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1.  Cognitive dysfunction and depression in adult kidney transplant recipients: Baseline findings from the FAVORIT Ancillary Cognitive Trial (FACT) 
Hyperhomocysteinemia and B-vitamin deficiency may be treatable risk factors for cognitive impairment and decline. Hyperhomocysteinemia, cognitive impairment and depression all are common in individuals with kidney disease, including kidney transplant recipient. Accordingly, we assessed the prevalence of cognitive impairment and depressive symptoms in transplant recipients and their association with kidney function, plasma total homocysteine (tHcy) and B-vitamin concentrations.
Cross sectional analysis of baseline data from the FAVORIT Ancillary Cognitive Trial, which included 183 participants in FAVORIT who underwent detailed neuropsychological assessment prior to the study intervention.
The mean age was 54.0 ± 9.5 yrs (range 7–386 months). Men comprised 55.2% of the cohort and the mean time between the current transplant and cognitive testing was 7.0 ± 5.8 yrs. 24% of participants reported neurological or psychiatric complaints and 30% exhibited symptoms of mild to severe depression. Testing revealed evidence of significant and selective deficits in this population: 33% performed more than 1 SD below normed means on a memory test, 58% fell lower than 1 SD below the norms on a test of attention and mental processing speed, and 33–42% fell lower than 1 SD below the norms on several tests of executive function. Lower estimated glomerular filtration rate and lower folate were associated with poorer performance on tests of memory and executive function.
These observations confirm previous reports of mood and cognitive impairments in adult kidney transplant recipients. Further research is needed to determine the benefit of B-vitamin supplementation and other interventions in this patient population.
PMCID: PMC4410356  PMID: 22153382
Transplantation; Cognition; Depression; Homocysteine; B-vitamins
2.  Arsenic is associated with reduced effect of folic acid in myelomeningocele prevention: a case control study in Bangladesh 
Environmental Health  2015;14:34.
Arsenic induces neural tube defects in several animal models, but its potential to cause neural tube defects in humans is unknown. Our objective was to investigate the associations between maternal arsenic exposure, periconceptional folic acid supplementation, and risk of posterior neural tube defect (myelomeningocele) among a highly exposed population in rural Bangladesh.
We performed a case–control study that recruited physician-confirmed cases from community health clinics served by Dhaka Community Hospital in Bangladesh, as well as local health facilities that treat children with myelomeningocele. Controls were selected from pregnancy registries in the same areas. Maternal arsenic exposure was estimated from drinking water samples taken from wells used during the first trimester of pregnancy. Periconceptional folic acid use was ascertained by self-report, and maternal folate status was further assessed by plasma folate levels measured at the time of the study visit.
Fifty-seven cases of myelomeningocele were identified along with 55 controls. A significant interaction was observed between drinking water inorganic arsenic and periconceptional folic acid use. As drinking water inorganic arsenic concentrations increased from 1 to 25 μg/L, the estimated protective effect of folic acid use declined (OR 0.22 to 1.03), and was not protective at higher concentrations of arsenic. No main effect of arsenic exposure on myelomeningocele risk was identified.
Our study found a significant interaction between drinking water inorganic arsenic concentration from wells used during the first trimester of pregnancy and reported intake of periconceptional folic acid supplements. Results suggest that environmental arsenic exposure reduces the effectiveness of folic acid supplementation in preventing myelomeningocele.
PMCID: PMC4404044  PMID: 25885259
Arsenic; Neural tube defect; Myelomeningocele; Birth defect; Folate deficiency
3.  The association between vitamin B12, albuminuria and reduced kidney function: an observational cohort study 
BMC Nephrology  2015;16:7.
Variants in CUBN, the gene encoding cubilin, a proximal tubular transport protein, have been associated with albuminuria and vitamin B12 (B12) deficiency. We hypothesized that low levels of B12 would be associated with albuminuria in a population-based cohort.
We analyzed participants from the Framingham Heart Study (n = 2965, mean age 58 years, 53% female) who provided samples for plasma B12. Logistic regression models adjusted for covariates including homocysteine were constructed to test the association between B12 and prevalent albuminuria (UACR ≥17 mg/g [men] and ≥25 mg/g [women]) and reduced kidney function (defined as an eGFR < 60 ml/min/1.73 m2, RKF). Because of a significant interaction between B12 and homocysteine in the prevalent RKF model (p = 0.005), the model was stratified by the median homocysteine levels. Logistic regression models were constructed to test the association between B12 and incident albuminuria and RKF. The results were replicated in 4445 participants from NHANES 2003–2004.
Baseline B12 levels ranged from 50-1690 pg/ml. Elevated B12 was associated with prevalent albuminuria (OR 1.44 per 1 SD increase, 95% CI 1.10-1.87) and RKF (OR 1.83, 95% CI 1.30-2.60). However after stratifying by median homocysteine levels, this relationship remained only in the higher homocysteine stratum. There was no association between B12 and incident albuminuria (OR 1.17, 95% CI 0.79 – 1.73) or RKF (OR 1.45, 95% CI 0.97 – 1.88). In the NHANES cohort, elevated B12 was associated with RKF after full covariate adjustment (OR 3.06, 95% CI 2.30-4.08). There was no association with albuminuria.
In participants with high baseline homocysteine levels, increased plasma B12 was associated with RKF.
