Western Norway B-vitamin Intervention Trial Research Articles

The association between dairy intake and risk of cardiovascular disease and mortality in patients with stable angina pectoris.

The association of dairy products with cardiovascular disease and mortality risk remains heavily debated. We aimed to investigate the association between intake of total dairy and dairy products and the risk of acute myocardial infarction (AMI), stroke, and cardiovascular and all-cause mortality. We included 1929 patients (80% men, mean age 62 years) with stable angina pectoris from the Western Norway B-vitamin Intervention Trial. Dietary data were obtained via a 169-item food frequency questionnaire. Risk associations were estimated using Cox proportional hazard regression models adjusted for relevant covariates. Non-linear associations were explored visually. The mean (±SD) dairy intake in the study population was 169 ± 108 g/1000 kcal. Median follow-up times were 5.2, 7.8, and 14.1 years for stroke, AMI, and mortality, respectively. Higher intake of total dairy and milk were positively associated with stroke risk [HR (95% CI): 1.14 (1.02, 1.27) and 1.13 (1.02, 1.27), cardiovascular mortality 1.06 (1.00, 1.12) and 1.07 (1.01, 1.13)] and all-cause mortality [1.07 (1.03, 1.11) and 1.06 (1.03, 1.10)] per 50 g/1000 kcal. Higher cheese intake was inversely associated with AMI risk [0.92 (0.83, 1.02)] per 10 g/1000 kcal. Butter was associated with increased AMI risk [1.10 (0.97, 1.24)] and all-cause mortality [1.10 (1.00, 1.20) per 5 g/1000 kcal. Higher dairy and milk consumption were associated with increased risk of mortality and stroke. Cheese was associated with decreased, and butter with increased, risk of AMI. Dairy is a heterogenous food group with divergent health effects and dairy products should therefore be investigated individually.

Within-person reproducibility of proteoforms related to inflammation and renal dysfunction.

Protein biomarkers and microheterogeneity have attracted increasing attention in epidemiological and clinical research. Knowledge of within-person reproducibility over time is paramount to determine whether a single measurement accurately reflects an individual’s long-term exposure. Yet, research investigating within-person reproducibility for proteoforms is limited. We investigated the reproducibility of the inflammatory markers C-reactive protein (CRP), serum amyloid A (SAA), and calprotectin (S100A8/9), and the renal function marker cystatin C (CnC) using a novel immuno-MALDI-TOF MS assay. Reproducibility, expressed as intraclass correlation coefficient (ICC), was calculated for 16 proteoforms using plasma samples of the Western Norway B Vitamin Intervention Trial (WENBIT) cohort collected 1–3 y apart from 295 stable angina pectoris (SAP) patients and 16 weeks apart from 38 subjects of the Intervention with Omega Fatty Acids in High-risk Patients with Hypertriglyceridemic Waist (OMEGA) trial with abdominal obesity but no other documented co-morbidities. ICCs for inflammatory markers were lower in WENBIT (CRP: 0.51, SAAt: 0.38, S100At: 0.31) compared to OMEGA subjects (CRP: 0.71, SAAt: 0.73, S100At: 0.48), while comparable for CnCt (WENBIT: 0.69, OMEGA: 0.67). Excluding SAP patients with elevated inflammation (CRP > 10 µg/ml) increased the ICC of SAAt to 0.55. Reduction of the time interval from 3 to 1 y in WENBIT group increased ICCs for all proteoforms. With a few exceptions ICCs did not differ between proteoforms of the same biomarker. ICCs were highest in OMEGA subjects with fair-to-good reproducibility for all markers. Reproducibility of SAA and S100A8/9 proteoforms in the WENBIT cohort was related to inflammation. This work will inform future clinical and epidemiological research which relies on single time point biomarker assessment to investigate inflammation and renal function.

The Association of Meat Intake With All-Cause Mortality and Acute Myocardial Infarction Is Age-Dependent in Patients With Stable Angina Pectoris.

Background: Red and processed meat intake have been associated with increased risk of morbidity and mortality, and a restricted intake is encouraged in patients with cardiovascular disease. However, evidence on the association between total meat intake and clinical outcomes in this patient group is lacking.Objectives: To investigate the association between total meat intake and risk of all-cause mortality, acute myocardial infarction, cancer, and gastrointestinal cancer in patients with stable angina pectoris. We also investigated whether age modified these associations.Materials and Methods: This prospective cohort study consisted of 1,929 patients (80% male, mean age 62 years) with stable angina pectoris from the Western Norway B-Vitamin Intervention Trial. Dietary assessment was performed by the administration of a semi-quantitative food frequency questionnaire. Cox proportional hazards models were used to investigate the association between a relative increase in total meat intake and the outcomes of interest.Results: The association per 50 g/1,000 kcal higher intake of total meat with morbidity and mortality were generally inconclusive but indicated an increased risk of acute myocardial infarction [HR: 1.26 (95% CI: 0.98, 1.61)] and gastrointestinal cancer [1.23 (0.70, 2.16)]. However, we observed a clear effect modification by age, where total meat intake was associated with an increased risk of mortality and acute myocardial infarction among younger individuals, but an attenuation, and even reversal of the risk association with increasing age.Conclusion: Our findings support the current dietary guidelines emphasizing a restricted meat intake in cardiovascular disease patients but highlights the need for further research on the association between meat intake and health outcomes in elderly populations. Future studies should investigate different types of meat separately in other CVD-cohorts, in different age-groups, as well as in the general population.

