Gut Microbiota Alterations and Circulating Imidazole Propionate Levels Are Associated With Obstructive Coronary Artery Disease in People With HIV.

BACKGROUND: The impact of gut microbiota and its metabolites on coronary artery disease (CAD) in people with human immunodeficiency virus (PWH) is unknown. Emerging evidence suggests that imidazole propionate (ImP), a microbial metabolite, is linked with cardiometabolic diseases. METHODS: Fecal samples from participants of the Copenhagen Comorbidity in HIV infection (COCOMO) study were processed for 16S rRNA sequencing and ImP measured with liquid chromatography-tandem mass spectrometry. CAD severity was investigated by coronary computed tomography-angiography, and participants grouped according to obstructive CAD (n = 60), nonobstructive CAD (n = 80), or no CAD (n = 114). RESULTS: Participants with obstructive CAD had a gut microbiota with lower diversity and distinct compositional shift, with increased abundance of Rumiococcus gnavus and Veillonella, known producers of ImP. ImP plasma levels were associated with this dysbiosis, and significantly elevated in participants with obstructive CAD. However, gut dysbiosis but not plasma ImP was independently associated with obstructive CAD after adjustment for traditional and HIV-related risk factors (adjusted odds ratio, 2.7; 95% confidence interval, 1.1-7.2; P = .048). CONCLUSIONS: PWH with obstructive CAD displays a distinct gut microbiota profile and increased circulating ImP plasma levels. Future studies should determine whether gut dysbiosis and related metabolites such as ImP are predictive of incident cardiovascular events.

Longitudinal associations of plasma kynurenines and ratios with fatigue and quality of life in colorectal cancer survivors up to 12 months post-treatment.

Fatigue is prevalent in colorectal cancer (CRC) survivors, impacting their health-related quality of life (HRQoL). Inflammation-induced activation of the kynurenine pathway may play a role in cancer-related fatigue and HRQoL, but evidence is scarce. Therefore, we aimed to investigate longitudinal associations of plasma tryptophan, kynurenines, and ratios with fatigue and HRQoL in CRC survivors up to 12 months post-treatment. Repeated measurements at 6 weeks, 6 months, and 12 months post-treatment were performed in 249 stage I-III CRC survivors. Plasma tryptophan and eight kynurenines were analyzed using liquid chromatography-tandem mass spectrometry (LC/MS-MS). Fatigue and HRQoL outcomes were evaluated using validated questionnaires. Confounder-adjusted linear mixed models were conducted to analyze longitudinal associations, with false discovery rate (FDR) correction. Higher tryptophan (Trp), kynurenic acid (KA), and xanthurenic acid (XA) concentrations, as well as a higher kynurenic acid-to-quinolinic acid ratio (KA/QA), were associated with less fatigue and better functioning, while a higher kynurenine-to-tryptophan ratio (KTR) and 3-hydroxykynurenine ratio (HKr) were associated with more fatigue and worse functioning. Finally, higher KA and XA concentrations and a higher KA/QA ratio were associated with a higher overall HRQoL summary score, while a higher HKr was associated with a lower overall HRQoL summary score. In conclusion, we observed that tryptophan and several kynurenines were longitudinally associated with fatigue and HRQoL in CRC survivors up to 12 months post-treatment. Future research is needed to validate our findings and explore the potential of the kynurenine pathway as intervention target for reducing fatigue and enhancing HRQoL after CRC treatment.

The effect of vitamin B12 supplementation during pregnancy on infant growth and development in Nepal: a community-based, double-blind, randomised, placebo-controlled trial.

BACKGROUND: Vitamin B12 is required for healthy infant growth and development, but low and marginal vitamin B12 status is endemic in low-income and middle-income countries. We aimed to measure the effect of vitamin B12 supplementation from early pregnancy until 6 months post partum on infant growth and neurodevelopment. METHODS: In this community-based, double-blind, placebo-controlled trial, we randomly assigned (1:1) 800 pregnant women (aged 20-40 years) who were up to 15 weeks pregnant-recruited from home visits and outpatient departments at three hospitals in Nepal-to daily supplementation with 50 µg oral vitamin B12 or placebo until 6 months postpartum. Independent scientists generated the list that linked allocation to participants' study identification number. Participants were masked to group assignment and all investigators were masked until data cleaning was completed. The primary outcomes were length-for-age Z score (LAZ) at age 12 months and the cognitive composite score of the Bayley Scales of Infant and Toddler Development (3rd edition) at age 6 months and 12 months. The primary and secondary outcomes, including adverse events, were assessed in the intention-to-treat population, for all participants with available outcome data. This trial is registered with ClinicalTrials.gov, NCT03071666. FINDINGS: 800 eligible pregnant women were enrolled in the trial between March 28, 2017, and Oct 15, 2020, with 400 women randomly assigned to each group. Follow-up was completed on May 18, 2022. At baseline, 569 (71%) of 800 women had plasma vitamin B12 indicating low or marginal status (<221 pmol/L). We found no effect of vitamin B12 on the primary outcomes. The mean LAZ at age 12 months were -0·57 (SD 1·03) in the B12 group and -0·55 (1.03) in the placebo group (366 infants in the vitamin B12 group vs 363 infants in the placebo group) with a mean difference of -0·02 (95% CI -0·16 to 0·13). The mean cognitive composite scores were 97·7 (SD 10·5) in the B12 group and 97·1 (10·2) in the placebo group, with a mean difference of 0·5 (95% CI -0·6 to 1·7) measured in 364 and 361 infants. Stillbirths or infant deaths occurred in three (1%) of 374 women in the vitamin B12 group and nine (2%) of 379 women in the placebo group. INTERPRETATION: Although vitamin B12 deficiency was prevalent in our study population and vitamin B12 supplementation from early pregnancy substantially improved vitamin B12 status, supplementation did not improve infant growth or neurodevelopment. Our findings support the current WHO recommendations of no routine vitamin B12 supplementation during pregnancy. FUNDING: Research Council of Norway.

