specific Ceramidase Present in the Intestinal Contents of Rats and Humans, Lipids 36, 807–812. 27. Kirby, R.J., Zheng, S., Tso, P., Howles, P.N., and Hui, D.Y. (2002) Bile Salt-Stimulated Carboxyl Ester Lipase Influences Lipoprotein Assembly and Secretion in Intestine: A Process Mediated via Ceramide Hydrolysis, J. Biol. Chem. 277, 4104–4109. 28. Nyberg, L., Duan, R.D., and Nilsson, Å. (2000) A Mutual Inhibitory Effect on Absorption of Sphingomyelin and Cholesterol, J. Nutr. Biochem. 11, 244–249. 29. Eckhardt, E.R., Wang, D.Q., Donovan, J.M., and Carey, M.C. (2002) Dietary Sphingomyelin Suppresses Intestinal Cholesterol Absorption by Decreasing Thermodynamic Activity of Cholesterol Monomers, Gastroenterology 122, 948–956. 30. Moschetta, A., Frederik, P.M., Portincasa, P., Van Berge-Henegouwen, G.P., and Van Erpecum, K.J. (2002) Incorporation of Cholesterol in Sphingomyelin-Phosphatidylcholine Vesicles Has Profound Effects on Detergent-Induced Phase Transitions, J. Lipid Res. 43, 1046–1053. 31. Liu, J.J., Nilsson, Å., and Duan, R.D. (2002) Effects of Phospholipids on Sphingomyelin Hydrolysis Induced by Intestinal Alkaline Sphingomyelinase: An In Vitro Study, J. Nutr. Biochem. 11, 192–197. 32. Liu, J.J., Nilsson, Å., and Duan, R.D. (2002) In Vitro Effects of Fat, FA, and Cholesterol on Sphingomyelin Hydrolysis Induced by Rat Intestinal Alkaline Sphingomyelinase, Lipids 37, 469–474. 33. Hertervig, E., Nilsson, Å., Nyberg, L., and Duan, R.D. (1997) Alkaline Sphingomyelinase Activity Is Decreased in Human Colorectal Carcinoma, Cancer 79, 448–453. 34. Hertervig, E., Nilsson, Å., Bjork, J., Hultkrantz, R., and Duan, R.D. (1999) Familial Adenomatous Polyposis Is Associated with a Marked Decrease in Alkaline Sphingomyelinase Activity: A Key Factor to the Unrestrained Cell Proliferation? Br. J. Cancer 81, 232– 236. 35. Hertervig, E. (2000) Alkaline Sphingomyelinase. A Potential Inhibitor in Colorectal Carcinogenesis, Thesis, University of Lund. 36. Cheng, Y., Tauschel, H.D., Nilsson, Å., and Duan, R.D. (1999) Ursodeoxycholic Acid Increases the Activities of Alkaline Sphingomyelinase and Caspase-3 in the Rat Colon, Scand. J. Gastroenterol. 34, 915–920. 37. Yang, L., Mutanen, M., Cheng, Y., and Duan, R.D. (2002) Effects of Fat, Beef, and Fiber in Diets on Activities of Sphingomyelinase, Ceramidase, and Caspase-3 in Rat Colonic Mucosa, Med. Princ. Pract. 11, 150–156.

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Chapter 8

Dietary Sphingolipids in the Prevention and Treatment of Colon Cancer Eva M. Schmelz Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201

Introduction The involvement of sphingolipid metabolites in processes that regulate cell growth, differentiation, and cell death has been demonstrated in many in vitro studies using a wide spectrum of cell lines, such as fibroblasts, epithelial cells, keratinocytes, and lymphoid, leukemia, and nerve cells (this is by far not a complete list). The results from these studies suggest that exogenous sphingolipids have regulatory functions in cells that lack tight regulation, such as cancer cells. In general, transformed cells display an increase in proliferation, a decrease in apoptosis, and/or a reduction in differentiation. Both endogenous and exogenous sphingolipids are growth inhibitory and have been shown to induce apoptosis or differentiation in many cell lines. Furthermore, sphingolipid metabolites have been shown to inhibit or circumvent multidrug resistance, inhibit cell motility, and inhibit angiogenesis. These functions make sphingolipids good candidates for cancer intervention and/or cancer prevention studies.

Requirements for the Use of Sphingolipid Metabolites as Anti-Cancer Agents To test the hypothesis that exogenous sphingolipids can be used as chemotherapeutic or chemopreventive agents, the bioactive metabolites have to be delivered directly to the cancer cells. The toxicity of the metabolites and their physical and chemical properties make this a difficult task. The colon is an apparent target because throughout the intestinal tract, complex sphingolipids from foods are digested to ceramide, sphingoid bases, and possibly other metabolites (see structure of sphingolipids, Fig. 8.1). These are the same metabolites that have been shown to change cell growth, differentiation, and death in vitro. Most of the dietary sphingolipids are hydrolyzed and taken up in the upper part of the small intestine; however, a small fraction of approximately 10% reaches the colon (1). Therefore, by eating foods rich in sphingolipids, the colon is exposed to bioactive sphingolipid metabolites.

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Fig. 8.1. Structure of complex sphingolipids containing ceramide with a sphingosine base as found in most mammalian cells. As shown, more complex sphingolipids contain a head group on position 1.

The complexity of the headgroups has no effect on the hydrolysis in the intestinal tract, and all complex sphingolipids tested so far released only ceramides and free sphingoid bases, comparable both in amounts and time course (2,3). It is not known how high the concentrations of sphingolipid metabolites in the colon are after ingestion of sphingolipid-containing foods, but it seems they are low enough not to have any obvious deleterious effects to the colonic epithelium. A study by Kobayashi et al. (4) showed that even high amounts of sphingolipids in the diet (1% by weight or 100 times more than the estimated average human consumption, or 10 to 40 times the amount we have used in our rodent studies) did not affect body weight or blood lipid levels in mature rats or their offspring. However, the concentration of the sphingolipid metabolites after ingestion of dietary complex sphingolipids is high enough to affect colon tumorigenesis. The lack of serious side effects is important for establishing sphingolipids as anti-cancer agents because it sets them apart from compounds that are already in clinical trials. Some of these compounds are very effective against colon cancer, but they are also toxic to nontransformed cells and thereby cause serious side effects either in the target organs or other sites in the body.

Dietary Sphingolipids Reduce Chemically Induced Colon Cancer in Rodents The number of sphingolipid-modulated proteins and the complexity of signaling pathways complicate the attempt to explicate how dietary sphingolipids could affect tumorigenesis. We therefore used a mouse model of chemically induced colon cancer with aberrant crypt foci (ACF) as early morphological markers of colon tumorigenesis. It is thought that adenomas and adenocarcinomas will develop over time from these early lesions, making ACF a suitable model to assess the potency of dietary sphingolipids as chemotherapeutic agents. Female CF1 mice were injected with dimethylhydrazine, a colon carcinogen. After tumor initiation, they were fed complex sphingolipids (sphingomyelin, glu-

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cosylceramide, lactosylceramide, and the gangliosides GM1 and GD3) at 0.025 to 0.1% of the diet for four weeks. The estimated daily intake of sphingolipids lies at approximately at 0.01% (5); therefore, these amounts could be achieved in the human diet. All sphingolipids reduced the number of ACF significantly by 50 to 80% (Fig. 8.2) (2,6,7). This is presumably due to the release of the same metabolites into the colon after removal of the headgroups and the amid-bound fatty acids by the colonic microflora. Sphingolipids that do not contain sphingosine but a sphinganine base also reduce ACF formation (8). Sphingolipids found in plants contain mostly sphingoid bases that differ from sphingosine (found in mammalian cells, see Fig. 8.1) in the number and the location of their double bonds. However, since the suppression of ACF by sphingolipids is not dependent on the size or type of the headgroup or the appearance of the 4,5-trans double bond, it is feasible that complex sphingolipids found in all food groups could be hydrolyzed in the colon and play a role in suppression of colon cancer. These early studies were designed as late intervention studies to determine if dietary sphingolipids can suppress tumorigenesis after the initial cell damage that leads to tumor formation had been accomplished. The suppression of ACF in this model strongly suggests that dietary sphingolipids exert inhibitory functions in early stages of colon carcinogenesis. In a follow-up study (Lemmonier et al., manuscript in preparation) this experimental design was used to determine if the suppression of ACF indeed leads to a reduced tumor incidence after 40 weeks of feeding sphingolipids. In this study, the mice were fed a diet supplemented with sphingomyelin after tumor initiation for 40 weeks, while the control group received only

Fig. 8.2. Suppression of aberrant crypt foci (ACF) by dietary sphingolipids.

Sphingomyelin (SM), lactosylceramide (LacCer), glucosylceramide (GlcCer), and ganglioside GD3 (GD3) were fed at 0.1% of the diet to CF1 mice after tumor initiation.

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the semi-purified AIN 76A diet. Of the control mice, 23% developed colon tumors; feeding sphingomyelin at 0.05% of the diet reduced the tumor incidence significantly to 4% (P < 0.001). We also tested in this study if dietary sphingolipids are more effective as chemopreventive agents and fed sphingolipids before tumor initiation with the colon carcinogen. Interestingly, we found a comparable reduction in tumor incidence (6%) as in the group fed the sphingolipids after tumor initiation. There was no statistically significant difference between the treatments, suggesting that dietary sphingolipids are equally effective when administered before or after tumor initiation. It is not known if this will also be true for later stages of colon carcinogenesis or if there is a change in the cancer cells that renders them resistant to the effects of dietary sphingolipids. This is clearly an important problem that still has to be investigated.

Dietary Sphingolipids Reduce Tumor Formation in Min Mice To our knowledge, there have been no clinical trials to date that tested the efficacy of dietary sphingolipids on colon cancer in humans. However, several rodent models that closely resemble the human disease are currently available. C57Bl/6JMin/+ (Multiple Intestinal Neoplasia, Min) mice carry the same mutation of the adenomatous polyposis coli (APC) gene that is found in all patients with familial adenomatous polyposis (FAP) and 40 to 80% of sporadic colon cancer (9,10). These mice spontaneously develop multiple tumors throughout the intestinal tract. Although these tumors are mostly developed in the small intestine, the phenotype of the disease is the same as in humans (11). Since sphingolipids are digested throughout the intestinal tract and the bioactive metabolites appear in the small intestine and the colon, this is a good model to test the effect of dietary sphingolipids on APC-related tumor formation. We fed a mixture of dietary sphingolipids (at 0.1% of the diet) as they appear in milk (sphingomyelin, lactosylceramide, glucosylceramide, and ganglioside GD3) to Min mice after weaning when the mice were 5 weeks old. The study was terminated when the mice were 100 days old because most of the mice become moribund after 120 days and die due to anemia. This was again a late intervention study because some tumors have already developed at this time. Feeding the sphingolipids for 65 days to Min reduced the number of tumors significantly by 40%. Adding ceramide to this mixture but maintaining 0.1% of the diet reduced the number of tumors even further (to 50% of the control). This was seen in all regions of the intestinal tract (3). These results are very promising and suggest that the effects of dietary sphingolipids are not limited to chemically induced colon cancer but also may suppress colon cancer in humans.

Reduction of Proliferation by Exogenous Sphingolipids As mentioned previously, cancer cells exhibit an enhanced cell growth, a reduced rate of apoptosis, or a combination of both. Exogenous sphingolipids reduce cell growth and induce apoptosis in vitro. We did not find an increase of apoptosis after

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feeding sphingolipids to the carcinogen-injected CF1 mice (3). This is not due to a resistance of colonic cells to sphingolipid-induced apoptosis because incubation even with low concentrations of sphingosine (10 µM) for 30 min increased the number of apoptotic cells (E.M. Schmelz and A.H. Merrill, Jr., unpublished results). This suggests that either an induction of apoptosis in damaged or transformed cells is not a mechanism of how dietary sphingolipids suppress colon cancer, or this is an early event and no longer apparent after 4 weeks of feeding the sphingolipids. We then determined the proliferation in mice injected with the carcinogen and found an increase in the number of proliferative cells and an elevation of the area of proliferation toward the luminal side of the colon. Feeding sphingolipids for 4 weeks reduced the rate of proliferation to the levels of mice not injected with the carcinogen, presumably the normal rate of colonic cell growth. Furthermore, the number of dividing cells in the upper half of the colonic crypts was drastically reduced (2). This reduction of cell growth to normal levels may be a key event in the suppression of colon cancer by dietary sphingolipids. We do not know which signaling pathways are modulated by the sphingolipid metabolites to reduce early and late stages of colon cancer. Nonetheless, APC mutations may be a common event in both chemical carcinogenesis and the Min mouse. Increasing evidence suggests that APC mutations are not only found in adenomas and carcinomas of the colon but also in chemically induced ACF (12–14). Since the APC mutations were only found in dysplastic ACF that eventually will progress toward tumor formation and not in hyperplastic ACF that may regress (15), it is possible that APC mutations may be the determining factor of the fate of ACF toward tumor formation versus regression. Tumor formation in the Min mice is preceded by the loss of the APC wild type allele and the concomitant accumulation of cytosolic β-catenin. We found a high expression of cytosolic β-catenin in the Min mice fed the control diet. In mice that were fed sphingolipids and developed a small number of tumors, the localization of the β-catenin was located mostly at the lateral membranes, the localization also found in the genetic background mice (3). This could also been seen in vitro in colon cancer cell lines that stably overexpress cytosolic β-catenin and suggests that exogenous sphingolipids may normalize β-catenin localization despite APC mutations. Since cytosolic β-catenin will translocate into the nucleus and activate transcription of proteins involved in cell growth, it is possible that the removal of cytosolic β-catenin is one way that sphingolipids reduce cell growth in cancer cells. This is the first time that an intracellular target for dietary sphingolipids has been identified, but it cannot be concluded from these data that β-catenin regulation is the only mechanism utilized by sphingolipid metabolites to suppress tumor formation in Min mice. However, given the importance of the dysregulation of β-catenin metabolism/catabolism with concomitant increase of cytosolic β-catenin not only in colon cancer but also a growing list of other cancers (prostate, breast, skin, kidney), the down-regulation of cytosolic βcatenin is clearly an important event and may directly contribute to the anti-cancer effects of dietary sphingolipids. This clearly warrants further investigation.

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Future Directions Our studies clearly demonstrate a beneficial effect of dietary sphingolipids on colon carcinogenesis. The fact that these effects are mediated by concentrations that can be achieved in the human diet and that are nontoxic (at least in rodent models), probably due to limited digestion of complex sphingolipids (16), sets sphingolipids apart from other chemopreventive and chemotherapeutic agents. These often have toxic side effects and require continuous administration. However, more research is necessary to determine the effects of dietary sphingolipids on human colon cancer and elucidate the mechanisms. The growing list of intracellular targets of sphingolipids as shown in Fig. 8.3 will direct these studies. These may be modulated as depicted here by regulating

Fig. 8.3. Possible signaling pathways modulated by dietary sphingolipids

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the MAPK (Mitogen activated protein kinase) or PI-3K (phosphoinositide 3-kinase) pathways directly or via modulation of growth factor receptors such as the epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor (PDGFR). A direct activation/inactivation of key players in these pathways is also feasible, as well as an indirect regulation via known targets of sphingolipid metabolites such as protein kinase C (PKC) isoforms or protein phosphatase 2A (PP2A). We hope that the elucidation of these pathways will lead to a new concept of colon cancer prevention and/or treatment and help to establish sphingolipids as chemotherapeutic and chemopreventive agents. References 1. Schmelz, E.M., Crall, K.L., LaRocque, R., Dillehay, D.L., and Merrill, A.H., Jr. (1994) Uptake and Metabolism of Sphingolipids in Isolated Intestinal Loops of Mice, J. Nutr. 124, 702–712. 2. Schmelz, E.M., Sullards, M.C., Dillehay, D.L., and Merrill, A.H., Jr. (2000) Inhibition of Colonic Cell Proliferation and Aberrant Crypt Foci Formation by Dairy Glycosphingolipids in 1,2-Dimethylhydrazine-Treated CF1 Mice, J. Nutr. 130, 522– 527. 3. Schmelz, E.M., Roberts, P.C., Kustin, E.M., Lemonnier, L.A., Sullards, M.C., Dillehay, D.L., and Merrill, A.H., Jr. (2001) Modulation of β-Catenin Localization and Intestinal Tumorigenesis in Vitro and in Vivo by Sphingolipids, Cancer Res. 61, 6723–6729. 4. Kobayashi, T., Shimizugawa, T., Osakabe, T., Watanabe, S., and Okuyama, H. (1997) A Long-Term Feeding of Sphingolipids Affected the Level of Plasma Cholesterol and Hepatic Triacylglycerol but Not Tissue Phospholipids and Sphingolipids, Nutr. Res. 17, 111–114. 5. Vesper, H., Schmelz, E.M., Nikolova-Karakashian, M., Dillehay, D.L., Lynch, D.V., and Merrill, A.H., Jr. (1999) Sphingolipids in Food and the Emerging Importance of Sphingolipids to Nutrition, J. Nutr. 129, 1239–1250. 6. Dillehay, D.L., Webb, S.K., Schmelz, E.M., and Merrill, A.H., Jr. (1994) Dietary Sphingomyelin Inhibits 1,2-Dimethylhydrazine-Induced Colon Cancer in CF1 Mice, J. Nutr. 124, 615–620. 7. Schmelz, E.M., Dillehay, D.L., Webb, S.K., Reiter, A., Adams, J., and Merrill, A.H., Jr. (1996) Sphingomyelin Consumption Suppresses Aberrant Colonic Crypt Foci and Increases the Proportion of Adenomas Versus Adenocarcinomas in CF1 Mice Treated with 1,2-Dimethylhydrazine: Implications for Dietary Sphingolipids and Colon Carcinogenesis, Cancer Res. 56, 4936–4941. 8. Schmelz, E.M., Bushnev, A.B., Dillehay, D.L., Liotta, D.C., and Merrill, A.H., Jr. (1997) Suppression of Aberrant Colonic Crypt Foci by Synthetic Sphingomyelins with Saturated or Unsaturated Sphingoid Base Backbones, Nutr. Cancer 28, 81–85. 9. Nagase, H., and Nakamura, Y. (1993) Mutations of the APC (adenomatous polyposis coli) Gene, Hum. Mut. 2, 425–434. 10. Sparks, A.B., Morin, P.J., and Kinzler, K.W. (1998) Mutational Analysis of the APC/β Catenin/TCF Pathway in Colorectal Cancer, Cancer Res. 58, 1130–1134. 11. Kinzler, K.W., and Vogelstein, B. (1996) Lessons from Hereditary Colon Cancer, Cell 87, 159–170.

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12. De Fillipo, C., Caderni, G., Bazzicalupo, M., Briani, A., Fazi, M., and Dolara, P. (1998) Mutations of the APC Gene in Experimental Colorectal Carcinogenesis Induced by Azoxymethane in F344 Rats, Br. J. Cancer 77, 2148–2152. 13. Malzman, T., Whittington, J., Driggers, L., Stephens, J., and Ahnen, D. (1997) AOMInduced Mouse Colon Tumors Do Not Express Full-Length APC, Carcinogenesis 18, 435–439. 14. Ochiai, M., Ubagai, T., Kawamori, T., Imai, H., Sugimura, T., and Nakagama, H. (2001) High Susceptibility of Scid Mice to Colon Carcinogenesis Induced by Azoxymethane Indicates a Possible Caretaker Role for DNA-Dependent Kinase, Carcinogenesis 22, 1551–1555. 15. Jen, J., Powell, S.M., Papdopoulos, N., Smith, K.J., Hamilton, S.R., Vogelstein, B., and Kinzler, K.W. (1994) Molecular Determinants of Dysplasia in Colorectal Lesions, Cancer Res. 54, 5523–5526. 16. Nyberg, L., Nilsson, Å., Lundgren, P., and Duan, R.-D. (1997) Localization and Capacity of Sphingomyelin Digestion in the Rat Intestinal Tract, J. Nutr. Biochem. 8, 112–118.

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Chapter 9

Compositional Analysis of Complex Mixtures of Sphingolipids by Liquid Chromatography– Tandem Mass Spectrometry M. Cameron Sullards, Elaine Wang, and Alfred H. Merrill, Jr. School of Chemistry and Biochemistry, and School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332

Introduction Since the discovery of “sphingosin” by Thudichum (1), sphingolipids have been shown to be a broad and diverse class of molecules. Sphingolipids help define structural properties of membranes and lipoproteins as well as participate in a wide variety of biological functions, including cell growth, development, and death (apoptosis) through participation in signaling, membrane trafficking, and other behaviors (2). The bioactivities of sphingolipids are of great interest and depend on their structure. For example, the free sphingoid base sphingosine (trans-4-sphingenine, So) inhibits protein kinase C in vitro and in intact cells and has been found to affect over 100 different cellular systems (2). Ceramide (Cer) is a building block in the biosynthesis of more complex sphingolipids and an intermediate in degradation, but is also a lipid second messenger (3,4) via activation of protein phosphatase(s), kinase(s), and other signal transduction pathways. Likewise, complex sphingolipids having differing polar headgroups are involved in a myriad of biological functions (5–9). Commonly used methods of identification and quantification of sphingolipids, such as gas chromatography (GC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and enzyme or antibody-based assays, do not readily provide complete details regarding the molecular species being anaThe sphingoid bases described herein share the core structure 2-amino, 1, 3-dihydroxy-octadecane (named sphinganine or d18:0, where “d” denotes a dihydroxy base) that may be substituted with an additional hydroxyl group at position 4 (4-hydroxy-sphinganine or t18:0, where “t” denotes a trihydroxy base) and/or has double bonds at positions 4 (commonly called sphingosine or trans-4-sphingenine, d18:1∆4), 8 (8-sphingenine or d18:1∆8), 4 and 8 (4,8-sphingadiene or d18:2∆4,∆8), or at position 8 with a hydroxyl at position 4 (4-hydroxy-8-sphingenine or t18:1∆8). The fatty acid portion amide linked to the sphingoid base in complex sphingolipids may, for example, contain no other substituents and may be denoted as c16:0. However, those which contain an α-hydroxy group are referred to as h16:0.

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lyzed. For example, they require extraction, hydrolysis, and chemical modification, which are not only laborious but also may introduce artifacts into the analyses. More importantly, these methods do not yield critical information about the pairing of specific head groups with various combinations of long-chain bases and fatty acids in the intact molecular species. Electron impact mass spectrometry (EIMS) was initially used to elucidate the structures of ceramides (10,11) and neutral glycolipid species (12,13). These early experiments permitted the analysis of sphingolipids as intact molecular species and yielded diagnostic fragmentations that distinguished isomeric sphingolipid structures (14–17). Use of EIMS, however, required derivitization of the sphingolipids to trimethylsilyl or permethyl ethers to increase their volatility to enter the gas phase for ionization. Furthermore, since EIMS is a high-energy ionization method that causes extensive fragmentation of molecular species, observation of molecular ions of larger species is precluded. Additionally, it is difficult to resolve complex mixtures of sphingolipids containing various head group, sphingoid base, and fatty acid combinations. The development of “softer” ionization techniques, such as fast atom bombardment (FAB) and liquid secondary ionization mass spectrometry (LSIMS) facilitated the generation of intact molecular ions without prior derivitization, which yielded numerous sphingolipid species in the resulting mass spectra. Structural information regarding individual molecular species could then be obtained using multiple stages of mass analysis (i.e., tandem mass spectrometry, MS-MS) to select an ion of interest, collisionally dissociate it, and detect the subsequently formed product ions. When either (M + H)+ or (M – H)– precursor ions fragment, they do so in specific positions to yield product ions distinctive of the head group, sphingoid base, or fatty acid (18). Additionally, mass spectral fragmentation pathways may be directed with ionization via alkali metal ions [(M + Me+)+ in which Me+ = Li, Na, K, Rb, or Cs] to improve structural determination of sphingolipids (19,20). These collections of work resulted in a system of nomenclature descriptive of the product ions observed (Fig. 9.1), and have been thoroughly reviewed elsewhere (18). FABMS and LSIMS do, however, have limitations with regard to sphingolipid analysis. One such drawback is the requirement of a matrix to solubilize and subsequently ionize the analyte. This results in a significant degree of background chemical noise arising from the matrix, which serves to limit sensitivity. Additionally, quantitating samples can be difficult as analyte is often introduced via a solid probe. Dynamic FAB (21,22) and LSIMS attempt to address these issues by continuously infusing a mixture of solvent and matrix onto the probe tip. Unfortunately, even when diluted by a factor of ~100, chemical noise from the matrix still presents a problem. Additionally, instrument vacuum systems severely limit the rate of sample introduction (≤10 µL min–1) with respect to typical HPLC flow rates (≥200 µL min–1). Thus, the flow from the HPLC must be split, reducing the amount of sample reaching the probe tip. In recent years, the field of mass spectrometry has undergone a revolution with the advent of electrospray ionization (ESI). This technique allows an analyte in solution to be infused directly into a specialized ion source, which consists of a metalized

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Fig. 9.1. Mass spectrometry nomenclature for cleavages of complex sphingolipids.

needle held at a high potential. At the needle tip, highly charged droplets containing both solvent and analyte are formed. The charged droplets are subsequently drawn into the orifice of the mass spectrometer via a potential difference. In the transition from atmosphere to vacuum, neutral solvent molecules are pumped away, resulting in the soft ionization of the analyte. Initially, ESI required low flow rates and multiple stages of pumping to remove excess solvent. This resulted in greatly reduced chemical noise and yielded sensitivity orders of magnitude lower than FAB. Subsequent improvements in pumping speed, heated ion sources, and the addition of heated nebulizing gas have allowed flow rates to increase greatly. Now it is possible to directly connect eluents from liquid chromatography (LC) columns to a mass spectrometer without splitting for the direct detection of complex biological materials. LC-MS/MS is emerging as a method of choice to obtain unambiguous data with regard to identification, structure elucidation, and quantitation of specific sphingolipid species. Recently, there have been some reports regarding the analyses of sphingolipids in biological materials by nano- (23) and LC-MS/MS (24). This is often accomplished by using known structure specific fragmentations to identify classes of sphingolipids (e.g., SM or Cer) present in complex mixtures. Quantitation was determined by addition of short carbon chain internal standards. However, details regarding kinetics of dissociation, duty cycle, and gas phase

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basicity were not addressed. These issues are of critical importance with regard to accurate quantitation of these species. Therefore, any quantitative data reported that does not take these factors into consideration must be considered suspect. In this article we describe the analytical methods for extraction, identification, and quantitation of sphingolipids using state-of-the-art LC-MS/MS. Biological samples are extracted and processed via liquid/liquid extraction techniques. Precursor ion scans are used to distinguish various molecular species of sphingolipids in crude extracts by their unique molecular decomposition products. Specific headgroup, sphingoid base, and fatty acid chain combinations can be readily determined from complex mixtures. Quantitation is achieved by using multiple reaction monitoring (MRM) experiments in conjunction with HPLC. MRM experiments provide much greater sensitivity by increasing duty cycle and provide lower limits of detection by monitoring a specific precursor/product ion pair. This yields the detection of dozens of individual molecular species under optimal ion formation and decomposition conditions for each species, thus eliminating any kinetic discrimination.

Experimental Methods Internal Standards Sphingolipid internal standards were purchased from Avanti Polar Lipids (Alabaster, AL). Internal Standards used for quantitation of complex sphingolipids contained 0.5 nmol each c12 SM, c8 GlcCer, and c12 Cer in ethanol. The internal standards used in the quantitation of free sphingoid bases contained 0.25 nmol each c20 So, c20 Sa, and c17 SoP in ethanol (solubility of stock solutions of sphingoid base-1-phosphates is increased by addition of dimethylamine if needed). Variant sphingoid base standards were added, typically 0.1 nmol each, as required for quantitation of Lyso-SM, Psy, Nmethyl So, N, N-dimethyl So, and N, N, N-trimethyl So in ethanol. Cellular Extracts Approximately 1 to 10 × 106 cells (ca 1 mg of protein) were transferred to 13 × 100 mm glass test tubes. Next, 0.5 mL of methanol, 0.25 mL of chloroform, and the internal standards were added. This mixture was then sonicated and incubated overnight at 48°C in heating block. The mixture was then allowed to cool, and 75 µL of 1 KOH in methanol was added. This was then sonicated and incubated for 2 h at 37°C. Afterward, the reaction mix was neutralized with 3 µL of glacial acetic acid. The test tubes were then centrifuged to settle insoluble material, and a 400µL aliquo was removed and dried for free sphingoid base analysis. This fraction was later reconstituted in 400 µL of methanol/water/acetic acid (50:50:1, by vol). The remaining reaction solution was transferred to clean test tubes and extracted by adding 1 mL of chloroform and 2 mL of water, mixed and centrifuged. The upper layer was carefully removed, leaving the interface (with some water). The lower layer (and remaining water) was then dried under reduced pressure (e.g.,

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Speed Vac). This fraction was later reconstituted in 400 µL of methanol/chloroform (25:75, vol/vol). Mass Spectrometry All experiments were performed on a PE Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo-ionspray source. Sphingolipid solutions were infused at a flow rate of 5 µL min–1. The ionspray needle was held at 5500 V while the inlet voltage (orifice) was kept low (<50 V) to minimize collisional decomposition of molecular ions prior to entry into the first quadrupole. Sphingolipid species in crude extracts were identified by precursor ion scans of unique molecular decompositions. Precursor ion spectra were acquired by scanning Q1 over a 200 u window around the estimated average molecular mass of the different sphingolipid species in 0.1 u steps with a dwell time of 1.0 ms. Typical scan ranges and associated collision energies are listed in Table 9.1. Nitrogen was used to collisionally activate precursor ion dissociation in Q2, which was offset from Q1 by differing collision energies to determine maximal formation of molecularly distinctive product ions. Q3 was then set to pass the product ions of interest to the detector. Data was acquired for a total of 5 min, resulting in the spectrum being a signal-averaged sum of approximately 150 scans. Detection of individual molecular species in the LC elution protocol was accomplished by using MRM. In these experiments Q1 was set to pass the precursor ion of interest to Q2, where it undergoes collision-induced dissociation, and Q3 was set to pass the structure-specific product ion of interest. Each individual ion dissociation pathway was optimized with regard to collision energy so that differences in rates of dissociation would not effect relative ion abundance. The dwell time for MRM transitions of each individual molecular species was 25 ms. HPLC Normal phase LC-MS/MS analysis of the complex sphingolipids was performed by taking a 10 µL aliquot of the extracted and base hydrolyzed sample and diluting TABLE 9.1 Typical Scan Ranges and Associated Collision Energiesa Species So, Sa, So-P, Sa-P Cer GlcCer SM

Scan range

Fragment m/z

Collision energy

280–480 500–700 675–875 650–850

264, 266 264, 266 264, 266 184, 000

25 eV 40 eV 50 eV 50 eV

aFragment

ions of m/z 264, 266 are indicative of a d18:1 and d18:0 sphingoid base chain, respectively. These represent the most commonly occurring sphingoid base chain length and unsaturation. Those sphingolipids, which have different chain lengths, branching, or unsaturation, will fragment to yield ions of different m/z. Thus, different precursor ion scans would be necessary to determine the corresponding molecular species. Furthermore, the scan range and collision energy will vary depending on the size and substitution of the various subspecies.

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to a final volume of 200 µL with solvent A. A total of 10 µL of this solution was injected onto a 15 cm × 2.1 mm Supelco silica column at a flow of 500 µL min–1. Solvent A was 97:2:1 CH3CN/CH3OH/CH3COOH and solvent B was 64:15:20:1 CH3OH/ H2O/BuOH/ CH3COOH both containing 5 mM ammonium acetate. In all cases the column eluent was directly infused into the turbo ionspray source, which was operated at 500°C with a 6 L min–1 nebulizing N2 gas flow. The elution protocol consisted of column pre-equilibration with 100% A for 3 min followed by sample injection, 3 min wash with 100% A, 3 min linear gradient to 100% B, and 6 min wash with 100% B. This protocol was also repeated on a 15 cm × 2.1 mm Supelco amino column. Reverse phase LC-MS/MS was used for analysis of the free sphingoid bases. A total of 50 µL of this solution was injected onto a 15 cm × 2.1 mm Supelco Discovery C18 column at a flow rate of 300 µL min–1. Solvent A was 69:30:1 H2O/CH3OH/ HCOOH and solvent B was 99:1 CH3OH/HCOOH; both contained 5 mM ammonium formate. The elution protocol consisted of column pre-equilibration with 50:50 A/B for 3 min followed by sample injection, 3 min wash with 50:50 A/B, 6 min linear gradient to 100% B, and 6 min wash with 100% B. Reverse phase LC-MS/MS analysis of soy was similarly performed by taking a 10-µL aliquot and diluting to a final volume of 200 µL with solvent A. A total of 30 µL of this solution was injected onto a 25 cm × 2.1 mm Supelco Discovery C18 column at a flow rate of 500 µL min –1. Solvent A was 41:58 H 2O/CH 3OH/ HCOOH and solvent B was 99:1 CH3OH/HCOOH; both contained 5 mM ammonium formate. The elution protocol consisted of column pre-equilibration with 30:70 A/B for 3 min followed by sample injection, 9 min wash with 30:70 A/B, 21 min linear gradient to 100% B, and 10 min wash with 100% B. Complex SL analysis was performed on a 5 cm × 2.1 mm Supelco amino column at a flow rate of 1500 µL min–1. Solvents A and B were the same, and the elution protocol consisted of column pre-equilibration with 98:2 A/B for 0.5 min followed by sample injection. The column was washed 0.6 min at 98:2 A/B, 0.2 min linear gradient to 82:18 A/B, 0.4 min hold at 82:18, A/B, and 0.8 min linear gradient to 100% B. Total run time including pre- and post-equilibration was 3 min. Free sphingoid base analysis was performed on a 5 cm × 2.1 mm Supelco Discovery C18 column at a flow rate of 1000 µL min–1. Solvent A was 41:58:1 H2O/CH3OH/HCOOH and solvent B was 99:1 CH3OH/HCOOH; both contained 5 mM ammonium formate. The elution protocol consisted of column pre-equilibration with 60:40 A/B for 0.3 min followed by sample injection, 0.6 min wash with 60:40 A/B, 1.8 min linear gradient to 100% B, and 0.6 min wash with 100% B. Total run time including pre- and post-equilibration was 3.6 min.

Results and Discussion Cellular Extracts Sphingosine (d18:1∆4) and sphinganine (d18:0) readily protonate to form (M + H)+ ions of m/z 300.3 and 302.3, respectively. Product ions scans show that they both

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dissociate primarily by neutral loss of H2O to yield carbocations. This decomposition is optimal at a collision energy of 25 eV. The single dehydration product ions are much more abundant than the double dehydration products over a range of collision energies. Furthermore, precursor ion scans of m/z 282/284 and 264/266 show that the single dehydration products yield approximately 10× more signal and suppress chemical noise to a greater extent than the double dehydration products. Optimization of ionization and collision conditions for sphingosine and sphinganine reveal two important points regarding accurate quantitation of these species. First, the free sphingoid bases are extremely susceptible to dehydration in the ion source. Decomposition of the analyte in the ion source may adversely effect any subsequent quantitation. Thus, atmospheric voltages must be kept low to avoid dissociation of (M + H)+ ions prior to mass analysis. Second, species containing a ∆4 double bond yield much more abundant dehydration products than do saturated “dihydro” species of similar abundance. The kinetic difference in reaction rate is a result of loss of the hydroxyl allylic to the double bond, which forms a stable conjugated carbocation. This difference in reaction rate requires an internal standard for both the saturated and unsaturated species so that they may be accurately quantitated. Phosphorylated sphingoid bases yield either (M + H)+ or (M – H)– ions when analyzed in positive or negative ion mode, respectively. The (M + H)+ ions of sphingosine-1-phosphate (m/z 380.3), for example, dissociate to form highly abundant product ions of m/z 264. Precursor ion scans of m/z 264, however, reveal only those phosphorylated sphingoid bases that have the specific structure d18:1 or t18:0. Saturation of the ∆4 double bond, chain length, or other structural modifications, therefore, will result in a corresponding shift in m/z of product ions of this type, as well as changes in the kinetics of dissociation. (M – H)– ions, however, dissociate to form abundant product ions of m/z 79, corresponding to PO3–. Precursor ion scans of this m/z yield all free sphingoid bases that have been phosphorylated regardless of substitution or structural modification. It should be noted that although the positive ions do not appear as revealing as the negative ions, they are often more sensitive. LC-MS/MS analysis of a cellular extract containing the free sphingoid bases reveals highly abundant sphingosine-1-phosphate, with sphingosine approximately 25% as abundant (Fig. 9.2). Sphinganine is detected in very low abundances and is ~10% of the base peak. Interestingly, sphinganine-1-phosphate is not detected. Ceramides, like the free sphingoid bases, easily protonate to form (M + H)+ species generally detected between m/z 500–700 (for d18:1 bases and c16:0-c24:0 fatty acids). It has been demonstrated that product ion scans of protonated ceramides undergo cleavage of the amide bond and dehydration to yield structurally distinctive O′′ ions (Fig. 9.1). The O′′ ions are characteristic of the sphingoid base and thus may vary in m/z depending on degree of unsaturation, chain, length, or other structural modifications of the base. For example, ceramides having d18:1, d18:0, t18:0, or d20:1 sphingoid bases will give rise to O′′ ions of m/z 264, 266, 264, and 292, respectively. Optimization of ionization conditions for ceramides showed that they are also susceptible to dehydration in the ion source. Therefore, atmospheric voltages must

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Ion current (cps)

Time (min) Fig. 9.2. High-performance liquid chromatography–tandem mass spectrometry extracted ion chromatogram of the free sphingoid bases sphingosine (black), sphingosine-1-phosphate (green), and sphinganine (red) from cells.

be kept low to avoid dissociation of (M + H)+ ions prior to mass analysis in order to yield accurate quantitative results. Likewise, ceramides containing a ∆4 double bond yield much more abundant O′′ ions (m/z 264) than do dihydro species (m/z 266) at similar concentrations. As before, the difference in reaction rate requires an internal standard for each species so that they may be quantitated accurately. The optimal collision energy for O′′ ion formation from ceramides is variable and directly proportional proportional to its degrees of freedom. For example, a small c2 ceramide (d18:1/c2:0) has an optimal collision energy of 25 eV, whereas 45 eV is optimal for a large ceramide (e.g., d18:1/c24:0). Thus, a precursor ion scan performed at 25 eV will yield different relative ion abundances than one performed at 45 eV. It is for this reason that precursor ion scans do not provide accurate quantitation of ceramides, even when internal standards are used, but rather reflect the kinetic differences in dissociation related to the mass of the ions. MRM scans, however, allow the collision energy to be optimized for each individual species monitored and are, therefore ideal for providing accurate quantitative data. Precursor ion scans of m/z 264 revealed ceramides having m/z 538.7, 622.8, 648.9, and 650.9 in the complex sphingolipid extract fraction of the extracted cells (data not shown). These ions correspond to ceramides having a d18:1 sphingoid base and c16:0, c22:0, c24:1, and c24:0 fatty acids, respectively. No other

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ceramide species were detected in precursor ion scans of m/z 266, 292, or 294 corresponding to d18:0, d20:1, and d20:0 sphingoid bases, respectively. The (M + H)+ ions of glucosylceramides are usually detected in the range m/z 675–875. It has been previously shown that these ions dissociate via two pathways. At low collision energies bond cleavage occurs at the glycosidic linkage. This results in the sugar head group being lost as a neutral species with charge retention on the remaining ceramide moiety forming the Yo/Zo type ions (Fig. 9.1). At higher collision energies both the sugar headgroup and the fatty acid acyl chain are cleaved with the charge retained on the dehydrated sphingoid base resulting in N′′ ions (Fig. 9.1). (Note: The N′′ ions are structurally identical to the O′′ ions of ceramides. The difference in nomenclature is the result of the glucosylceramides having a headgroup other than a hydrogen atom.) Precursor ions scans of m/z revealed GlcCer with m/z 700.5, 728.9, 784.6, 810.6, and 812.8 in the complex sphingolipid extract fraction of the cellular extract. These ions correspond to GlcCer with a d18:1 sphingoid base and c16:0, c18:0, c22:0, c24:1, and c24:0 fatty acids, respectively. No other GlcCer species were detected in precursor ion scans of m/z 266, 292, or 294 corresponding to d18:0, d20:1, and d20:0 sphingoid bases, respectively. Ionization conditions required for formation of (M + H)+ ions of glucosylceramides were not as sensitive as with either free sphingoid bases or ceramides. Dehydration of these ions in the source occurred only at high voltages. It was again observed that there were kinetic differences in the formation of N′′ ions depending on the degree and position of saturation in the sphingoid base. Likewise, the collision nergy required to maximize formation of N′′ ions from various glucosylceramides is directly proportional to degree of freedom. Accurate quantitation of these species can therefore be acquired using internal standards with the same sphingoid base backbone(s) as the analyte and using MRM scans individually optimized for each glucosylceramide present. Sphingomyelin forms highly abundant (M + H)+ ions typically between m/z 650–850. These ions are distinctive from the other sphingolipids studied for several reasons. First, they will have odd masses as a result of having an even number of nitrogen atoms and being an even electron ion (nitrogen rule). Second, upon collisional activation the phosphorylcholine headgroup is cleaved with the charge retained on this moiety yielding highly abundant and structurally distinctive C ions of m/z 184 (Fig. 9.1). Precursor ion scans of m/z 184 revealed SM with m/z 675.7, 689.4, 703.8, 717.7, 731.9, 759.9, and 787.7, 813.9, and 815.8 in the complex sphingolipid extract fraction of the cellular extract (Fig. 9.3). These ions correspond to SM with a d18:1 sphingoid base and c14:0, c15:0, c16:0, c17:0, c18:0, c20:0, c22:0, c24:1, and c24:0 fatty acids, respectively. No other SM species were detected. Optimization of ionization conditions for sphingomyelin shows little propensity for decomposition in the ion source. Dissociation of these ions prior to mass analysis is observed only at the highest potential differences. As with the other sphingolipids, optimal collision energy for formation of C ions is mass dependent.

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Relative ion abundance

m/z Fig. 9.3. A precursor ion spectrum of m/z 184.4 over the range m/z 650–850 that shows sphingomyelin in NIH-3T3 cells that have a d18:1 sphingoid base. (The various amidelinked fatty acids are labeled in the spectrum.)

However, we found no difference in the relative abundance of C ions for sphingomyelins that contain a saturated sphingoid base or a ∆4 double bond. This indicates that a single internal standard can be used in conjunction with inidividually optimized MRM scans to accurately quantitate these species. Precursor ion scans identified specific ceramide, glucosylceramide, and sphingomyelin species present in the cellular extracts. This provided the individual precursor/product ion m/z values, which were used as the MRM transitions to be detected. This allows each individual molecular species to be detected at optimized ionization and dissociation conditions. Thus, the MS/MS method described here may be coupled with liquid chromatography to provide a rapid, robust, and sensitive quantitative analysis of complex mixtures of sphingolipids. LC-MS/MS was performed on the complex SL-containing fraction of the cellular extract (Fig. 9.4). The results show that Cer, GlcCer, and SM are readily detected. Additionally, the order of elution of the various species is from hydrophobic to hydrophilic. Examination of the extracted ion chromatograms reveals that all chain length variants of a given species elute together, which makes quantitation straightforward. Precursor ion scans of m/z 262 were performed on commercially available soy GlcCer extract. The resulting spectrum, as expected, showed that the predominant GlcCer species contained a d18:2∆4,∆8, sphingoid base and an h16:0 fatty acyl chain at m/z 714.7. GlcCer species containing this same sphingoid base backbone were detect-

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Relative ion abundance

Time (min) Fig. 9.4. A high-performance liquid chromatography–tandem mass spectrometry total ion chromatogram of endogenous levels of the complex sphingoid bases ceramide (Cer), glucosylceramide (GlcCer), lactosylceramide (LacCer), and sphingomyelin (SM) from a cellular extraction.

ed at m/z 686.6, 798.7, and 826.8 and correspond to h14:0, h22:0, and h24:0 fatty acyl chains, respectively. Species having a t18:2∆8 sphingoid base with either h22:0 or h24:0 fatty acyl chains were detected at m/z 816.8 and 845.0, respectively. Subsequent LC-MS/MS analysis of the soy GlcCer extract by reverse phase revealed several interesting characteristics. As expected, the predominant GlcCer detected was the d18:2∆4,∆8/h16:0 species (Fig. 9.5). Interestingly, the extracted ion chromatogram for this MRM transition was observed to split into two peaks, indicating that there are two structural isoforms of this species. Analysis by NMR revealed that the ∆8 double bond was either a cis or trans isomer in a ratio of 35:65, respectively. Calculating the area under the peaks of the extracted ion chromatogram from the LC-MS/MS yields a 38:62 ratio for this species.

Conclusions Complex mixtures of sphingolipids are readily obtained from biological matrices using a liquid/liquid extraction technique. Identification of specific headgroup, sphingoid base, and fatty acid chain is accomplished via precursor ion scans. Ionization and dissociation conditions are then optimized to provide maximum signal intensity and minimal degradation of the analyte. MRM in conjunction with HPLC is used for quantitation. MRM experiments provide much greater sensitivity and lower limits of detection by monitoring a specific precursor/product ion pair.

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Relative ion abundance

Time (min) Fig. 9.5. A high-performance liquid chromatography–tandem mass spectrometry extracted ion chromatogram of glucosylceramide (GlcCer) from soybean. (Amide-linked fatty acids are labeled in the spectrum.)

This yields detection of dozens of individual molecular species under optimal ion formation and decomposition conditions for each, thus eliminating any discrimination that arises from kinetics of dissociation. Acknowledgments This work was supported by NIH grant # GM24856 and the Georgia Research Alliance.

References 1. Thudicuchum, J.L.W. (1884) A Treatise on the Chemical Constitution of Brain, Bailliere, Tindall, and Cox, London. 2. Merrill, A.H., Jr., Liotta, D.C., and Riley, R.T. (1996) in Handbook of Lipid Research, Volume 8: Lipid Second Messengers, Bell, R.M., Exton, J.H., and Prescott, J.M., Chapter 6, p. 205, Plenum Press, New York, 1996. 3. Hanun, Y.A. (1994) The Sphingomyelin Cycle and the Second Messenger Function of Ceramide, J. Biol. Chem. 269, 3125. 4. Kolesnick, R.N., and Kronke, M. (1998) Regulation of Ceramide Production and Apoptosis, Annu. Rev. Physiol. 60, 643. 5. Barenholz, Y., and Thompson, T.E. (1980) Sphingomyelins in Bilayers and Biological Membranes, Biochim. Biophys. Acta 604, 129. 6. Hakomori, S.I. (1991) Bifunctional Role of Glycosphingolipids. Modulators for Transmembrane Signaling and Mediators for Cellular Interactions, J. Biol. Chem. 265, 18713.

