Determination Of Folates Research Articles

A metal-complex based chemosensor for the detection of riboflavin and folate

The vitamins of riboflavin and folate are necessary nutrients for maintaining the proper functioning of human body. Riboflavin and folate deficiency will cause nerve damage, leading to peripheral neuritis, depression, tongue inflammation and other diseases. The sensitive detection of riboflavin and folate keeps its significance for patients and food quality control. In this study, a quinoline-pyridine-combined chemosensor (HQ-TPE) modified by tetraphenylethene was developed. After chelating Cd2+, the chemosensor exhibited high selectivity and sensitivity for riboflavin and folate. Moreover, it can discriminate the two different vitamins through different fluorescent responses, which should arise from the different intermolecular π-π interactions between the sensor HQ-TPE and the analyte upon coordination of riboflavin or folate with Cd2+. More importantly, the chemosensor could be used in visual semi-quantitative determination of riboflavin and folate in real samples (milk and lettuce). Therefore, the sensor presented here will not only be a powerful tool for the detection of riboflavin and folate, but also provides a new template for the design of metal complex as fluorescent sensor.

Folate Biofortification in Soybean: Challenges and Prospects

Folate deficiency is a significant global health issue that affects millions of people and causes severe adverse effects. Major staple crops, which provide significant amounts of calories, often contain inadequate folate levels. Synthetic fortification has contributed to a reduction in low-folate populations, but a more sustainable solution is needed. Biofortification, or the breeding of crops to naturally increase their nutrient content, is a promising alternative. Soybean is a highly nutritious crop and a good candidate for folate biofortification. However, studies on folate have been limited due to the challenges in folate analysis. The development of sensitive and selective tools, reference materials, and studies on the stability of folate vitamers in crops has facilitated the development of improved folate determination methods. Additionally, the soybean folate biofortification program can be improved by leveraging previous studies in major cereals, common bean and pea, as well as combining conventional breeding with new genomics approaches. In this review, we discuss the folate content, composition, and analytical challenges in soybean and suggest possible frameworks and strategies for folate biofortification in soybean. We also conducted an in silico analysis of key folate biosynthesis enzymes in soybean.

Dietary intake of folate and the frequency of its deficiency in children with type 1 diabetes mellitus and healthy children

Adequate folate intake is essential for a child’s growth. There is lack of information about the prevalence of this nutrient deficiency in the Ukrainian population, including children. The aim of the study was to evaluate the dietary intake of folate and determine the frequency of folate deficiency in children with Type 1 diabetes mellitus (T1D) and healthy children. Determination of folate in serum was performed by ELISA. Folate level <3 ng/ml was diagnosed as a folate deficiency. Among all observed children the folate deficiency was diagnosed in 23 (32.9%): in 6 (17.1%) patients with T1D and in 17 (48.6%) healthy children (P ≤ 0.01). The mean level of serum folate in patients with T1D was (5.09 ± 2.16) ng/ml and (3.72 ± 1.87) ng/ml in healthy children (P ≤ 0.01). The average daily intake of folate with food was (138.68 ± 70.37) µg, without difference between T1D (12.00 ± 3.51 yr.) and healthy groups (10.83 ± 3.24 yr.), and it was more than two times lower than age requirements (300 µg/day). However, it was self-reported that 15 (48.9%) children of T1D group received vitamin supplementation one time in six months, while in healthy children only 6 (17.1%) children received vitamins (P ≤ 0.01). In conclusion, the frequency of folate deficiency is high in the pediatric population. Nutrition does not provide the necessary intake of folate, which indicates the need for additional folate supplementation. Keywords: children., deficiency, diabetes mellitus, dietary intake, folate

A Novel Synthesis of a Magnetic Porous Imprinted Polymer by Polyol Method Coupled with Electrochemical Biomimetic Sensor for the Detection of Folate in Food Samples

