Measuring diet by metabolomics: a 14-d controlled feeding study of weighed food intake.

BACKGROUND: Metabolomics has the potential to enhance dietary assessment by revealing objective measures of many aspects of human food intake. Although metabolomics studies indicate that hundreds of metabolites are associated with dietary intake, correlations have been modest (e.g., r < 0.50), and few have been evaluated in controlled feeding studies. OBJECTIVES: The aim of this study was to evaluate associations between metabolites and weighed food and beverage intake in a controlled feeding study of habitual diet. METHODS: Healthy postmenopausal females from the Women's Health Initiative (N = 153) were provided with a customized 2-wk controlled diet designed to emulate their usual diet. Metabolites were measured by liquid chromatography tandem mass spectrometry in end-of-study 24-h urine and fasting serum samples (1293 urine metabolites; 1113 serum metabolites). We calculated partial Pearson correlations between these metabolites and intake of 65 food groups, beverages, and supplements during the feeding study. The threshold for significance was Bonferroni-adjusted to account for multiple testing (5.94 × 10-07 for urine metabolites; 6.91 × 10-07 for serum metabolites). RESULTS: Significant diet-metabolite correlations were identified for 23 distinct foods, beverages, and supplements (171 distinct metabolites). Among foods, strong metabolite correlations (r ≥ 0.60) were evident for citrus (highest r = 0.80), dairy (r = 0.65), and broccoli (r = 0.63). Among beverages and supplements, strong correlations were evident for coffee (r = 0.86), alcohol (r = 0.69), multivitamins (r = 0.69), and vitamin E supplements (r = 0.65). Moderate correlations (r = 0.50-0.60) were also observed for avocado, fish, garlic, grains, onion, poultry, and black tea. Correlations were specific; each metabolite correlated with one food, beverage, or supplement, except for metabolites correlated with juice or multivitamins. CONCLUSIONS: Metabolite levels had moderate to strong correlations with weighed intake of habitually consumed foods, beverages, and supplements. These findings exceed in magnitude those previously observed in population studies and exemplify the strong potential of metabolomics to contribute to nutrition research. The Women's Health Initiative is registered at clinicaltrials.gov as NCT00000611.

Serum Metabolomic Profiling of All-Cause Mortality: A Prospective Analysis in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study Cohort.

Tobacco use, hypertension, hyperglycemia, overweight, and inactivity are leading causes of overall and cardiovascular disease (CVD) mortality worldwide, yet the relevant metabolic alterations responsible are largely unknown. We conducted a serum metabolomic analysis of 620 men in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (1985-2013). During 28 years of follow-up, there were 435 deaths (197 CVD and 107 cancer). The analysis included 406 known metabolites measured with ultra-high-performance liquid chromatography/mass spectrometry-gas chromatography/mass spectrometry. We used Cox regression to estimate mortality hazard ratios for a 1-standard-deviation difference in metabolite signals. The strongest associations with overall mortality were N-acetylvaline (hazard ratio (HR) = 1.28; P < 4.1 × 10-5, below Bonferroni statistical threshold) and dimethylglycine, 7-methylguanine, C-glycosyltryptophan, taurocholate, and N-acetyltryptophan (1.23 ≤ HR ≤ 1.32; 5 × 10-5 ≤ P ≤ 1 × 10-4). C-Glycosyltryptophan, 7-methylguanine, and 4-androsten-3ß,17ß-diol disulfate were statistically significantly associated with CVD mortality (1.49 ≤ HR ≤ 1.62, P < 4.1 × 10-5). No metabolite was associated with cancer mortality, at a false discovery rate of <0.1. Individuals with a 1-standard-deviation higher metabolite risk score had increased all-cause and CVD mortality in the test set (HR = 1.4, P = 0.05; HR = 1.8, P = 0.003, respectively). The several serum metabolites and their composite risk score independently associated with all-cause and CVD mortality may provide potential leads regarding the molecular basis of mortality.

Nutritional metabolomics and breast cancer risk in a prospective study.

