Association between dietary macronutrient composition and plasma one-carbon metabolites and B-vitamin cofactors in patients with stable angina pectoris.

Elevated plasma concentrations of several one-carbon metabolites are associated with increased CVD risk. Both diet-induced regulation and dietary content of one-carbon metabolites can influence circulating concentrations of these markers. We cross-sectionally analysed 1928 patients with suspected stable angina pectoris (geometric mean age 61), representing elevated CVD risk, to assess associations between dietary macronutrient composition (FFQ) and plasma one-carbon metabolites and related B-vitamin status markers (GC-MS/MS, LC-MS/MS or microbiological assay). Diet-metabolite associations were modelled on the continuous scale, adjusted for age, sex, BMI, smoking, alcohol and total energy intake. Average (geometric mean (95 % prediction interval)) intake was forty-nine (38, 63) energy percent (E%) from carbohydrate, thirty-one (22, 45) E% from fat and seventeen (12, 22) E% from protein. The strongest associations were seen for higher protein intake, i.e. with higher plasma pyridoxal 5'-phosphate (PLP) (% change (95 % CI) 3·1 (2·1, 4·1)), cobalamin (2·9 (2·1, 3·7)), riboflavin (2·4 (1·1, 3·7)) and folate (2·1 (1·2, 3·1)) and lower total homocysteine (tHcy) (-1·4 (-1·9, -0·9)) and methylmalonic acid (MMA) (-1·4 (-2·0, -0·8)). Substitution analyses replacing MUFA or PUFA with SFA demonstrated higher plasma concentrations of riboflavin (5·0 (0·9, 9·3) and 3·3 (1·1, 5·6)), tHcy (2·3 (0·7, 3·8) and 1·3 (0·5, 2·2)) and MMA (2·0 (0·2, 3·9) and 1·7 (0·7, 2·7)) and lower PLP (-2·5 (-5·3, 0·3) and -2·7 (-4·2, -1·2)). In conclusion, a higher protein intake and replacing saturated with MUFA and PUFA were associated with a more favourable metabolic phenotype regarding metabolites associated with CVD risk.

Time-resolved concentrations of serum amino acids, one-carbon metabolites and B-vitamin biomarkers during the postprandial and fasting state: the Postprandial Metabolism in Healthy Young Adults (PoMet) Study.

Metabolomics has been utilised in epidemiological studies to investigate biomarkers of nutritional status and metabolism in relation to non-communicable diseases. However, little is known about the effect of prandial status on several biomarker concentrations. Therefore, the aim of this intervention study was to investigate the effect of a standardised breakfast meal followed by food abstinence for 24 h on serum concentrations of amino acids, one-carbon metabolites and B-vitamin biomarkers. Thirty-four healthy subjects (eighteen males and sixteen females) aged 20-30 years were served a breakfast meal (∼500 kcal) after which they consumed only water for 24 h. Blood samples were drawn before and at thirteen standardised timepoints after the meal. Circulating concentrations of most amino acids and metabolites linked to one-carbon metabolism peaked within the first 3 h after the meal. The branched-chain amino acids steadily increased from 6 or 8 hours after the meal, while proline decreased in the same period. Homocysteine and cysteine concentrations immediately decreased after the meal but steadily increased from 3 and 4 hours until 24 h. FMN and riboflavin fluctuated immediately after the meal but increased from 6 h, while folate increased immediately after the meal and remained elevated during the 24 h. Our findings indicate that accurate reporting of time since last meal is crucial when investigating concentrations of certain amino acids and one-carbon metabolites. Our results suggest a need for caution when interpretating studies, which utilise such biomarkers, but do not strictly control for time since the last meal.

Changes in Plasma Pyruvate and TCA Cycle Metabolites upon Increased Hepatic Fatty Acid Oxidation and Ketogenesis in Male Wistar Rats.