Influence of cardiorespiratory fitness on obesity-associated inflammation in women and men: The FATCOR study.

BACKGROUND AND AIMS: Cardiorespiratory fitness has been postulated to lower chronic inflammation in obesity. We assessed sex-specific associations of inflammation with cardiorespiratory fitness in overweight and obese persons. METHODS AND RESULTS: Peak oxygen uptake (VO2max) was measured by treadmill in 566 participants (age 48 ± 9 years, 60% women) with body mass index >27.0 kg/m2 in the FAT associated CardiOvasculaR dysfunction (FATCOR) study. Fitness was identified from age- and sex specific reference levels of VO2max. The inflammatory markers C-reactive protein (CRP), serum amyloid A (SAA), kynurenine:tryptophan ratio (KTR) and pyriodoxic acid ratio (PAr) were measured by mass spectrometry. In the total study population 63% had obesity and 74% were cardiorespiratory unfit. Unfit women had the highest fat percentage and the highest serum levels of CRP and SAA (p < 0.05). In multivariable linear regression analyses in women, higher CRP (ß -0.15, p = 0.001), SAA (ß -0.10, p = 0.03) and PAr (ß -0.09, p = 0.03) were associated with lower VO2max after adjusting for confounders. In multivariable analyses in men, higher PAr (ß -0.14, p = 0.02) was associated with lower VO2max. In multivariable analyses in obese women, higher CRP and PAr remained associated with lower VO2max (p < 0.05), while in obese men there was no significant association. When normalizing VO2max for fat-free mass (VO2maxFFM) higher CRP, SAA and PAr index were associated with lower VO2maxFFM in women, while only higher PAr index was associated with lower VO2maxFFM in men. CONCLUSION: The association of inflammation with lower cardiorespiratory fitness was more pronounced in women than men, in particular when obesity was present. CLINICAL TRIAL REGISTRATION: URL: http://www. CLINICALTRIALS: gov NCT02805478.

Plasma methylmalonic acid predicts risk of acute myocardial infarction and mortality in patients with coronary heart disease: A prospective 2-cohort study.

BACKGROUND: Elevated plasma methylmalonic acid (MMA) is reported in patients with established coronary heart disease (CHD) and is considered a marker of vitamin B12 deficiency. Moreover, MMA-dependent reactions have been linked to alterations in mitochondrial energy metabolism and oxidative stress, key features in the pathophysiology of cardiovascular diseases (CVDs). OBJECTIVES: We examined whether plasma MMA prospectively predicted the long-term risk of acute myocardial infarction (AMI) and mortality. METHODS AND RESULTS: Using Cox modeling, we estimated hazard ratios (HRs) for endpoints according to per 1-SD increment of log-transformed plasma MMA in two independent populations: the Western Norway Coronary Angiography Cohort (WECAC) (patients evaluated for CHD; n = 4137) and the Norwegian Vitamin Trial (NORVIT) (patients hospitalized with AMI; n = 3525). In WECAC and NORVIT, 12.8% and 18.0% experienced an AMI, whereas 21.8% and 19.9% died, of whom 45.5% and 60.3% from CVD-related causes during follow-up (range 3-11 years), respectively. In WECAC, age- and gender-adjusted HRs (95% confidence interval) were 1.18 (1.09-1.28), 1.25 (1.18-1.33), and 1.28 (1.17-1.40) for future AMI, total mortality, and CVD mortality, respectively. Corresponding risk estimates were 1.19 (1.10-1.28), 1.22 (1.14-1.31), and 1.30 (1.19-1.42) in NORVIT. These estimates were only slightly attenuated after multivariable adjustments. Across both cohorts, the MMA-risk association was stronger in older adults, women, and non-smokers. CONCLUSIONS: Elevated MMA was associated with an increased risk of AMI and mortality in patients with suspected or verified CHD.

