Betaine-homocysteine Methyltransferase Activity Research Articles

Inhibition of human betaine–homocysteine methyltransferase expression by S-adenosylmethionine and methylthioadenosine

BHMT (betaine-homocysteine methyltransferase) remethylates homocysteine to form methionine. SAM (S-adenosylmethionine) inhibits BHMT activity, but whether SAM modulates BHMT gene expression is unknown. Transcriptional regulation of the human BHMT is also unknown. The present study examined regulation of the human BHMT gene by SAM and its metabolite, MTA (5'-methylthioadenosine). To facilitate these studies, we cloned the 2.7 kb 5'-flanking region of the human BHMT gene (GenBank accession number AY325901). Both SAM and MTA treatment of HepG2 cells resulted in a dose- and time-dependent decrease in BHMT mRNA levels, which paralleled their effects on the BHMT promoter activity. Maximal suppression was observed with the BHMT promoter construct -347/+33, which contains a number of NF-kappaB (nuclear factor kappaB) binding sites. SAM and MTA treatment increased NF-kappaB nuclear binding and NF-kappaB-driven luciferase activities, and increased nuclear binding activity of multiple histone deacetylase co-repressors to the NF-kappaB sites. Overexpression of p50 and p65 decreased BHMT promoter activity, while blocking NF-kappaB activation increased BHMT expression and promoter activity, and prevented SAM but not MTA's ability to inhibit BHMT expression. The NF-kappaB binding site at -301 is responsible, at least in part, for this effect. Lower BHMT expression can impair homocysteine metabolism, which can induce ER (endoplasmic reticulum) stress. Indeed, MTA treatment resulted in increased expression ER stress markers. In conclusion, SAM and MTA down-regulate BHMT expression in HepG2 cells in part by inducing NF-kappaB, which acts as a repressor for the human BHMT gene. While SAM's mechanism is NF-kappaB-dependent, MTA has both NF-kappaB-dependent and -independent mechanisms.

Liver Choline Dehydrogenase and Kidney Betaine-Homocysteine Methyltransferase Expression Are Not Affected by Methionine or Choline Intake in Growing Rats

AbstractCholine dehydrogenase (CHDH) and betaine-homocysteine methyltransferase (BHMT) are 2 enzymes involved in choline oxidation. BHMT is expressed at high levels in rat liver and its expression is regulated by dietary Met and choline. BHMT is also found in rat kidney, albeit in substantially lower amounts, but it is not known whether kidney BHMT expression is regulated by dietary Met or choline. Similarly, CHDH activity is highest in the liver and kidney, but the regulation of its expression by diet has not been thoroughly investigated. Sprague Dawley rats (∼50 g) were fed, for 9 d in 2 × 3 factorial design (n = 8), an L-amino acid–defined diet varying in L-Met (0.125, 0.3, or 0.8%) and choline (0 or 25 mmol/kg diet). Liver and kidney BHMT and CHDH were assessed using enzymatic, Western blot, and real-time PCR analyses. Liver samples were also fixed for histological analysis. Liver BHMT activity was 1.3-fold higher in rats fed the Met deficient diet containing choline, which was reflected in corresponding increases in mRNA content and immunodetectable protein. Independent of dietary choline, supplemental Met increased hepatic BHMT activity ∼30%. Kidney BHMT and liver CHDH expression were refractory to these diets. Some degree of fatty liver developed in all rats fed a choline-devoid diet, indicating that supplemental Met cannot completely compensate for the lack of dietary choline in growing rats.