Altered hepatic mitochondrial fatty acid ß-oxidation and associated tricarboxylic acid (TCA) cycle activity contributes to lifestyle-related diseases, and circulating biomarkers reflecting these changes could have disease prognostic value. This study aimed to determine hepatic and systemic changes in TCA-cycle-related metabolites upon the selective pharmacologic enhancement of mitochondrial fatty acid ß-oxidation in the liver, and to elucidate the mechanisms and potential markers of hepatic mitochondrial activity. Male Wistar rats were treated with 3-thia fatty acids (e.g., tetradecylthioacetic acid (TTA)), which target mitochondrial biogenesis, mitochondrial fatty acid ß-oxidation, and ketogenesis predominantly in the liver. Hepatic and plasma concentrations of TCA cycle intermediates and anaplerotic substrates (LC-MS/MS), plasma ketones (colorimetric assay), and acylcarnitines (HPLC-MS/MS), along with associated TCA-cycle-related gene expression (qPCR) and enzyme activities, were determined. TTA-induced hepatic fatty acid ß-oxidation resulted in an increased ratio of plasma ketone bodies/nonesterified fatty acid (NEFA), lower plasma malonyl-CoA levels, and a higher ratio of plasma acetylcarnitine/palmitoylcarnitine (C2/C16). These changes were associated with decreased hepatic and increased plasma pyruvate concentrations, and increased plasma concentrations of succinate, malate, and 2-hydroxyglutarate. Expression of several genes encoding TCA cycle enzymes and the malate-oxoglutarate carrier (Slc25a11), glutamate dehydrogenase (Gdh), and malic enzyme (Mdh1 and Mdh2) were significantly increased. In conclusion, the induction of hepatic mitochondrial fatty acid ß-oxidation by 3-thia fatty acids lowered hepatic pyruvate while increasing plasma pyruvate, as well as succinate, malate, and 2-hydroxyglutarate.

Even chained acylcarnitines predict long-term cardiovascular prognosis in patients with chest pain and non-obstructive coronary artery disease.

Background: Acylcarnitines are essential for mitochondrial fatty acid oxidation. Earlier studies suggest that impaired energy metabolism may be implicated in the pathogenesis of microvascular angina. We explored metabolites from the carnitine pathway as predictors of cardiovascular disease (CVD) - and all-cause mortality among patients with non-obstructive coronary artery disease (NOCAD). Methods: A total of 1046 patients with suspected stable coronary syndrome underwent coronary angiography during 2000-2004, with findings of NOCAD. Serum levels of 8 selected carnitine metabolites were analyzed through liquid chromatography tandem mass spectrometry. Associations with CVD- and all-cause mortality were assessed by multivariable Cox regression models. Results: Median age at inclusion was 57 years. 51.5% were men. During median (25th- 75th percentiles), 14.1 (13.2-15.4) years of follow-up, 5.7% of the participants died from CVD and the incidence of all-cause mortality was 17.3%. Serum acetyl, octanoyl- and palmitoylcarnitine predicted CVD mortality with multivariable HR and 95% CI (per SD increment log transformed) of 1.36 (1.01-1.83), 1.49 (1.15-1.93) and 2.07 (1.49-2.85), p ≤ 0.04, respectively. Higher serum acetyl- and palmitoylcarnitines were also associated with increased risk of all-cause mortality (HR (95% CI): 1.27 (1.01-1.50), and 1.51 (1.26-1.81), p ≤ 0.007. Baseline levels of the precursors trimethyllysine and Æ´-butyrobetaine, carnitine or the odd chained propionylcarnitine and (iso)valerylcarnitine were not associated with adverse outcomes. Conclusion: Elevated serum even-chained acylcarnitines predicted adverse long-term prognosis in NOCAD. The strongest risk estimates were observed for palmitoylcarnitine, which predicted both CVD- and all-cause mortality after extensive multivariable adjustments. Underlying pathomechanisms should be further elucidated.

Plasma Amino Acids and Incident Type 2 Diabetes in Patients With Coronary Artery Disease.

OBJECTIVE: Altered plasma amino acid levels have been implicated as markers of risk for incident type 2 diabetes; however, amino acids are also related to established diabetes risk factors. Therefore, potential for confounding and the impact from competing risks require evaluation. RESEARCH DESIGN AND METHODS: We prospectively followed 2,519 individuals with coronary artery disease but without diabetes. Mixed Gaussian modeling identified potential for confounding. Confounding, defined as a change in effect estimate (≥10%), was investigated by comparing amino acid-incident diabetes risk in a Cox model containing age and sex with that in models adjusted for potential confounders (BMI, estimated glomerular filtration rate, HDL cholesterol, triacylglycerol, C-reactive protein), which were further adjusted for plasma glucose, competing risks, and multiple comparisons (false discovery rate = 0.05, Benjamini-Hochberg method). Finally, component-wise likelihood-based boosting analysis including amino acids and confounders was performed and adjusted for competing risks in order to identify an optimal submodel for predicting incident diabetes. RESULTS: The mean age of the source population was 61.9 years; 72% were men. During a median follow-up of 10.3 years, 267 incident cases of diabetes were identified. In age- and sex-adjusted models, several amino acids, including the branched-chain amino acids, significantly predicted incident diabetes. Adjustment for confounders, however, attenuated associations. Further adjustment for glucose and multiple comparisons rendered only arginine significant (hazard ratio/1 SD 1.21 [95% CI 1.07-1.37]). The optimal submodel included arginine and asparagine. CONCLUSIONS: Adjustment for relevant clinical factors attenuated the amino acid-incident diabetes risk. Although these findings do not preclude the potential pathogenic role of other amino acids, they suggest that plasma arginine is independently associated with incident diabetes. Both arginine and asparagine were identified in an optimal model for predicting new-onset type 2 diabetes.