Background: The epidemiologic evidence for associations between dietary factors and breast cancer is weak and etiologic mechanisms are often unclear. Exploring the role of dietary biomarkers with metabolomics can potentially facilitate objective dietary characterization, mitigate errors related to self-reported diet, agnostically test metabolic pathways, and identify mechanistic mediators.Objective: The aim of this study was to evaluate associations of diet-related metabolites with the risk of breast cancer in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial.Design: We examined prediagnostic serum concentrations of diet-related metabolites in a nested case-control study in 621 postmenopausal invasive breast cancer cases and 621 matched controls in the multicenter PLCO cohort. We calculated partial Pearson correlations between 617 metabolites and 55 foods, food groups, and vitamin supplements on the basis of the 2015 Dietary Guidelines for Americans and derived from a 137-item self-administered food-frequency questionnaire. Diet-related metabolites (P-correlation < 1.47 × 10-6) were evaluated in breast cancer analyses. ORs for the 90th compared with the 10th percentile were calculated by using conditional logistic regression, with body mass index, physical inactivity, other breast cancer risk factors, and caloric intake controlled for (false discovery rate <0.2).Results: Of 113 diet-related metabolites, 3 were associated with overall breast cancer risk (621 cases): caprate (10:0), a saturated fatty acid (OR: 1.77; 95% CI = 1.28, 2.43); γ-carboxyethyl hydrochroman (γ-CEHC), a vitamin E (γ-tocopherol) derivative (OR: 1.64; 95% CI: 1.18, 2.28); and 4-androsten-3ß,17ß-diol-monosulfate (1), an androgen (OR: 1.61; 95% CI: 1.20, 2.16). Nineteen metabolites were significantly associated with estrogen receptor (ER)-positive (ER+) breast cancer (418 cases): 12 alcohol-associated metabolites, including 7 androgens and α-hydroxyisovalerate (OR: 2.23; 95% CI: 1.50, 3.32); 3 vitamin E (tocopherol) derivatives (e.g., γ-CEHC; OR: 1.80; 95% CI: 1.20, 2.70); butter-associated caprate (10:0) (OR: 1.81; 95% CI: 1.23, 2.67); and fried food-associated 2-hydroxyoctanoate (OR: 1.46; 95% CI: 1.03, 2.07). No metabolites were significantly associated with ER-negative breast cancer (144 cases).Conclusions: Prediagnostic serum concentrations of metabolites related to alcohol, vitamin E, and animal fats were moderately strongly associated with ER+ breast cancer risk. Our findings show how nutritional metabolomics might identify diet-related exposures that modulate cancer risk. This trial was registered at clinicaltrials.gov as NCT00339495.

Serum Metabolomic Response to Long-Term Supplementation with all-rac-α-Tocopheryl Acetate in a Randomized Controlled Trial.

Background. The Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, a randomized controlled cancer prevention trial, showed a 32% reduction in prostate cancer incidence in response to vitamin E supplementation. Two other trials were not confirmatory, however. Objective. We compared the change in serum metabolome of the ATBC Study participants randomized to receive vitamin E to those who were not by randomly selecting 50 men from each of the intervention groups (50 mg/day all-rac-α-tocopheryl acetate (ATA), 20 mg/day ß-carotene, both, placebo). Methods. Metabolomic profiling was conducted on baseline and follow-up fasting serum (Metabolon, Inc.). Results. After correction for multiple comparisons, five metabolites were statistically significantly altered (ß is the change in metabolite level expressed as number of standard deviations on the log scale): α-CEHC sulfate (ß = 1.51, p = 1.45 × 10-38), α-CEHC glucuronide (ß = 1.41, p = 1.02 × 10-31), α-tocopherol (ß = 0.97, p = 2.22 × 10-13), γ-tocopherol (ß = -0.90, p = 1.76 × 10-11), and ß-tocopherol (ß = -0.73, p = 9.40 × 10-8). Glutarylcarnitine, beta-alanine, ornithine, and N6-acetyllysine were also decreased by ATA supplementation (ß range 0.40 to -0.36), but not statistically significantly. Conclusions. Comparison of the observed metabolite alterations resulting from ATA supplementation to those in other vitamin E trials of different populations, dosages, or formulations may shed light on the apparently discordant vitamin E-prostate cancer risk findings.

