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Methionine synthase (MS) and betaine-homocysteine methyltransferase (BHMT) are both zinc (Zn)-dependent methyltransferases and involved in the methylation of homocysteine. The objective of this study was to investigate the effects of dietary Zn supply on homocysteine levels and expression of the two enzymes in growing rats. Male weanling Sprague-Dawley rats were assigned randomly to four dietary groups (n=8/group) for 3 weeks: Zn deficient (ZD; <1mg Zn/kg); Zn control (ZC; 30mg Zn/kg); Zn supplemented (ZS; 300mg Zn/kg); pair fed (PF; 30mg Zn/kg) to the ZD group. Serum and femur Zn concentrations were 83% and 58% lower in ZD, and 49% and 62% higher in ZS compared to ZC (P<0.001), respectively. The ZD rats had lower feed intake (37%), body weight gains (45%), liver (43%) and kidney (31%) weights than those of ZC (P<0.001), but these parameters in ZD were not significantly different from the PF controls. Serum homocysteine concentrations were 65% higher in ZD compared to PF (P<0.05), and there was no significant difference in serum folate levels between ZD and PF groups. The mRNA expression of liver and kidney MS was 57% and 38% lower in ZD than PF (P<0.001), respectively. Hepatic and renal BHMT mRNA levels were not altered in ZD compared to controls. The aforementioned measurements were not significantly different between ZS and ZC groups, except Zn levels. These results demonstrated that homocysteine homeostasis appeared to be disturbed by Zn deficiency but not Zn supplementation, and elevated serum homocysteine might be due to reduced expression of MS during Zn deficiency., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Pyridoxal 5'-phosphate (PLP; a B₆ vitamer) serves as an important cofactor in a myriad of metabolic reactions, including the transsulfuration (TS) pathway, which converts homocysteine (Hcy) to cysteine. While overt vitamin B₆ deficiency is rare, moderate deficiency is common and may be exacerbated by anti-pyridoxine factors in the food supply. To this end, we developed a model of moderate B₆ deficiency and a study was conducted to examine the in vivo effect of 1-amino D-proline (1ADP), an anti-pyridoxine factor found in flaxseed, on indices of Hcy metabolism through the TS pathway in moderately B₆ deficient rats. Male weaning rats received a semi-purified diet containing either 7 mg/kg (control; CD) or 0.7 mg/kg (moderately deficient; MD) diet of pyridoxine·hydrochloride (PN∙HCl), each with 1 of 4 levels of 1ADP, viz. 0, 0.1, 1 and 10 mg/kg diet for 5 weeks. Perturbations in vitamin B₆ biomarkers were more pronounced in the MD group. Plasma PLP was significantly reduced, while plasma Hcy (8-fold) and cystathionine (11-fold) were increased in rats consuming the highest amount of 1ADP in the MD group. The activities of hepatic cystathionine β-synthase and cystathionine γ-lyase enzymes were significantly reduced in rats consuming the highest 1ADP compared to the lowest, for both levels of PN∙HCl. Dilation of hepatic central veins and sinusoids, mild steatosis and increased liver triglycerides were present in MD rats consuming the highest 1ADP level. The current data provide evidence that the consumption of an anti-pyridoxine factor linked to flaxseed may pose a risk for subjects who are moderate/severe vitamin B₆ deficient., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The sulfur amino acids, methionine and cysteine play crucial roles in cells as a substrate for protein synthesis, as a methyl donor, and for the synthesis of sulfur-containing compounds, including the key intracellular tripeptide, glutathione. Homocysteine is an intermediary metabolite formed during the metabolism of methionine to cysteine. Dysregulation of homocysteine metabolism is implicated in adverse clinical outcomes such as increased risk of cardiovascular disease, stroke, Alzheimer's disease dementia and osteoporosis. While hyperhomocysteinemia is commonly observed in those conditions, the impact on other related metabolites is condition-specific. Therefore, there exists a need to establish precise and sensitive analytical techniques that allow for the simultaneous measurement of homocysteine and related metabolites in biological samples. The current review outlines the development and use of liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) to simultaneously measure metabolites involved in sulfur amino acid metabolism. Additionally, extensions of the technique in relation to the measurement of sulfur amino acid and one-carbon kinetics in vivo are discussed. The LC-MS/MS technique has the capacity for unambiguous analyte identification and confirmation, due to its high specificity and sensitivity. It has the greatest potential of being accepted and utilized as a dedicated homocysteine and its related metabolite Standard reference method (SRM).