Dietary selenium (Se) but not folic acid (FA) affects the activity and mRNA of rat liver betaine homocysteine methyltransferase (BHMT)

Because of up-regulation of γ-glutamylcysteine synthetase (GGCS) rodents shunt homocysteine (Hcys) to the transsulfuration pathway resulting in low plasma Hcys when Se is low. However, humans with low Se status do not appear to up-regulate GGCS and therefore place more emphasis on remethylation of Hcys for removal. Thus, dietary factors that affect remethylation of Hcys are of critical importance. We have previously shown that the activity of liver BHMT is decreased by deficient or marginally deficient Se-fed rats. Gonzalez et al (J Nutr 2004) correlated low Se status with increased Hcys in humans and suggested that this may be mediated through the effect of Se on BHMT. Here we expand our previous studies on the influence of Se on BMHT in a rodent model. Weanling, Fischer-344 rats were fed diets containing 0, 0.2, or 2 μg Se (as selenite) and 0 or 2 μg FA/g in a 3 x 2 design. After 72 d, the activity of liver BHMT was significantly decreased (p<0.001) in rats fed no supplemental Se compared to rats fed adequate or supranutritional Se; there was no difference comparing adequate vs supranutritional Se. Supplementation of FA tended (p=0.08) to decrease the activity. As determined by real-time RT PCR, liver BHMT mRNA was significantly down-regulated (p<0.002) in rats fed no supplemental Se; FA had no effect. Although the effect of Se status on BHMT in humans is not known, our results are intriguing in light of work by Gonzalez et al who suggest that serum Se may have greater effect on regulating Hcys than FA in humans and that this may be mediated through the effect of Se on BHMT.

Inhibition of Betaine‐Homocysteine Methyltransferase (BHMT) increases plasma total homocysteine (tHcy) in mice

Elevation of tHcy is an independent risk factor for vascular disease. Hcy can be methylated by BHMT and folate-dependent methionine synthase (MS) to form methionine (Met), or it can be converted to cystathionine by cystathionine-β-synthase (CBS). Disruption of flux through CBS or MS elevates tHcy, but BHMT’s role in the tHcy regulation has not been characterized. We describe the effect of S-(δ-carboxybutyl) homocysteine (CBHcy), a specific inhibitor of BHMT, on tHcy, BHMT and CBS. Mice (n = 8/treatment) were given intraperitoneal injections of saline (control) or CBHcy (1mg) at 12 h intervals. Four animals within each group were euthanized after 28 h (3 doses), and 4 at 76 h (7 doses). tHcy was measured by HPLC, liver BHMT and CBS expression were assessed using enzymatic, Western blot and real-time PCR. tHcy increased several-fold at both 28 and 76 h in the CBHcy-treated animals. Compared to controls, BHMT activity was significantly lower in CBHcy-treated animals (77 ± 8.3, 45 ± 5.1 U/mg) at 28 h, but its activity increased to 114 ± 9.9 U/mg at 76 h. Increases in BHMT immunodetectable protein and a 4-fold increase in BHMT mRNA were also observed at 76 h. CBS activity decreased from 0.25 ± 0.13 U/mg (control) to 0.13 ± 0.01 U/mg in the CBHcy-treated mice. These data show that inhibition of BHMT activity by CBHcy elevates tHcy. The loss of BHMT activity is compensated for by an increase in expression. Reduction of CBS protein and activity may also contribute to the elevation of tHcy observed. This work was supported by NIDDK (DK52501); ADA (1-04-RA-96).

Effect of Dietary Methionine (Met) and Choline on Rat Liver and Kidney Betaine‐Homocysteine Methyltransferase (BHMT) and Choline Dehydrogenase (CHDH) Expression

