Folate and vitamin B12 status and predicted neural tube defects risk among nonpregnant women of reproductive age from the Malawi National Micronutrient Survey, 2015-2016.

BACKGROUND: Maternal folate and vitamin B12 deficiency can lead to serious adverse pregnancy outcomes. There are no nationally representative estimates on folate and vitamin B12 status among women of reproductive age (WRA) in Malawi. OBJECTIVE: We assessed folate and vitamin B12 status among nonpregnant WRA in Malawi and predicted the risk of folate-sensitive neural tube defects (NTDs) were they to become pregnant. METHODS: Using data from the cross-sectional, nationally representative 2015-2016 Malawi Micronutrient Survey, we calculated the proportion of folate and vitamin B12 deficiency and insufficiency by demographic characteristics among 778 nonpregnant WRA (15-49 years). We predicted NTD prevalence using red blood cell (RBC) folate distributions and a published Bayesian model of the association between RBC folate and NTD risk. Analyses accounted for complex survey design. RESULTS: Among WRA, 8.5% (95% CI: 6.2, 11.6) and 13.3% (10.0, 17.4) had serum (<7 nmol/L) and RBC folate (<305 nmol/L) deficiency, respectively. The proportion of vitamin B12 deficiency (<148 pmol/L) and insufficiency (≤221 pmol/L) was 11.8% (8.6, 16.0) and 40.6% (34.1, 47.4), respectively. RBC folate insufficiency (<748 nmol/L, defined as the concentration associated with the threshold for elevated NTD risk: >8 cases per 10,000 births) was widespread: 81.4% (75.0, 86.4). The predicted NTD risk nationally was 24.7 cases per 10,000 live births. RBC folate insufficiency and higher predicted NTD risk were more common among WRA living in urban areas or with higher education. CONCLUSIONS: These findings highlight the importance of nutritional and NTD surveillance in Malawi and the opportunity for improving folate and vitamin B12 nutrition among Malawian WRA.

Increasing Enzyme Mannose-6-Phosphate Levels but Not Miglustat Coadministration Enhances the Efficacy of Enzyme Replacement Therapy in Pompe Mice.

Pompe disease is a rare glycogen storage disorder caused by a deficiency in the lysosomal enzyme acid α-glucosidase, which leads to muscle weakness, cardiac and respiratory failure, and early mortality. Alglucosidase alfa, a recombinant human acid α-glucosidase, was the first approved treatment of Pompe disease, but its uptake into skeletal muscle via the cation-independent mannose-6-phosphate (M6P) receptor (CIMPR) is limited. Avalglucosidase alfa has received marketing authorization in several countries for infantile-onset and/or late-onset Pompe disease. This recently approved enzyme replacement therapy (ERT) was glycoengineered to maximize CIMPR binding through high-affinity interactions with ∼7 bis-M6P moieties. Recently, small molecules like the glucosylceramide synthase inhibitor miglustat were reported to increase the stability of recombinant human acid α-glucosidase, and it was suggested that an increased serum half-life would result in better glycogen clearance. Here, the effects of miglustat on alglucosidase alfa and avalglucosidase alfa stability, activity, and efficacy in Pompe mice were evaluated. Although miglustat increased the stability of both enzymes in fluorescent protein thermal shift assays and when incubated in neutral pH buffer over time, it reduced their enzymatic activity by ∼50%. Improvement in tissue glycogen clearance and transcriptional dysregulation in Pompe mice correlated with M6P levels but not with miglustat coadministration. These results further substantiate the crucial role of CIMPR binding in lysosomal targeting of ERTs. SIGNIFICANCE STATEMENT: This work describes important new insights into the treatment of Pompe disease using currently approved enzyme replacement therapies (ERTs) coadministered with miglustat. Although miglustat increased the stability of ERTs in vitro, there was no positive impact to glycogen clearance and transcriptional correction in Pompe mice. However, increasing mannose-6-phosphate levels resulted in increased cell uptake in vitro and increased glycogen clearance and transcriptional correction in Pompe mice, further underscoring the crucial role of cation-independent mannose-6-phosphate receptor-mediated lysosomal targeting for ERTs.

Dihydromyricetin supplementation improves ethanol-induced lipid accumulation and inflammation.

