Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-27 (of 27 Records) |
Query Trace: Fazili Z [original query] |
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Development of an improved standard reference material for folate vitamers in human serum
Camara JE , Pritchett JS , Daniels YC , Bedner M , Nelson MA , Lowenthal MS , Fazili Z , Pfeiffer CM , Phinney KW , Sharpless KE , Sander LC , Lippa KA , Yen JH , Kuszak AJ , Wise SA . Anal Bioanal Chem 2022 415 (5) 809-821 The US National Institute of Standards and Technology (NIST) developed a Standard Reference Material (SRM) 3949 Folate Vitamers in Frozen Human Serum to replace SRM 1955 Homocysteine and Folate in Human Serum. The presence of increased endogenous levels of folic acid and 5-methyltetrahydrofolate (5mTHF) in SRM 3949, enhanced folate stability via addition of ascorbic acid, and inclusion of values for additional minor folates are improvements over SRM 1955 that should better serve the clinical folate measurement community. The new SRM contains folates at three levels. To produce SRM 3949, pilot sera were collected from 15 individual donors, 5 of whom were given a 400-g folic acid supplement 1h prior to blood draw to increase serum levels of 5mTHF and folic acid for the high-level material. To stabilize the folates, 0.5% (mass concentration) ascorbic acid was added as soon as possible after preparation of serum. These pilot sera were screened for five folates plus the pyrazino-s-triazine derivative of 4--hydroxy-5-methyltetrahydrofolate (MeFox) at the US Centers for Disease Control and Prevention (CDC) by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Based on these results, a blending protocol was specified to obtain the three desired folate concentrations for SRM 3949. ID-LC-MS/MS analysis at the CDC and NIST was utilized to assign values for folic acid and 5mTHF, as well as several minor folates. |
Reduced kidney function is associated with increasing red blood cell folate concentration and changes in folate form distributions (NHANES 2011-2018)
Wang A , Yeung LF , Ríos Burrows N , Rose CE , Fazili Z , Pfeiffer CM , Crider KS . Nutrients 2022 14 (5) BACKGROUND: Current studies examining the effects of high concentrations of red blood cell (RBC) or serum folates assume that high folate concentrations are an indicator of high folic acid intakes, often ignoring the contributions of other homeostatic and biological processes, such as kidney function. OBJECTIVE: The current study examined the relative contributions of declining kidney function, as measured by the risk of chronic kidney disease (CKD), and usual total folic acid intake on the concentrations of RBC folate and serum folate (total as well as individual folate forms). DESIGN: Cross-sectional data from the National Health and Nutrition Examination Survey (NHANES) collected in 2-year cycles were combined from 2011 to 2018. A total of 18,127 participants aged ≥16 years with available folate measures, kidney biomarker data (operationalized as a categorical CKD risk variable describing the risk of progression), and reliable dietary recall data were analyzed. RESULTS: RBC folate concentrations increased as CKD risk increased: low risk, 1089 (95% CI: 1069, 1110) nmol/L; moderate risk, 1189 (95% CI: 1158, 1220) nmol/L; high risk, 1488 (95% CI: 1419, 1561) nmol/L; and highest risk, 1443 (95% CI: 1302, 1598) nmol/L (p < 0.0001). Similarly, serum total folate concentrations increased as CKD risk increased: low risk: 37.1 (95% CI: 26.3, 38.0) nmol/L; moderate risk: 40.2 (95% CI: 38.8, 41.7) nmol/L; high risk: 48.0 (95% CI: 44.3, 52.1) nmol/L; the highest Risk: 42.8 (95% CI: 37.8, 48.4) nmol/L (p < 0.0001). The modeled usual intake of folic acid showed no difference among CKD risk groups, with a population median of 225 (interquartile range: 108-390) µg/day. CONCLUSION: Both RBC and serum folate concentrations increased with declining kidney function without increased folic acid intake. When analyzing associations between folate concentrations and disease outcomes, researchers may want to consider the confounding role of kidney function. |
First things first: a step in the right direction for the preanalytical phase of thiamine measurements
