Hyperhomocysteinemia induced by guanidinoacetic acid is effectively suppressed by choline and betaine in rats

Rats were fed 25% casein (25C) diets differing in choline levels (0-0.5%) with and without 0.5% guanidinoacetic acid (GAA) or 0.75% L-methionine for 7 d to determine the effects of dietary choline level on experimental hyperhomocysteinemia. The effects of dietary choline (0.30%) and betaine (0.34%) on GAA- and methionine-induced hyperhomocysteinemia were also compared. Dietary choline suppressed hyperhomocysteinemia induced by GAA, but not by methionine, in a dose-dependent manner. GAA-induced enhancement of the plasma homocysteine concentration was suppressed by choline and betaine to the same degree, but the effects of these compounds were relatively small on methionine-induced hyperhomocysteinemia. Dietary supplementation with choline and betaine significantly increased the hepatic betaine concentration in rats fed a GAA diet, but not in rats fed a methionine diet. These results indicate that choline and betaine are effective at relatively low levels in reducing plasma homocysteine, especially under the condition of betaine deficiency without a loading of homocysteine precursor.     

Increased plasma homocysteine concentration in rats from a low casein diet

Rats were fed on a 10% casein (10C) diet, 30% casein (30C) diet, 10C+0.5% methionine diet, or 30C+0.5% methionine diet for 14 d to investigate the relationship between the dietary protein level and plasma homocysteine concentration. The plasma homocysteine concentration was significantly higher in the rats fed on the 10C diet than in the rats fed on the 30C diet, and this phenomenon persisted even under the condition of methionine supplementation. The activity of hepatic cystathionine beta-synthase (CBS) was significantly lower in the rats fed on the 10% casein diets than in the rats fed on the 30% casein diets, irrespective of methionine supplementation. This is the first demonstration of a low-protein diet increasing the plasma homocysteine concentration in experimental animals. It is suggested that the decreased CBS activity might be associated, at least in part, with the hyperhomocysteinemia caused by the low-casein diet.     

Increased Plasma Homocysteine Concentration in Rats from a Low Casein Diet

Rats were fed on a 10% casein (10C) diet, 30% casein (30C) diet, 10C+0.5% methionine diet, or 30C+0.5% methionine diet for 14 d to investigate the relationship between the dietary protein level and plasma homocysteine concentration. The plasma homocysteine concentration was significantly higher in the rats fed on the 10C diet than in the rats fed on the 30C diet, and this phenomenon persisted even under the condition of methionine supplementation. The activity of hepatic cystathionine β-synthase (CBS) was significantly lower in the rats fed on the 10% casein diets than in the rats fed on the 30% casein diets, irrespective of methionine supplementation. This is the first demonstration of a low-protein diet increasing the plasma homocysteine concentration in experimental animals. It is suggested that the decreased CBS activity might be associated, at least in part, with the hyperhomocysteinemia caused by the low-casein diet.     

Cysteine supplementation decreases plasma homocysteine concentration in rats fed on a low-casein diet in rats

The effect of dietary supplementation with cysteine on the plasma homocysteine concentration was investigated in rats fed on 10% casein (10C) and 30% casein (30C) diets. The 10C diet significantly increased the plasma homocysteine concentration as compared with the 30C diet. The hyperhomocysteinemia induced by the 10C diet was significantly suppressed by cysteine supplementation even at a 0.3% level, whereas cysteine did not decrease the plasma homocysteine concentration when added to the 30C diet. In contrast, 0.3% methionine supplementation of the 10C diet tended to increase the plasma homocysteine concentration. Cysteine supplementation to rats fed on the 10C diet did not alter the plasma cysteine concentration and the hepatic activities of cystathionine beta-synthase and betaine:homocysteine S-methyltransferase, whereas it significantly decreased the hepatic concentrations of S-adenosylmethionine and betaine. These results suggest that cysteine supplementation might be effective for suppressing the hyperhomocysteinemia induced by a low-protein diet.     

