Supplementary Figure Legends 1-3 from Mice Heterozygous for Germ-line Mutations in Methylthioadenosine Phosphorylase (MTAP) Die Prematurely of T-Cell Lymphoma

Supplementary Figures 1-3, Table 1 from Mice Heterozygous for Germ-line Mutations in Methylthioadenosine Phosphorylase (MTAP) Die Prematurely of T-Cell Lymphoma

DNA modifications are critical in fine-tuning the biological processes in model organisms. However, the presence of cytosine methylation (5mC) and the function of the putative DNA methyltransferase, PfDNMT2, in the human malaria pathogen, Plasmodium falciparum, remain controversial. Here, we revisited the 5mC in the parasite genome and the function of PfDNMT2. Low levels of genomic 5mC (0.1-0.2%) during asexual development were identified using a sensitive mass spectrometry procedure. Native PfDNMT2 displayed substantial DNA methylation activities, and disruption or overexpression of PfDNMT2 resulted in reduced or elevated genomic 5mC levels, respectively. PfDNMT2 disruption led to an increased proliferation phenotype, with the parasites having an extended schizont stage and producing a higher number of progenies. Consistent with PfDNMT2's interaction with an AP2 domain-containing transcription factor, transcriptomic analyses revealed that PfDNMT2 disruption led to a drastic alteration in the expression of many genes, some of which provided the molecular basis of enhanced proliferation after PfDNMT2 disruption. Furthermore, levels of tRNAAsp and its methylation rate at position C38, and the translation of a reporter containing an aspartate repeat were significantly reduced after PfDNMT2 disruption, while the levels of tRNAAsp and its C38 methylation were restored after complementation of PfDNMT2. Our study sheds new light on the dual function of PfDNMT2 during P. falciparum asexual development.

Background: In young adults blood pressure, hypertension rates, and CVD risk are lower in women than similarly aged men. However, soon after menopause hypertension and CVD rates converge with, and overtake those of men. Thus, by their mid-50s, most women are hypertensive, and CVD becomes the leading cause of death. While this age-related increase in blood pressure and hypertension is largely attributed to menopause, and the concomitant decrease in estradiol concentrations, there is little empirical evidence to support this hypothesis.Methods: Data from the National Health and Nutrition Examination Survey (NHANES) and Coronary Artery Risk Development in Young Adults (CARDIA) study was analyzed by segmented regression analysis to determine the relationship between systolic blood pressure and age. Data from NHANES was further stratified by Body Mass Index (BMI) and antihypertensive drug use to determine their impact on blood pressure. Estradiol concentrations (NHANES 2013-2016) were graphed against age to determine the start of menopause.Findings: Segmented regression analysis determined that the age-related increase in systolic blood pressure and systolic hypertension began when the women were in their early-30s, a decade before estrogen concentrations decline. BMI and antihypertensive drug use modified the response, but not the overall trend.Interpretation: Contrary to the commonly cited hypothesis, the age-related increase in hypertension is not related to menopause, nor decreased estradiol concentrations. Rather, the rise in systolic blood pressure and hypertension begins over a decade earlier, in a woman’s early-30s. These finding suggest that monitoring, and perhaps prophylactic treatment, of hypertension should begin at least a decade before menopause.Funding Statement: No funding was received for this study, nor for preparing and submitting the manuscript.Declaration of Interests: The Authors states that there is no conflict of interest.Ethics Approval Statement: Ethical approval for the study was provided by the University of Florida Institutional Review Board.

Objective A recent publication questioned the integrity of insulin purchased from U.S. retail pharmacies. We sought to independently validate the method employed, isotope dilution solid phase extraction (SPE) liquid chromatography (LC) mass spectrometry (MS) and expand analysis to two U.S. Pharmacopiea (USP) methods (high performance LC with ultra-violet detection (LC-UV) and LC-MS). Research Design and Methods Each method was used to evaluate nine insulin formulations, purchased at four pharmacies, within five geographic locations in the U.S. Results All human and analogue insulins measured by the USP methods (n=174) contained the expected quantity of active insulin (100±5 U/mL). When using isotope dilution SPE-LC-MS, U/mL values were well below product labeling due to unequal recovery of the internal standard compared to target insulin. Conclusions Insulin purchased from U.S. pharmacies is consistent with product labeling.

