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.
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.
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.
aged; cognition/*physiology; folic acid/blood; humans; methylmalonic acid/blood; vitamin B 12/*blood
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.
Folate; methylenetetrahydrofolate reductase (MTHFR); colorectal cancer
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.
biomarkers; epidemiology; magnetic resonance imaging; risk stratification; stroke prevention
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.
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.
oesophageal squamous cell carcinoma; gastric cardia cancer; hazard ratio; cysteine
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.
phosphatidylethanolamine N-methyltransferase; folate; vitamin B12; cysteine; bile
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).
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.
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)
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.
Elderly; metabolic syndrome; Ecuador; micronutrient deficiency; C-reactive protein
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.
carotid artery; intima-media thickness; homocysteine; atherosclerosis; Framingham Offspring Study
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.
atrial fibrillation; biomarkers; epidemiology; cohort; risk assessment
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.
Biomarkers; heart failure; risk; prediction
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.
folate; renal cell carcinoma; biological markers; nested case-control study; B vitamins
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.
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.
Background: Folic acid (FA) fortification of food created the need to determine whether fortification elevated concentrations of unmetabolized FA in plasma and whether this form of the vitamin in blood is associated with adverse health outcomes.
Objective: The objective of this research was to devise a simple, rapid method for the measurement of unmetabolized plasma FA in epidemiologic studies.
Design: We previously used the affinity/HPLC with electrochemical detection method to measure folate distribution in human plasma and red blood cells (RBCs). We modified this method with the inclusion of synthetic ethyltetrahydrofolate as an internal standard and with the use of 2 affinity columns connected in parallel to the analytic column through a switching valve to allow one column to be loaded while the other column was eluted into the analytic column.
Results: We identified FA and 5-methyltetrahydrofolate (5-mTHF) by retention time and characteristic response across the channels of the electrochemical detector. Limits of detection were 0.034 pmol for 5-mTHF and 0.027 pmol for FA per injection, and the recovery was 92.2% (5-mTHF) and 98.9% (FA). CVs for samples were 8.1% (within day) and 6.8% (between day) for 5-mTHF and 3.2% (within day) and 5.9% (between day) for FA. Total folate with the use of this method correlated highly (r2 = 0.98, P < 0.001) with values from the microbial assay. The run time for the method was 30 min per sample. Researchers can use this method with longer run times to measure the distribution of folate forms in RBCs.
Conclusion: This updated method allows efficient analysis of folate forms in human plasma and tissues without the loss of sensitivity or precision.
Cysteine is a glutathione precursor, but is also a homocysteine byproduct. We prospectively evaluated relationships between fasting plasma concentrations of total cysteine and total homocysteine, and subsequent myocardial infarction (MI) in women.
Among 32826 women who provided blood samples between 1989 and 1990, 239 were diagnosed with incident MI after blood collection, but before July 1998. Of these women, 144 had provided a post-fast sample. We matched controls to cases 2:1 by age, cigarette smoking status, and month and fasting status at the time of blood collection. We used conditional logistic regression to adjust for confounding.
Fasting total cysteine was positively related to MI risk in matching factor-adjusted analyses [RR for highest vs. lowest quartile 3.50 (95% CI 1.44, 8.52)]. However, after controlling for conventional risk factors of MI, it was not independently associated with risk [RR for highest vs. lowest quartile 1.32 (95% CI 0.42, 4.12; P trend=0.10)]. Fasting homocysteine was positively associated with MI risk; the multivariable adjusted rate ratio (RR) for the highest versus the lowest quartile was 3.37 (95% CI 1.30, 8.70; P trend=0.014).
Fasting plasma concentration of total homocysteine, but not total cysteine was positively associated with MI risk.
Homocysteine and cysteine are associated with oxidative damage and metabolic disorders, which may lead to carcinogenesis. Observational studies assessing the association between circulating homocysteine or cysteine and breast cancer are very limited and findings have been inconsistent. We prospectively evaluated plasma levels of homocysteine and cysteine in relation to breast cancer risk among 812 incident cases of invasive breast cancer and 812 individually matched control subjects from 28,345 women in the Women’s Health Study aged ≥45 years who provided blood samples and had no history of cancer or cardiovascular disease at baseline. Logistic regression controlling for matching factors and risk factors for breast cancer was used to estimate relative risks (RRs) and 95% confidence intervals (CIs). All statistical tests were two sided. Homocysteine levels were not associated with overall risk for breast cancer. However, we observed a positive association between cysteine levels and breast cancer risk; the multivariate RR for the highest quintile group relative to the lowest quintile was 1.65 (95% CI=1.04–2.61, p for trend=0.04). In addition, women with higher levels of homocysteine and cysteine were at a greater risk for developing breast cancer when their folate levels were low (p values for interaction were 0.04 and 0.002, respectively). Although our study offers little support for an association between circulating homocysteine and overall breast cancer risk, higher homocysteine levels may be associated with an increased risk for breast cancer among women with low folate status. The increased risk of breast cancer associated with high cysteine levels warrants further investigation.