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7. Spiegel, S., and Merrill, A.H., Jr. (1996) Sphingolipid Metabolism and Cell Growth Regulation, FASEB J. 10, 1388. 8. Harder, T., and Simons, K. (1997) Caveolae, DIGs, and the Dynamics of SphingolipidCholesterol Microdomains, Curr. Opin. Cell Biol. 9, 534. 9. Riboni, L., Viani, P., Bassi, R., Prinetti, A., and Tettamanti, G. (1997) The Role of Sphingolipids in the Process of Signal Transduction, Prog. Lipid Res. 36, 153. 10. Samuelsson, B., and Samuelsson, K. (1968) Gas–Liquid Chromatographic Separation of Ceramides as Di-O-trimethylsilyl Ether Derivatives, Biochem. Biophys. Acta 164, 421. 11. Samuelsson, B., and Samuelsson, K. (1969) Gas–Liquid Chromatography Mass Spectrometry of Synthetic Ceramides , J. Lipid Res. 10, 41. 12. Samuelsson, K., and Samuelsson, B. (1969) Gas-Liquid Chromatography Mass Spectrometry of Cerebrosides as Trimethylsilyl Ether Derivatives, Biochem. Biophys. Res. Commun. 37, 15. 13. Sweeley, C.C., and Dawson, G. (1969) Determination of Glycosphingolipid Structures by Mass Spectrometry, Biochem. Biophys. Res. Commun. 37, 6. 14. Samuelsson, K., and Samuelsson, B. (1970) Gas Chromatographic and Mass Spectrometric Studies of Synthetic and Naturally Occurring Ceramides, Chem. Phys. Lipids 5, 44. 15. Hammarstrom, S., Samuelsson, B., and Samuelsson, K. (1970) Gas–Liquid Chromatography Mass Spectrometry of Synthetic Ceramides containing 2-hydroxy Acids, J. Lipid Res. 11, 150. 16. Hammarstrom, S. (1970) Gas-Liquid Chromatography-Mass Spectrometry of Synthetic Ceramides Containing Phytosphingosine, J. Lipid Res. 11, 175. 17. Hayashi, A., and Matsura, F. (1973) 2-hydroxy Fatty Acid- and Phytosphingosine-containing Ceramide 2-N-methylaminoethyl-phosphonate from Turbo Cornutus, Chem. Phys. Lipids 10, 51. 18. Adams, J., and Ann, Q. (1993) Structure Determination of Sphingolipids by Mass Spectrometry, Mass Spectrom. Rev. 12, 51. 19. Ann, Q., and Adams, J. (1992) Structure Determination of Ceramides and Neutral Glycosphingolipids by Collisional Activation of (M + Li)+ Ions, J. Am. Soc. Mass Spectrom. 3, 260. 20. Ann, Q., and Adams, J. (1993) Structure-Specific Collision-Induced Fragmentations of Ceramides Cationized with Alkali-Metal Ions, Anal. Chem. 65, 7. 21. Suzuki, M., Sekine, M., Yamakawa, T., and Suzuki, A. (1989) High Performance Liquid Chromatography-Mass Spectrometry of Glycosphingolipids: I. Structural Characterization of Molecular Species of GlcCer and IV3Bgal-Gb4Cer, J. Biochem. 105, 829. 22. Suzuki, M., Sekine, M., Yamakawa, T., and Suzuki, A. (1990) High Performance Liquid Chromatography-Mass Spectrometry of Glycosphingolipids: II. Application to Neutral Glycolipids and Monosialogangliosides, J. Biochem. 108, 92. 23. Brugger, B., Erben, G., Sandhoff, R., Wieland, F.T., and Lehmann, W.D. (1997) Quantitative Analysis of Biological Membrane Lipids at the Low Picomole Level by Nanoelectrospray Ionization Tandem Mass Spectrometry, Proc. Natl. Acad. Sci. U.S.A. 94, 2339. 24. Mano, N., Oda, Y., Yamada, K., Asakawa, N., and Katayama, K. (1997) Simultaneous Quantitative Determination Method for Sphingolipid Metabolites by Liquid Chromatography/Ionspray Ionization Tandem Mass Spectrometry, Anal. Biochem. 244, 291.

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Chapter 10

Role of Dietary Gangliosides in Early Infancy Ricardo Ruedaa, Enrique Vázqueza, and Angel Gilb aR&D

Department, Ross Products Division, Abbott Laboratories, Camino de Purchil 68, Granada, Spain bDepartment of Biochemistry and Molecular Biology, University of Granada, Campus de Cartuja, 18071 Granada, Spain

Introduction Gangliosides are glycosphingolipids consisting of a hydrophobic ceramide and a hydrophilic oligosaccharide chain bearing one or more sialic-acid residues in addition to a number of sugars, namely, glucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine (1). Ceramide is an N-acylsphingosine in which the acyl residue is usually a saturated fatty acid with a chain length greater than 14 carbons; C14 to C18 predominate in certain sources and C20 to C26 in others (2). Although gangliosides were first detected in the brain, it is possible to find them in almost all vertebrate tissues and body fluids (3). The nomenclature developed by Svennerholm (4) continues to be the most commonly used because of its rationality, simplicity, and ease of remembering. The letter G is common to all gangliosides; this letter is followed by a Latin letter initial, M, D, T, Q, P, H, or S, corresponding to one, two, three, four, five, or exceptionally, six or seven sialic-acid residues present in the molecule. Thus, they are called monosialogangliosides, disialogangliosides, trisialogangliosides, etc. The two letters are followed by a subindex figure corresponding to a different number of sugar residues in the oligosaccharide moiety (1: four residues; 2: three residues; 3: two residues, and 4: one residue). An additional subindex letter (a, b, c, or d) indicates the pathway by which the molecule was biosynthetized. Thus, the abbreviation GD1b corresponds to a disialoganglioside different from GD1a by the position of the two sialic-acid residues within the molecule. The Nomenclature Commission of the International Union of Biochemistry has established a more detailed but more complex ganglioside nomenclature (5). The position of the sugar residue with respect to the ceramide is indicated by a Roman numeral (I to IV), whereas the position of the bond of the first sugar with the following residue is indicated by an exponent of the Roman numeral written as an Arabic numeral. Variations in sialic-acid structures also contribute to the diversity in gangliosides. The sialic-acid residues in gangliosides are present either as N-acetyl-neuraminic acid (NeuAc) or N-glycolyl-neuraminic acid (NeuGc). Occasionally, both types of sialic acid are present in the same structure.

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Localization and Biological Functions of Gangliosides Gangliosides are widely distributed in most vertebrate tissues. The ganglioside pattern in mammals is quite similar with respect to the main individual components. Within each species the ganglioside profile may vary from one tissue to another, and this could be related to a different function for each individual ganglioside. Gangliosides are especially abundant in neural tissues. Cerebral grey matter contains considerably higher concentrations of gangliosides than does white matter or peripheral nervous tissues. The major gangliosides in the brain of higher vertebrates are GM1, GD1a, GD1b, and GT1b, which account for 80–90% of the total gangliosides (6). Primate and avian species possess an additional ganglioside, GM4, which is particularly abundant in white matter (3). Gangliosides are preferentially located in the surface of cell membranes with the hydrophylic portion oriented to the outward cell environment. In the nervous system, gangliosides are constituents of the synaptic plasma membranes and synaptic vesicles (7). There have also been several reports on the soluble cytosolic pool of gangliosides in mammalian brains, although the concentration in this pool is extremely low (3). Brain-ganglioside concentration and distribution change significantly during development. Thus, a general increase in the total ganglioside concentration and the predominance of GM 3 and GD 3 during early embryonic ages have been described; however, at later ages the ganglio series of gangliosides predominates over the rest (3). The dynamic changes in ganglioside composition observed in cell proliferation and maturation are probably related to specific ganglioside functions. Gangliosides have also been isolated from other neural tissues (8,9) and body fluids, such as plasma (10), amniotic fluid (11), and milk (12). Cell behavior, comprising communication, growth and differentiation regulation, programmed cell death, immune response, and perhaps the development of malign tumors, is mediated mainly by the cell surface and more specifically by branched-chain sugar molecules universally present in plasma-cell membranes (13). These functions are mainly carried out by sphingolipids. The current paradigm for their action is that complex sphingolipids such as gangliosides interact with growth-factor receptors, the extracellular matrix, and neighboring cells, whereas the backbone sphingosine and other long-chain or sphingoid bases, ceramides, and sphingosine 1-phosphate activate or inhibit protein kinases or phosphatases, ion transporters, and other regulatory machinery (14). Recent studies suggest that gangliosides could be involved in the activation of T cells (15) and in the differentiation of different lymphocyte subpopulations (16,17–19). In fact, anti-GD3 antibodies deliver a potent co-stimulatory signal for antigen-induced proliferation of CD4+ T lymphocytes (20); T cell activation via GD3 results in phospholipase C (gamma) phosphorylation and Ca flux (21). These data suggest another mechanism of T-cell activation via a single, non-protein surface moiety. It is important to point out that gangliosides, as well as other sphingolipids, are present in foods. The total amounts of sphingolipids in foods vary considerably,

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ranging from a few micromoles per kilogram in the case of fruits to several millimoles per kilogram in rich sources, such as dairy products, eggs, and soybeans (22). Although nutritional requirements for sphingolipids are unknown, studies with animals suggest that sphingolipids represent a functional constituent of food (22). Taking into account this as well as the importance of milk, which is one of the main sources of gangliosides and other sphingolipids, as the main food during early infancy, the rest of the review will be devoted to describe the content and distribution of milk gangliosides as well as their functional role.

Milk Gangliosides Milk gangliosides are almost exclusively associated with the membrane fraction of the fat globule, which is derived mainly from the apical plasma membrane of the apocrine secretory cells in the lactating mammary gland (23,24). Bovine Milk Gangliosides Milk gangliosides were initially studied in bovine milk. The sialic acids of bovine milk gangliosides include both NeuAc and NeuGc (25). In most studies, GD3 was the major ganglioside of cow’s milk, and GM3 was the next most abundant. Other gangliosides amounted to no more than 20% of the total ganglioside content. Huang (26) found that bovine buttermilk is a rich source of gangliosides, GD3 and GM3 being the principal individual gangliosides. GD3 comprises 85% of the total gangliosides of buttermilk and consists of two types, one having long-chain (C22–C25) fatty acids and an equimolar proportion of C16 and C18 sphingosine bases and the other consisting mainly of C16 fatty acid and C18 sphingosine (27). Takamizawa et al. (28) reported that buttermilk contains 0.92 µmol of lipidbound sialic acid (LBSA) per gram of dry weight, 80% of which is in the form of GM3, GD3, and GT3. In addition, these authors identified a monosialoganglioside with a novel branched structure and a trisialoganglioside with the same branched oligosaccharide chain. The 9-O-acetyl form of GD3 has also been found in buttermilk (29). Finally, other O-acetyl derivatives of gangliosides have also been found, including 7-O-acetyl GD3 (1.2 mg/kg), 9-O-acetyl GD3 (22 mg/kg), and 7,9 di-Oacetyl GT3 (24 mg/kg) (30). All the above-mentioned studies were performed with mature milk. However, other works have pointed to the existence of variations in the ganglioside content and individual distribution in colostrum, transitional, and mature bovine milk. Puente et al. (31) found in Spanish-Brown cows that the ganglioside content was higher in colostrum (7.5 mg of LBSA/kg) than in transitional (2.3 mg) or mature milk (1.4 mg). The sialic acid content followed a profile similar to that of gangliosides with the highest content during the few postnatal days followed by a gradual decrease toward the end of lactation. Several changes were also found when the individual distribution of gangliosides was examined. GM 3, GD 3, and GT 3 accounted for 80–90% of the total ganglioside content, GD3 being the major single

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component (60–70%). GM3 increased from the first to the fifth day, whereas GD3 decreased during this period. However, GD3 increased from day 5 to the end of lactation as GM3 decreased. Lactational changes in the NeuGc content of bovine milk gangliosides have also been described (32). The NeuGc content of gangliosides, as well as that in total sialic acids, was high in colostrum and decreased thereafter until the end of lactation. Ganglioside and sialic-acid contents of goat milk also change during lactation (33). The highest ganglioside content appeared in colostrum (974 µg LBSA/kg) and then decreased to the end of lactation (175 µg LBSA/kg). The sialic acid content exhibited a trend similar to that of gangliosides; during early lactation sialic acid content (1147 mg/kg) was higher than in mature milk (203 mg/kg). As in cow milk, GM 3, GD 3, and GT 3 were the most abundant individual gangliosides (66–92%). Likewise, the content of GM3 decreased, whereas that of GD3 increased during lactation. The ganglioside content of sheep milk varied during lactation in a similar way to that of cow and goat milk, being high at the beginning of lactation and decreasing thereafter (34). Sheep milk had half the amount of gangliosides (expressed as LBSA) detected in goat milk and 6–7% of the ganglioside content of cow milk. The sialic acid content of sheep milk followed a trend similar to that of gangliosides, being higher in early lactation than in mature milk. Seasonal variations in the concentration of gangliosides have also been described in milk from different mammalian species: Holstein-Friesian cows, Murciana-Granadina goats, and Churra and Merina ewes (35). All species considered showed the highest ganglioside content in autumn, whereas the minimum ganglioside content was observed in summer milk for cows and goats but not for sheep. The distribution of bovine milk gangliosides, in relation with other sialoglycoconjugates, such as glycoproteins and oligosaccharides, during lactation has been recently reported. All the components showed a similar trend, with the highest values in colostrum, decreasing values in transitional and mature milks, and increasing values again in late-lactation milk (36). The same authors also described changes in ceramide moiety in bovine milk gangliosides with stage of lactation. The results pointed to a marked change in ceramide from colostrum to milk that was characterized by a dramatic decrease in saturated and the longest-chain fatty acids, as well as an increase in 18:1 and 18:2. The major long-chain base along lactation was a recently described structure, 3-ethoxy-15:0 sphinganine. According to the authors, these changes suggest differences in the fluidity of the fat globule membrane, reflecting physiological variations in cows with respect to milk production (37). Human Milk Gangliosides The content and distribution of gangliosides in human milk have been reported by several authors (38–42). Table 10.1 summarizes these findings and our own results

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Human milk samples

Pooled milk samples from 2 to 10 months postpartum

Pooled milk sample from 2 to 10 months postpartum

Individual milk samples, 2 to 390 days postpartum

Individual milk samples, 1 to 150 days postpartum

Individual milk samples, 2 to 46 days postpartum

Individual milk samples, 2 to 46 days postpartum

Reference

Laegreid et al. 1986 (Pediatr. Res. 20, 416–421)

Laegreid et al. 1986 (J. (Chromatog raphy 377, 59–67)

Takamizawa et al. 1986 (Biochim. Biophys. Acta 879, 73–77)

Rueda et al. 1995 (Biol. Chem, Hoppe Seyler 376, 723–727)

Pan & Izumi 1999 (Early Human Devel. 55, 1–8)

Pan & Izumi 2000 (Early Human Devel. 57, 25–31)

Changes in GM3 and GD3 concentration during lactation Changes in GM3 and GD3 concentration during lactation. Presence of previously unreported highly polar gangliosides in human milk, and changes in their concentration during lactation Changes in GM3 and GD3 concentration during lactation. Newly found gangliosides (no differences in percentage composition between colostrums and later milk), tentatively assumed to be gangliosides of the c-series Changes in GM3 and GD3 concentration during lactation. Newly found gangliosides (33–38% of total LBSA), tentatively assumed to be gangliosides of the c -series

Changes during (11.93–0.82 µg LBSA/g fresh LBSA/g fresh milk). Significant correlation (r = 0.5564; P = 0.0165) between ganglioside and total lipid contents in human milk 9.21 ± 1.32 µg LBSA/mL fresh milk

9.21 ± 1.32 µg LBSA/mL fresh milk

GM3 + GD3 (>95%), and traces of a monosialoganglioside close to GM2

Monosialoganglioside (74%) and diisialoganglioside (25%)

Ganglioside distribution

Changes during lactation (2.34–1.01 µmol LBSA/100 µL fresh milk)

0.33 mg/g dry fat

11 mg/L fresh milk

Ganglioside content

TABLE 10.1 Previously Published Data on the Content and Distribution of Human Milk Ganglioside

(43). Two within those studies were done with mature milk (38,39); the rest of the studies investigated samples from different periods of lactation. One of the previous studies of the ganglioside content in human mature milk (38) reported higher values than those we detected. However, the data are not comparable because in the former study, ganglioside content was corrected (multiplied by a factor of 2) to compensate for losses during the process of extraction. The ganglioside levels detected by Takamizawa and colleagues (40) were slightly higher than those detected by us (43). However, this group did not study any sample between 14 and 40 days, and we detected a significant increase in the ganglioside concentration in human milk during this period of lactation, especially between 14 and 21 days; 17 days showed the highest value (11.93 µg LBSA/g fresh milk). We also quantified the total lipid content of our milk samples and found a significant positive correlation between ganglioside and total lipid contents (43). Although this correlation was not strong, the increase in total lipid content in human milk during mid-lactation (44) may explain why we detected higher concentrations of gangliosides and total lipids in our samples from the third week of lactation. Like Takamizawa et al. (40), we observed a selective change in the relative concentrations of GM3 and GD3 between colostrum (days 1–5) and mature milk (43). The most abundant ganglioside in human milk at the beginning of lactation was GD3, whereas at the end of this period GM3 was the major ganglioside. A major finding in our studies was the detection of previously unreported highly polar gangliosides in human milk. Their high polarity suggests that they may be polysialogangliosides or complex gangliosides with branched oligosaccharide chains. Neither of these compounds has been found in earlier studies of milk gangliosides. These structurally complex gangliosides may play an important role within the mammary gland and in developing neonatal tissues, especially at the beginning of lactation. Pan and Izumi recently described changes in the ganglioside composition of human milk during lactation (41). They basically described the changes in GD3 and GM3 previously reported by us and other authors. GD3 predominated in colostrum (GD3, 42–56%; GM3, 2.22–6.5%), while GM3 increased sharply at eight days postpartum (GD3, 32.22%; GM3, 27.79%) and then increased gradually after eight days until seven weeks postpartum (GM3/GD3, 0.84–2.67). In addition, they detected four gangliosides, tentatively assumed to be gangliosides of the c-series (41). Our group studied the changes in the relative concentration of individual gangliosides in human milk from mothers delivering preterm and term infants during lactation (45). The relative content of GD3 was higher in colostrum than in mature milk and tended to be higher in preterm than in term colostrum, whereas the relative content of GM3 was higher in mature milk than in colostrum and was also higher in term than in preterm milk. As GD3 is usually detected in developing tissues, whereas GM3 is more abundant in mature tissues (3), these results suggest a

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relationship between the presence of individual gangliosides in human milk and immaturity of the mammary gland in mothers of preterm infants. The concentration and distribution of sialic acid in milk from mothers of fullterm and preterm infants at four stages of lactation (colostrum, transition, 1 month, and 3 months) was recently reported. Human milk from mothers of preterm infants contained 13–23% more sialic acid than did milk from mothers of full-term infants at three of the four lactation stages tested. Most of the sialic acid in human milk was bound to free oligosaccharides (46). Lactational changes in content and distribution of gangliosides in human milk from Spanish and Panamanian mothers delivering term newborns were also studied by our group to test the influences of different ethnic populations, dietary habits, and lifestyle (47). There were no statistically significant differences in the concentration of gangliosides between Spanish and Panamanian milk. Ganglioside content, expressed as a function of total milk lipids, tended to decrease as lactation progressed in both types of milk. There was a significant correlation between the ganglioside and total lipid contents in Panamanian milk. However, in Spanish milk, that correlation was not significant. We did not detect major differences in the relative concentration of individual gangliosides during lactation between milk from Spanish and Panamanian mothers. For both groups, GD3 was the most abundant ganglioside in colostrum, whereas the most abundant ganglioside in mature milk was GM3. Gangliosides in Infant Formulas Human and bovine milk present a different content and distribution of gangliosides (38). Since cow’s milk is used to manufacture infant formulas, the ganglioside distribution is similar in the two cases, although the absolute content is lower in formulas than in cow’s milk. Nevertheless, the content of gangliosides in human milk is higher than that found in milk formulas. According to this feature, and taking into account the potential role of human milk gangliosides, the supplementation of infant formulas with those molecules could influence neonatal physiology. Several authors have studied the composition of gangliosides and other sialoglycoconjugates in infant formulas in comparison to human and bovine milk. Sánchez-Díaz et al. (48) reported the total sialic acid and oligosaccharide, glycoprotein, and ganglioside sialic acid contents of bovine milk-based starter and follow-on formulas. Starter formulas had total sialic acid and oligosaccharide, glycoprotein, and ganglioside sialic acid contents of 36, 28, 50, and 20%, respectively, of those found in human colostrum or transitional milk. By contrast, follow-on formulas, used from 4 to 5 months of age, provided total sialic acid and oligosaccharide, glycoprotein, and ganglioside sialic acid contents similar to those supplied by mature human milk. The authors concluded that supplementation with sialic acidcontaining glycoconjugates of infant formulas recommended for the first days after delivery could be advisable when breast-feeding is not possible

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Pan and Izumi (42) recently reported the ganglioside composition of infant formulas in comparison to human and cow’s milk. Both the patterns and contents of gangliosides in human milk, cow’s milk, and infant formulas differed markedly. In human milk, the total lipid-bound sialic acid level was two times higher than those in cow’s milk and infant formulas. The major ganglioside in later human milk, GM3 (27.7%), was only a minor component in colostrum, cow’s milk, and infant formulas (3.3, 2.8. and 0.4–2.6%, respectively). GD3 represented 49.0, 61.0, and 72.4–86.6%, respectively, of the colostrum, cow’s milk, and infant formulas, compared to 31.8% of the later human milk gangliosides. The concentrations of oligosaccharide-bound, protein-bound, and free sialic acid in 21 different infant formulas were also recently described (46). The sialic acid content of most formulas was <25% of that found in mature human milk, and most of it (approximately 70%) was bound to glycoproteins.

Functional Roles of Gangliosides The role of gangliosides in human milk has not been well established. The distribution pattern of milk gangliosides selectively changes during lactation, as described above, and therefore, milk gangliosides might participate in the physiological processes that take place in the newborn development during lactation (8,9). A high concentration of GD3 has been detected in developing tissues (49) as well as in human colostrum, and this latter fact may reflect a biological role in the development of organs, such as the intestine in the neonate. However, biological studies on growth and differentiation of intestinal cells in the presence of gangliosides will be needed to test this hypothesis. As mentioned above, human milk contains a significant amount of highly polar gangliosides (43). This type of ganglioside has been detected in developing tissues (8,9). These gangliosides have also been detected in biological fluids, such as amniotic fluid (11), that are in contact with developing tissues; they may originate by being shed from these tissues (50,51). The function of these highly complex gangliosides in developing tissues is unknown, but it has been suggested that they act as mediators of specific types of cell-contact interactions during the early stages of mammalian development (52). Thus, the complex gangliosides in human milk may be shed by the lactating mammary gland and may play an important role in this organ or in developing infant tissues, particularly the small intestine, during early life. Characteristic expression of complex gangliosides during lactation in the murine mammary gland and in the milk-fat globule has been described (53). Human-milk gangliosides are also reportedly involved in the inhibition of Escherichia coli and Vibrio cholerae enterotoxins (54). This inhibitory action has been attributed to the monosialoganglioside GM1, which has been identified as the receptor for these enterotoxins (55). More recently, sialyllactose was identified as the responsible moiety for the inhibitory activity of milk on cholera toxin (56). GM1 is found in human milk only in very low concentrations, and immunological

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methods are necessary to detect it on high-performance and thin-layer chromatography plates (55). However, the concentration of GM1 is 10 times higher in human than in bovine milk (38). All these findings suggest that gangliosides play an important role as false receptors in the defense against infection during lactation. We found that the addition of gangliosides to an adapted milk formula at a concentration similar to that in human milk modifies the microbial composition of feces in preterm newborn infants. The fecal E. coli counts in preterm infants fed the ganglioside-supplemented formula were lower than that observed in infants fed the standard formula for the first month of life (Fig. 10.1); conversely, the fecal counts of bifidobacteria in the group having the ganglioside formula (Fig. 10.2) were higher, especially at 30 days of postnatal age (57). Although the exact mechanism by which dietary gangliosides reduce the fecal levels of Escherichia coli is unknown, in vitro experiments suggest that gangliosides interact with specific Escherichia coli strains (58). Gangliosides have been reported as calf and pig small-intestine receptors for K99 fimbriated enterotoxigenic E. coli (59,60). The inhibitory effects of human-milk gangliosides and their derivatives on the adhesion of enterotoxigenic and enteropathogenic E. coli to Caco-2 cells, a human intestinal carcinoma cell line, have been recently published

Postnatal age Fig. 10.1. Logarithmic Escherichia coli counts in feces of preterm newborn infants fed on milk formula (MF) and ganglioside-supplemented milk formula (GMF). Results are means ± SEM. Twenty samples were analyzed for each feeding group. Samples were collected at 3, 7 and 30 days of life. Kruskal Wallis nonparametric test was used to determine the effect of the diet as source of variation. ● P < 0.01; ● P < 0.001, with respect to MF.

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Postnatal age Fig. 10.2. Logarithmic bifidobacteria counts in feces of preterm newborn infants fed

on milk formula (MF) and ganglioside-supplemented milk formula (GMF). Results are means ± SEM. Twenty samples were analyzed for each feeding group. Samples were collected at 3, 7, and 30 days of life. Kruskal Wallis nonparametric test was used to determine the effect of the diet as source of variation. ● P < 0.05, with respect to MF.

(61). Likewise, the meconium and feces of breast-fed newborns have been reported to inhibit the adhesion of S-fimbriated E. coli to epithelial cells (62). The stronger inhibitory capacity found in meconium has been linked to the concentration of sialic acid. It was recently shown that meconium gangliosides are oncofetal gangliosides, and their structure resembles that of some human milk oligosaccharides and some buttermilk gangliosides (63). These data suggest that sialyloligosaccharides and other compounds with conjugated sialylated carbohydrates (glycoproteins and glycolipids) could function as receptor-analogous structures for bacterial adhesions. Such compounds could modify the intestinal microflora in the neonate and reduce the infectious capacity of these bacteria. Our recent data (57) suggests that colonization of bifidobacteria flora is faster in infants fed with a milk formula supplemented with gangliosides. These compounds, which are present in human milk but practically absent from milk formula, may be one of the components that promotes the growth of bifidobacteria. In fact, it has been recently reported that fortification of infant formula with N-acetylneuraminic-acid-containing substances may provide infants with a function of human milk—that is, the growth-promoting effect on bifidobacteria (64). The high content of sialic acid in breast-fed infants probably also contributes to low intestinal pH, which in turn favors bifidobacterial flora.

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Nakano et al. (65) recently reported that some sialylated compounds, in addition to inhibiting the adhesion of toxins, bacteria, and viruses to the receptors on the surface of epithelial cells, had growth-promoting effects on bifidobacteria and lactobacilli, predominantly in the intestinal flora of infants fed with human milk. The authors suggest that syalilated compounds in human milk possibly behave as a physiological component in the intestinal tract of infants to protect them against enteric infections. Another aspect that deserves special attention is the role of gangliosides as immunomodulators. Since recent studies suggest that gangliosides could be involved in the activation of T cells (15) and in the differentiation of different lymphocyte subpopulations (16,17–19), human milk gangliosides or gangliosides supplemented to infant formulas might contribute substantially to the process of proliferation, activation, and differentiation of immune cells, especially those from the intestine, in the neonate. Our group recently reported some findings related to the effect of dietary gangliosides on intestinal immunity in mice at weaning. For instance, dietary gangliosides positively modulate the percentages of Th1 and Th2 lymphocyte subsets in small intestine of mice at weaning (66). Animals fed with gangliosides showed an earlier development in the number of cytokine-secreting cells. In addition, mice fed with gangliosides showed a higher number of Th1 and Th2 cytokine-secreting lymphocytes in lamina propria and Peyer’s patch lymphocytes after 4 weeks of feeding (66). Although feeding with gangliosides modulated the percentage of both Th1 and Th2 secreting cells, when representing the ratio Th1/Th2 for lamina propria and Peyer’s patch lymphocytes, results indicated that the stimulation was higher for Th1 than for Th2 secreting cells (Fig. 10.3). These results suggest that dietary gangliosides influence the maturation process of the intestinal immune system that takes place during weaning. Another important finding was that dietary gangliosides increase the number of intestinal IgA-secreting cells (67) and the luminal content of secretory IgA in weanling mice (68). According to these results dietary gangliosides positively modulate the production and secretion of IgA at the intestinal level, which constitutes the main mechanism of defense against microorganisms entering through the gastrointestinal tract. The influence of gangliosides on in vitro intestinal lymphocyte proliferation was also recently reported by our group (69). Gangliosides elicited differential effects on intestinal lymphocyte proliferation depending on the composition and structure of individual gangliosides, their concentrations, and the type of lymphocute population. These data suggest that dietary gangliosides may have roles in the development of intestinal immunity by stimulating or inhibiting proliferative or inhibitory responses in intestinal lymphocytes during early infancy. In conclusion, dietary gangliosides may have an important role during early infancy modifying intestinal microflora and promoting the development of intestinal immunity in the neonate. However, further studies are required to clarify the mechanisms involved in these actions and the relevance of this finding to clinical outcome in neonates.

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Cytokine-secreting cells (%) Cytokine-secreting cells (%)

Feeding after weaning (d)

Feeding after weaning (d) Fig. 10.3. Ratio of percentage of Th1 (secreting IL-2 or γIFN) and Th2 (secreting IL-5 or IL-6) lymphocytes in small intestine lamina propria (LPL) and Peyer’s patches (PPL) in mice at 3, 7, 14, and 28 days after weaning fed with a Control diet (dotted bars) or a diet supplemented with gangliosides (solid bars). ● Significant differences between Control and DG for the same period of time (P < 0.05).

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References 1. Wiegandt, H. (1982) in Advances in Neurochemistry, Agranof, B.W., and Aprison, M.H., Plenum Press, New York, vol. 4, pp. 149–223 2. Cabezas, J.A., and Calvo, P. (1984) Gangliosidos, Sci. Am. (Spanish Ed) Jul, 86–95. 3. Yu, R.K., and Saito, M. (1989) in Neurobiology of Glycoconjugates, Margolis, R.V., and Margolis, R.K., Plenum Press, New York, pp.1–42. 4. Svennerholm, L. (1963) Chromatographic Separation of Human Brain Gangliosides, J. Neurochem. 10, 613–623. 5. Wiegandt, H. (1985) in Glycolipids, Wiegandt, H, Elsevier, Amsterdam, Vol. 10, pp. 199–260. 6. Ando, S., Chang, N.C., and Yu, R.K. (1978) High-Performance Thin-Layer Chromatography and Desitometric Determination of Brain Ganglioside Composition of Several Species, Anal. Biochem. 89, 437–450. 7. Leeden, R.W., and Yu, R.K. (1982) Gangliosides: Structure, Isolation, and Analysis, Methods of Enzymology 83, 139–191. 8. Rueda, R., and Gil, A. (1998) in Lipids in Infant Nutrition, Huang, Y-S, and Sinclair, A.J., AOCS Press, Champaign, Illinois, pp. 213–234. 9. Rueda, R., Maldonado, J., Narbona, E., and Gil, A. (1998) Neonatal Dietary Gangliosides, Early Hum. Dev. 53 Suppl., S135–S147. 10. Ladisch, S., and Gillard, B. (1987) Isolation and Purification of Gangliosides from Plasma, Methods Enzymol. 138, 300–306. 11. Rueda, R. Tabsh, K., and Ladisch, S. (1993) Detection of Complex Gangliosides in Human Amniotic Fluid, FEBS Lett. 328, 13–16. 12. Jensen, R.G., and Newburg, D.S. (1995) in Handbook of Milk Composition, Academic Press, New York, pp. 543–575. 13. Sharon, N., and Lis, H. (1989) Lectins as Cell Recognition Molecules, Science 246, 227–234. 14. Spiegel, S., and Merrill, A.H., Jr. (1996) Sphingolipid Metabolism and Cell Growth Regulation, FASEB J. 10, 1388–1397. 15. Yuasa, H., Scheinberg, D.A., and Houghton, A.N. (1990) Gangliosides of T Lymphocytes: Evidence for a Role in T-Cell Activation, Tissue Antigens 36, 47–56. 16. Ebel, F., Scmitt, E., Peter-Katalinic, J., Kniep, B., and Mühlradt, P.F. (1992) Gangliosides: Differentiation Markers for Murine T Helper Lymphocyte Subpopulations TH1 and TH2, Biochemistry 31, 12190–12197. 17. Nakamura, K., Suzuki, H., Hirabayashi, Y., and Suzuki, A. (1995) IV Alpha (NeuGc alpha 2-8NeuGc)-Gg4Cer is Restricted to CD4+ T Cells Producing Interleukin-2 and a Small Population of Mature Thymocytes in Mice, J. Biol. Chem. 270, 3876–3881. 18. Nashar, T.O., Webb, H.M., Eaglestone, S., Williams, N.A., and Hirst, T.R. (1996) Potent Immunogenicity of the B Subunit of Escherichia coli Heat-Labile Enterotoxin: Receptor Binding Is Essential and Induces Differential Modulation of Lymphocyte Subsets, Proc. Natl. Acad. Sci. USA 93, 226–230. 19. Taga, S., Tetaud, C., Mangeney, M., Tursz, T., and Wiels, J. (1995) Sequential Changes in Glycolipid Expression During Human B Cell Differentiation: Enzymatic Basis, Biochim. Biophys. Acta 1254, 56–65.

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20. Schlaak, J.F., Claus, C., Meyer-zum-Buschenfelde, K.H., and Dippold, W. (1995) AntiGD3 Antibodies Are Potent Activators of Human Gamma/Delta and Alpha/Beta Positive T Cells, Scand. J. Immunol. 41, 475–480. 21. Ortaldo, J.R., Mason, A.T., Longo, D.L., Beckwith, M., Creekmore, S.P., and McVicar, D.W. (1996) T Cell Activation Via the Disialoganglioside GD3: Analysis of Signal Transduction, J. Leukoc. Biol. 60, 533–539. 22. Vesper, H., Schmeiz, E.M., Nikolova-Karakashian, M.N., Dillehay, D.L., Lynch, D.V., and Merrill, A.H., Jr. (1999) Sphingolipids in Food and the Emerging Importance of Sphingolipids to Nutrition, J. Nutr. 129, 1239–1250. 23. Keenan, T.W., Huang, C.M., and Moore, D.J. (1972) Gangliosides: Non-Specific Localization in the Surface Membranes of Bovine Mammary Gland and Rat Liver, Biochim. Biophys. Res. Commun. 47, 1277–1283. 24. Keenan, T.W. (1974) Composition and Synthesis of Gangliosides in Mammary Gland and Milk of the Bovine, Biochim. Biophys. Acta 337, 255–270. 25. Bushway, A.A., and Keenan, T.W. (1978) Composition and Synthesis of Three Higher Ganglioside Analogs in Bovine Mammary Tissue, Lipids 13, 59–65. 26. Huang, R.T.C. (1973) Isolation and Characterization of the Gangliosides of Buttermilk, Biochim. Biophys. Acta 306, 82–84. 27. Hauttecoeur, B., Sonnino, S., and Ghidoni, R. (1985) Characterization of Two Molecular Species GD3 Ganglioside from Bovine Buttermilk, Biochim. Biophys. Acta 833, 303–307. 28. Takamizawa, K., Iwamori, M., Mutai, M., and Nagai, Y. (1986a) Gangliosides of Bovine Buttermilk. Isolation and Characterization of a Novel Monosialoganglioside with a New Branching Structure, J. Biol. Chem. 261, 5625–5630. 29. Bonafede, D.M., Macala, L.J., Constantine-Paton, M., and Yu, R.K. (1989) Isolation and Characterization of Ganglioside 9-O-Acetyl-GD3 from Bovine Buttermilk, Lipids 24, 680–684. 30. Ren, S., Scarsdale, J.N., Ariga, T., Zhang, Y., Klein, R.A., Hartmann, R., Kushi, Y., Egge, H., and Yu, R.K. (1992) O-Acetylated Gangliosides in Bovine Buttermilk, J. Biol. Chem. 267, 12632–12683. 31. Puente, R., García-Pardo, L.A., and Hueso, P. (1992) Gangliosides in Bovine Milk. Changes in Content and Distribution of Individual Ganglioside Levels During Lactation, Biol. Chem. Hoppe-Seyler 373, 283–288. 32. Puente, R., and Hueso, P. (1993) Lactational Changes in the N-Glycolylneuraminic Acid Content of Bovine Milk Gangliosides, Biol. Chem. Hoppe-Seyler 374, 475–478. 33. Puente, R., García-Pardo, L.A., Rueda, R., Gil, A., and Hueso, P. (1994) Changes in Ganglioside and Sialic Acid Contents of Goat Milk During Lactation, J. Dairy Sci. 77, 39–44. 34. Puente, R., García-Pardo, L.A., Rueda, R., Gil, A., and Hueso, P. (1995) Ewes’ Milk: Changes in the Contents of Ganglioside and Sialic Acid During Lactation, J. Dairy Res. 62, 651–654. 35. Puente, R., García-Pardo, L.A., Rueda, R., Gil, A., and Hueso, P. (1996) Seasonal Variations in the Concentration of Gangliosides and Sialic Acids in Milk from Different Mammalian Species, Int. Dairy Journal 6, 315–322. 36. Martín, M.J., Martín-Sosa, S., García-Pardo, L.A., and Hueso, P. (2001) Distribution of Bovine Milk Sialoglycoconjugates During Lactation, J. Dairy Sci 84, 995–1000.

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37. Martín, M.J., Martín-Sosa, S., and Hueso, P. (2001) Bovine Milk Gangliosides: Changes in Ceramide Moiety with Stage of Lactation, Lipids 36, 291–298. 38. Laegreid, A., Otnaess, A.B.K., and Fuglesang, J. (1986). Human and Bovine Milk: Comparison of Ganglioside Composition and Enterotoxin-Inhibitory Activity, Pediatr. Res. 20, 416–421. 39. Laegreid, A., and Otnaess, A.B.K. (1986) Purification of Human Milk Gangliosides by Silica Gel Chromatography and Analysis of Trifluoroacetate Derivatives by Gas Chromatography, J. Chromatogr. 377, 59–67. 40. Takamizawa, K., Iwamori, M., Mutai, M., and Nagai, Y. (1986) Selective Changes in Ganglioside of Human Milk During Lactation: A Molecular Indicator for the Period of Lactation, Biochim. Biophys. Acta 879, 73–77. 41. Pan, X.L., and Izumi, T. (1999) Chronological Changes in the Ganglioside Composition of Human Milk During Lactation, Early Hum. Dev. 55, 1–8. 42. Pan, X.L., and Izumi, T. (2000) Variation of the Ganglioside Compositions of Human Milk, Cow’s Milk, and Infant Formulas, Early Hum. Dev. 57, 25–31. 43. Rueda, R., Puente, R., Hueso, P., Maldonado, J., and Gil, A. (1995) New Data on Content and Distribution of Gangliosides in Human Milk, Biol. Chem. Hoppe-Seyler 376, 723–727. 44. Jensen, R.G. (1989) The Lipids of Human Milk, CRC Press, Boca Raton, pp. 7–23. 45. Rueda, R., García-Salmerón, J.L., Maldonado, J., and Gil, A. (1996a) Changes During Lactation in Ganglioside Distribution in Human Milk from Mothers Delivering Preterm and Term Infants, Biol. Chem. 377, 599–601. 46. Wang, B., Brand-Miller, J., McVeagh, P., and Petocz, P. (2001) Concentration and Distribution of Sialic Acid in Human Milk and Infant Formulas, Am. J. Clin. Nutr. 74, 510–515. 47. Rueda, R., Maldonado, J., and Gil, A. (1996b) Comparison of Content and Distribution of Human Milk Gangliosides from Spanish and Panamanian Mothers, Ann. Nutr. Metabolism 40, 194–201. 48. Sánchez-Díaz, A., Ruano, M.J., Lorente, F., and Hueso, P. (1997) A Critical Análisis of Total Sialic Acid and Sialoglycoconjugate Contents of Bovine Milk-Based Infant Formulas, J. Pediatr. Gastroenterol. Nutr. 24, 405–410. 49. Ando, S. (1983) Gangliosides in the Nervous System, Neurochem. Int. 5, 507–537. 50. Ladisch, S., Gillard, B., Wong, C., and Ulsh, L. (1983) Shedding and Immunoregulatory Activity of YAC-1 Lymphoma Cell Gangliosides, Cancer Res. 43, 3808–3813. 51. Li, R., and Ladisch, S. (1991) Shedding of Human Neuroblastoma Gangliosides, Biomed. Biochim. Acta 1083, 57–64. 52. Friedman S.J., Cheng, S., and Skehan, P. (1983). The Occurrence of Polysialogangliosides in a Human Trophoblast Cell Line, FEBS Lett. 152, 175–179. 53. Momoeda, M., Momoeda, K., Takamizawa, K., Matsuzawa, A., Hanaoka, K., Taketani, Y., and Iwamori, M. (1995) Characteristic Expression of GD1 Alpha Ganglioside During Lactation in Murine Mammary Gland, Biochim. Biophys. Acta 1256, 151–156. 54. Otnaess, A.B.K., Laegreid, A., and Ertresrag, K. (1983). Inhibition of Enterotoxin from Escherichia coli and Vibrio cholerae by Gangliosides from Human Milk, Infect. Immun. 40, 563–569. 55. Laegreid, A., and Otnaess, A.B.K. (1987) Trace Amounts of Ganglioside GM1 in Human Milk Inhibits Enterotoxin from Vibrio cholerae and Escherichia coli, Life Sci. 40, 55–62.

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56. Idota, T., Kawakami, H., Murakami, Y., and Sugawara, M. (1995) Inhibition of Cholera Toxin by Human Milk Fractions and Sialyllactose, Biosci. Biotech. Biochem. 59, 417–419. 57. Rueda, R., Sabatel, J.L., Maldonado, J., Narbona, E., and Gil, A. (1996) La Suplementación de una “Formula Infantil Adaptada con Gangliósidos Modifica la Microflora Fecal en Niños Recién Nacidos Pretérmino, I Congreso Nacional SENBA, Madrid. 58. Ettinger, A.C., U. S. Patent 4,762,822 (1988). 59. Teneberg, S., Willemsen, P., De Graaf, F.K., and Karlsson K.A. (1993) Calf Small Intestine Receptors for K99 Fimbriated Enterotoxigenic Escherichia coli, FEMS Microbiol. Lett. 109, 107–112. 60. Yuyama, Y., Yoshimatsu, K., Ono, E., Saito, M., and Naiki, M. (1993) Postnatal Change of Pig Intestinal Ganglioside Bound by Escherichia coli with K99 Fimbriae, J. Biochem. 113, 488–492. 61. Idota, T., and Kawakami, H. (1995) Inhibitory Effects of Milk Gangliosides on the Adhesion of Escherichia coli to Human Intestinal Carcinoma Cells, Biosci. Biotech. Biochem. 59, 69–72. 62. Schroten, H., Lethen, A., Hanisch, F.G., Plogmann, R., Hacker, J., Nobis-Bosch, R., and Wahn, V. (1992) Inhibition of Adhesion of S-Fimbriated Escherichia coli to Epithelial Cells by Meconium and Feces of Breast-Fed and Formula-Fed Newborns: Mucins Are the Major Inhibitory Component, J. Pediatr. Gastroenterol. Nutr. 15, 150–158. 63. Taki, T., Rokukawa, C., Kasama, T., Kon, K., Ando, S., Abe, T., and Handa, S. (1992) Human Meconium Gangliosides. Characterization of a Novel I-Type Ganglioside with the Neu-Acα2-6Gal Structure, J. Biol. Chem. 267, 11811–11817. 64. Idota, T., Kawakami, H., and Nakajima, I. (1994) Growth-Promoting Effects of NAcetylneuraminic Acid-Containing Substances on Bifidobacteria, Biosci. Biotech. Biochem. 58, 1720–1722. 65. Nakano, T., Sugawara, M., and Kawakami, H. (2001) Sialic Acid in Human Milk: Composition and Functions, Acta Paediatr. Taiwan 42, 11–17. 66. Vázquez, E., Gil, A., and Rueda, R. (2001) Dietary Gangliosides Positively Modulate the Percentages of Th1 and Th2 Lymphocyte Subsets in Small Intestine of Mice at Weaning, BioFactors 15, 1–9. 67. Vázquez, E., Gil, A., García-Olivares, E, and Rueda, R. (1999) Dietary Gangliosides Increase the Number of Intestinal IgA-Secreting Cells in Weanling Mice, Immunol. Letters 69, 447. 68. Vázquez, E., Gil, A., and Rueda, R. (2000) Dietary Gangliosides Increase the Number of Intestinal IgA-Secreting Cells and the Luminal Content of Secretory IgA in Weanling Mice, J. Pediatr. Gastroenterol. Nutr. 31, Suppl. 2, S133. 69. Vázquez, E., Gil, A., and Rueda, R. (2002) Low Concentration of Gangliosides Strongly Stimulate DNA Synthesis in Cultured Resting Intestinal Lymphocytes from Young Mice. 11th International Congress of Mucosal Immunology, 1301, Orlando, FL.

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Chapter 11

The Benefits of Lecithin on Cardiovascular Disease Debra L. Miller Central Soya Company Inc., 1946 West Cook Road, Fort Wayne, Indiana 46818

Introduction to Cardiovascular Disease Cardiovascular disease (CVD) accounts for 42% of all deaths in the United States and claims more lives each year than the next seven leading causes of death combined. CVD, also called coronary heart disease (CHD), is a term that encompasses many aspects of cardiac and arterial health, including myocardial infarction/angina pectoris, stroke, hypertension, congestive heart failure, and also congenital heart disorders. The process underlying most aspects of CVD, atherosclerosis, is characterized by a narrowing of the arteries supplying blood to the cardiac muscle due to the formation of plaques. Atherosclerotic plaque formation can progress to the ultimate inability to maintain adequate blood flow to vital organs including the heart, brain, and extremities. Plaques are formed when a denuding injury occurs to the inner lining or endothelium of an artery. Elevated blood cholesterol, specifically elevated LDL cholesterol, contributes to endothelial injury. When LDL become oxidized they act as free radicals, which can disrupt and permeate the endothelium. Monocytes initiate plaque formation by adhering to the injury site causing inflammation. Macrophages then invade the area and are unable to return to the blood stream after engulfing oxidized LDL. When macrophages engulf LDL and other debris they ultimately become foam cells imbedded in the artery wall. The process continues as platelets and other growth factors are drawn to the site and smooth muscle cells (from the deeper layers of the arterial wall) replicate and migrate to the injury site. Platelets then secrete collagen, which forms a crest-shaped fibrous plaque over the injury and other imbedded material. Ischemia (deficient blood flow) often occurs when plaques rupture and form a thrombus that blocks blood flow at the site or causes an embolism that travels to another site in the body. The ischemia causes tissue necrosis (infarction). An infarction in the cardiac area causes a heart attack, whereas infarction in the brain results in a stroke. Although cholesterol and other blood lipids are important risk factors in CVD, increased interest has recently been placed on specific functions of the endothelium itself. Under normal conditions, the endothelium and its components function to maintain normal vascular tone and blood fluidity and limit vascular inflammation and smooth muscle cell proliferation. However, when CVD risk factors are present, the endothelium can develop characteristics that facilitate inflammation, thrombosis, vasoconstriction, and atherosclerotic lesion formation. The purpose of

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this paper is to outline the role of phospholipids in the cardiovascular system and to discuss possible therapeutic benefits of phospholipids, primarily phosphatidylcholine (PC) and its component choline, to cardiovascular health. Risk factors, such as elevated blood cholesterol and homocysteine, and impairments in endothelial function as well as specific mechanisms (enzyme activity, methyl metabolism, oxidation, and inflammation) will be discussed.

The Role of Phospholipids on Lipoprotein Metabolism One of the important functions of phospholipids is their role in the formation of lipoproteins, which transport cholesterol. Following ingestion of dietary triglycerides, chylomicrons containing about 9% phospholipid and 2% protein are released into the lymph from the small intestine and VLDL are released from the liver. Chylomicrons and VLDL are reduced to remnants by lipoprotein lipases. The VLDL, in losing triglycerides, become denser and have a transient existence as intermediate-density lipoprotein (IDL). IDL are converted by the liver to more stable LDL in which the phospholipid has increased to about 20% and triglycerides have decreased to 10% of the particle. The LDL are characterized by a core enriched in cholesterol and cholesterol esters (8 and 37%, respectively) (1). LDL transport these core materials to tissue receptors and to a minor extent to the liver. HDL are produced principally by the liver and possibly by the small intestine (2). Lecithin is a critical component in the synthesis of HDL, which vary in composition but generally contain 24% phospholipid, 15% cholesterol esters, and only 4% triglycerides. Not only is lecithin a necessary component of HDL particle itself, it is required for the enzyme lecithin-cholesterol acyltransferase (LCAT). This enzyme allows HDL to sequester cholesterol in its interior, enabling it to pick up more damaging cholesterol-like LDL on its outer surface. Formation of HDL is thought to cause a redistribution of cholesterol among the plasma lipoproteins and provide a transport vehicle for the excretion of cholesterol into the bile. The importance of lecithin in the synthesis of LCAT and the metabolic sequels that may result will be discussed in more depth in a later section.