Introduction: Excessive alcohol consumption leads to a myriad of detrimental health effects, including alcohol-associated liver disease (ALD). Unfortunately, no available treatments exist to combat the progression of ALD beyond corticosteroid administration and/or liver transplants. Dihydromyricetin (DHM) is a bioactive polyphenol and flavonoid that has traditionally been used in Chinese herbal medicine for its robust antioxidant and anti-inflammatory properties. It is derived from many plants, including Hovenia dulcis and is found as the active ingredient in a variety of popular hangover remedies. Investigations utilizing DHM have demonstrated its ability to alleviate ethanol-induced disruptions in mitochondrial and lipid metabolism, while demonstrating hepatoprotective activity. Methods: Female c57BL/6J mice (n = 12/group) were treated using the Lieber DeCarli forced-drinking and ethanol (EtOH) containing liquid diet, for 5 weeks. Mice were randomly divided into three groups: (1) No-EtOH, (2) EtOH [5% (v/v)], and (3) EtOH [5% (v/v)] + DHM (6 mg/mL). Mice were exposed to ethanol for 2 weeks to ensure the development of ALD pathology prior to receiving dihydromyricetin supplementation. Statistical analysis included one-way ANOVA along with Bonferroni multiple comparison tests, where p ≤ 0.05 was considered statistically significant. Results: Dihydromyricetin administration significantly improved aminotransferase levels (AST/ALT) and reduced levels of circulating lipids including LDL/VLDL, total cholesterol (free cholesterol), and triglycerides. DHM demonstrated enhanced lipid clearance by way of increased lipophagy activity, shown as the increased interaction and colocalization of p62/SQSTM-1, LC3B, and PLIN-1 proteins. DHM-fed mice had increased hepatocyte-to-hepatocyte lipid droplet (LD) heterogeneity, suggesting increased neutralization and sequestration of free lipids into LDs. DHM administration significantly reduced prominent pro-inflammatory cytokines commonly associated with ALD pathology such as TNF-α, IL-6, and IL-17. Discussion: Dihydromyricetin is commercially available as a dietary supplement. The results of this proof-of-concept study demonstrate its potential utility and functionality as a cost-effective and safe candidate to combat inflammation and the progression of ALD pathology.

Estimating the serum folate concentration that corresponds to the red blood cell folate concentration threshold associated with optimal neural tube defects prevention: A population-based biomarker survey in Southern India.

BACKGROUND: RBC folate concentrations are monitored at the population level, with a recommended threshold for optimal neural tube defect (NTD) prevention. A corresponding threshold for serum folate has not been established. OBJECTIVES: This study aimed to estimate the serum folate insufficiency threshold corresponding to the RBC folate threshold for NTD prevention and examine how this threshold is modified by vitamin B12 status. METHODS: Participants were women (15-40 y; not pregnant or lactating; n = 977) from a population-based biomarker survey in Southern India. RBC folate and serum folate were measured via microbiologic assay. RBC folate deficiency (<305 nmol/L) and insufficiency (<748 nmol/L), serum vitamin B12 deficiency (<148 pmol/L) and vitamin B12 insufficiency (<221 pmol/L), elevated plasma MMA (>0.26 µmol/L), elevated plasma homocysteine (>10.0 µmol/L), and elevated HbA1c (≥6.5%) were evaluated. Bayesian linear models were used to estimate unadjusted and adjusted thresholds. RESULTS: Compared with adequate vitamin B12 status, the estimated serum folate threshold was higher in participants with serum vitamin B12 deficiency (72.5 vs. 28.1 nmol/L) or vitamin B12 insufficiency (48.7 vs. 24.3 nmol/L) and elevated MMA (55.6 vs. 25.9 nmol/L). The threshold was lower in participants with elevated HbA1c (HbA1c ≥6.5% vs. <6.5%; 21.0 vs. 40.5 nmol/L). CONCLUSIONS: The estimated serum folate threshold for optimal NTD prevention was similar to previous reports (24.3 vs. 25.6 nmol/L) among participants with sufficient vitamin B12 status. However, this threshold was more than 2-fold higher in participants with vitamin B12 deficiency and substantially higher across all indicators of insufficient vitamin B12 status (<221 pmol/L, elevated MMA, combined B12, impaired vitamin B12 status), and lower in participants with elevated HbA1c. Findings suggest a serum folate threshold for NTD prevention may be possible in some settings; however, it may not be appropriate in populations with high prevalence of vitamin B12 insufficiency. Am J Clin Nutr 2023;xx:xx-xx. This trial was registered at https://clinicaltrials.gov as NCT04048330.

