Cystathionine Synthase Activity Research Articles

Hepatic Activity and Transcription of Betaine-Homocysteine Methyltransferase, Methionine Synthase, and Cystathionine Synthase in Periparturient Dairy Cows Are Altered to Different Extents by Supply of Methionine and Choline

Compared with choline, Met enhances milk yield and feed intake, and elicits a better immuno-metabolic status in periparturient cows. It is unknown whether hepatic activity and transcription of betaine-homocysteine methyltransferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and cystathionine β-synthase (CBS) are responsive to Met and choline supply. This study sought to characterize hepatic BHMT, MTR, and CBS transcription and activity in response to Met and choline supplementation. Forty multiparous cows were used in a 2 × 2 factorial design from -21 d through 30 d around parturition to assess effects of dietary rumen-protected Met (0% or 0.08% dry matter basis) or rumen-protected choline (0 or 60 g · cow-1 · d-1). Liver tissue obtained on days -10, 7, 20, and 30 was used for analyses. Met-supplemented cows had greater methionine adenosyltransferase 1A (MAT1A) (0.38 compared with 0.27; SEM = 0.05; P = 0.02) and phosphatidylethanolamine methyltransferase (PEMT) (0.74 compared with 0.58; SEM = 0.08; P = 0.05) expression. Greater S-adenosylhomocysteine hydrolase (SAHH) (0.93 compared with 0.74; SEM = 0.05; P = 0.01) and CBS (1.16 compared with 1.02; SEM = 0.07; P = 0.04), as well as lower MTR activity (23.4 compared with 29.7 nmol product · h-1 · mg protein-1; SEM = 2.9; P = 0.04), also were detected in Met- but not choline-supplemented cows. Although BHMT and MTR expression and BHMT enzyme activity did not change (P > 0.05), MTR enzyme activity was lower in choline-supplemented cows (23.5 compared with 29.6 nmol product · h-1 · mg protein-1; SEM = 2.9; P = 0.05). These findings indicate that greater synthesis of phosphatidylcholine and antioxidants contribute to the better performance and immuno-metabolic status in Met-supplemented cows. Failure to generate a comparable amount of endogenous Met from choline could be one reason that choline-fed cows fail to achieve comparable performance and health benefits during the periparturient period.

Dietary crude protein has minimal effect on the activity of selected enzymes of methionine catabolism in kittens fed diets near‐limiting in methionine*

Previous experiments have shown that increasing the dietary crude protein (CP) of cats does not increase urea cycle enzymes or alanine amino transferase as occurs in rats. Also when an essential amino acid (EAA) is limiting in a diet for growing kittens, the kittens do not exhibit an amino acid imbalance when other EAAs are added to the diet. To study the metabolic basis for these observations which are different from that found in omnivores and herbivores, the hypothesis that increased dietary CP decreases methionine catabolism, so more is spared for growth, was tested. Fifteen male kittens were randomly assigned to one of three dietary treatments. Each diet contained 2.5 g l-methionine/kg diet and 200, 300 or 500 g CP/kg diet. The livers and kidneys were removed and assayed for methionine transaminase (MTA), cystathionase (CASE) and cystathionine synthase (CS). Free amino acid concentrations were determined in liver, kidney and plasma. The 300 and 500 g CP/kg groups had significantly greater kidney weights and body weight gains than the 200 g CP/kg group. Hepatic MTA activity was lower in the 300 than the 200 or 500 g CP/kg groups (p < 0.05). Renal MTA and CASE activities were 35% and 50% greater, respectively, for the 500 g CP/kg group than for the 200 g CP/kg diet group (p < 0.05). Renal CS activities for the 300 and 500 g CP/kg groups were 29% (p > 0.05) and 38% (p < 0.05) greater, respectively, than the 200 g CP/kg group. Cyst(e)ine concentrations were lower in the livers of the 500 g CP/kg group than the 200 g CP/kg group (p < 0.05). Cystathionine was lower in plasma and kidney from the 500 g CP/kg diet group than from the 200 g CP/kg diet group (p < 0.05). It was concluded that the metabolic basis for the increased growth of kittens fed diets marginally limiting in methionine, with increasing concentrations of dietary CP, was not mediated through decreased enzyme activity associated with the catabolism of methionine, but was the result of an increase in food (methionine) intake.

