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.

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 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.

Vitamin B12 status in infancy and the effect of a vitamin B12 injection in infants with subclinical vitamin B12 deficiency: study protocol for a register-based randomised controlled trial.

INTRODUCTION: Vitamin B12 (cobalamin) is crucial for optimal child development and growth, yet deficiency is common worldwide. The aim of this study is twofold; (1) to describe vitamin B12 status and the status of other micronutrients in Norwegian infants, and (2) in a randomised controlled trial (RCT), investigate the effect of vitamin B12 supplementation on neurodevelopment in infants with subclinical vitamin B12 deficiency. METHODS AND ANALYSIS: Infant blood samples, collected at public healthcare clinics, are analysed for plasma cobalamin levels. Infants with plasma cobalamin <148 pmol/L are immediately treated with hydroxocobalamin and excluded from the RCT. Remaining infants (cobalamin ≥148 pmol/L) are randomly assigned (in a 1:1 ratio) to either a screening or a control group. In the screening group, baseline samples are immediately analysed for total homocysteine (tHcy), while in the control group, the baseline samples will be analysed after 12 months. Screening group infants with plasma tHcy >6.5 µmol/L, are given an intramuscular injection of hydroxocobalamin (400 µg). The primary outcomes are cognitive, language and motor development assessed using the Bayley Scales of Infant and Toddler Development at 12 months of age. ETHICS AND DISSEMINATION: The study has been approved by the Regional Committee for Medical and Health Research Ethics (ref: 186505). Investigators who meet the Vancouver requirements will be eligible for authorship and be responsible for dissemination of study findings. Results will extend current knowledge on consequences of subclinical vitamin B12 deficiency during infancy and may inform future infant feeding recommendations. TRIAL REGISTRATION NUMBER: NCT05005897.

Vitamin B-6 and riboflavin, their metabolic interaction, and relationship with MTHFR genotype in adults aged 18-102 years.

BACKGROUND: The generation of the active form of vitamin B-6, pyridoxal 5'-phosphate (PLP), in tissues is dependent upon riboflavin as flavin mononucleotide, but whether this interaction is important for maintaining vitamin B-6 status is unclear. OBJECTIVE: To investigate vitamin B-6 and riboflavin status, their metabolic interaction, and relationship with methylenetetrahydrofolate reductase (MTHFR) genotype in adulthood. METHODS: Data from 5612 adults aged 18-102 y were drawn from the Irish National Adult Nutrition Survey (NANS; population-based sample) and the Trinity-Ulster Department of Agriculture (TUDA) and Genovit cohorts (volunteer samples). Plasma PLP and erythrocyte glutathione reductase activation coefficient (EGRac), as a functional indicator of riboflavin, were determined. RESULTS: Older (≥65 y) compared with younger (<65 y) adults had significantly lower PLP concentrations (P < 0.001). A stepwise decrease in plasma PLP was observed across riboflavin categories, from optimal (EGRac ≤1.26), to suboptimal (EGRac: 1.27-1.39), to deficient (EGRac ≥1.40) status, an effect most pronounced in older adults (mean ± SEM: 76.4 ± 0.9 vs 65.0 ± 1.1 vs 55.4 ± 1.2 nmol/L; P < 0.001). In individuals with the variant MTHFR 677TT genotype combined with riboflavin deficiency, compared with non-TT (CC/CT) genotype participants with sufficient riboflavin, we observed PLP concentrations of 52.1 ± 2.9 compared with 76.8 ±0.7 nmol/L (P < 0.001). In participants with available dietary data (i.e., NANS cohort, n = 936), PLP was associated with vitamin B-6 intake (nonstandardized regression coefficient ß: 2.49; 95% CI 1.75, 3.24; P < 0.001), supplement use (ß: 81.72; 95% CI: 66.01, 97.43; P < 0.001), fortified food (ß: 12.49; 95% CI: 2.08, 22.91; P = 0.019), and EGRac (ß: -65.81; 95% CI: -99.08, -32.54; P < 0.001), along with BMI (ß: -1.81; 95% CI: -3.31, -0.30; P = 0.019). CONCLUSIONS: These results are consistent with the known metabolic dependency of PLP on flavin mononucleotide (FMN) and suggest that riboflavin may be the limiting nutrient for maintaining vitamin B-6 status, particularly in individuals with the MTHFR 677TT genotype. Randomized trials are necessary to investigate the PLP response to riboflavin intervention within the dietary range. The TUDA study and the NANS are registered at www.ClinicalTrials.gov as NCT02664584 (27 January 2016) and NCT03374748 (15 December 2017), respectively.Clinical Trial Registry details: Trinity-Ulster-Department of Agriculture (TUDA) study, ClinicalTrials.gov no. NCT02664584 (January 27th 2016); National Adult Nutrition Survey (NANS), ClinicalTrials.gov no. NCT03374748 (December 15th 2017).