BHMT transfers a methyl group from betaine (Bet) to homocysteine to form Met and dimethylglycine. Bet is obtained from the diet or by the endogenous oxidation of choline by CHDH. BHMT is expressed in rat liver and is regulated by dietary Met and choline. BHMT is also found in rat kidney, albeit in substantially lower amounts, but it is not known whether it is regulated by dietary Met or choline. Whether diet has a role in the regulation of CHDH expression has not been investigated. Rats (~50g) were fed (9 days) in 3 x 2 factorial design (n = 8) a purified diet varying in Met (0.125, 0.3 or 0.8%) and choline (0 or 0.625%). Liver and kidney BHMT and CHCH expression were assessed using enzymatic, Western blot and real-time PCR analyses. Livers were also fixed in 10% buffered formalin for histological analysis. Animals fed low Met diets had significantly lower feed efficiencies. A degree of fatty liver was observed in all rats fed a choline-devoid diet, indicating that excess Met cannot compensate for low dietary choline. Compared to rats fed the 0.3% Met and 0.625% choline diet, liver BHMT activity was 113% higher in animals fed the 0.125% Met containing 0.625% choline and was also reflected in increases in mRNA content (2.7-fold) and immunodetectable protein. Excess dietary Met with or without choline increased liver BHMT activity 19.7% to 33.3%. Kidney BHMT was refractory to dietary treatment. Liver CHDH immunodetectable protein and mRNA content were not affected by dietary Met and choline availability. This work was supported by NIDDK (DK52501).

Effects of Betaine on Performence, Carcass Characteristics and Hepatic Betaine-homocysteine Methyltransferase Activity in Finishing Barrows

This experiment was conducted to determine the effect of dietary betaine (0, 0.125%) on performance, carcass composition, pork quality and hepatic betaine-homocysteine methyltransferase (BHMT) activity of crossbred finishing barrows. Three replicates often pigs were used for each treatment. The results showed that average daily gain, feed intake and feed conversion were not affected by betaine. Compared with the control group, pigs treated with betaine had a 8.17% (p<0.05) decrease in carcass fat percentage, and a 8.84% (p<0.05) reduction in 10th-rib backfat thickness, but dressing percentage, percentage lean, longissimus muscle area, and average backfat thickness were not affected. There were also no significant differences in muscle color score, marbling score, pork pH value and water loss rate between the control and betaine-treated groups. Hepatic betaine-homocysteine methyltransferase (BHMT) activity was significantly increased by 13.97% (p<0.05) when pigs were offered 0.125% betaine.

A nuclear-magnetic-resonance-based assay for betaine–homocysteine methyltransferase activity

Betaine–homocysteine methyltransferase (BHMT) activity can be measured directly and kinetically by 1H-nuclear magnetic resonance spectroscopy. The disappearance of substrates and the formation of products are monitored simultaneously. Alternative substrates, separately and when mixed with glycine betaine, can also be monitored. Each assay can be completed in 1 h. Using 2 mM glycine betaine and homocysteine as substrates in 20 mM phosphate buffer (pH 7.5) and measuring the production of N, N-dimethylglycine, the CV is 6.3% ( n=6) and the detection limit is 6 nkatal. An endpoint assay for BHMT activity was also developed, by measuring the N, N-dimethylglycine produced after incubation with 2 mM glycine betaine and homocysteine (CV=5.3%, n=6) with a detection limit of 2 nkatal. These assays were used to show that the natural betaines trigonelline, proline betaine, arsenobetaine, and l-carnitine are neither substrates nor significant inhibitors of rat liver BHMT, that the thetins dimethylthetin and dimethylsulfoniopropionate are substrates and inhibit methyl transfer from glycine betaine, and that the K m for glycine betaine is 0.19 ± 0.03 mM with a V max of 17 ± 0.7 nMol min −1 mg −1.

Effects of betaine in a murine model of mild cystathionine-β-synthase deficiency

Cystathionine-β-synthase (CBS) is required for transsulfuration of homocysteine, an amino acid implicated in vascular disease. We studied homocysteine metabolism in mice with mild hyperhomocysteinemia due to a heterozygous disruption of the Cbs gene. Mice were fed diets supplemented with betaine or dimethylsulfonioacetate (DMSA); betaine and DMSA provide methyl groups for an alternate pathway of homocysteine metabolism, remethylation by betaine:homocysteine methyltransferase (BHMT). On control diets, heterozygous mice had 50% higher plasma homocysteine than did wild-type mice. Betaine and DMSA had similar effects in both genotype groups: liver betaine increased dramatically, while plasma homocysteine decreased by 40% to 50%. With increasing betaine supplementation, homocysteine decreased by 75%. Plasma homocysteine and BHMT activity both showed a strong negative correlation with liver betaine. Homocysteinemia in mice is sensitive to a disruption of Cbs and to methyl donor intake. Because betaine leads to a greater flux through BHMT and lowers homocysteine, betaine supplementation may be beneficial in mild hyperhomocysteinemia.