Dietary Folate and Selenium Affect Dimethylhydrazine-Induced Aberrant Crypt Formation, Global DNA Methylation and One-Carbon Metabolism in Rats

Several observations suggest a role for DNA methylation in cancer pathogenesis. Although both selenium and folate deficiency have been shown to cause global DNA hypomethylation and increased cancer susceptibility, the nutrients have different effects on one-carbon metabolism. Thus, the purpose of this study was to investigate the interactive effects of dietary selenium and folate. Weanling, Fischer-344 rats (n = 23/diet) were fed diets containing 0 or 2.0 mg selenium (as selenite)/kg and 0 or 2.0 mg folate/kg in a 2 x 2 factorial design. After 3 and 4 wk of a 12-wk experiment, 19 rats/diet were injected intraperitoneally with dimethylhydrazine (DMH, 25 mg/kg) and 4 rats/diet were administered saline. Selenium deficiency decreased (P < 0.05) colonic DNA methylation and the activities of liver DNA methyltransferase and betaine homocysteine methyltransferase and increased plasma glutathione concentrations. Folate deficiency increased (P < 0.05) the number of aberrant crypts per aberrant crypt foci, the concentration of colonic S-adenosylhomocysteine and the activity of liver cystathionine synthase. Selenium and folate interacted (P < 0.0001) to influence one-carbon metabolism and cancer susceptibility such that the number of aberrant crypts and the concentrations of plasma homocysteine and liver S-adenosylhomocysteine were the highest and the concentrations of plasma folate and liver S-adenosylmethionine and the activity of liver methionine synthase were the lowest in rats fed folate-deficient diets and supplemental selenium. These results suggest that selenium deprivation ameliorates some of the effects of folate deficiency, probably by shunting the buildup of homocysteine (as a result of folate deficiency) to glutathione.

Altered methionine metabolism in long living Ames dwarf mice

Ames dwarf mice (df/df) are deficient in growth hormone, prolactin, and thyroid-stimulating hormone and live significantly longer than their normal siblings. In the current study, we found that the hormone deficiencies affect methionine metabolism. We previously reported that the dwarf mice exhibit enzyme activities and levels that combat oxidative stress more efficiently than those of normal mice. Moreover, methionine or metabolites of methionine are involved in antioxidative processes. Thus, we performed an experiment that compared various parameters of methionine metabolism between 18-month old male dwarf ( N=6) and wild type ( N=5) mice. The specific activity of liver methionine adenosyltransferase (MAT) was significantly elevated (205%, p<0.0001) in the dwarf mice, as were cystathionine synthase (50%, p<0.01), cystathionase (83%, p<0.001), and glycine N-methyltransferase (GNMT, 91%, p<0.001) activities. Even though the activities of MAT and GNMT were elevated, the concentration of liver S-adenosylmethionine was decreased (24%, p<0.001) and S-adenosylhomocysteine increased (113%, p<0.001) in the dwarf mice. These data indicate that dwarf mice, compared to wild type mice, have a markedly different metabolism of methionine. Altered methionine metabolism may partially explain earlier reports indicating less oxidative damage to proteins in dwarf mice. Taken together, the data suggest that methionine metabolism may play a role in oxidative defense in the dwarf mouse and should be studied as a potential mechanism of extended lifespan.