PMCID: PMC4361211  PMID: 25644490
Homocysteine; Albuminuria; Vitamin B12; Reduced kidney function
4.  Dietary vitamin B6 intake modulates colonic inflammation in the IL10−/− model of inflammatory bowel disease☆,☆☆ 
Pyridoxal-5-phosphate, the biologically active form of vitamin B6, is a cofactor for over 140 biochemical reactions. Although severe vitamin B6 deficiency is rare, mild inadequacy [plasma pyridoxal 5’-phosphate (PLP) <20 nmol/L] is observed in 19–27% of the US population. Plasma PLP concentrations are inversely related to markers of inflammation such as C-reactive protein. Furthermore, plasma PLP is diminished in those with inflammatory conditions and, in the case of inflammatory bowel disease (IBD), more so in those with active versus quiescent disease. Restricting B6 intake attenuates IBD pathology in mice; however, the effects of supplementation are unclear. We therefore sought to determine the effects of mild inadequacy and moderate supplementation of B6 on the severity of colonic inflammation. Weanling IL-10−/− (positive for Helicobacter hepaticus) mice were fed diets containing 0.5 (deficient), 6.0 (replete) or 24 (supplemented) mg/kg pyridoxine HCl for 12 weeks and then assessed for histological and molecular markers of colonic inflammation. Both low and high plasma PLP were associated with a significant suppression of molecular (TNFα, IL-6, IFN-γ, COX-2 and iNOS expression) and histological markers of inflammation in the colon. PLP is required for the breakdown of sphingosine 1-phosphate (S1P), a chemotactic lipid, by S1P lyase. Colonic concentrations of S1P and PLP were significantly and inversely correlated. If confirmed, vitamin B6 supplementation may offer an additional tool for the management of IBD. Although B6 is required in dozens of reactions, its role in the breakdown of S1P may explain the biphasic relationship observed between PLP and inflammation.
PMCID: PMC4199223  PMID: 24183308
Pyridoxal 5’ phosphate; Shingosine 1 phosphate; Inflammation; Colitis; Colon
5.  Prevalence of MTHFR C677T and MS A2756G polymorphisms in major depressive disorder, and their impact on response to fluoxetine treatment 
CNS spectrums  2012;17(2):76-86.
To examine the prevalence of the C677T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene and the A2756G polymorphism of methionine synthase (MS), and their impact on antidepressant response.
We screened 224 subjects (52% female, mean age 39 ± 11 years) with SCID-diagnosed major depressive disorder (MDD), and obtained 194 genetic samples. 49 subjects (49% female, mean age 36 ± 11 years) participated in a 12-week open clinical trial of fluoxetine 20–60 mg/day. Association between clinical response and C677T and A2756G polymorphisms, folate, B12, and homocysteine was examined.
Prevalence of the C677T and A2756G polymorphisms was consistent with previous reports (C/C=41%, C/T=47%, T/T=11%, A/A=66%, A/G=29%, G/G=4%). In the fluoxetine-treated subsample (n=49), intent-to-treat (ITT) response rates were 47% for C/C subjects and 46% for pooled C/T and T/T subjects (nonsignificant). ITT response rates were 38% for A/A subjects and 60% for A/G subjects (nonsignificant), with no subjects exhibiting the G/G homozygote. Mean baseline plasma B12 was significantly lower in A/G subjects compared to A/A, but folate and homocysteine levels were not affected by genetic status. Plasma folate was negatively associated with treatment response.
The C677T and A2756G polymorphisms did not significantly affect antidepressant response. These preliminary findings require replication in larger samples.
PMCID: PMC4117348  PMID: 22789065
Methylene Tetrahydrofolate Reductase; MTHFR; Methionine Synthase; MS; C677T; A2756G; depression; antidepressant; fluoxetine
6.  Prediagnostic plasma vitamin B6 (pyridoxal 5′-phosphate) and survival in patients with colorectal cancer 
Cancer causes & control : CCC  2013;24(4):10.1007/s10552-013-0152-x.
Higher plasma pyridoxal 5′-phosphate (PLP) levels are associated with a decreased incidence of colorectal cancer, but the influence of plasma PLP on survival of patients with colorectal cancer is unknown. We prospectively examined whether prediagnostic plasma PLP levels are associated with mortality among colorectal cancer patients.
We included 472 incident cases of colorectal cancer identified in the Nurses’ Health Study, the Health Professionals Follow-up Study, and the Physicians’ Health Study from 1984 to 2002. The patients provided blood samples two or more years before cancer diagnosis. Stratified Cox proportional hazards models were used to calculate hazard ratios (HR) with 95% confidence interval (CI) adjusted for other risk factors for cancer survival.
Higher plasma PLP levels were not associated with a significant reduction in colorectal cancer-specific (169 deaths) or overall mortality (259 deaths). Compared with patients who had less than 45 pmol/ml of plasma PLP (median: 33.6 pmol/ml), those who had 110 pmol/ml or higher levels (median: 158.8 pmol/ml) had multivariable HRs of 0.85 (95% CI 0.50–1.45, P trend = 0.37) and 0.87 (95% CI 0.56–1.35, P trend = 0.24) for colorectal cancer-specific and overall mortality. Higher plasma PLP levels, however, seemed to be associated with better survival among patients who had lower circulating 25-hydroxyvitamin D3 levels(< 26.5 ng/ml) (P interaction ≤ .005).
Higher prediagnostic plasma PLP levels were not associated with an improvement on colorectal cancer survival overall. Further research is needed to clarify the influence of vitamin B6 on colorectal cancer progression and survival.
