Alcohol Research Center, Section of Liver Disease and Nutrition, Bronx VA Medical Center and Mount Sinai School of Medicine, New York, NY, USA
Received 24 September 2002; in revised form 18 December 2002; accepted 7 January 2003
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ABSTRACT |
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INTRODUCTION |
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A mixture of polyenylphosphatidylcholines (PPC) corrects the oxidative stress and the hepatic GSH depletion resulting from long-term alcohol consumption in non-human pri-mates (Lieber et al., 1997) and at earlier stages of ALD in rats (Aleynik et al., 2000
), but whether PPC affects hepatic levels of SAMe remains unknown. In the present study we explored a rat model of alcohol-induced fatty liver to answer these questions.
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MATERIALS AND METHODS |
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Animals and diets
Thirty-two male rats of a SpragueDawley strain [CRL:CR(SD)BR], divided into four groups, were fed LieberDeCarli diets containing either ethanol or dextrose, with PPC or safflower oil (2.1 g/1000 kcal) for 8 weeks. The diets were purchased from Dyets Inc. (Bethlehem, PA, USA) and contained 1 kcal/ml, with 18% of energy as protein, 35% as fat, and 47% as carbohydrate in the controls, or 11% as carbohydrate in the rats fed ethanol (36% of energy). One rat escaped at the end of the experiments. PPC was given in the diet at a concentration of 3 g/1000 kcal. PPC contained 42% of dilinoleoyl-PC (18:2-18:2 PC), 24% of palmitoyl-PC (16:0-18:2 PC), 10% of oleoyllinoleoyl-PC (18:1-18:2 PC), 8% of linolenoyllinoleoyl-PC (18:3-18:2 PC), 6% of stearoyllinoleoyl-PC (18:0-18:2 PC), about 2% of palmitoyloleoyl-PC (16:0-18:1 PC) and stearoylarachidonoyl-PC (18:0-20:4 PC), as well as several other minor fractions, each less than 1%. The day before death, the animals received one-third of their daily ration in the morning and two-thirds in the evening.
SAMe assay
SAMe was measured in the liver by a high-performance liquid chromatographic (HPLC) method (Wise et al., 1997) with some modifications, including the use of S-adenosylethionine as an internal standard.
MAT assay
Liver cytosol was obtained by conventional differential centrifugation of liver homogenates and was incubated with 5 mM methionine according to Huang et al.(1998). The formed SAMe was measured by the HPLC procedure of Wise et al.(1997)
. MAT activity was expressed as the amount of SAMe produced per milligram of protein per minute.
Parameters of oxidative stress
4-HNE was measured by the GC/MS method of van Kuijk et al.(1995) with slight modifications. GSH was determined by HPLC with fluorometric detection according to Winters et al.(1995)
.
Statistical analysis
Statistical significance was assessed by t-test and one-way analysis of variance (ANOVA) with post hoc StudentNewman Keuls multiple comparison test. To estimate correlations, Pearsons linear coefficients were calculated. A P value of < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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SAMe synthesis from methionine and ATP is catalysed by MAT. Unlike the diminished activity reported in the advanced cirrhotic stage of ALD (Martin-Duce et al., 1988), we found no decrease in MAT activity at the earlier stage of liver disease in the model studied here (Fig. 4
), suggesting that alcohol-induced SAMe depletion was caused by another mechanism, possibly increased SAMe consumption. Indeed, lipid peroxidation plays a major role in the development of ALD, and enhanced oxidative stress increases GSH consumption. As a compensatory mechanism, more SAMe is converted to cystathionine and subsequently to cysteine to form GSH (vide supra). However, since SAMe synthesis, as assessed by the MAT reaction, remained unchanged, net SAMe depletion may have resulted (Fig. 1
). The strong positive correlation between SAMe and GSH and the negative one between SAMe and 4-HNE (Fig. 5
) are consistent with, and strengthen, the possibility that it is the alcohol-induced oxidative stress which results, at least in part, in an increase of GSH consumption, associated with SAMe depletion. Some other data are also corroborative. It is known that cystathionine ß-synthase (EC 4.2.1.22) is activated by oxidative stress. In such a case, more SAMe will be metabolized through transsulphuration, resulting in a decrease of the SAMe pool (Taoka et al., 1998
).