Methionine Supply to Growing Steers Affects Hepatic Activities of Methionine Synthase and Betaine-Homocysteine Methyltransferase, but Not Cystathionine Synthase

The effects of supplemental methionine (Met), supplied abomasally, on the activities of methionine synthase (MS), cystathionine synthase (CS) and betaine-homocysteine methyltransferase (BHMT) were studied in growing steers. Six Holstein steers (205 kg) were used in a replicated 3 x 3 Latin square experiment. Steers were fed 2.6 kg dry matter daily of a diet containing 83% soybean hulls and 8% wheat straw. Ruminal infusions of 180 g/d acetate, 180 g/d propionate, 45 g/d butyrate, and abomasal infusion of 300 g/d dextrose provided additional energy. An amino acid mixture (299 g/d) limiting in Met was infused into the abomasum to ensure that nonsulfur amino acids did not limit growth. Treatments were infused abomasally and included 0, 5 or 10 g/d L-Met. Retained N (20.5, 26.9 and 31.6 g/d for 0, 5 and 10 g/d L-Met, respectively) increased (P < 0.01) linearly with increased supplemental Met. Hepatic Met, vitamin B-12, S-adenosylmethionine and S-adenosylhomocysteine were not affected by Met supplementation. Hepatic folates tended (P = 0.07) to decrease linearly with Met supplementation. All three enzymes were detected in hepatic tissue of our steers. Hepatic CS activity was not affected by Met supplementation. Hepatic MS decreased (P < 0.01) linearly with increasing Met supply, and hepatic BHMT activity responded quadratically (P = 0.04), with 0 and 10 g/d Met being higher than the intermediate level. Data from this experiment indicate that sulfur amino acid metabolism may be regulated differently in cattle than in other tested species.

Effect of methionine supplementation on methionine metabolism in growing cattle (2002)

Methionine is often the first limiting amino acid for growing cattle. This study was conducted to determine how methionine metabolism is regulated in the liver of growing steers. Six ruminally cannulated steers were used in a replicated 3 x 3 Latin square experiment. Either 0, 5, or 10 g/day L-methionine was infused into the abomasum. These treatments were designed to be deficient, adequate, and in excess of the steers' requirements for methionine. Methionine supplementation linearly increased protein deposition and decreased the activity of methionine synthase (a methionine conserving enzyme). However, it had little effect on activity of cystathionine synthase (an enzyme that produces cysteine from methionine). Our results suggest that methionine metabolism and regulation in cattle may vary from that in monogastrics.

Saudi Experience with Classic Homocystinuria

Classic homocystinuria is an autosomal recessive disorder due to cystathionine ss-synthase deficiency. The clinical, radiological and neurophysiological findings of classic homocystinuria diagnosed at King Faisal Specialist Hospital and Research Centre (KFSH&RC) are presented in this report. Twenty-four patients (15 females and 9 males) were referred to KFSH&RC for work-up of mental retardation, seizures, thrombo-embolic episodes and dislocation of the ocular lenses. The common clinical findings included ectopia lentis (20 patients), skeletal system involvement (18 patients), vascular system involvement (9 patients), and mental retardation (all patients to varying degrees). Unusual findings consisted of a patient who developed severe lower gastrointestinal bleeding, a patient with insulin-dependent diabetes mellitus, probably due to vasculopathy, and another having severe bronchiectasis, which may have been due to fibrillin disruption, and required the resection of a lobe of the lung. The parents of 21 patients were first-degree relatives, and 19 patients had one or more family members affected by the same disease. All patients had markedly elevated plasma levels of methionine. Cystathionine synthase activity in the fibroblast was measured in 25% of the patients and was deficient. Only four patients responded to pyridoxine and their methionine level decreased to almost normal range. The aim of this study was to increase the awareness of this disease in the scientific and medical community, in particular in the general pediatrician working in Saudi Arabia who first encounters the clinical manifestations of the disease. Early detection through tandem mass spectrometry of blood spot screening and treatment are important, and may prevent the major complications of this disease.