A Randomized Trial of Quadruple-Fortified Salt for Anemia and Birth Defects Prevention in Southern India: Protocol Design and Methods.

Background: Women of reproductive age are at an increased risk of anemia and micronutrient deficiencies. Evidence supports the role of periconceptional nutrition in the development of neural tube defects (NTDs) and other pregnancy complications. Vitamin B12 deficiency is a risk factor for NTDs and may modify folate biomarkers that predict NTD risk at the population level. There is an interest in mandatory fortification with vitamin B12 and folic acid for anemia and birth defect prevention. However, there are limited population-representative data needed to inform policy and guidelines. Objectives: This randomized trial will be conducted to evaluate the efficacy of quadruple-fortified salt (QFS; iron, iodine, folic acid, vitamin B12) in 1,000 households in Southern India. Methods: Women 18 to 49 y who are not pregnant or lactating and reside within the catchment area of our community-based research site in Southern India will be screened and invited to participate in the trial. After informed consent, women and their households will be randomized to receive one of the following 4 interventions: 1) double-fortified salt (DFS; iron, iodine), 2) DFS + folic acid (iron, iodine, folic acid), 3) DFS + vitamin B12 (iron, iodine, vitamin B12), or 4) DFS + folic acid and vitamin B12 (QFS; iron, iodine, folic acid, vitamin B12) for 12 mo. Structured interviews will be conducted by trained nurse enumerators to collect sociodemographic, anthropometric, dietary, health, and reproductive history data. Biological samples will be collected at baseline, midpoint, and endpoint. Whole blood will be analyzed for hemoglobin using Coulter Counter. Total vitamin B12 will be measured by chemiluminescence; red blood cell folate and serum folate will be evaluated using the World Health Organization-recommended microbiologic assay. Conclusions: The results of this randomized trial will help to evaluate the efficacy of QFS to prevent anemia and micronutrient deficiencies. Clinical trial registration numbers: NCT03853304 and Clinical Trial Registry of India REF/2019/03/024479. Registration number: NCT03853304 and REF/2019/03/024479.

CDC's Vitamin A Laboratory-External Quality Assessment Program Shows Little Change in Laboratory Performance for Multiple Nutritional Biomarkers over a 10-Year Period (2008-2017).

BACKGROUND: The Vitamin A Laboratory-External Quality Assessment (VITAL-EQA) program operated by the CDC provides analytical performance assessment to low-resource laboratories conducting serum vitamins A (VIA), D (VID), B-12 (B12), and folate (FOL), as well as ferritin (FER) and CRP measurements for public health studies. OBJECTIVES: We aimed to describe the long-term performance of VITAL-EQA participants from 2008 to 2017. METHODS: Participating laboratories received 3 blinded serum samples biannually for duplicate analysis over 3 d. We assessed results (n = 6) for relative difference (%) from the CDC target value and imprecision (% CV) and conducted descriptive statistics on the aggregate 10-year and round-by-round data. Performance criteria were based on biologic variation and deemed acceptable (optimal, desirable, or minimal performance) or unacceptable (less than minimal performance). RESULTS: Thirty-five countries reported VIA, VID, B12, FOL, FER, and CRP results from 2008-2017. The proportion of laboratories with acceptable performance ranged widely by round: VIA 48%-79% (for difference) and 65%-93% (for imprecision), VID 19%-63% and 33%-100%, B12 0%-92% and 73%-100%, FOL 33%-89% and 78%-100%, FER 69%-100% and 73%-100%, and CRP 57%-92% and 87%-100%. On aggregate, ≥60% of laboratories achieved acceptable differences for VIA, B12, FOL, FER, and CRP (only 44% for VID), and over 75% of laboratories achieved acceptable imprecision for all 6 analytes. Laboratories participating continuously in 4 rounds (2016-2017) showed generally similar performance compared to laboratories participating occasionally. CONCLUSIONS: Although we observed little change in laboratory performance over time, on aggregate, >50% of the participating laboratories achieved acceptable performance, with acceptable imprecision being achieved more often than acceptable difference. The VITAL-EQA program is a valuable tool for low-resource laboratories to observe the state of the field and track their own performance over time. However, the small number of samples per round and the constant changes in laboratory participants make it difficult to identify long-term improvements.