Betaine homocysteine methyltransferase: gene cloning and expression analysis in rat liver cirrhosis

It has been known for over half a century that homocysteine levels are elevated in liver cirrhosis, but the basis for it is not fully understood. Using differential display, we identified betaine homocysteine methyltransferase (BHMT) as a gene down-regulated in rat liver cirrhosis and most likely involved in this dysregulation. A partial BHMT clone was isolated by screening of a cDNA library with the differential display fragment. The full-length gene was generated by primer extension of cDNA. Expression levels of BHMT in cirrhotic livers of bile duct ligated rats were compared to controls by Northern and Western blotting as well as by enzyme activity measurements. BHMT mRNA levels were reduced to 29±23% in established liver cirrhosis induced by bile duct ligation (BDL) as compared to controls. Enzyme assays in crude liver homogenates showed a similar reduction in BHMT activity in bile duct ligated rat livers. By Western blotting, BHMT could be detected in crude liver homogenates of control animals, but was reduced to below the limit of detection in cirrhotic livers. In conclusion, these findings establish a reduced BHMT enzyme activity in cirrhotic rat livers, which may explain the elevated plasma homocysteine levels in cirrhosis.

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.

Dimethylglycine accumulates in uremia and predicts elevated plasma homocysteine concentrations

Hyperhomocysteinemia is a risk factor for atherosclerosis that is common in chronic renal failure (CRF), but its cause is unknown. Homocysteine metabolism is linked to betaine-homocysteine methyl transferase (BHMT), a zinc metalloenzyme that converts glycine betaine (GB) to N,N dimethylglycine (DMG). DMG is a known feedback inhibitor of BHMT. We postulated that DMG might accumulate in CRF and contribute to hyperhomocysteinemia by inhibiting BHMT activity. Plasma and urine concentrations of GB and DMG were measured in 33 dialysis patients (15 continuous ambulatory peritoneal dialysis and 18 hemodialysis), 33 patients with CRF, and 33 age-matched controls. Concentrations of fasting plasma total homocysteine (tHcy), red cell and serum folate, vitamins B(6) and B(12), serum zinc, and routine biochemistry were also measured. Groups were compared, and determinants of plasma tHcy were identified by correlations and stepwise linear regression. Plasma DMG increased as renal function declined and was twofold to threefold elevated in dialysis patients. Plasma GB did not differ between groups. The fractional excretion of GB (FE(GB)) was increased tenfold, and FED(MG) was doubled in CRF patients compared with controls. Plasma tHcy correlated positively with plasma DMG, the plasma DMG:GB ratio, plasma creatinine, and FE(GB) and negatively with serum folate, zinc, and plasma GB. In the multiple regression model, only plasma creatinine, plasma DMG, or the DMG:GB ratio was independent predictors of tHcy. DMG accumulates in CRF and independently predicts plasma tHcy concentrations. These findings suggest that reduced BHMT activity is important in the pathogenesis of hyperhomocysteinemia in CRF.

Degradation of glycinebetaine by betaine-homocysteine methyltransferase in Aphanothece halophytica: effect of salt downshock and starvation.

We have investigated conditions leading to the degradation of glycinebetaine in Aphanothece halophytica and have shown the activity of betaine-homocysteine methyltransferase (BHMT). The intracellular glycinebetaine level was decreased approximately 50% after 36 h salt downshock from 2.0 m NaCl medium to 0.5 m NaCl medium. A slight additional decrease of glycinebetaine occurred when salt downshock was combined with dark treatment. The omission of carbon and nitrogen sources in the growth medium further decreased intracellular glycinebetaine. The activity of BHMT increased from 0 to 460 nmol h(-1)mg(-1) after 3 h salt downshock. Higher strength of salt downshock resulted in higher activity of the enzyme. Small increase of the enzyme activity was also observed when A. halophytica was deprived of carbon and nitrogen sources in the growth medium.