Lecithin and Cholesterol Metabolism The link between high cholesterol and CVD has been extensively studied. It is well documented that elevated blood cholesterol is associated with high risk of CVD. Many studies in the 1960s through the 1980s investigated the relationship between lecithin administration and serum cholesterol concentrations. Knuiman and colleagues’ review of 24 studies found the data equivocal (3). Early studies (4–8) administering moderate to large amounts of soy lecithin (12–54 g/d) resulted in marked reductions (20–58%) of total serum cholesterol in hyperlipidemic patients (8.9–19.0 mmol/L), whereas other investigations (9–17) of lecithin treatment resulted in small or no reductions. Also, four studies that controlled for the linoleic acid content in lecithin

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(fatty acids on the majority of position 1 and 2 of the glycerol molecule) resulted in no additional cholesterol-lowering benefits from the phospholipid complex (14,17–19). Although this review did not account for the PC content or specific phospholipids in soy lecithin, Knuiman and colleagues concluded that most of lecithin’s cholesterollowering benefits are due to its content of polyunsaturated fatty acids, primarily linoleic acid (soybean oil) (3). They state that soy lecithin, therefore, was no better than soybean oil in lowering total serum cholesterol (3). This review, however, based their conclusion on total cholesterol alone—failing to consider the effects of lecithin administration on subclasses of lipoproteins (3). Specifically, the analysis did not include the positive effects of lecithin on HDL cholesterol. Cholesterol transport in blood is carried out by LDL and HDL. LDL transport cholesterol from the liver to peripheral tissue, whereas HDL transport cholesterol from peripheral tissues to the liver for excretion as bile salts and to steroidogenic tissues for synthesis of steroid hormones. The HDL process of scavenging free cholesterol and returning for elimination or resynthesis is called “reverse cholesterol transport.” Because the HDL subclass of lipoproteins acts to clear cholesterol particles that could potentially cause an endothelial injury, it is considered to be protective against the development of CVD. To underscore the importance of serum HDL concentration in overall cardiovascular health, it is useful to put it into a relative context with other risk factors for CVD. In a comprehensive review of risk factors for atherosclerosis, Ridker and colleagues considered 11 biomarkers thought to be associated with increased or decreased risk of CVD (see Table 11.1) (20). They determined the strongest predictor of CVD to be an unfavorable ratio of total cholesterol to HDL cholesterol—meaning that those most likely to develop heart and vascular disease are those with low HDL cholesterol and high total cholesterol. Thus, simply considering effects of lecithin on total serum cholesterol tells only part of the story regarding risk factors for CVD. Other studies that have included measures of the other lipoprotein fractions show that lecithin’s effect on HDL cholesterol is independent from that of polyunsaturated fatty acids. Childs and colleagues (14) found no difference in total serum cholesterol in response to administration of soybean lecithin but did find a signifiTABLE 11.1 Serum Biomarkers of Cardiovascular Function • • • • • • • • •

Total Cholesterol HDL LDL TC:HDL Ratio Triglycerides Homocysteine C-reactive protein (CRP) Lipoprotein (a) Apolipoproteins (A-1 and B-100)

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cant increase in HDL cholesterol compared to an equivalent amount of polyunsaturated fatty esters ingested as corn oil. Another similar study by Galli and colleagues had similar findings (21). An animal study by O’Brien et al. investigated the effect of soybean phospholipids and egg phospholipids on total and HDL cholesterol concentrations in guinea pigs fed a high-cholesterol diet (22). Soybean phospholipids resulted in a 50% reduction in total serum cholesterol, whereas HDL cholesterol increased 23%. Egg phospholipids resulted in a 177% increase in HDL cholesterol with no effect on total cholesterol. Interestingly, the total cholesterol/HDL cholesterol ratio for the two treatments were similar (2.26 and 2.24 for soybean and egg, respectively), whereas the egg phospholipid had a low ratio of saturated to polyunsaturated fatty acid composition (P/S 0.38) compared to the soybean lecithin (P/S 3.5). The authors concluded that the cholesterolemic response to lecithin is not totally dependent on its fatty acid composition. This study, however, did not include comparisons to oils with similar fatty acid compositions. To look at the issue of lecithin’s effect on serum cholesterol apart from the effects of its component fatty acids, O’Brien and colleagues conducted a follow-up study in humans (23). They fed 10 normolipidemic men (aged 30–64) four test substances: • • • •

15 g of soy lecithin (78.3% PC, 19.3% phosphatidylethanolamine (PE)) 15 g of egg lecithin (75.4% PC, 23.4% PE) 12 g of soy lecithin fatty acids 12 g of egg lecithin fatty acids

Each test substance was fed for six weeks with three six-week washout periods between test periods. Although the responses in human serum lipid to the administration of the dietary phospholipids (both egg and soy) were similar to the responses to controls of comparable fatty acid composition; the data suggest that soy phospholipids selectively increase serum HDL cholesterol and serum phospholipids (23). This effect is most evident on the increase in the HDL2 particle, the most cardio-protective HDL subtype (24). HDL molecules have two main subclasses: HDL2 and HDL3 (24). The HDL3 molecule is synthesized in the liver and released into circulation to collect cholesterol. As the HDL3 molecule increases its cholesterol content, it becomes less dense and is classified as HDL2. HDL2 is then recycled in the liver, where the cholesterol is used to synthesize bile acids. The phospholipid (PL) and other components are repackaged into new HDL3, which are again released into circulation (24). Another study by Wojcicki and colleagues found that feeding 10 g/d of lecithin (23% PC) to hypercholesterolemic individuals resulted in a 36% reduction of LDL cholesterol and a 46% increase in HDL cholesterol (25). This study, however, was intended as a treatment observation and did not include a control group. The mechanism by which lecithin lowers cholesterol has received much investigation. The primary mechanism is thought to be decreasing the absorption of dietary

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cholesterol from the intestine to the bloodstream (26,27). There is also evidence that lecithin intake lowers cholesterol by increasing the amount of cholesterol used in the production of bile salts (28). As more cholesterol is used for bile salt synthesis, less is available to reach the blood stream and subsequently damage blood vessels. As mentioned, lecithin also contributes polyunsaturated fatty acids to the diet, which may help control blood cholesterol levels. The ability of soy lecithin to alter blood cholesterol via these mechanisms has led to some provocative research in adding lecithin to certain cholesterol-lowering drugs. One of the problems with drugs such as clofibrate is that, while it can lower levels of VLDL, thus lowering CVD risk, it can raise LDL levels, increasing CVD risk. One study found that, when administered with clofibrate, lecithin prevented the increase in LDL seen with clofibrate alone (29). The mechanism by which lecithin affects HDL cholesterol is less understood. It is commonly believed that HDL protect against the development of atherosclerosis by virtue of their ability to extract cholesterol from cells (reverse cholesterol transport). This view holds that HDL counteract the effects of LDL by removing cholesterol from cells in the artery endothelium and prevent the formation of foam cells. A critical step in reverse cholesterol transport is the esterification of cholesterol on the surface of HDL in a reaction catalyzed by LCAT. This reaction depletes the HDL surface of unesterified cholesterol and generates a concentration gradient that promotes the transfer of cholesterol from cell membranes to HDL.

Lecithin and LCAT Activity The role of LCAT in cardiovascular health is well established. Most LCAT is synthesized by the liver and circulates in blood reversibly bound to lipoproteins (30). Some LCAT is made in the brain, but the role of LCAT in brain tissue is not understood. Nascent HDL particles carry free cholesterol, apoproteins, and phospholipids. In blood, LCAT preferentially binds to these HDL particles and is activated by the major protein component of HDL, apolipoprotein A-I. The main function of LCAT is to convert free cholesterol and the PC into cholesteryl esters and lysophosphatidylcholine. LCAT removes excess unesterifed cholesterol from lipoproteins and tissues for transport back to the liver for excretion as bile salts and to steroidogenic tissues for synthesis of steroid hormones. The question relevant to this discussion is whether dietary lecithin can induce changes in LCAT activity that would be favorable for HDL synthesis and reverse cholesterol transport. There are contrasting reports, both clinical and experimental, on this issue. Day et al. (31) administered egg phospholipid (200 mg i.v. three times per week) to cholesterol-fed rabbits and showed acute plasma free cholesterol and phospholipid increases after each treatment. No variations in LCAT activity were found in either acute or chronic studies. However, in a study in humans, plasma HDL, HDL phospholipids, free cholesterol, and LCAT activity increased following i.v. administration of a single dose of egg phospholipid (32). A recent study indicates that a diet rich in linoleic acid may increase metabolism of serum cholesterol by LCAT in rats (33). This effect may not be due to elevated serum concentration of LCAT or of

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its apolipoprotein activators but most likely to changes in the chemical composition of endogenous lipoprotein substrates. Finally, an in vitro study found that the fatty acid in the 1-position of PC significantly influences cholesteryl ester formation, both by its direct involvement in the LCAT reaction and its contribution to the physiochemical properties of PC (34). These findings suggest that LCAT may be stimulated by the excess phospholipid in the serum—theoretically this may lead to an enhanced clearance of cholesteryl esters. Additional research is necessary to explore this issue further. One final connection between LCAT activity and cardiovascular health is a report by Vohl and colleagues, which proposes that LCAT may have antioxidant properties (35). Vohl and colleagues purified LCAT and found that it is not only capable of esterifiying cholesterol in the plasma but can also prevent the accumulation of oxidized lipids in LDL cholesterol. When pure human LCAT was added to LDL or palmitoyl-linoleoyl PC/sodium cholate micelles it inhibited the oxidationdependent accumulation of both conjugated dienes and lipid hydroperoxides. LCAT also inhibited the negative charge that occurs during LDL oxidation. This action appears to be enzymatic, and a serine residue may be the proton donor in the process. If this action occurs similarly in vivo, LCAT may play a significant role in preventing the accumulation of oxidized lipid in plasma lipoproteins.

Direct Effects of Lecithin on Atheroclerosis and Artery Health Data from the 1960s suggests that phospholipids may play a dual anti-atherosclerosis role by mobilizing cholesterol from arteries and by preventing the atherosclerogenic actions of cholesterol. Small plaque accumulations can begin in early adolescence and progress through life, thus both preventing or retarding plaque formation can have profound effects on cardiovascular health. Phospholipids administered intravenously reversed atherosclerotic lesions in several experimental animal species (36–41). In 1967, Adams and colleagues found that intravenous injection of lecithin given weekly to cholesterol-fed rabbits reduced the severity of both aortic atheroma and fatty liver, whereas injections of lecithin derived from eggs diminished fatty liver in cholesterol-fed rabbits but did not protect the aorta against atheroma (41). Another study found that intravenous lecithin administration retards the atherosclerotic plaques in baboons—even when the animals are fed a highly atherosclerotic diet (40). More recently, Hsia and colleagues tested the administration of PC transdermally to spontaneously hypercholesterolemic rabbits that develop atherosclerotic lesions in the aortic arch as early as two months of age (42). This method of administration circumvents alterations to the phospholipid that occur during digestion while avoiding the invasive nature to intravenous injection. This research group found that PC was well absorbed as assessed by elevated serum choline levels. Both total and LDL serum cholesterol were significantly reduced in the animals treated with PC compared to controls, and atherosclerotic lesions in the aortic arch were clearly less severe. There are a number of theories

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on the mechanisms of lecithin’s anti-atherosclerotic actions. Older theories attributed these effects to “detergent” effects and simply the lowering of serum cholesterol (41). More recently though, Hsia and colleagues suggested that PC is taken up by HDL to form PC-rich vesicles that have a greater capacity than native HDL for promoting cholesterol efflux leading to depletion of cellular cholesterol and increased LDL receptor activity and reduced arterial damage (42). However, exact mechanisms on endothelial repair and growth factors are yet to be determined.

Effects of Lecithin on Methyl Transfer and Homocysteine Metabolism Homocysteine is a sulfur-containing amino acid that is formed when a methyl group is removed from the essential amino acid methionine. A strong association exists between increasing homocysteine levels and increased CVD risk. Normal blood homocysteine levels range between 5–15 mmol/L (43). It is estimated that for every 5 mmol/L increase in blood homocysteine there is a corresponding 1.6-fold risk of CVD in men and 1.8-fold risk for women (44). In terms of risk, a 5 mmol/L change in blood homocysteine equates with a 20 mg/DL change in LDL cholesterol (44). According to the homocysteine theory of arteriosclerosis, elevated plasma homocysteine initiates arteriosclerotic plaques by endothelial injury, destruction of elastin, deposition of proteoglycans and lipoproteins, fibrosis, and calcification (45). Homocysteine metabolism involves two major metabolic pathways that utilize B vitamins, including choline (Fig. 11.1). Elevation of plasma homocysteine may be produced by deficiency of choline (46), pyridoxine (47), cobalamin, or folic acid (48). Choline deficiency also produces atheromatous changes in experimental animals (46). Choline is metabolized to betaine, which is a source of methyl groups, which remethylate homocysteine to methionine. This pathway is similar to the remethylation pathway of folate in which riboflavin is a co-factor in METHIONINE

glycine

HOMOCYSTEINE

Fig. 11.1. Methyl-donating metabolic pathways in the conversion of homocysteine to methionine.

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the conversion of methylenetetrahydrofolate to methyltetrahydrofolate, a direct precursor of methyl cobalamin, the coenzyme that methylates homocysteine to methionine. Although folic acid is another methyl donor known to reduce homocysteine (49), it does not act to lower cholesterol. Since 2000, the remethylation pathways of homocysteine have received considerable attention in the scientific literature, and most have focused on folate. However, much of the early work in this area included other B vitamins and choline. In 1989, Olszweski and colleagues investigated the effects of administering a vitamin cocktail containing pyridoxine, folate, cobalamin, choline, and riboflavin to a group of 12 male survivors of acute myocardial infarction for 21 days (50). The plasma concentrations of homocysteine and (α-amino adipic acid declined to 68% (P < 0.001) and 57% (P < 0.001) of the pretreatment values and the cholesterol, triglycerides, and LDL apo B declined to 79% (P < 0.001). Other studies have found that low concentrations of the free base form of homocysteine thiolactone, a compound that acts in reciprocal flux with homocysteine, are associated with atheroma development and thrombolytic activity (specifically platelet aggregation and release of thromboxane) (51). It has been speculated that reductions in plasma homocysteine via the remethylation by choline/betaine and other B vitamins can thus decrease susceptibility to platelet aggregation and thrombosis—two hallmark components of atherosclerosis. Much of the early work on homocysteine and CVD risk focused on genetic polymorphisms in genes that code for the enzymes cystathionone synthase or methylenetetrahydrofolate reductase resulting in impaired homocysteine metabolism. A number of studies in patients with such disorders demonstrated that folic and betaine can significantly lower plasma homocysteine levels during fasting and following methionine loading. In two studies by Wilcken and colleagues, treatment with 6 g/d of betaine in patients with cystathionine-synthase deficiency reduced plasma total homocysteine levels in patients both responsive (52) and nonresponsive to vitamin B6 therapy (53). Recent work in this area, however, has not focused on the CVD implications of genetic conditions that result in severe homocysteinemia and homocysteinuria, but rather has considered the relationship between mild hyperhomocysteinemia and arterial disease (43,54–56). While the impairment of homocysteine metabolism in cases of mild hyperhomocysteinemia may be also caused by some degree of deficient activity in cystathionone synthase, methylenetetrahydrofolate reductase, or other still unknown enzymes (43,54–56), it is more likely that the cause of the impairment is related to low dietary intake of vitamin B6, vitamin B12, folic acid, and choline (43,54). In patients under 50 years of age who are diagnosed with a variety of manifestations of arterial disease, excessive homocysteine concentrations after oral methionine loading has been observed with a prevalence varying from 8 to 42%. As with classic severe homocystinuria, treatment with methyl donors, such as choline/betaine or other cofactors, lowers the plasma homocysteine concentration in patients with moderately elevated homocysteine (55). In a review by Boushey et al. (44), a number of studies have shown strong inverse correlations between plasma

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levels of homocysteine and intake of vitamin B6 and folic acid. Dietary choline deficiency causes arteriosclerosis in rats (46), apparently due to hyperhomocysteinemia (57). A recent study by Olthof and colleagues investigated the level of betaine needed to suppress serum homocysteine in healthy participants (58). This study provided betaine in doses of 1.5, 3, and 6 g/d during both fasting and following a methionine load for six weeks. When measured in a fasted state, 3 g/d of betaine produced a reduction in plasma homocysteine of a similar magnitude as 6 g/d, whereas 1.5 g/d had a modest effect. Following methionine loading, each of the doses reduced plasma homocysteine compared to placebo. The 6 g/d of betaine reduced plasma homocysteine more than 3 and 1.5 g/d, both of which had intermediate effects (58). Overall, these studies provide evidence that a diet rich in lecithin and its metabolites, choline and betaine, might be beneficial in lowering the risk of CVD in humans with moderately elevated serum homocysteine levels.

Lecithin and Anti-Inflammatory Agents Finally, lecithin may also affect cardiovascular health by interactions with certain drugs. One such interaction is lecithin and nonsteroidal anti-inflammatory drugs (NSAIDS). NSAIDS (aspirin, ibuprofen, and naproxen) are currently being studied in a number of trials for their potential effect on reducing arterial disease by reducing inflammation by reducing the production of certain prostaglandins. However, because prostaglandins also act to protect stomach lining, these drugs can also cause upset stomach, gastrointestinal bleeding, and ulcers. The addition of lecithin to NSAIDS not only prevents the stomach irritation associated with these drugs (59) but also may potentiate their effects (60). A study in rats shows that free and lecithin-associated aspirin appear to be equally absorbed, but the latter form has greater antipyretic, antiinflammatory and analgesic efficacy. The improved efficacy may be due to increased uptake by target cells, increased binding to cyclooxygenase (the rate limiting enzyme in the synthesis of pro-inflammatory and other prostaglandins), or prolonged half-life (60). Lecithin liposomes have been studied as a delivery form for numerous drugs as well.

Lecithin and Endothelial Function: Future of Lecithin and CVD Research The vascular endothelium functions as a paracrine organ, secreting numerous factors regulating vascular tone, cell growth, platelet and leukocyte interactions, and thrombogenicity (61). The endothelium senses and responds to numerous endogenous and exogenous stimuli via a complex of cell membrane receptors and other mechanisms (61). Dysfunction in the endothelium is thought to be a factor in the development of atherosclerosis, hypertension, and other aspects of CVD. In recent years, flow-mediated dilation of the brachial artery has become a standard for assessing endothelial function. This noninvasive technique uses ultrasound technology to quantitate vasomotor

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changes in the brachial artery following an acute occlusion. As discussed in earlier sections, lecithin and phospholipids contain many compounds thought to have specific action on endothelial function. Currently, investigations are underway to determine the effects of lecithin (PC 40) as a chemical stimulus on the blood vessel ability to self-regulate tone and adjust blood flow.

Summary Lecithin and its metabolites have many actions that may facilitate cardiovascular health. Benefits of lecithin in the diet or as a supplement include cholesterol management— especially inducing increases in the protective HDL fraction of cholesterol. Actions of the critical enzyme LCAT may facilitate the HDL cholesterol in its function in reverse cholesterol transport as well as act as a protective antioxidant. The phospholipids that comprise lecithin can act directly to retard and even reverse atherosclerotic plaques when administered intravenously. Investigations of transdermal applications in this area are also very promising. Other metabolites of lecithin, choline, and betaine contribute to maintaining a proper balance in methylation pathways by lowering serum homocysteine levels. The synergistic effect of lecithin and NSAIDS and inflammatory aspects of CVD requires more research as does the effects of lecithin on endothelial function. References 1. Life Sciences Research Office (1981) Effects of Consumption of Choline and Lecithin on Neurological and Cardiovascular Systems, FASEB, Washington, DC. 2. Tall, A.R., and Small, D.M. (1978) Plasma High-Density Lipoproteins, N. Engl. J. Med. 299, 1232–1236. 3. Knuiman, J.T., Beynen, A.C., and Katan, M. B. (1989) Lecithin Intake and Serum Cholesterol, Am. J. Clin. Nutr. 49, 266–268. 4. Adlersberg, D., and Sobotka, H. (1943) Effect of Prolonged Lecithin Feeding on Hypercholesterolemia, J. Mt. Sinai Hosp. 9, 955–956. 5. Steiner, A., and Domanski, B. (1944) Effect of Feeding Soya Lecithin on Serum Cholesterol Level of Man, Proc. Soc. Exp. Biol. Med. 55, 236–238. 6. Morrison, L.M. (1958) Serum Cholesterol Reduction with Lecithin, Geriatrics 13, 12–19. 7. Varkoni, G. (1962) Beitrag zur therapeutischen Wirkung der oralen und intravenösen Verabreichung essentieller Cholinphospholipide, Zeitschr für die gesamte Innere Medizin und ihre Grenzgebiete 18, 1–8. 8. Funatzu, Y. (1966) Das Serumcholesterin bei Arteriosklerotiken unter der Bahnadlung mit Lipostabil, Med. Heute 15, 1–10. 9. Delevett, A.F., and Bruger, M. (1948) Plasma Lipids in Primary (Xanthomatosis) and Secondary Hypercholesterolemia, Arch. Int. Med. 81, 859–867. 10. Ter Welle, H.F., Van Gent, C.M., Dekker, W., and Willebrands, A.F. (1974) The Effect of Soya Lecithin on Serum Lipid Values in Type II Hyperlipoproteinemia, Acta. Med. Scand. 195, 267–271. 11. Svanberg, U., Gustafson, A., and Ohlson, R. (1974) Polyunsaturated Fatty Acids in Hyperlipoproteinemia. II. Administration of Essential Phospholipids in Hypertriglyceridemia, Nutr. Metab. 17, 338–346.

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12. Simons, L.A., Hickie, J.B., and Ruys, J. (1977) Treatment of Hypercholesterolemia With Oral Lecithin, Aust. N. Z. J. Med. 7, 262–266. 13. Cobb, M., Turkki, P., Linscheer, W., and Raheja, K. (1980) Lecithin Supplementation in Healthy Volunteers: Effect on Cholesterol Esterification and Plasma, and Bile Lipids, Nutr. Metab. 24, 228–237. 14. Childs, M.T., Bowlin, J.A., Ogilvie, J.T., Hazzard, W.R., and Albers, J.J. (1981) The Contrasting Effects of a Dietary Soya Lecithin Product and Corn Oil on Lipoprotein Lipids in Normolipidemic and Familial Hypercholesterolemic Subjects, Atherosclerosis 38, 217– 228. 15. Hawthorne, J.N., Hoccom, M., and O’Mullane J.E. (1981) Soya Lecithin and Lipid Metabolism. In Cairella, M., and Lekim, D., eds., Soya Lecithin: Nutritional and Clinical Aspects: Proceedings of the First International Colloquium on Soya Lecithin in Nutrition and Dietetics, Societa Editrice Universo, Rome, 67–73. 16. Vroulis, G., Smith, R.C., Schoolar, J.C., Dhalen, G., Katz, E., and Misra, C.H. (1982) Reduction of Cholesterol Risk Factors by Lecithin in Patients With Alzheimer’s Disease, Am. J. Psychiatry 139, 1633–1634. 17. Kesaniemi, Y.A., and Grundy, S.M. (1986) Effect of Dietary Polyenylphosphatidylcholine on Metabolism of Cholesterol and Triglycerides in Hypertriglyceridemic Pateints, Am. J. Clin. Nutr. 43, 98–107. 18. Greten, H., Raetzer, H., Stiehl, A., and Schettler, G. (1980) The Effect of Polyunsaturated Phosphatidylcholine on Plasma Lipids and Fecal Steroid Excretion, Atherosclerosis 36, 81– 88. 19. Prack, M., Sanborn, T., Waugh, D., Simkin, H., Bennett Clark, S., and Small, D.M. (1983) Effects of Polyunsaturated Lecithin on Plasma and Lipoprotein Cholesterol and Fatty Acids in Normal Men, In Perkins, E.G., and Visek, W.J., eds., Dietary Fats and Health, American Oil Chemist’s Society, Champaign, 689–697. 20. Ridker, P.M., Stampfer, M.J., and Rifai, N. (2001) Novel Risk Factors for Systemic Atherosclerosis: A Comparison of C-Reactive Protein, Fibrinogen, Homocysteine, Lipoprotein(a), and Standard Cholesterol Screening as Predictors of Peripheral Arterial Disease, JAMA 285, 2481–2485. 21. Galli, C., Tremboli, E., Giani, E., Maderna, P., Gianfranscheschi, G., and Sirtori, C.R., (1985) Oral Polyunsaturated Phosphatidylcholine Reduces Platelet Lipid and Cholesterol Contents in Healthy Volunteers, Lipids 20, 561–566. 22. O’Brien, B.C., and Corrigan, S.M. (1988) Influence of Dietary Soybean and Egg Lecithins on Lipid Responses in Cholesterol-Fed Guinea Pigs, Lipids 23, 647–650. 23. O’Brien, B.C., and Andrews, V.G. (1993) Influence of Dietary Egg and Soybean Phospholipids and Triacylglycerols on Human Serum Lipoproteins, Lipids 28, 7–12. 24. Durstine, J.L., and Haskell, W.L. (1994) Effects of Exercise Training on Plasma Lipids and Lipoproteins, Exerc. Sport Sci. Rev. 22, 477–521. 25. Wojcicki, J., Pawlik, A., Samochowiec, L., Kaldonska, M., and Mysliwiec, Z. (1995) Clinical Evaluation of Lecithin as a Lipid-Lowering Agent, Phytotherapy Res. 9, 597–599. 26. Beil, F.U. and Grundy, S.M. (1980) Studies on Plasma Lipoproteins During Absorption of Exogenous Lecithin in Man, J. Lipid Res. 21, 525–536. 27. Rampone, A.J., and Machida, C.M. (1981) Mode of Action of Lecithin in Suppressing Cholesterol Absorption, J. Lipid Res. 22, 744–752. 28. Mastellone, I.I., Polichetti, E., Gres, S., de la Maisonneuve, C., Domingo, N., Marin, V.V., Lorec, A., Farnarier, C., Portugal, H., Kaplanski, G., and Chanussot, F. (2000) Dietary Soybean Phosphatidylcholines Lower Lipidemia: Mechanisms at the Levels of Intestine, Endothelial Cell, and Hepato-Biliary Axis, J. Nutr. Biochem. 11, 461–466.

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29. Schneider, J., Muller, R., Buberl, W., Kaffarnik, H., Schubotz, R., Hausmann, L., Muhlfellner, G., and Muhlfellner, O. (1979) Effect of Polyenyl Phosphatidyl Choline on Clofibrate-Induced Increase in LDL Cholesterol, Eur. J. Clin. Pharmacol. 15, 15–19. 30. Jonas, A. (2000) Lecithin Cholesterol Acyltransferase, Biochim. Biophys. Acta 1529, 245– 256. 31. Day, A.J., Horsch, A., and Hudson, K. (1976) Effect of Polyunsaturated Lecithin on Serum and Aortic Lipids in Normal and Cholesterol-Fed Rabbits, Artery 2, 400–422. 32. Wallentin, L. (1976) Influence of Intravenous and Oral Administration of Phospholipids on Lecithin: Cholesteryl Acyltransfer Rate in Plasma, Artery 3, 40–51. 33. Romijn, D., Wiseman, S.A., Scheek, L.M., de Fouw, N.J., and van Tol, A. (1998) A Linoleic Acid Enriched Diet Increases Serum Cholesterol Esterification by Lecithin: Cholesterol Acyltransferase in Meal-Fed Rats, Ann. Nutr. Metab. 42, 244–250. 34. Assmann, G., Schmitz, G., Donath, N., and Lekim, D. (1978) Phosphatidylcholine Substrate Specificity of Lecithin: Cholesterol Acyltransferase, Scand. J. Clin. Lab. Invest. Suppl. 150, 16–20. 35. Vohl, M.C., Neville, T.A., Kumarathasan, R., Braschi, S., and Sparks, D.L. (1999) A Novel Lecithin-Cholesterol Acyltransferase Antioxidant Activity Prevents the Formation of Oxidized Lipids During Lipoprotein Oxidation, Biochemistry 38, 5979–5981. 36. Friedman, M., Byers, S.O., and Rosenman, R.H. (1957) Resolution of Oartic Atherosclerotic Infiltration in the Rabbit by Phosphatide Infusion, Proc. Soc. Exp. Biol. Med. 95, 586–588. 37. Byers, S.O. and Friedman, M. (1960) Effect of Infusions of Phosphatides Upon the Atherosclerotic Aorta In Situ and as an Ocular Aortic Implant, J. Lipid. Res. 1, 343–348. 38. Stafford, W.W. and Day, C.E. (1975) Regression of Atherosclerosis Affected by Intravenous Phospholipid, Artery 1, 106–114. 39. Sirtori, C.R. (1993) Phospholipids and Atherosclerosis: Mechanistic and Kinetic Aspects, Atherosclerosis Review 24, 175–198. 40. Howard, A.N., Patelski, J., Bowyer, D.E., and Gresham, G.A. (1971) Atherosclerosis Induced in Hypercholesterolaemic Baboons by Immunological Injury; and the Effects of Intravenous Polyunsaturated Phosphatidyl Choline, Atherosclerosis 14, 17–29. 41. Adams, C.W., Abdulla, Y.Y., Bayliss, O., and Morgan R.S. (1967) Modification of Aortic Atheroma and Fatty Liver in Cholesterol-Fed Rabbits by Intravenous Injection of Saturated and Polyunsaturated -Lecithin, J. Path. Bact. 94, 77–87. 42. Hsia, S.L., He, J.L., Nie, Y., Fong, K., and Milikowski, C. (1996) The Hypocholesterolemic and Antiatherogenic Effects of Topically Applied Phosphatidylcholine in Rabbits with Heritable Hyperchoesterolemia, Artery 22, 1–23. 43. Kang, S.S., Wong, P.W., and Manilow, M.R. (1992) Hyperhomocyst(e)inemia as a Risk Factor for Occlusive Vascular Disease, Annu. Rev. Nutr. 12, 279–298. 44. Boushey, C.J., Beresford, S.A., Omenn, G.S., and Motulsky, A.G. (1995) A Quantitative Assessment of Plasma Homocysteine as a Risk Factor for Vascular Disease. Probably Benefits of Increasing Folic Acid Intakes, JAMA 274, 1049–1057. 45. McCully, K.S., (1983) Homocysteine Theory of Arteriosclerosis: Development and Current Status, Atheroscler. Rev. 11, 157–60. 46. Hartroft, W.S., Ridout, J.H., Sellars, E.A., and Bost, C.H. (1952) Atheromatous Changes in Aorta, Carotid and Coronary Arteries of Choline Deficient Rats, Proc. Soc. Exp. Biol. Med. 81, 384–393. 47. Park, Y.K., and . Linkswiler, H. (1970) Effect of Vitamin B6 Depletion in Adult Man On the Excretion of Cystathionine and Other Methionine Metabolites, J. Nutr. 100, 110–116. 48. Kang, S.S., Wong, P.W., and Norusis, M. (1987) Homocysteinemia Due to Folate Deficiency, Metabolism 36, 458–462.

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49. Ubbink, J.B., Vermaak, W.J., van der Merwe, A., Becker, P.J., Delport, R., and Potgieter, H.C. (1994) Vitamin Requirements for the Treatment of Hyperhomocysteinemia in Humans, J. Nutr. 124, 1927–1933. 50. Olszewski, A.J., Szostak, W.B., Bialkowska, M., Rudnicki, S., and McCully, K.S. (1989) Reduction of Plasma Lipid and Homocysteine Levels by Pyridoxine, Folate, Cobalamin, Choline, Riboflavin, and Troxerutin in Atherosclerosis, Atherosclerosis 75, 1–6. 51. McCully, K.S., and Carvalho, A.C. (1987) Homocysteine Thiolactone, N-Homocysteine Thiolactonyl Retinamide, and Platelet Aggregation, Res. Commun Chem. Pathol. Pharmacol. 56, 349–360. 52. Wilcken, D.E., Wilcken, B., Dudman, N.P., and Tyrrell, P.A. (1983) Homocystinuria—The Effects of Betaine in the Treatment of Patients Not Responsive to Pyridoxine, N. Engl. J. Med. 309, 448–453. 53. Wilcken, D.E., Dudman, N.P., and Tyrrell, P.A. (1985) Homocystinuria Due to Cystathionine Beta-Synthase Deficiency—The Effects of Betaine Treatment in PyridoxineResponsive Patients, Metabolism 34, 1115–1121. 54. Ueland, P.M., Refsun, H., and Brattström, L. (1992) In R.B. Francis, The Hemostatic System, Endothelial Function and Cardiovascular Disease, Marcel Dekker, New York, 183–236. 55. Boers, G.H., Smals, A.G., Trijbels, F.J., Fowler, B., Bakkeren, J.A., Schoonderwaldt, H.C., Kleijer, W.J., and Kloppenborg, P.W. (1985) Heterozygosity for Homocystinuria in Premature Peripheral and Cerebral Occlusive Arterial Disease, N. Engl J. Med. 313, 709–715. 56. Clarke, R., Daly, L., Robinson, K., Naughten, E., Cahalane, S., Fowler, B., and Graham, I. (1991) Hyperhomocysteinemia: An Independent Risk Factor for Vascular Disease, N. Engl. J. Med. 324, 1149–1155. 57. McCully, K.S. (1969) Vascular Pathology of Homocysteinemia: Implications for the Pathogenesis of Arteriosclerosis, Am. J. Pathol. 56, 111–128. 58. Olthof, M.R., van Vliet, T., Boelsma, E., and Verhoef, P. (2002) Effect of Betaine on Plasma Homocysteine. (Abstract) ILPS Congress, Vienna, Austria. 59. Lichtenberger, L.M., Wang, Z.M., Romero, J.J., Ulloa, C., Perez, J.C., Giraud, M.N., and Barreto, J.C. (1995) Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Associate With Zwitterionic Phospholipids: Insight into the Mechanism and Reversal of NSAID-Induced Gastrointestinal Injury, Nat. Med. 1, 154–158. 60. Lichtenberger, L.M., Ulloa, C., Vanous, A.L., Romero, J.J., Dial, E.J., Illich, P.A., and Walters, E.T. (1996) Zwitterionic Phospholipids Enhance Aspirin’s Therapeutic Activity, As Demonstrated in Rodent Model Systems, J. Pharmacol. Exp. Ther. 277, 1221–1227. 61. Corretti, M.C., Anderson, T.J., Benjamin, E.J., Celermajer, D., Charbonneau, F., Creager, M.A., Deanfield, J., Drexler, H., Gerhard-Herman, M., Herrington, D., Vallance, P., Vita, J., and Vogel, R. (2002) Guidelines For the Ultrasound Assessment of EndothelialDependent Flow-Mediated Vasodilation of the Brachial Artery: A Report of the International Brachial Artery Reactivity Task Force, J. Am. Coll. Cardiol. 39, 257–265.

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Chapter 12

Is There a Rationale for Phospholipid Supplementation in Athletes? Fred Brouns Nutrition and Toxicology Research Institute Maastricht, Maastricht University, Maastricht, Netherlands, and Cerestar-Cargill, Vilvoorde R&D Centre, Havenstraat 84, Vilvoorde, Belgium

Introduction Very recently an international meeting (PASSCLAIM, ILSI, Berlin, Sept 2002) focused on the substantiation of Health Claims and Function Claims for functional foods and food components. The following criteria, which certainly will influence future regulations, were put forward: • Health and function benefit claims must be scientifically substantiated • Substantiation must be based on the totality of the evidence • Preferably, evidence should be derived from human intervention studies in the population at target for the claim benefit • Evidence should be based on a realistic/feasible amount of ingredient as consumed • There should be a plausible physiological/biochemical mechanism and/or a clear association with the function • The effect/benefit should be of physiological/psychological relevance for consumer It is in the light of these points that this paper will review the possible functions and benefits of choline supplementation for the support of performance and recovery from intensive physical exercise in humans. There are a number of theoretical reasons why phospholipids, in particular phosphatidylcholine (PC) and phosphatidylserine (PS) may play a role in the etiology of fatigue and the bodily limitations to perform long lasting exhausting physical and mental tasks. • Limitations in choline availability may impact choline content of tissues, actylcholine synthesis, and release in the central nervous system and, accordingly, neuronal signal transduction (12,20). • Choline acts as methyl donor in the synthesis of creatine. Theoretically limitations could reduce total creatine in muscle, a recently recognized performance-limiting factor (52).

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• The phospholipid content of membranes and the degree of polyunsaturation influences membrane fluidity. Accordingly, limitations in their availability may impact membrane functions, such as the production of intracellular messenger molecules diacylglycerol and ceramide, which are involved in the regulation of specific cellular responses by their influence on protein kinase C (PKC) for cell proliferation, differentiation, and cell growth (48,49,50). • Membrane fluidity also influences red blood cell (RBC) deformability when opposed to mechanical shear stress. The latter plays a role in RBC during circulation, in particular in contracting muscle. A reduced deformability may reduce erythrocyte’s lifetime by inducing hemolysis and also may reduce the ability to deliver oxygen in contracting muscle and, consequently, muscle’s ability to resynthesize ATP by oxidative phosphorylation pathways. • The fatty acid composition of phospholipids in muscle cell membranes influences insulin action through effects on glucose transporter externalization. Decreased concentrations of polyunsaturated fatty acids are associated with insulin insensitivity and reduced glucose transport (8) • PS has been tested in a number of studies, primarily regarding its effects on brain in elderly subjects. The content of PS in cell membranes of the central nervous system is suggested to play an important role in cell-cell communication, improving a number of cognitive functions as well as stress tolerance (21). Exercise was shown to reduce the PS content in red blood cells significantly (35), which was negatively correlated with maximal oxygen uptake. The secretion of stress hormones was observed to be reduced during exercise of low intensity (29,28). The latter findings are taken by marketers to suggest that PS may have a positive impact on exercise performance capacity.

Metabolic Backgrounds for a Role of Choline in Exercise Free choline is the precursor for acetylcholine (AC), a neurotransmitter of vital importance to optimal functioning of the central nervous system, including neuromuscular impulse transmission. During neural stimulation, the choline required for the release of AC comes from intracellular sources only. To maintain levels of free choline, cells take up choline from the blood via a carrier mechanism that is crucial at low plasma choline concentrations. When plasma choline is raised to higher levels, uptake also occurs by diffusion (11). Apart from free choline uptake, choline also arrives at the cells as PC, which is exchanged in the cell membrane. After incorporation in the membrane, cell-cell exchange by a flip-flop mechanism may occur. For the production of AC, choline may also be used from the cell membrane PC fraction (5,6,27,4). In this case choline is released from the membrane PC by action of the enzyme phospholipase C. During continuous or chronically repeated electrical nerve stimulation, the choline turnover in neural tissue is much higher than the rate at which free choline

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can be taken up from blood (11). This may result in a reduction in intracellular free choline (26,37). The longer the stimulation lasts, the more the reduction will be (5,6,42). Under such conditions choline liberation by degradation of PC in the cell membrane is the only way to avoid intracellular free choline depletion. The consequence of this kind of autocannibalism of the phospholipid pool in the membrane may have an effect on membrane function. Reductions in free choline have not been observed in resting muscle, which points to the fact that muscle choline requirements may be closely met by the synthesis rate in liver and uptake rate in muscle from blood (4,15). However, in chronically stimulated muscle, a drop in free choline concentration has been shown to induce a fall in AC release and a slowing (not impairment) of the transmission of the contraction-generating impulse (4,45). Animal studies suggest that a reduction in muscle AC production, caused by precursor inavailability, may contribute to muscle function impairment (22,30,25), but data from human studies are not available. Within the cells the formation of PC by the involvement of S-adenosyl methionine as methyldonor in the methylation of phosphatidylethanolamine proceeds only at a slow rate. Accordingly, prolonged utilization of choline from ongoing degradation of PC may, without appropriate resynthesis of PC, also result in a net loss of phosphatide from the cell, “a kind of autocannibalism” (7). The latter may, at least theoretically, additionally affect membrane function and cell viability. Supporting this assumption are the observations described by Blusztajn (7) that the PC content of neuroblastoma-glioma hybrid cells in culture can be modified by choline supply in the medium. It was shown that the cell membrane PC content (in relation to other phospholipids) effected both cell growth rate and cell survival rate. The question thus arises whether the exogenous supply of choline by oral intake of either choline salts or PC (purified lecithin, containing up to 40% w/w) may help to maintain required intracellular free choline concentration and AC synthesis in order to avoid a slow down of neuronal impulse transmission and possibly related muscle function impairments.

Choline Supply to Tissues Choline is to a large extent synthesized in the liver and to a lesser extent in other tissues, such as brain and mammary gland, by the methylation of phosphatidylethanolamine, using S-adenosylmethionine as methyl donor, resulting in the formation of PC. (50). Important quantities of the daily choline supply are ingested with the diet, mostly in the form of PC, especially from liver, egg, beef, and products including these foods (14). Increasing choline intake by normal dietary sources elevates circulating choline levels only slightly (51). However, raising dietary choline intake substantially by the intake of concentrated choline sources has been shown to increase the plasma choline level in a dose-dependent way (16,47) by two- to fourfold

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(5,40,51), which in turn has been shown not to be subject to product feedback inhibition (17,18,42) and to enhance brain choline levels (18). The latter was shown to result in an enhanced brain as well as muscle AC release during electrical stimulation (4,5,36). The firing rate of the neurons seems to be a crucial factor in such a response (41). This assumption is supported by the observation of Wecker et al. (39), who observed that choline supplementation in rats increased circulating choline levels by 52%. However, synthesis of AC in, and its release from, brain slices was not different compared to controls. In choline deficient slices, however, AC release was decreased. These findings suggest that oral choline supply will not beneficially impact neurotransmitter synthesis and release in the nondepleted state but may do so in a condition of relative shortage. Taking these observations all together, it appears that, at least theoretically, reduced choline availability may negatively impact neural processes in the central nervous system as well as neuromuscular impulse transmission. Accordingly, choline depletion may be involved in the etiology of both physical and mental fatigue as experienced by participants in stressful, exhausting conditions, such as elite endurance athletes and military soldiers during combat training. Two important questions then arise: (i) Does prolonged stimulation of nerves as takes place during intensive physical exercise indeed cause a local choline depletion, sufficient enough to result in impaired physical/mental performance? (ii) If the preceding is the case, does oral choline supplementation help maintain optimal performance?

Does Intense Physical Work Reduce Choline Availability? During a marathon run it was shown that the serum choline level of a group of participants decreased by 40% (10). Such a reduction is about comparable with the effect seen after ingestion of a choline-free diet for three weeks, as observed by Shephard (33), from 12.2 initially to 7.4 µM after three weeks. Such a reduction was shown also to lower brain choline levels and to suppress AC release (13). In another, but nonpublished, patent-related study it was observed that running 20 miles reduced plasma choline by 30% (32) and that choline supplementation resulted in a 5 min faster finishing time. However, the study was only published as an abstract. Full data are not available, and hence it is difficult to judge the validity of these results. In a more recent study (1) it was observed that 2 h of cycling at a speed of 35 km/h resulted in a fall of plasma choline by 16.9%. Warber (38), however, did not observe any effect on circulating choline levels in soldiers after 4 h of exhausting uphill walking with a 34-kg backpack nor did Spector (34) during short-term all-out cycling (about 2 min) or endurance cycling until exhaustion (72 min). Thus, the available literature is inconclusive with respect to the observation that prolonged physical stress reduces plasma choline. It may be that the combination of

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type, duration, and intensity of exercise is an important determinant in this respect. The cause of a reduction in circulating choline during exercise, when present, has not been elucidated thus far. There is some speculation about a shift into HDL that have been shown to be increased as a result of physical training and might, as such, be a choline sink. (44) However, it is doubtful that HDL synthesis will be increased during exhaustive physical work when protein synthesis is known to be inhibited and mobilization of substrates from glycogen, fat, and protein is increased. Also, a decreased release of choline from the liver, by a reduced blood flow during exercise has been suggested. However, the production and release of free choline from the liver is relatively small and may not explain a drop of plasma choline by 40% in only a few hours

Aspects of Choline Intake and Supplementation Choline Intake and Absorption Generally, choline from the diet (mainly as PC) has been shown to be absorbed rapidly in resting conditions. No data on the effects of exercise are available. It is suggested that the PC or lyso-PC is, in a dose-response manner, more rapidly taken up into blood and also into the brain, resulting in a larger enhancement of brain AC levels compared to salt choline chloride. (19,40). Choline-chloride is dose dependently partially degraded in the gut to di- and trimethylamine (15,46) The latter compounds cause a bad odor and are toxic (19). Accordingly, the source of choice for choline supplementation should be PC, which does not give rise to these compounds. In the gut PC is hydrolyzed by pancreatic lipase A2, resulting in the production of lyso-PC and a fatty acid. Both are taken up by the enterocyte, which reesterifies them to PC, or degraded to choline and glycerophosphate. PC is subsequently used for “chylomicron coating” and will enter the lymphatic system. The lyso-PC will be bound to albumin to be released in blood. Seventy percent of an oral dose of PC is absorbed within 3 h (23,24,43). It is not known whether exercise itself effects pancreatic lipase A2 release and subsequent PC hydrolysis, PC re-esterification in the enterocyte, or the rate of absorption of PC ingested during ultra-endurance exercise. However, available data suggest that ingestion and subsequent absorption do not seem to be a limitation in augmenting choline availability to the body. Effects of Supplementation on Performance Only a limited number of studies on the effects of choline supplementation on physical performance have been performed. Von Allwörden et al. (1,2) supplemented 10 triathletes with a dose of 0.2 g lecithin (this was 90% purified PC) 1 h before exercise. This resulted in the maintenance of pre-exercise plasma choline levels compared with the placebo group, which showed a decrease of 16.9%. Without exercise, the supplementation resulted in a 26.9% increase of plasma choline. Performance was not evaluated in this study.

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Two other studies were presented as a poster in a workshop and appeared in the workshop proceedings only: (i) Pascetta et al. (31) supplemented 45 college basketball players with 4.86 g/d of choline bitartrate or placebo for seven days in a bouble-blind, placebo-controlled, crossover experiment. After seven days of training (two sessions per day) pre-exercise fatigue was monitored by a self-administered profile of mood states (POMS). Pre-exercise fatigue on day 7 was found to be significantly decreased and vigor to be increased with the choline treatment. Performance was not measured in this study. (ii) Coates and Pascetta (9) supplemented 16 swimmers (9 males and 7 females) with either choline citrate (5.674 g) or placebo for five days in a randomized, crossover, placebo-controlled study. Plasma choline levels decreased with placebo by >20% and increased with choline citrate >25%. Fatigue and vigor levels after five days were determined with POMS and were reduced in the control trial and increased with the choline treatment, respectively. Performance of a 30-min all-out freestyle swim test was significantly improved by choline supplementation. These two observations show that prolonged choline supplementation either as PC or choline salt, does increase plasma choline during exercise and suggest that this may reduce subjective fatigue and increase performance. However, interpretation of these data should be done with great care since none of these studies were performed in controlled laboratory settings and the performance tests used were not experimentally validated. Moreover, full details were never published as far as we know. More recently, two well-designed trials were published. Spector (34) studied the effect of choline bitartrate supplementation during exercise of short and long duration in a double-blind crossover design. The athletes in the short duration group (n = 10) ingested a beverage with or without choline bitartrate (2.34 g) one hour before exercise. In the long exercise trial 10 other cyclists ingested the same amount one hour before cycling about one hour at an intensity of 70% maximal oxygen consumption (VO2 max) at a pedal cadence of 80–90 rpm. Neither group depleted choline during exercise under choline or placebo treatment. Increases in plasma choline of 37 and 52% were seen within one hour of ingestion for short and long exercise, respectively. There were no differences in fatigue time and performance. It may be that the duration of this work was too short to ellicit effects on choline availability for AC synthesis or that the type of exercise may play a role on choline availability. Warber (38) investigated the effect of choline supplementation on physical performance in soldiers in heavy physical training. In a double-blind crossover design 14 volunteers received either a placebo beverage or a treatment drink containing 8.425 g of choline citrate (equivalent to 3 g free choline) prior to and midway through a 4-h load carrier treadmill exercise (3% grade at 5.6 km/h, 20 km) carrying a total of 34.1 kg. Following this exercise all subjects performed a treadmill run ìtime to exhaustion testî as well as a squats test as a measure of muscle strength endurance capacity. Perceived exertion, circulating choline levels, and performance were measured. Plasma choline levels remained unchanged in the control group and rose by 128% in the choline-supplemented group. None of the metabolic, performance, or recovery parameters were affected by the choline supplementation.

