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Article

Addition of Arginine but Not Glycine to Lysine Plus Methionine–Enriched Diets Modulates Serum Cholesterol and Liver Phospholipids in Rabbits¹

Departments of Biochemistry and Pharmacology and Toxicology, The University of Western Ontario, London, ON, N6A 5C1, Canada

²To whom correspondence should be addressed.

	ABSTRACT

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Previous experiments from our laboratory showed that in rabbits fed an amino acid diet corresponding to 30% casein, enrichment of the diet with L-lysine and L-methionine caused a marked increase in serum total and LDL cholesterol levels as well as a substantial body weight loss. Both effects were partially prevented by supplementation with L-arginine. The present studies were designed to extend this earlier observation by assessing the role of different dietary amino acids in modulation of cholesterolemic responses and body weights. In the first experiment, the original lysine and methionine-enriched diet was supplemented with glycine in an attempt to modify methionine metabolism, and thus to reduce body weight loss. In addition, the mechanism of action of lysine and methionine was investigated by quantitation of major liver phospholipids. The results showed that glycine addition had no effect on weight loss or hypercholesterolemia, nor did it alter plasma levels of homocyst(e)ine, an intermediate in methionine metabolism. However, enrichment of the diet with lysine and methionine (with or without glycine) significantly increased liver levels of phosphatidylcholine and the ratio of phosphatidylcholine to phosphatidylethanolamine, apparently through increased enzymatic conversion. These changes were consistent with higher lipoprotein levels and thus may explain the hypercholesterolemia. A second experiment showed that similar effects on body weights and serum cholesterol could be obtained by adding lysine and methionine to a diet containing amino acids equivalent to only 15% casein, or 15% intact casein. This approach is more physiologic and also reduces the expense of experiments designed to study the effects of lysine and methionine in more detail.

KEY WORDS: • dietary amino acids • hypercholesterolemia • liver phospholipids • rabbits

	INTRODUCTION

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Previous studies on the effects of dietary proteins on cholesterolemia in rabbits have shown that high levels (30%) of casein or casein amino acids raise serum total and LDL cholesterol; however, this was not observed for soy protein and soy amino acids (Huff et al. 1977a , Kurowska et al. 1989 ). The hypercholesterolemic potential of casein appeared to be due to its high content of the essential amino acids (EAA),³ rather than nonessential amino acids (NEAA) (Kurowska and Carroll 1990 ). Further experiments demonstrated that an even more pronounced hypercholesterolemia can be induced in rabbits by selectively feeding high levels of all casein EAA except arginine (Arg). This suggested that Arg counteracted the elevation of serum total and LDL cholesterol induced by other EAA (Kurowska and Carroll 1992 ). Among the remaining EAA, lysine in combination with methionine [Lys + Met] but not Lys or Met separately, were the most hypercholesterolemic (Kurowska and Carroll 1994 , Giroux et al. 1999 ). Consistent with these results, relatively high levels of Lys and Met were found in dietary casein and other animal proteins that are hypercholesterolemic in animals, whereas high levels of Arg were found in soy protein and other plant proteins, previously reported to be normocholesterolemic (Carroll 1981 ). The higher ratio of Lys to Arg in casein had also been suggested earlier by Kritchevsky et al. (1987) as a reason for its hypercholesterolemic effects.

The mechanisms by which Lys, Met and Arg modulate cholesterolemia in rabbits have been investigated in our laboratory with emphasis on their effects on the liver. The results suggested that these amino acids could act directly in the liver, by regulating both synthesis of LDL protein, apolipoprotein B (apo B), and catabolism of LDL (Kurowska and Carroll 1992 and 1996 ). In support of this suggestion, our experiments in vitro showed that an excess of Arg in the medium of human hepatoma HepG2 cells reduced the amount of apo B released into the medium. The response of Arg in vitro as well as in vivo was apparently not mediated by nitric oxide, a product of Arg that is a potent metabolic mediator (Kurowska and Carroll 1998 ).

In contrast, an excess of Lys + Met in the medium of HepG2 cells did not produce any effects on apo B metabolism (Kurowska and Carroll 1996 ). However, feeding a diet enriched in Lys + Met to rabbits was associated with increased hepatic synthesis of phospholipids, and possibly also with the increased conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) (Giroux et al. 1999 , Kurowska and Carroll 1996 ). This suggested that PC and PE could play a role in the regulation of lipoprotein metabolism by Lys + Met, especially because PC is the main phospholipid required for assembly and secretion of apo B-containing lipoproteins (Yao and Vance 1988 ) and because PC synthesis from PE utilizes methyl groups from Met.