A 2-Year Randomized Controlled Trial With Low-Dose B-Vitamin Supplementation Shows Benefits on Bone Mineral Density in Adults With Lower B12 Status.

Folate, vitamins B12, B6, and riboflavin are required for one-carbon metabolism and may affect bone health, but no previous randomized trial has investigated all four nutrients in this context. We investigated the effect of low-dose B-vitamins for 2 years on bone mineral density (BMD) in a dual-centered, 2-year randomized controlled trial (RCT) in adults aged ≥50 years. Eligible participants not consuming B-vitamin supplements or fortified foods >4 times weekly were randomized to receive daily either combined folic acid (200 µg), vitamin B12 (10 µg), vitamin B6 (10 mg), and riboflavin (5 mg), or "active" placebo, whereby both the intervention and placebo groups received vitamin D (10 µg). BMD was assessed before and after intervention using dual-energy X-ray absorptiometry (DXA) scanning of the total hip, femoral neck, and lumbar spine (L1 to L4). Of 205 eligible participants randomized, 167 completed the trial in full. B-vitamin intervention resulted in increases in serum folate (p < 0.001), serum B12 (p < 0.001), and plasma pyridoxal-5-phosphate (p < 0.001) and decreases in functional biomarkers of B-vitamin status, erythrocyte glutathione reductase activation coefficient (p < 0.001), serum methylmalonic acid (MMA; p < 0.001), and serum total homocysteine (p < 0.001). B-vitamin intervention had no overall effect on BMD, which declined in both treatment groups by approximately 1% (ranging from -0.7% to -1.4%). However, in participants with lower baseline B12 status (serum B12 <246 pmol/L or MMA ≥0.22 µmol/L), B-vitamin intervention reduced the 2-year BMD decline versus placebo: adjusted mean (95% confidence interval [CI]) change of -0.003 (-0.008, 0.002) versus -0.015 (-0.021, -0.010) g/cm2 at the total hip and -0.004 (-0.010, 0.001) versus -0.013 (-0.018, -0.007) g/cm2 at the femoral neck. In conclusion, the findings indicate that although low-dose B-vitamin intervention for 2 years had no overall effect on BMD, improving B-vitamin status appears to have specific benefits for bone health in adults with lower B12 status. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Effects of vitamin B12 supplementation on neurodevelopment and growth in Nepalese Infants: A randomized controlled trial.