Dietary composition is associated with one-carbon metabolites and B-vitamin status in patients with stable angina – a cross-sectional study

AbstractIntroduction:Peroxisome Proliferator-Activated Receptor α (PPARα) is a known regulator of lipid and energy metabolism. In animal experiments of PPARα activation, we have reported on altered plasma concentration of metabolites related to one-carbon metabolism and B-vitamin status. Several of these metabolites, e.g. homocysteine and dimethylglycine, have been associated with increased cardiovascular disease risk. Diet can influence one-carbon metabolism, and this may partly be mediated through altered PPARα activity as dietary fatty acids can activate PPARα. As diet is a modifiable life style factor, the aim of this investigation is to explore potential associations between dietary composition and plasma concentration of one-carbon metabolites and markers of B-vitamin status.Materials and methods:The study population consisted of 1977 patients with stable angina (geometric mean age 61 years, 80% male) from the Western Norway B-vitamin Intervention Trial (WENBIT), who completed a self-administered food frequency questionnaire at baseline. Data on outcome variables were obtained from the baseline blood samples (35% fasting). Outcome variables of interest were metabolites related to one-carbon metabolism and B-vitamin status. Cross-sectional associations between protein, carbohydrate and fat intake with the outcome metabolites were analysed with linear regression, adjusted for age, sex, fasting status, BMI and reported energy intake. Estimates are given as % change in the outcome variable per isoenergetic increment of 1 energy % (E%) in the exposure nutrient.Results:The average distribution [95% prediction interval] of energy intake (E%) in the population was 48.5 (37.1, 63.4) from carbohydrate, 31.4 (21.9, 44.9) from fat and 16.5 (12.2, 22.3) from protein.The strongest associations were seen for increasing protein intake, while the associations with fat and carbohydrate intake were weaker. The most pronounced associations (% change [95% confidence interval]) with increasing protein intake were higher cobalamin (2.9 [2.1, 3.7]), PLP (2.7 [1.7, 3.7]) and riboflavin (2.3 [1.0, 3.6]), and lower tHcy (-1.4 [-1.9, -0.9]) and MMA (-1.3 [-1.9, -0.7]).Discussion:The current observations indicate that dietary macronutrient composition may influence plasma concentration of one-carbon metabolites and markers of B-vitamin status. The strongest associations are observed with increasing protein intake, but elucidating the importance of the nutrient substituted remains. Whether the observed associations are due to macronutrients per se, or reflect differences in food choices/nutrient intakes, or effects on physiological factors, i.e. inflammation or PPARα activation, is not known.

Adherence to the Healthy Nordic Food Index and the incidence of acute myocardial infarction and mortality among patients with stable angina pectoris.

The Healthy Nordic Food Index (HNFI) has been associated with beneficial effects on markers of cardiovascular disease (CVD). Whether such effects are present among patients with established coronary heart disease is unknown. In the present study, we investigated the association between adherence to the HNFI and the risk of acute myocardial infarction (AMI) (fatal or nonfatal) and death among patients with stable angina pectoris. In the Western Norway B-vitamin Intervention Trial, participants completed a 169-item semi-quantitative food frequency questionnaire. The HNFI was calculated from six food groups (fish, cabbage, apples/pears, root vegetables, whole grain bread and oatmeal), scoring 0-6. Three adherence groups were defined: 0-1 points (low), 2-3 points (medium) or 4-6 points (high). Cox regression analyses investigated associations between adherence to the HNFI and outcomes. Among 2019 men (79.7%) and women with mean age of 61.7years, 307 patients experienced an AMI event during a median (25th and 75th percentiles) follow-up of 7.5 (6.3 and 8.7) years. Median follow-up for total mortality was 10.5 (9.3 and 11.7) years; 171 patients died from CVD and 380 from any cause. No association between HNFI and the risk of AMI was detected. However, the HNFI was associated with a reduced risk of all-cause death, both by linear estimates [hazard ratio (95% confidence interval=0.91 (0.84-0.98)] and by comparison of the highest with the lowest adherence group [hazard ratio (95% confidence interval=0.70 (0.52-0.95)]. The results of the present study suggest that a Healthy Nordic diet may reduce mortality in patients with established CVD.