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Conclusions Based on the totality of the available evidence it can be concluded that • Choline is the precursor for the synthesis of the neurotransmittor AC. • A decline in circulating choline has been observed during some forms of intensive cyclic endurance exercise (marathon running, cycling) of long duration (>2 hours) but not during short-term exercise (3–72 min) or during exhausting military march exercise. • AC release has been shown to decrease significantly during a choline depleting diet that lasted three weeks, and to a similar degree a decrease was observed in some exercise studies. However, it may be that a choline depleting diet results in a cellular effect that differs from that induced by a short-term exercise-induced decrease. • Decreases in circulating serum choline levels have been suggested to play a role in the development of muscular fatigue, but the evidence is limited to muscle preparations. • Choline or lecithin (PC) supplementation has been shown to counteract a decrease in circulating choline levels during exercise. • However, currently there are no data from well-controlled studies to support a beneficial effect of choline or lecithin/PC supplementation on performance or recovery from it. • Effects of choline and PC on cell neuronal and muscle cell function during exhausting exercise in humans have not been studied to our knowledge. • Accordingly the only claim that can be supported thus far is that choline supplementation effectively compensates for a decline in circulating cholines if this occurs. The meaning of this with respect to performance is unclear thus far. Phosphatidyl Serine for Athletic Stress Reduction Recently, products containing PS have been put on the market with claims related to a decreased release of stress hormones and suggestions that this may be of benefit to physical performance capacity and recovery. This development may be related to two studies by Monteleone (28,29). This group supplied bovine brain cortex-derived PS to eight healthy male subjects who underwent three experiments on a bicycle ergometer. Fifty and 75 mg PS was administered intravenously 10 min prior to the start of exercise of short duration. The authors observed a blunted adrenocorticotropin (ACTH) and cortisol response during and after exercise. Others have used these findings to state that PS reduces exercise-induced stress. However, such interpretations should be viewed with great care. First of all, the duration of the exercise period (20 min) was so short that one may question the

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fate of the injected PS. Can such a short time really allow PS to incorporate in the cell membrane phospholipids pool in the central nervous system? One would assume that the PS would be distributed in the entire body and would enter the phospholipid pool in all cells. In that case a single dose of 50 or 75 mg PS would most probably be too small to exert any measurable effect on brain PS content. Secondly, the intensity of the exercise was low, as can be concluded from the low heart rates that did not exceed 140 beats/min. As such, the relevance of these findings remains unclear. Intensive physical exercise usually results in heart rates exceeding 170 beats/min and a hormonal system that is regulated to shift to an enhanced release of stress hormones. The latter is required for a redistribution of blood flow and for the mobilization of substrates from glycogen and fat pools. One may suggest that reducing the secretion of stress hormones may negatively impact substrate mobilization and ATP resynthesis rate. In a second study the same research group administered 800 mg/d of brain cortexderived PS to nine healthy men during a period of ten days. Again, ACTH and cortisol responses were observed to be reduced. The authors concluded that PS counteracts stress-induced activation of the hypothalamo-pituitary-adrenal axis. Also these data point to a possible role of PS in stress tolerance related to physical work. However, no other studies from other laboratories are available to validate these results. It is unclear whether stress hormone reduction during exercise may have any positive effect on athletic performance. Controlled performance experiments have not been performed thus far. The PS used in the studies of Monteleone and in most studies with elderly subjects concerns brain cortex-derived PS, which has never been launched on the market for reasons related to the Bovine Spongioform Encephalopathy (BSE) threat. Instead, use is made now of soy lecithin-derived PS (by enzymatic treatment). Efficacy of the latter in reducing stress hormone reductions or stress-related mental parameters in athletes or in elderly subjects has not been proven thus far (21). Accordingly, data from studies with non-brain cortex PS are required to substantiate benefit claims on PS in terms of stress reduction and performance in athletes. Very recently, Benton (3) studied the effect of soy lecithin-derived PS on mood and heart rate during and after an acute stressing arithmetic mental task in young adults with high neuroticism scores. Mood was found to be improved, but no effects on heart rate, as indicator of stress, were observed. According to these observations and taking into account the totality of the available evidence, it seems premature to make benefit claims related to enhanced performance or improved recovery. Clearly more studies focusing on the benefit endpoints are required to substantiate such claims. Some Directions for Future Research • What are the determinants of a depletion/reduction of circulating choline levels during exhaustive exercise? The observations that long endurance runs decrease the circulating choline levels but that short-term exercise, endurance

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exercise lasting about 70 min, or intensive walking or cycling exercise have no significant effect on serum choline levels points to a relationship between exercise type, intensity, and duration and choline requirements. Does exhausting exercise reduce free choline and PC levels and/or AC release in muscle and does choline supplementation induce an increased AC release during exercise-induced chronic stimulation of the neuromuscular system in favor of maintaining high-speed neuromuscular impulse transmission or a larger number of motor units that are able to be recruited at the same time in favor of a greater peak power contraction? Does choline supplementation effect impaired mental/cognitive processes that have been observed to coincide with exhausting all-out endurance events (e.g., by influencing brain precursor availability)? Can AC release during exhausting physical performance be influenced by supplying PC or EFA along with choline compared to choline salts alone? The observation that muscle membrane phospholipid contents effect insulin sensitivity may be very important for both athletes and type II diabetics. Studies on the effects of chronic lecithin supplementation on insulin action, substrate mobilization, and utilization and ultra-endurance performance would be in place The role of PS in metabolism related to physical stress is largely unknown. Generally, the role of PS is suggested to be related to cell membrane function. As such, the metabolic fate of ingested PS should be addressed to show that its concentration in target cells will increase with supplementation. The observed reduction in red blood cell membrane PS after physical loads requires further attention. Are similar effects present in neural cells? What are the effects of PS on stress hormones in real all-out exercise and on performance? The available studies on PS and exercise have been done with brain cortexderived PS. What are the effects with the currently available soy lecithinderived PS. Are there any differences in bioavailability, in membrane incorporation, or in functionality? Over-training studies in athletes, using stable isotopes and muscle biopsy techniques, may be an appropriate model to study the effects of phospholipids on muscle function and fatigue. Alternatively, such exhausting exercise studies can be done well using horses.

These questions may be addressed in future research References: 1. Von Allwörden, H.N., Horn, S., Kahl, J., and Feldheim, W. (1993) The Influence of Lecithin on Plasma Choline Concentrations in Triathletes and Adolescent Runners During Exercise, Eur. J. Appl. Physiol. 67, 87–91. 2. Von Allwörden, H.N., Horn, S., and Feldheim, W. (1995) The Influence of Lecithin on the Performance and the Recovery Process of Endurance Athletes, in Phospholipids: Characterization, Metabolism and Novel Biological Applications, Cevet, G., and Pallauf, F., AOCS Press, Champaign, IL, pp. 319–325.

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22. Krnjevic, K., and Miledi, R. (1959) Presynaptic Failure of Neuromuscular Propagation in Rats, J. Physiol. 149, 1–22. 23. Lekim, D., and Betzing, H. (1967) Intestinal Absorption of Polyunsaturated Phosphatidylcholine in the Rat, Hopp-Seyler's Z. Physiol. Chem 357, 1321–1331. 24. Lekim, D. (1981) The Resorption of Lecithin Administered Orally and Its Physiologic Implications, in Soya Lecithin: Nutritional and Clinical Aspects, Societa Editrice Universo, Rome, pp. 21–33. 25. Liley, A.W., and North, K.A.K. (1952) An Electrical Investigation of Effects of Repetitive Stimulation on Mammalian Neuromuscular Junction, J. Neurophysiol. 16, 509–527. 26. MacIntosh, F.C., and Collier, B. (1976) The Neurochemistry of Cholinergic Terminals, in Handbook of Experimental Pharmocology of Neuromuscular Junction, Springer Verlag, Berlin, pp. 99–228. 27. Maire, J.-C., and Wurtman, R.J. (1985) Effects of Electrical Stimulation and Choline Availability on Release and Contents of Acetylcholine and Choline in Superfused Slices from Rat Striatum, J. Physiologie 80, 189–195. 28. Monteleone, P., Maj, M., Beinat, L., Natale, M., and Kemali D. (1992) Blunting by Chronic Phosphatidylserine Administration of the Stress Induced Activation of the HypothalamoPituitary-Adrenal Axis in Healthy Men, Eur. J. Clin. Pharmacol. 42, 385–388. 29. Monteleone, P., Beinat, L., Tanzillo, C., Maj, M., and Kemali, D. (1990) Effects of Phosphatidylserine on the Neuroendocrine Response to Physical Stress in Humans, Neuroendocrinology 52, 243–248. 30. Pagala, M.K.D., and Namba, T. (1984) Failure of Neuromuscular Transmission and Contractility During Muscle Fatigue, Muscle Nerve 7, 454–464. 31. Pascetta, A., Fogel, K., Herenda, G., Blaney, G., Baker, B.J., and Sullivan, F. (1995) The Effect of Choline on Fatigue and Energy Levels in College Basketball Players (poster 35), Proceedings Workshop: Nutrition and Physical Activity to Optimize Performance and Well-Being, April 5–7, 1995, Buckhead, Atlanta, Georgia. 32. Sandage, B.W., Sabounjian, L., White, R., and Wurtman, R.J. (1992) Choline Citrate May Enhance Athletic Performance, Physiologist 35, 236. 33. Shepard, N.F., Tayek, J.A., Bistrian, B.R., Blackburn, G.L., and Zeisel, S.H. (1986) Plasma Choline Concentration in Humans Fed Parenterally, Am. J. Clin. Nutr. 43, 219–224. 34. Spector, S.A., Jackman, M.R., Sabounjian, L.A. Sakkas, C., Landers, D.M., and Willis, W.T. (1995) Effect of Choline Supplementation on Fatigue in Trained Cyclists, Med. Sci. Sports Exerc. 27, 668–673. 35. Sumikawa, K., Mu, Z., Inoue, T., Okochi, T., Yoshida, T., and Adachi, K. (1993) Changes in Erythrocyte Membrane Phospholipid Composition Induced by Physical Training and Physical Exercise, Eur. J. Appl. Physiol. Occup. Physiol. 67, 132–137. 36. Sved, A.F. (1983) Precursor Control of the Function of Monoaminergic Neurons, in Nutrition and the Brain, Wurtman, R.J., Raven Press, New York, pp. 223–275. 37. Tucek, S. (1978) Acetylcholine Synthesis in Neurons, Champman and Hall, London. 38. Warber, J.P., Patton, J.F., Tharion, W.J., Zeisel, S.H., Mello, R.P., Kemnitz, C.P., and Lieberman, H.R. (2000) The Effect of Choline Supplementation on Physical Performance, Int. J. Sport Nutr. Exerc. Metab. 10, 170–181. 39. Wecker, L. (1988) Influence of Dietary Choline Availability and Neuronal Demand on Acetylcholine Synthesis by Rat Brain, J. Neurochem. 51, 497–504. 40. Wurtman, R.J., Hirsch, M.J., and Growdon, J.H. (1977) Lecithin Consumption Raises Serum-Free-Cholin Levels, Lancet ii, 68–69.

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41. Wurtman, R.J., Hefti, F., and Melamed, E. (1981) Precursor Control of Neurotransmitter Synthesis, Pharmacol. Reviews 32, 315–335. 42. Wurtman, R.J. (1985) Alzheimer's Disease, Scient. Am. 252, 62–75. 43. Kim, L., and Benzig, H. (1984) Uptake and Metabolism of Polyene Phosphatidylcholine in Rat Brain, Arznei Mittel forschung/Drug Res. 34, 557–559. 44. Wurtman, R.J., and Lewis, M.C. (1991) Exercise, Plasma Composition, and Neurotransmission, in Advances in Nutrition and Topsport, Brouns, F., Medicine and Sport Science, Karger, Basel, vol. 32, pp. 99–104. 45. Xia, N. (1991) Effects of Dietary Choline Levels on Human Muscle Function, Boston University College of Engineering. 46. Zeisel, S.H, DaCosta, K.A., Mervat, Y., and Hensey, S. (1989) Conversion of Dietary Choline to Trimethylamine and Dimethylamine in Rats: Dose-Response Relationship, J. Nutr. 119, 800–804. 47. Zeisel, S.H., DaCosta, K.A., Franklin, P.D., Alexander, E.A., and Lamont, J.T. (1991) Choline, an Essential Nutrient for Humans, FASEB J 5, 2093–2098. 48. Zeisel, S.H., and Canty, D.J. (1993) Choline Phospholipids: Molecular Mechanisms for Human Disease: A Meeting Report, J. Nutr. Biochem. 4, 258–263. 49. Zeisel, S.H. (1993a) Choline Phospholipids: Signal Transduction and Carcinogenesis, FASEB J 7, 551–557. 50. Zeisel, S.H., and Blusztajn, J.K. (1994) Choline and Human Nutrition, Annu. Rev. Nutr. 14, 269–296. 51. Zeisel, S.H., Crowdon, J.H., Wurtman, R.J., Magil, S.G., and Logue L. (1980) Normal Plasma Choline Responses to Ingested Lecithin, Neurology 30, 1226–1229.

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Chapter 13

Effects of Phosphatidylcholine Intake on Liver Function and Liver Carcinogenesis David J. Canty Department of Nutrition and Food Studies, New York University, New York, New York

Introduction The role of choline in liver function and preventing liver carcinogenesis has been a major theme in the history of choline research and is the primary basis for human and animal dietary requirements for choline. Choline is unique in that it is the only nutrient for which a deficiency is directly linked to liver cancer in the absence of a carcinogen. The functions of choline in the liver also apply to phosphatidylcholine (PC, also referred to here as “lecithin”), which supplies about 60% of the choline in the diet. In addition, specific forms of lecithin appear to play other unique roles in protecting the liver from alcohol injury and, possibly, other states of liver injury.

History Choline was first recognized as present in mammalian tissues by Strecker in 1862 (1), but its importance as an essential nutrient and its function in the liver weren’t realized until studies on insulin in the 1930s (2,3). Though given insulin therapy, depancreatized rats and dogs nevertheless developed fatty livers and died; but when raw pancreas was administered, its lecithin content prevented fatty liver. As a result, choline and other substances that prevented fatty liver were designated “lipotropic.” In the 1940s research accumulated showing that choline deficiency produced cancer in several organ systems of rats and other species. Further investigations established the nutritional need for choline in many different species. In addition to fatty liver, choline deficiency in animals causes abnormal kidney function, infertility, growth impairment, bone abnormalities, decreased hematopoiesis, and hypertension (4,5). In the 1980s research diversified into several areas and continues today, focusing on the role of choline in methyl group metabolism, cell signaling, liver function and carcinogenesis, as well as memory, physical performance, cardiovascular health, and other areas (4–6). Choline was not recognized as essential for humans until 1998, when the United States Food and Nutrition Board of the National Academy of Sciences, which has established the recommended dietary allowances (RDA) for nutrients since 1941, established the first dietary reference intakes (DRI) for choline based

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on demonstration of a requirement for choline to maintain normal liver function in adult men (7).

Dietary Sources and Intake Chemically, choline is hydroxyethyl trimethyl ammonium hydroxide. PC, on average, contains about 13.5% choline by weight (Fig. 13.1). Granulated “commercial” lecithin is a mix of phospholipids extracted from soybeans and contains about 23% PC. One tablespoon of commercial lecithin granules provides about 1725 mg of PC and 250 mg of choline, a little less than the content in an egg. One capsule of commercial lecithin provides about 180 mg of PC and 24 mg of choline. Although lecithin and choline are present in a wide variety of foods, the richest sources tend to be foods high in cholesterol and fat. Though no major dietary surveys have included lecithin or choline, healthy adults in the United States are estimated to consume about 730 to 1040 mg per day of choline (8). These estimates, however, were based on food consumption data in the 1970s. Because consumption of fatty foods has decreased since then, the intake of lecithin and choline may be less than optimal (9). Lecithin appears to provide a more bioavailable source of choline than choline salts. This has been shown in studies in which lecithin sustained plasma choline at a higher level for a longer period of time compared to choline chloride (10–12). Lecithin is probably more bioavailable because less of its choline is lost to the formation of di- and trimethylamine by intestinal bacteria (13,14 ).

An Essential Nutrient Several lines of evidence support the idea that choline is essential for humans. Choline is required by human cells grown in culture, plays an important role in methyl group metabolism and cell signaling, is required for fetal and infant development, and deficiency produces abnormal liver function, even after just a few weeks. Although the Food and Nutrition Board used to define nutrient requirements as RDA, in 1998 they expanded the concept to DRI. This broader term includes CH2—Fatty acid | CH—Fatty acid | CH2—Fatty acid

CH2—Fatty acid | CH—Fatty acid | CH2OPO2-OCH2CH2N+(CH3)3

HOCH2CH2N+(CH3)3

Triglyceride

Lecithin (phosphatidylcholine)

Choline

(Average MW = 773)

(MW = 104.17)

Fig. 13.1. Molecular structures for a triglyceride, lecithin, and choline.

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RDA as well as “estimated average requirements” (EAR), the intake levels that meet the nutritional needs of 50% of individuals in different populations (e.g., various age and sex groups). An RDA is a nutrient intake value two standard deviations above the EAR and, therefore, meets the needs of about 98% of a given population. The DRI also include upper tolerable intake levels, representing the estimated highest amount that can be consumed without causing adverse effects. Rather than determining EAR or RDA for choline, the Food and Nutrition Board designated levels of “acceptable intake” (AI) because the critical data showing early signs of liver dysfunction with choline deficiency only existed for one population group: adult men. There was no dose-response data on the men to determine an exact requirement and no information on the effects in other population groups, such as women and children (7). Choline AIs range from 125 mg/d (for infants 0–6 months) to 550 mg/d (for males 14–70 years and lactating women); tolerable upper inakes are about 6 to 7fold higher (7).

Choline Deficiency: Findings in Animals More than 50 years ago it was shown that choline deficiency resulted in cancer in the liver and other organs in rats. Choline-deficient animals typically progress from fatty liver to fibrosis and, in many cases, to cancer. This has been definitively shown in studies by da Costa and coworkers: 15% of rats fed a choline-deficient diet developed liver cancer after one year and more than half had atypical hepatic foci; no control animals developed atypical foci or liver cancer (15,16). It is important to note that although animal studies suggest there is a progression from fatty liver to cirrhosis to cancer, these are probably somewhat distinct processes. Whereas severe choline deficiency induces fatty liver and cirrhosis, the enhancement of carcinogenesis requires only moderate deficiency that may not induce cirrhosis. In fact, severe choline or methyl group deficiency does not appear to promote hepatocarcinogenesis; it appears to arrest tumor cell cycling and increase tumor vulnerability to certain chemotherapeutic agents (17).

Choline Deficiency: Findings in Humans It has been known for some time that patients given total parenteral nutrition (TPN) formulas devoid of choline develop fatty liver. Evidence suggests TPNassociated liver disease occurs in more than 60% of long-term TPN patients, and end-stage liver disease occurs more commonly with TPN than previously thought, affecting 15–40% of patients (18). However TPN-related fatty liver can be prevented and reversed with supplemental choline (19). Choline has not been traditionally included in TPN formulas because it was thought—just as with healthy humans—that adequate amounts were synthesized

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through the hepatic transulfuration pathway. However, studies as far back as the 1970s and 1980s have shown this pathway is impaired or inactive in patients receiving intravenous nutrients. Alan Buchman and colleagues definitively showed that choline supplementation corrects fatty liver and liver dysfunction associated with TPN (18–20). One study (18) enrolled 15 patients who had been receiving TPN for more than 80% of their nutritional needs for an average of 12 years and had documented hepatic steatosis but were otherwise healthy. They were then randomized to receive their usual TPN or TPN + 2 g choline chloride daily for 24 weeks. CT scans showed the density of the livers increased in the choline-supplemented group compared to the nonsupplemented group after only four weeks: 13.3 versus 5.8 Hounsfield units, respectively (P = 0.04). Similarly, serum levels of liver function enzymes—alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AP)—showed significant improvements in the course of the study. These enzymes are used as markers of liver injury. The key data used by the Food and Nutrition Board in determining that choline was an essential nutrient was from a study by Steven Zeisel and co-workers (21). In a contolled hospital environment, 16 healthy adult male volunteers were fed a semisynthetic diet devoid of choline and given 500 mg supplemental choline per day for one week. Continuing on the choline-deficient diet, they were then randomly assigned to either continue receiving supplemental choline or no supplementation for three weeks. During that period, mean plasma choline concentration decreased 30%, and serum ALT increased by 52% (both changes were significant) among participants on the choline-deficient diet. These parameters normalized after choline supplementation was resumed at week 5.

Roles in Liver Function: Overview Choline serves two main biochemical roles in liver function: participation in methyl group metabolism and as the key component of lecithin. Choline is a major source of labile methyl groups, estimated to contribute about 60%, with much of the rest contributed by methionine. As described below, an inadequate supply of labile methyl groups can activate a number of mechanisms that can lead to cancer. Lecithin is an obligatory component for the manufacture of very-low-density lipoproteins (VLDL). Indeed, the long-recognized development of fatty liver during choline deficiency is thought to be largely due to the diminished capacity for the liver to manufacture and export triglycerides on VLDL, resulting in triglyceride accumulation. Lecithin is also an obligatory component of the enzyme lecithin-cholesterol acyl transferase, which allows high-density lipoproteins (HDL, the “good” cholesterol) to function in reverse cholesterol transport as a “clean-up crew,” taking cholesterol out of blood vessel walls and peripheral tissues and back to the liver or to other lipoproteins.

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Lecithin and other choline phospholipids are critical components in maintaining cell membrane integrity. They are also intimately involved in transmembrane signaling, serving as second messengers in signaling cascades.

Methyl Group Metabolism Fig. 13.2 shows how choline fits in methyl group metabolism. This is only a portion of the pathways involved, but it highlights some important points: • Methyl groups are provided through the diet mainly by choline and methionine and are "recycled" by folate and B12. Thus, the requirements for all these nutrients are inter-dependent. • A key end-product is S-adenosylmethionine (SAM), which provides methyl groups for methylation reactions, including DNA methylation and de novo synthesis of lecithin from phosphatidylethanolamine (PtdEtn). SAM is generated from methionine, which in turn can be generated from homocysteine through methyl donation from folate and B12, or from betaine, an oxidized choline metabolite. • With an inadequate intake of choline, lecithin can be synthesized de novo from PtdEtn and SAM, producing S-adenosylhomocysteine, which is converted to homocysteine, an atherogenic amino acid (reactions not shown). During choline deficiency, more SAM is shunted toward the synthesis of lecithin, which is required for membrane integrity and the choline it provides. That drains SAM and methionine, resulting in hypomethylation of DNA, which may lead to greater expression of growth-related genes and oncogenes. The diminished levels of lecithin lead to impairment of membrane function, signal transduction, and hepatic VLDL production and export, leading to fatty liver. Collectively, these conditions set the stage for the development of fatty liver and liver carcinogenesis.

Fig. 13.2. Methyl group metabolism.

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Cell Signaling Choline phospholipids, including lecithin, phosphatidylinositol, and sphingomyelin, reside in cell membranes and participate in cell signaling. In the signaling cascade, various phospholipases act upon these phospholipids, producing various second messengers, such as 1,2 diacylglycerol (DAG), arachidonic acid, ceramide, and sphingosine, which, in turn, modulate key regulatory enzymes, activate genes, and trigger processes, such as apoptosis (programmed cell death). Choline deficiency in rats has been shown to chronically over-produce DAG, which then over-stimulates a key regulatory enzyme, protein kinase C (PKC). This over-stimulation also may cause, downstream, increased expression of growth and tumor-related genes and increased cell growth and turnover, leading eventually to carcinogenesis (15,16).

Mechanisms of Hepatocarcinogenesis For obvious ethical reasons, no long-term deprivation studies have been performed in humans to determine if liver dysfunction progresses to hepatocarcinogenesis, as has been shown in animal studies. However, animal and in vitro studies have shed light on possible mechanisms of action of choline deficiency and the development of liver cancer, including the following: • The liver damage caused by choline deficiency causes accelerated regeneration, making the liver more vulnerable to chemical carcinogens that target rapidly reproducing cells. • The increased lipids in the liver could result in increased lipid peroxidation, in turn producing a greater load of DNA-damaging free radicals. • Choline or methyl deficiency may increase carcinogen activation to more potent species and/or limit their catabolism and excretion. In addition, various pathways may become activated that increase apoptosis during choline deficiency. The methyl group deficiency could result in hypomethylation of DNA, which could increase the activation of genes—including growth genes and oncogenes—and increase the amount of DNA that is more susceptible to damage. This would lead to a greater proliferation of abnormal cells and, as a result, increased apoptosis. This mechanism is a bit controversial, however, because decreased DNA methylation has been shown to occur late in choline deficiency after many cell changes have already happened. On the other hand, choline deficiency results in reduced synthesis of lecithin, reduced hepatic export of VLDL triglycerides, fat accumulation in the liver, and increased DAG levels. Normally, DAG would be re-esterified with phosphocholine, but in choline deficiency DAG accumulates, chronically over-stimulating PKC, as described above; this could increase the expression of genes promoting cell growth and turnover and, therefore, increased apoptosis.

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Regardless of the mechanism(s) involved, choline deficiency has been shown to be a potent inducer of apoptosis. Normally, apoptosis is thought to protect against cancer by triggering self-destruction of abnormal cells. However, some cells may adapt and become resistant to choline-deficiency-induced apoptosis (e.g., by developing a defect in their apoptotic pathways), escaping apoptotic elimination and progressing to cancer cells. Thus, just as chronic exposure of bacteria to an antibiotic can promote resistance, choline deficiency may create an environment with increased selective pressure for abnormal cells to develop resistance to apoptosis and progress to tumor cells (4,17). This hypothesis is supported by observations of a line of rat hepatocytes (CSWV-1) in which p53 protein is inactivated (22). P53 normally suppresses carcinogenesis, partly through apoptosis. When these cells were exposed to a lowcholine medium, they induced apoptosis despite lacking p53 protein. In 48 hours, only 1% of control group cells became apoptotic in normal medium containing 70 uM choline, compared to 16% of cells grown in choline-deficient medium (5 µM choline). After the CSWV-1 cells were gradually adapted to the choline-deficient medium, however, they became less vulnerable to choline-deficiency-induced apoptosis—only 7% of adapted cells became apoptotic compared to 19% of nonadapted cells. The adapted cells also exhibited increased anchorage-dependent growth and formed tumors when transplanted into nude mice. It is interesting to further note that, whereas choline deficiency is often equated with methyl deficiency, studies show when apoptosis is induced by choline, it is not suppressed by methyl supplementation from methionine, folate, B12, or even betaine, the choline metabolite. Thus, choline appears to fulfill specific, important functions not fulfilled by other methyl donors (4,23).

Lecithin: Additional Liver Protection Lecithin appears to play additional roles in protecting the liver, beyond merely supplying choline. Specifically, lecithin has been shown to protect the liver from damage due to alcohol and other agents (for a more detailed discussion of this topic, see Chapter 14 by Gundermann and Scheele). Charles Lieber’s group has performed a good deal of research on lecithin and liver protection from alcohol. In one study (24), baboons were fed either a control diet providing 100 µg/kcal choline or the same diet supplemented with 2.8 mg/kcal soy lecithin (94–96% pure PC) for up to eight years. Half the animals in each group received 50% of their calories from ethanol, and the other half received a calorically equivalent amount of carbohydrate. All animals given ethanol developed fatty liver, whether they received lecithin or not. However, 10 of the 12 animals fed the control diet with ethanol went on, over time, to develop septal fibrosis, and still later, 2 of those 10 developed cirrhosis; by contrast, no animals fed ethanol with supplemental lecithin developed septal

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fibrosis or cirrhosis (Fisher’s exact probability test, P < 0.001), though some developed a low-grade of earlier stage, perivenular fibrosis. A previous, similar study also showed that when animals in the lecithin + ethanol group were subsequently taken off lecithin, they rapidly progressed to more severe fibrosis (25). The reseachers also showed that one species of lecithin—dilinoleoylphosphatidylcholine (DLPC)—increased collagenase activity in vitro 60% over control values in hepatic lipocytes, the cells that can transform into collagen-producing cells (24). Further, lver biopsies showed that animals fed ethanol versus ethanol + lecithin had an average of 81% versus 48%, respectively, of hepatic lipocytes transformed into transitional cells that produce collagen. The investigators concluded that this PC species in particular may protect the liver by promoting hepatic collagen breakdown by collagenase and, possibly, decreasing collagen synthesis by transitional cells. A later study suggests it may protect also via antioxidant activity (26). These findings are especially interesting in light of the fact that previous studies by this group showed that alcoholic fibrosis was not prevented by even massive supplementation with choline (27) or methionine (28). Thus, there appears to be something unique to lecithin, particularly DLPC, beyond the contribution of choline, that protects the liver from alcoholic injury. It should be noted, however, that a small study showed that the choline metabolite betaine did improve nonalcoholic steatohepatitis (NASH) (29) Alcohol is known to have many effects on the liver, and additional findings suggest DLPC may protect against those assaults in a number of ways (30) in addition to increasing collagenase activity and decreasing collagen production: • Corrects deficiency in lecithin arising from ethanol-inhibition of PtdEtn methyltransferase. • Reduces oxidative stress. Interestingly, whereas polyunsaturated fats are generally more prone to oxidation, some evidence suggests DLPC may serve as an antioxidant (31). • Diminishes lipopolysaccharide-dependent generation of TNF-alpha, the overproduction of which can cause liver injury by activating Kupffer cells (32). • Decreases ethanol-induced increase in cytochrome P4502E1, which otherwise generates free radicals and promotes liver cell apoptosis (33,34). Lecithin has also been shown to protect the liver from injury due to carbon tetrachloride (35) and heterologous albumin, and other studies suggest it may be beneficial in treating hepatitis (36). A multicenter clinical trial of potential antifibrotic effects of DLPC in human alcoholics is near completion (Lieber, C.S., personal communication). Preliminary findings in a subset of heavy drinkers suggest a trend for DLPC to cause regression of fibrosis, improvement in serum levels of liver function enzymes, and a reduction in the incidence of ascites (37).

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Conclusions The evidence to date is fairly clear that: • Choline is required for normal liver function in humans and many animal species. • Choline serves multiple functions in the liver, including protection against carcinogenesis. • Lecithin protects the liver from alcoholic and, in some cases at least, nonalcoholic injury. • Lecithin serves multiple, unique functions as a liver protectant, beyond its contribution of choline. Some areas for further research include: • Quantitation of choline requirements for normal liver function among different populations; the only study to date was on adult men. • Interrelationships among choline, methionine, folate, B12, and B6 in liver function. These nutrients are metabolically linked and interdependent, so it’s difficult to know what the actual active agent is for a given beneficial or harmful effect. • The sequence of events and mechanism of action accounting for carcinogenesis in choline deficiency. Currently a good many links in the chain of events are known, but how they fit together—the sequence of their actions and their relative contributions to the carcinogenic process—is not understood. The need for greater understanding applies not only to the events following choline deficiency but also to the process of choline phospholipid signal transduction. • The mechanism of action of lecithin and choline as liver protectants. This would include determining whether DLPC is the only active form of PC and if lecithin’s protective effect comes, in part, from its contribution of choline or betaine. Whereas choline wasn’t effective in protecting against alcohol liver injury in baboons, might it be protective in humans, given the findings of benefit from betaine in NASH? Could betaine or choline act in NASH by sparing lecithin? Or is there something unique about the alcoholic state that makes lecithin effective and choline not? References 1. Strecker, A. (1862) Uber Einige Neue Bestandtheile der Schweingalle, Ann. Chem. Pharmacie 123, 353–360. 2. Best, C.H., and Huntsman, M.E. (1932) The Effects of the Components of Lecithin upon the Deposition of Fat in the Liver, J. Physiol. 75, 405–412. 3. Best, C.H., and Huntsman, M.E. (1935) Effect of Choline on Liver Fat of Rats in Various States of Nutrition, J. Physiol. 83, 255–274. 4. Zeisel, S.H. (1999) Choline and Phosphatidylcholine, in Modern Nutrition in Health and Disease, Shils, M.E., Olson, J.A., Shike, M., Ross, A.C., Williams & Wilkins, Baltimore, 513–523.

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5. Zeisel, S. (2000) Choline: An Essential Nutrient for Humans, Nutrition 16, 669–671. 6. Canty, D.J., and Zeisel, S.H. (1994) Lecithin and Choline in Human Health and Disease, Nutr. Rev. 52, 327–339. 7. Food and Nutrition Board, Institute of Medicine (1998) Choline, in Dietary Reference Intakes, Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin and Choline, Pitkin, R.M., Allen, L.H., Bailey, L.B., Bernfield, M., DeWals, P., Green, R., McCormick, D.B., Russell, R.M., Shane, B., Zeisel, S., and Rosenberg, I.H., Washington, D.C., National Academy Press, 390–422. 8. LSRO/FASEB (Life Sciences Research Office/Federation of American Societies for Experimental Biology) (1981) Effects of Consumption of Choline and Lecithin on Neurological and Cardiovascular Systems, PB-82-133257, Bethesda, MD, LSRO/ FASEB. 9. Wurtman, R.J. (1991) The Choline-Deficient Diet, FASEB J. 5, 2612. 10. Wurtman, R.J., and Hirsch, M.J., Growdon, J.H. (1977) Lecithin Consumption Raises Serum-Free-Choline Levels, Lancet 2, 68–69. 11. Hirsch, M.J., Growdon, J.H., and Wurtman, R.J. (1978) Relations Between Dietary Choline or Lecithin Intake, Serum Choline Levels, and Various Metabolic Indices, Metabolism 27, 953–960. 12. Zeisel, S.H., Growdon, J.H., Wurtman, R.J., Magil, S.G., and Logue, M. (1980) Normal Plasma Choline Responses to Ingested Lecithin, Neurology 30, 1226–1229. 13. De La Huerga, J., and Popper, H. (1951) Urinary Excretion of Choline Metabolites Following Choline Administration in Normals and Patients with Hepatobiliary Diseases, J. Clin. Invest. 30, 463–470. 14. de La Huerga, J., and Popper, H. (1952) Factors Influencing Choline Absorption in the Intestinal Tract, J. Clin. Invest. 31, 598–603. 15. da Costa, K.A., Cochary, E.F., Blusztajn, J.K., Garner, S.F., and Zeisel, S.H. (1993) Accumulation of 1,2-sn-Diradylglycerol with Increased Membrane-Associated Protein Kinase C May Be the Mechanism for Spontaeous Hepatocarcinogenesis in Choline Deficient Rats, J. Biol. Chem. 268, 2100–2105. 16. da Costa, K.A., Garner, S.F., Chang, J., and Zeisel, S.H. (1995) Effects of Prolonged (1 year) Choline Deficiency and Subsequent Re-Feeding of Choline on 1,2-sn-Diradylglycerol, Fatty Acids and Protein Kinase C in Rat Liver, Carcinogenesis 16, 327–334. 17. Rogers, A.E. (1995) Methyl Donors in the Diet and Responses to Chemical Carcinogens, Am. J. Clin. Nutr. 61, 659S–665S. 18. Buchman, A.L., Ament, M.E., Sohel, M., Dubin, M., Jenden, D.J., Roch, M., Pownall, H., Farley, W., Awal, W., and Ahn, C. (2001) Choline Deficiency Causes Reversible Hepatic Abnormalities in Patients Receiving Parenteral Nutrition: Proof of a Human Choline Requirement, A Placebo-Controlled Trial, JPEN 25, 260–268. 19. Buchman, A.L., Dubin, M., Jenden, D., Moukarzel, A., Roch, M.H., Rice, K., Gornbein, J., and Ament, M.E. (1992) Lecithin Increases Plasma Free Choline and Decreases Hepatic Steatosis in Long-Term Total Parenteral Nutrition Patients, Gastroenterology 102, 1363–1370. 20. Buchman, A.L., Dubin, M.D., Moukarzel, A.A., Jenden, D.J., Roch, M., Rice, K.M., Gornbein, J., and Ament, M.E. (1995) Choline Deficiency: A Cause of Hepatic Steatosis During Parenteral Nutrition That Can Be Reversed with Intravenous Choline Supplementation, Hepatology 22, 1399–1403. 21. Zeisel, S.H., da Costa, K.A., Franklin, P.D., Alexander, E.A., Lamont, J.T., Sheard, N.F., and Beiser, A. (1991) Choline: An Essential Nutrient for Humans, FASEB J. 5, 2093–2098. 22. Zeisel, S.H., Albright, C.D., Shin, O.H., Mar, M.H., Salganik, R.I., and da Costa, K.A. (1997) Choline Deficiency Selects for Resistance to p53-Independent Apoptosis

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23.

24.

25. 26.

27.

28. 29.

30. 31.

32.

33. 34.

35. 36.

37.

and Causes Tumorigenic Transformation of Rat Hepatocytes, Carcinogenesis 18, 731–738. Shin, O.H., Mar, M.H., Albright, C.D., Citarella, M.T., da Costa, K.A., and Zeisel, S.H. (1997) Methyl-Group Donors Cannot Prevent Apoptotic Death of Rat Hepatocytes Induced by Choline-Deficiency, J. Cell Biochem. 64, 196–208. Lieber, C.S., Robins, S.J., Li, J., De Carli, L.M., Mak, K.M., Fasulo, J.M., and Leo, M.A. (1994) Phosphatidylcholine Protects Against Fibrosis and Cirrhosis in the Baboon, Gastroenterology 106, 152–159. Lieber, C.S., De Carli, L.M., Mak, K.M., Kim, C.-I., and Leo, M.A. (1990) Attenuation of Alcohol-Induced Hepatic Fibrosis by Polyunsaturated Lecithin, Hepatology 12, 1390–1398. Lieber, C.S., Leo, M.A., Aleynik, S.I., Aleynik, M.K., and De Carli, L.M. (1997) Polyenylphosphatidylcholine Decreases Alcohol-Induced Oxidative Stress in the Baboon, Alcoholism, Clin. Exp. Res. 21, 375–379. Lieber, C.S., Leo, M.A., Mak, K.M., De Carli, L.M., and Sato, S. (1985) Choline Fails to Prevent Liver Fibrosis in Ethanol-Fed Baboons but Causes Toxicity, Hepatology 5, 561–572. Lieber, C.S., Leo, M.A., Mak, K.M., De Carli, L.M., and Sato, S. (1974) An Experimental Model of Alcohol Feeding and Liver Injury in the Baboon, J. Med. Primatol. 3, 153–163. Abdelmalek, M.F., Angulo, P., Jorgensen, R.A., Sylvestre, P.B., and Lindor, K.D. (2001) Betaine: A Promising New Agent for Patients with Nonalcoholic Steatohepatitis: Results of a Pilot Study, Am. J. Gastroenterol. 96, 2711–2717. Lieber, C.S. (2000) Alcohol: Its Metabolism and Interaction with Nutrients, Annu. Rev. Nutr. 20, 395–430. Aleynik, S.I., Leo, M.A., Takeshige, U., Aleynik, M.K., and Lieber, C.S. Dilinoleoylphosphatidylcholine Is the Active Antioxidant of Polyenylphosphatidylcholine, J. Investig. Med. 47, 507–512. Cao, Q., Mak, K.M., and Lieber, C.S. (2002) Dilinoleoylphosphatidylcholine Decreases LPS-Induced TNF-Alpha Generation in Kupffer Cells of Ethanol-Fed Rats: Respective Roles of MAPKs and NF-kappaB, Biochem. Biophys. Res. Commun. 294, 849–853. Aleynik, M.K., and Lieber, C.S. Dilinoleoylphosphatidylcholine Decreases EthanolInduced Cytochrome P4502E1, Biochem. Biophys. Res. Commun. 288, 1047–1051. Mi, L.J., Mak, K.M., and Lieber, C.S. Attenuation of Alcohol-Induced Apoptosis of Hepatocytes in Rat Livers by Polyenylphosphatidylcholine (PPC), Alcohol Clin. Exp. Res. 24, 207–212. Ma, X., Zhao, J., and Lieber, C.S. Polyenylphosphatidylcholine Attenuates Non-Alcoholic Hepatic Fibrosis and Accelerates Its Regression, J. Hepatology 24, 604–613. Archakov, A.I., Sel'tsovskii, A.P., Lisov, V.I., Tsyganov, D.I., Kniazhev, V.A., Ipatova, O.M., and Torkhovskaia, T.I. Phosphogliv: Mechanism of Therapeutic Action and Clinical Efficacy, Vopr. Med. Khim. 48, 139–153. Lieber, C.S., Weiss, D.G., Groszman, R., Paronetto, F., Schenker, S., Fye, C.L., Lowe, N., Feinman, L., Leo, M.A., Fimmel, C.J. et al. (2002) Effect of Moderation of Ethanol Consumption Combined with PPC Administration on Liver Injury in Alocholics: Prospective, Randomized, Placebo-Controlled, Multicenter VA TRial (CSP 391). Abstract 874. Presented at 2002 Annual Meeting of the American Association for the Study of Liver Diseases, Boston, MA, November 1–5.

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Chapter 14

Polyunsaturated Phosphatidylcholine in Chronic Liver Disease—Past and Present K.-J. Gundermanna and E.W. Scheeleb aDepartment

of Pharmacology and Toxicology, Pomeranian Medical Academy, Szczecin, Poland

bRadevormwald,

Germany

Polyunsaturated Phosphatidylcholine The term polyunsaturated phosphatidylcholine (PPC) indicates a highly purified extract of the semen of soybeans with a standardized content of 76 or 94% (3-snphosphatidyl)choline. PPC supplies the liver with nontoxic (3-sn-phosphatidyl)choline molecules with a high content of polyunsaturated fatty acids, in particular linoleic acid. Forty to fifty-two percent of the phosphatidylcholine molecules consist of 1,2-dilinoleoylphosphatidylcholine (DLPC) (1).

Mode of Action of PPC in Liver Disease The normal liver consists of about 300 billion hepatocytes. The total surface of all cellular and subcellular membranes amounts to approximately 33,000 square meters. At and in this membrane surface manifold biological reactions take place. Considerable disorders may occur due to toxic, inflammatory, allergic, metabolic, or immunological influences with subsequent morphological cell damages, which triggers a vicious circle. Liver damage is always associated with membrane damage, reduced phospholipid levels, and/or decreased membrane fluidity. Experimental and clinical results support the assumption that the therapeutic application of PPC has protective and even curative and regenerating effects on the biological membranes of sinus endothelial cells and hepatocytes. PPC allows damaged hepatocellular membrane structures to be restored quicker; membrane fluidity, immunomodulation, and cell protection are improved; membrane-associated liver function is enhanced; and peroxidative reactions are reduced, among others (2).

Pharmacology of PPC The hepato-protective and curative effect of PPC in the liver has been corroborated in 106 acute to chronic in vivo experiments (Table 14.1). Thirty different models with seven different animal species were investigated. These were either intoxica-

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TABLE 14.1 Cytoprotective Action of PPC in in vivo Investigations 106 Experiments

30 Different types of models

Intoxication with chemicals CCI4 CCI4 + Ethanol Ethanol + triton, INH/Rifampicin Cyanate Galactosamine Allyl alcohol Ethionine Organic solvents Carbon disulphide Thioacetamide Na-glutamate Hexachlorocyclohexane Ammoniumfluoride

Intoxication with drugs

acute/subacute chronic chronic acute/subacute chronic

17 10 1 14 16

acute/chronic acute/subacute acute subacute chronic chronic chronic chronic

4 9 5 1 2 1 1 1 1 1

chronic

7 Different species of animals

Paracetamol Tetracycline Rifampicin Cholic acid Indomethacin Choline deficiency Anesthetics INH Platidium ± CCI4 Reye syndrome

acute subacute subacute chronic acute subacute subacute subacute acute acute

Cholestasis intoxication Antigen-antibody-reaction Radiation-induced intoxication Lipid peroxidation by FeSO4 Endogenous oxidative stress

1 2 1 1 2 1 3 1 1 1 1 4 1 8 2 2

Status: December 2001.

tion with chemicals, such as carbon tetrachloride and ethanol, or intoxication with drugs, such as anaesthetics and tetracycline. Since the beginning of the 1990s, an increasing number of experimental investigations have been published, providing impressive evidence for the usefulness of PPC, especially in the field of alcoholic liver disease. In a first set of experiments, Lieber and co-workers demonstrated the influence of a lower purified phospholipid extract from soybean and, four to six years later, of PPC on alcoholic and non-alcoholic fibrogenesis in rats and baboons (1,3,4). Among others, 12 baboons each received a standard diet with or without an isocaloric replacement of carbohydrates by ethanol (1). Two further groups of animals received the same diets plus 2.8 g/1000 kcal PPC (n = 6 and n = 8). The treatment lasted 6.5 years on average. Noteworthy differences between the alcohol-treated groups were seen with respect to liver fibrosis: 10 of the 12 baboons fed alcohol without PPC developed septal fibrosis or cirrhosis. By contrast, none of the eight animals fed alcohol with PPC showed progression beyond the stage of perivenular fibrosis. Many (81 ± 3%) of the hepatic stellate cells transformed to collagen-producing myofibroblast-like cells in the control group, in comparison with 48 ± 9% when PPC was additionally administered. According to Poniachik et al. (5), DLPC seems to be the active ingredient. Brady et al. (6) isolated stellate cells from rats and stimulated these cells with platelet-derived growth factor with or without simultaneous use of PPC or DLPC. Both compounds inhibited in vitro the mitogen-induced stellate proliferation by

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inhibiting the mitogen-activated protein kinase signal transduction cascade and by reducing the binding of the transcription factor AP-1. In in vitro experiments with rat hepatic stellate cells, DLPC prevented the transforming growth factor-ß-1-induced accumulation of collagen type I (7). This effect was mediated through down-regulation of both α(I)procollagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression. The latter effect led to decreased TIMP-1 that, in the presence of unchanged matrix metalloproteinase13 (MMP-13) concentration, resulted in a larger MMP-13/TIMP-1 ratio, favoring collagen degradation and lesser collagen accumulation. Ethanol feeding also resulted in decreased liver phospholipids and phosphatidylcholines, both corrected by the supplementation with PPC (1). Part of these protective effects of PPC may be due to restoration of one of the key enzymes for phosphatidylcholine synthesis, the activity of the phosphatidylethanolamine-N-methyltransferase (8). The increase of hepatic DLPC from 0.075 ± 0.003 µmol/g in the ethanol group to 0.191 ± 0.032 µmol/g in the ethanol plus PPC group (control group: 0.120 ± 0.018 µmol/g) confirmed earlier pharmacokinetic investigations of Oette et al. (9), who showed that serum 1-linoleoylphosphatidylcholine is increased (in volunteers) by 32–40% after oral application of deuterium loaded DLPC. In 2000, Aleynik et al. (10) reported about a 1.4–2.5-fold postprandial increase of DLPC content in plasma and a 1.5-fold increase after six days of consecutive intake of 3 × 1.5 g PPC/d. As already mentioned, DLPC seems to be the main active ingredient in PPC. Only DLPC increased collagenase activity in vitro (1). Other investigated lipids, for example, saturated and monounsaturated phospholipids, free linoleate, or even 1.2dilinoleoylphosphatidylethanolamine, did not increase the activity of this enzyme. In 1997, Lieber and co-workers (11) showed that PPC strikingly attenuated ethanol-induced oxidative stress in liver tissue of baboons, as assessed by 4-hydroxynonenal (4-HNE), F2-isoprostanes (F2-IP), and glutathione, an effect associated with protection against alcohol-induced septal fibrosis and cirrhosis. F2-IP are derivatives of arachidonic acid and reflect in vivo peroxidation, and 4-HNE result from β-cleavage of fatty acid peroxides. These data and similar data with rats (12,13) confirmed earlier data with PPC and/or DLPC on CCl4-induced oxidative stress in rats (4). Last but not least, Navder et al. (14) showed that PPC, by markedly attenuating ethanolinduced increase in oxidation of LDL particles, apparently opposes one of the effects whereby alcohol promotes atherosclerosis (Fig. 14.1). The effects of PPC/DLPC are not limited to a correction of oxidative stress; these compounds were also effective in fibrosis and cirrhosis induced by injection of heterologous albumin in rats (an experimental model devoid of oxidative stress) (4). However, peroxidation belongs to the earliest events of liver damage and, consequently, has been intensively investigated (15). One way in which PPC opposes lipid peroxidation may be by inhibiting the activity of the cytochrome P450 2E1 (CYP2E1). Too much continuous alcohol consumption induces the microsomal ethanol detoxifying system (MEOS) and CYP2E1 as one

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Ethanol-fed baboons Lipid peroxides (nmoles/mg LDL protein ± SE)

Pair-fed controls Ethanol + PPC Controls + PPC

Fig. 14.1. Oxidation of LDL in baboons attenuated by polyunsaturated phosphatidylcholine (PPC) (14).

of its key enzymes, leading to too much reactive oxygen species, among others. Compared to linoleate from safflower oil, PPC and DLPC significantly and comparably decreased cytochrome b5, total cytochromes P450, CYP2E1 content, and its corresponding activities (Fig. 14.2) in rats (16,17). Furthermore, PPC and DLPC prevented the ethanol-diminished activity of mitochondrial cytochrome oxidase and the capacity of mitochondria to oxidize glutamate and palmitoyl-carnithine in rats (18). These results follow up those from Navder et al. from 1997 (19,20), in which mitochondrial dysfunction was corrected, too. PPC decreased postprandial VLDL-triglyceride and VLDL and LDL cholesterol levels, increased the HDL/LDL cholesterol ratio, and reduced alcoholic fatty liver formation in rats. Additionally, these results are consistent with those ones from Baraona et al. (21), who recently showed that PPC-attenuated chronic ethanol consumption increased activity of inducible nitric oxide synthetase and induced cytochrome P450 capable of producing both nitric oxide and superoxide. Consequently, PPC attenuated the associated increase in nitrotyrosine protein residues, products of peroxynitrite toxicity that occurred predominately in steatotic hepatocytes. Another approach to understanding the polyvalent action of PPC is to look into its influence on Kupffer cell activation. These cells not only induce stellate cell activation but also produce and release parameters of inflammation: chemokines, cytokines, eicosanoids, and adhesion molecules. DLPC selectively modulated lipopolysaccharide-induced Kupffer cell activation in vitro by decreasing the production of the

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Control Alcohol CYP2E1 content (pmol/mg protein) (mean)

PPC Alcohol + PPC

P < 0.001

P < 0.001

Fig. 14.2a. Inhibition of cytochrome P450 2E1 (CYP2E1) content by polyunsaturated

phosphatidylcholine (PPC) (17).