PMCID: PMC3884510  PMID: 23340905
vitamin B6; pyridoxal 5′ phosphate (PLP); colorectal cancer; mortality; survival; prospective study
7.  Prediagnostic plasma folate and the risk of death in patients with colorectal cancer 
Although previous studies have demonstrated an inverse relationship between folate intake and colorectal cancer risk, a recent trial suggests that supplemental folic acid may accelerate tumorigenesis among patients with a history of colorectal adenoma. Therefore, high priority has been given to research investigating the influence of folate on cancer progression in patients with colorectal cancer.
To investigate whether prediagnostic levels of plasma folate are associated with colorectal cancer-specific and overall mortality, we performed a prospective, nested observational study within two large U.S. cohorts, the Nurses’ Health Study and Health Professionals Follow-up Study. We measured folate levels among 301 participants who developed colorectal cancer 2 or more years after their plasma was collected and compared participants using Cox proportional hazards models by quintile of plasma folate.
Higher levels of plasma folate were not associated with an increased risk of colorectal cancer-specific or overall mortality. Compared with participants in the lowest quintile of plasma folate, those in the highest quintile experienced a multivariable-adjusted hazard ratio for colorectal-cancer-specific mortality of 0.42 (95% CI, 0.20–0.88) and overall mortality of 0.46 (95% CI, 0.24–0.88). When the analysis was limited to participants whose plasma was collected within 5 years of cancer diagnosis, no detrimental effect of high plasma folate was noted. In subgroup analyses, no subgroup demonstrated worse survival among participants with higher plasma folate levels.
In two large prospective cohorts, higher prediagnostic levels of plasma folate were not associated with an increased risk of colorectal cancer-specific or overall mortality.
PMCID: PMC3962289  PMID: 18591557
Colorectal cancer; cancer-specific mortality; folate; prospective cohort study
8.  Genome-Wide Meta-Analysis of Homocysteine and Methionine Metabolism Identifies Five One Carbon Metabolism Loci and a Novel Association of ALDH1L1 with Ischemic Stroke 
PLoS Genetics  2014;10(3):e1004214.
Circulating homocysteine levels (tHcy), a product of the folate one carbon metabolism pathway (FOCM) through the demethylation of methionine, are heritable and are associated with an increased risk of common diseases such as stroke, cardiovascular disease (CVD), cancer and dementia. The FOCM is the sole source of de novo methyl group synthesis, impacting many biological and epigenetic pathways. However, the genetic determinants of elevated tHcy (hyperhomocysteinemia), dysregulation of methionine metabolism and the underlying biological processes remain unclear. We conducted independent genome-wide association studies and a meta-analysis of methionine metabolism, characterized by post-methionine load test tHcy, in 2,710 participants from the Framingham Heart Study (FHS) and 2,100 participants from the Vitamin Intervention for Stroke Prevention (VISP) clinical trial, and then examined the association of the identified loci with incident stroke in FHS. Five genes in the FOCM pathway (GNMT [p = 1.60×10−63], CBS [p = 3.15×10−26], CPS1 [p = 9.10×10−13], ALDH1L1 [p = 7.3×10−13] and PSPH [p = 1.17×10−16]) were strongly associated with the difference between pre- and post-methionine load test tHcy levels (ΔPOST). Of these, one variant in the ALDH1L1 locus, rs2364368, was associated with incident ischemic stroke. Promoter analyses reveal genetic and epigenetic differences that may explain a direct effect on GNMT transcription and a downstream affect on methionine metabolism. Additionally, a genetic-score consisting of the five significant loci explains 13% of the variance of ΔPOST in FHS and 6% of the variance in VISP. Association between variants in FOCM genes with ΔPOST suggest novel mechanisms that lead to differences in methionine metabolism, and possibly the epigenome, impacting disease risk. These data emphasize the importance of a concerted effort to understand regulators of one carbon metabolism as potential therapeutic targets.
Author Summary
Elevated homocysteine (tHcy) is strongly associated with risk for common disorders such as stroke, cardiovascular disease and Alzheimer disease. Lowering tHcy levels has proven to have variable success in reducing clinical risk, so the question remains, “Are we correctly targeting these disorders by lowering tHcy?” Understanding folate one-carbon metabolism pathway (FOCM) genetic variation will aid us in developing new targets for therapy. The FOCM is essential in regulation of the epigenome, which controls genes in ways beyond nucleotide sequence. We present data generated from stroke-only and general populations where we identify strong association of genetic risk factors for variation in one-carbon metabolism function, characterized by the post-methionine load test. We show that GNMT harbors genetic and epigenetic differences that influence gene function, which may have downstream effects on the epigenome of the cell, affecting disease risk. We developed a genetic risk score that predicts post-methionine load homocysteine levels that may be useful in clinic. Finally, we identified a novel association between ischemic stroke and ALDH1L1, which emphasizes the clinical importance of this work. Our results highlight the importance of a concerted effort to target the FOCM (beyond tHcy) and parallel pathways in future pharmacogenetic work using the genetic variation we describe here.