B vitamin treatments modify the risk of myocardial infarction associated with a MTHFD1 polymorphism in patients with stable angina pectoris

BackgroundMethylenetetrahydrofolate dehydrogenase (MTHFD1) catalyzes three sequential reactions that metabolize derivatives of tetrahydrofolate (THF) in folate-dependent one-carbon metabolism. Impaired MTHFD1 flux has been linked to disturbed lipid metabolism and oxidative stress. However, limited information is available on its relation to the development of atherothrombotic cardiovascular disease. Methods and resultsWe explored the association between a MTHFD1 polymorphism (rs1076991 C > T) and acute myocardial infarction (AMI), and potential effect modifications by folic acid/B12 and/or vitamin B6 treatment in suspected stable angina pectoris patients (n = 2381) participating in the randomized Western Norway B Vitamin Intervention Trial (WENBIT). During the median follow-up of 4.9 years 204 participants (8.6%) suffered an AMI. After adjusting for established CVD risk factors, the MTHFD1 polymorphism was significantly associated with AMI (HR: 1.49; 95% CI, 1.23–1.81). A similar association was observed among patients allocated to treatment with vitamin B6 alone (HR: 1.53; 95% CI, 1.01–2.31), and an even stronger relationship was seen in patients treated with both vitamin B6 and folic acid/B12 (HR: 2.35; 95% CI, 1.55–3.57). However, no risk association between the MTHFD1 polymorphism and AMI was seen in patients treated with placebo (HR: 1.29; 95% CI, 0.86–1.93) or folic acid/B12 (1.17; 95% CI, 0.83–1.65). ConclusionA common and functional MTHFD1 polymorphism is associated with increased risk of AMI, although the risk seems to be dependent on specific B vitamin treatment. Further studies are warranted to elucidate the possible mechanisms, also in order to explore potential effect modifications by nutritional factors.

Dietary Intake of Saturated Fat Is Not Associated with Risk of Coronary Events or Mortality in Patients with Established Coronary Artery Disease1–3

AbstractBackground: Data from recent meta-analyses question an association between dietary intake of saturated fatty acids (SFAs) and risk of cardiovascular disease (CVD). Moreover, the prognostic effect of dietary SFA in patients with established CVD treated with modern conventional medication has not been extensively studied.Objective: We investigated the associations between self-reported dietary SFA intake and risk of subsequent coronary events and mortality in patients with coronary artery disease (CAD).Methods: This study included patients who participated in the Western Norway B-Vitamin Intervention Trial and completed a 169-item semiquantitative food-frequency questionnaire after coronary angiography. Quartiles of estimated daily intakes of SFA were related to risk of a primary composite endpoint of coronary events (unstable angina pectoris, nonfatal acute myocardial infarction, and coronary death) and separate secondary endpoints (total acute myocardial infarction, fatal coronary events, and all-cause death) with use of Cox-regression analyses.Results: This study included 2412 patients (81% men, mean age: 61.7 y). After a median follow-up of 4.8 y, a total of 292 (12%) patients experienced at least one major coronary event during follow-up. High intake of SFAs was associated with a number of risk factors at baseline. However, there were no significant associations between SFA intake and risk of coronary events [age- and sex-adjusted HR (95% CI) was 0.85 (0.61, 1.18) for the upper vs. lower SFA quartile] or any secondary endpoint. Estimates were not appreciably changed after multivariate adjustments.Conclusions: There was no association between dietary intake of SFAs and incident coronary events or mortality in patients with established CAD.

Evidence for increased catabolism of vitamin B-6 during systemic inflammation

Plasma concentrations of PL 5'-phosphate (PLP), which is the active coenzyme form of vitamin B-6, are reduced during inflammation. The underlying mechanisms may include altered tissue distribution or increased catabolism via pyridoxal (PL) to pyridoxic acid (PA). Recently, we showed that catabolic enzyme activity could be assessed by substrate product ratios measured in plasma. We evaluated the ratios PA:PL, PA:PLP, and PA:(PL + PLP) as possible markers of vitamin B-6 catabolism. Cross-sectional and longitudinal data were derived from the Western Norway B-Vitamin Intervention Trial. We analyzed associations of ratios with inflammatory markers and other clinical variables by using multiple linear regression and partial correlation. In addition, intraclass correlation coefficients (ICCs) were used to assess the ability of plasma indexes to differentiate between subjects. PA:(PL + PLP) had the highest ICC of all vitamin B-6 metabolites and ratios tested. In regression models, the inflammatory markers C-reactive protein, white blood cell count, neopterin, and kynurenine:tryptophan collectively accounted for 28% of the total and > 90% of the explained variation in PA:(PL + PLP). For individual B-6 metabolites, corresponding numbers were 19-25% and 20-44%, respectively, with vitamin supplement intake, smoking, and kidney function (estimated glomerular filtration rate) as additional predictors. In an analysis of receiver operating characteristics, PA:(PL + PLP) discriminated high inflammatory concentrations with an area under the curve (95% CI) of 0.85 (0.81, 0.89). Broad-specificity enzymes upregulated to reduce oxidative and aldehyde stress could explain increased catabolism of vitamin B-6 during inflammation. The ratio PA:(PL + PLP) may provide novel insights into pathologic processes and potentially predict risk of future disease.