Polyamines in testosterone-induced hypertrophic and antifolate-induced hyperplastic mouse kidney. Differential effect of α-difluoromethylornithine

In the testosterone-induced hypertrophic and antifolate ( N 10-propargyl,5,6-dideazafolic acid, CB 3717)-induced hyperplastic mouse kidney models, a marked increase of two diamine levels — putrescine and cadaverine — occurred which paralled induced ornithine decarboxylase (ODC) activity. Under these conditions the augmentation of spermidine levels was much smaller, while spermine levels were affected differentially — increased by testosterone and decreased by CB 3717; this resulted in an increase of spermidine/spermine ratio in hyperplastic, but not hypertrophic kidney. α-Difluoromethylornithine (DFMO) prevented testosterone- or CB 3717-induced increment of both diamine levels. Spermidine and spermine depletion in response to DFMO was significant in hyperplastic kidney only. DFMO also significantly affected the other biochemical markers of hyperplasia, namely lowered CB 3717-induced cell proliferation rate and increased S-adenosylmethionie decarboxylase (AdoMetDC) activity. In contrast, testosterone-induced hypertrophy was not influenced by DFMO, as judged by the lack of its effect on S-adenosylmethionine synthetase and cystathionine synthase activity. These results indicate that the increase of putrescine levels does not mediate testosterone-induced renal hypertrophy and possibly also antifolate-induced hyperplasia. The involvement of spermidine in mediation of renal hyperplasia is highly possible, while that of spermine is excluded.

Hyperhomocysteinemia in patients operated for lower extremity ischaemia below the age of 50—effect of smoking and extent of disease

Moderate hyperhomocysteinemia may be a risk factor for atherosclerotic peripheral vascular disease (PVD). In order to develop PVD at an early age risk factors are more strongly expressed and hyperhomocysteinemia may be one such factor. Homocysteine is derived from methionine and is metabolised by cystathionine-synthase to cystathionine or remethylated to methionine. Cystathionine-synthase activity is dependent on vitamin B6 while the remethylation of homocysteine is dependent on vitamin B12 and folate. The present study analyses homocysteine in patients operated on for lower extremity ischaemia before the age of 50. Homocysteine before and after loading with methionine, vitamin B6, B12 and folate were measured at follow-up. The patients were compared to age- and sex-matched controls. Significantly more patients than controls had hyperhomocysteinemia, 16/58 vs. 4/65, defined as fasting total homocysteine above 18.6 mumol/l. Loading with methionine did not further discriminate between patients and controls. Smoking patients had higher levels of homocysteine than non-smoking patients or smoking and non-smoking controls. Smoking patients also had lower levels of vitamin B6. When comparing patients with suprainguinal, infrainguinal and multilevel disease the highest homocysteine levels were seen in the latter group. Also, in this group smoking patients had higher homocysteine levels. Multivariate analysis revealed that homocysteine was associated with low levels of vitamin B12, folate and smoking. Smoking therefore seems to be connected to increased homocysteine levels in patients with early development of atherosclerosis, partly explained by decreased levels of B6, B12 and folate.

Quasi-Moyamoya Disease and Heterozygosity for Homocystinuria in a Five-Year-Old Girl

We report a patient with quasi-Moyamoya disease who presented with acute left hemiparesis. Routine metabolic evaluation was normal. However, by performing a methionine loading test and measurement of cystathionine synthase activity in cultured fibroblasts the diagnosis of heterozygosity for homocystinuria (HC) was established. Only recently it has been shown that heterozygosity for HC is a risk factor for premature cerebrovascular disease in adults. The present case suggests that also in children with cerebrovascular disorders the analysis of its pathogenesis should include extensive metabolic studies on homocysteine metabolism.

Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease: Boers GHJ, Smals AGH, Trijbels FJM, et al. N Engl J Med 1985; 313:709-15

Homozygous homocystinuria is associated with premature atherosclerosis and early death from associated complications. Metabolic manipulation of animals with induction of homocystinemia has been used as a model of endothelial injury for atherosclerosis research. In this study the authors investigated homocysteine metabolism in 75 patients with symptomatic occlusive vascular disease, all of whom were less than 50 years of age. The severity and extent of disease were determined by appropriate arteriography and resulted in the classification of patients into three different anatomic patterns: peripheral vascular occlusive disease, coronary artery disease, and cerebrovascular disease—intracerebral and extracranial. A calculated oral dose of methionine was administered to all patients and serum levels of homocysteine subsequently determined. Criteria for positive testing that indicated heterozygous homocystinuria were developed and previously reported by the authors. Careful control for potential variables influencing the homocysteine pathway was used. None of the 25 patients with coronary artery disease had abnormal serum homocysteine levels after methionine loading. Of the 25 patients with peripheral arterial occlusive disease, seven (28%) had abnormal loading tests. All of these patients were women and represented 50% of the female cohort in this group. Most lesions in this group occurred in the abdominal aorta and visceral vessels. In the 25 patients with occlusive cerebrovascular disease, seven (28%) had abnormal methionine loading tests with no sex preponderance (four men and three women). Most lesions occurred in the intracerebral vessels with only one patient having disease in the extracranial carotid system. Corroboration of this abnormality in homocysteine metabolism was also obtained from in vitro measurement of cystathionine synthase activity in cultured fibroblasts from skin biopsy specimens. This procedures was performed only in patients in whom the oral loading test was unequivocally positive and was positive in 12 of the 14 patients (85%). Laboratory values of mean fasting glucose, mean fasting cholesterol, and mean fasting triglycerides were performed in all patients, with no statistically significant difference in distribution between patients with normal and abnormal methionine loading studies. Although homozygous homocystinuria is relatively rare, this study provides strong evidence to suggest that abnormalities of homocysteine metabolism either in the heterozygous form or others may be an important predisposing factor to the premature development of atherosclerosis in patients less than 50 years of age. The potential importance of this finding is significant in large part because this abnormality can be treated with folic acid or pyridoxine, both of which provide a relatively innocuous means of enhancing homocysteine use through alternate pathways.

155 HUMAN LYMPHOCYTES AS AN INVESTIGATIVE TOOL OF SULFUR-CONTAINING AMINO ACID METABOLISM

The activities of five enzymes involved in methionine metabolism were determined in human lymphocytes at their resting phase and during blastogenesis in vitro. Blastogenesis was induced by purified phytohemagglutinin. Enzymes assayed were: 1) methionine adenosyltransferase (MAE) (EC 2.5.1.6); 2) cystathionine synthase (CS) (EC 4.2.1.22); 3) 5-methyltetrahydrofolate homocysteine-methyltransferase (MTHF) (EC 2.1.1.13); 4) cystathionase (EC 4.4.1.1.); 5) N5,N10-methylene-tetrahydrofolate reductase (CH2-THF) (EC 1.1.1.68). The results were as follows: The specific activity of MAE and MTHF increased two- to fivefold in human lymphocytes after 72 h in culture; thereafter a slight decrease in their specific activity was observed. The specific activity of CS was maximal in unstimulated human lymphocytes and slightly decreased during stimulation. Cystathionase and CH2-THF activities were present in human lymphocytes 96 h after stimulation. Their activity was not measured in the resting lymphocyte. These data suggest that cultured human lymphocytes may serve as a tool for studying inborn genetic errors involving methionine metabolism.

Effect of Dietary Cystine on Methionine Metabolism in Rat Liver

Cystine supplementation of adequate diets resulted in significantly higher hepatic levels of betaine-homocysteine methyltransferase. Other changes occurred but were a function of the basal diet. When the latter contained 0.25% methionine + 0.5% cystine, the additional cystine caused a markedly lower hepatic cystathionine synthase activity and lower levels of both adenosylmethionine and serine. The metabolic effect of these changes may be enhanced methionine retention and diminished transsulfuration.