Interaction between Dietary Methionine and Methyl Donor Intake on Rat Liver Betaine-homocysteine Methyltransferase Gene Expression and Organization of the Human Gene

We previously showed that rat liver betaine-homocysteine methyltransferase (BHMT) mRNA content and activity increased 4-fold when rats were fed a methionine-deficient diet containing adequate choline, compared with rats fed the same diet with control levels of methionine (Park, E. I., Renduchintala, M. S., and Garrow, T. A. (1997) J. Nutr. Biochem. 8, 541-545). A further 2-fold increase was observed in rats fed the methionine-deficient diet with supplemental betaine. The nutrition studies reported here were designed to determine whether other methyl donors would induce rat liver BHMT gene expression when added to a methionine-deficient diet and to define the relationship between the degree of methionine restriction and level of methyl donor intake on BHMT expression. Therefore, rats were fed amino acid-defined diets varying in methionine and methyl donor composition. The effect of diet on BHMT expression was evaluated using Northern, Western, and enzyme activity analyses. Similar to when betaine was added to a methionine-deficient diet, choline or sulfonium analogs of betaine induced BHMT expression. The diet-induced induction of hepatic BHMT activity was mediated by increases in the steady-state level of its mRNA and immunodetectable protein. Using methyl donor-free diets, we found that methionine restriction was required but alone not sufficient for the high induction of BHMT expression. Concomitant with methionine restriction, dietary methyl groups were required for high levels of BHMT induction, and a dose-dependent relationship was observed between methyl donor intake and BHMT induction. Furthermore, the severity of methionine restriction influenced the magnitude of BHMT induction. To study the molecular mechanisms that regulate the expression of BHMT, we have cloned the human BHMT gene. This gene spans about 20 kilobases of DNA and contains 8 exons and 7 introns. Using RNA isolated from human liver and hepatoma cells, a major transcriptional start site has been mapped using the 5' rapid amplification of cDNA ends technique, and this start site is 26 nucleotides downstream from a putative TATA box.

An assay for betaine-homocysteine methyltransferase activity based on the microbiological detection of methionine

Betaine-homocysteine methyltransferase (BHMT: EC 2.1.1.5) catalyzes a methyl transfer from betaine to homocysteine (Hcy) forming dimethylglycine and methionine (Met), respectively. We have developed an assay for BHMT activity based on the microbiological detection of one of its products, Met. The microbiological assay uses Escherichia coli J5-3, a Met auxotroph. When cultured in minimal media, J5-3 growth increases linearly with Met supplementation up to 50 μM (2.24 μg/mL). Although not sensitive enough for kinetic analyses, this assay is adequate for measuring activity levels in crude or purified enzyme preparations using saturating substrate concentrations. When BHMT activity was measured in crude liver extracts, using both the radioactive and microbiological assays, the values obtained show a high degree of correlation ( r 2 > 0.93) over an eight-fold range of activities. We have used the microbiological assay to identify stachydrine as a naturally occurring methyl donor substrate for BHMT. The sensitivity of detecting BHMT activity by the microbiological assay for Met can be increased at least an order of magnitude by using dimethylacetothetin, a sulfonium analog of betaine, as the methyl donor substrate.

Hepatic and renal betaine-homocysteine methyltransferase activity in pigs as affected by dietary intakes of sulfur amino acids, choline, and betaine.