Comparing metabolite profiles of habitual diet in serum and urine.

BACKGROUND: Diet plays an important role in chronic disease etiology, but some diet-disease associations remain inconclusive because of methodologic limitations in dietary assessment. Metabolomics is a novel method for identifying objective dietary biomarkers, although it is unclear what dietary information is captured from metabolites found in serum compared with urine. OBJECTIVE: We compared metabolite profiles of habitual diet measured from serum with those measured from urine. DESIGN: We first estimated correlations between consumption of 56 foods, beverages, and supplements assessed by a food-frequency questionnaire, with 676 serum and 848 urine metabolites identified by untargeted liquid chromatography mass spectrometry, ultra-high performance liquid chromatography tandem mass spectrometry, and gas chromatography mass spectrometry in a colon adenoma case-control study (n = 125 cases and 128 controls) while adjusting for age, sex, smoking, fasting, case-control status, body mass index, physical activity, education, and caloric intake. We controlled for multiple comparisons with the use of a false discovery rate of <0.1. Next, we created serum and urine multiple-metabolite models to predict food intake with the use of 10-fold crossvalidation least absolute shrinkage and selection operator regression for 80% of the data; predicted values were created in the remaining 20%. Finally, we compared predicted values with estimates obtained from self-reported intake for metabolites measured in serum and urine. RESULTS: We identified metabolites associated with 46 of 56 dietary items; 417 urine and 105 serum metabolites were correlated with ≥1 food, beverage, or supplement. More metabolites in urine (n = 154) than in serum (n = 39) were associated uniquely with one food. We found previously unreported metabolite associations with leafy green vegetables, sugar-sweetened beverages, citrus, added sugar, red meat, shellfish, desserts, and wine. Prediction of dietary intake from multiple-metabolite profiles was similar between biofluids. CONCLUSIONS: Candidate metabolite biomarkers of habitual diet are identifiable in both serum and urine. Urine samples offer a valid alternative or complement to serum for metabolite biomarkers of diet in large-scale clinical or epidemiologic studies.

Serum biomarkers of habitual coffee consumption may provide insight into the mechanism underlying the association between coffee consumption and colorectal cancer.

BACKGROUND: Coffee intake may be inversely associated with colorectal cancer; however, previous studies have been inconsistent. Serum coffee metabolites are integrated exposure measures that may clarify associations with cancer and elucidate underlying mechanisms. OBJECTIVES: Our aims were 2-fold as follows: 1) to identify serum metabolites associated with coffee intake and 2) to examine these metabolites in relation to colorectal cancer. DESIGN: In a nested case-control study of 251 colorectal cancer cases and 247 matched control subjects from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, we conducted untargeted metabolomics analyses of baseline serum by using ultrahigh-performance liquid-phase chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Usual coffee intake was self-reported in a food-frequency questionnaire. We used partial Pearson correlations and linear regression to identify serum metabolites associated with coffee intake and conditional logistic regression to evaluate associations between coffee metabolites and colorectal cancer. RESULTS: After Bonferroni correction for multiple comparisons (P = 0.05 ÷ 657 metabolites), 29 serum metabolites were positively correlated with coffee intake (partial correlation coefficients: 0.18-0.61; P < 7.61 × 10(-5)); serum metabolites most highly correlated with coffee intake (partial correlation coefficients >0.40) included trigonelline (N'-methylnicotinate), quinate, and 7 unknown metabolites. Of 29 serum metabolites, 8 metabolites were directly related to caffeine metabolism, and 3 of these metabolites, theophylline (OR for 90th compared with 10th percentiles: 0.44; 95% CI: 0.25, 0.79; P-linear trend = 0.006), caffeine (OR for 90th compared with 10th percentiles: 0.56; 95% CI: 0.35, 0.89; P-linear trend = 0.015), and paraxanthine (OR for 90th compared with 10th percentiles: 0.58; 95% CI: 0.36, 0.94; P-linear trend = 0.027), were inversely associated with colorectal cancer. CONCLUSIONS: Serum metabolites can distinguish coffee drinkers from nondrinkers; some caffeine-related metabolites were inversely associated with colorectal cancer and should be studied further to clarify the role of coffee in the cause of colorectal cancer. The Prostate, Lung, Colorectal, and Ovarian trial was registered at clinicaltrials.gov as NCT00002540.