PMCID: PMC3961178  PMID: 24651765
9.  Developmental consequences of in utero sodium arsenate exposure in mice with folate transport deficiencies 
Previous studies have demonstrated that mice lacking a functional folate binding protein 2 gene (Folbp2−/−) were significantly more sensitive to in utero arsenic exposure than were the wild-type mice similarly exposed. When these mice were fed a folate-deficient diet, the embryotoxic effect of arsenate was further exacerbated. Contrary to expectations, studies on 24-h urinary speciation of sodium arsenate did not demonstrate any significant difference in arsenic biotransformation between Folbp2−/− and Folbp2+/+ mice. To better understand the influence of folate pathway genes on arsenic embryotoxicity, the present investigation utilized transgenic mice with disrupted folate binding protein 1 (Folbp1) and reduced folate carrier (RFC) genes. Because complete inactivation of Folbp1 and RFC genes results in embryonic lethality, we used heterozygous animals. Overall, no RFC genotype-related differences in embryonic susceptibility to arsenic exposure were observed. Embryonic lethality and neural tube defect (NTD) frequency in Folbp1 mice was dose-dependent and differed from the RFC mice; however, no genotype-related differences were observed. The RFC heterozygotes tended to have higher plasma levels of S-adenosylhomocysteine (SAH) than did the wild-type controls, although this effect was not robust. It is concluded that genetic modifications at the Folbp1 and RFC loci confers no particular sensitivity to arsenic toxicity compared to wild-type controls, thus disproving the working hypothesis that decreased methylating capacity of the genetically modified mice would put them at increased risk for arsenic-induced reproductive toxicity.
PMCID: PMC3938173  PMID: 15694460
Arsenic; Teratogenicity; Biotransformation; Detoxification; Folbp1; RFC; Neural tube defects
10.  Effect of Combined Folic Acid, Vitamin B6, and Vitamin B12 on Colorectal Adenoma 
Folic acid, vitamin B6, and vitamin B12 act in concert in the one-carbon metabolism and may protect against colorectal neoplasia. We examined the effect of combined B-vitamin treatment on the occurrence of colorectal adenoma.
The Women’s Antioxidant and Folic Acid Cardiovascular Study was a randomized, double-blind, placebo-controlled trial of 5442 female health professionals at high risk for cardiovascular disease from April 1998 through July 2005. Participants were randomly assigned to receive a combination pill of folic acid (2.5mg), vitamin B6 (50mg), and vitamin B12 (1mg) or placebo. This study included 1470 participants who were followed up for as long as 9.2 years and underwent an endoscopy at any point during follow-up. We estimated relative risks using a generalized linear model with a natural logarithm link function and Poisson distributed errors. All statistical tests were two-sided.
The risk of colorectal adenoma was similar among participants receiving treatment (24.3%, 180 of 741 participants) vs placebo (24.0%, 175 of 729 participants) (multivariable adjusted relative risk = 1.00, 95% confidence interval = 0.83 to 1.20). Treatment was not associated with the risk of adenoma when data were analyzed by subsite, size, stage, and the number of adenomas. There was no statistically significant effect modification by alcohol intake, history of cancer or adenoma, or baseline plasma levels or intakes of folate, vitamin B6, or vitamin B12.
Our results indicate no statistically significant effect of combined folic acid, vitamin B6, and vitamin B12 treatment on colorectal adenoma among women at high risk for cardiovascular disease.
PMCID: PMC3611818  PMID: 23066166
11.  Circulating folic acid in plasma: relation to folic acid fortification 
The implementation of folic acid fortification in the United States has resulted in unprecedented amounts of this synthetic form of folate in the American diet. Folic acid in circulation may be a useful measure of physiologic exposure to synthetic folic acid, and there is a potential for elevated concentrations after fortification and the possibility of adverse effects.
We assessed the effect of folic acid fortification on circulating concentrations of folic acid and 5-methyltetrahydrofolate in the Framingham Offspring Cohort.
This is a cross-sectional study that used plasma samples from fasting subjects before and after fortification. Samples were measured for folate distribution with the use of an affinity-HPLC method with electrochemical detection.
Among nonsupplement users, the median concentration of folic acid in plasma increased from 0.25 to 0.50 nmol/L (P < 0.001) after fortification, and among supplement users the median increased from 0.54 to 0.68 nmol/L (P = 0.001). Among nonsupplement users, the prevalence of high circulating folic acid (≥85th percentile) increased from 9.4% to 19.1% (P = 0.002) after fortification. Among supplement users, the prevalence of high circulating folic acid increased from 15.9% to 24.3% (P = 0.02). Folic acid intake and total plasma folate were positively and significantly related to high circulating folic acid after adjustment for potential confounding factors (P for trend < 0.001).
Folic acid fortification has resulted in increased exposure to circulating folic acid. The biochemical and physiologic consequences of this are unknown, but these findings highlight the need to understand the effects of chronic exposure to circulating folic acid.
PMCID: PMC3763811  PMID: 18779294
12.  Vitamin B-12 and Folate Status in Relation to Decline in Scores on the Mini-Mental State Examination in the Framingham Heart Study 
Biochemical evidence of low vitamin B-12 status is common in seniors, but its clinical relevance is unclear. Vitamin B-12 deficiency can result in rapid, irreversible cognitive decline – a phenomenon that has been linked to high folate status. Our objective was to investigate the cognitive significance of low to low-normal plasma vitamin B-12 concentrations. Secondarily, we sought to shed light on the role that folate status plays in the association between vitamin B-12 status and cognitive decline.
We evaluated associations between plasma vitamin B-12 and folate and 8-year cognitive decline. We also assessed interactions between vitamin B-12 status and both folate status and supplemental folate use in relation to cognitive decline.
The Framingham Heart Study -- a prospective epidemiologic study
Five hundred forty-nine community-dwelling seniors (mean age 74.8±4.6 years).