Effects of Sulfur-containing Amino Acids and Glycine on Plasma Cholesterol Level in Rats Fed on a High Cholesterol Diet

The effects of the addition of individual amino acids on methionine-induced hypercholesterolemia (experiment 1), and the interacting effects of dietary protein level and sulfur-containing amino acids and glycine on plasma cholesterol concentration (experiment 2) were studied in growing rats fed on a high cholesterol diet. In experiment 1, rats were fed on a 25% casein-0.75% methionine (25CM) diet containing 2.5% of individual amino acids for 2 weeks. Methionine-induced hypercholesterolemia was prevented by the concurrent addition of glycine or serine, but the other amino acids tested (alanine, threonine, leucine, phenylalanine, lysine, arginine, and glutamic acid) had no effect. Histidine rather enhanced the hypercholesterolemia. In experiment 2, rats were fed on a 10%, 25%, or 50% casein diet containing 0.75% methionine, 0.60% cystine, 0.63% taurine, 2.5% glycine, or 0.75% methionine +2.5% glycine for 3 weeks. Dietary addition of 0.75% methionine increased the plasma cholesterol concentration for the 25% and 50% casein diets, but it decreased the plasma cholesterol for the 10% casein diet. When the addition level of methionine was doubled in the 10% casein diet, the plasma cholesterol concentration was significantly higher for the 1.5% methionine-added diet than for the 0.75% methionine-added diet. Cystine and taurine lowered plasma cholesterol for all dietary casein levels. Methionine-induced hypercholesterolemia with 25% and 50% casein diets was prevented by the glycine supplementation. These data suggest that sulfur-containing amino acids and glycine are important in plasma cholesterol regulation.     

Methionine and serine synergistically suppress hyperhomocysteinemia induced by choline deficiency, but not by guanidinoacetic acid, in rats fed a low casein diet

The effects of dietary supplementation with 0.5% methionine, 2.5% serine, or both on hyperhomocysteinemia induced by deprivation of dietary choline or by dietary addition of 0.5% guanidinoacetic acid (GAA) were investigated in rats fed a 10% casein diet. Hyperhomocysteinemia induced by choline deprivation was not suppressed by methionine alone and was only partially suppressed by serine alone, whereas it was completely suppressed by a combination of methionine and serine, suggesting a synergistic effect of methionine and serine. Fatty liver was also completely prevented by the combination of methionine and serine. Compared with methionine alone, the combination of methionine and serine decreased hepatic S-adenosylhomocysteine and homocysteine concentrations and increased hepatic betaine and serine concentrations and betaine-homocysteine S-methyltransferase activity. GAA-induced hyperhomocysteinemia was partially suppressed by methionine alone, but no interacting effect of methionine and serine was detected. In contrast, GAA-induced fatty liver was completely prevented by the combination of methionine and serine. These results indicate that a combination of methionine and serine is effective in suppressing both hyperhomocysteinemia and fatty liver induced by choline deprivation, and that methionine alone is effective in suppressing GAA-induced hyperhomocysteinemia partially.     

Methionine and Serine Synergistically Suppress Hyperhomocysteinemia Induced by Choline Deficiency,but Not by Guanidinoacetic Acid,in Rats Fed a Low Casein Diet

The effects of dietary supplementation with 0.5% methionine, 2.5% serine, or both on hyperhomocysteinemia induced by deprivation of dietary choline or by dietary addition of 0.5% guanidinoacetic acid (GAA) were investigated in rats fed a 10% casein diet. Hyperhomocysteinemia induced by choline deprivation was not suppressed by methionine alone and was only partially suppressed by serine alone, whereas it was completely suppressed by a combination of methionine and serine, suggesting a synergistic effect of methionine and serine. Fatty liver was also completely prevented by the combination of methionine and serine. Compared with methionine alone, the combination of methionine and serine decreased hepatic S-adenosylhomocysteine and homocysteine concentrations and increased hepatic betaine and serine concentrations and betaine-homocysteine S-methyltransferase activity. GAA-induced hyperhomocysteinemia was partially suppressed by methionine alone, but no interacting effect of methionine and serine was detected. In contrast, GAA-induced fatty liver was completely prevented by the combination of methionine and serine. These results indicate that a combination of methionine and serine is effective in suppressing both hyperhomocysteinemia and fatty liver induced by choline deprivation, and that methionine alone is effective in suppressing GAA-induced hyperhomocysteinemia partially.     