Abstract P452: Pathway Analysis Of One-carbon Metabolism In Association With Acute Myocardial Infarction In Patients With Stable Angina Pectoris

Background: One-carbon metabolism (OCM) is involved in RNA, DNA synthesis and in epigenetic regulation. Pathway gene analysis is more likely to capture potential gene-disease associations comparing to single gene analysis. We investigated whether genetic variants in folate network as well as their interactions would predict subsequent acute myocardial infarction (AMI) in patients undergoing elective coronary angiography for stable angina pectoris (SAP). Methods: A total of 2381 subjects (98% Caucasian) recruited to the Western Norway B Vitamin Intervention Trial (WENBIT) in 2000-2004 were studied. Patients were followed until a subsequent AMI or end of 2006. We genotyped 18 commonly investigated single nucleotide polymorphisms (SNPs) in 13 genes in the OCM network. Predictable variants were selected based on both out-of-bag importance scores from random survival forests and their incremental effects from recursive nested analysis. We then assessed gene-gene interactions among those predictive variants with generalized logistic model and calculated hazard ratio (HR), confidence interval (CI) for each predictor in Cox proportional hazard models. Results: There are 204 (8.57%) patients who experienced an AMI throughout 4.9 years (median) follow-up. We observed no violation from Hardy-Weinberg equilibrium in 15 SNPs in our population. Four SNPs (rs1076991, rs1021737, rs1801133 and rs3733890) in folate network were identified as promising candidates for predicting AMI risk. Interestingly, only rs1076991 associated significantly with AMI (HR=1.30, 95% CI: 1.10-1.52), indicating the other SNPs primarily mediate their risk bygene-gene interactions. There was a strong and stable interaction between the dominant model of rs3733890 and dominant model of rs34489327 (P for interaction: 0.003; HR =2.23, 95% CI: 1.27-3.92, adjusted for age, gender, smoking, diabetes mellitus, hypertension, extent of CAD by angiography (0-3), apolipoprotein (Apo) A1 and B, left ventricular ejection fraction and estimated glomerular filtration rate). Further adjustment for vitamins involved in OCM (folate, B2, B6, B12), the substrates (betaine and homocysteine) and products (methionine and dimethylglycine) of the betaine--homocysteine S-methyltransferase (BHMT) had minimal influence on the estimate. Conclusions: Prior studies show that BHMT transcription is associated with co-transcription of Apo B and that the rs3733890 of this gene is associated with higher dimethylglycine levels. We have recently shown that elevated plasma dimethylglycine is associated with increased risk of AMI and improves risk prediction in patients with SAP independent of Apo B levels. The current investigation extends previous knowledge, suggesting that a genetic variant in BHMT may modulate the risk of AMI by interfering with thymidylate synthesis independent of plasma dimethylglycine.

Plasma choline, smoking, and long-term prognosis in patients with stable angina pectoris

Plasma choline has been associated with cardiovascular disease and nonalcoholic steatohepatitis. We sought to study relations of plasma choline and its metabolite betaine to long-term risk of acute myocardial infarction (AMI) and all-cause mortality according to smoking status, in patients undergoing coronary angiography for stable angina pectoris. Samples were obtained before angiography from 2568 patients who were subsequently randomized in the Western Norway B-Vitamin Intervention Trial (WENBIT). Hazard ratios (HR) were calculated using multivariate Cox-regression and p-values were reported for trends over quartiles. Plasma concentrations of choline, but not betaine, were lower in smokers, and choline was positively associated with C-reactive protein and troponin T in nonsmokers, but not in smokers (p for interaction <0.03). During a follow up of 4.8 ± 1.4 (mean ± SD) years, 8.3% suffered from AMI and 6.1% died. In the total population, choline was not associated with AMI or all-cause mortality. However, comparing the highest vs. the lowest quartiles, plasma choline was associated with increased risk of AMI in nonsmokers (HR 2.63, 95% CI 1.56 to 5.51; p for trend = 0.013) and no risk in smokers (p for interaction < 0.001). Plasma choline significantly improved discrimination and reclassification when added to established cardiovascular risk factors. Plasma betaine was not associated with either endpoint. In patients with stable angina pectoris, elevated plasma choline is associated with elevated troponin levels and increased risk of AMI in nonsmokers. These results motivate further research into the relation between choline metabolism, smoking, and atherothrombosis.