BACKGROUND: Vitamin B12 deficiency is common and affects cell division and differentiation, erythropoiesis, and the central nervous system. Several observational studies have demonstrated associations between biomarkers of vitamin B12 status with growth, neurodevelopment, and anemia. The objective of this study was to measure the effects of daily supplementation of vitamin B12 for 1 year on neurodevelopment, growth, and hemoglobin concentration in infants at risk of deficiency. METHODS AND FINDINGS: This is a community-based, individually randomized, double-blind placebo-controlled trial conducted in low- to middle-income neighborhoods in Bhaktapur, Nepal. We enrolled 600 marginally stunted, 6- to 11-month-old infants between April 2015 and February 2017. Children were randomized in a 1:1 ratio to 2 µg of vitamin B12, corresponding to approximately 2 to 3 recommended daily allowances (RDAs) or a placebo daily for 12 months. Both groups were also given 15 other vitamins and minerals at around 1 RDA. The primary outcomes were neurodevelopment measured by the Bayley Scales of Infant and Toddler Development 3rd ed. (Bayley-III), attained growth, and hemoglobin concentration. Secondary outcomes included the metabolic response measured by plasma total homocysteine (tHcy) and methylmalonic acid (MMA). A total of 16 children (2.7%) in the vitamin B12 group and 10 children (1.7%) in the placebo group were lost to follow-up. Of note, 94% of the scheduled daily doses of vitamin B12 or placebo were reported to have been consumed (in part or completely). In this study, we observed that there were no effects of the intervention on the Bayley-III scores, growth, or hemoglobin concentration. Children in both groups grew on an average 12.5 cm (SD: 1.8), and the mean difference was 0.20 cm (95% confidence interval (CI): -0.23 to 0.63, P = 0.354). Furthermore, at the end of the study, the mean difference in hemoglobin concentration was 0.02 g/dL (95% CI: -1.33 to 1.37, P = 0.978), and the difference in the cognitive scaled scores was 0.16 (95% CI: -0.54 to 0.87, P = 0.648). The tHcy and MMA concentrations were 23% (95% CI: 17 to 30, P < 0.001) and 30% (95% CI: 15 to 46, P < 0.001) higher in the placebo group than in the vitamin B12 group, respectively. We observed 43 adverse events in 36 children, and these events were not associated with the intervention. In addition, 20 in the vitamin B12 group and 16 in the placebo group were hospitalized during the supplementation period. Important limitations of the study are that the strict inclusion criteria could limit the external validity and that the period of vitamin B12 supplementation might not have covered a critical window for infant growth or brain development. CONCLUSIONS: In this study, we observed that vitamin B12 supplementation in young children at risk of vitamin B12 deficiency resulted in an improved metabolic response but did not affect neurodevelopment, growth, or hemoglobin concentration. Our results do not support widespread vitamin B12 supplementation in marginalized infants from low-income countries. TRIAL REGISTRATION: ClinicalTrials.gov NCT02272842 Universal Trial Number: U1111-1161-5187 (September 8, 2014) Trial Protocol: Original trial protocol: PMID: 28431557 (reference [18]; study protocols and plan of analysis included as Supporting information).

Effect of Cod Residual Protein Supplementation on Markers of Glucose Regulation in Lean Adults: A Randomized Double-Blind Study.

Large quantities of protein-rich cod residuals, which are currently discarded, could be utilized for human consumption. Although fish fillet intake is related to beneficial health effects, little is known about the potential health effects of consuming cod residual protein powder. Fifty lean adults were randomized to consume capsules with 8.1 g/day of cod residual protein (Cod-RP) or placebo capsules (Control group) for eight weeks, in this randomized, double-blind study. The intervention was completed by 40 participants. Fasting glucose and insulin concentrations were unaffected by Cod-RP supplementation, whereas plasma concentrations of α-hydroxybutyrate, ß-hydroxybutyrate and acetoacetate all were decreased compared with the Control group. Trimethylamine N-oxide concentration in plasma and urine were increased in the Cod-RP group compared with the Control group. To conclude, the reduction in these potential early markers of impaired glucose metabolism following Cod-RP supplementation may indicate beneficial glucoregulatory effects of cod residual proteins. Trimethylamine N-oxide appears to be an appropriate biomarker of cod residual protein intake in lean adults.

Impact of the MTHFR C677T polymorphism on one-carbon metabolites: Evidence from a randomised trial of riboflavin supplementation.

Homozygosity for the C677T polymorphism in MTHFR (TT genotype) is associated with a 24-87% increased risk of hypertension. Blood pressure (BP) lowering was previously reported in adults with the TT genotype, in response to supplementation with the MTHFR cofactor, riboflavin. Whether the BP phenotype associated with the polymorphism is related to perturbed one-carbon metabolism is unknown. This study investigated one-carbon metabolites and their responsiveness to riboflavin in adults with the TT genotype. Plasma samples from adults (n 115) screened for the MTHFR genotype, who previously participated in RCTs to lower BP, were analysed for methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), betaine, choline and cystathionine by liquid chromatography tandem mass spectrometry (LC-MS/MS). The one-carbon metabolite response to riboflavin (1.6 mg/d; n 24) or placebo (n 23) for 16 weeks in adults with the TT genotype was also investigated. Plasma SAM (74.7 ± 21.0 vs 85.2 ± 22.6 nmol/L, P = 0.013) and SAM:SAH ratio (1.66 ± 0.55 vs 1.85 ± 0.51, P = 0.043) were lower and plasma homocysteine was higher (P = 0.043) in TT, compared to CC individuals. In response to riboflavin, SAM (P = 0.008) and cystathionine (P = 0.045) concentrations increased, with no responses in other one-carbon metabolites observed. These findings confirm perturbed one-carbon metabolism in individuals with the MTHFR 677TT genotype, and for the first time demonstrate that SAM, and cystathionine, increase in response to riboflavin supplementation in this genotype group. The genotype-specific, one-carbon metabolite responses to riboflavin intervention observed could offer some insight into the role of this gene-nutrient interaction in blood pressure.

