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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"The paper used in Ihi. booJ; i. acid·free and fall. wllmn the guiddilll'S established tOl ell.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

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