Introduction The systemic deleterious effects of End Stage Renal Disease (ESRD) are numerous and well established, however, advances in our understanding of the fundamental biology within the local small‐molecule environment has been limited. This study utilizes high‐throughput techniques to quantify global and targeted metabolomics of ESRD in comparison to healthy counterparts. We hypothesize that ESRD patients exhibit critical deviations in their metabolome, particularly within those elements surrounding nitric oxide (NO) production. These changes may provide a mechanistic explanation of previously established functional and structural vascular abnormalities associated with ESRD. Methods Following Institutional Review Board approval, human plasma samples were obtained from 5 patients with normal renal function and 5 patients with ESRD currently on hemodialysis. Global metabolomic analysis was performed by LC‐MS, and metabolites identified by their retention time and exact mass analysis using an in‐house library. Targeted metabolite analysis was performed by stable isotope dilution LC‐MS/MS on 31 NO‐associated metabolites of specific interest. Descriptive statistical analysis was performed to compare the respective metabolomes of ESRD to those with normal renal function. The discussion and inference of downstream effects was aided with the use of BioCyc™ pathway analysis tools. Results Targeted Metabolomics Patients with ESRD in comparison to control patients had significant differences p‐value <0.05 in 6 of the targeted 31 (19%) metabolites. Of those metabolites 5 of 6 (83%) were increased significantly in the ESRD population, with 1 of 6 (17%) significantly decreased (FIGURE). Global Metabolomics 3788 metabolite signatures were detected, of which 1099 (29%) were mapped to specific compounds. 523 of the identified 1099 metabolites (47.6%) were found to be different (p<0.05) in the ESRD population, with 212 (41%) significantly increased vs 311 (59%) significantly decreased. Discussion With the loss of glomerular filtration and replacement with 3×/week hemodialysis, we see significant alterations within the small‐molecule environment of those with ESRD. Targeted metabolomics provide insight into how these differences alter key regulators of vascular and cardiovascular homeostasis. Our data would suggest patients with ESRD have decreased production of NO. This is supported by our findings of ESRD patients exhibiting significant increases in both competitive NO inhibitors (SDMA, Methyl Arginine, and ADMA) and end products of alternative NO‐related pathways (Citrulline, and Ornithine). Expanding our understanding of the small‐molecule environment and its effects will provide important insights into the etiology of altered vascular pathophysiology observed in the ESRD population and offer potential novel targets of intervention to re‐establish normal homeostasis within the cardiovascular system. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Objectives: Assess the prevalence of hypogonadism in older male Veterans by comparing direct measurements of total testosterone (T) and bioavailable testosterone (BioT) versus indirect BioT values derived from existing and newly developed regression analyses.Design: Cohort study.Setting: Malcom Randall VA Medical Center, Gainesville, FL.Participants: Community-dwelling male Veterans aged 60 and older (n = 203).Measurements: Total T, BioT, albumin, sex hormone-binding globulin (SHBG), and body mass index were evaluated. Blood values were assessed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and clinical or commercially available immunoassays to compare accuracy among assessment techniques. Existing and newly developed multiple regression analyses were evaluated to assess accuracy in predicting BioT.Results: Total T was 13.80 +/- 6.25 nmol/L (398 +/- 180 ng/dL) and was low (<= 10.4 nmol/L or <= 300 ng/dL) in 34% of participants. SHBG was 58 +/- 35 nmol/L and elevated (>= 62 nmol/L) in 36% of participants. BioT was 1.94 +/- 0.97 nmol/L (56 +/- 28 ng/dL), with 72% of participants below the clinical cutoff (<= 2.43 nmol/L or <= 70 ng/dL). Albumin was within the normal clinical range. Total T and BioT measured via immunoassay and LC-MS/MS were moderately to highly correlated, with no differences between assessment methods. Several existing predictive equations overestimated BioT by 74% to 166% within our cohort (P < .001). A newly developed regression model that included total T, SHBG, albumin, and age more accurately predicted BioT, with values correlated (r = 0.508, P < .001) and comparable to LC-MS/MS.Conclusion: In our cohort, the prevalence of low total T was higher and low BioT was markedly higher than reported in the general age-matched population, indicating a greater incidence of hypogonadism in older male Veterans. In addition, existing empiric formulae, derived from other populations produced BioT values that were considerably greater than those directly measured, whereas our newly developed regression analysis provides improved predictive capabilities for older male Veterans. Published by Elsevier Inc. on behalf of AMDA - The Society for Post-Acute and Long-Term Care Medicine.