Metabolomic Evaluation of the Consequences of Plasma Cystathionine Elevation in Adults with Stable Angina Pectoris.

Background: An elevated circulating cystathionine concentration, which arises in part from insufficiencies of vitamin B-6, B-12, or folate, has been shown to be associated with cardiovascular disease (CVD) risk. Hydrogen sulfide (H2S) is a gasotransmitter involved in vasodilation, neuromodulation, and inflammation. Most endogenously produced H2S is formed by pyridoxal phosphate (PLP)-dependent enzymes by noncanonical reactions of the transsulfuration pathway that yield H2S concurrently form lanthionine and homolanthionine. Thus, plasma lanthionine and homolanthionine concentrations can provide relative information about H2S production in vivo.Objective: To determine the metabolic consequences of an elevated plasma cystathionine concentration in adults with stable angina pectoris (SAP), we conducted both targeted and untargeted metabolomic analyses.Methods: We conducted NMR and LC-mass spectrometry (MS) metabolomic analyses on a subset of 80 plasma samples from the Western Norway Coronary Angiography Cohort and selected, based on plasma cystathionine concentrations, a group with high cystathionine concentrations [1.32 ± 0.60 µmol/L (mean ± SD); n = 40] and a group with low cystathionine concentrations [0.137 ± 0.011 µmol/L (mean ± SD); n = 40]. Targeted and untargeted metabolomic analyses were performed and assessed with the use of Student's t tests corrected for multiple testing. Overall differences between the cystathionine groups were assessed by untargeted NMR and LC-MS metabolomic methods and evaluated by partial least squares discriminant analysis (PLS-DA) with significant discriminating metabolites identified with 99% confidence.Results: Subjects with high cystathionine concentrations had 75% higher plasma lanthionine concentrations (0.12 ± 0.044 µmol/L) than subjects with low cystathionine concentrations [0.032 ± 0.013 µmol/L (P < 0.001)]. Although plasma homolanthionine concentrations were notably higher than lanthionine concentrations, they were not different between the groups (P = 0.47). PLS-DA results showed that a high plasma cystathionine concentration in SAP was associated with higher glucose, branched-chain amino acids, and phenylalanine concentrations, lower kidney function, and lower glutathione and plasma PLP concentrations due to greater catabolism. The high-cystathionine group had a greater proportion of subjects in the postprandial state.Conclusion: These data suggest that metabolic perturbations consistent with higher CVD risk exist in SAP patients with elevated plasma cystathionine concentrations.

Smoking and body fat mass in relation to bone mineral density and hip fracture: the Hordaland Health Study.

Lower bone mineral density (BMD) in smokers may be attributable to lower body weight or fat mass, rather than to a direct effect of smoking. We analyzed the effects of smoking exposure, assessed by plasma cotinine, and body fat on BMD and the risk of subsequent hip fracture. In the community-based Hordaland Health Study (HUSK), 3003 participants 46-49 years and 2091 subjects 71-74 years were included. Cotinine was measured in plasma and information on health behaviors was obtained from self-administered questionnaires. BMD and total body soft tissue composition were measured by dual X-ray absorptiometry. Information on hip fracture was obtained from computerized records containing discharge diagnoses for hospitalizations between baseline examinations 1997-2000 through December 31st, 2009. In the whole cohort, moderate and heavy smokers had stronger positive associations between fat mass and BMD compared to never smokers (differences in regression coefficient (95% CI) per % change in fat mass = 1.38 (0.24, 2.52) and 1.29 (0.17, 2.4), respectively). In moderate and heavy smokers there was a nonlinear association between BMD and fat mass with a stronger positive association at low compared to high levels of fat mass (Davies segmented test, p<0.001). In elderly women and men, heavy smokers had an increased risk of hip fracture compared to never smokers (hazard ratio = 3.31, 95% CI: 2.05, 5.35; p<0.001). In heavy smokers there was a tendency of a lower risk of hip fracture with higher percentage of fat mass. The deleterious effect of smoking on bone health is stronger in lean smokers than in smokers with high fat mass.