The present study reports the development and application of a novel, sensitive, and selective voltammetric sensor for the quantitation of folate or vitamin B9 in foodstuffs. The sensor was made from magnetic molecularly imprinted polymers (MMIPs), which were synthesized by the core–shell method using magnetite nanoparticles obtained by the polyol method. The MMIP-based sensor was used for the selective and specific detection of folate in different food samples. The MMIP material was constructed using magnetic water-dispersible nanomaterial, which was prepared by immersing iron (III) acetylacetonate in tri-ethylene-glycol (TEG) solvent. The magnetic water-dispersible nanomaterial was then subjected to polymerization using allyl alcohol as a functional monomer, ethylene-glycol-dimethacrylate (EGDMA) as a cross-linking agent, and 2,2-Azobisisobutyronitrile (AIBN) as a radical initiator. The proposed magnetic materials were characterized by Brunauer–Emmett–Teller (BET), field emission gun scanning electron microscopy (FEG-SEM), thermogravimetric analysis (TGA), and vibrating sample magnetometer (VSM) analysis. The quantification of folate was performed by square wave voltammetry under optimized conditions using 15 mg of MMIPs and 85 mg of carbon paste. The modified electrode presented a linear dynamic range (LDR) of 2.0–12 µmol L−1 and a limit of detection (LOD) of 1.0 × 10−7 mol L−1 in 0.1 mol L−1 acetate buffer solution (pH 4.0). The proposed sensor was successfully applied for folate detection in different food samples, where recovery percentages ranging from 93 to 103% were obtained. Finally, the results obtained from the analysis of selectivity showed that the modified biomimetic sensor is highly efficient for folate determination in real food samples. Adsorption tests were used to evaluate and compare the efficiency of the MMIPs and magnetic non-molecularly imprinted polymer (MNIPs)—used as control material, through the application of HPLC as a standard method.

Simultaneous determination of folate and methotrexate metabolites in serum by LC-MS/MS during high-dose methotrexate therapy

High-dose methotrexate (HDMTX) is a central component in the treatment of acute lymphoblastic leukemia, osteosarcoma, and some lymphomas and brain tumors. MTX is given at lethal doses and then is followed by rescue treatment with folinic acid (FA). Despite FA rescue, many patients suffer severe toxicity. The pharmacokinetics of FA rescue have not been sufficiently studied. However, optimization of FA rescue could potentially increase anti-tumor effects, whilst decreasing organ toxicity. Here, we describe our efforts to establish and optimize a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the simultaneous determination of five essential components of the folate cycle, as well as MTX and its two metabolites. The method was applied to 6 individual patients receiving HDMTX, with 3 or 4 measurements for each patient. The method allows analysis of samples that were initially frozen. This notion, together with the test results in the 6 pilot patients, shows the feasibility of this method to study MTX and FA pharmacokinetics during HDMTX treatment. The method has the potential to optimize HDMTX and FA rescue treatment in individual patients.

Lipid profile and serum folate, vitamin B12 and homocysteine levels in patients with retinal vein occlusion

BackgroundRetinal vein occlusion (RVO) is mainly related with vascular risk factors (VRF). ObjectivesTo analyze the lipid profile and serum folate, vitamin B12 and homocysteine levels, in patients with RVO and a population-based control group. Patients and methodsCase–control study. Patients with RVO were assessed during an 11-year period. ResultsWe included 368 patients and 325 controls of similar age and sex. HDL cholesterol and folate levels were lower (52 [43–63]mg/dL vs. 55 [46–66]mg/dL; p=0.016 and 7 [5–10]ng/mL vs. 9 [7–13]ng/mL; p<0.0001, respectively) and non-HDL cholesterol and homocysteine levels higher (148.9±37.3mg/dL vs. 142.9±34.5mg/dL; p=0.03 and 13.4 [11.2–18.2]μmol/L vs. 11.1 [9.0–14.4]μmol/L; p<0.001) in patients with RVO than controls. Although total cholesterol, LDL-C, and triglyceride levels were higher and serum vitamin B12 levels were lower in RVO patients, these differences did not reach statistical significance. ConclusionsRVO-patients have lower serum HDL-C and folate levels and higher non-HDL-C and serum homocysteine levels than population-based controls of similar age and sex. In patients with RVO, apart from the lipid profile, determination of serum homocysteine, folate and vitamin B12 levels might be useful, as well as the treatment of their alterations.