Vitamin D status was not associated with ‘one-year’ progression of coronary artery disease, assessed by coronary angiography in statin-treated patients

Low vitamin D status is associated with increased risk of cardiovascular disease and may be involved in atherosclerosis. Our aim was to assess the association between vitamin D status and the progression of coronary artery disease (CAD). We measured 25-hydroxyvitamin D3 (25OHD3) by liquid chromatography tandem mass spectrometry (LC-MS/MS) in plasma from 348 participants with established CAD (84% males, mean ± standard deviation (SD) age 60 ± 10 years) of the Western Norway B-vitamin Intervention Trial (WENBIT, 1999-2006). The patients underwent invasive coronary angiography (CA) and percutaneous coronary intervention at baseline and a second CA after 302 ± 79 days of follow-up. From the angiograms, minimal lumen diameter (MLD) and diameter stenosis (DS) of atherosclerotic lesions were obtained. Significant CAD in non-intervened vessels was found in 309 coronary arteries from 183 participants either at baseline and/or at follow-up. To assess the association between levels of 25OHD3 and CAD progression in non-intervened vessels, we applied a linear quantile fitted mixed effects model with MLD or DS measured at follow-up as a function of continuous 25OHD3 concentrations. There were no statistically significant associations between plasma 25OHD3 concentrations (median: 63.9, 95% confidence interval (CI): 48.1-78.5 nmol/l) measured at baseline and the follow-up measures of either MLD (estimated effect per 10 nmol/l increase of 25OHD3 and 95% CI: -0.015 (-0.032-0.002) mm, p = 0.088) or DS (0.225 (-0.354-0.804) percentage points, p = 0.444). Multivariate adjustment did not alter these results. Plasma 25OHD3 levels were not associated with 'one-year' progression of CAD, assessed by CA in statin-treated patients.

Dietary intake of n-3 long-chain polyunsaturated fatty acids and risk of myocardial infarction in coronary artery disease patients with or without diabetes mellitus: a prospective cohort study

BackgroundA beneficial effect of a high n-3 long-chain polyunsaturated fatty acid (LCPUFA) intake has been observed in heart failure patients, who are frequently insulin resistant. We investigated the potential influence of impaired glucose metabolism on the relation between dietary intake of n-3 LCPUFAs and risk of acute myocardial infarction (AMI) in patients with coronary artery disease.MethodsThis prospective cohort study was based on the Western Norway B-Vitamin Intervention Trial and included 2,378 patients with coronary artery disease with available baseline glycosylated hemoglobin (HbA1c) and dietary data. Patients were sub-grouped as having no diabetes (HbA1c <5.7%), pre-diabetes (HbA1c ≥5.7%), or diabetes (previous diabetes, fasting baseline serum glucose ≥7.0, or non-fasting glucose ≥11.1 mmol/L). AMI risk was evaluated by Cox regression (age and sex adjusted), comparing the upper versus lower tertile of daily dietary n-3 LCPUFA intake.ResultsThe participants (80% males) had a mean age of 62 and follow-up of 4.8 years. A high n-3 LCPUFA intake was associated with reduced risk of AMI (hazard ratio 0.38, 95%CI 0.18, 0.80) in diabetes patients (median HbA1c = 7.2%), whereas no association was observed in pre-diabetes patients. In patients without diabetes a high intake tended to be associated with an increased risk (hazard ratio1.45, 95%CI 0.84, 2.53), which was significant for fatal AMI (hazard ratio 4.79, 95%CI 1.05, 21.90) and associated with lower HbA1c (mean ± standard deviation 4.55 ±0.68 versus 4.92 ±0.60, P = 0.02). No such differences in HbA1c were observed in those with pre-diabetes or diabetes.ConclusionsA high intake of n-3 LCPUFAs was associated with a reduced risk of AMI, independent of HbA1c, in diabetic patients, but with an increased risk of fatal AMI and lower HbA1c among patients without impaired glucose metabolism. Further studies should investigate whether patients with diabetes may benefit from having a high intake of n-3 LCPUFAs and whether patients with normal glucose tolerance should be careful with a very high intake of these fatty acids.Trial registrationThis trial is registered at clinicaltrials.gov as NCT00354081.