Pfeiffer CM , Fazili Z , Mineva EM , Ngac PK . Am J Clin Nutr 2021 114 (3) 829-830 The 2018 roadmap for global control programs for thiamine deficiency disorders calls out the lack of biochemical population data on thiamine status (1). This report states that “urgent public health responses are warranted in high-risk regions, considering the contribution of thiamine deficiency to infant mortality and research suggesting that even subclinical thiamine deficiency in childhood may have lifelong neurodevelopmental consequences.” This situation poses a bit of a chicken and egg dilemma, as investigators need suitable analytical methods to produce high-quality biomarker data and policymakers need comparable and interpretable biomarker data to develop policies and evaluate their impact. Furthermore, analytical methods need to be paired with practical preanalytical conditions that allow for the collection and generation of valid biological specimens. For years, the field of thiamine research has been hampered due to the labile physiochemical properties of thiamine compounds and the lack of simple, yet reliable, analytical methods. |
Folate Forms in RBC and Whole-Blood Lysates Appear Stable When Stored Frozen for 2 Years
Fazili Z , Paladugula N , Zhang M , Pfeiffer CM . J Nutr 2021 151 (9) 2852-2860 BACKGROUND: The use of RBC lysate (RBC-Lys) eliminates the need for serum folate and hematocrit (Hct) measurement to calculate RBC folate. Information on the long-term frozen storage stability of RBC-Lys is missing. OBJECTIVES: We aimed to assess the comparability of RBC folate forms in whole-blood lysate (WB-Lys) and RBC-Lys and the folate stability in both matrices. METHODS: We prepared conventional WB-Lys (1:11 dilution with 1% ascorbic acid) and RBC-Lys (1:11 dilution of washed and saline-diluted RBCs with 1% ascorbic acid) from EDTA blood (n = 60 adult donors) and stored lysates at -70°C until analysis at baseline (1 wk), 3, 6, 12, and 24 mo. Before analysis by HPLC-tandem MS, we incubated the WB-Lys (4 h at 37°C) and treated the RBC-Lys with human recombinant γ-glutamyl hydrolase for folate polyglutamate deconjugation. We analyzed RBC-Lys samples for hemoglobin (Hb) (same aliquot) to normalize for the preanalytical dilution; Hb-folate was converted to RBC folate for each folate form using the mean corpuscular Hb concentration. We analyzed Hct as well as folate forms in matching serum samples for traditional RBC folate calculation. We conducted descriptive data analyses (correlation, Bland-Altman plot, Deming regression). RESULTS: At baseline, results for RBC folate forms derived from WB-Lys compared with RBC-Lys samples showed excellent correlation (Pearson r ≥ 0.97). Mean ± SD concentrations compared well for total folate (WB-Lys: 886 ± 255 compared with RBC-Lys: 899 ± 271 nmol/L), 5-methyltetrahydrofolate (WB-Lys: 831 ± 258 compared with RBC-Lys: 843 ± 276 nmol/L), and nonmethyl folate (WB-Lys: 53.3 ± 74.4 compared with RBC-Lys: 52.9 ± 70.7 nmol/L), but were 17% higher in RBC-Lys for pyrazino-s-triazine derivative of 4α-hydroxy-5-CH3-H4folate (MeFox) (WB-Lys: 147 ± 44.1 compared with RBC-Lys: 172 ± 53.5 nmol/L). Frozen storage of WB-Lys and RBC-Lys samples for ≤24 mo showed ≤5%, ≤5%, ≤13%, and ≤11% change in total folate, 5-methyltetrahydrofolate, nonmethyl folate, and MeFox, respectively. CONCLUSIONS: Erythrocyte folate forms appear to be stable in RBC-Lys samples stored frozen at -70°C for ≤2 y. The relatively small changes in folate concentrations over time were comparable between RBC-Lys and conventionally prepared WB-Lys samples. |
Demographic, physiologic, and lifestyle characteristics observed with serum total folate differ among folate forms: Cross-sectional data from fasting samples in the NHANES 2011-2016
Fazili Z , Sternberg MR , Potischman N , Wang CY , Storandt RJ , Yeung L , Yamini S , Gahche JJ , Juan W , Qi YP , Paladugula N , Gabey G , Pfeiffer CM . J Nutr 2019 150 (4) 851-860 BACKGROUND: Serum folate forms were measured in the US population during recent NHANES to assess folate status. OBJECTIVE: We describe post-folic acid-fortification concentrations of serum folate forms in the fasting US population >/=1 y from the NHANES 2011-2016. METHODS: We measured 5 biologically active folates and 1 oxidation product (MeFox) of 5-methyltetrahydrofolate (5-methyl-THF). We calculated geometric means of 5-methyl-THF, unmetabolized folic acid (UMFA), nonmethyl folate (sum of tetrahydrofolate, 5-formyltetrahydrofolate, and 5,10-methenyltetrahydrofolate), total folate (sum of above biomarkers), and MeFox by demographic, physiologic, and lifestyle variables; estimated the magnitude of variables on biomarker concentrations after covariate adjustment; and determined the prevalence of UMFA >2 nmol/L. RESULTS: After demographic adjustment, age, sex, and race-Hispanic origin were significantly associated with most folate forms. MeFox increased with age, while 5-methyl-THF, UMFA, and nonmethyl folate displayed U-shaped age patterns. Compared with non-Hispanic whites, non-Hispanic blacks had 23% lower predicted 5-methyl-THF but comparable UMFA; non-Hispanic