Control CYP2E1 activity (nmol/min/mg protein) (mean)

Alcohol PPC Alcohol + PPC

P < 0.001

P < 0.001

Fig. 14.2b. Inhibition of cytochrome P450 2E1 (CYP2E1) activity by polyunsaturated phosphatidylcholine (PPC) (17).

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cytotoxic tumor necrosis factor α (TNF-α) while increasing that of the protective interleukin-1β (22). Last but not least, DLPC showed effects on alcohol-induced apoptosis (23,24). Apoptosis is an active, inherently regulated, cell death program that enables organisms to actively eliminate damaged, senescent, or unwanted cells. The alcoholinduced apoptosis appears to be mediated by the induction of CYP2E1 and its generation of free radicals, which results in enhanced lipid peroxidation that initiates apoptosis. Furthermore, TNF-α is known to provide apoptotic signals to the liver cells, and mitochondria play an important role in the apoptotic pathways. DLPC down-regulated such alcohol induced apoptosis (Fig. 14.3). Table 14.2 summarizes the results of the Lieber’s study group.

Clinical Results with PPC Between 1978 and 1997, nine randomized double-blind trials were published to describe the clinical efficacy of PPC in comparison with placebo in patients with chronic liver disease. A formal meta-analysis of these double-blind studies was published in 1998 (25). This analysis summarized and compared subjective, clinical, biochemical, and histological data on the liver as well as responder rates, the differences in these responder rates, and the corresponding interference statistical evaluation, assessing an overall effect. The 409 hospitalized patients suffered from either chronic active hepatitis or fatty degeneration of the liver. Control

Percentage of apoptotic hepatocytes (mean ± SE, n = 7)

P < 0.01

Alcohol

P < 0.01

No PPC

+ PPC

Fig. 14.3. Effect of polyunsaturated phosphatidylcholine (PPC) on alcohol-induced apop-

tosis of hepatocytes in rats (23).

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TABLE 14.2 PPC/DLPC in Alcoholic Fibrogenesisa Results found by the study group of Lieber et al., New York • • • • • • • • • • •

Reduced fibrogenesis (no septal fibrosis, no cirrhosis); accelerated regression of pre-existing cirrhosis (1 model) Prevented or attenuated phosphatidylethanolamine-N-methyltransferase activity Reduced stellate cell activation/less myofibroblast-like cells Corrected phospholipid and phosphatidylcholine depletions Direct influence on membrane fluidity (hepatic DLPC increased) Regenerated mitochondrial, membrane-bound cytochrome oxidase activity; decreased hepatic fat accumulation Reduced oxidative and immunological stress by toxins such as ethanol and heterologous albumins Inhibited free radical generation via cytochrome P450 2E1 and by products of peroxynitrite hepatotoxicity Decreased activation of Kupffer cells by endotoxin Prevented collagen accumulation, by blocked transformation growth factor-B1 and stimulated collagenase activity; improved collagen degradation Attenuated apoptosis

aPPC,

polyunsaturated phosphatidylcholine; DLPC, 1-2,-dilinoleoylphosphatidylcholine.

(%)

The overall effect of these studies showed a mean difference in the responder rates by 26.6% in favor of PPC with a 95% confidence interval of 18.2 to 35.1% (P < 0.0001) (Fig. 14.4). This superiority in the responder rate clinically represents a significant dimension in liver disease, inasmuch as in chronic liver disease reliable parameters for the assessment of statistically significant alterations of progression or status of the disease are missing.

Fig. 14.4. Differences in responder rates of polyunsaturated phosphatidylcholine (PPC)

double-blind trials in chronic liver disease (means and 95% confidence intervals + lower/upper limits) (25).

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In 1998, Niederau et al. (26) published results of an international, multicenter, randomized, double-blind, placebo-controlled study about PPC and α-interferon (IFN) for treatment of chronic hepatitis B and C. Thirty-two study centers in Germany, Austria, Poland, and the Czech Republic were included in the trial. All patients received subcutaneous injections of 5 million I.U. IFN in hepatitis B or 3 million I.U. IFN in hepatitis C, thrice weekly for 24 weeks. Additional trial medication was either 3 × 2 capsules PPC/d (1.8 g/d) or 3 × 2 capsules placebo/d. After cessation of IFN therapy, the responders continued to receive PPC or placebo for a second sequence of 24 weeks; 176 patients completed the trial, 22/25 with chronic hepatitis B, and 70/59 with chronic hepatitis C. Positive results were observed for chronic hepatitis C; 50 of the 70 patients responded with alanine aminotransferase (ALT) decrease >50% (71%), when compared to patients who received placebo in addition to IFN (30 of 59 patients; 51%) (P = 0.016) (Fig. 14.5). No significant differences were seen in the patients with chronic hepatitis B. The percentage of patients with sustained, >50%, ALT reduction was 41% in the PPC group compared to 15% in the control group and approached statistical significance (P = 0.064). In conclusion, PPC increased the α-interferon response rate in chronic hepatitis C and reduced the relapse rate after α-interferon therapy. The PPC long-time therapy was well tolerated, too. In 2000, Lieber (27) reported his results of a first randomized, double-blind, placebo-controlled clinical trial in alcohol-induced hepatic fibrosis without complete cirrhosis. Eighteen alcoholic patients were randomized to receive either 3 × 1.5 g/d PPC chewable tablets or a corresponding placebo. Both groups continued to drink but showed high compliance with the protocol. Duration of treatment was two years. For the nine patients on PPC, liver histology was unchanged or slightly

Responder Nonresponder

Verum (n = 70)

Placebo (n = 59)

Fig. 14.5. Percent of responders and nonresponders in 129 patients with hepatitis (26).

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improved. However, five of the nine patients on placebo showed fibrotic progression on liver biopsy. In the meantime, a second, much larger double-blind trial at 17 Veterans Administration Medical Centers in the United States has finished, and the data have been evaluated. A paper has been submitted for publication.

Summary According to the published pharmacological data, PPC is effective in acute and chronic hepatic intoxication, especially in the field of alcoholic fibrogenesis. A first double-blind trial gives hope to treat patients with alcoholic fibrosis with an innocuous and polyvalent compound, even when they cannot stop drinking. A meta-analysis of nine of ten double-blind trials showed a significant overall effect of PPC on chronic liver disease. References 1. Lieber, C.S., Robins, S.J., Li, J., De Carli, L.M., Mak, K.M., Fasulo, J.M., and Leo, M.A. (1994) Phosphatidylcholine Protects Against Fibrosis and Cirrhosis in the Baboon, Gastroenterology 106, 152. 2. Kuntz, E., and Kuntz, H.-D. (2002) Hepatology—Principles and Practice, Springer Press, Berlin-Heidelberg, New York, pp. 789–791. 3. Lieber, C.S., DeCarli, L.M., Mak, K.M., Kim, C.-I., and Leo, M.A. (1990) Attentuation of Alcohol-Induced Hepatic Fibrosis by Polyunsaturated Lecithin, Hepatology 12, 1390. 4. Ma, X., Zhao, J., and Lieber, C.S. (1996) Polyenylphosphatidylcholine Attenuates NonAlcoholic Hepatic Fibrosis and Accelerates Its Regression, J. Hepatol. 24, 604. 5. Poniachik, J., Baraona, E., Zhao, J., and Lieber, C.S. (1999) Dilinoleoylphosphatidylcholine Decreases Hepatic Stellate Cell Activation, J. Lab. Clin. Med. 133, 342. 6. Brady, L.M., Fox, E.S., and Fimmel, C.J. (1998) Polyenylphosphatidylcholine Inhibits PDGF-Induced Proliferation in Rat Hepatic Stellate Cells, Biochem. Biophys. Res. Commun. 248, 174. 7. Cao, Q., Mak, K.M., and Lieber, C.S. (2002) Dilinoleoylphosphatidylcholine Prevents Transforming Growth Factor-β1-Mediated Collagen Accumulation in Cultured Rat Hepatic Stellate Cells, J. Lab. Clin. Med. 139, 202. 8. Lieber, C.S., Robins, S.J., and Leo, M.A. (1994) Hepatic Phosphatidylethanolamine Methyltransferase Activity Is Decreased by Ethanol and Increased by Phosphatidylcholine, Alcohol. Clin. Exp. Res. 18, 592. 9. Oette, K., Kühn, G., Römer, A., Niemann, R., Gundermann, K.-J., and Schumacher, R. (1995) The Absorption of Dilinoleoyl-Phosphatidylcholine After Oral Administration, Drug Res. 45, 875. 10. Aleynik, S.I., Leo, M.A., and Lieber, C.S. (1999) Polyenylphosphatidylcholine Intake Increases Dilinoleoylphosphatidylcholine Content and Antioxidant Capacity in Human Plasma, Hepatology 25(Suppl.), 544A. 11. Lieber, C.S., Leo, M.A., Aleynik, S.I., Aleynik, M.K., and DeCarli, L.M. (1997) Polyenylphosphatidylcholine Decreases Alcohol-Induced Oxidative Stress in Baboon, Alcohol. Clin. Exp. Res. 21, 375.

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12. Aleynik, S.I., Leo, M.A., Ma, X, Aleynik, M.K., and Lieber, C.S. (1997) Polyenylphosphatidylcholine Prevents Carbon-Tetrachloride-Induced Lipid Peroxidation While It Attenuates Liver Fibrosis, J. Hepatol. 27, 554. 13. Aleynik, S.I., Leo, M.A., Aleynik, M.K., and Lieber, C.S. (2000) Polyenylphosphatidylcholine Protects Against Alcohol but not Iron-Induced Oxidative Stress in the Liver, Alcohol. Clin. Exp. Res. 24, 196. 14. Navder, K.P., Baraona, E., Leo, M.A., and Lieber, C.S. (1999) Oxidation of LDL in Baboons Is Increased by Alcohol and Attenuated by Polyenylphosphatidylcholine, J. Lipid Res. 40, 983. 15. Kuntz, E. (1991) The “Essential” Phospholipids in Hepatology—50 Years of Experimental and Clinical Experiences, Z. Gastroenterol. 29(Suppl.), 7. 16. Aleynik, M.K., Leo, M.A., Aleynik, S.I., and Lieber, C.S. (1999) Polyenylphosphatidylcholine Opposes the Increase of Cytochrome P-4502E1 by Ethanol and Corrects Its IronInduced Decrease, Alcohol. Clin. Exp. Res. 23, 96. 17. Aleynik, M.K., and Lieber, C.S. (2001) Dilinoleoylphosphatidylcholine Decreases Ethanol-Induced Cytochrome P4502E1, Biochem. Biophys. Res. Commun. 288, 1047. 18. Navder, K.P., and Lieber, C.S. (2002) Dilinoleoylphosphatidylcholine Is Responsible for the Beneficial Effects of Polyenylphosphatidylcholine on Ethanol-Induced Mitochondrial Injury in Rats, Biochem. Biophys. Res. Commun. 291, 1109. 19. Navder, K.P., Baraona, E., and Lieber, C.S. (1997) Polyenylphosphatidylcholine Decreases Alcoholic Hyperlipemia Without Affecting the Alcohol-Induced Rise of HDLCholesterol, Life Sciences 61, 1907. 20. Navder, K.P., Baraona, E., and Lieber, C.S. (1997) Polyenylphosphatidylcholine Attenuates Alcohol-Induced Fatty Liver and Hyperlipidemia in Rats, J. Nutr. 127, 1800. 21. Baraona, E., Zeballos, G.A., Shoichet, L., Mak, K.M., and Lieber, C.S. (2002) Ethanol Consumption Increases Nitric Oxide Production in Rats, and Its Peroxynitrite-Mediated Toxicity Is Attenuated by Polyenylphosphatidylcholine, Alcohol. Clin. Exp. Res. 26, 883. 22. Oneta, C.M., Mak, K.M., and Lieber, C.S. (1999) Dilinoleoylphosphatidylcholine Selectively Modulates Lipopolysaccaride-Induced Kupffer Cell Activation, J. Lab. Clin. Med. 134, 466. 23. Mi, L.-J., Mak, K.M., and Lieber, C.S. (2000) Attentuation of Alcohol-Induced Apoptosis of Hepatocytes in Rat Livers by Polyenylphosphatidylcholine (PPC), Alcohol. Clin. Exp. Res. 24, 207. 24. Katz, G.G., Shear, N.H., Malkiewicz I.M., Valentino K., and Neumann, M.G. (2001) Signaling for Ethanol-Induced Apoptosis and Repair In Vitro, Clin. Biochem. 34, 219. 25. Gundermann, K.-J., and Lehmacher, W. (1998) The Essential Phospholipids as Liver Therapeutic—A Meta-Analysis of Double-Blind Trials in Chronic Liver Disease, Gastroenterol. Polska 5, 553. 26. Niederau, C., Strohmeyer, G., Heintges, T., Peter, K., and Göpfert, E. (1998) Polyunsaturated Phosphatidyl-Choline and Interferon Alpha for Treatment of Chronic Hepatitis B and C: A Multi-Center, Randomized, Double-Blind, Placebo-Controlled Trial, Hepato-Gastroenterology 45, 797. 27. Lieber, C.S. (2000) Increased Circulating level of Dilinoleoylphosphatidylcholine Is Associated with Protection Against School Induced Oxidative Stress and Liver Fibrosis in Man.

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Chapter 15

Cyclic Phosphates Originating from Degradation of Phospholipids M. Shinitzky and A. Pelah Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel

Phospholipase C and phospholipase D are specific phosphodiesterases that presumably operate as classical hydrolases. Upon the initial formation of the phospholipid– enzyme complex, the free energy of binding is tunneled into the bond to be cleaved and energizes it. Two hydrolysis mechanisms can then follow, as illustrated for phospholipase C in Figure 15.1. In the first, a water molecule cleaves the energized bond to yield simultaneously the alcoholic product (i.e., 1,2 diglyceride) and the free phosphate derivative, as proposed by Sundell et al. (1). In the second mechanism a reactive nucleophilic side chain in the enzyme active site (e.g., serine) cleaves off the alcoholic product by binding to the phosphoryl residue through transphosphorylation. The bond of the phosphoryl-enzyme intermediate is highly reactive and, classically speaking, will be rapidly cleaved off by a water molecule, which will liberate the phosphate headgroup. In principle, these two mechanisms can be discerned by kinetic analysis of the rate of liberation of the diglyceride and the phosphate derivative. Some natural phospholipids contain hydroxyl or amine in their headgroup, which can act as nucleophiles. In the action of phospholipase C on these phospholipids, an intramolecular attack of the nucleophilic residue on the energized phospho-ester bond can compete with the hydrolysis process to yield a cyclic phosphate (2). The cyclic phosphate formed is prone to further hydrolysis, yielding the expected linear phosphoryl product (see Fig. 15.1). A list of cyclic phosphates, either established or putative, obtained upon phospholipase C cleavage of natural phospholipids is presented below. 1,2 Cyclic Inositol Phosphate Cleavage of phosphatidyl inositol by a specific phospholipase C was found to liberate 1,2 cyclic D-myo-inositol phosphate (5). In this process the hydroxyl in position 2 of the inositol residue acts as a nucleophile, competing with the water hydrolysis (see Fig. 15.1). No specific biochemical or physiological function has been so far assigned to 1,2 cyclic inositol phosphate. Yet, a specific phosphodiesterase that hydrolyzes this product to D-myo-inositol 1-phosphate was found in human placenta (7,8). This enzyme might act as a deactivator of 1,2 cyclic inositol phosphate in a specific putative cellular signaling.

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Phospho-enzyme intermediate

Fig. 15.1. Cleavage of phospholipids bearing a nucleophile X in their headgroup by

phospholipase C, yielding a cyclic phosphate intermediate. Two alternative mechanisms are presented.

Cyclic Glycerophosphates 1,3 Cyclic glycerophosphate (1,3 cGP) is a product of phospholipase C cleavage of phosphatidylglycerol (2). The structure of this cyclic phosphate can be either a “boat” or “chair” configuration, and in each the β-OH can be either in axial or equatorial positions. Out of these putative four isomers the chair with equatorial OH is presumably the predominant one and is probably the one liberated in the enzymic cleavage (2). The free β-OH in 1,3 cGP can react with the neighboring phospho-esters in the α or γ positions to form 1,2 cyclic glycerophosphate (1,2 cGP) by transphosphorylation. However under normal pH conditions this reaction is slow and for most cases can be considered as negligible. Transphosphorylation can also take place under controlled enzymatic (3) or basic hydrolysis of phospholipids (4). However, 1,2 and 1,3 cGP

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A list of cyclic phosphate obtained by cleavage of natural phospholipids with phospholipase C Cyclic phosphate Origin Reference

Phosphatidylglycerol cleavage by phospholipase C basic degradation of phospholipids

2

1,3 cyclic glycerophosphate Enzymatic or basic degradation of phospholipids

3,4

Phosphatidylinositol cleavage by phospholipase C

5,6

Phosphatidylethanolamine cleavage by phospholipase C

2

Phosphatidylserine cleavage by phospholipase C

2

1,2 cyclic glycerophosphate

1,2 cyclic phosphoinositol

Cyclic phosphoryl ethanolamine

Cyclic phosphoserine

Scheme 15.1

that are then formed are only intermediates of the final products, α and β-glycerophosphates. The progression of basic hydrolysis of phospholipids via 1,2 and 1,3 cGP is indicated by the production of β-glycerophosphate and by racemization of the final α-glycerophosphate product.

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Chemical synthesis of 1,2 or 1,3 cyclic glycerophosphates is expected to be relatively simple and to follow conventional routes. However, only scarce reports on the synthesis of these compounds appear in the literature (9,10) and without extrapolation to any biological implications.

Analogues of 1,3 cGP Substitution of the β-OH in glycerol will provide derivatives that can form cyclic phosphate analogues of 1,3 cGP. Furthermore, such glycerol derivatives can be hooked to a phosphatidyl backbone by a transphosphatidylation reaction to form non-biological phospholipids such as phosphatidyl 1,3 propanediol and phosphatidyldihydroxyacetone. Upon cleavage with phospholipase C, these phospholipids indeed yielded the expected cyclic phosphate analogues of 1,3 cGP (2).

Cell Signaling of 1,3 cGP and Its Analogues It is of intriguing interest that 1,3 cGP is actually the active residue of cyclic AMP, one of the most important signaling molecules in nature. The open form, α-glycerophosphate, is equivalent to the inactive residue in AMP. These analogies led us to search for biological activities of 1,3 cGP and its analogues in comparison to the inactive α-glycerophosphate. In a series of studies with various analogues and derivatives of 1,3 cGP we could demonstrate intracellular signaling in CHO and NIH-313 cells by such cyclic phosphates when applied extracellularly in the range of 0.1–10 µM. A series of protein phosphorylations, some of them belonging to the MAP kinase cascade, were thus identified (11). Other routes of signaling triggered by cyclic phosphates, presumably operating simultaneously to the MAP kinase cascade, were identified and are awaiting uncovering. Among the tested 1,3 cGP analogues, 1,3 cyclic propanediol phosphate (1,3 cPP) was found to be superior, and in the subsequent studies on physiological functions 1,3 cPP was the lead compound. The target protein that binds cyclic phosphates and then induces the signaling cascade is as yet unidentified. The current indications are that this putative 1,3 cGP-receptor belongs to the spectrin family. The overt physiological changes induced by 1,3 cGP are only partially delineated and are outlined in the following discussion.

Neuronal Outgrowth The rat pheochromocytoma cell line PC12 can be transformed to a sympathetic neuronlike phenotype in response to neurotrophins and as such has become a leading model in nerve differentiation studies. In a chronic presence of 1,3 cGP, 1,3 cPP, and other analogues, PC12 was found to develop neuronal networks (12). An example is presented in Figure 15.2. Neuronal differentiation of PC12 cells with neuronal growth factor (NGF) followed by NGF deprivation results in massive neuronal retraction and cell death, a

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Fig. 15.2. Morphology of PC12 cells after eight days in tissue culture in the presence of 50 ng/mL NGF (c) and 0.5 µM 1,3 cGP (d). Control cultures were grown in the presence of 0.5 µM αGP (b) or in the absence of additive (a).

well-documented process that is believed to take place in vivo after neuronal injury. Our cyclic phosphates clearly demonstrated a strong capacity for nerve rescue in situations of NGF deprivation (12). This finding may bear important physiological and pharmacological implications, which are currently under investigation. Analogous experiments with rat embryo hypocampus cells, designated to become neuronal cells, revealed a distinct stimulatory effect of 1,3 cGP under similar conditions to those applied in the experiments with PC12 cells (Shinitzky et al., to be published).

Differentiation Therapy of Breast Cancer The promotion of morphological differentiation in PC12 cells (see previous) led us to test 1,3 cPP on human breast cancer cells in vitro. Breast cancer cells at their virulent low differentiation states are characterized by low levels of estrogen and progesterone receptors. In a recent study with the human breast cancer cell line MCF-7 (13), we could demonstrate a marked increase in these receptors upon in vitro application

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of 1,3 cPP in a dose-dependent manner. Furthermore, the growth of MCF-7 cells in nude mice could be completely inhibited by injections of 1,3 cPP. “Differentiation therapy” of human breast cancer with various cyclic phosphates is currently under investigation in our laboratory. The therapeutic potential associated with this approach, in addition to the nontoxic status and stability of 1,3 cGP and its analogues, renders our regimen high potential for future treatment of human breast cancer.

Cyclic Phosphoramidates Putative cyclic phosphoramidates can be liberated by the cleavage of phosphatidyl ethanolamine or phosphatidyl serine by phospholipase C. However, such fivemembered rings are expected to be highly unstable because of bond constraint (14) and the high sensitivity of the P-N bond to hydrolysis in such structures. As a matter of fact, the cyclic phosphoramidates presented above have never been synthesized despite a series of attempts and successful synthesis of some of their analogues (15–17). It is tempting to hypothesize that such short-lived cyclic phosphoramidates are indeed liberated in the previously mentioned enzymic reactions and serve as pulse signaling molecules that fade spontaneously. Six-membered cyclic phosphoramidates are relatively stable and can be readily synthesized (18,19). We thus found that cleavage of the non-natural phosphatidylpropanolamine by phospholipase C indeed liberated the relatively stable 1,3 cyclic phosphorylpropanolamine (unpublished).

Cyclic Phosphates Liberated by Phospholipase D Some enzymes of the phospholipase D family operate on lysophospholipids. There is also a specific enzyme for lysophospholipid substrates, lysophospholipase D (20,21). Following the arguments presented for phospholipase C, the initial step in the cleavage mechanism with these enzymes is the release of the alcoholic moiety of the head group. The free β-hydroxyl group on the glycerol back bone can then compete with water in the subsequent reaction with the phosphate radical. The reaction with this hydroxyl group will lead to cyclic lysophosphatidic acid (cyclic LPA), which contains a five-membered ring of phosphodiester (22). Subsequent hydrolysis will lead to LPA of α and β mixed isomers. The level of the non-natural β-LPA can serve as an estimate for the ratio of cyclic LPA to LPA in the previously mentioned lysophospholipase D reactions. On the other hand, the level of cyclic LPA can be evaluated by the unique PNMR signal of the five-membered ring, cyclic phosphate (22). LPA is a well-documented cell activator that operates by binding to a specific receptor that then activates various cellular functions (23,24). It is of interest that cyclic LPA promotes a series of cellular activities that are different than those induced by LPA (25). An open question that remains to be studied is whether LPA and cyclic LPA are inter-convertible by a specific enzyme cycle of cyclase-phosphodiesterase analogous to those operating in the cyclic AMP-AMP cycles.

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A specific cyclic phosphatidic acid with a special acyl chain that contains a cyclopropyl residue was isolated from a slime mold. This specific cyclic LPA was found to possess a series of biological functions other than those established for linear LPA or observed for conventional cyclic LPA (reviewed in Ref. 25). In principle, sphingosyl phosphorylcholine, which includes a free amine at the βposition, can produce the analogous long-chain cyclic phosphoramidate upon the reaction with phospholipase D. Such a compound, which has not yet been discovered, is expected to be as highly unstable as the other five-membered ring cyclic phosphoramidates (see previous). It will rapidly be hydrolyzed to the linear sphingosyl phosphate.

Concluding Remarks The dominant role of phospholipids in the structural aspects of biological membranes is now well characterized. In this territory the phospholipid acyl chains are the major players. They dictate the phospholipid distribution, from close to homogeneity to segregated domains, and to a major extent the overall membrane fluidity, the dynamics and function of membrane enzymes and receptors, as well as various physiological functions. The phospholipid headgroups, which have only a minor contribution to membrane structure, were presumably selected as precursors of signaling molecules released upon cleavage by phospholipase C or phospholipase D. The activation of these enzymes, which could belong to a subclass specific to the phospholipid substrate (e.g., phosphorylated phosphatidylinositol), may be linked to receptor binding and thus integrated into intra- or intercellular signaling cascades. The cyclic phosphates described in this article probably belong to such cascades—an exciting area that by and large is unexplored. References 1. Sundell, S., Hansen, S., and Hough, E. (1994) A Proposal for the Catalytic Mechanism in Phospholipase C Based on Interaction Energy and Distance Geometry Calculations, Prot. Eng. 7, 571–577. 2. Shinitzky, M., Friedman, P., and Haimovitz, R. (1993) Formation of 1,3-cyclic Glycerophosphate by the Action of Phospholipase C on Phosphatidylglycerol, J. Biol. Chem. 268, 14109–14115. 3. Clarke, N., and Dawson, R.M.C. (1976) Enzymic Formation of Glycerol 1:2-cyclic Phosphate, Biochem. J. 153, 745–747. 4. Ukita, T., Bates, N.A., and Carter, H.E. (1955) Studies on the Alkaline Hydrolysis of Lecithin: Synthesis of Cyclic 1,2 Glycerophosphate, J. Biol. Chem. 216, 867–874. 5. Dawson, R.M.C., Freinkel, N., Jungalwala, F.B., and Clarke, N. (1971) The Enzymic Formation of Myoinositol 1:2-cyclic Phosphate from Phosphatidylinositol, Biochem. J. 122, 605–607. 6. Griffith, O.H., Volwerk, J.J., and Kuppe, A. (1991) Phosphatidylinositol-Specific Phospholipases C from Bacillus cereus and Bacillus thuringiensis, Method Enzymol. 197, 493–499. 7. Majerus, P.W., Connolly, T.M., Dechmyn, H., Ross, T.S., Bross, S.E., Ishii, H., Bansal, V.S., and Willson, D.B. (1986) The Metabolism of Phosphoinositide-Derived Messenger Molecules, Science 234, 1519–1526.

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8. Ross, T.S., and Majerus, P.W. (1986) Isolation of D-myo-inositol 1:2-cyclic Phosphate 2-inositolphosphohydrolase from Human Placenta, J. Biol. Chem. 261, 11119–11123. 9. Bailly, O. (1922) Sur l’Action de l’Epichlorhydrine sur le Phosphate Neutre de Sodiumen Solution Aquese et sur la Stabilité d’um Diéther Diglycéromonophosphorique, Bull. Soc. Chim. Fr. 31, 848–862. 10. Khorana, H.G., Tener, G.M., Wright, R.S., and Moffatt, J.K. (1957) Cyclic phosphates. III. Some General Observations on the Formation and Properties of Five-, Six- and SevenMembered Cyclic Phosphate Esters, J. Am. Chem. Soc. 79, 430–436. 11. Shinitzky, M., Haimovitz, R., Nemas, M., Cahana, N., Mamillapalli, R., and Seger, R. (2000) Induction of Intracellular Signaling by Cyclic Glycerophosphates and Their Deoxy Analogues, Eur. J. Biochem. 267, 2547–2554. 12. Haimovitz, R., and Shinitzky, M. (2001) Neuronal Outgrowth and Rescue Induced by Cyclic Phosphates in PC12 Cells, Life Sci. 69, 2711–2723. 13. Adan, Y., Goldman, Y., Haimovitz, R., Mammon, K., Eilon, T., Tal, S., Tene, A., Karmel, and Shinitzky, M. (2002) Phenotypic Differentiation of Human Breast Cancer Cells by 1,3 cyclic Propanediol Phosphate, Cancer Lett., in press. 14. Kugel, L., and Halmann, M. (1967) Hydrolysis of Glycero-1,2-cyclic Phosphate, J. Am. Chem. Soc. 89, 4125–4128. 15. Jones, A.S., Mcguigan, C., Walker, R.T., Balzarini, J., and De Clercq, E. (1984) Synthesis, Properties and Biological Activity of Some Nucleoside Cyclic Phosphoramidates, J. Chem. Soc. Perkin Trans. I, 1471–1474. 16. Euerby, M.R., Partridge, L.Z., Learmonth, M.P., Ball, H.L., and Gibbons, W.A. (1987) The Use of 1,3,2-oxazaphospholidin-2-ones in the Synthesis of Alkoxy- and Aryloxyphosphorylated Derivatives, J. Chem. Res. S, 74–75. 17. Euerby, M.R., Partridge, L.Z., and Gibbons, W.A. (1988) The Use of 1,3,2-oxazaphospholidin-2-ones in the Synthesis of Phosphorylethanolamine Derivatives from “Lower Animals,” J. Chem. Res. S, 394–395. 18. Sato, T., Ueda, H., Nakagawa, K., and Bodor, N. (1983) Asymmetric Synthesis of Enantiomeric Cyclophosphamides, J. Org. Chem. 40, 98–101. 19. Gilard, V., Martino, R., Malet-Martino, M.C., Niemeyer, U., and Pohl, J. (1999) Chemical Stability and Fate of the Drug Ifosfamide and Its N-Dechloroethylated Metabolites in Acidic Aqueous Solutions, J. Med. Chem. 42, 2542–2560. 20. Tokumura, A., Harada, K., Fukuzawa, K., and Tsukatani, H. (1986) Involvement of Lysophospholipase D in the Production of Lysophosphatidic Acid in Rat Plasma, Biochim. Biophys. Acta 875, 31–38. 21. Wykle, R.L., and Straum, J.C. (1991) Lysophospholipase D, Methods Enzymol. 197, 583–590. 22. Friedman, P., Haimovitz, R., Markman, O., Roberts, M.F., and Shinitzky, M. (1996) Conversion of Lysophosphatidic Acid by Phospholipase D, J. Biol. Chem. 271, 953–957. 23. van-Corven, E.J., Van Rijswijk, A., Jalink, Van der Bend, R.L., Van Bliterswijk, W.J., and Moolenaar, W.H. (1992) Mitogenic Action of Lysophosphatidic Acid and Phosphatidic Acid on Fibroblasts. Dependence on Acyl-Chain Length and Inhibition by Suramin, Biochem. J. 281, 163–169. 24. Jalink, K., Hordijk, P.L., and Moolenaar, W.H. (1994) Growth Factor-Like Effects of Lysophosphatidic Acid, a Novel Lipid Mediator, Biochim. Biophys. Acta 1198, 185–196. 25. Murakami-Murofushi, K., Uchiyama, A., Fujiwara, Y., Kobayashi, T., Kobayashi, S., Mukai, M., Murafushi, H., and Tigyi, G. (2002) Biological Function of a Novel Lipid Mediator, Cyclic Phosphatidic Acid, Biochim. Biophys. Acta 1582, 1–7.

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Chapter 16

Effect of Two Diets in Children and Adolescents with Familial Hypercholesterolemia: Soy-Protein Diet Versus Low Saturated Fat Diet K. Widhalm and E. Reithofer Department of Pediatrics, Division of Neonatalogy, Intensive Care and Inborn Errors, University of Vienna, Austria

Introduction Familial hypercholesterolemia (FH) is one of the most common lipoprotein disorders caused by mutations in the LDL-receptor gene, with an incidence of approximately 1:500 in the general population. Due to the fact that most affected subjects show symptoms (cardiovascular diseases in the fourth decade of life) (1), it is generally accepted that children and adolescents should be treated as early as possible in order to prevent later cardiovascular diseases (2). The basis of treatment is a diet characterized by low amounts of saturated fat and high amounts of unsaturated fats. However, most studies in children and adolescents show that diet can lower cholesterol and LDL cholesterol in the range between 6–20% (3,4). Despite the fact that recently published data on children and adolescents underline the safety of cholesterol-lowering drugs, such as statins (4–7), it is obvious that all dietary measures to lower elevated LDL levels should be used before a decision for longterm drug therapy is established. In recent years few reports have been published showing that substitution of soy protein for animal protein is able to act as an additional blood cholesterol-lowering factor. So far, only studies for a period of several weeks and months have been published (8,9). The aim of our study was to investigate the effect of a soy protein substituted diet on blood lipids and lipoproteins in children and adolescents with FH compared with a usual low-fat, high-unsaturated fat diet. The evaluation of each diet period was, on average, 3 and 5 mon.

Patients For this study, 12 adolescents (boys: n = 3, girls: n = 9, age: 9 years) with proven FH according to the American Academy of Pediatrics were studied (LDL cholesterol > 130 mg/dL; one parent affected with cardiovascular disease or hypercholesterolemia). All patients were referred to our clinic from other pediatric hospitals for further diagnosis and treatment.

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Diet All patients and their families were involved in a strict diet-education program. This included a basis dietary record for at least 3 d after information by a trained dietitian. Afterward, a diet low in saturated fats and high in monounsaturated fats was recommend (Diet I). Details of dietary treatment have been described elsewhere (9). Then a break (return to the usual diet) was performed and a second 5-month diet period was started. During this period animal protein was partially substituted by soy protein, thus the subjects had an intake of approx. 17–20 g/d soy protein (Diet II). Soy protein was purchased from Protein Technologies Int., Brussels. Actual diet was calculated according to the records by the same dietitian and data are given in Table 16.1.

Laboratory Methods Blood was drawn in the morning from a sitting position from a cubital vein after a 12-hr fast. Cholesterol, triglycerides, and HDL cholesterol levels were obtained according to conventional enzymatic methods; LDL cholesterol levels were obtained according to the Friedewald formula.

Results All children and adolescents kept their diet records very strictly and were seen every four weeks by one of us and the dietitian. They tolerated the diet well and did not complain of any discomfort caused by the diet. Body weight did not change more than +1 kg within the study periods. As seen in Table 16.1 the habitual diet was characterized by a high fat content and a relatively low carbohydrate content. Diets I and II had a similar fat percentage within the range recommended for this age (10). The content of monounsaturated fatty acids was higher in diet I + II, and protein content was considerably higher in Diet II due to the addition of soy protein powder. The results of serum lipid and lipoprotein measurements show a clear reduction of total cholesterol and LDL cholesterol during both diets, but the effect during Diet II was more pronounced (Table 16.2). TABLE 16.1 Diet Energy (%) Before intervention Diet I Diet II (incl. 17–20 g soyprotein)

Fat (%)

MUFAS (%)

CHO (%)

Protein (%)

41 32 31

28 31 38

43 49 45

16 19 24

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TABLE 16.2 Resultsa

Diet I % Change Diet II % Change aDiet

I vs. Diet II

Begin End Begin End

Chol mg/dL

TG mg/dL

LDL-C mg/dL

HDL-C mg/Dl

244.6 ± 40.0 223.7 –8 246.0 ± 41.0 217.0 ± 37.0 –12

94.0 ± 45.0 63.0 ± 15.0 –33 93.0 ± 25.0 70.6 ± 21.0 –25

175.0 ± 41.0 159.4 ± 41.0 –9 176.0 150.6 –15

49.6 49.3 –1 51,5 48.6 –6

P < 0.05

P < 0.05

In regard to serum triglycerides it is noteworthy that the reduction was stronger under diet I, however not reaching statistical difference.

Discussion It could be shown that both the low-fat diet and the soy-protein diet are able to lower elevated total cholesterol and LDL cholesterol levels; however, the soy-protein diet was able to lower to a higher extent. In previous studies a conventional diet (Step I-Diet) was able to lower cholesterol and LDL cholesterol by approximately 10–20% as it has been shown in some other short-term studies. However it is not quite clear from the literature how many pediatric patients do not respond to dietary therapy. In several studies in adults, substitution of soy protein had an additional cholesterol and LDL cholesterol-lowering effect (11). So far, it is not quite clear by which mechanism soy protein is able to lower LDL cholesterol and which component (i.e., isoflavones, etc.) is the effective substance (12). However, it seems to be very important to use all dietary measures that could support the cholesterol-lowering effect without using drugs. Even in children the long-term use of drugs should be avoided as long as possible in order to prevent possible side effects and also to prevent becoming a “drug user.” It is our experience that people who are placed on drug treatment do not want to adhere to dietary regulation because they think that the drug will do everything. Further studies seem to be necessary in order to investigate the long-term effect of those dietary regimes containing soy protein. References 1. Slack, J. (1969) Risks of Ischemic Heart Disease in Familial Hyperlipoproteinemia States, Lancet 2, 1380–1382. 2. American Academy of Pediatrics (1992) National Cholesterol Education Program: Report on the Expert Panel on Blood Cholesterol Levels in Children and Adolescents, Pediatrics 89, 525–584 3. Tonstad, S. (1997) A Rational Approach to Treating Hypercholesterolemia in Children, Drug Safety 16, 330–341.

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4. Glassmann, M., Spach, A., Berezin, S., Schwarz, S., Medolo, M., and Newman, L.J. Treatment of Type IIa Hyperlipidemia in Childhood by a Simplified American Heart Association Diet and Fiber Supplementation, Am. J. Dis. Child 194, 973–976. 5. Stein, E.A., Illingworth, D.R., Kwiterovich, Jr., P.O., Liacouras, C.A., Siimes, M.A., Jacobsen, M.S., Brewster, T.G., Hopkins, P., Davidson, M., Graham, K. et al. (1999) Efficacy and Safety of Lovastatin in Adolescent Males with Heterozygous Familial Hypercholesterolemia, J. Am. Med. Ass. 281, 137–144. 6. Jongh, de S., Ose, L., Szamosi, T., Gagné, C., Lambert, M., Scott, R., Ferron, P., Dobblelaere, D., Saborio, M., Tuohy, M.B. et al. (2002) Efficacy and Safety of Statin Therapy in Children with Familial Hypercholesterolemia. A Randomized, Double-Blind, Placebo-Controlled Trial with Simvastatin, Circulation 106, 2231–2237. 7. Dirisamer, A., and Widhalm, K. The Effect of Low-Dose Simvastatin in Children and Adolescents with Familial Hypercholesterolemia: 1 Year Observation, Eur. J. Pediatr. in press. 8. Gaddi, A., and Descovich, G.C. (1987) Hypercholesterolemia Treated by Soybean Diet, Arch. Dis. Child 62, 274–278. 9. Widhalm, K., Brazda, G., Schneider, B., and Kohl, S. (1993) Effect of Soy Protein Diet vs. Standard Low Fat, Low Cholesterol Diet on Lipid and Lipoprotein Levels in Children with Familial or Polygenic Hypercholesterolemia, J. Pediatr. 123, 30–34. 10. American Academy of Pediatrics: Committee on Nutrition (1998) Cholesterol in Childhood, Pediatrics 101, 141–147. 11. Anderson, J.W., Johnstone, B.M., and Cook-Newell, M.E. (1995) Meta-Analysis of the Effects of Soy Protein Intake on Serum Lipids, N. Engl. J. Med. 333, 276–282. 12. Messina, M.J. (1999) Legumes and Soybeans: Overview of Their Nutritional Profiles and Health Effects, Am. J. Clin. Nutr. 70 (Suppl.), 4395–4505.

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Chapter 17

Essential Polyunsaturated Fatty Acids in Mothers and Their Neonates Gerard Hornstra Nutrition and Toxicology Research Institute, Maastricht University, NutriScience Research and Consultancy, PO Box 616, 6200 MD Maastricht, The Netherlands

Essential Fatty Acid Biochemistry Essential Fatty Acids and Long-Chain Polyenes Certain fatty acids are indispensable for human development and health but cannot be synthesized de novo by humans. Therefore, they need to be consumed with the diet. These fatty acids are collectively known as essential polyunsaturated fatty acids (PUFA) and comprise the “parent” essential fatty acids (EFA) and their longer-chain, more unsaturated derivatives, the long-chain polyenes (LCPUFA). EFA and LCPUFA are important structural and functional membrane components. In addition, some LCPUFA are precursors of prostanoids (prostaglandins and thromboxanes) and leukotrienes, local hormonelike substances with important bioregulatory functions (1). There are two essential PUFA families, the n-6 and the n-3 families. The essentiality of the n-6 family has been recognized for decades (2), but that of the n3 family has been a matter of debate for some time. However, at present there is no longer any doubt that n-3 fatty acids are essential for reproductive, brain, and visual functions (3). The parent fatty acids of both EFA families are linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), respectively. These EFA, which are mainly present in seed oils (LA + ALA) and green leafs (ALA), can be desaturated and elongated in the human body to a series of longer-chain, more unsaturated derivatives, the LCPUFA. Functionally, the most important LCPUFA are arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3). AA is involved in the regulation of a large variety of metabolic and physiological processes, whereas DHA is the major LCPUFA in the central nervous system (4). In humans, the endogenous formation of LCPUFA from their respective EFA precursors is relatively slow. Since the two parent EFA compete for the same desaturation and elongation enzymes and the habitual Western diet usually contains much more LA than ALA, endogenous DHA formation is particularly low (5). Therefore, an adequate LCPUFA status requires the direct consumption of DHA and possibly AA, which are present in fatty fish (mainly DHA), egg yolk (mainly AA), lean meat, and dietary supplements.

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Essential PUFA Status and Functional Status Markers For the assessment of the essential PUFA status of an individual, the total amount of the various EFA and LCPUFA in plasma or erythrocyte phospholipids is a useful indicator (6). It should be realized, however, that the plasma content of essential PUFA does not necessarily guarantee the proper use of these fatty acids by cells and tissues. Therefore, additional “status markers” are required to reliably assess the functional PUFA status of a given individual. If insufficient essential PUFA are available to meet the PUFA requirements, the body starts to synthesize certain fatty acids with a comparable molecular structure but lacking the specific essential functions. These “surrogate” fatty acids are hardly present under normal conditions and can, therefore, be used as essential PUFA status markers. The bestknown marker is Mead acid (20:3n-9), the increased presence of which indicates a general shortage of all essential PUFA. Another suitable indicator of the overall essential PUFA status is the essential PUFA status index, which is the ratio between all essential PUFA and all non-essential unsaturated fatty acids. The higher the essential PUFA status index, the better the essential PUFA status. Finally, if there is a functional shortage of DHA, the body increases the synthesis of Osbond acid (22:5n-6, Ref. 7). Therefore, under steady-state conditions, the ratio between DHA and Osbond acid is a reliable indicator of the functional DHA status.