Oleuropein supplementation increases urinary noradrenaline and testicular testosterone levels and decreases plasma corticosterone level in rats fed high-protein diet

The effects of oleuropein, a phenolic compound in extra virgin olive oil, on protein metabolism were investigated by measuring testicular testosterone and plasma corticosterone levels in rats fed diets with different protein levels. In Experiment 1, rats were fed experimental diets with different protein levels (40, 25 and 10 g/100 g casein) with or without 0.1 g/100 g oleuropein. After 28 days of feeding, the testosterone level in the testis was significantly higher and the plasma corticosterone level was significantly lower in rats fed the 40% casein diet with oleuropein than in those fed the same diet without oleuropein. The urinary noradrenaline level, nitrogen balance and hepatic arginase activity were significantly higher in rats fed the 40% casein diet with oleuropein supplementation than in those fed the 40% casein diet without oleuropein supplementation. In Experiment 2, the effects of oleuropein aglycone (a major phenolic compound in extra virgin olive oil and the absorbed form of oleuropein ingested in the gastrointestinal tracts) on the secretion of luteinizing hormone (LH) from the pituitary gland, which regulates testosterone production in the testis, were investigated in anesthetized rats. Plasma LH level increased dose dependently after the administration of oleuropein aglycone (P<.001, r= 0.691). These findings suggest that dietary supplementation with 0.1 g/100 g oleuropein alters the levels of hormones associated with protein anabolism by increasing urinary noradrenaline and testicular testosterone levels and decreasing plasma corticosterone level in rats fed a high-protein diet.     

Dietary methionine effects on plasma homocysteine and HDL metabolism in mice

The effects of dietary manipulation of folate and methionine on plasma homocysteine (Hcy) and high-density lipoprotein cholesterol (HDL-C) levels in wild-type and apolipoprotein-E-deficient mice were determined. A low-folate diet with or without folate and/or methionine supplementation in drinking water was administered for 7 weeks. Fasted Hcy rose to 23 microM on a low-folate/high-methionine diet, but high folate ameliorated the effect of high methionine on fasted plasma Hcy to approximately 10 microM. Determination of nonfasted plasma Hcy levels at 6-h intervals revealed a large diurnal variation in Hcy consistent with a nocturnal lifestyle. The daily average of nonfasted Hcy levels was higher than fasted values for high-methionine diets but lower than fasted values for low-methionine diets. An acute methionine load by gavage of fasted mice increased plasma Hcy 2.5 h later, but mice that had been on high-methionine diets had a lower fold induction. Mice fed high-methionine diets weighed less than mice fed low-methionine diets. Based on these results, two solid-food diets were developed: one containing 2% added methionine and the other containing 2% added glycine. The methionine diet led to fasted plasma Hcy levels of >60 microM, higher than those with methionine supplementation in drinking water. Mice on methionine diets had >20% decreased body weights and decreased HDL-C levels. An HDL turnover study demonstrated that the HDL-C production rate was significantly reduced in mice fed the methionine diet.     

Effects of protein level, methionine supplementation and carbohydrate type of the diet on liver lipid and plasma free threonine contents in the lactating rat

Eight groups of 13-15 female rats were fed purified diets after littering. Four groups received a low protein (8% casein) diet (groups 8) and the others, a normal protein (20% casein) diet (groups 20). Carbohydrates were supplied either as starch (groups S) or as starch plus 40% fructose (groups F). Half the animals received a 0.4% methionine supplementation (groups M). Four or five dams per group were sacrificed on days 2, 7 and 14 after littering. The diet intake was increased by methionine supplementation, substitution of starch for fructose and increased protein content, mainly during the second week of lactation. This influenced weight variation of the dams and litter growth. On all days, the plasma levels of cholesterol esters, triglycerides and phospholipids were positively correlated with the dietary protein level. On days 7 and 14, the liver neutral lipid content was increased in rats fed the low protein diets supplemented with methionine (groups 8SM and 8FM) and the normal protein diets containing 40% fructose (groups 20F and 20FM). The plasma free threonine content was positively correlated with the protein level in the diet. On day 14, rats fed a low protein diet had a threonine deficiency, except those in groups 8S and 8F. The plasma free threonine content of these rats was not reduced, possibly due to an impaired utilization of this amino acid. The liver lipidosis observed during lactation, in contrast to that observed during growth with a low protein diet, was not due to a threonine deficiency.     