Testing multiple biological mediators simultaneously.

MOTIVATION: Modern biomedical and epidemiological studies often measure hundreds or thousands of biomarkers, such as gene expression or metabolite levels. Although there is an extensive statistical literature on adjusting for 'multiple comparisons' when testing whether these biomarkers are directly associated with a disease, testing whether they are biological mediators between a known risk factor and a disease requires a more complex null hypothesis, thus offering additional methodological challenges. RESULTS: We propose a permutation approach that tests multiple putative mediators and controls the family wise error rate. We demonstrate that, unlike when testing direct associations, replacing the Bonferroni correction with a permutation approach that focuses on the maximum of the test statistics can significantly improve the power to detect mediators even when all biomarkers are independent. Through simulations, we show the power of our method is 2-5× larger than the power achieved by Bonferroni correction. Finally, we apply our permutation test to a case-control study of dietary risk factors and colorectal adenoma to show that, of 149 test metabolites, docosahexaenoate is a possible mediator between fish consumption and decreased colorectal adenoma risk. AVAILABILITY AND IMPLEMENTATION: R-package included in online Supplementary Material.

Metabolomic profile of response to supplementation with ß-carotene in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study.

BACKGROUND: Two chemoprevention trials found that supplementation with ß-carotene increased the risk of lung cancer and overall mortality. The biologic basis of these findings remains poorly understood. OBJECTIVE: The objective was to compare the on-study change in metabolomic profiles of men randomly assigned to receive or not receive ß-carotene supplements in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study. DESIGN: The ATBC Study was a randomized, double-blind, placebo-controlled, primary cancer prevention trial; participants were Finnish male smokers assigned to 1 of 4 intervention groups: 1) α-tocopherol, 2) ß-carotene, 3) both, or 4) placebo. Fifty participants with both baseline and follow-up fasting serum samples were randomly selected from each of these groups. Metabolomic profiling was conducted by mass spectrometry. The association between change in each metabolite over time and trial assignment (ß-carotene or no ß-carotene) was estimated by linear regression. RESULTS: We measured 489 metabolites, and 17 changed significantly (P < 0.05) in response to ß-carotene supplementation. More of these 17 metabolites were of xenobiotic origin than would be expected by chance (9 of 60, or 15%; P = 0.00004). We also found a suggestive association with 1,5-anhydroglucitol-a marker of glycemic control (ß = -0.379, P = 0.0071). CONCLUSIONS: Male smokers supplemented with ß-carotene developed metabolomic profiles consistent with the induction of cytochrome P450 enzymes, the primary metabolizers of xenobiotics in humans. These findings may shed light on the increased mortality associated with ß-carotene supplementation in the ATBC Study and suggest the need to explore potential interactions between medication use and dietary supplements, particularly among smokers. This trial was registered at clinicaltrials.gov as NCT00342992.

Association of variants in two vitamin e transport genes with circulating vitamin e concentrations and prostate cancer risk.

