Indicator Of Folate Status Research Articles

Development of Simple Method for the Determination of 5-Methyl Tetrahydrofolate in Dried Blood Spot by High-Performance Liquid Chromatography

Folate (Reduced) or folic acid (oxidized), is a water-soluble vitamin necessary for thymidylate synthase, methionine synthase, serine hydroxymethyltransferase, and folate deficiency associated with megaloblastic anemia, neural tube defect, and coronary heart disease. The available folate data is minimal due to a dearth of suitable, accurate, field-friendly blood collection and estimation methods. Conventional assays like microbiological and competitive protein-binding radioassay methods are used to estimate the total folate in serum and whole blood. In erythrocytes, folate exists mainly as 5-methyltetrahydrofolate polyglutamate and indicates the long-term storage of folate status. Therefore, the study aimed to develop a simple method for estimating 5-methyl tetrahydrofolate (5-MTHF) by high-performance liquid chromatography (HPLC) in dried blood spots (DBS) as an indicator of folate status. The present study results indicate standard 5-methyltetrahydrofolate with different concentrations with linear regression was showed R2 -0.98, and the minimum detection limit of a standard was 20 pg. The inter-and intra-assay variations were found to be less than 10%. A good correlation (R2 -0.964) between the DBS and blood and the recovery of added standard in the DBS sample was more than 80%. A minimum of three DBS punches equivalent to 20mL of blood was required for the analysis, and the competitive protein-binding radioassay showed slightly high folate compared to the HPLC method. The stability of 5-MTHF in DBS was also tested periodically. DBS technique and HPLC method are simple, sensitive, not expansive, and safe compared to the microbiological and competitive binding assay for assessing sub-clinical folate status in a field survey on a large population.

Population RBC folate concentrations can be accurately estimated from measured whole blood folate, measured hemoglobin, and predicted serum folate—cross-sectional data from the NHANES 1988–2010

RBC folate (RBF) is an indicator of folate status and risk of neural-tube defects. It is calculated from whole blood folate (WBF), serum folate (SFOL), and hematocrit (Hct). SFOL and/or Hct are sometimes unavailable; hemoglobin (Hb) is generally available in surveys. We assessed the ability of different RBF approximations to generate population data in women aged 12-49 y. Using SFOL, RBF, Hct, Hb, and mean corpuscular Hb content (MCHC) from prefortification (1988-1994) and postfortification (1999-2006, 2007-2010) NHANES we applied 6 approaches: #1) assume SFOL=0; #2) impute SFOL (population median); #3) impute Hct (population median); #4) estimate Hct (Hb/MCHC); #5) assume SFOL=0 and estimate Hct; and #6) predict SFOL (from WBF) and estimate Hct. For each approach, we calculated the paired percentage difference to the "true" RBF and estimated various statistics. For 2007-2010 (unweighted data), the median relative difference from "true" RBF was lowest for approaches #2 (-0.74%), #4 (-0.96%), and #6 (-1.15%), intermediate for #3 (-3.36%), and highest for #5 (4.96%) and #1 (5.78%). The 95% agreement limits were smallest for approach #1 (2.33%, 13.0%) and largest for #3 (-20.8%, 11.3%). Approach #2 showed concentration-dependence (negative compared with positive differences at low compared with high RBF). Using weighted data, we found similar patterns across approaches for mean relative differences by demographic subgroup for all 3 time periods. We obtained the best agreement between estimated and "true" RBF when we predicted SFOL using a regression equation obtained from a subset of samples (approach #6). Alternatively, the consistent overestimation of RBF when assuming SFOL=0 (∼6%) could be addressed by adjusting the data (approach #5). Similar observations for pre- and postfortification periods suggest applicability to low and high folate status situations, but should be confirmed elsewhere. To estimate RBF, at least WBF and Hb are needed.

Folate status and concentrations of serum folate forms in the US population: National Health and Nutrition Examination Survey 2011-2.