Transcriptomic characterization of clinical skeletal muscle biopsy from late-onset Pompe patients.

Pompe disease is a rare lysosomal storage disorder arising from recessive mutations in the acid α-glucosidase gene and resulting in the accumulation of glycogen, particularly in the cardiac and skeletal muscle. The current standard of care is administration of enzyme replacement therapy in the form of alglucosidase alfa or the recently approved avalglucosidase alfa. In order to better understand the underlying cellular processes that are disrupted in Pompe disease, we conducted gene expression analysis on skeletal muscle biopsies obtained from late-onset Pompe disease patients (LOPD) prior to treatment and following six months of enzyme replacement with avalglucosidase alfa. The LOPD patients had a distinct transcriptomic signature as compared to control patient samples, largely characterized by perturbations in pathways involved in lysosomal function and energy metabolism. Although patients were highly heterogeneous, they collectively exhibited a strong trend towards attenuation of the dysregulated genes following just six months of treatment. Notably, the enzyme replacement therapy had a strong stabilizing effect on gene expression, with minimal worsening in genes that were initially dysregulated. Many of the cellular process that were altered in LOPD patients were also affected in the more clinically severe infantile-onset (IOPD) patients. Additionally, both LOPD and IOPD patients demonstrated enrichment across several inflammatory pathways, despite a lack of overt immune cell infiltration. This study provides further insight into Pompe disease biology and demonstrates the positive effects of avalglucosidase alfa treatment.

Microglia ferroptosis is regulated by SEC24B and contributes to neurodegeneration.

Iron dysregulation has been implicated in multiple neurodegenerative diseases, including Parkinson's disease (PD). Iron-loaded microglia are frequently found in affected brain regions, but how iron accumulation influences microglia physiology and contributes to neurodegeneration is poorly understood. Here we show that human induced pluripotent stem cell-derived microglia grown in a tri-culture system are highly responsive to iron and susceptible to ferroptosis, an iron-dependent form of cell death. Furthermore, iron overload causes a marked shift in the microglial transcriptional state that overlaps with a transcriptomic signature found in PD postmortem brain microglia. Our data also show that this microglial response contributes to neurodegeneration, as removal of microglia from the tri-culture system substantially delayed iron-induced neurotoxicity. To elucidate the mechanisms regulating iron response in microglia, we performed a genome-wide CRISPR screen and identified novel regulators of ferroptosis, including the vesicle trafficking gene SEC24B. These data suggest a critical role for microglia iron overload and ferroptosis in neurodegeneration.

Dihydromyricetin ameliorates social isolation-induced anxiety by modulating mitochondrial function, antioxidant enzymes, and BDNF.

Stress has been implicated in the etiology of neurological and psychological illnesses. Chronic social isolation (SI) is a psychological stressor that provokes neurobehavioral changes associated with psychiatric disorders, including anxiety disorders. Mitochondria dysfunction and oxidative stress are hallmarks of anxiety pathogenesis. Here we demonstrate the effects of SI-induced stress on mitochondrial function, antioxidative enzymes, autophagy, and brain derivative neurotrophic factor (BDNF). SI induced a reduction in electron transport chain subunits C-I, C-II, and C-VI and an increase in hydrogen peroxide. Treatment with dihydromyricetin (DHM), extracted from Ampelopsis grossedentata, counteracted these changes. A dramatic increase in several primary mitochondrial antioxidative enzymes such as superoxide dismutase 2 (SOD2), heme oxygenase-1 (HO-1), peroxiredoxin-3 (PRDX3), and glutathione peroxidase 4 (GPX4) was observed after SI and a repeated episode of SI. Both SI and repeated SI induced a reduction in sequestosome 1 (SQSTM1/p62). However, only repeated SI modulated autophagy primary protein beclin-1 (Bcl-1). In addition, SI and repeated SI modulated the BDNF-TrkB signaling pathway and the phosphorylation of the downstream extracellular signal-regulated MAP kinase1/2 (p-Erk p42 and p-Erk p44) cascade. DHM treatment ameliorated these changes. Collectively, we demonstrated that DHM treatment counteracted the effects of SI and repeated SI on antioxidative enzymes, autophagy, and the BDNF-TrkB signaling pathway. These findings highlight the molecular mechanisms that partially explain the anxiolytic effects of DHM.