Dietary Intake and Biomarkers of Folate and Cobalamin Status in Norwegian Preschool Children: The FINS-KIDS Study.

BACKGROUND: Folate and cobalamin (vitamin B-12) are essential for growth and development. However, few population-based studies have investigated B-vitamin status in children. OBJECTIVES: This study aimed to assess biomarkers of folate and vitamin B-12 status and to explore their dietary determinants in healthy Norwegian children. METHODS: Using baseline data obtained from a randomized controlled trial on the effect of fish intake on neurodevelopment in children aged 4-6 y, we measured the plasma concentrations of folate, cobalamin, total plasma homocysteine (tHcy), and methylmalonic acid (MMA). Food-frequency questionnaires (FFQs) were used to assess dietary intake. We used unadjusted and multiple linear regression models to explore the determinants of biomarker concentrations. RESULTS: The median (IQR) of plasma folate (n = 197) and plasma cobalamin (n = 195) concentrations were 15.2 (12.2-21.1) nmol/L and 785 (632-905) pmol/L, respectively. Plasma folate concentrations of <10 nmol/L were observed in 13% of the children. No child had a cobalamin concentration <148 pmol/L. Two children were identified with elevated plasma MMA concentrations (>0.26 µmol/L) and 8 children had elevated tHcy concentrations (>6.5 µmol/L). Plasma folate concentration was inversely correlated with tHcy (ρ = -0.24, P < 0.001); we found no correlation between tHcy and cobalamin (ρ = -0.075, P = 0.30). Children who consumed vitamin supplements had 51% higher plasma folate concentrations (P < 0.0001) than those who did not. Consumption of red meat for dinner more than twice a week was associated with 23% lower plasma folate (P < 0.01). No other significant associations between dietary intake and the biomarkers were observed. CONCLUSIONS: The Norwegian preschool children from this cohort had adequate vitamin B-12 status. Poor folate status was common and associated with elevated tHcy. The implications of poor folate status during childhood should be a prioritized research question. This trial was registered at ClinicalTrials.gov as NCT02331667.

Using metabolic profiling and gene expression analyses to explore molecular effects of replacing saturated fat with polyunsaturated fat-a randomized controlled dietary intervention study.

BACKGROUND: Replacing dietary saturated fatty acids (SFAs) with polyunsaturated fatty acids (PUFA) reduces the plasma low-density lipoprotein (LDL) cholesterol and subsequently the risk of cardiovascular disease. However, beyond changes in LDL cholesterol, we lack a complete understanding of the physiologic alterations that occur when improving dietary fat quality. OBJECTIVES: The aim of this study was to gain knowledge of metabolic alterations paralleling improvements in the fat quality of the diet. METHODS: We recently conducted an 8-wk, double-blind, randomized controlled trial replacing SFAs with PUFAs in healthy subjects with moderate hypercholesterolemia (n = 99). In the present substudy, we performed comprehensive metabolic profiling with multiple platforms (both nuclear magnetic resonance- and mass spectrometry-based technology) (n = 99), and analyzed peripheral blood mononuclear cell gene expression (n = 95) by quantitative real-time polymerase chain reaction. RESULTS: A large number of lipoprotein subclasses, myristoylcarnitine and palmitoylcarnitine, and kynurenine were reduced when SFAs were replaced with PUFAs. In contrast, bile acids, proprotein convertase subtilisin/kexin type 9, acetate, and acetoacetate were increased by the intervention. Some amino acids were also altered by the intervention. The mRNA levels of LXRA and LDLR were increased, in addition to several liver X receptor α target genes and genes involved in inflammation, whereas the mRNA levels of UCP2 and PPARD were decreased in peripheral blood mononuclear cells after replacing SFAs with PUFAs. Partial least squares-discriminant analysis showed that the 30 most important variables that contributed to class separation spanned all classes of biomarkers, and was in accordance with the univariate analysis. CONCLUSIONS: Applying metabolomics in randomized controlled dietary intervention trials has the potential to extend our knowledge of the biological and molecular effects of dietary fat quality. This study was registered at clinicaltrials.gov as NCT01679496.