BACKGROUND The three branched amino acids (valine, leucine, and isoleucine) and two aromatic amino acids (tyrosine and phenylalanine) have been associated with many adverse metabolic pathways, including diabetes. However, these associations have been identified primarily in otherwise healthy Caucasian populations. We aimed to investigate the association of this five-amino-acid signature with metabolic syndrome and impaired fasting glucose (IFG) in a hypertensive cohort of Caucasian and African Americans. METHODS We analyzed data from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) studies PEAR and PEAR2 conducted between 2005 and 2014. Subjects were enrolled at the University of Florida (Gainesville, FL), Emory University (Atlanta, GA), and Mayo Clinic (Rochester, MN). A total of 898 patients with essential hypertension were included in this study. Presence of metabolic syndrome and IFG at baseline were determined on the basis of measurements of demographic and biochemical data. Levels of the five amino acids were quantified by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). RESULTS With a multiple logistic regression model, we found that all five amino acids were significantly associated with metabolic syndrome in both Caucasian and African Americans. IFG and the five amino acids were associated in the Caucasian Americans. Only valine was significantly associated with IFG in African Americans. CONCLUSION In both Caucasian and African Americans with uncomplicated hypertension, plasma levels of the five-amino-acid signature are associated with metabolic syndrome. Additionally, in Caucasians we have confirmed the five-amino-acid signature was associated with IFG.

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.

Low vitamin B-6 nutritional status is associated with increased risk for cardiovascular disease and certain cancers. Pyridoxal 5'-phosphate (PLP) serves as a coenzyme in many cellular processes, including several reactions in one-carbon (1C) metabolism and the transsulfuration pathway of homocysteine catabolism. To assess the effect of vitamin B-6 deficiency on these processes and associated pathways, we conducted quantitative analysis of 1C metabolites including tetrahydrofolate species in HepG2 cells cultured in various concentrations of pyridoxal. These results were compared with predictions of a mathematical model of 1C metabolism simulating effects of vitamin B-6 deficiency. In cells cultured in vitamin B-6-deficient medium (25 or 35 nmol/l pyridoxal), we observed >200% higher concentrations of betaine (P < 0.05) and creatinine (P < 0.05) and >60% lower concentrations of creatine (P < 0.05) and 5,10-methenyltetrahydrofolate (P < 0.05) compared with cells cultured in medium containing intermediate (65 nmol/l) or the supraphysiological 2,015 nmol/l pyridoxal. Cystathionine, cysteine, glutathione, and cysteinylglycine, which are components of the transsulfuration pathway and subsequent reactions, exhibited greater concentrations at the two lower vitamin B-6 concentrations. Partial least squares discriminant analysis showed differences in overall profiles between cells cultured in 25 and 35 nmol/l pyridoxal vs. those in 65 and 2,015 nmol/l pyridoxal. Mathematical model predictions aligned with analytically derived results. These data reveal pronounced effects of vitamin B-6 deficiency on 1C-related metabolites, including previously unexpected secondary effects on creatine. These results complement metabolomic studies in humans demonstrating extended metabolic effects of vitamin B-6 insufficiency.