The hypercholesterolemia produced in rabbits by the Lys + Met supplements was also associated with a marked inhibition of body weight gain that occurred in spite of adequate food intake (Giroux et al. 1999 , Kurowska and Carroll 1994 ). The body weight losses, as hypercholesterolemia, were reversed in part by the addition of Arg (Giroux et al. 1999 ). The toxicity of the [Lys + Met]-enriched amino acid diets and their influence on liver phospholipid metabolism suggested that the responses could be mediated by Lys in combination with a toxic metabolite of Met. Some products of Met catabolism have been implicated previously in weight losses and/or modulation of cholesterolemia. In rats fed an excess of dietary Met, an accumulation of the Met deamination product, methanethiol, induced body weight losses, and this was reversed by the addition of glycine (Gly), which facilitates the Met demethylation pathway (Benevenga and Steele 1984 ). Other evidence suggests that the Met catabolic intermediate, S-adenosylmethionine (SAM), could be involved in inducing toxicity (Regina et al. 1993 ). An excess of Met demethylation products such as homocyst(e)ine could also play a role in [Lys + Met]-induced responses because elevated levels of homocyst(e)ine have been associated with an increased risk of vascular disease in humans (Meleady and Graham 1998 ).

Another possible cause for toxicity of the Lys + Met supplement could be an overall high level of nitrogen in the experimental diets. In our previous studies, the amino acid diets containing an excess of all or selected EAA were made up to contain 29.5% amino acids by addition of casein NEAA (Kurowska and Carroll 1994 ). Because the excess of NEAA had previously been found to have little effect on serum cholesterol (Kurowska and Carroll 1990 ), it is possible that reducing the level of these amino acids in the [Lys + Met]-supplemented, casein amino acid-based or casein-based diet could help to alleviate the overall toxicity without affecting hypercholesterolemia.

In the first study, we investigated whether an accumulation of toxic Met deamination metabolites, such as methanethiol, or an increased formation of the Met demethylation product, homocyst(e)ine, could play a role in the mechanism of [Lys + Met]-induced hypercholesterolemia and body weight loss. The possible involvement of Met deamination products was evaluated by monitoring changes in cholesterolemia and body weights in rabbits fed [Lys + Met]-enriched amino acid diets to which a supplement of Gly was added to facilitate Met demethylation (Benevenga and Steele 1984 ). The importance of a Met demethylation product, homocyst(e)ine, was tested by measuring homocyst(e)ine in plasma of animals given control vs. [Lys + Met]-enriched amino acid diets. Liver PC and PE levels, and activity of liver enzymes involved in PC synthesis were also determined in this experiment to shed more light on the mechanisms involved in the effects of the dietary amino acids.

The purpose of our second study was to determine whether the toxicity of the 29.5% casein amino acid diet containing an excess of Lys + Met as well as a high level of NEAA can be prevented either by reducing the amount of NEAA or by feeding intact casein instead of casein amino acids with supplements of Lys + Met. In this experiment, the levels of liver phospholipids and the activity of principal enzymes involved in PC synthesis were also monitored.

	MATERIALS AND METHODS

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Animals and diets.

The animal protocol was in accordance with the Canadian Council of Animal Care guidelines and was approved by the University Council of Animal Care, The University of Western Ontario. Male New Zealand White rabbits (Reimen's Fur Ranches, Guelph, Canada) of 9 wk and weighing ~1.8 ± 0.1 kg (mean ± SD) were housed individually in stainless steel cages in an animal room maintained at 21–24°C with a 12-h light:dark cycle. Upon arrival, they were fed for 5 d ground High Fiber Rabbit pellets (Agway, Syracuse, NY). They were then randomly divided into groups and gradually transferred to the experimental diets over a 6-d period. The diets were pair-fed to the [Lys + Met]-enriched diet over a period of 3.5 wk. Free access to water was provided. Body weights and food consumption were monitored.