Mini-Mental State Examination (MMSE), plasma folate, vitamin B-12, methylmalonic acid, homocysteine, demographic factors, and body mass index.
MMSE scores declined by 0.24 points/year over the 8-year follow-up period. Decline was significantly accelerated among cohort members in the bottom two plasma vitamin B-12 quintile categories, and no apparent cognitive advantage was associated with plasma vitamin B-12 from 187–256.8 pmol/L versus <186 pmol/L. Among cohort members with plasma vitamin B-12<258 pmol/L, having a plasma folate concentration>20.2 nmol/L was associated with an approximate 1-point/year decline, as was use of supplemental folate.
Plasma vitamin B-12 from 187–256.8 pmol/L predicts cognitive decline. High plasma folate and supplemental folate use identify subgroups in this vitamin B-12 range and below who are prone to especially rapid cognitive decline.
PMCID: PMC3419282  PMID: 22788704
aged; cognition/*physiology; folic acid/blood; humans; methylmalonic acid/blood; vitamin B 12/*blood
13.  Plasma folate, methylenetetrahydrofolate reductase (MTHFR), and colorectal cancer risk in three large nested case-control studies 
Cancer causes & control : CCC  2012;23(4):537-545.
Few prospective studies have examined the associations between blood levels of folate, in conjunction with methylenetetrahydrofolate reductase (MTHFR) polymorphisms, and colorectal cancer. We evaluated the associations between plasma folate, MTHFR C677T and A1298C, and colorectal cancer in three large prospective studies: the Nurses’ Health Study, the Health Professionals Follow-up Study, and the Physicians’ Health Study. A total of 602 incident cases were identified and individually matched to controls who provided blood specimens. We used conditional logistic regression to calculate the relative risk (RR) and 95% confidence interval (95% CI) and then pooled the estimates using a random effects model. We found a lower risk of colorectal cancer among participants with low plasma folate levels: compared with the lowest quartile, RRs (95% CIs) for each successively higher quartile of plasma folate levels were 1.55 (1.14–2.11), 1.37 (1.00–1.88), and 1.47 (1.07–2.01; P for trend = 0.10). For the MTHFR polymorphisms, RRs (95% CIs) were 0.62 (0.44–0.90) for the 677TT vs. CC/CT and 0.68 (0.31–1.51) for the 1298CC vs. AC/AA, and these lower risk genotypes were associated with lower circulating plasma folate levels. When we partitioned the variation in plasma folate levels, variation due to folate intake was not positively associated with colorectal cancer risk. We found that low plasma folate levels were associated with lower risk of colorectal cancer. The reasons underlying a lower risk of colorectal cancer with low plasma folate levels require elucidation because plasma folate levels can reflect dietary intake, genetic influences, and other factors.
PMCID: PMC3721151  PMID: 22367721
Folate; methylenetetrahydrofolate reductase (MTHFR); colorectal cancer
14.  Multiple Biomarkers and Risk of Clinical and Subclinical Vascular Brain Injury: The Framingham Offspring Study 
Circulation  2012;125(17):2100-2107.
Several biomarkers have been individually associated with vascular brain injury but no prior study has explored the simultaneous association of a biologically plausible panel of biomarkers with the incidence of stroke/TIA, and the prevalence of subclinical brain injury.
Methods and Results
In 3127 stroke-free Framingham Offspring (59±10 yrs, 54%F), we related a panel of 8 biomarkers assessing inflammation(C-reactive protein[CRP]), hemostasis(D-dimer and plasminogen activator inhibitor-1), neurohormonal activity(aldosterone-to renin ratio, B-type natriuretic peptide[BNP] and N-terminal pro-atrial natriuretic peptides) and endothelial function (homocysteine and urinary albumin/creatinine ratio[UACR]) measured at the 6th examination(1995–98) to risk of incident stroke/TIA. In a subset of 1901 participants with available brain MRI (1999–2005), we further related these biomarkers to total cerebral brain volume (TCBV), covert brain infarcts (CBI), and large white matter hyperintensity volume(LWMHV).
During a median follow-up of 9.2 years, 130 participants experienced incident stroke/TIA. In multivariable analyses adjusted for stroke risk factors, the biomarker panel was associated with incident stroke/TIA and with TCBV (p<0.05 for both), but not with CBI or LWMHV (p >0.05). In backwards elimination analyses higher log-BNP (hazards ratio [HR] 1.39/SD, p=0.002) and log-UACR (HR1.31/SD, p=0.004) were associated with increased risk of stroke/TIA and improved risk prediction over using the Framingham stroke risk profile alone; using <5%, 5–15% or >15% 10-year risk categories the net reclassification index was 0.109;p=0.037). Higher CRP (β=−0.21/SD,p=0.008), D-dimer(β==−0.18/SD,p=0.041), tHcy(β=−0.21/SD,p=0.005), and UACR(β=−0.15/SD,p=0.042) were associated with lower TCBV.
In a middle-aged community sample, we identified multiple biomarkers that were associated with clinical and subclinical vascular brain injury and could improve risk stratification.
PMCID: PMC3427730  PMID: 22456473
biomarkers; epidemiology; magnetic resonance imaging; risk stratification; stroke prevention
15.  Pre-Diagnostic Leukocyte Genomic DNA Methylation and the Risk of Colorectal Cancer in Women 
PLoS ONE  2013;8(4):e59455.
Abnormal one-carbon metabolism may lead to general genomic (global) hypomethylation, which may predispose an individual to the development of colorectal neoplasia.