Accumulation of the homocysteine (Hcy) precursor S-adenosylhomocysteine (AdoHcy) may cause cellular hypomethylation in the setting of hyperhomocysteinemia because of cystathionine β-synthase (CBS) deficiency, an inborn error of metabolism. To test this hypothesis, DNA and protein arginine methylation status were assessed in liver, brain, heart, and kidney obtained from a previously described mouse model of CBS deficiency. Metabolite levels in tissues and serum were determined by high-performance liquid chromatography or liquid chromatography-electrospray ionization-tandem mass spectrometry. Global DNA and protein arginine methylation status were evaluated as the contents of 5-methyldeoxycytidine in DNA and of methylarginines in proteins, respectively. In addition, histone arginine methylation was assessed by Western blotting. CBS-deficient mice exhibited increased (>6-fold) Hcy and AdoHcy levels in all tissues examined compared with control levels. In addition, global DNA methylation status was not affected, but global protein arginine methylation status was decreased (10-35%) in liver and brain. Moreover, asymmetric dimethylation of arginine 3 on histone H4 (H4R3me2a) content was markedly decreased in liver, and no differences were observed for the other histone arginine methylation marks examined. Our results show that CBS-deficient mice present severe accumulation of tissue Hcy and AdoHcy, protein arginine hypomethylation in liver and brain, and decreased H4R3me2a content in liver. Therefore, protein arginine hypomethylation arises as a potential player in the pathophysiology of CBS deficiency.

Purpose) The inflammasomes trigger the biological maturation of proinflammatory cytokines such as interleukin-1 beta or interleukin 18.They induce inflammation as expected, and also, they mediate host defence against microbial pathogens.Three Nod-like receptors (NLR family; NLRP1, NLRP3, NLRC4) are involved in the assembly of multiprotein complex inflammasomes.Recent advances in understanding pathological organ network such as intestine-to-liver revealed that intestinal inflammasomes have important role in the liver inflammation and fibrosis.It has been reported that intesitinal inflammasome activation plays an important role in the progression of NASH (Nature.2012).The intestinal inflammasome activation induces recovery of NASH and the somatic deletion of inflammasome gene results in progression of NASH.Anti-oxidants, such as vitamin E and pioglitazone have favorable effects in NASH.L-Carnitine plays an important role in mitochondrial beta-oxidation and has been reported to be effective in NASH.In this study, we examined the effects of vitamin E and L-carnitine as antioxidant supplementations on liver pathology and regulation of intestinal inflammasome activation in NASH mouse model.. (Methods) We used streptozotocin(STZ)-treated mouse diabetes model with high fat diet supplementation, known as the NASH hepatocarcinogenesis model (STAM mice).Eight-week-old STAM mice were divided into 3 experimental groups and fed for 4 weeks as follows:(1) high fat diet (S group) (2) high fat diet + L-Carnitine (SC group) (3) high fat diet + vitamin E (SE group).After 4 weeks mice were sacrificed.Following data were compared between these three groups; hepatic histological findings, hepatic 8-OHdG concentration, expression of hepatic inflammatory and hepatic lipogenic genes, and intestinal inflammasome related genes by real time PCR.(Results) In histological analysis, SC group and SE group showed low inflammation and fibrosis compared with S group.The concentration of 8-OHdG was reduced in SC and SE group compared with S group.The hepatic gene expression of inflammatory cytokine TNF-α was down-regulated in SC and SE group.The beta oxidation pathway related genes AOX and MCAD, the peroxisomal proliferator-activated receptor PPAR-α and PPAR-γ were up-regulated in SC and SE group.On the other hand, the intestinal inflammasome related genes were up-regulated in SC and SE group compared with S group.(Conclusion) Vitamin E and L-carnitine may prevent progression of non-alcoholic steatohepatitis with up-regulation of intestinal inflammasome activation.