Perfil lipídico y concentraciones séricas de ácido fólico, vitamina B12 y homocisteína en pacientes con obstrucción venosa retiniana

BackgroundRetinal vein occlusion (RVO) is mainly related with vascular risk factors (VRF). ObjectivesTo analyze the lipid profile and serum folate, vitamin B12 and homocysteine levels, in patients with RVO and a population-based control group. Patients and MethodsCase-control study. Patients with RVO were assessed during an 11-year period. ResultsWe included 368 patients and 325 controls of similar age and sex. HDL cholesterol and folate levels were lower (52 [43-63] mg/dL vs. 55 [46-66]; p = 0.016 and 7 [5-10] ng/mL vs. 9 [7-13]; p < 0.0001, respectively) and non-HDL cholesterol and homocysteine levels higher (148.9 ± 37.3 mg/dL vs. 142.9 ± 34.5; p = 0.03 and 13.4 [11.2-18.2] μmol/L vs. 11.1 [9.0-14.4]; p < 0.001) in patients with RVO than controls. Although total cholesterol, LDL-C, and triglyceride levels were higher and serum vitamin B12 levels were lower in RVO patients, these differences did not reach statistical significance. ConclusionsRVO-patients have lower serum HDL-C and folate levels and higher non-HDL-C and serum homocysteine levels than population-based controls of similar age and sex. In patients with RVO, apart from the lipid profile, determination of serum homocysteine, folate and vitamin B12 levels might be useful, as well as the treatment of their alterations.

Comprehensive Vitamer Profiling of Folate Mono- and Polyglutamates in Baker's Yeast (Saccharomyces cerevisiae) as a Function of Different Sample Preparation Procedures.

Folates are a group of B9 vitamins playing an important role in many metabolic processes such as methylation reactions, nucleotide synthesis or oxidation and reduction processes. However, humans are not able to synthesize folates de novo and thus rely on external sources thereof. Baker’s yeast (Saccharomyces cerevisiae) has been shown to produce high amounts of this vitamin but extensive identification of its folate metabolism is still lacking. Therefore, we optimized and compared different sample preparation and purification procedures applying solid phase extraction (SPE). Strong anion exchange (SAX), C18 and hydrophilic–lipophilic-balanced (HLB) materials were tested for their applicability in future metabolomics studies. SAX turned out to be the preferred material for the quantitative purification of folates. Qualification of several folate vitamers was achieved by ultra-high pressure liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-ToF-MS) measurements and quantification was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) applying stable isotope dilution assays (SIDAs). The oxidation product s-pyrazino-triazine (MeFox) was included into the SIDA method for total folate determination and validation. Applying the best protocol (SAX) in regard to folate recovery, we analyzed 32 different vitamers in different polyglutamate states up to nonaglutamates, of which we could further identify 26 vitamers based on tandem-MS (MS2) spectra. Total folate quantification revealed differences in formyl folate contents depending on the cartridge chemistry used for purification. These are supposedly a result of interconversion reactions occurring during sample preparation due to variation in pH adjustments for the different purification protocols. The occurrence of interconversion and oxidation reactions should be taken into consideration in sample preparation procedures for metabolomics analyses with a focus on folates.

Determination of Folates Synthesized by Lactic Acid Bacteria

Eight strains of lactic acid bacteria, including Bifidobacterium longum B6 and 15708, Lactobacillus acidophilus N-1 and 4356, Lactobacillus bulgaricus 448 and 449, and Streptococcus thermophilus MC and 573 were investigated for their folate synthesizing ability by applying the high-performance liquid chromatographic method. The results showed that lactic acid bacteria synthesized maximum levels of folate in six hours in fermented milk. B. longum B6 demonstrated the highest folate synthesizing ability, and B. longum 15708 also produced folate satisfactorily. These two strains increased folate 3.5 and 2.4 fold, respectively, after 6h fermentation at 37°C. 5-Methyltetrahydrofolate was the main derivative of folate found in fermented milk.

Advances in Methods for the Determination of Folate in Foods

Advances in Methods for the Determination of Folate in Foods

Folate monoglutamate in cereal grains: Evaluation of extraction techniques and determination by LC-MS/MS