Total homocysteine (tHcy) and cysteine (tCys) concentrations in biological fluids are routinely used in the clinical diagnosis of genetic and metabolic diseases, and this necessitates the development of rapid and sensitive methods for quantification. Liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) was used to measure tHcy and tCys in 23 plasma and 21 urine samples from healthy adults and 14 urine samples from healthy children. The results were compared with a standard high-performance liquid chromatography (HPLC) method. The coefficient of variation (CV) for the LC-MS/MS method ranged from 2.9% to 6.1% for the intraassay and 4.8% to 6.4% for the interassay. Mean recoveries were close to 100% for both plasma and urinary tHcy and tCys. The mean plasma tHcy and tCys concentrations in healthy adults were 8.62 and 261.40 micromol/L, respectively. The mean urinary tHcy and tCys in adults were 0.98 and 22.60 micromol/mmol creatinine, respectively. The mean urinary tHcy and tCys in children were 1.17 and 27.43 micromol/mmol creatinine, respectively. Bland-Altman difference plots of method comparison between LC-MS/MS and HPLC showed good agreement in plasma and urinary tHcy and tCys concentrations. Our method is suitable for rapid measurements, and the reported urinary values in children will help to develop a pediatric reference range for clinical use.

Plasma total homocysteine (tHcy) concentrations are associated with atherogenesis in adults and increased risk of stroke in infants and children. After a series of experiments to compare the methionine (Met) requirement and cysteine (Cys)-sparing capacity in piglets that were parenterally or enterally fed, we examined the effects of route of feeding and dietary Cys on plasma tHcy concentrations. Piglets (n = 60; 6-8 d old) were fed elemental diets, intragastrically (n = 28) or intravenously (n = 32), with 0.55 g. kg(-1). d(-1) dietary Cys (n = 28) or without dietary Cys (n = 32). Dietary Met ranged from deficient to excess. Increasing Met intake increased (P < 0.01) plasma tHcy in all treatment groups. Plasma tHcy concentrations were higher (P < 0.05) in the enterally fed piglets that did not receive dietary Cys than in all other groups, which did not differ from each other. Therefore, both route of feeding and dietary supply of Met and Cys significantly affected the concentrations of plasma tHcy. These dramatic and rapid alterations in plasma tHcy warrant further studies of sulfur amino acid metabolism in neonatal animals.

The objectives of the current studies included the characterization of the temporal changes in indices of folate status and amino acid concentrations during both folate depletion and repletion phases. In trial 1, a 6 week folate depletion protocol was employed, using 60 weanling rats assigned to receive an amino acid-defined diet with or without 1 mg/kg folic acid. A 4 week folate depletion period was judged to be optimal on the basis of the development of nadirs in both plasma and hepatic folate stores and elevated (>6-fold relative to folate-adequate controls) concentrations of plasma homocysteine and glycine. In trial 2, 54 weanling rats, previously maintained on a folate-devoid diet for 4 weeks, were assigned to receive 0.25 mg/kg folate as either crystalline folic acid or folate from a folate-enriched egg yolk powder. Both forms of folate supported similar rates of gain, increases in plasma and hepatic folate stores, and reductions in plasma glycine concentrations, whereas the folate in egg yolk powder lowered plasma homocysteine concentrations further than the crystalline folic acid (P < 0.05). These data support the use of both plasma glycine and homocysteine as sensitive response criteria for folate status in a rat bioassay of folate depletion and repletion and establish appropriate temporal end-points for such studies.

We have used a combination of in vivo and in vitro techniques to measure factors regulating homocysteine metabolism and the plasma concentration of this atherogenic amino acid. The germane findings include: 1. Homocysteine metabolism in rat kidney proceeds predominantly through the transsulfuration pathway, whose enzymes are enriched within the proximal cells of kidney tubules. Furthermore, the rat kidney possesses significant reserve capacity to handle both acute and chronic elevations in plasma homocysteine concentrations. 2. Plasma homocysteine concentrations are lower in diabetic rats. Insulin administration corrects this perturbation. Therefore, insulin and/or one of its counter-regulatory hormones affects homocysteine metabolism, possibly through an increased flux in the hepatic transsulfuration pathway. In support of these data, glucagon administration to rats produced similar results. Further support was provided by studies with isolated rat hepatocytes, from which homocysteine export was reduced when incubated in the presence of glucagon.