Serum and erythrocyte (RBC) total folate are indicators of folate status. No nationally representative population data exist for folate forms. We measured the serum folate forms (5-methyltetrahydrofolate (5-methylTHF), unmetabolised folic acid (UMFA), non-methyl folate (sum of tetrahydrofolate (THF), 5-formyltetrahydrofolate (5-formylTHF), 5,10-methenyltetrahydrofolate (5,10-methenylTHF)) and MeFox (5-methylTHF oxidation product)) by HPLC-MS/MS and RBC total folate by microbiologic assay in US population ≥1 year (n approximately 7500) participating in the National Health and Nutrition Examination Survey 2011-2. Data analysis for serum total folate was conducted including and excluding MeFox. Concentrations (geometric mean; detection rate) of 5-methylTHF (37·5nmol/l; 100%), UMFA (1·21nmol/l; 99·9%), MeFox (1·53nmol/l; 98·8%), and THF (1·01nmol/l; 85·2%) were mostly detectable. 5-FormylTHF (3·6%) and 5,10-methenylTHF (4·4%) were rarely detected. The biggest contributor to serum total folate was 5-methylTHF (86·7%); UMFA (4·0%), non-methyl folate (4·7%) and MeFox (4·5%) contributed smaller amounts. Age was positively related to MeFox, but showed a U-shaped pattern for other folates. We generally noted sex and race/ethnic biomarker differences and weak (Spearman's r<0·4) but significant (P<0·05) correlations with physiological and lifestyle variables. Fasting, kidney function, smoking and alcohol intake showed negative associations. BMI and body surface area showed positive associations with MeFox but negative associations with other folates. All biomarkers showed significantly higher concentrations with recent folic acid-containing dietary supplement use. These first-time population data for serum folate forms generally show similar associations with demographic, physiological and lifestyle variables as serum total folate. Patterns observed for MeFox may suggest altered folate metabolism dependent on biological characteristics.

Discussion: Effects of Folate and Vitamin B12 Deficiencies During Pregnancy on Fetal, Infant, and Child Development

The essentiality of folate and vitamin B12 for the synthesis of DNA may interfere with a successful pregnancy outcome when the mother is deficient in these micronutrients. The objective of this paper is to assess the effects of folate and vitamin B12 deficiencies on pregnancy outcomes, other than neural tube defects (NTD), and the effects of these deficiencies on infant and child development. Supplementation studies identified by two Cochran Reviews were selected to assess the impact of folate deficiency on pregnancy outcomes, and a systematic review of the literature was used to assess their effects on infant and child developments. Folate supplementation consistently resulted in improvement of hematological and folate status indicators. Seven supplemental studies consistently found no differences in the risk of total fetal loss, early or late miscarriage, stillbirth, preeclampsia, perinatal death, neonatal death, preterm birth, small-for-gestational age, or infant death in supplemented women compared with their controls. Two of those studies found greater placental weights (difference 96 g; 95% CI, 30.7 to 161.2 g), birthweights (difference 312 g; 95% CI, 108.5 to 515.4 g), and a lower risk for newborns weighing &lt; 2,500 g in supplemented women (RR = 0.94; 95% CI, 0.90 to 0.99). Abnormal vitamin B12 and homocysteine serum concentrations were more readily associated with poor pregnancy outcomes. The very few studies addressing the effects of folate and vitamin B12 deficiencies on infant and child development were inconclusive. Early supplementation with folate to pregnant women improves hematological and folate status indicators, but has little or no effect on pregnancy outcomes, other than on NTD. Vitamin B12 deficiencies and low homocysteine are more readily associated with poor pregnancy outcomes.

Abstract 2098: The effect of in utero folic acid supplementation on colorectal cancer risk in the offspring in a chemical carcinogen rodent model