Substrate product ratios of enzymes in the kynurenine pathway measured in plasma as indicators of functional vitamin B-6 status

Tryptophan metabolism through the kynurenine pathway includes 2 vitamin B-6 [pyridoxal 5'-phosphate (PLP)]-dependent enzymes. We recently showed that plasma 3-hydroxykynurenine (HK) was elevated at low PLP concentrations. We further evaluated and characterized kynurenine-based indexes as possible markers of functional B-vitamin status in plasma. Cross-sectional and longitudinal data were derived from the Western Norway B-vitamin Intervention Trial, including PLP, kynurenine, HK, kynurenic acid (KA), anthranilic acid, xanthurenic acid (XA), and 3-hydroxyanthranilic acid (HAA) measured in plasma at 2 time points. Partial Spearman's correlation, generalized additive models, and receiver operating characteristic (ROC) analysis were used to assess associations of kynurenines with PLP. Ratios HK:XA, HK:HAA, and HK:KA showed markedly stronger negative correlations with PLP than did HK alone (Spearman's ρ = -0.36, -0.29, and -0.31 compared with -0.18, respectively). All associations were nonlinear, with the strongest relation at low PLP. In the ROC analysis, areas under the curve for discriminating low PLP (less than the fifth percentile; 18.6 nmol/L) were 0.78, 0.78, and 0.74, respectively, compared with 0.65 for HK. Oral treatment with 40 mg pyridoxin hydrochloride for 28 d reduced the ratios by up to 60%, with strongest reductions for subjects with low plasma PLP at baseline. Whereas HK was associated with kidney function and several inflammatory markers, such associations were abolished or attenuated for the ratios. Plasma values of HK:XA and HK:HAA, which are substrate-product pairs for kynurenine transaminase and kynureninase, respectively, may reflect the intracellular availability of the cofactor (PLP) and, therefore, present as potential markers of functional vitamin B-6 status.

Assessment of Urinary Betaine as a Marker of Diabetes Mellitus in Cardiovascular Patients

Abnormal urinary excretion of betaine has been demonstrated in patients with diabetes or metabolic syndrome. We aimed to identify the main predictors of excretion in cardiovascular patients and to make initial assessment of its feasibility as a risk marker of future diabetes development. We used data from 2396 patients participating in the Western Norway B-vitamin Intervention Trial, who delivered urine and blood samples at baseline, and in the majority at two visits during follow-up of median 39 months. Betaine in urine and plasma were measured by liquid-chromatography-tandem mass spectrometry. The strongest determinants of urinary betaine excretion by multiple regression were diabetes mellitus, age and estimated glomerular filtration rate; all p<0.001. Patients with diabetes mellitus (n = 264) had a median excretion more than three times higher than those without. We found a distinct non-linear association between urinary betaine excretion and glycated hemoglobin, with a break-point at 6.5%, and glycated hemoglobin was the strongest determinant of betaine excretion in patients with diabetes mellitus. The discriminatory power for diabetes mellitus corresponded to an area under the curve by receiver-operating characteristics of 0.82, and betaine excretion had a coefficient of reliability of 0.73. We also found a significant, independent log-linear relation between baseline betaine excretion and the risk of developing new diabetes during follow-up. The good discriminatory power for diabetes, high test-retest stability and independent association with future risk of new diabetes should motivate further investigation on the role of betaine excretion in risk assessment and long-term follow-up of diabetes mellitus.

The Association between Progression of Atherosclerosis and the Methylated Amino Acids Asymmetric Dimethylarginine and Trimethyllysine

ObjectiveWe previously showed that treatment with folic acid (FA)/B12 was associated with more rapid progression of coronary artery disease (CAD). High doses of FA may induce methylation by increasing the availability of S-adenosyl-methionine (SAM). Asymmetric dimethylarginine (ADMA) and trimethyllysine (TML) are both produced through proteolytic release following post-translational SAM–dependent methylation of precursor amino acid. ADMA has previously been associated with CAD. We investigated if plasma levels of ADMA and TML were associated with progression of CAD as measured by quantitative coronary angiography (QCA).Methods183 patients from the Western Norway B Vitamin Intervention Trial (WENBIT) undergoing percutaneous coronary intervention (PCI) were randomized to daily treatment with 0.8 mg FA/0.4 mg B12 with and without 40 mg B6, B6 alone or placebo. Coronary angiograms and plasma samples of ADMA and TML were obtained at both baseline and follow-up (median 10.5 months). The primary end-point was progression of CAD as measured by diameter stenosis (DS) evaluated by linear quantile mixed models.ResultsA total of 309 coronary lesions not treated with PCI were identified. At follow-up median (95% CI) DS increased by 18.35 (5.22–31.49) percentage points per µmol/L ADMA increase (p-value 0.006) and 2.47 (0.37–4.58) percentage points per µmol/L TML increase (p-value 0.021) in multivariate modeling. Treatment with FA/B12 (±B6) was not associated with ADMA or TML levels.ConclusionIn patients with established CAD, baseline ADMA and TML was associated with angiographic progression of CAD. However, neither ADMA nor TML levels were altered by treatment with FA/B12 (±B6).Trial RegistrationControlled-Trials.com NCT00354081

Abstract 18411: Dietary Intake of n-3 Long-Chain Polyunsaturated Fatty Acids, Diabetes Mellitus and Risk of Myocardial Infarction in Patients with Suspected Coronary Artery Disease