The composition of the cholesterol-free, low fat, semipurified diets was similar to that used in earlier experiments (Kurowska and Carroll 1990 ) (Table 1 ). The protein and amino acid composition of the different diets is given in Table 2 . In Experiment 1, the low amino acid control diet [14.7% amino acid (AA)] contained casein essential and nonessential amino acids at levels equivalent to 14.7% casein. Among the four supplemented diets, the high amino acid control diet (14.7% AA + NEAA) also contained 14.7% casein amino acids but had a total level of amino acids adjusted to 29.5% with casein NEAA, which have previously been shown to have little effect on cholesterolemia in rabbits (Kurowska and Carroll 1990 ). The remaining three formulations contained 14.7% casein amino acids and supplements of Lys and Met (+LM), Lys, Met and Gly (+LMG) or Lys, Met and Arg (+LMA) at levels corresponding to a 45% casein amino acid diet. The total level of nitrogen in these three diets was adjusted to 29.5% by addition of NEAA. Dextrose was added at the 60% level to a low amino acid control diet and at the 45% level to the amino acid–supplemented diets. Diets used in Experiment 2 contained 14.7% casein AA or intact casein, with a 3% supplement of NEAA (two control diets, 17.7% AA and 17.7% casein) or with equivalent 3% supplements of Lys + Met at levels as in the 45% casein amino acid diet (two experimental diets, 17.7% AA + LM^aa and 17.7% cas + LM^c). Dextrose was added at the 57% level to these diets.

At the end of the feeding period, rabbits were deprived of food overnight and blood samples were collected from the marginal ear vein. Blood hematocrit and hemoglobin were measured immediately at the time of blood collection. The percentage hematocrit was determined in fresh blood, using standard microhematocrit tubes. Blood hemoglobin concentration was determined by measuring the optical absorbance of oxyhemoglobin in oxygenated hemolyzed blood. Serum creatinine was assayed, using a Vet Test 8008 automatic analyzer (Idexx Technical Services, Markham, Canada). The activity of liver -glutamyltransferase and serum aspartate aminotransferase was determined with enzymatic tests (GGTP and AST/GOT kits from Sigma Diagnostics, St. Louis, MO).

VLDL (d < 1.006 kg/L), LDL (1.006 kg/L < d < 1.063 kg/L) and HDL (1.063 kg/L < d < 1.21 kg/L) fractions were isolated from serum by discontinuous density gradient ultracentrifugation (Redgrave et al. 1975 ). Cholesterol concentration was measured in serum and lipoprotein fractions, using an enzymatic-colorimetric test (CHOD-PAP kit from Boehringer-Mannheim, Montreal, Canada). Triacylglycerol levels were determined in serum with an enzymatic-colorimetric assay (GPO-PAP kit from Randox, Ardmore, Crumlin, UK).

ApoB was isolated from the LDL fraction by isopropanol precipitation; the apoB content was determined by subtracting the protein concentration of the supernatant from the total LDL protein concentration (Huff et al. 1992 ). Protein concentration was measured by the method of Markwell et al. (1978) .

Plasma total homocyst(e)ine analysis.

Plasma total homocyst(e)ine levels were determined by the fluorometric HPLC determination method of Jacobsen et al. (1994) in the laboratory of Dr. David J. Freeman at the Robarts Research Institute (London, ON).

Analysis of liver lipids.

After the blood samples were collected, the animals were killed by an overdose of Euthanyl (Canada Packers, Cambridge, Canada). Livers were excised at autopsy and rinsed in cold saline. Pieces of tissue were stored at -20°C for lipid extraction by the method of Folch et al. (1957) . The remaining tissue was immediately processed to prepare microsomes for measurement of phosphatidylethanolamine N-methyltransferase (PEMT, EC 2.1.1.17), choline phosphotransferase (CPT, EC 2.7.8.2) and choline phosphate cytidylyltransferase (CT, EC 2.7.7.15). Liver cholesterol and triacylglycerol concentrations were measured in Folch extracts, using enzymatic-colorimetric methods (CHOD-PAP kit from Boehringer-Mannheim and GPO-PAP kit from Randox). Liver phospholipids were digested with perchloric acid (Edmond 1974 ) before phosphorus determination (Inorganic phosphorus kit from Sigma Diagnostics). PC and PE were separated by thin-layer chromatography with the solvent system chloroform/methanol/ammonia/water (65:35:4:4, v/v/v/v) (Rymerson and Carroll 1992 ), and the phosphorus content of the PC and PE fractions was determined enzymatically (Inorganic phosphorus kit from Sigma Diagnostics) after extraction with chloroform/methanol (2:1, v/v).

Activity of enzymes involved in liver PC synthesis.