We evaluated the association between pre-diagnostic leukocyte genomic DNA methylation level and the risk of colorectal cancer in a nested case-control study of 358 colorectal cancer cases and 661 matched controls within the all-female cohort of the Nurses’ Health Study (NHS). Among control subjects, we further examined major plasma components in the one-carbon metabolism pathway in relation to genomic DNA methylation level. Liquid chromatography/tandem mass spectrometry was used to examine leukocyte genomic DNA methylation level. We calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) using logistic regression.
Overall genomic DNA methylation level was not associated with the risk of colorectal cancer (p for trend, 0.45). Compared with women in the lowest quintile of methylation, the multivariate OR of colorectal cancer risk was 1.32 (95% CI, 0.82–2.13) for those in the highest quintile. We did not find significant associations between major plasma components of one-carbon metabolism or risk factors for colorectal cancer and genomic DNA methylation level (all p for trend >0.05). Also, neither one-carbon metabolism-related plasma components nor well-known risk factors for colorectal cancer modified the association between genomic DNA methylation level and the risk of colorectal cancer (all p for interaction >0.05).
We found no evidence that hypomethylation of leukocyte genomic DNA increases risk of colorectal cancer among women. Additional studies are needed to investigate the association between pre-diagnostic genomic DNA methylation level and colorectal cancer risk among diverse populations.
PMCID: PMC3613344  PMID: 23560049
16.  Prospective Study of Serum Cysteine Levels and Oesophageal and Gastric Cancers in China 
Gut  2011;60(5):618-623.
Cancers of the upper gastrointestinal tract remain a significant cause of morbidity and mortality. Cysteine, known to be involved in a myriad of immuno-modulatory, anti-oxidant, and anti-carcinogenic pathways, has not been investigated in the aetiology of oesophageal or gastric cancers. To examine the relationship between serum cysteine concentration and risk of these cancers we conducted a nested case-cohort study within the General Population Nutrition Intervention Trial in Linxian, China.
498 oesophageal squamous cell carcinomas (OSCC) and 255 gastric cardia adenocarcinomas (GCA) were matched by age and sex to 947 individuals from the wider cohort. We calculated hazard ratios (HR) and 95% confidence intervals (95% CI) using the case-cohort estimator for the Cox proportional hazards models, stratified on age and sex, with adjustment for potential confounders.
Higher concentrations of serum cysteine were significantly associated with a lower risk of both OSCC and GCA. For those in the highest quartile of serum cysteine, compared to those in the lowest, the multivariate HRs were 0.70 for OSCC (95% CI: 0.51, 0.98) and 0.59 for GCA (95% CI: 0.38, 0.91). These associations were dose dependent (P for trend = 0.006 and 0.008, respectively). These inverse associations were not significantly modified by other risk factors, with the exception of age, where a stronger association was noted among persons in the older age strata.
Higher serum concentrations of cysteine were associated with a significantly reduced risk of OSCC and GCA. Cysteine should be further investigated for its potential as a chemopreventive agent for upper gastrointestinal cancers.
PMCID: PMC3428021  PMID: 21242262
oesophageal squamous cell carcinoma; gastric cardia cancer; hazard ratio; cysteine
17.  Hyperhomocysteinemia from trimethylation of hepatic phosphatidylethanolamine during cholesterol cholelithogenesis in inbred mice 
Hepatology (Baltimore, Md.)  2011;54(2):697-706.
Because hyperhomocysteinemia can occur in cholesterol gallstone disease, we hypothesized that this may result from trimethylation of phosphatidylethanolamine (PE), which partakes in biliary phosphatidylcholine (PC) hypersecretion during cholesterol cholelithogenesis. We fed murine strains C57L/J, C57BL/6J, SWR/J, AKR/J, PE N-methyltransferase (PEMT) knockout (KO), PEMT heterozygous (HET), and wild type (WT) mice a cholesterol/cholic acid lithogenic diet (LD) for up to 56 days and documented biliary lipid phase transitions and secretion rates. We quantified plasma total homocysteine (tHcy), folate, and vitamin B12 in plasma and liver, as well as biliary tHcy and cysteine secretion rates. Rate-limiting enzyme activities of PC synthesis, PEMT and cytidine triphosphate: phosphocholine cytidylyltransferase (PCT), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in liver homogenates. Other potential sources of plasma tHcy, glycine N-methyltransferase (GNMT) and guanidinoacetate N-methyltransferase (GAMT), were assayed by gene expression. Plasma tHcy and PEMT activities became elevated during cholelithogenesis in gallstone-susceptible C57L, C57BL/6, and SWR mice, but not in the gallstone-resistant AKR mice. Persisting in C57L mice, which exhibit the greatest Lith gene burden, these increases were accompanied by elevated hepatic SAM/SAH ratios and augmented biliary tHcy secretion rates. Counter-regulation included remethylation of Hcy to methionine concurrent with decreased folate and vitamin B12 levels and Hcy transsulfuration to cysteine. Concomitantly, methylenetetrahydrofolate reductase (Mthfr), betaine-homocysteine methyltransferase (Bhmt), and cystathionine-β-synthase (Cbs) were upregulated, but Gnmt and Gamt genes were downregulated. PEMT KO and HET mice displayed biliary lipid secretion rates and high gallstone prevalence rates similar to WT mice without any elevation in plasma tHcy levels.
This work implicates upregulation of PC synthesis by the PEMT pathway as a source of elevated plasma and bile tHcy during cholesterol cholelithogenesis.