A multicenter analytical performance evaluation of a multiplexed immunoarray for the simultaneous measurement of biomarkers of micronutrient deficiency, inflammation and malarial antigenemia.

A lack of comparative data across laboratories is often a barrier to the uptake and adoption of new technologies. Furthermore, data generated by different immunoassay methods may be incomparable due to a lack of harmonization. In this multicenter study, we describe validation experiments conducted in a single lab and cross-lab comparisons of assay results to assess the performance characteristics of the Q-plex™ 7-plex Human Micronutrient Array (7-plex), an immunoassay that simultaneously quantifies seven biomarkers associated with micronutrient (MN) deficiencies, inflammation and malarial antigenemia using plasma or serum; alpha-1-acid glycoprotein, C-reactive protein, ferritin, histidine-rich protein 2, retinol binding protein 4, soluble transferrin receptor, and thyroglobulin. Validations included repeated testing (n = 20 separately prepared experiments on 10 assay plates) in a single lab to assess precision and linearity. Seven independent laboratories tested 76 identical heparin plasma samples collected from a cohort of pregnant women in Niger using the same 7-plex assay to assess differences in results across laboratories. In the analytical validation experiments, intra- and inter-assay coefficients of variation were acceptable at <6% and <15% respectively and assay linearity was 96% to 99% with the exception of ferritin, which had marginal performance in some tests. Cross-laboratory comparisons showed generally good agreement between laboratories in all analyte results for the panel of 76 plasma specimens, with Lin's concordance correlation coefficient values averaging ≥0.8 for all analytes. Excluding plates that would fail routine quality control (QC) standards, the inter-assay variation was acceptable for all analytes except sTfR, which had an average inter-assay coefficient of variation of ≥20%. This initial cross-laboratory study demonstrates that the 7-plex test protocol can be implemented by users with some experience in immunoassay methods, but familiarity with the multiplexed protocol was not essential.

Anemia and Vitamin B-12 and Folate Status in Women of Reproductive Age in Southern India: Estimating Population-Based Risk of Neural Tube Defects.

BACKGROUND: Women of reproductive age (WRA) are a high-risk population for anemia and micronutrient deficiencies. However, there are few representative population-level data from India, which could help inform evidence-based recommendations and policy. OBJECTIVE: To conduct a population-based biomarker survey of anemia and vitamin B-12 and folate status in WRA as part of a periconceptional surveillance program in southern India. METHODS: Participants were WRA (15-40 y) who were not pregnant or lactating. Whole blood (n = 979) was analyzed for hemoglobin via a Coulter counter (Coulter HMX). Plasma, serum, and RBCs were processed and stored at -80°C or less until batch analysis. Vitamin B-12 concentrations were measured via chemiluminescence; RBC and serum folate concentrations were evaluated via microbiological assay. Anemia and severe anemia were defined as hemoglobin <12.0 g/dL and <8.0 g/dL, respectively. Vitamin B-12 deficiency and insufficiency were defined as total vitamin B-12 <148 pmol/L and <221 pmol/L, respectively. Folate deficiency and insufficiency were defined as RBC folate <305 nmol/L and <748 nmol/L. A previously developed Bayesian model was used to predict neural tube defect (NTD) prevalence per 10,000 births. RESULTS: A total of 41.5% of WRA had anemia and 3.0% had severe anemia. A total of 48.3% of WRA had vitamin B-12 deficiency and 74.3% had vitamin B-12 insufficiency. The prevalence of RBC folate deficiency was 7.6%, and 79.3% of WRA had RBC folate <748 nmol/L, the threshold for optimal NTD prevention. Predicted NTD prevalence per 10,000 births based on RBC folate concentrations was 20.6 (95% uncertainty interval: 16.5-25.5). CONCLUSIONS: The substantial burden of anemia, vitamin B-12 deficiency, and RBC folate insufficiency in WRA in this setting suggests an opportunity for anemia and birth defects prevention. Findings will directly inform the development of a randomized trial for anemia and birth defects prevention in southern India.This study was registered at clinicaltrials.gov as NCT04048330.