Maternal LCPUFA Status During Pregnancy and Thereafter Changes of Maternal LCPUFA Levels During Pregnancy (Table 17.1) Pregnancy is associated with a generalized lipidemia (8,9), and from a longitudinal study (10) it appeared that between early pregnancy (10th week) and delivery, the plasma amounts (mg/L) of the phospholipid (PL)-associated essential PUFA increase by about 40%. For AA and DHA, these figures are 23 and 52%, respectively. These pregnancy-associated fatty acid changes have been confirmed under highly different dietary and cultural conditions and, therefore, seem to be a rather general phenomenon (11–15). Most LCPUFA changes start very early in pregnancy and cannot be explained by a changing LCPUFA intake (16). Therefore, the pregnancy-associated LCPUFA increase may be caused by an enhanced enzymatic conversion of the EFA precursor fatty acids, by LCPUFA mobilization from maternal stores, or by a metabolic LCPUFA shift from energy production to structural use. The amounts of the non-essential unsaturated fatty acids increase considerably stronger than those of the essential PUFA (65 vs. 40%). Actually, the general PUFA status marker Mead acid and the specific DHA status marker Osbond acid increase by 92 and 125%, respectively (10). This indicates that under the present dietary conditions, pregnancy is associated with a reduction of the functional PUFA status, and of the functional DHA status in particular. This is also suggested

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TABLE 17.1 Changes in Long-Chain Polyenes Content (mg/L or g Tissue) and Status (Ratio) of Maternal Plasma Phospholipids During Pregnancy and At Deliverya Pregnancy weekb Prepregnancy 4 10 14 18 22 26 30 34 38 Delivery UP UV UA

22:6n-3

2:5n-6

32.2 37.4 47.1 55.8 61.3 63.8 66.8 69.1 67.8 69.8 65.0 36.1 0.82 0.82

2.5 2.9 4.1 5.1 6.2 6.9 7.5 7.8 8.2 8.9 8.7 5.1 0.43 0.48

DHA status 12.9 12.9 11.5 10.9 9.9 9.2 8.9 8.9 8.3 7.8 7.5 7.1 1.9 1.7

20:4n-6 104 116 119 127 132 133 137 138 138 147 141 95.7 2.94 2.13

20:3n-9

AA status

2.5 2.8 3.2 3.6 4.0 4.4 4.7 5.0 5.4 6.1 6.1 3.9 0.07 0.44

41.6 41.4 37.2 35.3 33.0 30.2 29.1 27.6 25.6 24.1 23.1 24.5 42.0 4.8

aData bUP,

derived from studies described in (10), (16), and (42). umbilical plasma; UV, umbilical venous walls; UA, umbilical arterial walls.

from the significant reduction in plasma PL of the relative concentrations (% of total PL-associated fatty acids) of AA, DHA, and most other essential PUFA (17). Normalization of Maternal LCPUFA Status After Delivery: Effect of Breastfeeding (Fig. 17.1) After delivery, normalization of the essential PUFA status in maternal plasma PL takes place, but this is a relatively slow process taking about 32 weeks (10,18). Since human milk contains LCPUFA, lactating women continue to transfer their own LCPUFA to their infants. As a result, normalization of the maternal DHA status takes longer for lactating than for nonlactating mothers. Moreover, the relative DHA levels in plasma and erythrocyte PL become significantly lower in lactating as compared to the nonlactating women, which cannot be explained by differences in essential PUFA intakes. Finally, the DHA values in maternal plasma and erythrocyte PL become lower the longer the duration of breastfeeding. After weaning the infant, the maternal DHA values increase rapidly to values comparable to those of nonlactating women (18). Pregnancy and Maternal DHA Depletion In a cross-sectional study it was demonstrated that throughout pregnancy the DHA content of plasma PL of primigravida is significantly higher than that of multigravida. Actually, a significant, negative relationship was observed between this DHA content at delivery and the parity number (19). This indicates that certain maternal DHA

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22:6n-3, (%) Fig. 17.1. Breastfeeding causes lower DHA concentrations in maternal plasma phos-

pholipids than formula feeding. Data from references 10, 16, and 18. Prepregnancy DHA concentration: 2.98% wt/wt ~100%.

stores may not be fully replenished after pregnancy, as a result of which DHA mobilization during pregnancy is compromised. Alternatively, DHA synthesis from precursor fatty acids may become diminished as a result of repeated pregnancies. This is suggested from the significant negative relationship between the n-6 LCPUFA/LA ratio of nonpregnant women (a proxy for the efficiency of the EFA-LCPUFA conversion) and the number of pregnancies completed by these women. Moreover, this ratio is significantly lower in mothers than in nonmothers (20). Whatever the reason, in pregnant women the plasma PL DHA content is lower. Since a highly significant and positive relationship exists between the LCPUFA status of the neonate and that of its mother (see below), first-born infants have a significantly higher DHA status than their laterborn siblings (19).

The Essential PUFA Status During Fetal Development and at Birth Relation Between Maternal and Neonatal LCPUFA Status As mentioned before, EFA and their LCPUFA cannot be synthesized de novo by humans and, therefore, the fetal essential PUFA supply will strongly depend on maternal essential PUFA consumption and metabolism, as well as on the placental transport of these fatty acids. This dependence is convincingly illustrated by the significant, positive maternal-fetal correlations for most EFA and their LCPUFA (10,17,21). However, results from an international comparative study involving differences in

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habitual diets (11) suggest that the dependence of the fetal on the maternal LCPUFA status is considerably stronger for n-3 than for n-6 fatty acids (22). This relative autonomy of the fetal n-6 LCPUFA status may be due to the fact that the habitual intake of n-6 PUFA is usually much higher than that of n-3 PUFA (5, 11). No matter the strong correlations between mothers and their term neonates with respect to the essential PUFA levels, plasma and erythrocyte PL fatty acid profiles of neonates are very different from that of their mothers. In general, relative LCPUFA values (% of total PL-associated fatty acids) are considerably higher, whereas the concentrations of the parent EFA are greatly reduced in neonates as compared to their mothers (10,11,21,23,24). When expressed in absolute figures (mg/L plasma), however, all fatty acid amounts are much lower in neonatal than in maternal plasma, which is due to considerably smaller neonatal plasma PL pools (6) (see also Table 17.1). Essential PUFA Changes During Fetal Development Preterm infants were shown to have an essential PUFA status significantly lower than that of term neonates (25). However, the EFA and LCPUFA amounts in cord plasma of preterm infants at birth are not lower than that in cord plasma obtained by fetal blood sampling of ongoing pregnancies at a comparable gestational age (26). Therefore, the low essential PUFA status of preterm infants is most probably a physiological situation and not a pathological condition. These comparative studies also demonstrate that the essential PUFA status of the fetus is not stable during its development, but changes with gestational age in a fatty acid-specific way. Thus, the fetal LA content strongly decreases during early gestation (27), after which it increases slightly during the second and third trimester (26). Fetal AA levels, however, slowly decrease throughout gestation, whereas DHA concentrations rise strongly during the last two months of fetal development (26). Since maternal fatty acid values also change during pregnancy (10,11), comparative studies in which maternal or neonatal fatty acid data are not corrected for pregnancy duration/gestational age are difficult, if not impossible, to interpret. In preterm infants, positive relationships were observed between the amount of DHA in umbilical artery PL and birth weight, head circumference, and birth length. In addition, the essential PUFA status at birth appeared the strongest determinant of the essential PUFA status at the expected date of delivery (28). Therefore, a higher DHA status may be of benefit to preterm neonates, not only for their intrauterine development but also for their postnatal development as well. The Neonatal Essential PUFA Status May Be Sub-Optimal The usually observed declines of the maternal EFA and LCPUFA status occurring during pregnancy (10,11) may imply a suboptimal PUFA status of the newborn infants. This view is supported by the observation that the PUFA status (and the AA status in particular, see Table 17.1) of the walls of the umbilical vein (the supplying

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blood vessel) is higher than that of the umbilical arteries, which carry the blood away from the fetus back to the placenta. Although certain tissues may be preferred sites of EFA/LCPUFA uptake (29), the essential PUFA status of umbilical venous and arterial walls likely reflect the PUFA status of “upstream” and “downstream” fetal tissue, respectively. Consequently, the typical fatty acid profiles of umbilical veins and arteries indicate that the EFA status of the developing fetus is relatively low, and is lower in “downstream” as compared to “upstream” areas. A suboptimal neonatal EFA/LCPUFA status is also suggested from our observation that newborn singletons have a higher essential PUFA status than infants born after multiple pregnancies (30,31). As mentioned previously, the relative amounts of most essential PUFA in maternal plasma PL decrease during pregnancy. In a study comprising 627 mother-infant pairs, Rump and coworkers (32) observed that this decrease is more pronounced the higher the neonatal birth weight. Nonetheless, in these term neonates the LCPUFA contents of umbilical plasma PL are negatively related to birth weight. This indicates that maternal-to-fetal LCPUFA transfer is limited. Since the relationships between birth weight and the neonatal levels of the PUFA shortage markers Mead acid and Osbond acid were positive (32), it seems that maternal-to-fetal LCPUFA transfer, although increased in heavier fetuses, is insufficient to keep the fetal LCPUFA status independent of fetal size. This may possibly result in a suboptimal neonatal LCPUFA status.

Relation Between Habitual EFA and LCPUFA Intake During Pregnancy and Maternal and Neonatal LCPUFA Status Humans are unable to synthesize essential fatty acids de novo, and LCPUFA synthesis from EFA precursors is inefficient in man. Therefore, the essential PUFA status of pregnant women is most likely determined by their intake of EFA and LCPUFA. Several investigators have now confirmed this suggestion. Thus, Al and coworkers (33) observed a significant, positive correlation between the dietary intake and the plasma PL contents of linoleic acid. It is frequently thought that AA levels in plasma and tissue are directly dependent on the habitual LA intake. However, in a group of 288 pregnant women, the LA intake in mid-gestation was not significantly related to the AA content of maternal or neonatal plasma PL at delivery/birth (5,17). Interestingly, a significant, negative relationship was observed between the maternal LA intake and the amounts of the n-3 LCPUFA 20:5n-3 (eicosapentaenoic acid, EPA), 22:5n-3, and DHA in maternal as well as neonatal plasma PL. This may be due to an inhibitory effect of linoleic acid on the incorporation of n-3 PUFA in plasma and tissue PL, as has been demonstrated for DHA (34,35). In the same 288 pregnant women, a significant, positive relationship was observed between the maternal ALA consumption and the ALA amounts in maternal plasma PL (5) as well as the neonatal EPA concentrations (17). A higher maternal ALA consumption was not associated with a higher maternal or neonatal DHA status.

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In nonpregnant subjects, the habitual intake of n-3 LCPUFA is reliably reflected by the n-3 LCPUFA content of plasma and erythrocyte PL (36,37). This also holds for pregnant women (38,39).

Maternal LCPUFA Status and Pregnancy Outcome Pregnancy-Induced Hypertension From observational studies it has been suggested that a reduced n-3 LCPUFA status may contribute to pregnancy-induced hypertension (PIH) (40,41). However, in a prospective nested case control study, Al and coworkers observed a slightly higher n-3 LCPUFA status in women with PIH (42). Kesmodel and colleagues found no significant association between fish intake and the occurrence of PIH (43). Moreover, in a series of prophylactic and therapeutic trials it was demonstrated that supplementation during pregnancy with up to 6.1 g/d of n-3 LCPUFA does not lower PIH risk (44–46). Therefore, a causal role of LCPUFA in the etiology of PIH seems unlikely. Postpartum Depression Hibbeln observed that higher seafood consumption is associated with a lower prevalence of postpartum depression (47). Interestingly, a higher DHA content in mother’s milk also predicted a lower prevalence of postpartum depression, whereas seafood consumption significantly correlated with the DHA content of mother’s milk. These findings suggest that a low DHA status may be involved in the prevalence of postpartum depression. Further studies are indicated to substantiate this suggestion. Preterm Delivery Olsen and his group extensively studied the relationship between the maternal n-3 LCPUFA intake and preterm delivery. Until recently, their results were inconsistent (43,48–50). However, their most recent prospective cohort study among 8729 pregnant women clearly demonstrated that length of gestation is positively related to the intake of n-3 LCPUFA and that low fish consumption is a strong risk factor for preterm delivery (51). This finding is consistent with results of intervention studies performed by the same group (45,52). Birth Weight Using dietary history data obtained in a group of 372 pregnant women during their 22nd week of pregnancy and after adjustment for potential confounders, Badart and colleagues observed that birth weight and Ponderal Index (birth weight/cube of birth length) are not significantly related to maternal PUFA consumption midway in gestation (53). In a later study with 627 mother-infant pairs, Rump and co-workers (32) confirmed that birth weight is not closely associated with maternal PUFA

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consumption during pregnancy as represented by the amounts of n-6 and n-3 fatty acids in maternal plasma PL. Relationships between maternal fish intake during pregnancy and infant birth weight have been found inconsistent, but tend to be positive (48,50,51). Fish consumption during pregnancy has also been reported to reduce the risk of intrauterine growth retardation (51). However, fish oil supplementation did not reduce this risk (45). Other Birth Dimensions Length of infants at birth appeared significantly and positively associated with maternal consumption of total PUFA minus LA (53). However, length at birth was not significantly associated with fish intake (48,50). Placental weight and neonatal head circumference were shown to be associated with maternal fish consumption in a positive way (48).

Relationship Between Fetal Essential PUFA Status and Pregnancy Outcome Gestational Age at Birth Al and coworkers (10) observed a highly significant, negative correlation between fetal LA availability (reflected by cord plasma PL LA concentrations) and gestational age at birth (g.a.), where the amounts and concentrations of DHA and the sum of all n-3 fatty acid were positively correlated with g.a. In a cohort of 780 infants, these DHA findings were confirmed by Rump and Hornstra (17), who also observed a positive association between g.a. and the fetal availability of 22:5n-3 and adrenic acid (22:4n-6). Mead acid concentrations, on the other hand, were negatively related with g.a. In a recent study performed at the Faroer Islands (where the habitual intake of marine fatty acids is high), Grandjean and co-workers (54) observed a positive association between cord serum DHA concentrations and g.a. The correlation with adrenic acid was positive also. Head Circumference In a group of 110 normal neonates, Al and colleagues observed that head circumference was significantly and negatively correlated with the LA percentage in umbilical plasma PL (10). This finding could imply that neonatal head circumference is negatively influenced by maternal LA intake. Indeed, maternal LA consumption mid-gestation was negatively related with neonatal head circumference (53). Head circumference is an excellent predictor of brain weight (55), and AA and DHA are major “building blocks” of the brain. Under the present dietary conditions, maternal LA intake during pregnancy is negatively related to neonatal LCPUFA amounts (17). Therefore, the negative association between LA intake and head circumference could possibly be explained by an overabundant LA availability, resulting in substrate inhi-

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bition of the ∆6-desaturation reaction required for a proper EFA-to-LCPUFA conversion (5). In addition, LA has also been shown to inhibit LCPUFA incorporation in plasma and tissue PL (34,35). This suggests that the ratio between the amounts of n-3 and n-6 PUFA in the present diet is too low and needs readjustment. Birth Weight Although there are some indications that maternal fish consumption may promote infant birth weight (see section 5) and maternal consumption of fish or fish oil increases the neonatal n-3 LCPUFA status (54,56,57), birth weight has not been shown to be positively related to fetal n-3 LCPUFA levels. On the contrary, negative relationships have been reported between birth weight and the concentrations of various n-3 LCPUFA in cord plasma and cord serum PL (32,54) (see also Table 17.2). In one of these studies (32), negative associations with birth weight were also observed for AA, whereas the relation with dihomo-gamma linolenic acid (20:3n-6) was positive. Correlations between birth weight and the umbilical amounts of the essential PUFA shortage markers Mead acid and Osbond acid were positive and significant in both studies, suggesting that the maternal-to-fetal LCPUFA transfer is too limited to secure an adequate, birth-weight independent neonatal LCPUFA status (see also previous discussion).

Early LCPUFA Availability and Later Neurodevelopment Suggestions that LCPUFA are important for early brain and cognitive development resulted from observational studies with infants reared on either mother’s milk (contains LCPUFA) or formula without LCPUFA. These studies invariably show higher LCPUFA concentrations in blood of breast-fed as compared to bottle-fed TABLE 17.2 Relationship Between Neonatal Long-Chain Polyene Concentrations (% of Plasma Phospholipid-Associated Fatty Acids, Unadjusted) and Birth Weighta Weight-for-gestational-age percentile (n) Fatty acid 22:5n-3 22:6n-3 22:5n-6 20:3n-6 20:4n-6 20:3n-9

<10 (81)

10–25 (95)

25–75 (339)

75–90 (71)

>90 (41)

P for trend (adjb)

0.51 6.56 0.78 4.73 17.61 0.35

0.47 6.28 0.85 5.03 17.01 0.43

0.46 6.13 0.85 5.18 16.68 0.48

0.48 6.32 0.86 5.18 16.60 0.49

0.45 5.74 0.95 5.35 16.23 0.61

0.0003 <0.0001 0.0001 <0.0001 <0.0001 <0.0001

aData

derived from (32). for maternal age, weight at entry, weight increase during pregnancy, smoking, parity, pregnancy duration, and mode of delivery and for infant sex and 5-min Apgar score. Trend for 20:5n-3 (mean within percentile classes 0.22–0.24%) not significant.

bAdjusted

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infants (for a review on DHA, see Ref. 3). In addition, in many observational studies breast-fed infants show a long-term intellectual advantage over formula-fed infants (58). However, higher quality studies (optimum control of confounding, minimization of susceptibility bias) appeared less persuasive (59). Intervention studies comparing developmental or visual outcomes of young infants fed with formula with and without added LCPUFA confirmed the importance of these fatty acids for short-term brain development and function of preterm infants (60–62). At present, longer-term benefits have not yet been reported. Results for infants born at term are not conclusive, since only in a minority of the studies significant benefits have been observed (63–67) and longer-term benefits have not been reported at all. Therefore, further studies are certainly warranted (68). Nonetheless, an expert panel already concluded (69) that, although breastfeeding is the preferred option, formulas for term and preterm infants should contain DHA and AA. Since the brain has its growth spurt during the third trimester of pregnancy and in the neonatal period, it seems feasible to suggest that the maternal LCPUFA status during pregnancy and lactation could affect infant cognitive development. We recently investigated the potential importance of maternal LCPUFA status during pregnancy for later infant development by relating the fetal availability of AA and DHA (as represented by their concentrations in umbilical plasma PL) and cognitive, motor, and visual functions at 7 to 8 years of age (70). Children (n = 305) with known neonatal fatty acid profiles were investigated at 7 years of age for their cognitive performance (Kaufman-Assessment Battery of Children) and their quantitative and qualitative motor skills (Movement-ABC and Maastricht Motor Test). In addition, visual function of an ad random selection of 60 of these infants was assessed by measuring visual acuity after optimal eye correction and visual evoked potentials and by electroretinography (n = 33) at 8 years of age. By means of multiple regression analyses, neonatal fatty acid values were related to the functional outcomes, after correction for social class, maternal intelligence, parenting skills, and maternal smoking and drinking habits during pregnancy, as well as for the child’s duration of breastfeeding, birth order, birth weight, gender, gestational age, and age at measurement. No significant associations were observed between either DHA or AA at birth and cognitive performance at 7 years of age (71). Likewise, no significant relationship was observed between cognitive performance at 3.5 years of age and LCPUFA status of neonatal erythrocytes (72). However, DHA status at birth was significantly and positively related to movement quality and to visual acuity at 7–8 years of age. Speed of visual information processing, measured by visual evoked potentials and electroretinograms at follow-up, were also positively related to DHA levels at birth. None of the functional outcome measures were significantly associated with DHA or AA levels at follow-up. These results indicate that a higher perinatal DHA availability may promote later neurodevelopment and function. The results also suggest that an adequate prenatal DHA supply, and consequently maternal DHA intake during pregnancy, may be of at least equal importance for cognitive, motor, and visual development as dietary LCPUFA later in life.

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Biochemical and Functional Effects on Neonates and Breast-Fed Infants of Maternal Essential PUFA Supplementation During Pregnancy and Lactation Since the essential PUFA status of pregnant women is determined by their intake of these fatty acids and the essential PUFA status of neonates is strongly correlated with that of their mothers, it is likely that the essential PUFA status of neonates can be influenced by nutritional intervention of their mothers during pregnancy. Intervention studies demonstrated that it is feasible indeed to increase the essential PUFA status of neonates or breast-fed infants by dietary supplementation of their mothers. Maternal supplementation with LA increased the neonatal n-6 PUFA status at the expense of the n-3 PUFA status (73), whereas maternal supplementation with fish oil (56,57,74) results in an increase of the neonatal n-3 PUFA status. However, this is often associated with a lower n-6 PUFA status (5). Therefore, it seems that an overall increase of the maternal and, consequently, neonatal LCPUFA status would require an increased maternal consumption of both n-6 and n-3 fatty acids. For the maternal LCPUFA status this has been confirmed by a series of studies with single cell oils rich in DHA or AA (75,76). It has amply been demonstrated that—maybe with the exception of AA (77)— the essential PUFA content of human milk can be influenced by dietary supplementation of lactating women with essential PUFA (78–84). Therefore, it can also be expected that the essential PUFA status of breast-fed infants can be influenced by modulating the essential PUFA content of breast milk via supplementation of lactating women. Indeed, for DHA it was shown that the infant levels after 8–12 weeks of lactation were significantly and positively related to the DHA dose their mothers were supplemented with (81,83), independent of the source of DHA (fish oil, single cell oil, or DHA-enriched eggs). In one study (81), infant plasma and erythrocyte AA contents reduced significantly in the supplemented groups and in proportion to the maternal DHA supplementation doses. No matter these fatty acid changes, no neurodevelopment differences were observed between the various groups (85), but it should be realized that group sizes (8–12) might have been too small for a reliable assessment, considering the many potential sources of variability (86). Effects of maternal fatty acid interventions during pregnancy and lactation on infant development have been reported only once so far (57). Thus, as compared to a placebo, maternal fish oil supplementation during pregnancy did not significantly affect mental development of the infants measured at 6 and 9 months of age. Brain maturity, as reflected by an electroencephalogram (EEG), at 3 months of age was not significantly influenced either, although neonates with mature EEG scores had higher n-3 LCPUFA levels at birth than infants with immature EEG scores. The authors suggest that the relatively small contrast between the two groups might have been due to the high habitual fish (oil) intake of their (Norwegian) study population.

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The use of cod liver oil supplements during pregnancy has also been reported to be associated with a lower risk of Type I (insulin-dependent) diabetes mellitus in the offspring, both unadjusted and after adjustment for age, sex, breastfeeding, maternal education, and maternal use of other supplements (87).

Implications for Nutrition During Pregnancy Should the Essential PUFA Content of the Maternal Diet Be Increased? From the results summarized previously, it may be felt necessary to increase the dietary EFA and/or PUFA intake of pregnant women in order to prevent the decrease of their essential PUFA status during pregnancy and to optimize the fetal PUFA status. This latter may be of particular importance for infants born preterm because they have a significantly lower PUFA status than term neonates (25). In addition, their LCPUFA status drops considerably during the first postnatal weeks, even when given breast milk (88,89), whereas during intrauterine life the fetal EFA status increases considerably during the same postconceptional period (26). Consequently, during the growth spurt of the brain, the availability of LCPUFA is much lower for infants born preterm than for the intrauterine fetus of comparable postconceptional age. Whether or not this contributes to the well-known developmental disadvantage of preterm versus term infants needs careful consideration. As discussed previously, the DHA content of maternal plasma PL is significantly lower in multiparous as compared to primiparous women and infants born to multiparous women have significantly less DHA in umbilical tissue PL than infants born to primiparous women. Whether or not this has functional consequences for these infants is not known as yet. However, there is now good evidence that the pre- and early postnatal DHA status has important consequences for growth and function of the central nervous system and, consequently, for neurologic and cognitive development (see previous sections). Therefore, a lower pre- and perinatal DHA availability may—at least in part—present an explanation for observations that first born children, in general, do better than their younger siblings on several developmental, behavioral, and intelligence tests (90,91). Finally, if supplementation with essential PUFA during pregnancy is considered, it should be recalled that the two PUFA families compete for the same metabolic enzymes. Therefore, the supplement of choice should contain a mixture of n6 and n-3 (LC)PUFA. Recommended LCPUFA Intake During Pregnancy, Lactation, and Infancy Several advisory bodies and expert panels have formulated recommendations for the intake of LCPUFA by young infants. All agree that breastfeeding is to be preferred, but if—for whatever reason—this is not an option, LCPUFA-containing formula should be used, not only for babies born preterm (69,92–95), but also for

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term infants (69,94,95). An expert panel of the American FDA (Food and Drug Administration) did not follow this general trend, because they felt that too little data is available for such an advice. The panel, therefore, recommended that “the question of requiring the addition of specific LCPUFA to infant formulas be reassessed within five years” (68). Recently, the FDA has acknowledged certain DHA and AA preparations as GRAS (Generally Recognized As Safe) when used in specified ratios in infant formula (96). So far, no official recommendations have been made for the LCPUFA intake of pregnant and lactating women. It is felt that this would require more functional studies (69). However, since pregnant and lactating mothers are the major source of LCPUFA for their infants and pregnancy and lactation are associated with a reduced (biochemical) LCPUFA status, it seems prudent for pregnant and lactating women to increase their LCPUFA intake. Are PL the Preferred LCPUFA Carriers? The major natural LCPUFA sources for human nutrition are fatty fish (mainly n-3 LCPUFA, but also some AA), egg yolk (mainly AA but also DHA in eggs from specially fed chickens), meat, and breast milk (DHA and AA). In addition, DHA and AA are available as dietary supplements, such as fish oil concentrates (mainly DHA and its precursors 20:5n-3 and 22:5n-3) and single cell oils (DHA and AA). In fatty fish, breast milk, fish oils, and single cell oils, the LCPUFA are mainly present in triacylglycerol (triglyceride, TG) form; in lean fish, meat, and egg yolk, PL are the major LCPUFA carriers, whereas in certain supplements n-3 LCPUFA are present as ethyl esters. Short-term absorption of n-3 LCPUFA seems highest for free fatty acids, intermediate for TG, and lowest for ethyl esters (97,98), although the differences between both ester forms are less pronounced or absent on longer administration (99,100). In addition, no functional differences have been reported for n-3 LCPUFA administered as either TG or EE (101). Although in preterm infants the LCPUFA absorption from formula was initially shown to be comparable to that from breast milk (102), later studies demonstrated that n-3LCPUFA are better absorbed when present as PL as compared to TG. This difference was not observed for n-6 LCPUFA (103). Studies with infant piglets fed with formula containing AA and DHA either as TG or PL demonstrated that the AA and/or DHA concentrations in certain plasma lipid and lipoprotein fractions depend on the LCPUFA carrier. Thus, AA concentrations in plasma PL, cholesterylesters, VLDL, and LDL were significantly higher when the LCPUFA were administered as PL than as TG, whereas DHA levels were not different. Interestingly, AA and DHA proportions in LDL PL were higher when the dietary LCPUFA were available as TG instead of PL, whereas the opposite was true for LCPUFA proportions in HDL PL (104). Since LDL and HDL serve different functions, which may be modulated by their fatty-acid composition (105–109), these

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effects of different dietary LCPUFA sources on lipoprotein LCPUFA content need further study. Peroxidative stability of DHA has been reported to be highest when carried by PL, intermediate when present as ethyl ester, and lowest in TG form (110). The group of Lagarde presented evidence that in young rats lyso-phosphatidylcholine (lysoPC) is the preferred carrier for DHA uptake in the brain (111). In an elegant series of studies in baboon neonates, Wijendran and colleagues demonstrated that PL was about twofold more effective as an AA carrier than TG for brain AA accretion. PL-associated AA was also preferentially incorporated in blood, liver, and lung (112). These results suggest that formulas with LCPUFA carried by PL may be more effective for tissue LCPUFA accretion than formulas that provide LCPUFA as TG. However, further confirmatory studies in humans are needed before this view can be accepted.

Summary EFA and their longer-chain, more unsaturated derivatives, the so-called LCPUFA, are indispensable for human development and health. Since these essential PUFA cannot (EFA) or hardly (LCPUFA) be synthesized by man, they need to be consumed with the diet. Consequently, the LCPUFA status of the developing fetus depends on that of its mother, as is also suggested from the positive relation observed between maternal PUFA consumption and neonatal PUFA status. Pregnancy is associated with increasing amounts of LCPUFA in maternal plasma PL. However, a relatively stronger increase in the amounts of LCPUFA deficiency markers indicates that the increased LCPUFA demand during pregnancy is not adequately covered. A reduction of the maternal LCPUFA status during pregnancy is also suggested by the decrease in the relative amounts of most maternal LCPUFA during pregnancy. For the most important LCPUFA, AA and DHA, this decrease is more pronounced the higher the neonatal birth weight. Nonetheless, in term neonates the AA and DHA contents of umbilical plasma PL are negatively related to birth weight. This suggests that the maternal-to-fetal fatty acid transfer is limited, as a result of which the neonatal essential PUFA status may not be optimal. This latter suggestion is supported by the high amounts of LCPUFA deficiency markers in neonatal blood and tissue and by the lower neonatal LCPUFA status after multiple as compared to single birth. For most maternal PUFA levels, normalization after delivery is complete within 32 weeks. However, maternal DHA concentrations are lower with each following pregnancy, suggesting a long-lasting effect of pregnancy on LCPUFA metabolism or mobilization. As a result, the amount of DHA in plasma PL of a first-born neonate is higher than that of its later-born siblings. Breastfeeding retards normalization of the maternal LCPUFA status after delivery and is associated with a lower maternal DHA status than bottle-feeding. Breastfed infants have a higher LCPUFA status than formulafed infants, but this effect is temporary only. Nonetheless, these infants have a

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long-term cognitive benefit as compared with infants reared on formula without LCPUFA. Neonatal DHA levels and head circumference are negatively related with maternal linoleic acid consumption during pregnancy. This indicates that for an optimum perinatal DHA status, the essential PUFA balance of the maternal diet needs to be optimized. Maternal and neonatal LCPUFA status are thought to be associated with certain pregnancy complications and certain aspects of the pregnancy outcome, but data are not consistent. Postnatal supplementation with LCPUFA temporarily promotes neurodevelopment of the infants, after preterm birth in particular. Longerterm follow-up data have not yet been published. The growth spurt of the developing brain takes place during late pregnancy and early extrauterine life. However, no significant associations were observed between the perinatal DHA or AA availability to infants and their cognitive performance at 3.5 and 7 years of age. On the other hand, movement quality (which is another marker of brain maturity and has been shown to be a significant predictor of later developmental problems like ADHD) was significantly and positively related to DHA status at birth, as was visual acuity and speed of visual information processing. None of the functional outcome measures were significantly associated with neonatal AA levels or with DHA or AA concentrations at follow-up. Although in adult volunteers dietary LCPUFA administered as triacylglycerol or as PL are absorbed to a comparable extent, evidence is emerging that for their absorption and tissue incorporation in young infants, LCPUFA should preferably be administered as PL.

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of Breast Milk Lipids and Maternal and Infant Plasma Phospholipids, Am. J. Clin. Nutr. 71, 292S–299S. Thijs, C., Houwelingen, A., Poorterman, I., Mordant, A., and Van den Brandt, P. (2000) Essential Fatty Acids in Breast Milk of Atopic Mothers: Comparison with Non-Atopic Mothers and Effect of Borage Oil Supplementation, Eur. J. Clin. Nutr. 54, 234–238. Gibson, R.A., Neumann, M.A., and Makrides, M. (1997) Effect of Increasing Breast Milk Docosahexaenoic Acid on Plasma and Erythrocyte Phospholipid Fatty Acids and Neural Indices of Exclusively Breast Fed Infants, Eur. J. Clin. Nutr. 51, 578–584. Tolley, E.A., and Carlson, S.E. (2000) Considerations of Statistical Power in Infant Studies of Visual Acuity Development and Docosahexaenoic Acid Status, Am. J. Clin. Nutr. 71, 1–2. Stene, L.C., Ulriksen, J., Magnus, P., and Joner, G. (2000) Use of Cod Liver Oil during Pregnancy Associated with Lower Risk of Type I Diabetes in the Offspring, Diabetologia 43, 1093–1098. Foreman-van Drongelen, M.M.H.P., Van Houwelingen, A.C., Kester, A.D.M., De Jong, A.E.P., Blanco, C.E., Hasaart, T.H.M., and Hornstra, G. (1995) Long-Chain Polyene Status of Preterm Infants with Regard to the Fatty Acid Composition of Their Diet: Comparison between Absolute and Relative Fatty Acid Levels in Plasma and Erythrocyte Phospholipids, Brit. J. Nutr. 73, 405–422. Foreman-van Drongelen, M.M.H.P., Van Houwelingen, A.C., Kester, A.D.M., Blanco, C.E., Hasaart, T.H.M., and Hornstra, G. (1996) Influence of Feeding Artificial Formulas Containing Docosahexaenoic and Arachidonic Acids on the Postnatal, Long-Chain Polyunsaturated Fatty Acid Status of Healthy Preterm Infants, Brit. J. Nutr. 76, 649–667. Belmont, L., and Marolla, F.A. (1973) Birth Order, Family Size and Intelligence, Science 182, 1096–1101. Gale, C.R., and Martyn, C.N. (1996) Breast Feeding, Dummy Use and Intelligence, Lancet 347, 1072–1075. ESPGAN Committee on Nutrition. (1991) Comment on the Content and Composition of Lipids in Infant Formula, Acta Paed. Scand. 80, 887–896. British Nutrition Foundation (1992) Unsaturated Fatty Acids: Nutritional and Physiological Significance. Task and Force Report, Chapman and Hall, London. FAO/WHO (1994) Fats and Oils in Human Nutrition (Report of a Joint Expert Consultation), Fat and Nutrition Paper 57, Rome. ISSFAL (1995) Recommendations for the Essential Fatty Acid Requirements for Infant Formulas, J. Amer. Coll. Nutr. 14, 213–214. Food and Drug Administration: Opinion of an Expert Panel on the Generally Recognized as Safe (GRAS) Status of ARA and DHA Single Cell Oils for Infants and Children. Lawson, L.D., and Hughes, B.G. (1988) Human Absorption of Fish Oil Fatty Acids as Triacylglycerols, Free Fatty Acids, or Ethyl Esters, Biochem. Biophys. Res. Comm. 152, 328–335. Beckermann, B., Beneke, M., and Seitz, I. (1990) [Comparative Bioavailability of Eicosapentaenoic Acid and Docosahexaenoic Acid from Triglycerides, Free Fatty Acids, and Ethyl Esters in Volunteers, in German], Arzneimittelforschung 40, 700–704. Nordøy, A., Barstad, L., Connor, W.E., and Hatcher, L. (1991) Absorption of the n-3 Eicosapentaenoic and Docosahexaenoic Acids as Ethyl Esters and Triglycerides by Humans, Am. J. Clin. Nutr. 53, 1185–1190.

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100. Krokan, H.E., Bjerve, K.S., and Mork, E. (1993) The Enteral Bioavailability of Eicosapentaenoic Acid and Docosahexaenoic Acid is as Good from Ethyl Esters as from Glyceryl Esters in Spite of Lower Hydrolytic Rates by Pancreatic Lipase in Vitro, Biochim. Biophys. Acta. 1168, 59–67. 101. Hansen, J.B., Olsen, J.O., Wilsgard, L., Lyngmo, V., and Svensson, B. (1993) Comparative Effects of Prolonged Intake of Highly Purified Fish Oils as Ethyl Ester or Triglyceride on Lipids, Haemostasis and Platelet Function in Normilipaemic Men, Eur. J. Clin. Nutr. 47, 497–507. 102. Boehm, G., Muller, H., Kohn, G., Moro, G., Minoli, I., and Bohles, H.J. (1997) Docosahexaenoic and Arachidonic Acid Absorption in Preterm Infants Fed LCP-Free or LCP-Supplemented Formula in Comparison to Infants Fed Fortified Breast Milk, Ann. Nutr. Metab. 41, 235–241. 103. Carnielli, V.P., Verlato, G., Pederzini, F., Luijendijk, I., Boerlage, A., Pendrotti, D., and Sauer, P.J.J. (1998) Intestinal Absorption of Long-Chain Polyunsaturated Fatty Acids in Preterm Infants Fed Breast Milk or Formulas, Am. J. Clin. Nutr. 67, 97–103. 10.4 Amate, L., Gil, A., and Ramírez, M. (2001) Feeding Infant Piglets Formula with LongChain Polyunsaturated Fatty Acids as Triacylglycerols or Phospholipids Influences the Distribution of these Fatty Acids in Plasma Lipoprotein Fractions, J. Nutr. 131, 1250– 1255. 105. De Graaf, J., Hak-Lemmers, H.L., Hectors, M.P., Demacker, P.N., Hendriks, J.C., and Stalenhoef, A.F. (1991) Enhanced Susceptibility to in Vitro Oxidation of the Dense Low Density Lipoprotein Subfraction in Healthy Subjects, Arterioscler. Thromb. 11, 298–306. 106. Davidson, W.S., Gillotte, K.L., Lund-Katz, S., Johnson, W.J., Rothblat, G.H., and Phillips, M.C. (1995) The Effect of High Density Lipoprotein Phospholipid Acyl Chain Composition on the Efflux of Cellular Free Cholesterol, J. Biol. Chem. 270, 5882–5890. 107. Galeano, N.F., Al-Haideri, M., Keyserman, F., Rumsey, S.C., and Deckelbaum, R.J. (1998) Small Dense Low Density Lipoprotein has Increased Affinity for LDL ReceptorIndependent Cell Surface Binding Sites: A Potential Mechanism for Increased Atherogenicity, J. Lipid Res. 39, 1263–1273. 108. Vakkilainen, J., Makimattila, S., Seppala-Lindroos, A., Vehkavaara, S., Lahdenpera, S., Groop, P.H., Taskinen, M.R., and Yki-Jarvinen, H. (2000) Endothelial Dysfunction in Men with Small LDL Partcles, Circulation 102, 716–721. 109. Montoya, M.T., Porres, A., Serrano, S., Fruchart, J.C., Mata, P., Gerique, J.A., and Castro, G.R. (2002) Fatty Acid Saturation of the Diet and Plasma Lipid Concentrations, Lipoprotein Particle Concentrations, and Cholesterol Efflux Capacity, Am. J. Clin. Nutr. 75, 484–491. 110. Song, J.H., Inoue, Y., and Miyazawa, T. (1997) Oxidative Stability of Docosahexaenoic Acid-Containing Oils in the Form of Phospholipids, Triacylglycerols, and Ethyl Esters, Biosci. Biotechnol. Biochem. 61, 2085–2088. 111. Lagarde, M., Bernoud, N., Brossard, N., Lemaitre-Delaunay, D., Thies, F., Croset, M., and Lecerf, J. (2001) Lysophosphatidylcholine as a Preferred Carrier Form of Docosahexaenoic Acid to the Brain, Neurosci. 16, 201–204; discussion 215–221. 112. Wijendran, V., Huang, M.-C., Diau, G.-Y., Boehm, G., Nathanielz, P.W., and Brenna, T. (2002) Efficacy of Dietary Arachidonic Acid Provided as Triglyceride or Phospholipid as Substrate for Brain Arachidonic Acid Accretion in Baboon Neonates, Pediatr. Res. 51, 265–272.

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Chapter 18

Liposomes in Nutrition B.C. Keller BioZone Laboratories, Inc., Pittsburg, California

Introduction With a prolific rise in dietary supplement ingredient availability more attention and effort is now focusing on product formulation and efficient delivery systems to enhance product and ingredient stability and assist or enhance absorption. The quantity and rate at which a nutrient reaches the bloodstream is directly proportional to the effectiveness of the ingredient on its target. Oral dosing is the most common, and the most efficient, route of obtaining proper nutrition. Food and other biologically active ingredients are digested and adsorbed in the gastrointestinal (GI) tract, facilitated by this route of entry. Although our existence clearly demonstrates that this is an effective process, we must be aware that many molecules are poorly adsorbed. The nutritional supplement industry has grown rapidly over the past decade and is now a $12.7 billion (USD) industry (1). Nutritionally beneficial ingredients that were not even in our nutritional lexicon 10 years ago are consumed daily by health conscious men and women wishing to enhance their health and quality of life. The passage of favorable legislation in the United States in the form of the Dietary Supplement Health and Education Act of 1994 (DSHEA) has fueled an explosion of new nutritional supplement ingredients entering the marketplace, which in turn has allowed raw material suppliers to venture internationally. GI absorption takes place in two ways, in either a passive or an active transport process. Passive transport allows an active ingredient to enter the intestinal lumen by a passive diffusion across the enteric cells and distribution between aqueous and membrane phase. Active transport is facilitated by cell membranes that contain specific receptor proteins that take up, in an ATP-driven process, interacting molecules such as amino acids and cholesterol (2). Absorption of molecules depends, therefore, on their aqueous solubility (function of pK of molecules), its lipophilicity (typically measured as octanol/water distribution coefficient, or more recently, liposome/water distribution coefficient), as well as its secretion rate and chemical stability against different enzymes (metabolism) and various values of pH. (3) With a rare exception of the uptake of larger particles (up to several micrometers), which occurs through M cells (which sample the lumen for antigens) on Peyer’s patches, molecules are adsorbed only when they are dissolved (4).

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Oils and other nonpolar molecules, including cholesterol and triglycerides, are dissolved by bile salt detergents and transported by amphiphilic colloidal particles, such as micelles, mixed micelles, and vesicles (5).

Liposomes Liposomes are microscopic, spherical particles that encapsulate a fraction of the solvent in which they freely diffuse (float) into their interior. They can have one, several, or multiple concentric membranes. Liposomes are constructed of polar lipids, which are characterized by having a lipophilic and hydrophilic group on the same molecule (6,7). Upon interaction with water, polar lipids self-assemble and form self-organized colloidal particles. Simple examples are detergents, which form micelles, whereas polar lipids with bulkier hydrophobic parts cannot associate into micelles with high curvature radii but form bilayers that can self-close into liposomes or lipid vesicles. A cross-section of a liposome (Fig. 18.1) depicts the hydrophilic heads of the amphiphile orienting toward the water compartment and the lipophilic tails away from the water toward the center of the vesicle, thus forming a bilayer. Consequently, water-soluble compounds are entrapped in the water compartment and lipid soluble compounds in the lipid section. Uniquely, liposomes can encapsulate both hydrophilic and lipophilic materials. Liposomes resemble the lipid membrane part of cells. Numerous biological processes in living organisms depend on the action of small unilamellar liposomes. Typical compositions include lecithin (phosphatidyl cholines) and kephalins (phosphatidylethanolamines), often containing negatively charged lipids, such as phos-

Lipid soluble ingredients (drugs, nutrients and vitamins)

Amphiphilic lipids

MLV

Water-soluble ingredients (drugs, nutrients and vitamins)

H2O layer Fig. 18.1. Cross-section of a liposome.

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Lipid layer

phatidyl serine and phosphatidyl inositol. In addition to ceramides, such as sphingomyelin, sterols (cholesterol, ergosterol, sitosterol, etc.) are also included. Recently, the use of synthetic, most often nonionic, lipids is starting to increase (7,8). With respect to the number of lamellae and size one typically distinguishes small (<0.1 µm) and large unilamellar vesicles (0.1–1 µm) and large multilamellar vesicles (MLV), which can be up to 500 µm in diameter and can contain hundreds of concentric bilayers (9). Because of their structure and composition liposomes are used extensively in basic research as a model for biological membranes and, due to their biocompatibility, biodegradability, and, in general, absence of toxicity, in applications from drug and gene delivery to diagnostics, cosmetics, long-lasting immunocontraception (10), and the food industry. (11) Rational liposome design can be done by selecting appropriate composition and geometry. Lipids are normally mixed in organic phase, which is evacuated either by rotary evaporation, lyophilization (typically from tertiary butanol), or spray drying. Dry lipid film, cake, or powder can be hydrated by addition of aqueous phase during agitation. Alternatively, organic lipid solution can be injected into aqueous phase and removed by dialysis (ethanol, propylene glycol) or evaporation (ether, Freons) (12). The use of supercritical CO2, a potent solvent, has not been thoroughly investigated. Hydration of lipid normally results in the formation of large multilamellar vesicles (13). Typically, size distribution ranges from very small unilamellar vesicles to large multilamellar liposomes in the 1–10 µm size range, possibly with some liposomes, which can be observed by naked eye. In the case of highly charged lipid bilayers, which, especially in low ionic strength media, swell and disperse better, a larger fraction of unilamellar liposomes with larger encapsulated volume is formed. In general, these liposomes are too heterogeneous and have too small internal volume for many applications and are therefore sized down. In a small-scale setting, this can be done by sonication or extrusion. Homogenization is a method of choice to produce liposomes on a large scale.

Liposome Stability and Interaction Characteristics Liposome stability includes physical, chemical and biological aspects. For a given industrial application this translates into “shelf-life” stability, which is measured by constancy of size distribution and encapsulation efficiency of the active component on the colloidal side and chemical degradation of lipids and ingredients on the molecular. Freezing or freeze drying can increase liposome stability (14). This can be achieved if appropriate cryoprotectants are added. Typically glucose, lactose, or mannitol is used for freezing and sucrose for freeze-drying (7). In the test tube liposomes can aggregate and fuse. This can be paralleled or followed by flocculation and precipitation. Increasing intraparticle repulsion, either electrostatically or sterically, enhances their colloidal stability. The former is achieved by increasing liposome charge density, i.e., their surface- and, conse-

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quently, zeta-potential, whereas the latter lipids with bulky nonionic groups, such as (polyethylene) glycol-lipids, are included into the bilayer (15). Many substances can destabilize liposomes. Detergents and other surfactants can dissolve liposomes into mixed micelles, whereas oppositely charged multivalent counter ions and macromolecules can aggregate and fuse or restructure liposomes. Mechanical stabilization, which can be achieved either by polymerizing liposomes or by choosing lipid compositions, which give rise to very strong interbilayer cohesiveness, significantly increases the stability of liposomes against hydrophobic insertion of detergents and proteins. In biological systems liposomes are quickly covered with various proteins, which dictate their biological fate. Upon parenteral administration into the body this is mostly by uptake by phagocytic cells. Coating liposome surface with polymers can substantially reduce these interactions and different biodistribution of liposomes are obtained. This effect is due to steric stabilization, which results in reduced absorption of macromolecules on the liposome surface of liposomes, and therefore these liposomes are called sterically stabilized liposomes. Due to their invisibility to the body’s immune system, they are often referred to as stealth liposomes (16). Currently a very promising application is reacting positively charged liposomes with nucleic acids and using the resulting complexes in the gene delivery. DNA-liposome/lipid complexes (genosomes) show a variety of structures, which are a consequence of thermodynamic and kinetic factors during their preparation (17). Sterility is also very important in liposome applications. Pharmaceutical formulations are typically sterilized by 0.2-µm sterile filtration, whereas for cosmetic and oral applications, preservatives are added. These include benzyl alcohol, methylparaben, and ethyparaben or alcohol (for topical applications). Obviously, in the methods where liposomes are manufactured from alcoholic solutions, and where solvents, such as propylene glycol or ethanol, are present at concentrations below 5–10%, the remaining solvents can act as a preservative, simplifying the preparation procedure by eliminating the solvent removal step.

Liposome Applications Due to their lipophilic/hydrophilic properties, colloidal size, and ability to compartmentalize space (i.e., encapsulation efficiency), liposomes are widely used as delivery vehicles for active agents in pharmaceutics, cosmetics, food, and the nutrition industry, as well as in the coating industry and ecology. Liposome formulations can be injected intravenously, intramuscularly, or subcutaneously (liquid suspensions); inhaled (aerosol of liposome suspension or lyophilized powder); applied to the skin (suspension, cream, gel, ointment); or ingested (any of the physical forms), (7,18). The major modes of action of liposomes in pharmaceutics and drug delivery are enhanced drug solubilization, protection of sensitive drug molecules, enhanced intracellular uptake, and altered pharmacokinetics and biodistribution (7). In many

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cases effective chemotherapy is severely limited due to the toxic side effects of the drugs. Liposome encapsulation can change spatial and temporal distribution of the encapsulated drug molecules in the body that may significantly reduce unwanted toxic side effects and increase efficacy of the treatment. Similar concepts and, additionally, the potential for the enhanced skin penetration effect of liposome-associated drug/agents apply also in their applications in cosmetics. Because liposomes can entrap hydrophilic molecules in their interior, and hydrophobic molecules in their lipophilic membrane, they have been used as a delivery system for many different molecules in agricultural industry and nutrition. Liposomes were used in the food industry for acceleration of cheese ripening (19). Liposomes offer better and more uniform distribution of hydrophilic enzymes in hydrophobic environment. Similarly, many hydrophilic and hydrophobic compounds, including various vitamins and antioxidants, can be better dispersed in various matrices by their encapsulation into liposomes. As a sustained release system, liposomes are also being tested for improved delivery of pesticides and functional ingredients in food (20).