Suboptimal vitamin B-6 status, as reflected by low plasma pyridoxal 5'-phosphate (PLP) concentration, is associated with increased risk of vascular disease. PLP plays many roles, including in one-carbon metabolism for the acquisition and transfer of carbon units and in the transsulfuration pathway. PLP also serves as a coenzyme in the catabolism of tryptophan. We hypothesize that the pattern of these metabolites can provide information reflecting the functional impact of marginal vitamin B-6 deficiency. We report here the concentration of major constituents of one-carbon metabolic processes and the tryptophan catabolic pathway in plasma from 23 healthy men and women before and after a 28-d controlled dietary vitamin B-6 restriction (<0.35 mg/d). liquid chromatography-tandem mass spectrometry analysis of the compounds relevant to one-carbon metabolism showed that vitamin B-6 restriction yielded increased cystathionine (53% pre- and 76% postprandial; P < 0.0001) and serine (12% preprandial; P < 0.05), and lower creatine (40% pre- and postprandial; P < 0.0001), creatinine (9% postprandial; P < 0.05), and dimethylglycine (16% postprandial; P < 0.05) relative to the vitamin B-6-adequate state. In the tryptophan pathway, vitamin B-6 restriction yielded lower kynurenic acid (22% pre- and 20% postprandial; P < 0.01) and higher 3-hydroxykynurenine (39% pre- and 34% postprandial; P < 0.01). Multivariate ANOVA analysis showed a significant global effect of vitamin B-6 restriction and multilevel partial least squares-discriminant analysis supported this conclusion. Thus, plasma concentrations of creatine, cystathionine, kynurenic acid, and 3-hydroxykynurenine jointly reveal effects of vitamin B-6 restriction on the profiles of one-carbon and tryptophan metabolites and serve as biomarkers of functional effects of marginal vitamin B-6 deficiency.

Vitamin B12, a co-factor in methyl-group transfer, is important in maintaining DNA (deoxycytidine) methylation. Using two independent assays we examined the effect of vitamin B12-deficiency (plasma vitamin B12<148 pmol/L) on DNA methylation in women of childbearing age. Coagulated blood clot DNA from vitamin B12-deficient women had significantly (p<0.001) lower percentage deoxycytidine methylation (3.23±0.66%; n = 248) and greater [3 H]methyl-acceptance (42,859±9,699 cpm; n = 17) than DNA from B12-replete women (4.44±0.18%; n = 128 and 26,049±2,814 cpm; n = 11) [correlation between assays: r = -0.8538; p<0.001; n = 28]. In contrast, uncoagulated EDTA-blood cell pellet DNA from vitamin B12-deficient and B12-replete women exhibited similar percentage methylation (4.45±0.15%; n = 77 vs. 4.47±0.15%; n = 47) and [3 H]methyl-acceptance (27,378±4,094 cpm; n = 17 vs. 26,610±2,292 cpm; n = 11). Therefore, in simultaneously collected paired blood samples, vitamin B12-deficiency was associated with decreased DNA methylation only in coagulated samples. These findings highlight the importance of sample collection methods in epigenetic studies, and the potential impact biological processes can have on DNA methylation during collection.

BACKGROUND:The methylenetetrahydrofolate reductase (MTHFR) genotype is associated with modification of disease and risk of neural tube defects. Plasma and red blood cell (RBC) folate and plasma homocysteine concentrations change in response to daily intakes of folic acid supplements, but no large-scale or population-based randomized trials have examined whether the MTHFR genotype modifies the observed response.OBJECTIVE:We sought to determine whether the MTHFR 677C→T genotype modifies the response to folic acid supplementation during and 3 mo after discontinuation of supplementation.DESIGN:Northern Chinese women of childbearing age were enrolled in a 6-mo supplementation trial of different folic acid doses: 100, 400, and 4000 μg/d and 4000 μg/wk. Plasma and RBC folate and plasma homocysteine concentrations were measured at baseline; after 1, 3, and 6 mo of supplementation; and 3 mo after discontinuation of supplementation. MTHFR genotyping was performed to identify a C→T mutation at position 677 (n = 932).RESULTS:Plasma and RBC folate and homocysteine concentrations were associated with MTHFR genotype throughout the supplementation trial, regardless of folic acid dose. MTHFR TT was associated with lower folate concentrations, and the trend of TT < CC was maintained at even the highest doses. Folic acid doses of 100 μg/d or 4000 μg/wk did not reduce high homocysteine concentrations in those with the MTHFR TT genotype.CONCLUSION:MTHFR genotype was an independent predictor of plasma and RBC folate and plasma homocysteine concentrations and did not have a significant interaction with folic acid dose during supplementation. This trial was registered at clinicaltrials.gov as NCT00207558.