Background: A high intake of n-3 long-chain polyunsaturated fatty acids (LCPUFAs) is usually recommended in secondary prevention of coronary heart disease, particularly in patients with high triglyceride levels. Furthermore, a beneficial effect has been observed in patients with heart failure, who are frequently insulin resistant. Objective: The aim was to study the influence of impaired glucose metabolism and diabetes mellitus on the relation between dietary intake of n-3 LCPUFAs and risk of acute myocardial infarction (AMI) in patients undergoing coronary angiography for suspected coronary artery disease in 2000-2004. Design: This study included 2378 participants of the Western Norway B-Vitamin Intervention Trial (WENBIT). Daily intakes of n-3 LCPUFAs were estimated based on average dietary intakes during the last year as reported in a food-frequency questionnaire at baseline. Hazard ratios (HRs (95% CI)) of AMI (fatal and non-fatal) according to n-3 LCPUFA intake were calculated using Cox-regression adjusted for cardiovascular risk factors by comparing upper vs. lowest tertile and as trend across tertiles. Patients were followed until 31 December 2006. Results: Mean age of the participants was 62 years and 80% were male. Patients were sub-grouped into non-diabetics (HbA1c &lt;5.7%), pre-diabetics (HbA1c ≥5.7%), and diabetics (diagnosed), and were also stratified according to median triglyceride levels. Among patients with diabetes there was a significantly reduced risk of AMI in the upper vs. the lowest tertile of n-3 LCPUFA intake, multivariate HR (95% CIs) 0.34 (0.14, 0.82), P for trend=0.008. In non-diabetic patients with HbA1c &lt;5.7% there was an increased risk of AMI in the upper tertile of n-3 LCPUFA intake, 1.97 (1.05, 3.67), P for trend=0.02. Risk estimates were strengthened in their respective directions in both diabetic and non-diabetic patients with triglyceride levels above median. No risk associations were seen in pre-diabetics. Conclusions: Among patients with established coronary artery disease, a high intake of n-3 LCPUFAs was associated with a reduced risk of AMI in diabetic patients and an increased risk of AMI in non-diabetic patients. The effects were particularly pronounced in those with elevated triglyceride levels.

Coronary blood flow in patients with stable coronary artery disease treated long term with folic acid and vitamin B12

Plasma concentration of total homocysteine is associated with risk of cardiovascular disease in epidemiological studies. We wanted to examine the effects of B-vitamin therapy, which may lower total homocysteine, on coronary flow and vascular function in patients with established coronary artery disease (CAD). Forty patients with stable CAD, mean (standard deviation) aged 57.8 (9.0) years, recruited into the Western Norway B-Vitamin Intervention Trial, were randomly assigned to daily oral treatment with 0.8 mg of folic acid and 0.4 mg of vitamin B12 or placebo, and 40 mg of vitamin B6 or placebo, using a 2 × 2 factorial design. At baseline, and after 9 and 24 months, coronary blood flow was assessed by coronary angiography and Doppler flow-wire measurements during intracoronary infusion of saline (basal), incremental doses of acetylcholine, adenosine, and nitroglycerin. We found a significant increase in basal (P < 0.02) and adenosine-induced (P < 0.05) coronary blood flow in patients who received folic acid/vitamin B12 for 24 months, compared with placebo or vitamin B6 alone. Folic acid/vitamin B12 or vitamin B6 treatment did not change endothelial-dependent response after acetylcholine infusion or flow-dependent proximal dilatation in response to adenosine-induced maximal hyperemia (P ≥ 0.45). Long-term treatment with a combination of folic acid and vitamin B12 increase basal and adenosine-induced maximal coronary blood flow. This may reflect improved microvascular function in patients with stable CAD.

Effect of Homocysteine-Lowering B Vitamin Treatment on Angiographic Progression of Coronary Artery Disease: A Western Norway B Vitamin Intervention Trial (WENBIT) Substudy