Vitamin B12 and Folate Status in Pregnant Females and Their Infants in Norway: Secondary Analysis from the Mommy's Food Study.

BACKGROUND: Vitamin B12 and folate are essential micronutrients important for normal infant growth and development. OBJECTIVES: The aims were to describe vitamin B12 and folate status in pregnant females and their infants according to commonly used status cutoffs and examine the associations between maternal status, maternal supplement use, and breastfeeding and infant status. METHODS: Pregnant females were recruited at 18 wk gestation in Bergen, Norway. Maternal vitamin B12 and folate status were measured at gestational weeks 18 (n = 136) and 36 (n = 116), and infant status was measured at ages 3 (n = 73) and 6 (n = 74) mo. RESULTS: At gestational weeks 18 and 36, respectively, 4.4% and 2.6% of the mothers had plasma cobalamin concentrations <148 pmol/L, 0.7% and 6.9% had methylmalonic acid (MMA) concentrations >0.26 µmol/L, and 3.7% and 30% had folate concentrations <10 nmol/L. None of the females had total homocysteine (t-Hcy) concentrations >13 µmol/L or 3 combined indicator of vitamin B12 (cB12) < -0.5. At 3 and 6 mo, respectively, 4.1% and 5.4% of the infants had cobalamin concentrations <148 pmol/L, 63% and 74% had t-Hcy concentrations >6.5 µmol/L, 59% and 66% had MMA concentrations >0.26 µmol/L, and 47% and 60% had cB12 > -0.5. None of the infants had folate concentrations <10 nmol/L. Several of the vitamin B12 biomarkers in infants were associated with maternal vitamin B12 status during pregnancy. Breastfed infants had lower vitamin B12 status (as indicated by plasma cobalamin, t-Hcy, and cB12) than nonbreastfed infants at both 3 and 6 mo. Use of supplements during pregnancy was associated with better vitamin B12 status among infants at 3 and 6 mo, as indicated by infants' cobalamin and t-Hcy concentrations. CONCLUSIONS: Subclinical vitamin B12 deficiency among infants was common and associated with maternal vitamin B12 status during pregnancy and breastfeeding. Among the mothers, an increase in biochemical folate deficiency was discovered toward the end of gestation. Further studies are needed to investigate clinical consequences. This trial was registered at clinicaltrials.gov as NCT02610959.

The Effect of Vitamin B12 Supplementation on Leukocyte Telomere Length in Mildly Stunted Nepalese Children: A Secondary Outcome of a Randomized Controlled Trial.

BACKGROUND: Vitamin B12 is essential for deoxyribonucleic acid synthesis and genome stability. A deficiency of vitamin B12 is associated with telomere shortening, genomic aging, and increased risk of chronic disease and mortality. OBJECTIVES: The study aims to determine the effect of vitamin B12 supplementation on leukocyte telomere length (LTL) in infants at risk of vitamin B12 deficiency. METHODS: The study was a predefined secondary analysis of a randomized controlled trial enrolling 600 Nepalese infants aged 6 -11 mo, who were supplemented with 2 µg (2-3 recommended daily allowances) vitamin B12 or placebo daily for 1 y. At the end of the study, LTL was measured in 497 participants. Mean LTL was compared between the treatment arms in the full sample and predefined subgroups based on markers of vitamin B12 status, hemoglobin, sex, and growth indices. RESULTS: LTL at end-study did not differ between the vitamin B12 and placebo arm with a standardized mean difference (95% confidence interval) of 0.04 (-0.14, 0.21). There was no effect of vitamin B12 on LTL in any of the subgroups. CONCLUSIONS: Providing daily vitamin B12 for 1 y during infancy in a population at risk of vitamin B12 deficiency does not affect LTL. This trial was registered at clinicaltrials.gov as NCT02272842.

Correlations between kynurenines in plasma and CSF, and their relation to markers of Alzheimer's disease pathology.