Metabolic profiling of the effects of a marginal B‐6 deficiency can provide insight into the sensitivity of 1C metabolism to B‐6 status, and the level of stress placed on different pathways due to this deficiency. It also can add to our knowledge regarding vitamin B‐6 requirement for the optimal performance of the 1C cycle and may generate new hypotheses to be tested. This study aimed to assess the impact of marginal vitamin B‐6 deficiency (plasma PLP 20 –30 nM) on the plasma profile of one‐carbon metabolites in humans. Twenty‐two healthy men and women were placed on a 28‐d controlled dietary vitamin B‐6 restriction. Fasting plasma samples were collected at baseline and at day 28. Targeted analysis of known substrates and products of 1C metabolism and transsulfuration were performed by LC/MS‐MS. A one‐step extraction allowed simultaneous analysis of the concentrations of fifteen metabolites in a small volume of plasma. Results show a significant increase in plasma cystathionine (p<0.01) at marginal B‐6 deficiency, consistent with previous findings from our group. Plasma concentrations of serine and glycine also were significantly higher (p<0.05) at low B‐6 status. Postprandial plasma samples were collected as well and will allow evaluation of substrate availability in the fed versus fasted state on the 1C pathway in a marginal B‐6 deficiency.Supported by NIH grant DK072398 and NIH National Center for Research Resources (NCRR) CTSA grant 1UL1RR029890.

Methionine is the precursor for S-adenosylmethionine (SAM), the major 1-carbon donor involved in >100 transmethylation reactions. Homocysteine produced from SAM must be metabolized either by remethylation for recycling of methionine or transsulfuration to form cystathionine and then cysteine. Pyridoxal 5'-phosphate (PLP) serves as a coenzyme in enzymes involved in transsulfuration as well as for primary acquisition of 1-carbon units used for remethylation and other phases of 1-carbon metabolism. Because the intake of vitamin B-6 is frequently low in humans and metabolic consequences of inadequacy may be amplified in the postprandial state, we aimed to determine the effects of marginal vitamin B-6 deficiency on the postprandial rates of remethylation, transmethylation, overall transsulfuration, and cystathionine synthesis. Healthy, young adults (4 male, 5 female; 20-35 y) received a primed, constant infusion of [1-(13)C]methionine, [methyl-(2)H(3)]methionine, and [5,5,5-(2)H(3)]leucine to quantify in vivo kinetics at normal vitamin B-6 status and after a 28-d dietary vitamin B-6 restriction. Vitamin B-6 restriction lowered the plasma PLP concentration from 49 +/- 4 nmol/L (mean +/- SEM) to 19 +/- 2 nmol/L (P < 0.0001). Mean remethylation, transsulfuration, and transmethylation rates did not change in response to vitamin B-6 restriction; however, the responses to vitamin B-6 restriction varied greatly among individuals. The plasma cystathionine concentration increased from 142 +/- 8 to 236 +/- 9 nmol/L (P < 0.001), whereas the fractional cystathionine synthesis rate increased by a mean of 12% in 8 of 9 participants. Interrelationships among plasma concentrations of glycine and cystathionine and kinetic results suggest that individual variability occurs in normal postprandial 1-carbon metabolism and in the response to vitamin B-6 restriction. J. Nutr. 141: 835-842, 2011.