In Exp. 1, young pigs were fed a basal diet containing .17% methionine (Met) (.14% digestible Met), and .48% cystine (.38% digestible cystine) for 14 d (34 to 48 d of age). Treatment additions were .25% DL-Met, .34% betaine, .30% choline, or .25% DL-Met and .34% betaine. Methionine, but not betaine or choline supplementation, increased (P < .05) weight gain and feed efficiency. Hepatic betaine-homocysteine methyltransferase (BHMT) activity was increased (P < .05) by betaine and choline supplementation but was not affected by Met deficiency. Renal BHMT activity was increased (P < .05) by Met deficiency and was further increased (P < .05) by betaine supplementation. In Exp. 2, 10-kg pigs were fed the basal diet from Exp. 1 supplemented with enough DL-Met to bring the total basal Met to .24% (.20% digestible Met). Treatment additions consisted of .20% DL-Met or .34% betaine, and diets were fed for 16 d (34 to 50 d of age). Feed efficiency increased (P < .05) in response to Met, but not to betaine, supplementation. Hepatic BHMT activity increased (P < .05) in response to betaine and Met, but no changes in renal BHMT activity occurred. Although statistically significant changes in hepatic and renal BHMT activity occurred in both experiments, the magnitude of the responses was probably not physiologically important. Therefore, in contrast to previous findings with rats and chicks, it does not seem that hepatic and renal BHMT activity in pigs is influenced substantially by Met deficiency, or by surfeit levels of choline or betaine.

Diet-Induced Changes in Hepatic Betaine-Homocysteine Methyltransferase Activity Are Mediated By Changes in the Steady-State Level of Its mRNA

Liver betaine-homocysteine methyltransferase (EC 2.1.1.5) activity fluctuates with changes in the dietary intake of sulfur amino acids, choline, and betaine. The purpose of this study was to determine whether dietary-induced changes in the activity of hepatic betaine-homocysteine methyltransferase are mediated by changes in the level of its mRNA. The hepatic activity and mRNA content of betaine-homocysteine methyltransferase were measured in rats fed one of five amino acid–defined diets: basal (1 g/kg methionine, 3 g/kg cystine, 1.25 g/kg choline bitartrate), control (basal plus 2 g/kg methionine), betaine supplemented (basal plus 3 g/kg betaine), cystine supplemented (basal plus 3 g/kg cystine), or betaine and cystine supplemented (basal plus 3 g/kg betaine plus 3 g/kg cystine). The basal diet was deficient solely in methionine, and the control diet was adequate in all nutrients. When compared with rats consuming the control diet, rats fed the methionine-deficient diet exhibited a 4-fold increase in the steady-state level of betaine-homocysteine methyltransferase mRNA ( p < 0.05). Betaine addition to the methionine-deficient diet elevated mRNA level even further, resulting in a nearly 10-fold higher mRNA levels compared with the methionine-adequate control diet ( p < 0.05). Dietary cystine had no effect on betaine-homocysteine methyltransferase mRNA levels. Liver betaine-homocysteine methyltransferase activity mirrored its mRNA levels. We conclude that dietary-induced changes of liver betaine-homocysteine methyltransferase activity are mediated by changes in the steady-state levels of its mRNA.

Betaine effects on hepatic methionine metabolism elicited by short-term ethanol feeding

Previous studies in this laboratory have shown that feeding of ethanol to rats produces prompt inhibition of methionine synthetase (MS) as well as a subsequent increase in activity of betaine homocysteine methyltransferase (BHMT). Further studies have shown that supplemental dietary betaine enhanced methionine metabolism and S-adenosylmethionine (SAM) generation in control and ethanol-fed rats. Because MS and BHMT are both involved in the formation of SAM, this study was conducted to determine early effects of ethanol on hepatic SAM levels and the influence of betaine supplementation on parameters of methionine metabolism during the early periods of MS inhibition and enhanced BHMT activity. Results showed that ethanol feeding produced a significant loss in SAM in the first week with a return to normal SAM levels in the second week. Betaine feeding enhanced hepatic betaine pools in control as well as ethanol-fed animals. This feeding attenuated the early loss of SAM in ethanol-fed animals, produced an early increase in BHMT activity, and generated increased levels of SAM in both control and ethanol-fed groups. Furthermore, betaine lowered significantly the accumulation of hepatic triglyceride produced by ethanol after 2 weeks of ingestion.