Significant reductions in prostate cancer incidence and mortality were observed in men randomized to receive 50 mg supplemental vitamin E (alpha-tocopherol) per day in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study. We hypothesized that variation in key vitamin E transport genes might directly affect prostate cancer risk or modify the effects of vitamin E supplementation. Associations between prostate cancer risk and 13 polymorphisms in two genes, TTPA and SEC14L2, were examined in 982 incident prostate cancer cases and 851 controls drawn from the ATBC Study. There was no association between the genetic variants and prostate cancer risk. Significant interactions were observed, however, between two variants in SEC14L2 (IVS11+931A>G and IVS11-896A>T) and the trial alpha-tocopherol supplement such that vitamin E supplementation reduced prostate cancer risk among men who were homozygous for either common allele [odds ratios (OR) and 95% confidence intervals (95% CI), 0.52 (0.30-0.90) and 0.64 (0.46-0.88), respectively] and nonsignificantly increased risk among those who carried one or two copies of either variant allele [ORs and 95% CIs, 1.27 (0.90-1.79) and 1.21 (0.96-1.52), respectively; both P for interaction < 0.05]. Genotype-phenotype analyses revealed significant but modest differences in baseline circulating concentrations of alpha-tocopherol and serum responses to the vitamin E supplementation for several polymorphisms. This study shows that genetic variation in TTPA and SEC14L2 is associated with serum alpha-tocopherol but does not have a direct effect on prostate cancer. Our results do, however, suggest that polymorphisms in SEC14L2 may modify the effect of vitamin supplementation regimens on prostate cancer risk.

Healthy lifestyle and the risk of stroke in women.

BACKGROUND: Healthy lifestyle has been associated with decreased risk of coronary heart disease. In contrast, little is known about its association with stroke risk. METHODS: This is a prospective cohort study among 37 636 women 45 years or older participating in the Women's Health Study. Stroke was self-reported and confirmed by means of medical record review. We considered the following self-reported lifestyle factors: smoking, alcohol consumption, exercise, body mass index, and diet. The health index was calculated from these variables by assigning scores from 0 to 4 to the respective variable categories, with a higher score indicating healthier behavior. Healthy behavior was defined as never smoking, alcohol consumption between 4 and 10.5 drinks per week, exercise 4 or more times per week, body mass index (calculated as weight in kilograms divided by the square of height in meters) less than 22, and a diet high in cereal fiber, folate, and omega-3 fatty acids, with a high ratio of polyunsaturated to saturated fat, and low in trans fat and glycemic load. RESULTS: During 10 years of follow-up, 450 strokes (356 ischemic, 90 hemorrhagic, and 4 undefined) were confirmed. Compared with participants with 0 to 4 health index points (4.3%), women with 17 to 20 health index points (4.7%) had multivariable-adjusted hazard ratios (95% confidence interval) of 0.45 (0.24-0.83; P<.001 for trend) for total stroke, 0.29 (0.14-0.63; P<.001 for trend) for ischemic stroke, and 1.27 (0.37-4.29; P = .62 for trend) for hemorrhagic stroke. CONCLUSIONS: In this large prospective cohort of apparently healthy women, a healthy lifestyle consisting of abstinence from smoking, low body mass index, moderate alcohol consumption, regular exercise, and healthy diet was associated with a significantly reduced risk of total and ischemic stroke but not of hemorrhagic stroke. Our findings underscore the importance of healthy behaviors in the prevention of stroke.

Folate, vitamin B6, multivitamin supplements, and colorectal cancer risk in women.

The authors evaluated associations between intakes of folate and vitamin B(6) and colorectal cancer risk among women enrolled in a randomized trial on aspirin and vitamin E in disease prevention. At baseline (1992-1995), 37,916 US women aged >or=45 years who were free of cancer and cardiovascular disease provided dietary information. During an average of 10.1 years of follow-up (through February 20, 2004), 220 colorectal adenocarcinoma cases were documented. Total folate and vitamin B(6) intakes were not significantly associated with the risk of colorectal cancer. However, dietary intakes of folate and vitamin B(6) were significantly inversely associated with colorectal cancer risk among women who were not taking supplements containing folate and vitamin B(6). Multivariable relative risks among women in the highest quintiles of intake versus the lowest were 1.16 (95% confidence interval (CI): 0.76, 1.79) for total folate, 1.14 (95% CI: 0.77, 1.69) for total vitamin B(6), 0.46 (95% CI: 0.26, 0.81) for dietary folate, and 0.69 (95% CI: 0.41, 1.15) for dietary vitamin B(6). The use of multivitamin supplements was not related to colorectal cancer risk. These findings suggest that higher dietary intakes of folate and vitamin B(6) may reduce the risk of colorectal cancer in women. An alternative explanation is that other factors related to dietary intakes of folate and vitamin B(6) account for the inverse associations.