Periconceptional surveillance for prevention of anaemia and birth defects in Southern India: protocol for a biomarker survey in women of reproductive age.

INTRODUCTION: Women of reproductive age (WRA) are a high-risk population for anaemia and micronutrient deficiencies. Evidence supports the role of periconceptional nutrition in the development of adverse pregnancy complications. However, in India, there are limited population-based data to guide evidence-based recommendations and priority setting. The objective of this study is to conduct a population-based biomarker survey of anaemia and vitamin B12 and folate status in WRA as part of a periconceptional surveillance programme in Southern India. METHODS: WRA (15-40 years) who are not pregnant or lactating and reside within 50 km2 of our community research site in Southern India will be screened and invited to participate in the biomarker survey at our research facility at Arogyavaram Medical Centre. After informed consent/assent, structured interviews will be conducted by trained nurse enumerators to collect sociodemographic, dietary, anthropometry, health and reproductive history data. Venous blood samples will be collected at enrolment; whole blood will be analysed for haemoglobin. Plasma, serum and red blood cells (RBCs) will be processed and stored <-80°C until batch analysis. Vitamin B12 concentrations will be measured via chemiluminescence, and RBC and serum folate concentrations will be evaluated using the World Health Organisation (WHO)-recommended microbiological assay at our laboratory in Bangalore. A WHO surveillance system will also be established to determine the baseline prevalence of birth defects in this setting. ETHICS AND DISSEMINATION: This study has obtained clearance from the Health Ministry Screening Committee of the Indian Council of Medical Research. The study protocol was reviewed and approved by the Institutional Review Board at Cornell University and the Institutional Ethics Committees at Arogyavaram Medical Centre and St. John's Research Institute. Findings from this biomarker survey will establish the burden of anaemia and micronutrient deficiencies in WRA and directly inform a randomised trial for anaemia and birth defects prevention in Southern India. The results of this study will be disseminated at international research conferences and as published articles in peer-reviewed journals. TRIAL REGISTRATION NUMBERS: Clinical trials registration number NCT04048330, NCT03853304 and Clinical Trials Registry of India (CTRI) registration number REF/2019/03/024479.

Folate status in the US population 20 y after the introduction of folic acid fortification.

BACKGROUND: Enriched cereal-grain products have been fortified in the United States for >20 y to improve folate status in women of reproductive age and reduce the risk of folic acid-responsive neural tube birth defects (NTDs). OBJECTIVES: Our objectives were to assess postfortification changes in folate status in the overall US population and in women aged 12-49 y and to characterize recent folate status by demographic group and use of folic acid-containing supplements. METHODS: We examined cross-sectional serum and RBC folate data from the NHANES 1999-2016. RESULTS: Serum folate geometric means increased from 2007-2010 to 2011-2016 in persons aged ≥1 y (38.7 compared with 40.6 nmol/L) and in women (35.3 compared with 37.0 nmol/L), whereas RBC folate showed no significant change. Younger age groups, men, and Hispanic persons showed increased serum and RBC folate concentrations, whereas non-Hispanic black persons and supplement nonusers showed increased serum folate concentrations. The folate insufficiency prevalence (RBC folate <748 nmol/L; NTD risk) in women decreased from 2007-2010 (23.2%) to 2011-2016 (18.6%) overall and in some subgroups (e.g., women aged 20-39 y, Hispanic and non-Hispanic black women, and supplement nonusers). After covariate adjustment, RBC folate was significantly lower in all age groups (by ∼10-20%) compared with persons aged ≥60 y and in Hispanic (by 8.2%), non-Hispanic Asian (by 12.1%), and non-Hispanic black (by 20.5%) compared with non-Hispanic white women (2011-2016). The 90th percentile for serum (∼70 nmol/L) and RBC (∼1800 nmol/L) folate in supplement nonusers aged ≥60 y was similar to the geometric mean in users (2011-2014). CONCLUSIONS: Blood folate concentrations in the US population overall and in women have not decreased recently, and folate insufficiency rates are ∼20%. Continued monitoring of all age groups is advisable given the high folate status particularly in older supplement users.

Age-specific reference ranges are needed to interpret serum methylmalonic acid concentrations in the US population.