有氧运动对高蛋氨酸饮食大鼠血浆NO、ET和NO/ET系统的影响

目的：探讨有氧运动对高蛋氨酸饮食大鼠血浆总一氧化氮合成酶（T-NOS）、一氧化氮（NO）、内皮素（ET）和NO/ET系统的影响。方法：雄性Wistar大鼠随机分为正常饮食对照组（对照组）、高蛋氨酸饲料组（高蛋氨酸组）和有氧运动＋高蛋氨酸饮食组（运动干预组）。对照组喂饲普通饲料,高蛋氨酸组和运动干预组喂饲含3%蛋氨酸的高蛋氨酸饲料,运动干预组同时每日同时进行90 min无负重游泳运动,实验共8周。分别测定血浆同型半胱氨酸（Hcy）、ET、NO和T-NOS含量。结果：高蛋氨酸组血浆Hcy含量显著高于对照组达2倍以上（P〈0.01）,T-NOS和NO含量显著降低,ET含量显著升高（P〈0.01）,且NO/ET比值均显著降低（P〈0.05）;与高蛋氨酸组相比,运动干预组血浆Hcy含量显著下降（P〈0.05）,T-NOS,NO含量和NO/ET比值显著升高（P〈0.05）,且与对照组相比上述各项指标无显著差异。结论：高蛋氨酸饮食可诱发大鼠高同型半胱氨酸血症,血浆NO/ET失衡;有氧运动可降低高蛋氨酸饮食大鼠血浆Hcy水平,改善NO/ET失衡,预防高同型半胱氨酸血症。     

Metabolic Effects of Arginine and Methionine Supplement on Rats Fed Excess Glycine Diets

The contents of glycogen, lipid, urea and amino acids, and some enzyme activities in plasma, liver muscle and urine were determined with rats fed 10 to 12 g of 100 g body weight per day of the 10% casein diet (control) and 10% casein diets containing 7% glycine with or without 1.4% l-arginine HC1 and l-methionine for 7 days.

Nine hours after the final feeding, the amount of liver glycogen was high in the order of rats fed 10% casein diet containing 7% glycine, 10% casein diet containing 7% glycine with l-arginine and l-methionine, and the control. The amount of muscle glycogen was decreased only in those fed the control diet. The amount of liver lipid was increased by the addition of l-arginine and l-methionine to the excess glycine diet. Plasma and urinary urea was increased in animals given the excess glycine diets with or without both amino acids. In plasma liver, and muscle of animals given either of both the excess glycine diets 3 and 9 hr after the feeding, in general, glycine and serine were increased, and threonine and alanine were decreased as compared with those of rats given the control diet. However, the increase of glycine in plasma, liver and muscle detected at 9 hr after feeding the excess glycine diet was slightly prevented by the supplementation of both amino acids to the excess glycine diet. The activities of liver glycine oxidase and ornithine δ-aminotransferase of rats given the excess glycine diet with both amino acids were higher than those of other dietary groups. Liver serine dehydratase and glutamate-oxalacetate transaminase activities were high in the order of the animals fed the control, the excess glycine diet and the excess glycine diet containing both amino acids. Glutamate-pyruvate transaminase activity in the liver of rats fed the excess glycine diets with or without both amino acids were markedly higher than that of those fed the control. The activities of phosphopyruvate carboxylase and aconitase in the liver of animals given the excess glycine diet were higher than those of other dietary groups. Liver pyruvate kinase and glutamate dehydrogenase activities were similar among those dietary groups.     

Suppression of methionine-induced hyperhomocysteinemia by glycine and serine in rats

The hyperhomocysteinemia induced by a dietary addition of 1% methionine was significantly suppressed by the concurrent addition of 1% glycine or 1.4% serine to the same degree. The methionine-induced increase in the hepatic concentration of methionine metabolites was significantly suppressed by glycine and serine, but the hepatic cystathionine beta-synthase activity was not enhanced by these amino acids. When the methionine-supplemented diet was changed to the methionine plus glycine or serine diet, the plasma homocysteine concentration rapidly decreased during and after the first day. The hyperhomocysteinemia induced by an intraperitoneal injection with methionine was also suppressed by concurrent injection with glycine or serine, although the effect of serine was significantly greater than that of glycine. These results indicate that glycine and serine were effective for suppressing methionine-induced hyperhomocysteinemia: serine and its precursor glycine are considered to have elicited their effects mainly by stimulating cystathionine synthesis by supplying serine, another substrate for cystathionine synthesis.     