Liposomes in Milk Liposomes have been used in drug delivery and in a wide range of applications from chemotherapy to blood surrogates to skin treatment products (7,21,22). In addition liposomes can be effective carriers for nutritionally valuable ingredients. For the first example of liposomes in nutrition we turn to human milk. Human milk has been studied for years. The composition of breast milk and the relationship to infant nutrition and development are, indeed, well understood. However, the physical structure of human milk and the relationship of its “microstructure” to nutritional and immunologic activity are still not completely understood. Analyses of the chemical and nutrient composition of human milk reveal it to be an elaborate suspension that contains more than 200 fat-soluble and water-soluble ingredients. Various analyses show predominately emulsion droplets and casein micelles. In recent electron microscopic studies lipid vesicles (liposomes) were discovered (23). Fig. 18.2 supports the presence of liposomes in breast milk taken from five healthy mothers. In Fig. 18.2A a freeze fracture micrograph shows a large unilamellar liposome with a diameter of about 500 nm. In Fig. 18.2B a large oligolamellar liposome is seen; at least three concentric bilayers can be observed (arrow). Liposomes fracture in the middle of the bilayers, and therefore, smooth concave or convex fractures with typical shadowing, consistent with literature data, are detected. In contrast, emulsions droplets fracture irregularly, which is shown in Fig. 18.2C and 18.2D. To distinguish the difference from a lipid vesicle (liposome) and an emulsion, one can observe the irregular fracture pattern in these two freezefracture micrographs of emulsion droplets. In Fig. 18.2C smaller emulsion droplets (arrow) as well as smaller unilamellar vesicles (double arrow) can be observed.

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Fig. 18.2. Electron micrographs of liposomes (A and B) and emulsion droplets (C and D) in human milk.

Part of the emulsion droplet shown in Fig. 18.2D contains densely packed lipid bilayers forming a multilamellar liposome associated with the emulsion droplet (arrow). Liposomes as a micro-structural component of breast milk may play an important role in enhanced nutrient absorption, colloidal stability, and immunogenicity.

Oral Liposomes Oral liposomes were first formulated in the early 1970s. Experiments resulted in several failed attempts to deliver the drug substance across the GI tract, but the major problems were identified (7,24). The biggest barriers to successful oral liposome drug delivery were the low pH of the stomach, the presence of bile salts, and the presence of lipases that all eventually destabilize the drug-liposome complex. Their stability in this environment can be increased by preparing more stable bilay-

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ers (mechanic stabilization, by including cholesterol), coating their surface with protecting polymers (steric stabilization against proteins), and putting them into gelatin capsules. In addition to its simplicity, oral administration can improve drug absorption. Polypeptides and proteins, which cannot be administered by the oral route, have been shown to be delivered in liposomal forms into bloodstream (25). The mechanism has been explained by the uptake of whole liposomes by M cells on Peyer’s patches. Because the amount of the delivered drug is not large, this approach is mostly used for vaccination, where small doses are required. Additional liposome benefit seems to be in the enhanced shuttling of agents with poor aqueous solubility from the intestinal lumen fluids into enteric cells via bile salt—polar lipid mixed micelles and vesicles. In addition to the drug uptake in and past the stomach, oral administration also offers direct uptake into the blood by the sublingual mucosal membranes, which bypasses the first pass liver clearance and metabolism. Further, the therapeutic effect of a nutrient is directly related to the quantity and rate at which the unchanged nutrient reaches the bloodstream. For many nutritional supplements, formulation and route of administration have a great effect on both of these parameters. Due to the disadvantages of an oral delivery and potential for enhancing nutrient delivery, an inter-oral liposome delivery system has been developed (called LipoSpray®). To bypass the the destabilization of liposomes and nutritional ingredients in the lower GI tract, BioZone Laboratories Inc. developed a delivery system that targets inter-oral tissue and lymphatic ducts in efforts to enhance product performance by increasing bioavailability, nutrient solubility, and stability. In addition, there is a segment of the user population that cannot, or will not, use a conventional dosage form, i.e., a compressed tablet, two-piece hard gelatin capsule, or soft-gel capsule, because of swallowing difficulties or personal preference. In one recent study, which was designed to determine the effects of a delivery system on bioavailability of a dietary supplement, a two-piece gelatin capsule containing powderized CoEnzyme Q10 (CoQ10) (Ubiquinone), which typically has poor solubility (26), was compared to a liposomal oral spray (26). The plasma versus time profiles of the two dosage forms, and respective routes of administration, show the variation of CoQ10 above baseline levels over a 24-hr period. An increase of 100% of CoQ10 over endogenous levels was observed with the sublingual spray compared to a 50% increase over baseline levels with a two-piece gelatin capsule as measured by area-under-the-curve (AUC), the best determination of absolute bioavailability (27) (Fig. 18.3). The time of onset of the liposome spray formulation was twice as fast as the capsule; this can be an important aspect when immediate onset is desired, as in the case of diet aids; performance enhancing formulas; cardiogenic supplements, such as CoQ10; cough treatments; and when treating pain, fever, and insomnia. Other data that supports increased bioavailability, as demonstrated by AUC, is a 24-hour study of plasma levels of melatonin, a pineal hormone used for blind entrainment, jet lag, insomnia, cancer protection, and oral contraception (28). In

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Mean plasma concentration (ug/mL)

Time (h) Fig. 18.3. Bioavailability of CoEnzymeQ10.

Mean plasma melatonin (pg/mL)

this study a compressed tablet was compared to a liposomal spray over 24 hours. Absolute bioavailability, determined by calculating AUC using the trapezoid rule (29) was greater for the liposomal encapsulated melatonin by 50% (Fig. 18.4). The time of onset was remardably faster for the sublingual spray that showed increased melatonin levels immediately and a peak plasma level at two hours, which was sustained for six hours. The tablet dosage form peaked four hours after administration and the elimination phase began shortly thereafter. The beneficial action of liposomes in oral delivery of nutrients is due to several modes of their action. These are improved nutrient solubilization and protection, possibly enhanced absorption (of small hydrophobic moieties), and direct entry into the bloodstream. More needs to be learned about liposome interactions in the GI tract and subsequent absorption.

Time (h) Fig. 18.4. Bioavailability of melatonin 3 mg.

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Several synthetic lipids and lipid-polymer combinations are currently being studied and show promise of prolonging GI transit time and enhancing absorption. With phospholipids, there is always the beneficial effect of the carrier itself. Lecithin is the major component of cell membranes and has been shown to lower serum cholesterol levels, whereas some reports on improved memory function are more difficult to verify. In any case, the value of liposomes has been proven as customer satisfaction is reflected in the rapid growth of the market and emergence of many novel companies in this field. References 1. U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition (1999) Economic Characterization of the Dietary Supplement Industry, Final Report, Sec. 1, March. 2. Yeh, P.Y., Ellens, H., and Smith P.L. (1998) Physiological Considerations in the Design of Particulate Dosage Forms for Oral Vaccine Delivery, Adv. Drug Del. Rev. 34, 123–133. 3. Niebergall, P.I., in Remington: The Science and Practice of Pharmacy, Ionic Solutions and Electrolytic Equilibrium, Mack Publishing Company, Easton, PA 18042, Chapter 17, pp. 230. 4. Norris, D.A., Puri, N., and Sinko, P.J. (1998) The Effect of Physical Barriers and Properties in the Oral Absorption of Particulates, Adv. Drug Del Rev. 34, 135–154. 5. Shulthess, G., Compassi, S., Boffelli, D., Werder, M., Weder, F.E., and Hauser, H. (1996) A Comparative Study of Sterol Absorption in Different Small-Intestinal Brush Border Membrane Models, Lipid Res. 37, 2405. 6. Bangham, A.D., Standish, M.M., and Watkins, J.D. (1965) Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids, J. Mol. Biol. 13, 238. 7. Lasic, D.D. (1993) Liposomes: From Physics to Applications, Elsevier, Amsterdam, London, New York, Tokyo. 8. Vanlerberghe, G. (1996) in Handbook of Nonmedical Applications of Liposomes, Vol. IV: Liposomes: from Gene Therapy and Diagnostics to Ecology, Lasic, D.D., Barenholz, Y., CRC Press, Boca Raton, pp. 88. 9. Helfrich, W. (1986) Size Distributions of Vesicles: The Role of the Effective Rigidity of Membranes, J. Phys. 47, 321. 10. Brown, R.G., Bowen, W.D., Eddington, W.D., Kimmins, W.E., Mezei, M., Parsons, J.L., and Pohajdak, B. (1997) Evidence From a Long Lasting Single Administration Contraceptive Vaccine in Wild Gray Seals, J. Repro. Immun. 35, 43–51. 11. Dufour, P., Laloy, E., Vuillemard, J.-C., and Simard, R.E. (1996) Liposomes and Cheesemaking, in Handbook of Nonmedical Applications of Liposomes, Vol. IV: Liposomes in Cheesemaking, from Gene Delivery and Diagnosis to Ecology, Lasic, D., and D., Barenholz, Y., CRC Press, Boca Raton, pp. 158–164. 12. Kriftner, R.W. (1993) Liposome Production: The Ethanol Injection Technique and the Development of the First Approved Liposome Dermatic, Liposome Dermatics, (BraunFalco, O., Korting, H.C., and Maibach, H.I., eds.), Springer-Verlag, pp. 91–100. 13. Pidgeon, C. (1993) in Liposome Technology, Gregordias, G., CRC Press, Boca Raton, pp. 99–110.

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14. Strauss, G., Schurtenberger, P., and Hauser, H. (1986) The Interaction of Saccharides with Lipid Bilayer Vesicles: Stabilization During Freeze-Thawing and Freeze-Drying, Biochim. Biophys. ACTA 858, pp. 169–180. 15. Glass, R.L. (1991) Effect of Herbicides on Phase Transition of DPPC Vesicles, Chem Phys Lip. 59, pp. 91–95. 16. Lasic, D.D., and Martin, F.J. (1995) Stealth Liposomes, CRC Press, Boca Raton. 17. Lasic D.D. (1997) Liposomes in Gene Delivery, CRC Press, Boca Raton. 18. Abra, R.M., and Lasic, D.D. (1992) Liposomes, Agro-Food Industry Hi-Tech, Nov/Dec, 12–16. 19. Dufour, P., Laloy, E., Vuillemard, J.C., and Simard, R. (1996) in Liposomes in Cheesemaking, in Nonmedical Applications of Liposomes, Vol. IV, from Gene Delivery and Diagnosis to Ecology, Lasic, D., and Barenholz, Y., CRC Press, Boca Raton, vol. 4, pp. 153–164. 20. Kirby, C.J. (1993) in Liposome Technology, Gregoriadis, G., CRC Press, Boca Raton, pp. 215–231. 21. Mezei, M. (1992) in Liposome Dermatics, Korting, Braun-Falco, (Braun-Falco, O., Korting, H.C., and Maibach, eds.), Springer-Verlag, Berlin, 206–214. 22. Lasic, D.D., Papahadjopoulos, D. (1998) Medical Applications of Liposomes, Elsevier, Amsterdam, NewYork, Tokyo. 23. Keller B.K., Lasic, D.D., Faulkner, G. (2000) Liposomes in Breast Milk, Agro-Food Industry Hi-Tech. 11, 3, 6–8. 24. Roger, J.A., Anderson, K.E. (1998) The Potential of Liposomes in Oral Drug Delivery, Crit. Rev. Ther. Drug. Carr. Syst. 15, 421, 480. 25. Maitani, Y., Hazama, M., Tojo, Y., Shimoda, N., and Nagai, T. (1996) Oral Administration of Recombinant Human Erythropeitin in Liposomes in Rats: Influence of Lipid Composition and Size of Liposomes on Bioavailability, J. Pharm. Sci. 85, 440. 26. Folkers, C. (1981) Biomedical and Clinical Research on the Aspects of Coenzyme Q, Biomedical Press, Elsevier, Amsterdam, vol.3. 27. Gibaldi, M. (1991) Biopharmaceutics Clinical Pharmacokinetics, 4th ed., Lea & Febiger, Philadelphia, pp. 377. 28. The Review of Natural Products, Melatonin Monograph 1996, Facts and Comparisons, St Louis, 2000. 29. Rowland, M., Towzer, T.N. (1995) Clinical Pharmacokinetics: Concepts and Applications, 3rd ed. Williams & Williams, Baltimore, pp. 17. 30. The Review of Natural Products, Respective Monographs, Facts and Comparisons, St Louis, 2000.

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Chapter 19

Lower Incidence of Necrotizing Enterocolitis in Infants Fed a Pre-term Formula with Egg Phospholipids Susan E. Carlson, Michael B. Montalto, Debra L. Ponder, Susan H. Werkman, and Sheldon B. Korones University of Kansas Medical Center, Department of Dietetics and Nutrition, 4019 Delp, Kansas City, Missouri 66160

Hospitalized pre-term infants are watched closely for signs of feeding intolerance, which can signal the onset of necrotizing enterocolitis (NEC) or infection. These conditions cause significant mortality and morbidity in this population. NEC occurs in approximately 10% of infants of <1500 g birth weight (1) and has been estimated to be responsible for approximately 4000 deaths per year in the United States alone (2). The long-term growth and development of pre-term infants who survive NEC may be compromised by prolonged delays in enteral feeding (3). Even pre-term infants who do not develop NEC may have poor nutrient intake and suboptimal growth and development if enteral feeding is delayed or provided by formulas too low in nutrients for fear of NEC. Typically, NEC occurs suddenly and without warning in infants who have tolerated enteral feeding for 2–6 wk. NEC is associated with many conditions that reduce mesenteric blood flow. These include immaturity, polycythemia, intrauterine growth retardation, asphyxia, exposure to cocaine, respiratory distress syndrome, exchange transfusions, intravascular catheter placement, proinflammatory cytokines, cold stress, fluid overload, hyperosmolar solutions, abdominal distention, and portal hypertension (1,4–10). Poor mesenteric blood flow may lead to intestinal hypoxia and injury and decreased mucosal surface area and loss of barrier function (11,12). Overgrowth of atypical organisms, enteral feedings, and inflammatory cytokines may cause additional injury to the intestinal mucosa (13,14). NEC has been reported to decrease with antenatal steroids (15,16), human milk feeding (17), low pH formula (18), enteral IgA (19), and antibiotics (20,21). The putative mechanisms include enhanced barrier function (antenatal steriods) (22), immunoprotection (human milk feeding, IgA) (23–25), cell growth (human milk) (26), and bacteriostasis (antibiotics, low pH formula) (18,20,21). None of these interventions

Reprinted with permission from Pediatric Research 44, 491–498 (1998)

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has eliminated NEC, and each is associated with some problem, such as poor availability, metabolic acidosis (18), antibiotic resistance (27,28), or the inability to delay pre-term birth. The addition of egg phospholipids to formula feedings produced a near-total reduction in NEC in a nursery population of very-low-birth-weight infants historically at high risk for the disease (1).

Methods Subject Selection Study infants were enrolled continuously between September 1992 and March 1997 under a protocol approved by the University of Tennessee, Memphis, Institutional Review Board, and infants received care in the Newborn Center of the same institution. Infants ≤32 weeks of gestation (29) weighing 725–1375 g and ≥5th percentile for weight (30) at birth whose mothers chose formula feeding were eligible for enrollment. No infant received human milk. Other exclusion criteria were periventricular/intraventricular hemorrhage greater than grade 2; cardiac, renal, or hepatic dysfunction; maternal history of alcohol or drug abuse; congenital malformations; sepsis at birth; and pulmonary disease that did not improve over the first days of life. Except for one infant who developed an intraventricular hemorrhage, infants were removed after enrollment only if enteral feeding was discontinued for more than seven cumulative days after full external feeding (100 kcal or 416 kJ per kg body weight/day). The exclusion and removal criteria were included to minimize confounding of the main study outcomes of first year growth and development that will be reported when the postdischarge portion of the study is complete. A total of 120 infants were randomized to diet, but one infant was transferred after seven days because his insurance company required that his care be given in another hospital. All remaining 119 randomized infants were included in the in-hospital evaluation reported here. Experimental Design The study was a randomized, double-masked clinical trial with infants assigned to one of three feeding regimens. The main purpose of the study was to look at the effects of providing pre-term infants with a diet containing arachidonic acid (AA) and docosahexaenoic acid (DHA) in amounts typically reported for milk of American women (31) from birth or after discharge home. However, diseases occurring after assignment to diet were prospectively monitored as part of risk benefit assessment of the diets. Two regimens received a commercial pre-term formula (Similac Special Care, Ross Laboratories, Columbus, OH) during their nursery days. They constituted the control group in this report. One of these two regimens changed to experimental formula after discharge from the hospital, the other continued to receive control formula. The group assigned to the third regimen, which constituted the experimental group in this report, was fed experimental formula in

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the hospital and after discharge home. Both the control and experimental formulas were masked to avoid identification. Only the lipid blends of the two formulas differed (Table 19.1), resulting in different amounts of formula total phospholipids (Table 19.1), fatty acids (Table 19.2), and choline (Table 19.3). The randomization to diet was successful as illustrated in Table 19.4, which shows the neonatal and perinatal characteristics of the groups. Both formulas were manufactured by Ross Products Division of Abbott Laboratories. Infants were fed Similac Special Care under its commercial label until randomized to their treatment regimen at a mean of 4.9 days of age. All infants were fed by nasogastric infusion (6 h on and 2 h off) until they weighed 1.5 kg and then at three-hour intervals by nipple or bolus infusion until hospital discharge. Eligible infants were stratified in three birth weight categories (725–925, 926–1150, and 1151–1375 g), and each weight category was balanced for gender. Gender was unmasked to permit enrollment of eligible infants regardless of gender. To guarantee that the groups remained balanced for the planned post-discharge outcomes, infants who were lost from the study were replaced by the next infant who met the same gender and weight criteria. Enrollment continued until 30 infants per regimen could be followed through four months of corrected age. In-Hospital Monitoring and Diagnosis All 119 infants (115 singletons, 4 twins) were evaluated for diseases occurring during their hospitalization regardless of their availability or eligibility for the post-discharge phase of the study. Infants were weighed, and total energy intake was recorded daily while in the hospital. Starting with the first study day, the energy per kg body weight from parenteral and enteral sources was summed and averaged for each subsequent complete seven-day interval. Weekly weight gains were calculated for the same seven-day intervals. Only infants remaining in the study for the entire week were included in the calculations of weekly energy intake and growth reported here because the purpose of including these data was to evaluate TABLE 19.1 Lipids in Control and Experimental (Egg Phospholipid) Formulas Ingredientsa MCT Coconut oil Soy oil Egg phospholipid Soy lecithins Cholesterol aMCT,

Control formula (g/100 g total lipid)b

Experimental formula (g/100 g total lipid)b

50 30 20 0 1 NDc

50 21 20 9 1 NDc

medium chain triglycerides from fractionated coconut oil. g total lipid/L formula. cND, not detected. b44.1

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TABLE 19.2 Average Fatty Acid Profiles in g/100 g Total Fatty Acids in the Control and Experimental Formulas Fatty acid 6:0 caproic 8:0 caprylic 10:0 capric 12:0 lauric 14:0 myristic 15:0 and 14:1 16:0 palmitic 16:1 palmitoleic 17:0 margaric 18:0 stearic 18:1-9 oleic 18:2n-6 linoleic 18:3n-6 γ-linolenic 18:3n-3 α-linolenic 18:4n-6 20:0 arachidic 20:1n-9 20:2n-9 20:3n-9 20:4n-6 AA 20:5-3 eicosapentaenoic 22:0 behenic 22:5n-6 22:5n-3 22:6n-3 DHA 24:0 Total Σ 20n-3 Total Σ 20n-6 aND,

Control formulaa

Experimental formula

0.71 30.56 19.61 9.69 3.85 0.04 5.51 0.03 0.04 2.68 8.31 16.36 2.30 2.24 ND 0.12 0.04 0.02 ND ND ND 0.07 ND ND ND 0.04

0.27 23.11 16.44 10.24 4.08 0.01 7.65 0.12 0.09 3.69 11.25 18.87 ND 2.45 0.02 0.14 0.09 0.02 0.05 0.41 ND 0.12 0.07 0.07 0.13 0.07

ND ND

0.21 0.48

not detected.

TABLE 3 Sources of Choline (mg/L) in Study Formulasa

Free choline Esterified choline Total choline aFree

Control formula

Experimental formula

95 79 174

97 585 682

Choline (85) and total choline (Engelhardt, R., and Vojacek, M., unpublished results) were analyzed and esterified choline determined by the difference between total and free choline. One mg = 8.25 µmol.

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TABLE 19.4 Characteristics of Infants Fed Control and Experimental (Egg Phospholipid) Formula in the Hospital

Gender (M/F) Birth weight (g) <1000 g at birth Gestational age (wk) Maternal age (yr)a Mother <25 yr Delivery (cesarean-section/vaginal) Gravida Apgar 1 min Apgar 5 min Race (B/W) Weight at enrollment (g) First enteral feed (h)a First study feed/age at enrollment (d) Full enteral feeds (d)b Ventilation (h)a,b Total oxygen (h)a,b In the hospital (d)a,b

Control formula (n = 85)c

Experimental formula (n = 34)c

43/42 1115 ± 163 21/85 29.4 ± 1.7 22.0 ± 5.6 58/85 38/47 2.8 ± 1.4 5.1 ± 2.4 7.3 ± 1.6 79/6 1033 ± 171 56.4 ± 1.6 4.9 ± 1.9 22.6 ± 8.5 8.7 ± 4.90 53.1 ± 6.3 34.5 ± 1.4

17/17 1123 ± 152 9/34 29.2 ± 1.8 22.9 ± 5.8 21/34 19/14 2.5 ± 2.1 4.4 ± 1.8 7.0 ± 1.3 31/3 1055 ± 164 60.4 ± 1.8 4.9 ± 1.9 22.8 ± 7.10 13.3 ± 4.40 61.7 ± 6.7 38.4 ± 1.4

aIndividual

values were log transformed to adjust for a nonrandom distribution. lost from the study because of NEC are not included. Had they been included, the mean value for the control group would have increased. cValues are expressed as mean ± SD (range) or, for binary data as ratios. bInfants

nutritional support before the onset of NEC. Hours of total oxygen supplementation and mechanical ventilation were recorded. Infants with feeding intolerance, gastrointestinal bleeding, dysmotility, abnormal bowel sounds, abdominal distention or tenderness, or bilious emesis were evaluated for NEC. Diagnosis of NEC stage II or III was made by radiographic observations, including intestinal pneumatosis, portal venous air, and peritoneal free air. Modified (32) Bell’s criteria for staging of NEC (33) were used to distinguish NEC stages. Two neonatologists confirmed the diagnosis of NEC. All infants diagnosed with NEC were noted to have elevated C-reactive protein (>0.9 mg/dL), which is added evidence of ongoing disease. Septicemia, bronchopulmonary dysplasia, and retinopathy of prematurity were other diseases that occurred after randomization. Sepsis was confirmed by positive blood cultures and elevated C-reactive protein (34). The diagnosis of bronchopulmonary dysplasia was dependent upon a need for supplemental oxygen on day 28 of life and radiographic changes as described by Northway et al. (35). Retinopathy of prematurity was graded by standardized criteria (36) and categorized according to the more severely affected eye (Table 19.5).

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TABLE 19.5 Diseases of Pre-Term Infants by in-Hospital Diet Assignment Number (% incidence) experimental formula NEC stages II and III Male infants with NEC Female infants with NEC Bronchopulmonary dysplasiad Documented septicemia None 1 episode 2 episodes 3 episodes Retinopathy of prematurity None/none charted Stage 1 Stage 2 Stage 3 Stage 4 Deaths (all causes)

Control formula (n = 85) number (% incidence)

Experimental formula (n = 34) Experimental formula

15 (17.6) 11/43 (25.6) 4/42 (9.5)c 19 (23.4) 24 (28.2) 59 (69)25 22 (25.9) 2 (2.4) 2 (2.4) 34 (40.0) 51 (60) 29 (34.1) 1 (1.2) 3 (3.5)1 1 (1.2) 5 (6)

1(2.9)a 0/17 (0)b 1/17 (5.9) 8 (23.5) 9 (26.5) 25 (73.5) 6 (17.6) 2 (5.9) 1 (2.9) 13 (38.2) 21 (61.8) 12 (35.3) 0 (0) (1.2) 0 (0) 1 (3)

aSignificantly

different from the equivalent group of infants fed control formula, P < 0.05. < 0.01. cSignificantly different from male infants fed control formula, P < 0.05. dFour control infants who died before BPD could be diagnosed at 28 days of age were not included in the denominator. bP

Plasma Phospholipid Concentration and Fatty Acid Composition Plasma phosphatidylcholine (PC) and phosphatidlethanolamine (PE) AA and DHA concentrations were determined after extracting total plasma lipids, separating the individual phospholipids by thin layer chromatography, transesterifying phospholipid fatty acids with boron trifluoride-methanol to yield fatty acid methyl esters, and separating and quantifying the individual fatty acids by gas–liquid chromatography on a 0.25-mm × 30m fused silica column with a stationary liquid phase (SP 2330, Supelco, Inc., Bellafonte, PA). C17:0 was added as an internal standard to the phospholipids isolated by thin layer chromatography. The detailed methods used for plasma and formula fatty acid analysis have been described previously (37). The molar concentrations of PC and PE were estimated from the molar concentration and percent of total fatty acids as AA in each fraction by assuming less than one molecule of AA per molecule of phospholipid. Statistical Analysis Fisher’s test (38) was used to determine whether diet effected the incidence of disease. Repeated-measures ANOVA was used to compare in-hospital weight gain and energy intake before NEC and the effects of diet on concentrations of plasma phospholipids and their concentrations of AA and DHA.

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Results Incidence of Disease Of the 85 infants in the control group, 15 (17.6%) developed NEC stage II (n = 9) or III (n = 6) (Table 19.5). Their NEC was diagnosed at a mean of 18.4 days of age (range 12–35 d). Four of the fifteen infants with NEC died from the disease. One other control infant without NEC died after surgery for intestinal strictures. Only 1 of 34 infants (2.9%) fed the experimental formula developed NEC. That infant developed Stage III NEC at 18 days of age (Table 19.5), 12 days after starting the experimental formula, and died. The incidence of NEC in the experimental group was significantly lower than in the control group (P < 0.05) (Table 19.5). The incidence of NEC in the control group (17.6%) did not appear different from the 22.4% incidence of NEC among very-low-birth-weight infants cared for in our nursery before this study (1). On the other hand, the low incidence of NEC in the experimental group was unprecedented for our nursery. Male infants accounted for a disproportionate number of NEC cases. When male infants fed the control formula were compared with those fed the experimental formula, the effect of diet was highly significant (P < 0.01). Male infants fed the control formula also had a higher incidence of NEC than did female infants fed the control formula (P < 0.05) (Table 19.5). Although NEC has been associated with many neonatal and perinatal characteristics, six independent risk factors were identified in an earlier multicenter study that included our nursery: (i) black male, (ii) vaginal delivery, (iii) mother <25 yr, (iv) birthweight < 1000 g, (v) 5-min Apgar <7, and (vi) prolonged ruptured membranes. We reasoned that the infant who developed NEC on the experimental formula might carry a higher number of these risk factors that infants fed the control formula. In fact, the single case of NEC in the experimental group had five of these six risk factors, whereas the control infants with NEC had a range of one to five risk factors and carried a lower mean number of risk factors (Table 19.6). Antenatal steroids did not appear to reduce the incidence of NEC. Sixty-nine percent of infants with NEC received antenatal steroids compared with 54% of infants who did not develop NEC. We also looked at patent ductus arteriosis (PDA) and indomethacin exposure because these have been associated with NEC in several studies. Twelve infants developed NEC (11 control, 1 experimental subject) among the 103 (73 control, 30 experimental) infants who did not have PDA and were not exposed to indomethacin (11.7%). Four infants developed NEC (all control subjects) of the 13 infants with PDA (10 control, 3 experimental subjects) (30.8%), two among seven infants who were given indomethacin (5 control, 2 experimental subjects), and two among six infants (5 control, 1 experimental subject) who were not given indomethacin or surgical ligation. Three other infants (2 control, 1 experimental subject) exposed to indomethacin in utero had neither PDA nor NEC. The type of diet fed in the hospital had no influence on the incidence or apparent severity of documented septicemia, retinopathy of prematurity, or bronchopulmonary dysplasia (BPD) (Table 19.5).

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TABLE 19.6 Risk Factors for Necrotizing Enterocolitis Identified in Uauy et al. (1) as Present in Study Cases Case no.

Black male

Vaginal delivery

Mother < 25 yr >

Birth weight < 1000 g

5-min Apgar

Prolonged ROMb

No. of risk factors present

1 2 3 4 5a 6 7 8 9 10 11 12 13 14 15 16

Y Y N Y N Y N Y N Y Y Y Y Y Y N

N Y N Y Y Y Y Y Y N Y Y Y Y N Y

N Y Y Y Y Y Y Y Y N Y N Y Y N Y

N N Y N Y N N Y Y N N Y Y N Y N

N N N Y Y N N N N N N Y N N Y N

N N N N Yc Y N N Y N Y Y Y Y N Y

1 3 2 4 5 4 2 4 4 1 4 5 5 4 3 3

aFed

experimental formula. rupture of membranes. cDesignated as prolonged ROM because a circlage was placed at 20 wk of postmenstrual age to prevent delivery. bROM,

Energy Intake and Growth Table 19.7 shows the energy intake from parenteral and enteral nutrition during hospitalization. There were no differences in total energy intake or progression of enteral feeding between the diet groups that suggested more rapid progression of enteral intake in the group with the higher incidence of NEC (control subjects). Furthermore, diet did not influence weight gain among infants who could be maintained on enteral feeding (Table 19.8). If anything, enteral intake as a proportion of total energy intake was somewhat higher in the experimental group beginning in the third week of study (Table 19.7). Because enteral intakes were advanced as tolerated, the somewhat higher ratio of enteral to parenteral intake that developed in the experimental group well into the study could suggest generally better feeding tolerance before the development of overt disease in some infants. Concentration of Selected Lipids Although AA and DHA were components of the experimental formula, plasma PC AA and DHA concentration changed little between enrollment and two weeks after full enteral feeding (Fig. 19.1, A and B). In contrast, the concentrations of plasma PC AA and DHA decreased by approximately 40% in the control group. During

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TABLE 19.7 In-Hospital Energy Intake from Parenteral and Enteral Nutrition (kcal/kg per d ± SD, Number) of Infants Fed Control and Experimental (Egg Phospholipid) Formulaa Control formula Study 1 2 3 4 5 6 7

Experimental formula

Parenteral

Enteral

Parenteral

Enteral

63 ± 17 (83) 32 ± 26 (82) 18 ± 29 (76) 16 ± 29 (71) 15 ± 28 (49) 12 ± 25 (36) 5 ± 10 (20)

32 ± 17 (83) 67 ± 29 (82) 84 ± 38 (76) 91 ± 42 (71) 94 ± 41 (49) 102 + 38 (36) 107 ± 27 (20)

60 ± 17 (34) 34 ± 29 (32) 13 ± 20 (32) 7 ± 16 (28) 3 ± 58 (23) 2 ± 7 (18) 1 ± 2 (13)

31 ± 20 (34) 66 ± 30 (32) 91 ± 31 (32) 104 ± 31 (28) 111 ± 24 (23) 116 ± 22 (18) 128 ± 13 (13)

aParenteral

and enteral energy intakes were calculated for each complete week beginning with the day of enrollment. Infants lost from the study for any reason that precluded enteral feeding for a complete week were excluded in that week and in subsequent weeks. Infants lost or discharged home were treated as missing values. There were no statistical differences between formula groups but a highly significant effect of time by repeated measures analysis of variance. Kcal + 4.16 kJ.

the same interval, the total concentration of plasma PC increased by 27.7% in the experimental group but was virtually unchanged in the control group (Fig. 19.1C). The concentration of plasma PE AA increased by 98% between enrollment and two weeks after full enteral feeding in the experimental group, whereas there was little change in the concentration of PE AA in the control group (Fig. 19.1D). DHA declined in both groups but the decrease was larger in the control group (Fig. 19.1E). The total concentration of PE in the experimental group increased by 40% but there was no effect on PE in the control group (Fig. 19.1F). The relative effects of diet on the concentrations of PC and PE and on AA and DHA in these phospholipids were TABLE 19.8 In-Hospital Weight Gain (g/wk ± SD, Number) of Infants Fed Control and Experimental (Egg Phospholipid) Formulaa Study week

Control formula

Experimental formula

1 2 3 4 5 6 7

53 ± 61 (83) 117 ± 58 (82) 143 ± 68 (76) 147 ± 67 (71) 164 ± 74 (49) 193 ± 82 (36) 185 ± 72 (20)

33 ± 64 (34) 111 ± 50 (32) 146 ± 46 (32) 152 ± 78 (28) 168 ± 63 (23) 184 ± 48 (18) 192 ± 44 (13)

aWeight

gains were calculated for each complete week beginning with the day of enrollment. Infants lost from the study for any reason that precluded enteral feeding for a complete week were excluded in the week they were lost and in subsequent weeks. Infants lost or discharged home were treated as missing values. There were no statistical differences between formula groups but a highly significant effect of time by repeated measures analysis of variance.

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Fig. 19.1. (A) Plasma PC AA concentrations. (B) Plasma PC DHA concentrations. (C) Plasma total PC concentrations. (D) Plasma PE AA concentrations. (E) Plasma PE DHA concentrations. (F) Plasma total PE concentrations. Control group (●) and experimental group (▲). Study enrollment (E, ~5d of age), full enteral feeding (FF), and two weeks after FF (FF + 2). Data are expressed as the mean ± SD. *Diet groups differ, P < 0.05. (AA, arachidonic acid; DHA, docosahexanoic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine.

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similar and highly significant. However, as indicated here and in Fig. 19.1, the specific effects of diet were influenced by the particular phospholipid.

Discussion The egg phospholipids fed in this study were 75% PC, and they provided nearly equimolar amounts of AA and choline as well as small amounts of DHA. Because AA, choline, and their metabolic products play important roles in gastrointestinal function, it is plausible that one or more of these factors may have protected our study infants against NEC. For example, eicosanoids derived from AA are important regulators of normal gastrointestinal function (39). They act as vasodilators in the mesenteric vascular bed (40), thereby increasing blood flow to the intestine (41). Eicosanoids derived from AA also function as homeostatic regulators of intestinal motility and secretion (39) in cytoprotection of gastrointestinal mucus (42) and by increasing mucosal growth (43), mucus secretion (44), phospholipid synthesis (45), and the density of surfactantlike particles in the mucus gel layer (46). Compared with term infants, pre-term infants have lower plasma phospholipid AA (47). A relationship between the concentration of AA in plasma phospholipids and somatic growth of pre-term infants has been used to suggest that at least some formula-fed pre-term infants have suboptimal AA status due to their early birth and diets that do not include AA (48). The suggestion that AA could have been involved in protection against NEC is further supported by an earlier study that found more NEC (though not a statistically significant increase) (49) in pre-term infants whose phospholipid AA was decreased by feeding an experimental formula with long-chain n-3 fatty acids (50). It is well known that n-3 fatty acids and their lipid-derived mediators have different physiologic functions than do AA and mediators derived from AA. Prostaglandin E2 production depends upon tissue PC AA concentration (51). When the diet contains a balance of AA and DHA, such as the experimental formula fed in this study, there is evidence that tissue eicosanoid profiles favor the n-6 (e.g., AA) rather than n-3 (e.g., DHA) fatty acid family (52). The data of Huang and Craig-Schmidt (52) could be used to suggest that n-6 fatty acid derived prostaglandins were lower (though not measured) in our previous study (49) relative to the study presented here. In further support of the hypothesis that prostaglandins from AA may have had a protective role in this study, prostaglandin E1 has been shown to counteract the reduction in mesenteric blood flow and ameliorate the bowel injury in experimental NEC caused by platelet-activating factor (PAF) (53). Some reports show that pre-term infants given indomethacin to permit closure of a PDA have a higher incidence of NEC (54,55). However, the incidence of NEC was not affected when indomethacin was given prophylactically before PDA developed (56) and was lower among indomethacin-treated infants with clinically significant PDA (57). The variability among these reports suggests that a mediating variable, e.g., mesen-

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teric blood flow, may influence the response to indomethacin. More infants with PDA in this study developed NEC than did infants without PDA; however, the proportions of infants on each diet who developed PDA or who were treated for PDA with indomethacin were similar. The experimental formula provided four times as much choline as the control formula, nearly all as PC. The choline phospholipids (PC and sphingomyelin) are structurally important for all cell membranes, and lysoPC and lysosphingomyelin modulate protein kinase C activity and mediate growth-factor actions, respectively [see Zeisel (58) for review]. Choline is the source of acetylcholine, which is important for intestinal vasodilation and motility (59), mediating gastric mucus phospholipid secretion (60), and increasing intestinal fluid and ion transport (61). One mechanism by which small arteries in the mesentery dilate is by the binding of acetylcholine to muscarinic M-3 receptors followed by the release of nitric oxide from the arterial endothelium (62). Both nitric oxide (63) and arginine (64), a substrate of nitric oxide synthase, have been shown to protect against intestinal injury in experimental models of NEC caused by, respectively, PAF and intraluminal acidified casein. In choline deficiency, tissue acetylcholine is reduced (65,66). Subsequently, the unmodulated vasoconstrictive effects of adrenergic neurotransmitters may lead to tissue hypoxia and necrosis (65). Instances of heart, liver, kidney, and pancreatic necrosis have been reported in choline-deficient animals (67). Intestinal acetylcholine also decreases in choline deficiency (65), but neither low choline nor low acetylcholine has been associated with intestinal necrosis (67). Even if the intestine were less susceptible than other organs to choline deficiency, choline might be limited for intestinal functions under circumstances that increase the need for choline. Choline requirement is influenced by a number of physiologic and nutritional variables, including rapid growth/high energy intakes, male gender, intestinal flora, and nutritional status of protein, zinc, folic acid, vitamin B12, and antioxidants (67). Although the control formula had a choline concentration (174 mg or 1436 µmol/L) similar to mature human milk (158 mg or 1351 µmol/L) (68), conditions known to increase the need for choline commonly occur among pre-term infants. These include relatively higher energy intakes and growth, marginal protein status and marginal nutritional status in general, and overgrowth of atypical intestinal organisms. Additionally, low vitamin B12 and folic acid status characterize many pregnancies in the lower socioeconomic class (69), especially those ending in pre-term birth (70,71), such as the lower socioeconomic class pre-term infants studied here. Anther factor that might have influenced the incidence of NEC was that most choline in the experimental formula was PC. Homes-McNary et al. (68) showed that human milk contains less free choline and more esterified choline than cow’s milk-derived infant formulas, although human milk contained mainly phosphocholine and glycerophosphocholine. A study reported later by these authors found that these various sources of choline had different bioavailability in young rats (72).

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The experimental formula contained approximately seven times as much choline-containing phospholipids as human milk, mostly PC (68). PC is a major component of pulmonary surfactant, the deficiency of which can lead to respiratory distress syndrome. It is well known that pre-term infants have relatively low pulmonary conversion of phosphocholine to CDP-choline, the rate-limiting step in the enzymatic conversion of choline to PC (73). As a result, exogenous surfactants are routinely administered to the airways of pre-term infants to treat surfactant deficiency. Recent studies have demonstrated a gastrointestinal surfactant with many similarities to lung surfactants (45,74–77) that protects the gastric mucosa against damage from low pH (77–79). Like administration of exogenous surfactant into the airways with prevention against respiratory distress syndrome, exogenous phospholipids have been administered enterally and shown to protect against and promote the healing of gastric ulcers (79,80). At present, the function of intestinal surfactant and the ability of pre-term infant to synthesize or otherwise maintain intestinal surfactant are unknown. It is interesting to consider that the egg phospholipid-containing formula might have protected the luminal surface of the intestine in some manner (81), thereby reducing in the incidence of NEC. Antenatal steroids did not appear to protect against NEC as suggested by an earlier study (15), nor were they protective in several other recent reports (1,82,83). Human milk has been shown to lower the incidence of NEC (17), but none of the infants in this study received any human milk as the study was designed to compare infant formulas. The risk factors for NEC identified in an earlier study from the NICHD Neonatal Network (1) were present in the same proportion in each diet group even though NEC was not. Other evidence that the randomization led to comparison of equivalent groups may be found in Table 19.4 (similar neonatal/perinata characteristics), Table 19.5 (similar occurrence of disease other than NEC), and Tables 19.7 and 19.8 (similar enteral and parenteral nutrition and growth before developing NEC). Because the diet groups appeared to be similar, it is reasonable to conclude that the lower incidence of NEC in experimental formula was most likely due to the presence of egg phospholipid in that formula. In summary, we studied diseases in a select group of hospitalized pre-term infants to assess the risk/benefit of feeding an infant formula with egg phospholipids. Egg phospholipid-containing formula reduced the incidence of NEC but had no effect on the incidence or severity of other common diseases of hospitalized pre-term infants compared with the control formula. The number of infants studied was relatively small, but the effect of diet reached statistical significance because the reduction in NEC with the experimental formula was very large. A type I error cannot be ruled out from a single study; however, there is strong evidence for the importance of several components of egg phospholipids as well as dietary phospholipids themselves in maintenance of intestinal blood flow and cytoprotection of the intestinal mucosa. In pre-term infants, these physiologic functions of gastrointestinal tract and the roles that nutrients play in modulating them are poorly understood (84). However, the association between physiologic immaturity and NEC is well known. We speculate that one

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or more components of egg phospholipids enhanced one or more immature intestinal functions to lower the incidence of NEC in this study. Based on these data, a large prospective randomized trial to test the effects of this egg phospholipid-containing formula on NEC seems warranted, because NEC and the fear of NEC remain important causes of morbidity and mortality among pre-term infants.

Acknowledgments The authors thank Jeanette Peeples of the University of Tennessee, Memphis, for the plasma phospholipid fatty acid analyses and Rene Englehardt of Ross Products Division for choline analyses.

References 1. Uauy, R.D., Fanaroff, A.A., Korones, S.B., Phillips, E.A., Phillips, J.B., and Wright, L.L. (1991) Necrotizing Enterocolitis in Very Low Birth Weight Infants: Biodemographic and Clinical Correlates, J. Pediatr. 119, 630–638. 2. Brown, B.G., and Sweet, A.Y. (1982) Neonatal Necrotizing Enterocolitis, Pediatr. Clin. North Am. 29, 1149–1170. 3. Tobiansky, R., Lui, K., Roberts, S., and Beddovi, M. (1995) Neurodevelopmental Outcome in Very Low Birthweight Infants with Necrotizing Enterocolitis Requiring Surgery, J. Pediatr. Child Health 31, 233–236. 4. Rand, T., Weninger, M., Kohlhauser, C., Bischof, S., Heine-Peer, G., Tratting, S., Popow, C., and Salzer, H.R. (1996) Effect of Umbilical Arterial Catheterization on Mesenteric Hemodynamics, Pediatr. Radiol. 26, 435–438. 5. Lopez, S.L., Tacusch, H.W., Findlay, R.D., and Walther, F.J. (1995) Time of Onset of Necrotizing Enterocolitis in Newborn Infants with Known Prenatal Cocaine Exposure, Clin. Pediatr. 34, 424–429. 6. Tesley, A.M., Merritt, T.A., and Dixon, S.D. (1988) Cocaine Exposure in a Term Neonate. Necrotizing Enterocolitis as a Complication, Clin. Pediatr. 37, 547–550. 7. Kuscheid, T., and Holschneider, A.M. (1993) Necrotizing Enterocolitis Mortality and Long Term Results, Eur. J. Pediatr. Surg. 3, 139–143. 8. Kliegman, R.M., and Walsh, M.C. (1992) Pathophysiology and Epidemiology of Necrotizing Enterocolitis, in Fetal and Neonatal Physiology, Polin, R.A., Fox, W.W., WB Saunders, Philadelphia, vol. II, pp. 1078–1084. 9. Ohman, U. (1984) The Effects of Luminal Distension and Obstruction on the Intestinal Circulation, in Physiology of the Intestinal Circulation, Shepard, A.P., and Granger, D.N., Raven Press, York, pp. 321–334. 10 Clark, D.A., and Miller, M.J.S. (1992) Development of the Gastrointestinal Circulation in the Fetus and Newborn, in Fetal and Neonatal Physiology, Polin, R.A., and Fox, W.W., WB Saunders, Philadelphia, vol. I, pp. 690–694. 11. Beach, R.C., Menzies, I.S., Clayden, G.S., and Scopes, J.W. (1982) Gastrointestinal Permeability Changes in the Pre-Term Neonate, Arch. Dis. Child 57, 141–145. 12. Edelstone, D., and Holzman, I. (1984) Fetal and Neonatal Intestinal Circulation, in Physiology of the Intestinal Circulation, Shepherd, A.P., and Granger, D.N., Raven Press, New York, pp. 179–190. 13. Hoy, C., Millar, M.R., MacKay, P., Godwin, P.G.R., Langdale, V., and Levene, M.I. (1990) Quantitative Changes in Fecal Microflora Preceding Necrotizing Enterocolitis in Premature Neonates, Arch. Dis. Child 65, 1057–1059.

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14. Caplan, M.S., and MacKendrick, W. (1993) Necrotizing Enterocolitis: A Review of Pathogenetic Mechanisms and Implications for Prevention, Pediatr. Pathol. 13, 3576–369. 15. Bauer, C.R., Morrison, J.C., Poole, W.K., Korones, S.B., Boehm, J.J., Rigatto, H., and Zachman, R.D. (1984) A Decreased Incidence of Necrotizing Enterocolitis After Prenatal Glucocorticoid Therapy, Pediatrics 73, 682–688. 16. Halac, E., Halac, J., Begue, B.F., Casanas, J.M., Indiveri, D.R., Petit, J.F., Figueroa, M.J., Olmas, J.M., Rodriguez, J.A, Obregon, R.J., Martinez, M.V., Grinblat, D.A., and Vilarrodona, H.O. (1990) Prenatal and Postnatal Corticosteroid Therapy to Prevent Neonatal Necrotizing Enterocolitis: A Controlled Trial, J. Pediatr. 117, 132–138. 17. Lucas, A., and Cole, T.J. (1990) Breastmilk and Neonatal Necrotizing Enterocolitis, Lancet 336, 1519–1523. 18. Carrion, V., and Egan, E.A. (1990) Prevention of Neonatal Necrotizing Enterocolitis, J. Pediatr. Gastroenterol. Nutr. 11, 317–323. 19. Eibl, M.M., Wolf, H.M., Furnkranz, H., and Rosenkranz, A. (1988) Prevention of Necrotizing Enterocolitis in Low Birth-Weight Infants by IgA-IgG Feeding, N. Engl. J. Med. 319, 1–7. 20. Grylack, L.F., and Scanlon, J.W. (1978) Oral Gentamicin Therapy in the Prevention of Neonatal Necrotizing Enterocoliltis, Am. J. Dis. Child 132, 1192–1194. 21. Egan, E.A., Mantilla, G., Nelson, R.M., and Eitzman, D.V. (1976) A Prospective Controlled Trial of Oral Kanamycin in the Prevention of Neonatal Necrotizing Enterocolitis, J. Pediatr. 89, 467–470. 22. Israel, E.F., Schiffrin, E.J., Carter, B.A., Frieberg, B., and Walker, W.A. (1991) Cortisone Strengthens the Intestinal Mucosal Barrier in a Rodent Necrotizing Enterocolitis Model, Adv. Exp. Med. Biol. 310, 375–380. 23. Goldman, A.S., and Smith, C.S. (1979) Host Resistance Factors in Human Milk, J. Pediatr. 94, 295–296. 24. Carver, J.D., Cox, W.I., and Barness, L.A. (1990) Dietary Nucleotide Effects Upon Murine Natural Killer Cell Activity and Macrophage Activation, J. Parenter. Enternal Nutr. 14, 18–22. 25. Insoft, R.M., Sanderson, I.R., and Walker, W.A. (1996) Development of Immune Function in the Intestine and Its Role in Neonatal Diseases, Pediatr. Clin. North AM 43, 551–571. 26. Carver, J.D., and Barness, L.A. (1996) Trophic Factors for the Gastrointestinal Tract, Clin. Perinatol. 23, 265–285. 27. Egan, E.A., Nelson, R.M., Mantila, G., and Eitzman, D.V. (1977) Additional Experience with Routine Use of Oral Kanamycin Prophylaxis for Necrotizing Enterocolitis in Infants Under 1000 g, J. Pediatr. 93, 31–32. 28. Boyle, R., Nelson, J.S., Stonestreet, B.S., Peter, G., and Oh, W. (1978) Alterations in Stool Flora Resulting from Oral Kanaycin Prophylaxis of Necrotizing Enterocolities, J. Pediatr. 93, 857–861. 29. Ballard, J.L., Khoury, J.C., Wedig, K., Want, I., Eilers-Walsman, B.L., and Lipp, R. (1991) New Ballard Score Expanded to Include Extremely Premature Infants, J. Pediatr. 119, 417–423. 30. Lubchenco, L.O., Hansman, C., and Boyd, E. (1996) Intrauterine Growth in Length and Head Circumference as Estimated from Live Births at Gestational Ages from 26 to 42 Weeks, Pediatrics 37, 403–408. 31. Putman, J.C., Carlson, S.E., DeVoe, F.W., and Barness, L.A. (1982) The Effect of Variations in Dietary Fatty Acids on the Fatty Acid Composition of Erythrocyte Phosphatidylcholine and Phosphatidylethanolamine in Human Infants, Am. J. Clin. Nutr. 36, 106–114.