An elevation in the concentration of total plasma homocysteine is known to be an independent risk factor for the development of vascular disease. Alterations in homocysteine metabolism have also been observed clinically in diabetic patients. Patients with either type 1 or type 2 diabetes who have signs of renal dysfunction tend to exhibit elevated total plasma homocysteine levels, whereas type 1 diabetic patients who have no clinical signs of renal dysfunction have lower than normal plasma homocysteine levels. The purpose of this study was to investigate homocysteine metabolism in a type 1 diabetic animal model and to examine whether insulin plays a role in its regulation. Diabetes was induced by intravenous administration of 100 mg/kg streptozotocin to Sprague-Dawley rats. We observed a 30% reduction in plasma homocysteine in the untreated diabetic rat. This decrease in homocysteine was prevented when diabetic rats received insulin. Transsulfuration and remethylation enzymes were measured in both the liver and the kidney. We observed an increase in the activities of the hepatic transsulfuration enzymes (cystathionine beta-synthase and cystathionine gamma-lyase) in the untreated diabetic rat. Insulin treatment normalized the activities of these enzymes. The renal activities of these enzymes were unchanged. These results suggest that insulin is involved in the regulation of plasma homocysteine concentrations by affecting the hepatic transsulfuration pathway, which is involved in the catabolism of homocysteine.

Background: Elevated plasma total homocysteine, an independent risk factor for cardiovascular disease, is commonly observed in renal patients. We have previously shown that the kidney is a major site for the removal of plasma homocysteine in the rat. The present investigation was performed to further characterize the capacity of the kidney to handle acute elevations in plasma homocysteine concentrations., Methods: Acute hyperhomocysteinemic conditions (4- to 7-fold > controls) in rats were produced by either a primed-continuous infusion of L-homocysteine or exposure to 80:20% nitrous oxide:oxygen, which results in the inhibition of methionine synthase., Results: At physiological homocysteine concentrations, approximately 15% of the arterial plasma homocysteine was removed on passage through the kidney. Renal homocysteine uptake was approximately 85% of the filtered load. The urinary excretion of homocysteine was negligible (<2%). During acute hyperhomocysteinemia produced by the infusion of L-homocysteine, renal homocysteine uptake was increased fourfold and was equivalent to 50% of the infused dose, while urinary excretion remained negligible. Renal homocysteine uptake during nitrous oxide-induced hyperhomocysteinemia increased threefold, with urinary excretion remaining negligible., Conclusions: These results provide strong evidence that the kidney has a significant capacity for metabolizing acute elevations in plasma homocysteine, and support a very limited role for the re-methylation pathway in renal homocysteine metabolism.

Epidemiological studies have provided strong evidence that an elevated plasma homocysteine concentration is an important independent risk factor for cardiovascular disease. We have shown, in the rat, that the kidney is a major site for the removal and subsequent metabolism of plasma homocysteine [Bostom, Brosnan, Hall, Nadeau and Selhub (1995) Atherosclerosis 116, 59-62]. To characterize the role of the kidney in homocysteine metabolism further, we measured the disappearance of homocysteine in isolated renal cortical tubules of the rat. Renal tubules metabolized homocysteine primarily through the transulphuration pathway, producing cystathionine and cysteine (78% of homocysteine disappearance). Methionine production accounted for less than 2% of the disappearance of homocysteine. Cystathionine, and subsequently cysteine, production rates, as well as the rate of disappearance of homocysteine, were sensitive to the level of serine in the incubation medium, as increased serine concentrations permitted higher rates of cystathionine and cysteine production. On the basis of enrichment profiles of cystathionine beta-synthase and cystathionine gamma-lyase, in comparison with marker enzymes of known location, we concluded that cystathionine beta-synthase was enriched in the outer cortex, specifically in cells of the proximal convoluted tubule. Cystathionine gamma-lyase exhibited higher enrichment patterns in the inner cortex and outer medulla, with strong evidence of an enrichment in cells of the proximal straight tubule. These studies indicate that factors that influence the transulphuration of homocysteine may influence the renal clearance of this amino acid.