Comparable Performance Characteristics of Plasma Thiamine and Erythrocyte Thiamine Diphosphate in Response to Thiamine Fortification in Rural Cambodian Women.

Background: Traditionally, vitamin B1 status is assessed by a functional test measuring erythrocyte transketolase (ETK) activity or direct measurement of erythrocyte thiamine diphosphate (eThDP) concentration. However, such analyses are logistically challenging, and do not allow assessment of vitamin B1 status in plasma/serum samples stored in biobanks. Using a multiplex assay, we evaluated plasma concentrations of thiamine and thiamine monophosphate (TMP), as alternative, convenient measures of vitamin B1 status. Methods: We investigated the relationships between the established biomarker eThDP and plasma concentrations of thiamine and TMP, and compared the response of these thiamine forms to thiamine fortification using samples from 196 healthy Cambodian women (aged 18-45 years.). eThDP was measured by high performance liquid chromatography with fluorescence detection (HPLC-FLD) and plasma thiamine and TMP by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Plasma thiamine and TMP correlated significantly with eThDP at baseline and study-end (p < 0.05). Among the fortification groups, the strongest response was observed for plasma thiamine (increased by 266%), while increases in plasma TMP (60%) and eThDP (53%) were comparable. Conclusions: Plasma thiamine and TMP correlated positively with eThDP, and all thiamine forms responded significantly to thiamine intervention. Measuring plasma concentrations of thiamine forms is advantageous due to convenient sample handling and capacity to develop low volume, high-throughput, multiplex assays.

Circulating concentrations of biomarkers and metabolites related to vitamin status, one-carbon and the kynurenine pathways in US, Nordic, Asian, and Australian populations.

Background: Circulating concentrations of biomarkers that are related to vitamin status vary by factors such as diet, fortification, and supplement use. Published biomarker concentrations have also been influenced by the variation across laboratories, which complicates a comparison of results from different studies.Objective: We robustly and comprehensively assessed differences in biomarkers that are related to vitamin status across geographic regions.Design: The trial was a cross-sectional study in which we investigated 38 biomarkers that are related to vitamin status and one-carbon and tryptophan metabolism in serum and plasma from 5314 healthy control subjects representing 20 cohorts recruited from the United States, Nordic countries, Asia, and Australia, participating in the Lung Cancer Cohort Consortium. All samples were analyzed in a centralized laboratory.Results: Circulating concentrations of riboflavin, pyridoxal 5'-phosphate, folate, vitamin B-12, all-trans retinol, 25-hydroxyvitamin D, and α-tocopherol as well as combined vitamin scores that were based on these nutrients showed that the general B-vitamin concentration was highest in the United States and that the B vitamins and lipid soluble vitamins were low in Asians. Conversely, circulating concentrations of metabolites that are inversely related to B vitamins involved in the one-carbon and kynurenine pathways were high in Asians. The high B-vitamin concentration in the United States appears to be driven mainly by multivitamin-supplement users.Conclusions: The observed differences likely reflect the variation in intake of vitamins and, in particular, the widespread multivitamin-supplement use in the United States. The results provide valuable information about the differences in biomarker concentrations in populations across continents.

Maternal Folate Intake during Pregnancy and Childhood Asthma in a Population-based Cohort.