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51. Kawashima, Y., Mizuguch, H., and Kozuka, H. (1994) Modulation by Dietary Oils and Clofibric Acid of Arachidonic Acid Content in Phosphatidylcholine in Liver and Kidney of Rat: Effects on Prostaglandin Formation in Kidney, Biochem. Biophys. Acta 1210, 187–104. 52. Huang, M.-C., and Craig-Schmidt, M.C. (1996) Arachidonate and Docosahexaenoate Added to Infant Formula Influenced Fatty Acid Composition and Subsequent Eicosanoid Production in Neonatal Pigs, J. Nutr. 126, 2199–2208. 53. Zhang, C., and Hsueh, W. (1991) PAF-Induced Bowel Necrosis: Effects of Vasodilators, Dig. Dis. Sci. 36, 634–640. 54. Norton, M.R., Merrill, J., Cooper, B.A., Kuller, J.A., and Clyman, R.I. (1993) Neonatal Complications After the Administration of Indomethacin for Pre-Term Labor, N. Engl. J. Med. 329, 1602–1607. 55. Major, C.A., Lewis, D.F., Harding, J.A., Porto, M.A., and Garite, T.J. (1994) Tocolysis with Indomethacin Increases the Incidence of Necrotizing Enterocolitis in the LowBirth Weight Neonate, Am. J. Obstet. Gynecol. 170, 102–106. 56. Couser, R.J., Ferrera, T.B., Wright, G.B., Caballa, A.K., Schilling, C.G., Hoekstra, R.E., and Payne, N.R. (1996) Prophylactic Indomethacin Therapy in the First Twenty-Four Hours of Life for the Prevention of Patent Ductus Arteriosus in Pre-Term Infants Treated Prophylactically with Surfactant in the Delivery Room, J. Pediatr. 128, 631–637. 57. Robie, D.K., Waltrip, T., Garcia-Prats, J.A., Pokorny, W.J., and Jaksic, T. (1996) Is Surgical Ligation of a Patent Ductus Arteriosus the Preferred Initial Approach for the Neonate with Extremely Low Birth Weight? J. Pediatr. Surg. 31, 1134–1137. 58. Zeisel, S.H., (1991) Choline and Human Nutrition, Annu. Rev. Nutr. 14, 269–296. 59. Bean, J.W., and Sidky, M.M. (1958) Intestinal Blood Flow as Influenced by Vascular and Motor Reactions to Acteylcholine and Carbon Dioxide, Am. J. Physiol. 194, 512–518. 60. Sengupta, S., Piotrowski, E., Slomiany, A., and Slomiany, B.L. (1991) Role of Adrenergic and Cholinergic Mediators in Gastric Mucus Phospholipid Secretion, Biochem. Int. 24, 1145–1153. 61. Cooke, H.J. (1987) Neural and Humoral Regulations of Small Intestine Electrolyte Transport, in Physiology of Gastrointestinal Tract, 2nd edn., Johnson, L.R., Raven Press, New York, pp. 1307–1350. 62. Wu, C.C., Chen, S.J., and Yen, M.H. (1997) Loss of Acetylcholine-Induced Relaxation by M-3 Receptor Activation in Mesenteric Arteries of Spontaneously Hypertensive Rats, J. Cardiovasc. Pharmacol. 30, 245–252. 63. MacKendrick, W., Caplan, M., and Hsueh, W. (1993) Endogenous Nitric Oxide Protects Against Platelet Activation Factor-Induced Bowel Injury in the Rat, Pediatr. Res. 32, 222–238. 64. Di Lorenzo, M., Bass, J., and Krantis, A. (1995) Use of L-Arginine in the Treatment of Experimental Necrotizing Enterocolitis, J. Pediatr. Surg. 30, 235–241. 65. Naglar, A.L., Dettbarn, W.-D., Seifter, E., and Levenson, S.M. (1968) Tissue Levels of Acetylcholine and Acetyl Cholinesterase in Weaning Rats Subjected to Acute Choline Deficiency, J. Nutr. 94, 13–19. 66. Nagler, A.L., Dettbarn, W.-D., and Levenson, S.M. (1968) Tissue Levels of Acetylcholine and Acetyl Cholinesterase in Weanling and Germfree Rats Subjected to Acute Choline Deficiency, J. Nutr. 95, 603–606. 67. Wilson, R.B. (1978) Nutrition and Food, Section E: Nutritional Disorders, CRC Press, West Palm Beach, FL, vol. II, pp. 95–121. 68. Holmes-McNary, M.Q., Cheng, W.-L., Mar, M.-H., Fussel, S., and Zeisel, S.H. (1996) Choline and Choline Esters in Human Milk and Rat Milk and in Infant Formulas, Am. J. Clin. Nutr. 64, 572–576. 69. Bailey, L.M., Mahan, C.S., and Dimperio, D. (1997) Folacin and Iron Status in LowIncome Pregnant Adolescents and Mature Women, Am. J. Clin. Nutr. 33, 1997–2001.

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70. Carlson, S.E., Palmer, S.M., Caldwell, E.I., and Rhodes, P.O. (1985) Folate Status and Length of Gestation, Am. J. Clin. Nutr. 41, 844. 71. O’Scholl, T., Hediger, M.U., Schal, J.I., Khoo, C.-S., and Fisher, R.L. (1996) Dietary and Serum Folate: Their Influence, Am. J. Clin. Nutr. 63, 520–525. 72. Cheng, W.-L., Holmes-McNary, M.Q., Mar, M.-H., Lien, E.L., and Zeisel, S.H. (1996) Bioavailability of Choline and Choline Esters from Milk in Rat Pups, J. Nutr. Biochem. 7, 457–464. 73. Farrell, P.M., Epstein, M.F., Fleischman, A.K., Oakes, G.K., Chez, R.A. (1976) Lung Lecithin Biosynthesis in the Nonhuman Primate Fetus: Determination of the Primary Pathway in Vivo, Biol. Neonate 29, 238–246. 74. Wassef, M.K., Lin, Y.N., and Horowitz, M.J. (1997) Molecular Species of Phosphatidylcholine from Rat Gastric Mucosa, Biochim. Biophys. Acta 573, 222–226. 75. Butler, B.D., Lichtenberger, L.M., and Hills, B.A. (1983) Distribution of Surfactants in the Canine GI Tract and Their Ability to Lubricate, Am. J. Physiol. 7, G645–G651. 76. Mach, D.R., Neumann, A.W., Policova, Z., and Sherman, P.M. (1992) Surface Hydrophobicity of the Intestinal Tract, Am. J. Physiol. 262, G171–G177. 77. Lichtenberger, L.M. (1995) The Hydrophobic Barrier Properties of Gastrointestinal Mucus, Ann. Rev. Physio. 57, 565–583. 78. Goddard, P.J., Lichtenberger, L.M. (1987) Does Aspirin Damage the Canine Gastric Mucosa by Reducing Its Surface Hydrophobicity? Am. J. Physiol. 15, G421–G430. 79. Lichtenberger, L.M., Graziani, L.A., Dial, E.F., Butler, B.D., and Hills, B.A. (1983) Role of Surface-Active Phospholipids in Gastric Cytoprotection, Science 219, 1327–1329. 80. Swarm, R.A., Ashley, S.W., Soybel, D.I., Ordway, F.S., and Cheung, L.Y. (1987) Protective Effect of Exogenous Phospholipid on Aspirin-Induced Gastric Mucosal Injury, Am. J. Surg. 153, 48–53. 81. Bengmark, S., and Jeppsson, G. (1995) Gastrointestinal Surface Protection and Mucosa Reconditioning, J. Parenter. Enterol. Nutr. 19, 410–415. 82. Moise, A.A., Weardon, M.F., Kozinetz, C.A., Gest, A.J., Welty, S.E., Hanson, T.N. (1995) Antenatal Steroids Are Associated with Less Need for Blood Pressure Support in Extremely Premature Infants, Pediatrics 95, 845–850. 83. Tucker, L., Hoff, C., Peevy, K., Brost, B., Holland, S., and Calhoun, B.C. (1995) The Effects of Antenatal Steroids Use in Premature Rupture of Membranes, Aust. NZ J. Obstet. Gynrcol. 35, 390–392. 84. Kleigman, R.M., Walker, W.A., and Yolken, R.H. (1993) Necrotizing Enterocolitis: Research Agenda for a Disease of Unknown Etiology and Pathogenesis, Pediatr. Res. 34, 701–708. 85. Engelhardt, R., and Winzeler, J. (1993) Automated and Rapid Determination of Choline in Nutritional Products Employing Immobilized Choline Oxidase and Electrochemical Detection of Hydrogen Peroxide, Institute of Food Technologist Annual Meeting Technical Program Book of Abstracts, p. 120.

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Chapter 20

Perinatal Supply and Metabolism of Long-Chain Polyunsaturated Fatty Acids Hans Demmelmair, Elvira Larque, and Berthold Koletzko Division of Metabolism and Nutrition, Kinderklinik and Kinderpoliklinik, Dr. von Hauner Children’s Hospital, Ludwig-Maximilians-University of Munich, D-80337 München, Germany

Introduction The potential of the early diet for modulation of the normal trajectory of brain development is of great interest. Long-chain polyunsaturated fatty acids (LCPUFA), especially arachidonic (AA) and docosahexaenoic acid (DHA), are preferentially deposited in relatively high concentrations in developing neural cells, and they modulate the structure, fluidity, and function of brain membranes (1,2). DHA promotes intercellular signaling and via the flexibility of its acyl chain positively influences the biophysical properties of membranes. Furthermore, there is an influence on apoptosis and gene expression (3). The prenatal and postnatal accretion of LCPUFA determines myelination and synaptogenesis during the postnatal brain growth spurt (4). Recent studies have also provided evidence that DHA is involved in dopamine and serotonin metabolism (2). In preterm babies the availability of AA has been associated with weight at birth and growth during the first year of life (5). AA and DHA are synthesized by desaturation and elongation of the essential fatty acids linoleic acid (LA, C18:2 n-6) and α-linolenic acid (ALA, C18:3 n-3), respectively (Fig. 1). The metabolism of LA and ALA uses the same enzymes, resulting in competition between n-6 and n-3 fatty acids (6–8). In the central nervous system, neurons appear unable to carry out fatty acid desaturation. In contrast, glial cells, astrocytes, and cerebral endothelium can elongate and desaturate precursors of LCPUFA and accumulate DHA for maintaining a brain environment enriched in LCPUFA (9). However, the accumulation of preformed DHA and AA in the brain is far more efficient than the desaturation and elongation of the precursors (10). In humans the fetal and infant brain DHA content is relatively more affected by the diet than AA content, suggesting that endogenous metabolic regulation of AA contents is more effective (11). This article discusses some aspects of fatty acid metabolism during the perinatal period, focusing on LCPUFA. As sources of fatty acids during this period, the metabolism of fatty acids in pregnant women and the transfer from mothers to their babies via the placenta before birth and the involvement of various body

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Fig. 20.1. Simplified scheme of the conversion of the precursor esential fatty acids to their long-chain polyunsaturated metabolites.

stores in lipid metabolism during lactation are discussed. Furthermore, some aspects of infantile LCPUFA synthesis are addressed.

Sources of Essential- and Long-Chain Polyunsaturated Fatty Acids in the Prenatal Period The intrauterine development of the fetus depends on an adequate nutrient supply via the placenta. This cannot be achieved by simple diffusion of substrates, but the transfer is regulated by the placenta as it metabolizes substrates, and the transport proteins appear to be adapted to fetal demands (12). Nevertheless, for all transported substrates or metabolites the transplacental concentration gradient is an important influencing factor (13). In all mammals glucose, amino acids, and fatty acids are transferred from mother to fetus, but particularly for fatty acids there are considerable differences between species (14). Corresponding to a relatively high fat deposition in the human fetus, there is a relatively high placental transfer of lipids in humans (15). Lipids are transferred as the fetus requires essential LA and ALA, and additionally, not all other fatty acids can be synthesized to a sufficient extent by the fetus itself. Umbilical vessel walls of intrauterine growth-retarded infants showed significantly lower proportions of LCPUFA, which the authors ascribed to a deteriorated placental transfer of fatty acids (16). Although glucose is the main source of energy for the fetus, lipids are used for the gain of energy. Furthermore there is a considerable deposition of lipids in the fetal tissues. The accreted adipose tissue contains mainly saturated and monounsaturated fatty acids, but newly formed membranes and nervous tissue are rich in

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lipids and mainly contain LCPUFA, especially DHA (17,18). The importance of intrauterine LCPUFA transfer is emphasized by the decreasing portion of LCPUFA in plasma phospholipids postpartum, even in breastfed neonates (19), which suggests that intrauterine acquired lipid stores are used for postnatal development. Consequently, prematurely born infants are at an elevated risk for adverse effects of a suboptimal supply, as they lack adequate lipid stores (20). Comparisons of the fatty acid compositions of infantile and maternal total plasma lipids and individual lipid classes have shown a lower proportion of essential fatty acids, but a higher proportion of LCPUFA in infantile relative to maternal plasma (21) suggesting a preferential placental transfer of LCPUFA that appears to correlate to fetal requirements. Several studies have shown that increased dietary n-3 LCPUFA intake during pregnancy causes increased n-3 LCPUFA levels in maternal plasma, which is reflected in higher levels of n-3 LCPUFA in cord plasma and umbilical vessel walls (22). This demonstrates that diet affects fetal supply, but no conclusions on the mechanisms involved in the transfer are possible. Deeper insight into the mechanisms of placental transfer has been obtained by using the perfused placenta model. In these experiments it was shown that triglycerides, phospholipids, and cholesterol esters cannot pass the placenta as such, while nonesterified fatty acids pass the placenta in both directions (23,24). Fatty acids are liberated from lipids by the action of placental lipoprotein lipase and taken up by throphoblasts, where they may be partially esterified into triglycerides, but the low triglyceride concentration in the cells suggests a high turnover of triglycerides (25). At the fetal side fatty acids are released mainly as nonesterified fatty acids, but additionally mono-, di-, and triglycerides and phospholipids are found (24). Interestingly, AA was preferentially bound in fetal phospholipids relative to LA, although the overall transfer of LA was higher (26). As intact PL apparently cannot pass the placenta, this channelling of AA into PL by the placental metabolism might contribute to the increased LCPUFA content on the fetal side. Recently a major role of fatty acid binding proteins (FABP) with respect to the modification of the fatty acid composition during the transplacental transfer has been recognized (27). Albumin-bound fatty acids or fatty acids hydrolyzed from triglycerides can be bound by FABP, as fatty acids show a significantly higher affinity to FABP than to albumin. Mediated by the FABP, whose preferential allocation at the maternal side of trophoblasts has been shown (28), fatty acids enter the trophoblast and, due to the concentration gradient, are released at the fetal side, where they may be accepted by albumin or α-fetoprotein (29). As the affinity toward placental FABP differs between fatty acids, lipid composition can be modified during the transfer. In vitro perfusions of human placentas have shown that placental FABP, in contrast to hepatic FABP, preferentially bind LCPUFA (30). As free fatty acids have been used for the perfusion studies, a potential influence of lipoprotein lipase was not investigated, but an influence of the maternal fatty acid composition could be demonstrated. By inclusion of D2O as a freely diffusible

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marker, absolute transfer rates were determined, which strongly depend on the concentrations on the maternal side. An increase of the maternal DHA concentration from 0.3 to 2.3% caused an increase of the transfer of 0.03 nmol to 0.38 nmol per mL perfusion fluid (31). However, under these in vitro conditions an open circuit for the fetal side was applied, which means that the concentration gradient did not respond to the increased transfer as one would expect to occur in vivo.

Essential Fatty Acid and LCPUFA Supply with Breastfeeding After birth, breastfed infants receive appreciable amounts of preformed AA and DHA with human milk lipids. The fatty acid concentration of milk is related to the maternal diet, maternal plasma fatty acid composition, length of breastfeeding, and other factors (32). Whereas LA values appear to be related to maternal dietary LA intake, AA values in milk are within a rather narrow range. In contrast, there are more than fourfold differences between the lowest and highest ALA and DHA values, respectively, which indicates a larger relative variability of n-3 than of n-6 fatty acid contents in human milk (33). Although milk LCPUFA content is influenced by the maternal diet, studies investigating milk composition indicate some metabolic control of milk PUFA contents. We studied n-6 PUFA metabolism in lactating women using uniformly 13Clabeled LA (34). The collection of milk and breath samples over a period of 5 days after the tracer application revealed that about 30% of milk LA is directly transferred from the diet, whereas about 11% of milk dihomo-γ-linolenic acid and 1.2% of milk AA originate from endogenous conversion of dietary LA (Fig. 20.2). Therefore, the major portion of PUFA in human milk lipids is derived from maternal body stores and not directly from the maternal diet. This results in a relatively constant milk AA supply to the recipient infant, which might be of biological benefit. In a more recent study, we evaluated the contribution of dietary and endogenously synthesized AA to its milk secretion in 10 Mexican women on a habitual diet with a very low fat content (35). The accumulated 72-hour recovery of 13C-LA in milk was 16.3 ± 6.4% of the dose, but only 0.01% of the label was found as 13C-AA. The AA stemming from conversion of dietary linoleic acid contributed only 1.1% to the total milk AA secreted. In this population 70% of LA secreted in milk were not derived from direct intestinal absorption. Moreover, if the transfer intensity for dietary AA is similar to that of dietary LA, almost 90% of milk AA are not derived directly from the diet. Thus, as only a minor fraction of milk AA stemmed from conversion of LA, maternal body stores must be the major sources of human milk LA and AA.

LCPUFA Status in Infants Fed Human Milk or Formula Without and with LCPUFA In contrast to human milk, conventional milk formulas with fat derived from vegetable oils do not provide appreciable amounts of LCPUFA. Infants fed such formulas

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CO2

Gut Linoleic acid in human milk

20% Liver

12.5% 30% from the diet, 70% from maternal depots Adipose tissue and other depots

Fig. 20.2. Schematic depiction of linoleic acid turnover in human lactation. Stable isotope

studies with oral intake of 13C-labeled linoleic acid indicate that 12.5% of dietary linoleic acid is transferred into milk, 20% is oxidized, and about 67% is deposited in maternal stores with slow turnover, such as adipose tissue (34). Only 30% of the infant linoleic acid supply in human milk is derived directly from the maternal diet, but 70% originates from maternal body depots with slow turnover. Thus, breastfed infants receive a relatively constant PUFA supply even if there are short-term changes of maternal dietary intake.

depend on the utilization of body stores, or on endogenous LCPUFA synthesis, for tissue deposition. A number of studies have evaluated the fatty acid composition of plasma and erythrocyte membrane lipid classes in full-term infants fed human milk or formula without LCPUFA in order to estimate their LCPUFA status. Markedly lower values of AA and DHA in plasma and red blood cells were found in infants fed formula not providing preformed LCPUFA as compared with breastfed infants at different ages (36,37). Moreover, not only plasma and red blood cell but also tissue LCPUFA contents are affected (38). The proportion of DHA in the brain cortex of breastfed infants was higher compared to those fed formula without LCPUFA (39). Although infantile LCPUFA synthesis has been demonstrated in newborns during the first week of life with refined stable isotope techniques, a limited ∆6desaturate activity and a high utilization of LCPUFA for decomposition, oxidation, and metabolic conversion to eicosanoids seems to result in an inadequacy of endogenous n-6 and n-3 LCPUFA synthesis to prevent LCPUFA depletion in infants fed conventional formulas without preformed (40,41). Supplementation studies were carried out with fat supplements, such as a fish oil with low eicosapentaenoic acid (EPA) content, fractionated egg yolk phospholipids, and single cell oils from algae and fungi (1). The studies showed that a balanced supplementation with both DHA and AA can normalize LCPUFA status in infants relative to reference groups fed human milk. As the initial ∆6-desaturation seems to be the limiting step in endogenous AA synthesis, it is tempting to supplement the infantile diet with γ-linolenic acid

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(GLA), which is readily available from plant oils, such as borage oil or black currant seed oil, which show high contents of GLA (42). We have performed a feeding study in preterm infants, comparing a standard formula (without LCPUFA and GLA) to a formula with 0.6% GLA, to a formula with 0.6% GLA + 0.1% EPA + 0.3% DHA, to a formula with 0.6% GLA + 0.2% EPA + 0.3% DHA and to human milk (43). In agreement with other studies (44,45) we observed after four weeks on the corresponding diets that AA content in plasma phospholipids was not significantly different between the formula groups. As the AA content was lower than in the breastfed group (0.5% AA), this clearly shows that dietary GLA was not endogenously converted to AA in a sufficient amount to replace exogenous AA. Although the formulas did not provide appreciable amounts of dietary dihomo-γlinolenic acid (DGLA) the plasma phospholipid percentage of DGLA tended to be higher in the infants who received a formula with GLA than in the breast fed infants (0.4% DGLA of total breast milk fatty acids. Thus elongation was very efficient, but the ∆5-desaturation step or the incorporation into phospholipids seemed to limit the AA percentage in the GLA-fed infants. The different EPA contents in the study formulas were clearly translated into correspondingly different EPA contents in infantile plasma PL but did not significantly influence the AA content; thus competatitive inhibition of in corporation by EPA does not seem to be the reason for the absence of a GLA effect. Considering that a certain part of the exogenous GLA was oxidized (46), there seems to be a stronger affinity toward incorporation of DGLA in phospholipids than toward further desaturation. Thus an increased addition of GLA to the diet might somewhat improve AA status, but GLA and DGLA would accumulate and quantitative relations between n-6 fatty acids would differ from breast fed infants. This observation is in contrast to the results of a tracer study, showing that only a small portion of dietary 13C-LA was converted to 13C-DGLA in breastfed neonates, whereas a considerable portion of the 13C-DGLA was desaturated to AA (19). Thus it is tempting to speculate that there is a difference in the metabolic disposal between endogenously produced GLA and dietary GLA. More sophisticated tracer studies would be required to resolve this question. As a balanced supply of AA and DHA appears to be required for optimal infantile development, at present supplementation with both AA and DHA is the preferred choice. The available trials support the efficacy of n-3 LCPUFA intake on the early development of the visual system, which was not achieved to a similar extent with formulas providing the n-3 precursor PUFA ALA. In a meta-analysis of the previously published results, San Giovanni and co-workers concluded that DHA-supplemented formula versus DHA-free formula showed significant differences in visual resolution acuity at two and four months of age, with combined estimates of behaviorally based visual resolution acuity differences at these ages of 0.47 ± 0.14 octaves and 0.28 ± 0.08 octaves, respectively, at two and four months (47). A oneoctave difference equals a reduction in the width of the stimulus elements by 50%.

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Conclusions There is accumulating evidence for effects of perinatal LCPUFA supply and metabolism on early nervous system development. Although some studies could not detect effects of DHA provision on visual function in healthy infants, other trials have clearly documented that the addition of preformed DHA to infant formulas improves visual acuity in preterm infants. Recommendations for the dietary requirements of n-6 and n3 fatty acids for brain development are complex and involve both the amounts and balance of the precursors LA and ALA and the amounts and balance of AA and DHA. Variability of human milk fatty acid composition highlights the need for caution in using these data as the basis to define infant substrate requirements (48). References 1. Koletzko, B., and Decsi, T. (2001) The Role of Long-Chain Polyunsaturated Fatty Acids for Infant Growth and Development, in Preventive Nutrition, Bendlich, A., and Deckelbaum, R.J., Preventive Nutrition. 2. Innis, S.M. (2000) The Role of Dietary n-6 and n-3 Fatty Acids in the Developing Brain, Dev. Neurosci. 22, 474–480. 3. Salem, N. Jr., Litman, B., Kim, H.Y., and Gawrisch, K. (2001) Mechanisms of Action of Docosahexaenoic Acid in the Nervous System, Lipids 36, 945–959. 4. Fernstrom, J.D. (1999) Effects of Dietary Polyunsaturated Fatty Acids on Neuronal Function, Lipids 32, 161–169. 5. Koletzko, B., and Braun, M. (1991) Arachidonic Acid and Early Human Growth: Is There a Relation? Ann. Nutr. Metab. 35, 128–131. 6. Sprecher, H. (2000) Metabolism of Highly Unsaturated n-3 and n-6 Fatty Acids, Biochim. Biophys. Acta 1486, 219–231. 7. Sauerwald, T.U., Hachey, D.L., Jensen, C.L., Chen, H., Anderson, R.E., and Heird, W.C. (1997) Intermediates in Endogenous Synthesis of C22:6ω3 and C20:4ω6 by Term and Preterm Infants, Pediatr. Res. 41, 183–187. 8. Sprecher, H., Luthria, D.L., Mohammed, and Baykousheva, S.P. (1995) Reevaluation of the Pathways for the Biosynthesis of Polyunsaturated Fatty Acids, J. Lipid Res. 36, 2471–2477. 9. Moore, S.A., Yoder, E., Murphy, S., Dutton, G.R., and Spector, A.A. (1991) Astrocytes, Not Neurons, Produce Docosahexaenoic Acid and Arachidonic Acid, J. Neurochem. 6, 518–524. 10. Greiner, R.C., Winter, J., Nathanielsz, P.W., and Brenna, J.T. (1997) Brain Docosahexaenoate Accretion in Fetal Baboons: Bioeqivalence of Dietary Alpha-Linolenic and Docosahexaenoic Acids, Pediatr. Res. 42, 826–834. 11. Makrides, M., Neumann, M., Byard, J., Simmer, K., and Gibson, R. (1994) Fatty Acid Composition of Brain, Retina, and Erythrocytes in Breast- and Formula-Fed Infants, Am. J. Clin. Nutr. 60, 189–194. 12. Schneider, H. (1991) The Role of the Placenta in Nutrition of the Human Fetus, Am. J. Obstet. Gynecol. 164, 967–973. 13. Schneider, H. (1991) Placental Transport Function, Reprod. Fertil. Dev. 3, 345–353. 14. Thomas, C.R. (1987) Placental Transfer of Nonesterified Fatty Acids in Normal and Diabetic Pregnancy, Biol. Neonate 51, 94–101. 15. Battaglia, F.C., and Meschia, G. (1986) Introduction to Fetal Physiology, Academic Press, Orlando.

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16. Felton, C.V., Chang, T.C., Crook, D., Marsh, M., Robson, S.C., and Spencer, J.A.D. (1994) Umbilical Vessel Wall Fatty Acids After Normal and Retarded Fetal Growth, Arch. Dis. Child 70, F36–F39. 17. Clandinin, M.T. (1999) Brain Development and Assessing the Supply of Polyunsaturated Fatty Acids, Lipids 34, 131–137. 18. Crawford, M.A., Costeloe, K., Ghebremeskel, K., Phylactos, A., Skirvin, L., and Stacey, F. (1997) Are Deficits of Arachidonic and Docosahexaenoic Acids Responsible for the Neural and Vascular Complications of Preterm Babies? Am. J. Clin. Nutr. 66, 1032S–1041S. 19. Szitanyi, P., Koletzko, B., Mydlilova, A., and Demmelmair, H. (1999) Metabolism of 13C-Labelled Linoleic Acid in Newborn Infants During the First Week of Life, Pediatr. Res. 45, 669–673. 20. Koletzko, B. (1998) Lipid Supply and Metabolism in Infancy, Current Opinion in Clinical Nutrition and Metabolic Care 1, 171–177. 21. Berghaus, T.M., Demmelmair, H., and Koletzko, B. (1998) Fatty Acid Composition of Plasma Lipid Classes in Maternal and Cord Plasma: Conclusions for Placental Transfer, Eur. J. Pediatr. 157, 31–36. 22. Van Houwelingen, A.C., Dalby Sorensen, J., Hornstra, G., Simonis, M.M.G., Boris, J., Olsen, S.F., and Secher, N. (1995) Essential Fatty Acid Status in Neonates After FishOil Supplementation During Late Pregnancy, Br. J. Nutr. 74, 723–731. 23. Dancis, J., Jansen, V., Kayden, H.J., Schneider, H., and Levitz, M. (1973) Transfer Across Perfused Human Placenta. II. Free Fatty Acids, Pediatr. Res. 7, 192–197. 24. Coleman, R.A. (1986) Placental Metabolism and Transport of Lipid, Federation Proc. 45, 2519–2523. 25. Szabo, A.J., De Lellis, R., and Grimaldi, R.D. (1973) Triglyceride Synthesis by the Human Placenta, Am. J. Obstet. Gynecol. 15, 257–262. 26. Kuhn, D.C., and Crawford, M. (1986) Placental Essential Fatty Acid Transport and Prostaglandin Synthesis, Prog. Lipid Res. 25, 345–353. 27. Campbell, F.M., Gordon, M.J., and Dutta-Roy, A.K. (1996) Preferential Uptake of Long-Chain Polyunsaturated Fatty Acids by Isolated Human Placental Membranes, Mol. Cell Biochem. 155, 77–83. 28. Campbell, F., and Dutta-Roy, A.K. (1995) Plasma Membrane Fatty Acid-Binding Protein (FABPpm) is Exclusively Located in the Maternal Facing Membranes of the Human Placenta, FEBS Letters 375, 227–230. 29. Neville, M.C. (1999) Adaption of Maternal Lipid Flux to Pregnancy: Research Needs, Eur. J. Clin. Nutr. 53, S120–S123. 30. Campbell, F., Gordon, M.J., and Dutta-Roy, A.K. (1998) Placental Membrane Fatty Acid-Binding Protein Preferentially Binds Arachidonic and Docosahexaenoic Acids, Life Sciences 4, 235–240. 31. Haggerty, P., Ashton, J., Joynson, M., Abramovich, D.R., and Page, K. (1999) Effect of Maternal Polyunsaturated Fatty Acid Concentration on Transport by the Human Placenta, Biol. Neonate 75, 350–359. 32. Rodriguez-Palmero, M., Koletzko, B., Kunz, C., and Jensen, R. (1999) Nutritional and Biochemical Properties of Human Milk, Part II: Lipids, Micronutrients, and Bioactive Factors, Clin. Perinatol. 26/2, 335–359. 33. Lauritzen, L., Horby Jorgensen, M., Hansen, H.S., and Michaelson, K.F. (2002) Fluctuations in Human Milk Long-Chain PUFA Levels in Relation to Dietary Fish Intake, Lipids 37, 237–244.

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34. Demmelmair, H., Baumheuer, M., Koletzko, B., Dokoupil, K., Kratl, G. (1998) Metabolism of U13C-Labeled Linoleic Acid in Lactating Women, J. Lipid Res. 39, 1389–1396. 35. Del Prado, M., Villalpando, S., Elizondo, S., Rodriguez, M., Demmelmair, H., and Koletzko, B. (2001) Contribution of Dietary and Newly Formed Arachidonic Acid to Human Milk Lipids in Women Eating a Low-Fat Diet, Am. J. Clin. Nutr. 74, 242–247. 36. Koletzko, B., Edenhofer, S., Lipowsky, G., and Reinhardt, D. (1995) Effects of a Low Birthweight Infant Formula Containing Human Milk Levels of Docosahexaenoic and Arachidonic Acids, J. Pediatr. Gastroenterol. Nutr. 21, 200–208. 37. Decsi, T., Kelemen, B., Minda, B., Burus, I., and Kohn, G. (2000) Effect of Type of Early Infant Feeding on Fatty Acid Composition of Plasma Lipid Classes in Full-Term Infants During the Second 6 Months of Life, J. Pediatr. Gastroenterol. Nutr. 30, 547–551. 38. Koletzko, B., Knoppke, B., Von Schenck, U., Demmelmair, H., and Damli, A., (1999) Noninvasive Assessment of Essential Fatty Acid Status in Preterm Infants by Mucosal Cell Phospholipid Analysis, J. Pediatr. Gastroenterol. Nutr. 29, 467–474. 39. Gibson, R.A., Neumann, M.A., and Makrides, M. (1996) Effect of Dietary Docosahexaenoic Acid on Brain Composition and Neural Function in Term Infants, Lipids 31, S177–S181. 40. Demmelmair, H., Von Schenck, U., Behrendt, E., Sauerwald, T., and Koletzko, B. (1995) Estimation of Arachidonic Acid Synthesis in Full Term Neonates Using Natural Variation of 13C Content, J. Pediatr. Gastroenterol. Nutr. 21, 31–36. 41. Carnielli, V.P., Wattimea, D.J.L., Luijendijk, I.H.T., Boerlage, A., Degenhart, H.J., and Sauer, P.J.J. (1996) The Very Low Weight Premature Infant Is Capable of Synthesizing Arachidonic and Docosahexaenoic Acids from Linoleic and Linolenic Acids, Pediatr. Res. 40, 169–174. 42. Fan, Y.Y., and Chapkin, R.S. (1998) Importance of Dietary γ-Linolenic Acid in Human Health and Nutrition, J. Nutr. 128, 1411–1414. 43. Demmelmair, H., Feld, F., Horvath, I., Niederland, T., Ruszinko, V., Raederstorff, D., De Min, C., Muggli, R., and Koletzko, B. (2001) Influence of Formulas with Borage Oil or Borage Oil Plus Fish Oil on the Arachidonic Acid Status in Premature Infants, Lipids 36, 555–566. 44. Horby Jorgensen, M., Holmer, G., Lund, P., Hernell, O., and Fleischer Michaelsen, K. (1998) Effect of Formula Supplemented with Docosahexaenoic Acid and γ-Linolenic Acid on Fatty Acid Status and Visual Acuity in Term Infants, JPGN 26, 412–421. 45. Makrides, M., Neumann, M., Simmer, K., Pater, J., and Gibson, R. (1995) Are LongChain Polyunsaturated Fatty Acids Essential Nutrients in Infancy? Lancet 345, 1463–1468. 46. Leyton, J., Drury, P.J., and Crawford, M.A. (1987) Different Oxidation of Saturated and Unsaturated Fatty Acids in Vivo in the Rat, Br. J. Nutr. 57, 383–393. 47. SanGiovanni, J. P., Parra-Cabrera, S., Colditz, G.A., Berkey, C.S., and Dwyer, J.T. (2000) Meta-Analysis of Dietary Essential Fatty Acids and Long-Chain Polyunsaturated Fatty Acids As They Relate to Visual Resolution Acuity in Healthy Preterm Infants, Pediatrics 105, 1292–1298. 48. Koletzko, B., Agostoni, C., Carlson, S.E., Clandinin, T.M., Hornstra, G., Neuringer, M., Uauy, R., Yamashiro, Y., and Willatts, P. (2001) Long-Chain Polyunsaturated Fatty Acids (LC-PUFA) and Perinatal Development, Acta Paediatr. 90, 460–464.

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Chapter 21

Phosphatidylcholine as Drug Substance and as Excipient—the Mechanism of Biological Activity Miklos Ghyczya and Mihály Borosb aPhospholipid bInstitute

GmbH, Nattermannalle 1, D 50829 Cologne, Germany

of Surgical Research, University of Szeged, Pécsi u. 4, 6720 Szeged, Hungary

Introduction Phosphatidylcholine (PC) from soybean or egg, in both native and saturated form, is used in drug formulations either as an active drug substance or as an inert excipient (1). The annual demand is approximately 300 tons per year in each field of application with significant growth potential. At present the only important medical application of PC derived from egg lecithin is the use of lipid emulsions for intravenous infusion (2). PC is one of the most important structure-forming biomolecules in vivo. In aqueous environment PC forms micelles, bilayers, and monolayers, allowing for the preparation of liposomes, protein–lipid complexes, lipid emulsions, and monolamellar structures as lung surfactant. These systems can be used in formulations to influence fat and drug solubility, enhance the skin penetration of drug substances, and alter the transport and ameliorate the side effects of drugs by incorporation into vesicles, such as liposomes or micelles. In all these cases the excipient PC is believed to be a nontoxic, stable, and inert molecule. PC is being consumed as a nutritional supplement, either alone or in combination with other ingredients. Nevertheless, experiments and clinical experiences suggest that PC functions as an active substance under certain in vivo conditions. It should be noted that PC has been used as a drug substance for the treatment of liver diseases and atherosclerosis for several decades (3). Despite this long experience, the biological mode of action of PC is still not understood and this has restricted the scope for relevant clinical protocols and hindered the worldwide acceptance of PC and related biomolecules as drugs and dietetic supplements. The aim of this paper is to propose a mode of action for the preventive and curative efficacy of PC.

Biological Efficacy of PC In the last decades several biological studies were performed with PC and its metabolites. Concomitantly, several studies extended the knowledge about the

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consequences of choline- or methyl-deficient diet (4). The different studies and protocols may be arranged into four groups of experiments focusing on the protective effects of PC. Ethanol and Carbon Tetrachloride Intoxication The protective effect of PC is documented in a study on baboons maintained on a liquid diet of ethanol or isocaloric carbohydrates with or without PC supplementation for eight years (5). The ethanol-fed animals developed septal fibrosis and cirrhosis, and the transformation of lipocytes into transitional cells occurred in almost all cases. Animals in the PC-supplemented group developed no septal fibrosis and after discontinuation of PC in the diet progressed to cirrhosis within 18–21 months. In a similar study, baboons were fed a high-ethanol diet with or without the PC supplement (6). In the group without the PC supplement nearly all animals developed septal fibrosis or cirrhosis with the transformation of the hepatic lipocytes into the collagen-producing transitional cells. In the group that received the PC supplement, the lipocyte transformation was rare, and septal fibrosis and cirrhosis did not develop. In a double-blind, randomized, placebo-controlled trial with patients suffering from alcohol-induced hepatitis, the survival rate in the PC-supplemented group was 69% as compared to 49% in the placebo group (7). Phosphatidylethanolamine methyltransferase (PEMT) plays a key role in the pathway for the synthesis of membrane PC. Alcohol intake significantly decreases PEMT activity in baboons with a corresponding reduction in liver PC levels. It has been demonstrated that a PC-enriched diet ameliorated the ethanolinduced decrease in PEMT activity (8). Additionally, PC protected the gastric mucosa in ethanol-induced injury in rats (9,10). Although PC is not an antioxidant (as antioxidants are chemically defined as electron donors), it prevents carbon tetrachlorideinduced hepatic lipid peroxidation (11). F2-isoprostanes and 4-hydroxynonenal, breakdown products of lipid peroxidation, were significantly increased in baboons fed alcohol, but this was fully prevented by 2.8 g per 1000 calories PC supplementation (12). Hypoxia PC has been shown to have a protective effect in the ischemic isolated rat heart (13). When added to an isolated rat heart prior to ischemia, PC significantly enhanced the chances of recovery, reduced the reperfusion-induced arrhythmia, and improved sub-sarcolemmal mitochondrial oxidative phosphorylation. The protective effect of PC was also demonstrated during ischemia-reperfusion in isolated ventricular tissue (14). Redox Cycling Substances PC significantly reduced acute toxicity of doxorubicin when the latter was administered in association with (15), encapsulated in (16), or complexed with (17) PCbased liposomes. The ameliorated toxicological profile resulted in prolonged survival, reduced severity of cardiomyopathy and nephropathy (16), and reduced body

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and organ weight losses. At a dose of 7.5 mg/kg doxorubicin, 100% of mice receiving liposome-associated doxorubicin survived a cumulative dose of 60 mg/ kg administered over 98 days, whereas 92% of mice receiving the free drug died (15). In addition, PC-based liposomes significantly decreased edema, monocytic infiltration, and cellular necrosis (17). The prerequisite for the improved tolerance was related to the nonspecific association of PC and doxorubicin. This indicates that the presence of PC—and not of the liposomes—was the decisive factor for decreased toxicity of doxorubicin. Nonsteroidal Anti-Inflammatory Drugs and Cyclosporin PC was found to protect the intestinal mucosa in rats against nonsteroidal antiinflammatory drug (NSAID)-induced damage. The effect was independent of the fatty acid composition in the PC molecules and was documented for different ratios of NSAID to PC (9,18–21). PC improved the tolerance of volunteers to cyclosporin A when the two substances were applied as an aerosol. Compared to the formulation containing cyclosporin A only, this mixture diminished tracheal irritation and coughing (22).

Biological Efficacy of Biomolecules Related to PC Until recently it was widely believed that biological efficacy depends on the fatty acid moiety present in the PC molecule (12,23,24). In contrast to this, some of the studies compiled show that the protective role of PC is independent of the fatty acids, and it may be assumed that the active principle is the choline moiety. PC is metabolized by phospholipase D under the impact of a great variety of stress factors to choline (25) and betaine. The methyl group on the tertiary, positively charged nitrogen moiety of betaine is transferred to S-adenosylmethionin (SAM) and to 5,10-methylene-THF (26). These latter molecules are central methylating agents for biological amines, such as carnitine. To prove the hypothesis that the active principle of PC is the choline moiety, we have evaluated literature data and reported that betaine, SAM, and carnitine show comparable protective efficacy as PC (27).

Oxidative and Reductive Stress Redox imbalance in cells and subcellular structures can lead either to oxidative or to reductive stress. Oxidative stress has been extensively studied for many years. Reductive stress, by contrast, has not been widely recognized. Yet reductive stress is probably both common and of clinical importance; indeed, reductive stress plus oxygen rather than oxidative stress may be the most common mechanism leading to the generation of reactive oxygen species (ROS). One possible link between the two may be the reduction of Fe3+ and its liberation from ferritin. The reduced metal could catalyze ROS generation (28).

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There are, among a few others, four different experimental methods for the generation of reductive stress: 1. Ethanol intoxication can be followed by the elevation of the lactate/pyruvate ratio (29). 2. Hypoxia can prevent the oxidation of NADH to NAD+ (30). 3. Reductive stress can result from the dislocation of electrons by redox cycling substances, such as doxorubicin (31). 4. Uncouplers can interrupt the flow of electrons down the electron transport chain (32). Recognition of reductive stress as a potentially common cause of pathological states raises the question of the nature of protective mechanisms in the same way as recognition of oxidative stress led to the study of antioxidants many years ago. The previous review of literature, theoretical molecular considerations, and experiments now in progress point to the key role of electrophilic biomolecules capable of oxidizing NADH to NAD+. They may be assumed to have a positively charged nitrogen or sulphur atom in their structure, rendering the adjacent methyl group electron deficient. We termed these biomolecules electrophilic methyl group (EMG) compounds (27).

Biomolecules with Electrophilic Methyl Groups and Reductive Stress The previous short review of literature shows that PC and other biomolecules with an EMG moiety possess preventive efficacy in experimentally induced reductive stress. The four biomolecules with EMG moiety (PC, SAM, betaine, and carnitine) differ in their chemical structures and their currently recognized functions as biomolecules. At the same time they are similar in terms of their EMG moiety. They are used as drug substances for similar indications, and they are part of our diet. They form a pool of EMG-containing molecules, which suggests a supply of methyl groups from a common source for a common demand. Under normal conditions, the pool is in a dynamic balance: EMG are continuously replenished by EMG from the diet. However, there are two sets of conditions under which the size of the pool will decrease: (i) pathological reductive stress with the accompanying increased demand for EMG, and (ii) pathological deficiency of EMG in the diet. Experiments with EMG-deficient diets indicate that in human subjects the size of the pool is depressed to a pathological level after two weeks (33) and in rats after several days (34).

PC as a Unique Excipient for Drug Formulations Drug formulations contain excipients to make the active substance more acceptable to the patient and to make the drug bioavailable to the organism. According to

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today’s expectations, excipients should not only be functional but also nontoxic, inert, stable for years, tasteless, and colorless chemical substances. PC is a new type of excipient because (i) under nonpathological conditions it meets the expectations placed upon a normal excipient, but (ii) in pathological states it normalizes redox imbalance conditions and prevents the formation of highly toxic oxygen radicals. As redox imbalance is a common outcome of adverse drug effects, PC may represent a new approach to the enhancement of the therapeutic index.

PC as Drug or Dietetic Supplement in Preventing ROS-Induced Toxicity The reduction of oxygen to water is the basic energy-supplying reaction for the sustenance of aerobic life. The disturbance of the steady state between oxygen uptake and oxygen metabolism leads to the generation of oxygen radicals and to diminished energy supply. As the oxygen uptake is a function of the constant concentration of oxygen in the earth’s atmosphere, the only possible cause for the generation of ROS and for the ensuing toxicity is reductive stress, originating from abnormal reducing equivalent in the interior of the cell. The prerequisite for the relief of reductive stress is the presence of an oxidant, an electron acceptor, and not an antioxidant. The logic of this reasoning is further supported by the disappointing experience with antioxidants. Recent clinical studies suggest that the potential of antioxidants to prevent or to cure ROS-involved diseases is limited (35–37). In fact, extensive clinical and experimental work over the past 30 years has failed to reveal a single abnormal clinical state that could be confidently ascribed to oxidative stress or, more importantly, that has convincingly benefited from antioxidants. The superior efficacy of EMG compared to antioxidants may be explained by the facts that (i) oxidative stress is a secondary condition to reductive stress and (ii) NADH has a longer half-life than ROS and therefore its removal is more feasible. The long experience with PC and the results presented here suggest that limitation of reductive stress with PC is a superior concept to avoid ROS-induced damages in biological systems as compared to conventional antioxidants (Fig. 21.1).

Summary PC is a ubiquitous membrane-forming entity and an emulsifier in aerobic organism. It is used as an inert, nontoxic excipient. Additionally, PC is used as a drug substance. Recent biological and clinical findings furnish the basis for the understanding of the mechanism of PC in vivo. Additional supporting evidence can be gained from studies with methyl deficient diets. We propose that the EMG bound on positively charged nitrogen in PC and related biomolecules functions as an electron acceptor and normalizes elevated reducing equivalent, a condition named

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NADH Fe++

Oxidants electro philic methyl groups

Antioxidant

Antioxidant

Antioxidant

Antioxidant

Damage to biomolecules

Fig. 21.1. Mechanism of action of antioxidants and proposed role for phosphatidylcholine and electrophilic methyl group-containing biomolecules.

reductive stress. This abnormal redox condition is recognized as the most common cause for the formation of oxygen radicals. PC is a unique substance as it combines protective efficacy in redox imbalance conditions in vivo with the inherent structureforming property. Making use of this combination in new formulations opens up the possibility of developing drugs with enhanced therapeutic index. This long experience and biological findings suggest that PC and metabolites with an EMG moiety are superior to antioxidants in preventing ROS-induced or related damages in humans. References 1. Wendel, A. (1995) in Encyclopedia of Chemical Technology, Othmer, K., John Wiley & Sons, vol. 15. 2. Ferezou, J., Gulik, A., Domingo, N., Milliat, F., Dedieu, J.C., Dunel-Erb, S., Chevalier, C., and Bach, A.C. (2001) Intralipid 10%: Physicochemical Characterization, Nutrition 17, 930–933. 3. Peeters, H. (1976) Phosphatidylcholine, Springer Verlag, Berlin Heidelberg, New York. 4. Blusztajn, J.K. (1998) Choline, a Vital Amine, Science 281, 794–795. 5. Lieber, C.S., De Carli, L.M., Mak, K.M., Kim, C.I., and Leo, M.A. (1990) Attenuation of Alcohol-Induced Hepatic Fibrosis by Polyunsaturated Lecithin, Hepatology 12, 1390–1398.

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