INTRODUCTION: Altered levels of kynurenines in blood and cerebrospinal fluid (CSF) have been reported in Alzheimer's disease (AD). However, it is still largely unknown whether peripheral kynurenine concentrations resemble those found in CSF and how they relate to AD pathology. We therefore studied correlations between kynurenines in plasma and CSF and their associations with CSF amyloid-beta (Aß1-42) and tau levels in patients from the memory clinic spanning the whole cognitive spectrum. METHODS: The Biobank Alzheimer Center Limburg study is a prospective cohort study of consecutive patients referred to the memory clinic of the Alzheimer Center Limburg. Plasma and CSF concentrations of tryptophan (TRP), eight kynurenines and neopterin from 138 patients were determined by means of LC-MS/MS. Additionally, CSF Aß1-42, total-tau (t-tau) and phosphorylated tau (p-tau) concentrations were determined using commercially available single-parameter ELISA methods. Partial correlations were used to analyze cross-sectional associations between kynurenines in plasma and CSF and their relation to AD related CSF-biomarkers adjusted for age, sex, educational level, and kidney function. RESULTS: Moderate to strong correlations were observed between plasma and CSF levels for quinolinic acid (QA; r = 0.63), TRP (r = 0.47), anthranilic acid (r = 0.59), picolinic acid (r = 0.55), and the kynurenine (KYN)/TRP ratio (KTR; r = 0.55; all p < 0.0001), while other kynurenines correlated only weakly with their corresponding CSF values. No correlations were found between plasma and CSF levels of KA/QA. Several kynurenines were also weakly correlated with Aß1-42, t-tau or p-tau. Plasma levels of KA/QA were negatively correlated with Aß1-42 (r = -0.21, p < 0.05). Plasma levels of TRP were negatively correlated with t-tau (r = -0.19) and levels of KYN with p-tau (r = -0.18; both p < 0.05). CSF levels of KYN (r = 0.20, p < 0.05), KA (r = 0.23, p < 0.01), and KTR (r = 0.18, p < 0.05) were positively correlated with Aß1-42. Finally, TRP and KYN were negatively (r = -0.22 and r = -0.18, respectively), and neopterin positively (r = 0.19) correlated with p-tau (all p < 0.05). CONCLUSIONS: Plasma concentrations of TRP, KP metabolites, KTR, and neopterin all significantly correlated positively with their corresponding CSF concentrations, but many correlations were weak. Additionally, our results suggest a relation between higher kynurenine levels and lower AD pathology load. These results need verification in future studies and require more research into (shared) underlying mechanisms.

Healthy Nordic diet and associations with plasma concentrations of metabolites in the choline oxidation pathway: a cross-sectional study from Northern Sweden.

BACKGROUND: The choline oxidation pathway and metabolites involved have been linked to diseases including cardiovascular disease, type 2 diabetes and cancer. A healthy Nordic diet is a recently defined dietary pattern associated with decreased risk for these diseases. Our aim was to explore associations between adherence to a healthy Nordic diet and plasma concentrations of metabolites of the choline oxidation pathway. METHODS: The Healthy Nordic Food Index (HNFI) and Baltic Sea Diet Score (BSDS) were applied to cross-sectional data (n = 969) from the Västerbotten Intervention Programme in Northern Sweden to score adherence to a healthy Nordic diet. Data included responses to a dietary questionnaire and blood sample analyses (1991-2008). Associations of diet scores with plasma concentrations of metabolites of the choline oxidation pathway and total homocysteine (tHcy), seven metabolites in total, were evaluated with linear regression, adjusting for age, BMI, education and physical activity. RESULTS: HNFI scores showed linear relationships with plasma choline (ß = 0.11), betaine (ß = 0.46), serine (ß = 0.98) and tHcy (ß = - 0.38), and BSDS scores with betaine (ß = 0.13) and tHcy (ß = - 0.13); unstandardized beta coefficients, all significant at P < 0.05. The regression models predicted changes in plasma metabolite concentrations (± 1 SD changes in diet score) in the range of 1-5% for choline, betaine, serine and tHcy. No other statistically significant associations were observed. CONCLUSIONS: A healthy Nordic diet was associated with plasma concentrations of several metabolites of the choline oxidation pathway. Although relationships were statistically significant, effect sizes were moderate. Further research is warranted to explore the underlying mechanisms and associations with health outcomes.

Altered Gut Microbial Metabolism of Essential Nutrients in Primary Sclerosing Cholangitis.

BACKGROUND & AIMS: To influence host and disease phenotype, compositional microbiome changes, which have been demonstrated in patients with primary sclerosing cholangitis (PSC), must be accompanied by functional changes. We therefore aimed to characterize the genetic potential of the gut microbiome in patients with PSC compared with healthy controls (HCs) and patients with inflammatory bowel disease (IBD). METHODS: Fecal DNA from 2 cohorts (1 Norwegian and 1 German), in total comprising 136 patients with PSC (58% with IBD), 158 HCs, and 93 patients with IBD without PSC, were subjected to metagenomic shotgun sequencing, generating 17 billion paired-end sequences, which were processed using HUMAnN2 and MetaPhlAn2, and analyzed using generalized linear models and random effects meta-analyses. RESULTS: Patients with PSC had fewer microbial genes compared with HCs (P < .0001). Compared with HCs, patients with PSC showed enrichment and increased prevalence of Clostridium species and a depletion of, for example, Eubacterium spp and Ruminococcus obeum. Patients with PSC showed marked differences in the abundance of genes related to vitamin B6 synthesis and branched-chain amino acid synthesis (Qfdr < .05). Targeted metabolomics of plasma from an independent set of patients with PSC and controls found reduced concentrations of vitamin B6 and branched-chain amino acids in PSC (P < .0001), which strongly associated with reduced liver transplantation-free survival (log-rank P < .001). No taxonomic or functional differences were detected between patients with PSC with and without IBD. CONCLUSIONS: The gut microbiome in patients with PSC exhibits large functional differences compared with that in HCs, including microbial metabolism of essential nutrients. Alterations in related circulating metabolites associated with disease course, suggesting that microbial functions may be relevant for the disease process in PSC.