Folates are essential micronutrients for human health. To determine the total folate content, the extraction and quantification of seven monoglutamate folate derivatives in cereals (maize, rice, and wheat) were optimised and validated in this study. Di-enzyme treatment with α-amylase and rat conjugase was proved ideal for folate extraction from the cereal grains. The quantification method by liquid chromatography-tandem mass spectrometry was validated based on its matrix effect, linearity, sensitivity, recovery, inter-day and intra-day precision. The limits of detection and quantification of folate derivatives ranged from 0.03–0.88 and 0.1–1.0 μg/100 g among the three cereal samples. The absolute recoveries of most folate derivatives were 72–96 % for these cereal samples, with the exception of dihydrofolate, tetrahydrofolate, and folic acid (44–65 %). The inter-day and intra-day precisions were < 12 % for the three cereals. Analysis of folate content and composition in several cereal grains showed that the total folate levels were approximately 26−37 μg/100 g, with 5-methyl-tetrahydrofolate and 5-methyl-tetrahydrofolate as the dominant. MeFox, an oxidation product of 5-methyltetrahydrofolate, was detected at concentrations 20–39-fold higher than those of total folates in rice and wheat grains. This validated method is an efficient approach for folate determination in cereal crops.

Folate determination in livers of different animal species

The liver is the main storage organ for folate. In the study the folate content in chicken, turkey, pig, and beef livers was evaluated with the use of the HPLC (high performance liquid chromatography) method which allowed various folate vitamers differentiation. The total folate content in the tested livers ranged from 419 to 1289 µg 100 g–1 in pork and chicken livers, respectively, and was several times higher than in folate rich vegetables, which are the most common folate sources in the human diet. Additionally, good stability of two folate abundant forms, tetrahydrofolate and 5-methyltetrahydrofolate, under frying was shown in the chicken liver samples. The obtained results contribute well to the general need of promoting the folate intake from various natural sources, not only of plant origin.&lt;br /&gt;&lt;br /&gt;

Determination of Total Folates in Complex Nutritional Drinks and Supplements Using a Tri-Enzyme Microbiological Method and Michaelis-Menten Kinetics.

Background: Recent development of LC methods for the determination of total folates (vitamin B9) in complex matrixes have been hindered by vitamer interconversion and yield variability. The official microbiological method (AOAC Official Methods of Analysis 944.12 and 960.46) uses an end point turbidity reading to determine folate concentration. However, when measuring complex matrixes, shifts are observed in the growth curves of the microorganism and inaccuracies are introduced to this quantification method. Objective/Methods: In addition to the tri-enzyme digestion of the standard microbiological method, we have applied enzyme modeling of the initial velocity of bacterial growth using Michaelis-Menten kinetics to achieve more accurate and reproducible determinations of total folates. Results/Conclusions: Accuracy determined through spike recovery in Infant/Adult Nutritional Drink and a complex vitamin matrix gave values acceptable to AOAC standards of 85-110%. Repeatability of the low mass fraction analyte measured at micrograms per 100 g yielded relative standard deviations <15% for all matrixes tested, including three standard reference materials.

Clinical determination of folates: recent analytical strategies and challenges.

Since the introduction of liquid chromatography tandem mass spectrometry in clinical laboratories, folate analysis has shifted from microbiological or protein-binding assays to chromatographic methods. Now, it is possible to sensitively and selectively determine several folate species in clinical samples where only a total folate content could be quantified using a microbiological or a binding assay. Although several chromatographic methods have been developed, validated, and published, interlaboratory variability limits the comparability of the results. In this review, we provide an overview of the latest strategies for sampling, sample treatment, and analysis and how these may influence the final analytical result. Among the variables covered are the effect of pH, temperature, and storage and the use of antioxidants and anticoagulants on analyte stability. In addition, we highlight the importance of correct assay calibration and the use of (labeled) certified reference materials in order to obtain correct and comparable results among different laboratories. Graphical abstract ᅟ.

Human serum folate can be measured using folate binding protein linked to enzyme-labeled protein ligand binding assay (ELPLBA) as well as ELISA

Background: Folate is an important substance used for purine and pyrimidine nucleotide synthesis. One measurement of folate that already establishes is using ELISA (Enzyme-linked immunosorbent assay) method. Folate binding protein is a protein that can bind folate, therefore it considered can be used as a tool that can replace antibody dependent ELISA method.&#x0D; Objectives: The aim of this research was to create a method for folate measurement in serum called Enzyme-labeled protein ligand binding assay (ELPLBA) by replacing antibody as used in ELISA method with folate binding protein (FBP) that purified from the whey of milk.&#x0D; Methods: The method is tested using 20 serum samples and compared to ELISA. Folate binding protein was purified from bovine’s milk using ammonium sulfate up to 90% saturated, DEAE-cellulose anion exchange chromatography and affinity chromatography. SDS-PAGE and western blot were used to establish the protein band of FBP that has molecular weight of ~25-35 kDa. ELPLBA was arranged with stationary phase using aminohexyl-agarose, and folic acid linked on it using carbodiimide.&#x0D; Results: The result show there was no significant difference of folate concentration between ELPLBA (14.804 ± 2.795) and ELISA method (13.859 ± 3.638), p = 0.363.&#x0D; Conclusion: ELPLBA method show similarity for determination of folate in serum which was the same as standard folate measurement (ELISA).