PMCID: PMC3145001  PMID: 21567442
phosphatidylethanolamine N-methyltransferase; folate; vitamin B12; cysteine; bile
18.  Methotrexate Increases Skeletal Muscle GLUT4 Expression and Improves Metabolic Control in Experimental Diabetes 
Long-term administration of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) mimics the effects of endurance exercise by activating AMP kinase and by increasing skeletal muscle expression of GLUT4 glucose transporter. AICAR is an intermediate in the purine de novo synthesis, and its tissue concentrations can be increased, in vivo, by low doses of methotrexate (MTX) through the inhibition of the enzyme AICAR transformylase. We report here the first evidence that, in experimental type 2 diabetes, chronic treatment with low doses of MTX increases skeletal muscle GLUT4 expression and improves metabolic control. MTX (0.5 mg/kg body weight) or vehicle was administered intraperitoneally, once a week for 4 weeks, to genetically diabetic female C57BL/KsJ-m+/+Leptdb mice (db+/db+) and their normoglycemic littermates (db+/+m). In the db+/db+ mice, MTX treatment was associated with a ∼2-fold increase in skeletal muscle GLUT4 protein concentration and a >4-fold increase in GLUT4 mRNA expression (P < 0.01, all), as compared to vehicle-treated mice; no significant differences were noted in controls. MTX treatment was also associated with a significant reduction of glucose and insulin serum concentrations in diabetic mice (P < 0.001), and glucose levels only (P < 0.05) in controls. These data indicate a different route to increase skeletal muscle GLUT4 expression, through the potential inhibition of the enzyme AICAR transformylase.
PMCID: PMC3384889  PMID: 22778921
19.  Metabolic syndrome in elderly living in marginal peri-urban communities in Quito, Ecuador 
Public health nutrition  2010;14(5):758-767.
The proportion of Latin American population > 60 y is expected to double during the next few decades. Metabolic syndrome (MetS) is associated with high morbidity and mortality worldwide. However, little is known about MetS in Latin America in general, and in Ecuador in particular.
To examine the prevalence of MetS and its association with blood micronutrient, homocysteine (tHcy) and CRP concentrations in elderly living in a low-income urban area.
We performed a cross-sectional study. MetS, using the International Diabetes Federation definition, dietary intake and plasma micronutrient, CRP and tHcy concentrations were assessed.
352 elderly (≥65 y)
Quito, Ecuador.
MetS was prevalent (40 %)—considerably more so among women (81%) than men (19%) (X2 = 32.6, P<0.0001). Further, 53 % of those without MetS exhibited two or more of its components. Micronutrient deficiencies were prevalent including those of vitamin C, zinc, B12, and folate. Vitamin C and E concentrations were inversely (OR= 0.78, CI = 0.71 – 0.86; OR= 0.16, CI =0.03 – 0.81, respectively), and CRP (OR=1.79, CI =1.04 – 3.06) was positively associated with MetS.
The co-existence of MetS with micronutrient deficiencies suggests that elderly Ecuadorians suffer from the double burden of diseases that is increasingly observed in less-developed countries. More research is needed to determine causal factors, but results presented suggest that these older adults would benefit from interventions to reduce the risk factors for MetS, in particular, higher consumption of micronutrient-rich foods.
PMCID: PMC3025090  PMID: 20955641
Elderly; metabolic syndrome; Ecuador; micronutrient deficiency; C-reactive protein
20.  Segment-specific association between plasma homocysteine and carotid artery intima-media thickness in the Framingham Heart Study 
Higher plasma total homocysteine (tHcy) is an established risk factor for cardiovascular disease. The relation between tHcy and carotid artery intima-media thickness (IMT) at the internal carotid artery (ICA)/bulb-IMT and common carotid artery (CCA)-IMT has not been systematically examined. Since the ICA/bulb segment is more prone to plaque formation than the CCA segment, differential associations with tHcy at these sites might suggest mechanisms of tHcy action.
We examined the cross-sectional segment-specific relations of tHcy to ICA/bulb-IMT and CCA-IMT in 2,499 participants from the Framingham Offspring Study, free of cardiovascular disease.
In multivariable linear regression analysis, ICA/bulb-IMT was significantly higher in the fourth tHcy quartile category compared to the other quartile categories, in both the age- and sex-adjusted and in the multivariable-adjusted model (P for trend <0.0001 and <0.01, respectively). We observed a significant age by tHcy interaction for ICA/bulb-IMT (P=0.03) and therefore stratified the analyses by median age (58 years). There was a significant positive trend between tHcy and ICA/bulb-IMT in individuals 58 years of age or older (P-trend <0.01), but not in the younger individuals (P-trend=0.24). For CCA-IMT, no significant trends were observed in any of the analyses.
The segment-specific association between elevated tHcy levels and ICA/bulb-IMT suggests an association between tHcy and plaque formation.
PMCID: PMC3011043  PMID: 20580253
carotid artery; intima-media thickness; homocysteine; atherosclerosis; Framingham Offspring Study
21.  Relations of Biomarkers of Distinct Pathophysiological Pathways and Atrial Fibrillation Incidence in the Community 
Circulation  2010;121(2):200-207.
Biomarkers of multiple pathophysiological pathways have been related to incident atrial fibrillation (AF), but their predictive ability remains controversial.