Circulating trimethylamine N-oxide levels do not predict 10-year survival in patients with or without coronary heart disease.

BACKGROUND: Trimethylamine N-oxide (TMAO) is an amine oxide generated by gut microbial metabolism. TMAO may contribute to atherothrombosis and systemic inflammation. However, the prognostic value of circulating TMAO for risk stratification is uncertain. METHODS: We assessed prospective relationships of plasma TMAO with long-term risk of all-cause, cardiovascular (CV), and non-CV mortality in the Western Norway Coronary Angiography Cohort (WECAC; 4132 patients with suspected coronary artery disease) and the Hordaland Health Study (HUSK; 6393 community-based subjects). Risk associations were examined using Cox regression analyses. RESULTS: Mean follow-up was 9.8 and 10.5 years in WECAC and HUSK, respectively. Following adjustments for established CV risk factors and indices of renal function in WECAC, the hazard ratios (HRs) (95% confidence intervals [CIs]) per one standard deviation increase in log-transformed plasma TMAO were 1.04 (0.97-1.12), 1.06 (0.95-1.18), and 1.03 (0.93-1.13) for all-cause, CV, and non-CV mortality, respectively. Essentially similar results were obtained in patients with angiographically significant coronary artery disease and patients with reduced left ventricular ejection fraction. Corresponding HRs (95% CIs) in the HUSK cohort were 1.03 (0.96-1.10), 1.01 (0.89-1.13), and 1.03 (0.95-1.12) for all-cause-, CV, and non-CV mortality, respectively. CONCLUSIONS: Circulating TMAO did not predict long-term all-cause, CV, or non-CV mortality in patients with coronary heart disease or in community-based adults. This large study does not support a role of TMAO for patient risk stratification in primary or secondary prevention.

Consumption of a light meal affects serum concentrations of one-carbon metabolites and B-vitamins. A clinical intervention study.

The transfer of one-carbon units between molecules in metabolic pathways is essential for maintaining cellular homeostasis, but little is known about whether the circulating concentrations of metabolites involved in the one-carbon metabolism are affected by the prandial status. Epidemiological studies do not always consistently use fasting or non-fasting blood samples or may lack information on the prandial status of the study participants. Therefore, the main aim of the present study was to investigate the effects of a light breakfast on serum concentrations of selected metabolites and B-vitamins related to the one-carbon metabolism; i.e. the methionine-homocysteine cycle, the folate cycle, the choline oxidation pathway and the transsulfuration pathway. Sixty-three healthy adults (thirty-six women) with BMI ≥ 27 kg/m2 were included in the study. Blood was collected in the fasting state and 60 and 120 min after intake of a standardised breakfast consisting of white bread, margarine, white cheese, strawberry jam and orange juice (2218 kJ). The meal contained low amounts of choline, betaine, serine and vitamins B2, B3, B6, B9 and B12. Serum concentrations of total homocysteine, total cysteine, flavin mononucleotide, nicotinamide and pyridoxal 5'-phosphate were significantly decreased, and concentrations of choline, betaine, dimethylglycine, sarcosine, cystathionine and folate were significantly increased following breakfast intake (P < 0·05). Our findings demonstrate that the intake of a light breakfast with low nutrient content affected serum concentrations of several metabolites and B-vitamins related to the one-carbon metabolism.

Associations between plasma kynurenines and cognitive function in individuals with normal glucose metabolism, prediabetes and type 2 diabetes: the Maastricht Study.

AIMS/HYPOTHESIS: Studies investigating associations between kynurenines and cognitive function have generally been small, restricted to clinical samples or have found inconsistent results, and associations in the general adult population, and in individuals with type 2 diabetes in particular, are not clear. Therefore, the aim of the present study was to investigate cross-sectional associations between plasma kynurenines and cognitive function in a cohort of middle-aged participants with normal glucose metabolism, prediabetes (defined as impaired fasting glucose and/or impaired glucose tolerance) and type 2 diabetes. METHODS: Plasma kynurenines were quantified in 2358 participants aged 61 ± 8 years. Cross-sectional associations of kynurenines with cognitive impairment and cognitive domain scores were investigated using logistic, multiple linear and restricted cubic spline regression analyses adjusted for several confounders. RESULTS: Effect modification by glucose metabolism status was found for several associations with cognitive impairment, hence analyses were stratified. In individuals with prediabetes, 3-hydroxykynurenine (OR per SD 0.59 [95% CI 0.37, 0.94]) and 3-hydroxyanthranilic acid (0.67 [0.47, 0.96]) were associated with lower odds of cognitive impairment after full adjustment. In individuals with type 2 diabetes, kynurenine (0.80 [0.66, 0.98]), 3-hydroxykynurenine (0.82 [0.68, 0.99]), kynurenic acid (0.81 [0.68, 0.96]), xanthurenic acid (0.73 [0.61, 0.87]) and 3-hydroxyanthranilic acid (0.73 [0.60, 0.87]) were all associated with lower odds of cognitive impairment. Kynurenic acid (ß per SD 0.07 [95% CI 0.02, 0.13]) and xanthurenic acid (0.06 [0.01, 0.11]) were also associated with better executive function/attention. No associations were observed in individuals with normal glucose metabolism. CONCLUSIONS/INTERPRETATION: Several kynurenines were cross-sectionally associated with lower odds of cognitive impairment and better cognitive functioning in type 2 diabetes, while less widespread associations were seen in prediabetes. Low levels of kynurenines might be involved in the pathway of type 2 diabetes and cognitive decline but this needs further studies.