Microsomes were isolated as described by Ridgway and Vance (1992) for measurement of PEMT activity, according to Kanoh and Ohno (1981) for CPT activity and according to Sugiyama et al. (1996) for CT activity. The microsomes were stored in liquid nitrogen and used within 1 wk. Microsomal protein concentration was measured by the method of Lowry et al. (1951) after protein precipitation with trichloroacetic acid (Markwell et al. 1978 ). The in vitro PEMT activity was assayed as described by Ridgway and Vance (1992) , using [methyl-¹⁴C]-adenosylmethionine (SA = 2.1 TBq/mol, Amersham, Oakville, Canada) and dimethyl-phosphatidylethanolamine as substrates. The in vitro activity of CPT in liver microsomes was measured according to Cornell (1992) , using the substrates [methyl-¹⁴C]-cytidine 5'diphosphocholine (CDP-choline) (SA = 1.9 TBq/mol, Amersham) and sn-1,2-diolein (Sigma). The [¹⁴C]-phosphatidylcholine formed was extracted by the method of Folch et al. (1957) . The activities of PEMT and CPT were expressed as picomoles of [¹⁴C]-phosphatidylcholine formed per minute incubation per milligram of microsomal protein. Liver microsomal CT activity was assayed in vitro by the method of Weinhold and Feldman (1992) , using phosphoryl [methyl-¹⁴C] choline (SA = 2.1 TBq/mol, Amersham) and cytidine triphosphate as substrates. [¹⁴C]-CDP-choline was isolated by thin-layer chromatography according to Vance et al. (1981) , with CDP-choline used as a cold carrier. The activity of CT was defined as picomoles [¹⁴C]-CDP-choline formed per minute incubation per milligram microsomal protein.

Statistical analysis.

ANOVA was used to compare the results of the different diet groups in Experiment 1. When the ANOVA was significant, a Student-Newman-Keuls procedure was used to test differences among the groups. Student's t tests were used in Experiment 2 to compare the results of the 17.7% AA diet with the 17.7% AA + LM^aa diet and of the 17.7% casein diet to 17.7% casein + LM^c diet. The analysis was performed using the Sigma Stat Statistical Software (Jandel Corporation, San Rafael, CA). The effects of the diets were considered to be significant if P < 0.05. Data were expressed as means ± SEM

	RESULTS

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Growth and body functions.

As in our earlier experiments (Kurowska and Carroll 1994 and 1996 ), the [Lys + Met]-enriched diets induced substantial weight loss, whereas a similar effect was not observed in rabbits that were pair-fed other diets (Table 3 , Experiments 1 and 2). In Experiment 1, three rabbits fed the 14.7% AA + LM diet had to be killed before the end of the study because they lost too much weight. The addition of Arg to the 14.7% AA + LM diet partially counteracted the weight loss, but the addition of Gly had no protective effect. Feeding the low and high casein amino acid control diets resulted in normal weight gains. In Experiment 2, both groups fed [Lys + Met]-enriched diets lost weight, whereas control groups gained weight normally.

Serum lipids and lipoproteins.

In Experiment 1, the 29.5% amino acid control diet (14.7% AA + NEAA diet) resulted in no change in serum total cholesterol levels and a significant rise in LDL cholesterol and apoB levels, compared with the low amino acid control diet (14.7% AA diet; Table 4 ). Enrichment with Lys + Met significantly increased serum total and LDL cholesterol levels as well as LDL apoB levels, compared with the 14.7% AA and 14.7% AA + NEAA diets. Gly had no effect on these variables when added to the 14.7% AA + LM diet. However, the addition of Arg to the 14.7% AA + LM diet partly counteracted the increase in serum total and LDL cholesterol and the increase in LDL apoB levels, the latter effect being significant. The 14.7% AA + LMG diet resulted in lower HDL cholesterol levels (0.3 ± 0.1 mmol/L) than did the 14.7% AA + NEAA diet (0.7 ± 0.1 mmol/L) (P < 0.05); the other diets gave intermediate levels. Feeding the amino acid diets to the rabbits had no effects on their VLDL cholesterol concentrations (data not shown).

Plasma homocyst(e)ine levels.

In Experiment 1, there were no significant differences in plasma homocyst(e)ine levels among the different dietary groups (data not shown).

Liver lipids.