Supplementation of orotic acid to the casein, but not to egg protein, soy protein, or wheat gluten diets, increases serum ornithine carbamoyltransferase activity

Effects of dietary supplementation of orotic acid to a diet containing the casein protein were compared with diets containing egg protein, soy protein, or wheat gluten on lipid levels in the liver and serum and activities of ornithine carbamoyltransferase (OCT) and alanine aminotransferase in the serum of rats. We found that supplementation of orotic acid to each diet increased the contents of the liver total lipids, triacylglycerol, and phospholipids compared with those not supplemented. The contents of liver total lipids, triacylglycerol, cholesterol, and phospholipids in rats fed the casein diet were significantly higher than those of rats fed the other three diets when orotic acid was supplemented. The levels of triacylglycerol, cholesterol, and phospholipids in the serum of rats fed the casein diet were markedly decreased by addition of orotic acid. The supplementation of orotic acid significantly increased the activities of both serum OCT and alanine aminotransferase in rats fed the casein diet, but not in rats fed the other diets. In conclusion, liver lipid accumulation induced by dietary orotic acid depends on the type of dietary protein. The enhancement of serum OCT activity may result from liver lipid accumulation in rats fed the casein diet supplemented with orotic acid, demonstrating hepatic damage.     

高蛋氨酸饮食对大鼠血管内皮细胞分泌功能的影响

目的:探讨高蛋氨酸饮食对大鼠血管内皮细胞分泌功能的影响。方法:雄性Wistar大鼠随机分为正常饮食对照组(对照组)和高蛋氨酸饮食组(高蛋氨酸组)。对照组喂饲普通饲料,高蛋氨酸组大鼠喂饲含3%蛋氨酸的饲料,共8周。采用高效液相色谱法测定大鼠血浆同型半胱氨酸含量,以MDA、SOD、NO/ET和t-PA/PAI平衡等指标建立研究平台和内皮功能评价体系,同时以扫描电镜观察主动脉弓血管内皮细胞形态。结果:与对照组相比,高蛋氨酸组血浆Hcy含量显著高于对照组,是对照组的2倍以上;血浆MDA和SOD活力显著升高(P<0.05),t-PA/PAI-1和NO/ET比值均显著降低(P<0.05)。扫描电镜显示高蛋氨酸组大鼠内皮细胞呈典型虫蛀样损害,伴有附壁血栓形成和脂质沉积。结论:高蛋氨酸饮食可诱发大鼠高半胱氨酸血症,Hcy可通过氧化应激机制损伤血管内细胞分泌功能,血浆NO/ET和t-PA/PAI-1系统失衡。     

Relationship between dietary tyrosine and plasma cholesterol in the rat

The present study was undertaken to measure the effects of dietary tyrosine added to fish protein and peanut meal on plasma cholesterol and plasma thyroid hormone levels in the rat. These dietary proteins were chosen because they contain similar amounts of tyrosine but release it at different rates during enzymatic hydrolysis. Casein was chosen as the reference protein. Supplementation was used to obtain tyrosine levels similar to that of casein. Male Sprague-Dawley rats were fed cholesterol-enriched diets containing 15% protein. After 3 weeks of experimental feeding, total postprandial plasma cholesterol was similar in the casein and peanut meal groups and significantly lower in the fish group. When added to the fish diet, tyrosine caused an increase in plasma cholesterol to a level similar to that of the casein group, whereas supplementation had no effect on plasma cholesterol of rats fed the peanut meal diet. The effects of dietary proteins or of tyrosine supplementation on cholesterol levels of the (density less than 1.006 g/mL) lipoprotein fraction were comparable, but not all significant, to those observed on total plasma cholesterol. In addition, casein and fish diets induced significantly higher levels of plasma triiodothyronine (T3) and lower levels of plasma thyroxine (T4) than did the peanut meal diet. However, the addition of tyrosine to the fish or the peanut meal diet did not modify the plasma thyroid hormone levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased response of liver microsomal delta 6-desaturase activity to dietary methionine in rats