Abstract Background: Epidemiologic studies suggest that dietary intake and blood levels of folate are inversely related to colorectal cancer (CRC) risk. However, animal studies and recent human intervention trials have suggested that although folate supplementation may decrease the risk of developing de novo CRC, folate supplementation may promote the progression of existing, undiagnosed preneoplastic lesions in the colorectum to CRC. Total intake and blood levels of folate in North America have dramatically increased over the past decade owing to mandatory folic acid (FA; the synthetic form of folate) fortification and 30-40% of the population taking multivitamins containing FA. It is unknown whether high folate in the intrauterine environment may affect CRC risk in the offspring. We therefore investigated the effect of FA supplementation provided in utero and postnatally on CRC risk in the offspring in the azoxymethane (AOM) rat model of CRC. Methods: Female Sprague-Dawley rats were placed on either a control diet (2mg FA/kg diet) or a supplemented diet (5mg FA) for 3 weeks prior to breeding, and remained on the diet throughout pregnancy and lactation. Male pups from each maternal diet group were randomized to either the control or supplemented diet (n=55 per group) at weaning (21 days of age). At 5 and 6 weeks of age, 2 weekly s.c. injections of AOM were given. At 34 weeks of age, all rats were sacrificed and tumor incidence, multiplicity and burden as well as plasma folate and homocysteine (Hcy; an accurate inverse indicator of folate status) and liver folate concentrations were determined. Results: Plasma and liver folate concentrations accurately reflected dietary FA levels (p&amp;lt;0.001), and a significant interaction was found between maternal and pup diets (p&amp;lt; 0.02). Plasma Hcy was lower in pups fed the 5mg diet (p=0.036). Maternal FA supplementation significantly decreased the risk of developing CRC in the offspring (p=0.003) whereas postnatal FA supplementation had no significant effect. Pups from dams on the control diet had a nearly 3-fold increased risk of developing CRC compared with those from dams on FA supplementation (odds ratio=2.78; 95% CI, 1.41-5.50). There was no significant effect of interaction between maternal and pup diets on CRC incidence. Total number of tumors and sum of tumor diameter were significantly different among the diet groups (p=0.014 and p=0.006, respectively) with FA supplemented pups from dams on the control diet bearing a higher number of tumors and a larger tumor diameter than other groups (p&amp;lt;0.03). Conclusions: Notwithstanding the limitations associated with this model, our data suggest that FA supplementation provided in utero, but not postnatally, may protect the offspring from developing CRC. Although FA supplementation may promote the progression of established precursors of CRC, FA supplementation may decrease the risk of developing de novo CRC.

A comparison of a high‐throughput LC‐MS/MS method and two dietary intake survey instruments for erythrocyte folate status

The primary goal of the mandatory national folate fortification policy was to optimize folate nutritional status for the prevention of neural tube defects. While red blood cell (RBC) folate is considered the best indicator of folate status, concerns about the quality of the measurement and its relation to folate intake remain. The results of two folate intake instruments, a food frequency questionnaire (FFQ) and a folate-targeted semi-quantitative Block Dietary Folate Equivalents (DFE) Screener were compared to a newly developed high-throughput LC-MS/MS method for 370 normal adults. RBC folate was 1178 ± 259 nmol/L (mean ± SD). Folate intakes were 556 ± 265 μg/day by the FFQ and 520 ± 272 μg/d by the DFE. Intakes by both instruments were highly correlated (r = 0.607, p < 0.01) and folate intakes by the DFE were approximately 36 μg less than by the FFQ (p < 0.01). The RBC folate values determined by the LC-MS/MS method were compared with the DFE (r = 0.335) and FFQ (r = 0.283). In conclusion, the RBC folate concentrations by LC-MS/MS validated the folate intake assessment instruments. This study demonstrates that the DFE Screener produces estimates that are similar to an established FFQ.

Red blood cell folate concentrations increase more after supplementation with [6 S]-5-methyltetrahydrofolate than with folic acid in women of childbearing age

For the primary prevention of neural tube defects (NTDs), public health authorities recommend women of childbearing age to take 400 mug folic acid/d 4 wk before conception and during the first trimester. The biologically active derivate [6S]-5-methyltetrahydrofolate ([6S]-5-MTHF) could be an alternative to folic acid. We investigated the effect of supplementation with [6S]-5-MTHF compared with that of folic acid on red blood cell folate concentration, an indicator of folate status. The study was designed as a double-blind, randomized, placebo-controlled intervention trial. Healthy women (n = 144) aged 19-33 y received 400 microg folic acid, the equimolar amount of [6S]-5-MTHF (416 microg), 208 microg [6S]-5-MTHF, or placebo as a daily supplement for 24 wk. Red blood cell and plasma folate concentrations were measured at baseline and at 4-wk intervals. The increase in red blood cell folate over time was significantly higher in the group receiving 416 microg [6S]-5-MTHF/d than in the groups receiving 400 microg folic acid/d or 208 microg [6S]-5-MTHF/d (P < 0.001). No plateau was reached in red blood cell folate concentration in the 3 treatment groups during 24 wk of intervention; however, plasma folate plateaued after 12 wk. We showed that administration of [6S]-5-MTHF is more effective than is folic acid supplementation at improving folate status. In addition, the study indicates that the recommended period for preconceptional folic acid supplementation should be extended to >4 wk for maximal prevention of NTDs based on folate concentrations. [6S]-5-MTHF might be an efficient and safe alternative to folic acid.