Synthesis of GSH is highly dependent on availability of intracellular cysteine and, in the liver, cysteine originating from transsulphuration of SAMe is of particular importance. In chronic liver disease, the decrease in GSH was attributed, in part, to diminished synthesis of SAMe (Lu, 1998) and supplementation with SAMe normalized GSH levels in both animal models and patients (Lieber et al., 1990
; Corrales et al., 1992
; Mato et al., 1997
). Therefore, one could anticipate a parallelism in the changes of SAMe and GSH. This hypothesis is strengthened by known effects of alcohol and/or PPC on hepatic GSH levels in rats (Aleynik et al., 2000
) and, at more advanced stages of ALD, in baboons (Lieber et al., 1997
). In the present study, changes of SAMe levels in the various experimental groups (Fig. 1
) show the same trend as for GSH (Fig. 2
). Furthermore, hepatic SAMe and GSH levels were strongly correlated (Fig. 5B
) and the feeding of PPC resulted in a significant blockade of the alcohol-induced SAMe decrease (Fig. 1
).
The degree of SAMe depletion found in this study may lead to a decreased capacity to sustain normal levels of phosphatidylethanolamine N-methyltransferase (PEMT, EC 2.1.1.17)-dependent PC synthesis, as described in more advanced stages of ALD (Lieber et al., 1994), providing an additional explanation for the alcohol-induced reduction in hepatic phospholipids, particularly arachidonate-containing PC species, as reported before (Aleynik et al., 2000
). Indeed, SAMe is a universal methyl donor that provides a methyl group to phosphatidylethanolamine, thereby forming PC, the backbone of cellular membranes. This reaction is catalysed by PEMT, and represents an alternative pathway to that of cytidyldiphosphocholine for PC synthesis. It is particularly active in the liver (Sundler and Akesson, 1975
), and generates PCs with long-chain polyunsaturated fatty acids containing a higher percentage of arachidonate (DeLong et al., 1999
). This pathway of PC synthesis requires a considerable amount of SAMe, compared with other transmethylation reactions, since, for each mole of PC formed, 3 mol of SAMe are needed. Therefore, we hypothesize that PPC, by providing unsaturated long-chain PCs, spares the substantial utilization of SAMe for PC synthesis catalysed by PEMT. This could explain, at least in part, the PPC-induced GSH replenishment (Fig. 2
) and also the correction of the alcohol-induced oxidative stress (Fig. 3
). This hypothesis pre-supposes that, unlike in long-lasting chronic experiments (Lieber et al., 1994
), PEMT activity is not as yet decreased after only 8 weeks of alcohol feeding as carried out in the present study. In any event, the hypothesis may also explain the statistically significant increase of SAMe and GSH levels above control values when PPC was given without alcohol (Figs 1
and 2
).
The findings of this study have some practical implications. Some essential nutrients, including methionine, must first be activated in the liver before they can exert their key functions. This activating process, however, is altered by liver disease and, as a consequence, nutritional requirements change. Experimentally, it has been shown that even a 7-fold increase in the normal dietary methionine content fails to significantly alter hepatic SAMe (Finkelstein and Martin, 1986). This is exacerbated when there is significant liver disease, which is commonly associated with impairment of the enzyme activating methionine to SAMe (Martin-Duce et al., 1988
). Therefore, supplementation with methionine is useless in most such circumstances and may even result in toxicity, because of its accumulation as a result of non-utilization (Kinsell et al., 1947
). Indeed, elevated concentrations of circulating methionine in patients with liver disease have been reported (Iber et al., 1957
; Fischer et al., 1974
; Montanari et al., 1988
), and excess methionine was shown to have toxic effects (Finkelstein and Martin, 1986
), including a decrease in hepatic ATP (Hardwick et al., 1970
). Accordingly, treatment with SAMe instead of methionine has been advocated (reviewed in Lieber, 2002
). However, it has been claimed that the liver does not take up SAMe from the bloodstream (Hoffman et al., 1980
), but other studies indicate its uptake by isolated hepatocytes (Pezzoli et al., 1978
; Travers et al., 1984
; Engstrom and Benevenga, 1987
). Results in baboons also clearly showed hepatic uptake of exogenous SAMe in vivo, associated with beneficial effects on liver function and structure (Lieber et al., 1990
). In these baboons, correction of the ethanol-induced hepatic SAMe depletion with oral SAMe administration resulted in a corresponding attenuation of ethanol-induced liver injury. Furthermore, a significant therapeutic success in ALD was achieved in a long-term randomized, placebo-controlled, double-blind, multicentre clinical trial of SAMe in patients with alcoholic liver cirrhosis in whom SAMe improved survival or delayed liver transplantation (Mato et al., 1999
). The results of the present study indicate that SAMe should not only be used for the treatment of alcoholic cirrhosis (Mato et al., 1999
), but that it should now also be considered for the therapy of earlier stages of the disease, and that its beneficial effects could be potentiated by concomitant administration of PPC.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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