BACKGROUND: Serum vitamin B-12 is measured to evaluate vitamin B-12 status. Serum methylmalonic acid (MMA) is a specific functional indicator of vitamin B-12 status; however, concentrations increase with impaired renal function. OBJECTIVE: The aim of this study was to describe the distribution of serum vitamin B-12 and MMA in US adults, and estimate age-specific reference intervals for serum MMA in a healthy subpopulation with replete vitamin B-12 status and normal renal function. METHODS: We examined cross-sectional data for serum vitamin B-12 and MMA in adults participating in the NHANES from 2011 to 2014. Vitamin B-12 was measured by electrochemiluminescence assay and MMA by isotope-dilution liquid chromatography-tandem mass spectrometry. RESULTS: In both bivariate and multivariate analyses, age, race/Hispanic origin, and vitamin B-12 supplement use were generally significantly associated with serum vitamin B-12 and MMA concentrations. Serum MMA concentrations increased with age, particularly in persons aged ≥70 y. Non-Hispanic white persons had lower vitamin B-12 and higher MMA concentrations than non-Hispanic black persons. Shorter fasting times and impaired renal function were significantly associated with higher serum MMA concentrations, but not with serum vitamin B-12 concentrations after controlling for covariates. The central 95% reference intervals for serum vitamin B-12 and MMA concentrations were widest for persons aged ≥70 y compared with younger age groups. Compared with the overall population, the central 95% reference intervals for serum MMA concentrations were considerably narrower for a vitamin B-12-replete subpopulation with normal renal function, but still age-dependent. Serum vitamin B-12 showed little, whereas serum MMA showed notable, increases with impaired renal function. CONCLUSIONS: The higher serum MMA concentrations throughout the entire distribution in older persons (especially persons aged ≥70 y) who are vitamin B-12-replete and have normal renal function indicate the need for age-specific MMA reference intervals to better interpret vitamin B-12 status in epidemiologic research.

Framework for laboratory harmonization of folate measurements in low- and middle-income countries and regions.

The measurement of serum and red blood cell folate, two commonly used biomarkers of folate status in populations, is complicated by analytical and data interpretation challenges. Folate results show poor comparability across laboratories, even using the same analytical technique. The folate microbiologic assay produces accurate results and requires simple instrumentation. Thus, it could be set up and maintained in low- and middle-income country laboratories. However, the assay has to be harmonized through the use of common critical reagents (e.g., microorganism and folate calibrator) in order to produce comparable results across laboratories and over time, so that the same cutoff values can be applied across surveys. There is a limited need for blood folate measurements in a country owing to the periodic nature of surveys. Having a network of regional resource laboratories proficient in conducting the folate microbiologic assay and willing and able to perform service work for other countries will be the most efficient way to create an infrastructure wherein qualified laboratories produce reliable blood folate data. Continuous participation of these laboratories in a certification program can verify and document their proficiency. If the resource laboratories conduct the work on a fee-for-service basis, they could become self-sustaining in the long run.

Quantitative reproducibility analysis for identifying reproducible targets from high-throughput experiments.

BACKGROUND: High-throughput assays are widely used in biological research to select potential targets. One single high-throughput experiment can efficiently study a large number of candidates simultaneously, but is subject to substantial variability. Therefore it is scientifically important to performance quantitative reproducibility analysis to identify reproducible targets with consistent and significant signals across replicate experiments. A few methods exist, but all have limitations. METHODS: In this paper, we propose a new method for identifying reproducible targets. Considering a Bayesian hierarchical model, we show that the test statistics from replicate experiments follow a mixture of multivariate Gaussian distributions, with the one component with zero-mean representing the irreproducible targets. RESULTS: A target is thus classified as reproducible or irreproducible based on its posterior probability belonging to the reproducible components. We study the performance of our proposed method using simulations and a real data example. CONCLUSION: The proposed method is shown to have favorable performance in identifying reproducible targets compared to other methods.

Differential Ly6C Expression after Renal Ischemia-Reperfusion Identifies Unique Macrophage Populations.

Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear. In this study, we used a renal bilateral ischemia-reperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b(+) cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies. Macrophage populations were isolated from kidney homogenates by fluorescence-activated cell sorting for whole genome microarray analysis. The CD11b(+)/Ly6C(high) population associated with the onset of renal injury and increase in proinflammatory cytokines, whereas the CD11b(+)/Ly6C(intermediate) population peaked during kidney repair. The CD11b(+)/Ly6C(low) population emerged with developing renal fibrosis. Principal component and hierarchical cluster analyses identified gene signatures unique to each population. The CD11b(+)/Ly6C(intermediate) population had a distinct phenotype of wound healing, confirmed by results of studies inhibiting the macrophage colony-stimulating factor 1 receptor,whereas the CD11b(+)/Ly6C(low) population had a profibrotic phenotype. All populations, including the CD11b(+)/Ly6C(high) population, carried differential inflammatory signatures. The expression of M2-specific markers was detected in both the CD11b(+)/Ly6C(intermediate) and CD11b(+)/Ly6C(low) populations, suggesting these in vivo populations do not fit into the traditional classifications defined by in vitro systems. Results of this study in a renal ischemia-reperfusion injury model allow phenotype and function to be assigned to CD11b(+)/Ly6C(+) monocyte/macrophage populations in the pathophysiology of disease after AKI.

Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA.

Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.

Biomarkers of Nutrition for Development-Folate Review.

The Biomarkers of Nutrition for Development (BOND) project is designed to provide evidence-based advice to anyone with an interest in the role of nutrition in health. Specifically, the BOND program provides state-of-the-art information and service with regard to selection, use, and interpretation of biomarkers of nutrient exposure, status, function, and effect. To accomplish this objective, expert panels are recruited to evaluate the literature and to draft comprehensive reports on the current state of the art with regard to specific nutrient biology and available biomarkers for assessing nutrients in body tissues at the individual and population level. Phase I of the BOND project includes the evaluation of biomarkers for 6 nutrients: iodine, iron, zinc, folate, vitamin A, and vitamin B-12. This review represents the second in the series of reviews and covers all relevant aspects of folate biology and biomarkers. The article is organized to provide the reader with a full appreciation of folate's history as a public health issue, its biology, and an overview of available biomarkers (serum folate, RBC folate, and plasma homocysteine concentrations) and their interpretation across a range of clinical and population-based uses. The article also includes a list of priority research needs for advancing the area of folate biomarkers related to nutritional health status and development.

Folate and Vitamin B12 Deficiency Among Non-pregnant Women of Childbearing-Age in Guatemala 2009-2010: Prevalence and Identification of Vulnerable Populations.

INTRODUCTION: Information on folate and vitamin B12 deficiency rates in Guatemala is essential to evaluate the current fortification program. The objectives of this study were to describe the prevalence of folate and vitamin B12 deficiencies among women of childbearing age (WCBA) in Guatemala and to identify vulnerable populations at greater risk for nutrient deficiency. METHODS: A multistage cluster probability study was designed with national and regional representation of nonpregnant WCBA (15-49 years of age). Primary data collection was carried out in 2009-2010. Demographic and health information was collected through face-to-face interviews. Blood samples were collected from 1473 WCBA for serum and red blood cell (RBC) folate and serum vitamin B12. Biochemical concentrations were normalized using geometric means. Prevalence rate ratios were estimated to assess relative differences among different socioeconomic and cultural groups including ethnicity, age, education level, wealth index and rural versus urban locality. RESULTS: National prevalence estimates for deficient serum [<10 nmol per liter (nmol/L)] and RBC folate (<340 nmol/L) concentrations were 5.1 % (95 % CI 3.8, 6.4) and 8.9 % (95 % CI 6.7, 11.7), respectively; for vitamin B12 deficiency (<148 pmol/L) 18.5 % (95 % CI 15.6, 21.3). Serum and RBC folate deficiency prevalences were higher for rural areas than for urban areas (8.0 vs. 2.0 % and 13.5 vs. 3.9 %, respectively). The prevalence of RBC folate deficiency showed wide variation by geographic region (3.2-24.9 %) and by wealth index (4.1-15.1 %). The prevalence of vitamin B12 deficiency also varied among regions (12.3-26.1 %). CONCLUSIONS: In Guatemala, folate deficiency was more prevalent among indigenous rural and urban poor populations. Vitamin B12 deficiency was widespread among WCBA. Our results suggest the ongoing need to monitor existing fortification programs, in particular regarding its reach to vulnerable populations.