LC-MS/MS Determination of Mono-Glutamate Folates and Folic Acid in Beer

A reliable LC-MS/MS method was developed for determination of folates in beer. Clean-up steps and chromatographic conditions were optimized to remove interferences and decrease the matrix effect. The work showed that an immuno-affinity column designed for folic acid (FA) was also suitable for the oxidized forms 10-formylfolic acid (10-HCO-FA) and 7,8-dihydrofolic acid (DHF), obtaining clean extract and improving the limit of detection. For the other vitamer forms, an Oasis MAX column was used. For chromatographic conditions, a RP-18 column with low ligand density showed satisfactory retention times; moreover, the use of methanol instead of acetonitrile lowered the limit of detection up to 0.1 μg l−1. The most popular beers in Italy were collected and analyzed; mainly oxidized mono-glutamate forms, in particular 10-HCO-FA and FA, were found, at levels from 12.4 to 98.6 μg l−1 and 1.4 and 11.9 μg l−1, respectively. The vitamers 5-formyl-tetrahydrofolate and 5-methyl-tetrahydrofolate were also detected.

Association between seasonal serum folate levels and ultraviolet radiation

UV radiation has been consistently reported to cause folate photodegradation in vitro and in human skin. Seasonal variations in UV radiation might explain seasonal changes in folate levels in blood. Yet, few studies have addressed this phenomenon. The main objective of this study was to investigate whether a relationship exists between seasonal variations in serum folate levels in a population of Spain (Latitude: 36° 41′ 6.88″; Longitude: 4° 30′ 0.64″) and the annual variations of solar ultraviolet reached in the localization.From a sample of 118,831 serum folate determinations, two studies were performed. The first one, which included all subjects, showed a decreased in mean folate concentrations in all seasons with respect to winter with lower values in summer. The risk of folate deficiency was 1.37 times greater in summer than in winter (95%CI: 1.29–1.46). In the second study, subjects with a first folate determination in winter and a second one in summer were 3.32 more likely to develop folate deficiency than those with a first folate determination in summer and a second one in winter (95%CI: 1.55 to 7.11).Folate levels showed a seasonal variation inversely related to solar total UV radiation reached in our location, with maximum daily doses of 5000 J m−2 reached in June. A gradual increase in percentage of folate deficiency is observed since spring. So, patients with folate levels close to deficiency are at a higher risk of having folate deficiency in summer.

Determination of vitamin B12, folate, and ferritin levels of inpatients in a psychiatry clinic: A one-year retrospective study

Amac: Bu calismada bir psikiyatri kliniginde yatan hastalarin B12 vitamini, folat ve ferritin duzeyleri karsilastirildi. Gerec ve yontemler: Calismaya 01 Ocak 2017 ve 31 Aralik 2017 tarihleri arasinda Adiyaman Universitesi Egitim ve Arastirma Hastanesi psikiyatri servisinde yatarak tedavi goren 286 hasta (178 erkek, 108 kadin; ort. yas 35.4±12.9 yil; dagilim, 15-76 yil) dahil edildi. Hastalarin tanilari ve B12 vitamini, folat ve ferritin duzeyleri retrospektif olarak incelendi. Bulgular: En sik gorulen tanilar sizofreni (%23.4), bipolar affektif bozukluk (%23.1) ve major depresif bozukluk (%11.5) idi. Cinsiyete gore B12 vitamini duzeylerinde anlamli farklilik yokken (p=0.850) erkeklerde folat duzeyi (p=0.001) ve kadinlarda ferritin duzeyi (p=0.001) anlamli olarak dusuk idi. Tanilar arasindaki karsilastirmada; B12 vitamini, folat ve ferritin duzeyleri arasinda farklilik tespit edilmedi. Sonuc: B12 vitamini, folat ve ferritin duzeylerinde eksiklik psikiyatrik semptomlara neden olabilir. Calismamizda B12 vitamini duzeyi dusuk olan hasta sayisinin fazla olmasina bagli olarak B12 vitamini replasmaninda daha dikkatli olunabilir. Kadinlardaki ferritin eksikligi ve erkeklerdeki folat eksikligi dikkat cekicidir. Bu calismanin bulgularini desteklemek ve psikiyatrik sendromu olan hastalara verilen bakimin optimize edilmesi icin B12 vitamini, folat ve ferritine iliskin daha spesifik ve ayrintili algoritmalari belirlemek amaciyla ileri arastirmalar gerekmektedir.