In Experiment 1, the rabbits fed the high amino acid control diet (14.7% AA + NEAA diet) had liver total phospholipid and PE levels as well as PC to PE ratios not different than their counterparts fed the low amino acid control diet (14.7% AA diet), but higher liver PC levels. High levels of Lys + Met did not raise liver total phospholipids and significantly raised liver PC levels and the ratio of PC to PE, compared with both the 14.7% AA and 14.7% AA + NEAA diets. Liver PE levels were unaffected by Lys + Met supplementation (Table 5 ). Arg supplementation of the 14.7% AA + LM diet resulted in no change in levels of liver total phospholipids, significantly lower liver PC and PC to PE ratio, and significantly higher PE levels compared with the 14.7% AA + LM diet. Thus, rabbits fed the 14.7% AA + LMA diet had liver total phospholipid and PC levels not different from those fed the 14.7% AA + NEAA diet, and higher PE levels than those fed the other diets. Liver phospholipids were not affected by the addition of Gly to the 14.7% AA + LM diet. There were no effects on liver cholesterol and triacylglycerol levels (data not shown).

Activity of liver phospholipid enzymes.

In Experiment 1, liver PEMT and CPT activities were increased by the 14.7% AA + LM and 14.7% AA + LMG diets, compared with the 14.7% AA + NEAA and 14.7% AA diets, although the difference in the PEMT activity between the low amino acid control and [Lys + Met]-supplemented diet was not significant. The addition of Arg to the 14.7% AA + LM diet reversed the effect on PEMT activity and partially counteracted the effect on CPT activity (Table 6 ).

	DISCUSSION

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The results of Experiments 1 and 2 confirmed an earlier observation that high dietary levels of Lys + Met produce a marked elevation of total and LDL cholesterol in rabbits, and that Arg tends to counteract the hypercholesterolemia produced by feeding high levels of all other EAA (Kurowska and Carroll 1994 ), especially high levels of Lys + Met (Giroux et al. 1999 ). The results obtained also supported the hypothesis of Kritchevsky et al. (1987) that casein is more hypercholesterolemic in rabbits than soy protein because casein has a higher ratio of Lys to Arg.

The hypercholesterolemia and weight loss induced by feeding [Lys + Met]-enriched amino acid diets failed to be alleviated by a supplementation with Gly, an amino acid known to facilitate metabolism of Met through the demethylation rather than deamination pathway. This observation did not support our original hypothesis and was inconsistent with previous reports showing hypocholesterolemic effects of Gly supplements in rabbits and rats (Katan et al. 1982 , Sugiyama et al. 1986 ). Our results suggested, therefore, that effects of Lys + Met were not mediated by toxic Met deamination products such as methanethiol. The possible involvement of the Met demethylation intermediate, homocyst(e)ine, in the mechanism was also highly unlikely because plasma homocyst(e)ine levels were not significantly different among the dietary groups. The lack of importance of homocyst(e)ine was consistent with the data obtained by Sugiyama et al. (1996) who reported that liver levels of S-adenosyl-homocyst(e)ine, the precursor of homocyst(e)ine, were not significantly different in rats fed either casein or soy protein.

Although previous studies suggested that in rabbits, chronic supplementation with Met could cause hepatitis due to increased lipid peroxidation and free radical formation (Toborek et al. 1996 ), the animals fed [Lys + Met]-enriched diets did not show any evidence of liver or kidney damage, as confirmed by the observation that blood hematocrit, hemoglobin, serum creatinine and liver function tests were all normal. In addition, liver histology was normal at autopsy (data not shown). Our results also demonstrated that the [Lys + Met]-induced toxicity and hypercholesterolemia were not counteracted by reducing the overall high level of amino acids present in the diet from 29.5 to 17.7%, or by replacing the 14.7% casein amino acid mixture with an equivalent level of intact casein (Experiment 2). These results mean that the overall high level of amino acids in the [Lys + Met]-enriched diet did not contribute to the toxicity of this diet and that the mechanism of action of Lys + Met can now be explored further without the expense of relatively large amounts of costly amino acids for the dietary experiments.

Because both hypercholesterolemia and weight loss induced by dietary Lys + Met were alleviated in part by the addition of Arg, it is possible that the observed toxicity was due largely to Lys rather than Met. Lys is known to be a potent competitive inhibitor of liver arginase, an enzyme responsible for the conversion of Arg into ornithine and urea in the urea cycle (Cynober et al. 1995 ). The inhibition of arginase by excess dietary Lys could therefore reduce the efficiency of the urea cycle leading to accumulation of ammonia, increased excretion of orotic acid and, consequently, signs and symptoms of toxicity (Reyes and Klahr 1994 ). Also, it cannot be excluded that other metabolites of Met not measured in this study, especially SAM (Regina et al. 1993 ), could contribute to the toxicity of [Lys + Met]-enriched diets.