Improved identification of heterozygotes for homocystinuria due to cystathionine synthase deficiency by the combination of methionine loading and enzyme determination in cultured fibroblasts

Previous data on tentative identification of the carrier state for homocystinuria due to cystathionine synthase deficiency using methionine loading or measurement of cystathionine synthase activity in tissue extracts are conflicting. We studied the results of standardized oral methionine loading in 20 obligate heterozygotes and compared them with those of determination of cystathionine synthase activity in cultured fibroblasts. Special attention was devoted to our recently reported observation on the small but striking differences in methionine metabolism between healthy pre- and postmenopausal women and men. Fasting and after load peak levels of methionine in serum did not discriminate the carriers from the control subjects. The mean fasting level of total homocysteine was only significantly higher in the group of premenopausal heterozygotes than in the corresponding control group. Nevertheless, the individual values overlapped with the normal range in 4 of 12 premenopausal heterozygotes. After loading peak levels of total homocysteine in 18 out of the 20 obligate heterozygotes exceeded the upper limit of the ranges in the three control groups. Thus, this parameter discriminated 90% of the obligate carriers. Measurement of cystathionine synthase activity in cultured fibroblasts from a skin biopsy identified the obligate heterozygotes to a similar degree (85%). No significant correlation between the measurements of cystathionine synthase activity and the after load peak levels of total homocysteine in the individual heterozygotes was established. Combination of both methionine loading and determination of cystathionine synthase activity in cultured fibroblasts identified all of these carriers.

Variation of cysteine level by chlorobenzene-induced pertubation of glutathione metabolism in rat liver.

Effects of chlorobenzene-induced alteration in glutathione levels on cysteine metabolism in rat liver were investigated. Male Wistar rats were intraperitoneally injected with chlorobenzene (0.2, 0.5, 1.0 or 2.0 mmol/kg body weight). Both hepatic glutathione and cysteine levels were dose-dependently decreased by the chlorobenzene 6 h after the injection. However, at 24 h, the glutathione in the rats with chlorobenzene increased significantly as compared to that in the rats without chlorobenzene. Concomitant to the elevation in glutathione levels, hepatic glutathione synthesis activities were increased by the chlorobenzene by 68-111%. On the other hand, no significant difference between the rats with and without chlorobenzene was observed as regards cysteine levels at 24 h. Hepatic glutamate, glycine, methionine and serine levels were unaltered but hepatic taurine levels were significantly decreased by the chlorobenzene at both 6 and 24 h. Chlorobenzene administration had no effect on hepatic cystathionine synthase and cystathionase activities. These results indicate that a transient loss of hepatic glutathione, caused by the administration of chlorobenzene, resulted in an acceleration of glutathione synthesis and an increase of cysteine demand in the liver.

Factors Affecting the Accumulation of Homocyst(e)ine in Rats Deficient in Vitamin B-6

Factors that may affect the concentration of accumulated homocyst(e)ine in rats deficient in vitamin B-6 were studied. Hepatic cystathionine synthase activity in vitamin B-6 deficient rats was 41.5% of control and plasma protein-bound and free homocyst(e)ine concentrations were significantly increased; however, among deficient rats there was no correlation between the concentration of homocyst(e)ine and the activity of hepatic cystathionine synthase. The plasma protein-bound homocysteine concentration did not fluctuate with time of day, but a decrease in bound homocysteine was found when blood was sampled repeatedly from the same animal during a 24-hour period. A 24-hour fast resulted in a reduction in plasma homocyst(e)ine to concentrations not different from those of control animals. The protein content of the diet affected growth but not food intake of rats. Feeding a 10% versus a 60% casein diet did not change the concentration of accumulated homocyst(e)ine. Of the factors examined, only food restriction significantly affected plasma homocyst(e)ine concentration in rats deficient in vitamin B-6. It is speculated that decreased and sporadic food intake in rats deficient in vitamin B-6 may be responsible for the large fluctuations in homocyst(e)ine concentration observed in these animals over time.