Quantifying Precision Loss in Targeted Metabolomics Based on Mass Spectrometry and Nonmatching Internal Standards.

In mass spectrometry, reliable quantification requires correction for variations in ionization efficiency between samples. The preferred method is the addition of a stable isotope-labeled internal standard (SIL-IS). In targeted metabolomics, a dedicated SIL-IS for each metabolite of interest may not always be realized due to high cost or limited availability. We recently completed the analysis of more than 70 biomarkers, each with a matching SIL-IS, across four mass spectrometry-based platforms (one GC-MS/MS and three LC-MS/MS). Using data from calibrator and quality control samples added to 60 96-well trays (analytical runs), we calculated analytical precision (CV) retrospectively. The use of integrated peak areas for all metabolites and internal standards allowed us to calculate precision for all matching analyte (A)/SIL-IS (IS) pairs as well as for all nonmatching A/IS pairs within each platform (total n = 1442). The median between-run precision for matching A/IS across the four platforms was 2.7-5.9%. The median CV for nonmatching A/IS (corresponding to pairing analytes with a non-SIL-IS) was 2.9-10.7 percentage points higher. Across all platforms, CVs for nonmatching A/IS increased with increasing difference in retention time (Spearman's rho of 0.17-0.93). The CV difference for nonmatching vs matching A/IS was often, but not always, smaller when analytes and internal standards were close structural analogs.

One-Carbon Metabolism in Nepalese Infant-Mother Pairs and Child Cognition at 5 Years Old.

BACKGROUND: One-carbon metabolism (OCM) refers to the transfer of methyl groups central to DNA methylation and histone modification. Insufficient access to methyl donors and B-vitamin cofactors affects epigenetic maintenance and stability, and when occurring in early life may impact future health and neurodevelopment. OBJECTIVE: The objective of this study was to examine the relative associations between one-carbon metabolites in Nepalese mother-infant pairs and child cognition measured at 5 y of age. METHODS: This is a cross-sectional study from Bhaktapur, Nepal, in a population at high risk of subclinical B-vitamin deficiencies and cumulative infection burden. Venous blood samples from 500 mother-infant pairs were collected when the infants were 2 to 12 mo old, and metabolite concentrations measured by microbiological assays and GC-tandem MS. We re-enrolled 321 of these children at 5 y and assessed cognition by the Ages and Stages Questionnaire, 3rd edition, and subtests from the Developmental Neuropsychological Assessment, 2nd edition (NEPSY-II). The associations of the independent metabolites or unobserved metabolic phenotypes (identified by latent class analysis) with the cognitive outcomes were estimated by seemingly unrelated regression. We explored direct and indirect relations between the OCM pathway and the cognitive outcomes using path analysis. RESULTS: Infant cystathionine concentration was inversely associated with 4 cognitive outcomes (standardized ßs ranging from -0.22 to -0.11, P values from <0.001 to 0.034). Infants with a metabolic phenotype indicating impaired OCM and low vitamin B-12 status had poorer cognitive outcomes compared with infants with normal OCM activity and adequate vitamin B-12 status (standardized ßs ranging from -0.80 to -0.40, P < 0.001 and 0.05). In the path analysis, we found several OCM biomarkers were associated with affect recognition through infant plasma cystathionine. CONCLUSIONS: Elevated plasma cystathionine during infancy reflects a metabolic phenotype of impaired OCM and low vitamin B-12 status and is associated with poorer cognitive function when the children are 5 y old.

Biomarkers and Fatty Fish Intake: A Randomized Controlled Trial in Norwegian Preschool Children.