Plasma choline, nicotine exposure, and risk of low bone mineral density and hip fracture: the Hordaland health study.

Choline, obtained from diet and formed by biosynthesis, is the immediate precursor of betaine. Animal studies suggest an impact of choline on bone metabolism. We examined the associations of plasma choline and betaine with bone mineral density (BMD), the risk of hip fractures, and possible effect-modification by nicotine exposure. The Hordaland Health Study (1998 to 2000) included 7074 women and men (ages 46 to 49 or 71 to 74 years). In 5315, BMD was measured. The oldest (n = 3311) were followed for hip fractures through 2009. Risk associations were studied by logistic and Cox regression by comparing the lowest and middle tertiles with the highest, as well as trends across tertiles of plasma choline and betaine. In analyses adjusted for sex and age, participants in the lowest (odds ratio [OR] = 2.00, 95% confidence interval [CI] 1.69-2.37) and middle (OR = 1.39, CI 1.17-1.66) tertiles of plasma choline had an increased risk of low BMD (lowest quintile) (p trend < 0.001). Separate analyses for sex and age groups revealed the strongest relations in elderly women (lowest tertile: OR = 2.84, CI 1.95-4.14; middle tertile: OR = 1.80, CI 1.22-2.67, p trend < 0.001), and highest OR among those in the lowest tertile who were exposed to nicotine (OR = 4.56, CI 1.87-11.11). Low plasma choline was also associated with an increased risk of hip fracture in elderly women and men (lowest tertile: hazard ratio [HR] = 1.45, CI 1.08-1.94; middle tertile: HR = 1.13, CI 0.83-1.54, p trend = 0.012). In elderly women, the HR for hip fracture was 1.90 (CI 1.32-2.73) and 1.36 (CI 0.92-1.99) (p trend < 0.001) for lowest and middle tertiles of choline, and the highest HR was found among women in the lowest tertile exposed to nicotine (HR = 2.68, CI 1.16-6.19). Plasma betaine was not related to BMD or hip fracture. Low plasma choline was associated with low BMD in both sexes and increased the risk of hip fracture in elderly women. These results should motivate further studies on choline, nicotine exposure, and bone metabolism.

Relative importance of risk factors for coronary heart disease--the Hordaland Homocysteine study.

OBJECTIVES: The aim was to rank coronary heart disease (CHD) risk factors according to their importance in predicting CHD morbidity and mortality using a scale-independent statistical approach. DESIGN: We studied 15 515 community-dwelling adults in a population-based cohort established during 1992-93 in Western Norway. Participants were 40-42 and 65-67 years old at baseline and were followed through 2006. Endpoints were non-fatal/fatal acute myocardial infarction (AMI) and CHD death. Each factor was rank transformed and scaled to the range 0-5 before estimation of Cox models. Hazard ratios (HR) may thus be interpreted as HR per quintile increment for each factor, and the magnitude of the HR was used to rank the risk factors according to strength. RESULTS: Total cholesterol and triglycerides were important risk factors for both CHD death and non-fatal/fatal AMI only in the middle-aged group. Risk factors were generally stronger in the middle-aged, except total homocysteine which was significantly associated with CHD death in the oldest group only. The only significant difference between men and women was found for single living which was an important risk factor for non-fatal/fatal AMI in middle-aged women but not in middle-aged men. CONCLUSIONS: We have demonstrated a simple method for direct and scale-independent comparison of the strength of both categorical and continuous risk factors. The importance of individual risk factors differed substantially between the two age groups.

Competing risk analysis of events 10 years after revascularization.