Transsulfuration in parenterally nourished beagle pups

Interactions of plasma and tissue sulfur-containing amino acid were studied in beagle pups that received either total parenteral nutrition (TPN) or intravenous (IV) glucose from 21 to 35 days of age and compared with those in pups nursed from birth to 35 days of age. The TPN infusate provided 6 g/kg/day of crystalline amino acids (2 mmol/kg/day of ni.-methionine: less than 0. 1 mmol/�g/day of cysteine) and 180 kcal/kg/day of glucose. The IV glucose regimen provided 60 kcal/kg/day. Plasma methionine, cystathionine, and cysteine concentrations of the pups that received TPN were similar to those of controls. Cerebral methionine and cystathionine concentrations (jsmol/ 100 g tissue), however, were greater while cerebral cyst(e)ine concentration was less than controls. The hepatic content (jzmol/organ) of all three amino acids in these animals was similar to that of controls. Although plasma methionine, cystathionine, and cyst(e)ine concen- trations of pups that received the IV glucose regimen were significantly less than control values, both cerebral concentrations and hepatic contents were similar. Cerebral specific activity and hepatic total cystathionine synthase activity as well as total hepatic cystathionase activity of animals that receivedTPN were similar to those of control animals. The activities of these enzymes in animals that received only IV glucose, however, were lessthan those of controls.These data demonstrate that the relationship between plasma and tissue concentrations of the sulfur-containing amino acids during a period of altered nutrition in beagle pups is neither simple nor direct. During TPN, cerebral methionine and cystathionine concentrations increase and cerebral cysteine concen- tration falls, due in part to the inability of the brain to metabolize methionine further than cystathionine: competition among cysteine and other plasma amino acids for transport from plasma to brain may also contribute to the low cerebral cysteine concentration. In contrast, both methionine and cystathionine are metabolized readily by the liver, and hepatic cyst(e)ine content is main- tained. Am. J. Clin. Nutr. 34: 1520-1525, 1981.

Growth and liver enzyme response in growing rats to graded levels of methionine plus cystine in fortified-barley diets. Response at constant methionine:cystine.

1. Twenty-eight male rats of initial age 27 d were fed on fortified-barley diets for 3 weeks. In all experimental diets, both crude protein (nitrogen x 6.25) and methionine:cystine were constant at 120.0 g/kg dry matter (DM) and 2:1 respectively. The basal diet contained 4.5 g methionine plus cystine/kg DM with L-methionine plus L-cystine (2:1, w/w) added in increments of 0.5 g/kg DM to a final level of 7.0 methionine plus cystine/kg DM. A 'positive-control' diet of barley plus 193.7 g soya-bean meal/kg DM contained 6.0 g methionine plus cystine/kg DM. 2. Weight gain, food conversion efficiency (FCE), urinary urea-N excretion, carcass composition and activities of liver cystathionine synthase (EC 4.2.1.22) and N5-methyltetrahydrofolate-homocysteine-methyltransferase (EC 2.1.1.13) were determined. 3. Weight gain, food consumption, FCE and carcass composition measurements of rats showed either small or no differences between the experimental diets containing 4.5--7.0 g methionine plus cystine/kg DM. For the over-all period, weight gain and FCE of rats receiving the 'positive control' diet were significantly higher than values obtained with rats receiving any of the experimental diets. 4. Cystathionine synthase activity (mumol/mg protein per 60 min; units) increased from 13.38 at 4.5 g dietary methionine plus cystine/kg DM to 18.81 at 5.0 g dietary methionine plus cystine/kg DM. The activity was then inhibited to reach a minimum value of 10.16 units at the 6.0 g/kg DM dietary level. Thereafter the activity increased to a value of 30.00 units at 7.0 g dietary methionine plus cystine/kg DM. 5. The activity of N5-methyltetrahydrofolate-methyltransferase was constant at 0.70--0.74 nmol/mg protein per 60 min between dietary levels of 4.5 and 5.0 g methionine plus cystine/kg DM. The activity then increased to a maximum value of 2.32 nmol/mg protein per 60 min at the 6.0 g/kg DM level. Thereafter the activity decreased, reaching a minimum value of 0.70 nmol/mg protein per 60 min at the 7.0 g methionine plus cystine/kg level. 6. Urinary urea-N excretion decreased significantly from 1.07 g/kg DM intake at the 4.5 g dietary methionine plus cystine/kg DM level to 1.05 g/kg DM at the 5.0 g/kg dietary level, then dropped significantly to a level of 1.01--1.00 g/kg DM intake for the higher levels of dietary methionine plus cystine.