Methods and Results
In 3120 Framingham cohort participants (mean age 58.4±9.7, 54% women), we related 10 biomarkers representing inflammation (C-reactive protein [CRP], fibrinogen), neurohormonal activation (B-type natriuretic peptide [BNP], N-terminal pro-atrial natriuretic peptide), oxidative stress (homocysteine), renin-angiotensin-aldosterone system (renin, aldosterone), thrombosis and endothelial function (D-dimer, plasminogen-activator inhibitor 1 [PAI-1]), and microvascular damage (urine albumin excretion, n=2673) with incident AF (n=209, 40% women) over a median follow-up of 9.7 years (range 0.05–12.8 years).
In multivariable-adjusted analyses, the biomarker panel was associated with incident AF (P<0.0001). In stepwise selection models (P<0.01 for entry and retention), log-transformed BNP, hazard ratio [HR] per standard deviation 1.62 (95% confidence interval [CI] 1.41–1.85, P<0.0001), and CRP, HR 1.25 (95% CI 1.07–1.45, P=0.004), were chosen.
The addition of BNP to variables recently combined in a risk score for AF increased the C-statistic from 0.78 (95%CI 0.75–0.81 to 0.80 (95% CI 0.78–0.83), and showed an integrated discrimination improvement of 0.03 (95% CI 0.02–0.04, P<0.0001) with 34.9% relative improvement in reclassification analysis. The combined analysis of BNP and CRP did not appreciably improve risk prediction over the model incorporating BNP in addition to the risk factors.
BNP is a predictor of incident AF and improves risk stratification based on well-established clinical risk factors. Whether knowledge of BNP concentrations may be used to target individuals at risk of AF for more intensive monitoring or primary prevention needs further investigation.
PMCID: PMC3224826  PMID: 20048208
atrial fibrillation; biomarkers; epidemiology; cohort; risk assessment
22.  A Multimarker Approach for Prediction of Heart Failure Incidence in the Community 
Circulation  2010;122(17):1700-1706.
Several biological pathways are activated in ventricular remodeling and in overt heart failure (HF). There are no data, however, on the incremental utility of a parsimonious set of biomarkers (reflecting pathways implicated in HF) for predicting HF risk in the community.
Methods and Results
We related a multi-biomarker panel to the incidence of a first HF event in 2754 Framingham Heart Study participants (mean age 58 years; 54% women), who were free of HF and underwent routine assays for 6 biomarkers (c-reactive protein, plasminogen activator inhibitor-1, homocysteine, aldosterone-to-renin ratio, b-type natriuretic peptide [BNP] and urinary albumin-to-creatinine ratio [UACR]). We estimated model c-statistic, calibration and net reclassification improvement (NRI) to assess the incremental predictive usefulness of biomarkers. We also related biomarkers to incidence of non-ischemic HF in participants without prevalent coronary heart disease.
On follow-up (mean 9.4 years), 95 first HF events occurred (54 in men). In multivariable-adjusted models, the biomarker panel was significantly related to HF risk (p=0.00005). Upon backwards elimination, BNP and UACR emerged as key biomarkers predicting HF risk: hazards ratio (HR; confidence interval [CI]) per standard deviation increment in log-marker were 1.52 (1.24-1.87) and 1.35 (1.11-1.66), respectively. BNP and UACR significantly improved the model c-statistic (CI) from 0.84 (0.80-0.88) in standard models to 0.86 (0.83-0.90), enhanced risk reclassification (NRI = 0.13; p=0.002), and were also independently associated with non-ischemic HF risk.
Using a multimarker strategy, we identified BNP and UACR as key risk factors for new-onset HF with incremental predictive utility over standard risk factors.
PMCID: PMC2993157  PMID: 20937976
Biomarkers; heart failure; risk; prediction
23.  A prospective study of one-carbon metabolism biomarkers and risk of renal cell carcinoma 
Cancer causes & control : CCC  2010;21(7):1061-1069.
Previous studies have found associations between one-carbon metabolism factors and risk of several cancers, but little is known regarding renal cell carcinoma (RCC). We conducted a nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, a prospective study of Finnish male smokers aged 50-69 at baseline.
Prediagnostic folate, vitamin B6, vitamin B12, cysteine, riboflavin and homocysteine concentrations were measured in fasting serum from 224 incident RCC cases and 224 controls (matched on age and date of serum collection). Conditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), adjusted for potential confounders.
Serum folate tended to be inversely associated with RCC; compared to the first quartile, the odds ratios (95% CI) for subsequent quartiles were 0.62 (0.35-1.08), 0.52 (0.29-0.93), and 0.67 (0.37-1.20) (P-trend = 0.19). When modeled as a threshold effect, subjects in the lowest serum folate quartile (≤ 6.64 nmol/L), which corresponds to deficient folate status, had a significant increased RCC risk (OR = 1.68, 95% CI 1.06-2.65) compared to those with higher serum folate. The other one-carbon metabolism biomarkers were not associated with RCC.
This study in male smokers suggests that deficient folate status may increase risk of RCC, but confirmation is needed in other epidemiologic studies that include women and non-smokers.
PMCID: PMC2902168  PMID: 20383577
folate; renal cell carcinoma; biological markers; nested case-control study; B vitamins
24.  Biomarkers of folate status in NHANES: a roundtable summary123456 
A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999–2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991–1994 and NHANES 1999–2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007–2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.
PMCID: PMC3127517  PMID: 21593502
25.  Biomarkers of vitamin B-12 status in NHANES: a roundtable summary123456 
A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12–related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES—serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)—and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12–related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.
PMCID: PMC3127527  PMID: 21593512

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