Ethnicity and Race Influence the Folate Status Response to Controlled Folate Intakes in Young Women

Population-based studies report differences in folate status indicators among Mexican American (MA), African American (AA) and Caucasian (CA) women. It is unclear, however, whether these differences are due to variations in dietary folate intake. The present study was designed to investigate the influence of ethnicity/race on folate status parameters in MA, AA, and CA women (18-45 y; n = 14 in each group) under conditions of strictly controlled folate intake. In addition, the adequacy of the 1998 folate U.S. recommended dietary allowance (RDA), 400 micro g/d as dietary folate equivalents (DFE), for non-Caucasian women was assessed. Subjects (n = 42) with the methylenetetrahydrofolate reductase 677 CC genotype consumed a low-folate diet (135 micro g DFE/d) for 7 wk followed by repletion with 400 (7 MA, 7 AA, 7 CA) or 800 micro g DFE/d (7 MA, 7 AA, 7 CA) for 7 wk. AA women had lower (P </= 0.05) blood folate concentrations and excreted less (P </= 0.05) urinary folate throughout folate depletion and repletion with 400 and/or 800 micro g DFE/d compared with MA and/or CA women. MA women had lower (P </= 0.05) plasma total homocysteine (tHcy) throughout folate depletion and during repletion with 400 micro g DFE/d relative to the other ethnic/racial groups. Repletion with the 1998 folate U.S. RDA led to normal blood folate and plasma tHcy for all 3 ethnic/racial groups. Collectively, these data demonstrate that ethnicity/race is an important determinant of folate status under conditions of strictly controlled dietary folate intake and support the adequacy of the 1998 folate U.S. RDA for the 3 largest ethnic/racial groups in the United States.

Folate catabolite excretion is responsive to changes in dietary folate intake in elderly women

The major route of folate turnover is by catabolic cleavage of the C9-N10 bond producing p-aminobenzoylglutamate (pABG) and its primary excretory form, p-acetamidobenzoylglutamate (ApABG). We hypothesize that total pABG (ApABG + pABG) excretion parallels both the mass of body folate pools from which these catabolites originate and the folate-status indicators. The objective was to determine whether urinary folate catabolite excretion reflects body pool size and parallels the static and functional measures of folate status. Urinary folate catabolite excretion was measured in women (aged 60-85 y) consuming controlled amounts of folate for 14 wk. A low-folate diet (120 microg/d) was consumed (n = 33) for 7 wk, and then subjects were randomly assigned to consume either 200 (n = 14) or 400 (n = 16) microg folate/d. Urinary pABG and ApABG concentrations were measured by HPLC at 0, 7, and 14 wk. Urinary excretion of total pABG was significantly lower (P = 0.001) after depletion (73.9 +/- 4.7 nmol/d) than at baseline (115 +/- 12.7 nmol/d). This rate of decline (approximately 0.7% per day) is consistent with the kinetically measured rate of turnover of total body folate at moderate folate intakes. The average percentage increase in total pABG in response to folate repletion with 400 microg/d (75%) was significant (P = 0.02). Folate catabolite excretion was significantly (P = 0.0001) associated with serum and red blood cell folate, plasma homocysteine, and DNA hypomethylation after depletion and with serum folate (P = 0.001) and plasma homocysteine (P = 0.0002) after repletion with 400 microg folate/d. Total urinary pABG excretion reflects total body folate pool size and is a long-term indicator that parallels functional measures of folate status.

Depression and Folate Status in the US Population

Background: Folate deficiency and low folate status have been linked in clinic studies to depression, persistent depressive symptoms, and poor antidepressant response. These relationships have not been demonstrated in general populations. This study examined associations between depression and folate status indicators in an ethnically diverse general US population sample aged 15–39 years. Methods: Healthy subjects whose red blood cell (RBC) folate concentrations had been measured were determined to have no depression (n = 2,526), major depression (n = 301 ), or dysthymia (n = 121) using a diagnostic interview schedule. Serum concentrations of folate and total homocysteine (tHcy) were also measured. Results: After adjustment for sociodemographic factors, serum vitamin B₁₂ concentration, alcohol consumption over the past year and current status as to overweight and use of vitamin/mineral supplements, cigarettes and illegal drugs, subjects who met criteria for a lifetime diagnosis of major depression had folate concentrations in serum and RBCs that were lower than those of subjects who had never been depressed. Subjects who met criteria for dysthymia alone had lower RBC folate concentrations than never-depressed subjects, but the serum folate concentrations of the two groups were comparable. Serum tHcy concentration was not related to lifetime depression diagnoses. Low folate status was found to be most characteristic of recently recovered subjects, and a large proportion of such subjects were folate deficient. Conclusions: Low folate status was detectable in depressed members of the general US population. Folate supplementation may be indicated during the year following a depressive episode.