Aging; Amino acid; Caloric restriction; Demography; Dietary restriction; Drosophila; Longevity; Methionine; Mortality; Nutrition
The relationship of circulating levels of high sensitivity C-reactive protein (CRP) with cardiovascular disease (CVD) risk, particularly with consideration of effects at intermediate levels of risk, has not been fully assessed.
Among 3006 Offspring participants in the Framingham Heart Study free of CVD (mean age 46 years at baseline), there were 129 Hard coronary heart disease (CHD) events and 286 Total CVD events during 12 years of follow up. Cox regression, discrimination with area under the receiver operating characteristic curve, and net reclassification improvement were used to assess the role of CRP on vascular risk.
In an age-adjusted model that included both sexes the hazard ratios for new Hard CHD and Total CVD were significantly associated with higher CRP levels. Similar analyses according to increasing homocysteine (Hcys) level showed significant protective associations for Hard CHD but not for Total CVD. In multivariable analyses that included age, sex, systolic blood pressure, total cholesterol, HDL-cholesterol, diabetes mellitus, current smoking, hypertension treatment and homocysteine, the log CRP level remained significantly related to developing Hard CHD and Total CVD and provided moderate improvement in the discrimination of events. The net reclassification improvement when CRP was added to traditional factors was 5.6% for Total CVD (P=0.014) and 11.8% for Hard CHD (P=0.009).
Circulating levels of CRP help to estimate risk for initial cardiovascular events and may be used most effectively in persons at intermediate risk for vascular events, offering moderate improvement in reclassification of risk.
risk factors; coronary disease; homocysteine; C-reactive protein
Low plasma B-vitamin levels and elevated homocysteine have been associated with cancer, cardiovascular disease and neurodegenerative disorders. Common variants in FUT2 on chromosome 19q13 were associated with plasma vitamin B12 levels among women in a genome-wide association study in the Nurses’ Health Study (NHS) NCI-Cancer Genetic Markers of Susceptibility (CGEMS) project. To identify additional loci associated with plasma vitamin B12, homocysteine, folate and vitamin B6 (active form pyridoxal 5′-phosphate, PLP), we conducted a meta-analysis of three GWA scans (total n = 4763, consisting of 1658 women in NHS-CGEMS, 1647 women in Framingham-SNP-Health Association Resource (SHARe) and 1458 men in SHARe). On chromosome 19q13, we confirm the association of plasma vitamin B12 with rs602662 and rs492602 (P-value = 1.83 × 10−15 and 1.30 × 10−14, respectively) in strong linkage disequilibrium (LD) with rs601338 (P = 6.92 × 10−15), the FUT2 W143X nonsense mutation. We identified additional genome-wide significant loci for plasma vitamin B12 on chromosomes 6p21 (P = 4.05 × 10−08), 10p12 (P-value=2.87 × 10−9) and 11q11 (P-value=2.25 × 10−10) in genes with biological relevance. We confirm the association of the well-studied functional candidate SNP 5,10-methylene tetrahydrofolate reductase (MTHFR) Ala222Val (dbSNP ID: rs1801133; P-value=1.27 × 10−8), on chromosome 1p36 with plasma homocysteine and identify an additional genome-wide significant locus on chromosome 9q22 (P-value=2.06 × 10−8) associated with plasma homocysteine. We also identified genome-wide associations with variants on chromosome 1p36 with plasma PLP (P-value=1.40 × 10−15). Genome-wide significant loci were not identified for plasma folate. These data reveal new biological candidates and confirm prior candidate genes for plasma homocysteine, plasma vitamin B12 and plasma PLP.