Effect of Excess Dietary Methionine on the Catabolism of Cysteine in Rats

The effect of excess methionine (MET) on cysteine (CYS) catabolism was investigated in rats prefed either a control (10% casein + 0.3% L-MET) or high-MET (10% casein + 3.0% L-MET) diet for 50 or 20 days. The activities of cysteine dioxygenase, cysteine desulfhydrase and cystathionase were increased in high-MET rats to levels 5.9, 2.7 and 2.7 times, respectively, those of control rats after 5 days and to 2.9, 2.0 and 2.7 times control levels after 20 days. Cysteine aminotransferase and cystathionine synthase activities were increased to 1.5 and 1.7 times control values after 5 days but were not significantly different from control values at 20 days. Following gastric intubation of 5 g of an L-amino acid diet containing 0.2% L-[35S]CYS, the 24-hour urinary exretion of 35SO4, [35S]taurine and total 35S (% of administered dose) and the [35S]taurine:35S and [35S]taurine:35SO4 ratios were increased in rats prefed excess MET for 5 or 20 days. When 2.6% L-[35S]CYS was administered similarly, no significant differences between high-MET and control rats were observed. However, the [35S]taurine:35S and [35S]taurine:35SO4 ratios were elevated in both high-MET and control rats given 2.6% L-[35S]CYS over those for control rats fed 0.2% L-[35S]CYS. The increase in cysteine dioxygenase activity and the increase in [35S]taurine:35SO4 ratio in rats fed excess MET or given a load dose of CYS suggest that the cysteine sulfinic acid pathway plays a major role in the regulation of CYS degradation.

Growth and liver enzyme response in growing rats to graded levels of methionine and cystine in fortified-barley diets: response at constant cystine concentration in the diet.

1. Thirty-six male rats of initial age 27 d were fed on diets based on barley grain for 3 weeks. Crude protein (nitrogen × 6.25) was fixed at 120.0 g/kg dry matter (dm) and cystine at 2.0 g/kg dm in all diets. The basal diet contained 3.5 g methionine plus cystine/kg dm with l-methionine added in increments of 0.5 g/kg dm up to a final level of 7.0 g methionine plus cystine/kg dm. A ‘positive control’ diet of barley plus 193.7 g soya-bean meal/kg dm contained 6.0 g methionine plus cystine/kg dm.2. Weight gain, food conversion efficiency (FCE), urinary urea-N excretion, carcass composition and activities of liver cystathionine synthase (EC 4.2.1.22) and N5-methyltetrahydrofolate-homocysteine-methyltransferase (EC 2.1.1.13) were determined.3. Weight gain, FCE and urinary urea-N excretion indicated that levels of 3.5 and 4.0 g methionine plus cystine/kg dm were inadequate for optimal growth of the rats. These variables indicated 4.7 g/kg dm to be the requirement for methionine plus cystine. Carcass composition results gave no clear indication of optimal requirements for methionine plus cystine.4. Cystathionine synthase and N5-methyltetrahydrofolate-homocysteine-methyltransferase activities (/mg protein per 60 min) were constant at 24.32 μmol and 0.64 μmol respectively, when methionine plus cystine level was between 3.5 and 5.0 g/kg dm. Cystathionine synthase was then inhibited at the 6.0 g/kg dm level to a minimum activity of 16.12 μmol/mg protein per 60 min, whereas activity of the re-methylation enzyme increased to a maximum level of 2.92 μmol/mg protein per 60 min at the 6.0 g/kg dm level of methionine plus cystine. At the levels of methionine plus cystine above 6.0 g/kg dm cystathionine synthase activity increased while the activity of N5-methyltetrahydrofolate-homocysteine-methyltransferase decreased.