RATIONALE: A potential adverse effect of high folate intake during pregnancy on children's asthma development remains controversial. OBJECTIVES: To prospectively investigate folate intake from both food and supplements during pregnancy and asthma at age 7 years when the diagnosis is more reliable than at preschool age. METHODS: This study included eligible children born 2002-2006 from the Norwegian Mother and Child Cohort Study, a population-based pregnancy cohort, linked to the Norwegian Prescription Database. Current asthma at age 7 was defined by asthma medications dispensed at least twice in the year (1,901 cases; n = 39,846) or by maternal questionnaire report (1,624 cases; n = 28,872). Maternal folate intake was assessed with a food frequency questionnaire validated against plasma folate. We used log-binomial and multinomial regression to calculate adjusted relative risks with 95% confidence intervals. MEASUREMENTS AND MAIN RESULTS: Risk of asthma was increased in the highest versus lowest quintile of total folate intake with an adjusted relative risk of 1.23 (95% confidence interval, 1.06-1.44) that was similar for maternally reported asthma. Mothers in the highest quintile had a relatively high intake of food folate (median, 308; interquartile range, 241-366 µg/d) and nearly all took at least 400 µg/d of supplemental folic acid (median, 500; interquartile range, 400-600 µg/d). CONCLUSIONS: In this large prospective population-based cohort with essentially complete follow-up, pregnant women taking supplemental folic acid at or above the recommended dose, combined with a diet rich in folate, reach a total folate intake level associated with a slightly increased risk of asthma in children.

Inflammation, vitamin B6 and related pathways.

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), serves as a co-factor in more than 150 enzymatic reactions. Plasma PLP has consistently been shown to be low in inflammatory conditions; there is a parallel reduction in liver PLP, but minor changes in erythrocyte and muscle PLP and in functional vitamin B6 biomarkers. Plasma PLP also predicts the risk of chronic diseases like cardiovascular disease and some cancers, and is inversely associated with numerous inflammatory markers in clinical and population-based studies. Vitamin B6 intake and supplementation improve some immune functions in vitamin B6-deficient humans and experimental animals. A possible mechanism involved is mobilization of vitamin B6 to the sites of inflammation where it may serve as a co-factor in pathways producing metabolites with immunomodulating effects. Relevant vitamin B6-dependent inflammatory pathways include vitamin B6 catabolism, the kynurenine pathway, sphingosine 1-phosphate metabolism, the transsulfuration pathway, and serine and glycine metabolism.

Combined Measurement of 6 Fat-Soluble Vitamins and 26 Water-Soluble Functional Vitamin Markers and Amino Acids in 50 µL of Serum or Plasma by High-Throughput Mass Spectrometry.

Targeted metabolic profiling characterized by complementary platforms, multiplexing and low volume consumption are increasingly used for studies using biobank material. Using liquid-liquid extraction, we developed a sample workup suitable for quantification of 6 fat- and 26 water-soluble biomarkers. 50 µL of serum/plasma was mixed with dithioerythritol, ethanol, and isooctane/chloroform. The organic layer was used for analysis of the fat-soluble vitamins all-trans retinol (A), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, α-tocopherol (E), γ-tocopherol (E), and phylloquinone (K1) by LC-MS/MS. The remaining aqueous fraction was mixed with ethanol, water, pyridine, and methylchloroformate (in toluene) to derivatize the water-soluble biomarkers. The resulting toluene layer was used for GC-MS/MS analysis of alanine, α-ketoglutarate, asparagine, aspartic acid, cystathionine, total cysteine, glutamic acid, glutamine, glycine, histidine, total homocysteine, isoleucine, kynurenine, leucine, lysine, methionine, methylmalonic acid, ornithine, phenylalanine, proline, sarcosine, serine, threonine, tryptophan, tyrosine, and valine. Isotope-labeled internal standards were used for all analytes. Chromatographic run times for the LC-MS/MS and GC-MS/MS were 4.5 and 11 min, respectively. The limits of detection (LOD) for the low-concentration analytes (25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and phylloquinone) were 25, 17, and 0.33 nM, respectively, while all other analytes demonstrated sensitivity significantly lower than endogenous concentrations. Recoveries ranged from 85.5-109.9% and within- and between-day coefficients of variance (CVs) were 0.7-9.4% and 1.1-17.5%, respectively. This low-volume, high-throughput multianalyte assay is currently in use in our laboratory for quantification of 32 serum/plasma biomarkers in epidemiological studies.