In our studies, the [Lys + Met]-induced hypercholesterolemia and body weight loss appear to be closely associated. However, results obtained by others suggested otherwise; a substantial hypercholesterolemia and normal growth were achieved in rabbits by feeding high dietary levels of casein, all casein amino acids or casein EAA (Hamilton and Carroll 1976 , Huff et al. 1977b , Kurowska and Carroll 1990 ). This apparent discrepancy suggests that different compounds might produce hypercholesterolemia and body weight loss.

Our studies on the mechanisms of hypercholesterolemia provided support for the association with changes in the metabolism of liver phospholipids. Supplementation with Lys + Met increased the level of hepatic PC, and this effect was partially counteracted by Arg, but not by Gly. It also persisted when the total nitrogen level in the diet was reduced or when Lys + Met were added to intact casein. The observed changes tended to parallel the effects on serum cholesterol levels in both Experiments 1 and 2. The dietary [Lys + Met]- and Arg-induced alterations in liver phospholipids were consistent with our previous results, which also showed increases in PC and in the PC/PE ratio after feeding [Lys + Met]-enriched diets and decreases of both variables after the addition of Arg (Giroux et al. 1999 ).

Consistent with changes in hepatic PC and PE was the observation that [Lys + Met]-enriched diets increased in vitro activities of CPT, a last enzyme in the pathway of PC biosynthesis from choline, and PEMT, an enzyme responsible for conversion of PE into PC via a secondary pathway. On the other hand, the activity of CT, a rate-limiting enzyme in biosynthesis of PC from choline (Vance 1996 ), was not affected, indicating a lack of involvement of this pathway in the mechanism. The results suggest that high dietary levels of Met could possibly contribute to [Lys + Met]-induced hypercholesterolemia by increasing the availability of methyl groups derived from SAM for synthesis of PC via a secondary, PEMT-mediated pathway. The accumulation of PC could in turn stimulate synthesis of apo B-containing lipoproteins (Noga et al. 1998 ). In support, Sugiyama et al. (1996 and 1997) reported that levels of SAM and the ratio of PC to PE in the liver were both higher in rats fed a casein diet or a Met-enriched soy protein diet compared with a soy protein diet. An excess of SAM induced by high dietary intake of Met was also previously suggested to cause toxicity in rats (Regina et al. 1993 ).

The role of Arg in counteracting the PC accumulation and raising PE levels is not clear. It is possible that in the presence of Arg, the methyl groups from SAM are not used preferentially for the formation of PC from PE. Instead, SAM and Arg could be both utilized for other reactions, such as biosynthesis of polyamines (Grillo 1985 ). In this case, Arg could also help to provide the polyamine substrate, ornithine, by counteracting the Lys-induced inhibition of arginase.

In summary, our results indicate that supplementation with Arg, but not Gly, counteracts in part the hypercholesterolemia and weight loss induced by high levels of Lys + Met in rabbits. The hypercholesterolemic effect of the [Lys + Met]-supplemented diet may be mediated in part by an increase in liver PC synthesis because an elevation of hepatic PC was also counteracted partially by Arg, but not by Gly. In addition, our data show that the responses produced by Lys + Met supplementation can be reproduced by replacing high levels of dietary casein amino acids with physiologically adequate, lower amounts of either casein amino acids or casein. This suggests that in the future, the expense of dietary studies in rabbits could be considerably reduced.

	ACKNOWLEDGMENTS

 
The palm oil used to prepare the diets for these experiments was kindly provided by Can Amera Foods (Toronto, Canada) and the amino acids were provided at cost by the Ajinomoto Company U.S.A. (New York, NY).

	FOOTNOTES

 
¹ Supported by the Heart and Stroke Foundation of Ontario, grant # B-3333 and by the Ontario Soybean Growers' Marketing Board. I.G. received support from Les Fonds pour les Chercheurs et l'Aide à la Recherche du Québec.

³ Abbreviations used: AA, amino acid; ApoB, apolipoprotein B; CDP-choline, [methyl-¹⁴C]-cytidine 5'diphosphocholine; CPT, choline phosphotransferase; CT, choline phosphate cytidylyltransferase; EAA, essential amino acids; NEAA, nonessential amino acids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase; SAM, S-adenosylmethionine.

Manuscript received August 28, 1998. Initial review completed March 18, 1999. Revision accepted July 4, 1999.

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