BACKGROUND: Biomarkers such as omega-3 (n-3) PUFAs, urinary iodine concentration (UIC), 1-methylhistidine (1-MH), and trimethylamine N-oxide (TMAO) have been associated with fish intake in observational studies, but data from children in randomized controlled trials are limited. OBJECTIVES: The objective of this exploratory analysis was to investigate the effects of fatty fish intake compared with meat intake on various biomarkers in preschool children. METHODS: We randomly allocated (1:1) 232 children, aged 4 to 6 y, from 13 kindergartens. The children received lunch meals of either fatty fish (herring/mackerel) or meat (chicken/lamb/beef) 3 times a week for 16 wk. We analyzed 86 biomarkers in plasma (n = 207), serum (n = 195), RBCs (n = 211), urine (n = 200), and hair samples (n = 210). We measured the effects of the intervention on the normalized biomarker concentrations in linear mixed-effect regression models taking the clustering within the kindergartens into account. The results are presented as standardized effect sizes. RESULTS: We found significant effects of the intervention on the following biomarkers: RBC EPA (20:5n-3), 0.61 (95% CI: 0.36, 0.86); DHA (22:6n-3), 0.43 (95% CI: 0.21, 0.66); total n-3 PUFAs, 0.41 (95% CI: 0.20, 0.64); n-3/n-6 ratio, 0.48 (95% CI: 0.24, 0.71); adrenic acid (22:4n-6, -0.65 (95% CI: -0.91, -0.40), arachidonic acid (20:4n-6), -0.54 (95% CI: -0.79, -0.28); total n-6 PUFAs, -0.31 (95% CI: -0.56, -0.06); UIC, 0.32 (95% CI: 0.052, 0.59); hair mercury, 0.83 (95% CI: 0.05, 1.05); and plasma 1-MH, -0.35 (95% CI: -0.61, -0.094). CONCLUSIONS: Of the 86 biomarkers, the strongest effect of fatty fish intake was on n-3 PUFAs, UIC, hair mercury, and plasma 1-MH. We observed no or limited effects on biomarkers related to micronutrient status, inflammation, or essential amino acid, choline oxidation, and tryptophan pathways.The trial was registered at clinicaltrials.gov (NCT02331667).

Combined Supplementation with Vitamin B-6 and Curcumin is Superior to Either Agent Alone in Suppressing Obesity-Promoted Colorectal Tumorigenesis in Mice.

BACKGROUND: Obesity increases the colorectal cancer risk, in part by elevating colonic proinflammatory cytokines. Curcumin (CUR) and supplemental vitamin B-6 each suppress colonic inflammation. OBJECTIVES: We examined whether the combination of CUR and vitamin B-6 amplifies each supplement's effects and thereby suppress obesity-promoted tumorigenesis. METHODS: Male Friend Virus B (FVB) mice (4-week-old; n = 110) received 6 weekly injections of azoxymethane beginning 1 week after arrival. Thereafter, they were randomized to receive a low-fat diet (10% energy from fat), a high-fat diet (HFD; 60% energy from fat), a HFD containing 0.2% CUR, a HFD containing supplemental vitamin B-6 (24 mg pyridoxine HCl/kg), or a HFD containing both CUR and supplemental vitamin B-6 (C + B) for 15 weeks. Colonic inflammation, assessed by fecal calprotectin, and tumor metrics were the primary endpoints. The anti-inflammatory efficacy of the combination was also determined in human colonic organoids. RESULTS: HFD-induced obesity produced a 2.6-fold increase in plasma IL-6 (P < 0.02), a 1.9-fold increase in fecal calprotectin (P < 0.05), and a 2.2-fold increase in tumor multiplicity (P < 0.05). Compared to the HFD group, the C + B combination, but not the individual agents, decreased fecal calprotectin (66%; P < 0.01) and reduced tumor multiplicity and the total tumor burden by 60%-80% (P < 0.03) in an additive fashion. The combination of C + B also significantly downregulated colonic phosphatidylinositol-4,5-bisphosphate 3-kinase, Wnt, and NF-κB signaling by 31%-47% (P < 0.05), effects largely absent with the single agents. Observations that may explain how the 2 agents work additively include a 2.8-fold increased colonic concentration of 3-hydroxyanthranillic acid (P < 0.05) and a 1.3-fold higher colonic concentration of the active coenzymatic form of vitamin B-6 (P < 0.05). In human colonic organoids, micromolar concentrations of CUR, vitamin B-6, and their combination suppressed secreted proinflammatory cytokines by 41%-93% (P < 0.03), demonstrating relevance to humans. CONCLUSIONS: In this mouse model, C + B is superior to either agent alone in preventing obesity-promoted colorectal carcinogenesis. Augmented suppression of procancerous signaling pathways may be the means by which this augmentation occurs.

Inflammation-Related Marker Profiling of Dietary Patterns and All-cause Mortality in the Melbourne Collaborative Cohort Study.