OBJECTIVES: To evaluate the influence of competing risk (CR) non-cardiac death during long-term follow-up of revascularized patients on the interpretation of the cardiac outcomes. METHODS: Retrospectively, we compared outcomes estimated with the Kaplan-Meier and the cumulative incidence function (CIF) methods after a median 10.8 years follow-up in 1,234 consecutive patients (594 CABG, 640 PCI) undergoing first time non-emergent revascularization in a community cohort. RESULTS: Overall 301 (24.4%) patients died (27.3% in the CABG vs. 21.7% in the PCI group, p = 0.02). The causes of death were cardiac (10.3%) and non-cardiac (14.1%). CR analysis showed a similar probability of cardiac death (CIF 0.10 (95% CI 0.092, 0.18) vs. 0.093 (0.07, 0.12)) in the CABG and PCI treated patients, respectively. The probability for acute myocardial infarction (CIF 0.12 vs. 0.16 p < 0.001), congestive heart failure (CIF 0.15 vs. 0.09 p = 0.007) in the CABG and PCI group respectively, differed. The differences were also statistically significant after multivariate adjustment for the competing risks of death. For all outcomes the Kaplan-Meier method overestimated risk estimates. CONCLUSIONS: The competing risk adjusted probability for cardiac death, but not other cardiac endpoints are comparable in patients treated with either CABG or PCI after very long-term follow-up. The risk for all-cause death was mainly predicted by the occurrence of non-cardiac diseases.

Plasma vitamin B6 vitamers before and after oral vitamin B6 treatment: a randomized placebo-controlled study.

BACKGROUND: Vitamin B(6) has attracted renewed interest because of its role in homocysteine metabolism and its possible relation to cardiovascular risk. We examined the plasma B(6) vitamers, pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), and 4-pyridoxic acid (4-PA) before and after vitamin B(6) supplementation. METHODS: Patients (n = 90; age range, 38-80 years) undergoing coronary angiography (part of the homocysteine-lowering Western Norway B-Vitamin Intervention Trial) were allocated to the following daily oral treatment groups: (A), vitamin B(12) (0.4 mg), folic acid (0.8 mg), and vitamin B(6) (40 mg); (B), vitamin B(12) and folic acid; (C), vitamin B(6); or (D), placebo. EDTA blood was obtained before treatment and 3, 14, 28, and 84 days thereafter. RESULTS: Before treatment, PLP (range, 5-111 nmol/L) and 4-PA (6-93 nmol/L) were the predominant B(6) vitamers identified in plasma. During the 84-day study period, the intraindividual variation (CV) in patients not treated with vitamin B(6) (groups B and D) was 45% for PLP and 67% for 4-PA. Three days after the start of treatment, the increases in concentration were approximately 10-, 50-, and 100-fold for PLP, 4-PA, and PL, respectively. No significant additional increase was observed at the later time points. The PLP concentration correlated to the concentrations of 4-PA and PL before treatment, but not after treatment. The PL concentration correlated with 4-PA before and after treatment. CONCLUSIONS: Vitamin B(6) treatment has an immediate effect on the concentrations and the forms of B(6) vitamers present in plasma, and the changes remain the same during prolonged treatment. Our results suggest that the B(6) vitamers in plasma reflect vitamin B(6) intake.

Holo-transcobalamin is an early marker of changes in cobalamin homeostasis. A randomized placebo-controlled study.

BACKGROUND: We examined the effect of oral vitamin B(12) treatment on fluctuations in plasma total cobalamin and its binding proteins transcobalamin (TC) and haptocorrin (HC). METHODS: Patients (n = 88; age range, 38-80 years) undergoing coronary angiography (part of the homocysteine-lowering Western Norway B-Vitamin Intervention Trial) were allocated to daily oral treatment with (a) vitamin B(12) (0.4 mg), folic acid (0.8 mg), and vitamin B(6) (40 mg); (b) vitamin B(12) and folic acid; (c) vitamin B(6); or (d) placebo. EDTA blood was obtained before treatment and 3, 14, 28, and 84 days thereafter. RESULTS: The intraindividual variation for patients not treated with B(12) was approximately 10% for plasma total cobalamin, total TC, apo-TC, and apo-HC, and <20% for holo-TC and TC saturation. In B(12)-treated patients, the maximum change in concentrations was observed already after 3 days for total TC (-16%), holo-TC (+54%), and TC saturation (+82%). At this time holo-HC (+20%) and plasma total cobalamin (+28%) showed an initial burst, but had increased further at 84 days. All changes were highly significant compared with the control group (P <0.0001). CONCLUSIONS: Oral vitamin B(12) treatment produces maximal effects on total TC, holo-TC, and TC saturation within 3 days, whereas maximal increases in holo-HC and plasma total cobalamin occur later. The results support the view that holo-TC is an early marker of changes in cobalamin homeostasis.