Association of folate intake and serum homocysteine in elderly persons according to vitamin supplementation and alcohol use

The serum total homocysteine concentration (tHcy), an indicator of folate status and a possible risk factor for vascular disease, is elevated with impaired renal function and poor vitamin B-12 status, which are common in the elderly. Our objective was to determine the association between tHcy, folate intake, alcohol consumption, and other lifestyle factors in elderly persons. This cross-sectional study used linear regression to model changes in tHcy. Subjects were 278 men and women aged 66-94 y studied in 1993. Total folate intake was negatively associated with tHcy in models adjusted for age, sex, serum creatinine, and serum albumin. We found an interaction between food folate intake and supplement use. Food folate intake had an inverse dose-response relation with tHcy that was limited to nonusers of supplements. Predicted tHcy was 1.5 micromol/L lower in users of supplements containing folate and vitamin B-12 than in nonusers and was independent of food folate intake. We found a positive dose-response relation of coffee and tea intake with tHcy, a positive association for alcohol intake of > or = 60 drinks/mo compared with low intake, and an interaction of alcohol use with folate intake and supplement use. Compared with alcohol users, nonusers had higher predicted tHcy and a lower inverse dose-response relation of food folate intake with tHcy. The inverse association between folate intake and tHcy was strongest among nonusers of supplements and among alcohol drinkers. Identifying modifiable factors related to tHcy, a possible risk factor for vascular disease, is especially important in elderly persons.

Potentiated decrease of plasma folate levels caused by the coadministration of folic acid in rats treated with methotrexate.

The decrease of plasma 5-methyltetrahydrofolic acid (5-MF) levels, postulated as an indicator of folate status, was studied following the administration of both methotrexate (MTX) alone and MTX with folic acid (FA) using rats as our experimental model. Blood and urine samples were serially collected over a 9 hr period after the administration of MTX, MTX with FA and from a control group to examine the plasma kinetics and the renal clearance of 5-MF. The pharmacokinetics of MTX and the plasma protein binding of 5-MF were also examined. The concentrations of these analytes were assayed using high performance liquid chromatography (HPLC). MTX administration produced decreased plasma 5-MF levels. This observed decrease was potentiated by oral FA administration, suggesting that the folate status was more severely altered by the coadministration of FA. The renal clearance of 5-MF also increased dose-dependently with FA (0.05-5 mg/kg) coadministration. The plasma protein binding of 5-MF was not affected by the FA administration, which indicates that the fraction of 5-MF that was filtered through the glomerular apparatus appeared to be unchanged. In addition, the pharmacokinetic profiles of MTX also appeared not to be affected by the addition of FA. We conclude that the inhibition of reabsorption of 5-MF in the renal tube by concurrent administration of MTX and FA must be one of the causal factors for the demonstrated decrease in the plasma 5-MF levels in rats.

Use of the deoxyuridine suppression test to evaluate localized folate deficiency in rat colonic epithelium

In this study the deoxyuridine suppression test (dUST) was performed on isolated rat colonocytes to establish its value as an indicator of folate status in the colonic epithelium. [3H]thymidine incorporation into DNA was suppressed greater than 90% by deoxyuridine (dU) concentrations greater than 2.5 mumol/L. Preincubation of cells with 5-fluorouracil (1-100 mumol/L) but not methotrexate (10-100 mumol/L) resulted in a significant decrease in the degree of suppression. The dUST performed on colonocytes from folate-deficient animals displayed less suppression than on colonocytes from folate-replete animals (P less than 0.05). The abnormal degree of suppression was corrected by adding 100 mumol folinic acid/L. There was a negative correlation between the degree of suppression and the folate concentration of the colonic epithelium (P less than 0.001). These data indicate that the dUST is useful for detecting folate deficiency in the colonic epithelium and may therefore be valuable in assessing a deficiency state localized to that epithelium.