BACKGROUND: Nutritional epidemiology research using self-reported dietary intake is prone to measurement error. Objective methods are being explored to overcome this limitation. OBJECTIVES: We aimed to examine 1) the association between plasma markers related to inflammation and derive marker scores for dietary patterns [Mediterranean dietary score (MDS), energy-adjusted Dietary Inflammatory Index (E-DIITM), Alternative Healthy Eating Index 2010 (AHEI)] and 2) the associations of these marker scores with mortality. METHODS: Weighted marker scores were derived from the cross-sectional association between 30 plasma markers and each dietary score (assessed using food-frequency questionnaires) using linear regression for 770 participants in the Melbourne Collaborative Cohort Study (aged 50-82 y). Prospective associations between marker scores and mortality (n = 249 deaths) were assessed using Cox regression (median follow-up: 14.4 y). RESULTS: The MDS, E-DII, and AHEI were associated (P < 0.05) with 9, 14, and 11 plasma markers, respectively. Healthier diets (higher MDS and AHEI, and lower anti-inflammatory, E-DII) were associated with lower concentrations of kynurenines, neopterin, IFN-γ, cytokines, and C-reactive protein. Five of 6 markers common to the 3 dietary scores were components of the kynurenine pathway. The 3 dietary-based marker scores were highly correlated (Spearman ρ: -0.74, -0.82, and 0.93). Inverse associations (for 1-SD increment) were observed with all-cause mortality for the MDS marker score (HR: 0.84; 95% CI: 0.72-0.98) and the AHEI marker score (HR: 0.76; 95% CI: 0.66-0.89), whereas a positive association was observed with the E-DII marker score (HR: 1.18; 95% CI: 1.01-1.39). The same magnitude of effect was not observed for the respective dietary patterns. CONCLUSIONS: Markers involved in inflammation-related processes are associated with dietary quality, including a substantial overlap between markers associated with the MDS, the E-DII, and the AHEI, especially kynurenines. Unfavorable marker scores, reflecting poorer-quality diets, were associated with increased mortality.

Associations of neopterin and kynurenine-tryptophan ratio with survival in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Biomarkers of inflammation may be of clinical utility in primary sclerosing cholangitis (PSC). We aimed to investigate the interferon gamma-related biomarkers neopterin and kynurenine-tryptophanratio (KT-ratio) in PSC. METHODS: Circulating neopterin, tryptophan and kynurenine were measured with LC-MS/MS in multiple cross-sectional cohorts comprising in total of 524 PSC patients and 100 healthy controls from Norway, Germany and Sweden. RESULTS: Neopterin and KT-ratio were significantly increased in PSC patients compared with controls in both a discovery and a validation cohort from Norway. Furthermore, high neopterin and KT-ratio levels were associated with a shorter transplantation-free survival in the PSC patients in the Norwegian discovery cohort and the German validation cohort. However, in the validation PSC cohort from Sweden, no relationship between neopterin and KT-ratio and liver transplantation-free survival was observed. The correlations between neopterin and KT-ratio were moderate to strong and similar in all cohorts (rho 0.50-0.67). Neopterin and KT-ratio also correlated with C-reactive protein (rho 0.17-0.63) and revised Mayo risk score (rho 0.23-0.42) in all cohorts. CONCLUSIONS: Neopterin and KT-ratio were elevated in PSC and associated with liver transplantation-free survival in two independent PSC cohorts, highlighting a possible role of interferon gamma-driven inflammation in the pathogenesis. However, the lack of association with survival in one of the cohorts reduces the potential clinical value of neopterin and KT-ratioas biomarkers and highlights the need to validate new biomarkers in PSC in multiple cohorts.

Maternal Vitamin B12 Status and Risk of Cleft Lip and Cleft Palate Birth Defects in Tamil Nadu State, India.

BACKGROUND AND OBJECTIVE: The causal role of maternal nutrition in orofacial clefts is uncertain. We tested hypotheses that low maternal vitamin B12 and low folate status are each associated with an increased risk of isolated cleft lip with or without cleft palate (CL±P) in a case-control study in Tamil Nadu state, India. METHODS: Case-mothers of CL±P children (n = 47) and control-mothers of unaffected children (n = 50) were recruited an average of 1.4 years after birth of the index child and plasma vitamin B12, methylmalonic acid (MMA), total homocysteine (tHcy), and folate were measured at that time. Logistic regression analyses estimated associations between nutrient biomarkers and case-control status. RESULTS: Odds ratios (ORs) contrasting biomarker levels showed associations between case-mothers and low versus high plasma vitamin B12 (OR = 2.48, 95% CI, 1.02-6.01) and high versus low plasma MMA, an indicator of poor B12 status (OR = 3.65 95% CI, 1.21-11.05). Case-control status was not consistently associated with folate or tHcy levels. Low vitamin B12 status, when defined by a combination of both plasma vitamin B12 and MMA levels, had an even stronger association with case-mothers (OR = 6.54, 95% CI, 1.33-32.09). CONCLUSIONS: Mothers of CL±P children in southern India were 6.5 times more likely to have poor vitamin B12 status, defined by multiple biomarkers, compared to control-mothers. Further studies in populations with diverse nutritional backgrounds are required to determine whether poor maternal vitamin B12 or folate levels or their interactions are causally related to CL±P.