Homocysteine catabolism: levels of 3 enzymes in cultured human vascular endothelium and their relevance to vascular disease

Elevated plasma homocysteine enhances the risk of thrombosis and premature arteriosclerosis. We have assessed the activity of the 3 prime enzymes of homocysteine metabolism in cultured human venous endothelial cells, in a study of their possible protective roles. In cells from 4 individuals, cultured in Dulbecco's modified Eagle medium, the mean activity ± S.D. of cystathionine β-synthase (nmol of product/h per mg of cell protein, at 37°C) was 3.58 ± 3.11 at pH 8.6. The assay used was our newly developed amino acid analyser-based procedure. The activity of 5-methyltetrahydrofolate:homocysteine methyltransferase at pH 7.4 was 4.12 ± 1.25 and betaine:homocysteine methyltransferase (BHMT) was undetectable (< 1.4 nmol/h per mg protein). Cells were also cultured in a medium aimed at stimulating methionine biosynthesis, containing methionine-deficient Dulbecco's modified Eagle medium to which L-homocystine (100 μmol/1) and methylcobalamin (1 μmol/1) had been added. In these cells 5-methyltetrahydrofolate:homocysteine methyltransferase activity increased to 7.95 ± 1.45, P < 0.001, there was a non-significant decrease in cystathionine β-synthase activity to 2.16 ± 1.52 and BHMT activity was still undetectable. These cells were more resistant to in vitro homocysteine-induced detachment than were cells from the same line cultured in Dulbecco's modified Eagle medium alone. Our findings establish that human endothelial cells express 2 of the 3 primary enzymes of homocysteine catabolism. They suggest that persons who are deficient in cystathionine β-synthase or 5-methyltetrahydrofolate:homocysteine methyltransferase activity may not only develop homocysteinemia, but also have vascular endothelium which is more susceptible to damage by homocysteine than persons with normal enzyme levels.

Effect of vitamin B12-deficiency on the activity of hepatic cystathionine beta-synthase in rats.

The effect of vitamin B12(B12)-deficiency on the activities of hepatic methionine synthase, homocysteine methyltransferase, and cystathionine beta-synthase was investigated in rats. The rats bred from B12-deficient dams were fed the B12-deficient diets for 150 days after weaning. Growth retardation of the B12-deficient rats was already observed on day 30 and continued through 150 days. But dietary supplementation of 0.5% DL-methionine slightly improved the growth retardation. Urinary excretion of methylmalonic acid increased to about 15 mg/mg creatinine and hepatic B12 concentration declined to about 2 ng/g liver after a 150-day feeding of the B12-deficient diets. Hepatic methionine synthase activity in rats fed the B12-deficient diets supplemented with or without methionine decreased to about 5% of B12-supplemented controls. Hepatic betaine-homocysteine methyltransferase activity showed no significant change caused by B12-deficiency. Hepatic cystathionine beta-synthase activity in rats fed the B12-deficient diets supplemented with or without methionine decreased to about 61% and 27% of their B12-supplemented controls, respectively, but the decrease was partially improved by methionine supplementation. In conclusion, the rats bred from B12-deficient dams showed a severe B12-deficiency after a 150-day feeding of the B12-deficient diets. The decrease of hepatic cystathionine beta-synthase activity was supposed to be due to the adaptation by the defect of methionine resynthesis.

Betaine metabolism in human neonates and developing rats

Proton nuclear magnetic resonance spectroscopy has been used to demonstrate the presence of high concentrations of betaine (up to 0.75 mol/mol creatinine) in the urine of normal healthy human neonates. Betaine is not normally excreted in adults. Excretion of betaine from birth to 7 days old was monitored. The excretion of betaine in rats from 21 days after birth to 40–45 days old was also monitored. A peak in excretion in the rats of 1.5-3 mol/mol creatinine occurred between days 30–35. The presence of a high concentration of betaine in the urine is unlikely to be caused by a relative lack of betaine homocysteine methyl transferase activity compared with adults but may relate to the disposal of dietary choline during development.