Total plasma homocysteine (tHcy) is an independent risk factor for coronary artery disease, and tHcy is lowered by B vitamins. To assess the effect of homocysteine-lowering B-vitamin treatment on angiographic progression of coronary artery disease, this substudy of the Western Norway B Vitamin Intervention Trial (WENBIT) included patients who had undergone percutaneous coronary intervention. The patients were randomized to daily oral treatment with folic acid, vitamin B(12), and vitamin B(6) or placebo in a 2 x 2 factorial design. The coronary angiograms obtained at baseline and follow-up were evaluated. The primary angiographic end points were the changes in minimum lumen diameter and diameter stenosis. A total of 348 subjects (288 men) with a mean +/- SD age of 60 +/- 10.2 years were followed up for a median of 10.5 months (twenty-fifth, seventy-fifth percentile 9.2, 11.8). The baseline median plasma tHcy level was 10.0 mumol/L (twenty-fifth, seventy-fifth percentile 8.1, 11.0), and treatment with folic acid/vitamin B(12) lowered the tHcy levels by 22%. At follow-up, we found 309 lesions with a significant decrease from baseline in the minimum lumen diameter of a mean of -0.16 +/- 0.4 mm and an increase in the diameter stenosis of 4.4 +/- 0.7%. Treatment with folic acid/vitamin B(12) or vitamin B(6) was not associated with a change in diameter stenosis or minimum lumen diameter. In a post hoc analysis, folic acid/vitamin B(12) treatment was significantly associated with rapid progression (odds ratio 1.84, 95% confidence interval 1.07 to 3.18). In conclusion, vitamin B treatment showed no beneficial effect on the angiographic progression of coronary artery disease, and the post hoc analyses suggested that folic acid/vitamin B(12) treatment might promote more rapid progression.

Homocysteine‐lowering therapy does not affect inflammatory markers of atherosclerosis in patients with stable coronary artery disease

A high level of total homocysteine (tHcy) is a risk marker for cardiovascular disease (CVD), and is related to inflammation. We wanted to test the effect of homocysteine-lowering B-vitamin therapy, as used in the Western Norway B-vitamin Intervention Trial (WENBIT), on inflammatory markers associated with atherosclerosis. Single centre, prospective double-blind clinical interventional study, randomised in a 2 x 2 factorial design. Ninety patients (21 female) with suspected coronary artery disease (CAD), aged 38-80 years, were blindly randomised into one of four groups of daily oral treatment with (A) folic acid (0.8 mg)/vitamin B12 (0.4 mg)/vitamin B6 (40 mg), (B) folic acid/vitamin B12, (C) vitamin B6 alone or (D) placebo. Blood samples were collected before and after 6 months of treatment. Before intervention, median levels of the analytes were: tHcy 11.0 micromol L(-1), neopterin 8.1 nmol L(-1), soluble CD40 ligand (sCD40L) 3.9 ng mL(-1), interleukin (IL)-6 1.9 pg mL(-1), C-reactive protein (CRP) 1.9 mg L(-1) and low-density lipoprotein (LDL) cholesterol 3.3 mmol L(-1). tHcy was significantly associated with neopterin (r = 0.49, P < 0.001) and with IL-6 (r = 0.29, P = 0.01), but not with CRP or sCD40L. Neither treatment with folic acid/B12 nor with B6 induced significant changes in any of these inflammatory biomarkers (P >or= 0.14). In patients receiving folic acid/B12 (groups A and B), tHcy was reduced with 33% (P < 0.001). In patients with stable CAD, homocysteine-lowering therapy with B-vitamins does not affect levels of inflammatory markers associated with atherogenesis. Failure to reverse inflammatory processes, may partly explain the negative results in clinical secondary B-vitamin intervention trials.

Changes in basal and postmethionine load concentrations of total homocysteine and cystathionine after B vitamin intervention

Vitamin B-6 is necessary for the metabolism of homocysteine and is often used in combination with folic acid and vitamin B-12 in clinical trials that investigate whether the lowering of plasma total homocysteine (tHcy) can prevent vascular disease. We compared the effects of vitamin B-6 with the effects of folic acid and vitamin B-12, as used in the Western Norway B-vitamin Intervention Trial (WENBIT), on basal and postmethionine load (PML) tHcy and cystathionine concentrations. Ninety patients with suspected coronary artery disease were randomly assigned to 1 of 4 groups to receive daily oral treatment with 1) 0.8 mg folic acid, 0.4 mg vitamin B-12, and 40 mg vitamin B-6 (group A); 2) 0.8 mg folic acid and 0.4 mg vitamin B-12 (group B); 3) 40 mg vitamin B-6 (group C); or 4) placebo (group D). For the first 2 wk, groups A and B received additional folic acid (5 mg/d). A methionine-loading test was performed at baseline and after 3 mo. Treatment with folic acid and vitamin B-12 caused a rapid and significant lowering of basal (31%) and PML tHcy concentrations (22%), with no effect on cystathionine. Vitamin B-6 did not change basal tHcy and had a significant but limited effect on PML tHcy concentrations. However, vitamin B-6 treatment markedly lowered basal and PML cystathionine by 31% and 42%, respectively. The folic acid and vitamin B-12 combination applied in WENBIT provides rapid, substantial, and long-term tHcy-lowering effects, whereas the effect of vitamin B-6 on tHcy was relatively small and confined to PML tHcy. However, vitamin B-6 treatment caused a marked reduction in plasma cystathionine. Cystathionine could be a useful marker for assessment of the vitamin B-6 effect and should, together with tHcy, be related to clinical outcome in ongoing trials.