The effects of dietary casein level (5-40%) on the liver microsomal phospholipid profile, delta 6-desaturase activity and related variables were investigated in rats to examine whether the dietary protein level affected the delta 6-desaturase activity through an alteration of the liver microsomal phospholipid profile. The effects of supplementing a 10% casein diet with certain amino acids were also investigated. The concentration of hepatic S-adenosylmethionine (SAM), the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) and the delta 6-desaturase activity in liver microsomes, and the ratio of arachidonate to linoleate of microsomal PC increased with increasing dietary casein level. There were significant correlations between the dietary methionine content and hepatic SAM concentration, hepatic SAM concentration and microsomal PE concentration, and microsomal PE concentration and delta 6-desaturase activity. Supplementation of the 10% casein diet with methionine significantly increased the hepatic SAM concentration, PC/PE ratio, delta 6-desaturase activity, and arachidonate/linoleate ratio, whereas cystine supplementation had no or little effect on these variables. These increases induced by methionine were significantly suppressed by additional glycine. The results obtained here, together with those in our previous report, suggest that quantity and type of dietary protein might affect the delta 6-desaturase activity through an alteration of the liver microsomal profile of phospholipids, especially PE, and that the alteration of phospholipid profile might be mediated by a hepatic SAM concentration that reflects the dietary methionine level.     

Increased dietary protein elevates plasma uric acid and is associated with decreased oxidative stress in rapidly-growing broilers

Uric acid is an important antioxidant and methods to elevate its plasma concentration may be important in animal health. In a first study, the effect of dietary protein on plasma uric acid (PUA) and glucose concentrations were determined in 3-week-old chicks. Twenty-four broiler chicks were randomly assigned to four diets: a commercial control diet (C, 20% crude protein), low protein (LP) containing 10% casein, medium protein (MP) containing 20% casein or high protein (HP) containing 45% casein for a 3-week experiment. PUA concentration increased (P<0.05) in chicks fed HP diet and declined (P<0.05) in chicks fed LP while plasma glucose concentrations were lower (P<0.05) in chicks fed the LP diet at the end of the study. In a second study, PUA and leukocyte oxidative activity (LOA) were determined in broilers fed C, LP, MP or HP diets for 4 weeks. As in the first study, dietary protein directly affected PUA concentrations. In birds consuming HP diets, PUA was negatively correlated (P=0.06) with lowered LOA. These data support the view that increases in dietary protein can increase PUA concentrations, which can ameliorate oxidative stress.     

Folate and homocysteine metabolism in copper-deficient rats.

To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague-Dawley rats were fed semi-purified diets containing either 0. 1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group (P<0.05). Copper deficiency resulted in a 21% reduction in hepatic methionine synthase activity as compared to the control group (P<0.01). This change most likely caused the increased hepatic 5-methyltetrahydrofolate and plasma homocysteine concentrations in the copper-deficient group. Our results indicate that hepatic methionine synthase may be a cuproenzyme, and plasma homocysteine concentrations are influenced by copper nutriture in rats. These data support the concept that copper deficiency can be a risk factor for cardiovascular disease.     

Age-related effects of methionine-enriched diet on plasma homocysteine concentration and methylation of hepatic DNA in rats

In the present study we determined the age-related effect of methionine-enriched diet, a model of hyperhomocysteinemia, on the level of plasma homocysteine and hepatic global DNA methylation in rats. Feeding methionine diet to middle-aged rats for only 14 days resulted in a significant increase in plasma homocysteine level and DNA hypomethylation. In contrast, feeding the methionine-containing diet for 2 weeks to juvenile or post-pubertal animals did not alter the level of plasma homocysteine or hepatic DNA methylation. Supplementation of the methionine-enriched diet with vitamins B6, B12 and folic acid prevented both hepatic DNA hypomethylation and an increase of plasma homocysteine concentration in the middle-aged rats. These findings indicate that the elevated level of plasma homocysteine may be indicative of much broader and deeper alterations in intracellular methylation dysfunction, and suggest that dietary enrichment with B-vitamins is essential for the metabolism of homocysteine, especially in adult animals.