Isolation of folate-producing Weissella spp. from Thai fermented fish (Plaa Som Fug)

Folate-producing lactic acid bacteria (LAB) have received a great deal of attention since they are generally used as starter cultures. When they produce folate, fermented products obtained are beneficial to health. This study sought to screen new metabolites from Weissella spp. isolated from Thai fermented fish (Plaa Som Fug). Among 52 lactic acid bacteria isolates, 11 strains were identified as Weissella spp. based on 16S rRNA gene sequences, six of which were W. confusa (SF2-1, SF2-3, SF4-4, SF5-1, SF6-2 and PL13-2); five of which were W. cibaria (SF1-1, SF1-2, SF4-3, PL 5–4 and PL17-3). All strains of Weissella produced lactic acid at a concentration of 8.75–32.30 mg mL −1. Determination of folate by high-performance liquid chromatography (HPLC) revealed that these strains were also capable of producing folate in a folate-free medium, with a maximum concentration of 4.14 μg mL−1 yielded by W. cibaria PL17-3. The average folate concentration produced among the 11 strains was 2.47 μg mL−1. This is the first study to report the isolation of folate-producing W. cibaria. The discovery of folate-producing Weissella may help improve the nutrition of fermented food.

Folate Deficiency Is Prevalent in Women of Childbearing Age in Belize and Is Negatively Affected by Coexisting Vitamin B-12 Deficiency: Belize National Micronutrient Survey 2011

Background: Folate deficiency, vitamin B-12 deficiency, and anemia can have adverse effects on birth outcomes. Also, low vitamin B-12 reduces the formation of metabolically active folate.Objectives: We sought to establish the baseline prevalence of and factors associated with folate deficiency and insufficiency, vitamin B-12 deficiency, and anemia among women of childbearing age (WCBA) in Belize.Methods: In 2011, a national probability-based survey was completed among Belizean nonpregnant WCBA aged 15-49 y. Blood samples for determination of hemoglobin, folate (RBC and serum), and vitamin B-12 (plasma) and sociodemographic and health information were collected from 937 women. RBC and serum folate concentrations were measured by microbiologic assay (MBA). Folate status was defined based on both the WHO-recommended radioproteinbinding assay and the assay adjusted for the MBA.Results: The national prevalence estimates for folate deficiency in WCBA, based on serum and RBC folate concentrations by using the assay-matched cutoffs, were 11.0% (95% CI: 8.6%, 14.0%) and 35.1% (95% CI: 31.3%, 39.2%), respectively. By using the assay-matched compared with the WHO-recommended cutoffs, a substantially higher prevalence of folate deficiency was observed based on serum (6.9% absolute difference) and RBC folate (28.9% absolute difference) concentrations. The prevalence for RBC folate insufficiency was 48.9% (95% CI: 44.8%, 53.1%). Prevalence estimates for vitamin B-12 deficiency and marginal deficiency and anemia were 17.2% (95% CI: 14.2%, 20.6%), 33.2% (95% CI: 29.6%, 37.1%), and 22.7% (95% CI: 19.5%, 26.2%), respectively. The adjusted geometric means of the RBC folate concentration increased significantly (P-trend < 0.001) in WCBA who had normal vitamin B-12 status relative to WCBA who were vitamin B-12 deficient.Conclusions: In Belize, the prevalence of folate and vitamin B-12 deficiencies continues to be a public health concern among WCBA. Furthermore, low folate status co-occurred with low vitamin B-12 status, underlining the importance of providing adequate vitamin B-12 and folic acid intake through approaches such as mandatory food fortification.