B vitamins, such as folate, vitamin B6 and vitamin B12, play an important role as co-enzymes in one-carbon metabolism and may affect colorectal cancer (CRC) risk. We aimed to comprehensively investigate the relationships of plasma folate, pyridoxal-5’-phosphate (PLP, the active form of vitamin B6), vitamin B12, methylmalonic acid, homocysteine and cysteine with CRC risk, accounting for suspected modifiers [alcohol intake, MTHFR C677T genotype and plasma C-reactive protein (CRP)] and potential confounders. We conducted a case-control study nested within the Multiethnic Cohort study and analyzed prospectively collected blood samples from 224 incident CRC cases and 411 controls matched on age, sex, race/ethnicity, study site, date/time of blood draw, and hours of fasting. We found an inverse association between plasma PLP levels and CRC, with odds ratios and 95% confidence intervals for increasing quartiles of 1.00, 0.84 (0.51–1.40), 0.62 (0.37–1.03), 0.49 (0.29–0.83), p trend: 0.009. This association was not explained by an association with plasma folate, appeared to be stronger at low levels of alcohol intake and among individuals with the MTHFR 677TT genotype, and was independent of plasma CRP levels. An inverse association with plasma folate was also observed among individuals with a low level of alcohol intake. These data suggest an independent role for vitamin B6 in reducing CRC risk.
Vitamin B; folate; vitamin B6; colorectal cancer
Several studies have examined serum creatinine as a marker for prostate cancer stage, recurrence, and prognosis. We evaluated whether serum creatinine concentration was associated with risk of developing prostate cancer in a prospective cohort of male smokers.
A nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of 50–69 year old Finnish men was conducted. Two controls (n=464) were matched to each case (n=232) on study center, intervention group, date of baseline blood draw (±45 days), and age (±5 years). Conditional logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI). All p-values were two-sided.
Cases had significantly higher pre-diagnostic serum creatinine concentrations compared with controls (medians of 1.13 vs 1.10 mg/dL, respectively; p=0.004). Serum creatinine was associated with a significantly greater risk of prostate cancer (multivariate OR=2.23, 95% CI 1.33–3.75 for highest vs. lowest quartile), with a significant trend (p-trend = 0.0008). Exclusion of subjects with a reported history of diabetes, benign prostatic hyperplasia, or hypertension, or whose cancer was diagnosed within the first five years of follow-up, did not alter the association. Risk did not differ by disease stage or time from blood draw to diagnosis.
Prospectively measured serum creatinine, within normal ranges, is positively related to prostate cancer risk. Future research should reexamine the association in other populations, including any inter-relationship with serum prostate-specific antigen.
prostate cancer; creatinine
Vitamin B6 is widely involved in amino acid metabolism and is a modulator of several reactions important to cardiovascular health. We prospectively evaluated relationships between fasting plasma levels of vitamin B6, as pyridoxal phosphate (PLP), to subsequent myocardial infarction risk in women. We also evaluated the predictors of fasting plasma concentration of pyridoxal phosphate.
Participants were adult nurses who completed questionnaires, and updated exposures every 2 years since 1976. Subjects for this analysis were selected by a nested case control design. Blood samples were collected between 1989 and 1990. We restricted our analysis to those women who had provided fasting blood samples (≥10 hours since last meal). During follow-up through June 1998, 144 were diagnosed with myocardial infarction (fatal and non-fatal). Cases were matched 1:2 by age, cigarette smoking status, and month and fasting status at the time of blood collection. Conditional logistic regression was used to adjust for potential confounders, including anthropometric factors, dietary intake, and selected biomarkers. Linear regression was used to determine which variables predict fasting total PLP concentration among control women.
Median age at blood collection was 63. Among controls, lower estimated creatinine clearance, plasma total homocysteine and body mass index were statistically significant predictors of higher plasma PLP, as were higher dietary vitamin B6, and folate intake (all P <0.05). Plasma levels of pyridoxal phosphate were inversely associated with risk of MI, the multivariable adjusted rate ratio (RR) between extreme quarters was 0.22 (95% CI 0.09,0.55; Ptrend=0.05). The effect of plasma PLP varied by age. Among women who were aged less than 60 at blood sampling, the RR (95%CI) comparing top vs. bottom quarter was 0.03 (0.002,0.48), whereas among older women the corresponding RR (95%CI) was 0.43 (0.15,1.25).
Fasting plasma concentration of pyridoxal phosphate was inversely associated with MI risk. Plasma PLP is positively correlated with dietary vitamin B6, and is inversely correlated with renal function and body mass index. Future studies are needed to better understand both dietary and non-dietary determinants of plasma and tissue vitamin B6 status, and how these can be optimized to prevent MI and other diseases.
vitamins; nutrition; women; myocardial infarction; risk factors