Asians had comparable 5-methyl-THF but 28% lower UMFA; Hispanics, non-Hispanic Asians, and non-Hispanic blacks had approximately 20% lower MeFox. After additional physiologic and lifestyle adjustment, predicted UMFA and MeFox concentrations were 43% and 112% higher, respectively, in adults with chronic kidney disease and 17% and 15% lower, respectively, in adults consuming daily 1-<2 alcoholic beverages; 5-methyl-THF concentrations were 20% lower in adult smokers. The prevalence of UMFA >2 nmol/L was highest in persons aged >/=70 y (9.01%) and lowest in those aged 12-19 y (1.14%). During 2011-2014, the prevalence was 10.6% in users and 2.22% in nonusers of folic acid-containing supplements. CONCLUSIONS: In fasting persons >/=1 y, the demographic, physiologic, and lifestyle characteristics observed with serum total folate differed among folate forms, suggesting biological and/or genetic influences on folate metabolism. High UMFA was mostly observed in supplement users and older persons. |
Serum folate forms are stable during repeated analysis in the presence of ascorbic acid and during frozen sample storage
Paladugula N , Fazili Z , Sternberg MR , Gabey G , Pfeiffer CM . J Appl Lab Med 2019 3 (6) 993-1002 BACKGROUND: Serum folate forms, and particularly tetrahydrofolate, are sensitive to oxidation. METHODS: Using a repeated measures design, we investigated the stability of folate forms in convenience samples with added ascorbic acid (AA; 5 g/L) analyzed initially and after variable (approximately 1-33 weeks) storage time at -70 degrees C. We examined the recovery of tetrahydrofolate added at different spiking levels to serum with and without AA (5 g/L). We also assessed the long-term frozen storage stability of folate forms. RESULTS: Repeat analysis produced consistent results with the initial analysis; the mean relative change (95% CI; Lin's concordance correlation between initial and repeat result; sample size) was 0.08% (-0.24% to 0.39%; r c = 0.999; n = 301) for 5-methyltetrahydrofolate, 4.23% (2.44%-6.05%; r c = 0.984; n = 211) for pyrazino-s-triazine derivative of 4alpha-hydroxy-5-methyltetrahydrofolate (MeFox), -0.22% (-1.90% to 1.49%; r c = 0.986; n = 214) for folic acid, and 1.49% (-2.71% to 5.88%; r c = 0.889; n = 81) for tetrahydrofolate. Linear regression testing for a time trend indicated an estimated average percent change of less than +/-5% for samples retested after 4 months: 5-methyltetrahydrofolate P trend = 0.0007, folic acid P trend < 0.0001, MeFox P trend = 0.38, and tetrahydrofolate P trend = 0.0256. The mean +/- SD tetrahydrofolate spiking recovery was 96.7% +/- 9.4% for serum with added AA, but <50% for serum without added AA. We observed </=10% loss for most serum folate forms during 4 years of storage at -70 degrees C. CONCLUSIONS: Serum containing added AA showed acceptable stability of folate forms during repeat analysis from the same vial within 4 months, complete spiking recovery of tetrahydrofolate during sample processing, and long-term frozen storage stability of folate forms. |
Folate status in the US population 20 y after the introduction of folic acid fortification
Pfeiffer CM , Sternberg MR , Zhang M , Fazili Z , Storandt RJ , Crider KS , Yamini S , Gahche JJ , Juan W , Wang CY , Potischman N , Williams J , LaVoie DJ . Am J Clin Nutr 2019 110 (5) 1088-1097 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 approximately 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 ( approximately 70 nmol/L) and RBC ( approximately 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 approximately 20%. Continued monitoring of all age groups is advisable given the high folate status particularly in older supplement users. |
Daily supplementation with 5 mg of folic acid in Brazilian patients with hereditary spherocytosis
Paniz C , Lucena MR , Bertinato JF , Lourenco FR , Barros BCA , Gomes GW , Figueiredo MS , Cancado RD , Blaia DAvila VLN , Pfeiffer CM , Fazili Z , Green R , Carvalho VM , Guerra-Shinohara EM . J Investig Med 2019 67 (8) 1110-1117 Patients with hereditary spherocytosis (HS) have increased rates of erythropoiesis and higher folate requirements. In a case-control study of patients with HS, we evaluated the associations between the use of 5 mg folic acid (FA) daily and serum concentrations of folate, unmetabolized folic acid (UMFA), interleukin (IL)-6, IL-8, IL-10, interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha); and mRNA expression of dihydrofolate reductase (DHFR), methylene tetrahydrofolate reductase (MTHFR), IL8, IFNG and TNFA genes. Total serum folate and folate forms were measured in 27 patients with HS (21 users [HS-U] and 6 non-users [HS-NU] of supplemental FA) and 54 healthy controls not consuming 5 mg/day supplemental FA. Each patient was matched to two controls based on age, sex and body mass index. The mononuclear leucocyte mRNA expression of relevant genes and their products were determined. Serum folate, UMFA, 5-methyl-tetrahydrofolate (5-methyl-THF) and tetrahydrofolate (THF) concentrations were significantly higher in HS-U compared with matched healthy controls (p<0.001, n=42). HS-NU had lower serum folate concentrations than matched healthy controls (p=0.044, n=12). HS-U and HS-NU presented similar hematological and biochemical markers profiles. No differences were found between HS-U and HS-NU for cytokine serum concentrations and mRNA expression genes. DHFR mRNA expression was higher in HS-U than in HS-NU. The use of high daily doses of FA for treatment of patients with HS may be excessive and is associated with elevated serum UMFA and elevated DHFR mRNA expression. It is not known whether long-term high-dose FA use by patients with HS might have adverse health effects. |
Addition of exogenous gamma-glutamyl hydrolase eliminates the need for lengthy incubation of whole-blood lysate for quantitation of folate vitamers by high-performance liquid chromatography-tandem mass spectrometry
Stamm RA , Fazili Z , Pfeiffer CM . Curr Dev Nutr 2018 2 (1) 1-9 Background: Measurement of folate monoglutamates by HPLC-tandem mass spectrometry (HPLC-MS/MS) in whole-blood lysate (WBL) requires lengthy incubation before analysis, risking degradation of labile folate vitamers. Objective: We explored whether the addition of a commercially available recombinant exogenous gamma-glutamyl hydrolase (exoGGH) enzyme reduced the required incubation time of WBL for measurement of folate as monoglutamates. Methods: For conventional deglutamylation of polyglutamates, WBL was incubated for 4 h at 37 degrees C. Alternatively, we added exoGGH to WBL at varying concentrations (1-10 microg/mL) and incubation times (0-90 min). We also investigated modifications to the sample diluent (pH, ascorbic acid compared with sodium ascorbate, and ascorbate concentration). Finally, we tested the effect of the enzyme in different sample types: WBL from frozen whole blood compared with frozen WBL or with frozen washed RBCs. Samples (n </= 15/experiment) were analyzed by HPLC-MS/MS for 6 folate monoglutamates and 5-methyltetrahydrofolate diglutamate. Results: Optimal deconjugation of folate polyglutamates was achieved by using 1% ascorbic acid and 5 microg enzyme/mL WBL, requiring </=30 min incubation time to achieve complete folate recovery as monoglutamates. This treatment resulted in similar folate concentrations as conventional deglutamylation (4 h at 37 degrees C). The exoGGH enzyme was effective in samples stored frozen as whole blood and as WBL. However, the extended thaw time of whole blood resulted in 5-methyltetrahydrofolate loss and unacceptable changes to the non-methyl folate concentration. Total folate (with exoGGH) measured in washed RBCs was approximately 15% lower than RBC folate calculated from WBL concentrations (conventional deglutamylation). Conclusions: The use of exoGGH minimized incubation time and thus may avoid degradative losses of labile folate forms during sample preparation. The lower folate results in washed RBCs may be due to inadequate packing of RBCs, among other unidentified factors. A larger study is required to confirm the lack of differences in folate concentrations determined with and without the use of exoGGH. |
Association between serum unmetabolized folic acid concentrations and folic acid from fortified foods
Palchetti CZ , Paniz C , de Carli E , Marchioni DM , Colli C , Steluti J , Pfeiffer CM , Fazili Z , Guerra-Shinohara EM . J Am Coll Nutr 2017 36 (7) 1-7 OBJECTIVE: To investigate the association between serum unmetabolized folic acid (UMFA) concentrations and folic acid from fortified foods and nutrients known as dietary methyl-group donors (folate, methionine, choline, betaine and vitamins B2, B6 and B12) in participants exposed to mandatory fortification of wheat and maize flours with folic acid. METHODS: Cross-sectional study carried out with 144 healthy Brazilian participants, both sexes, supplement nonusers. Serum folate, UMFA, vitamin B12 and total plasma homocysteine (tHcy) were biochemically measured. Dietary intake was assessed by 2 non-consecutive 24-hour dietary recalls (24-HRs) and deattenuated energy-adjusted nutrient data were used for statistical analysis. RESULTS: Ninety eight (68.1%) participants were women. Median (interquartile range) age was 35.5 (28.0-52.0) years. Elevated serum folate concentrations (>45 nmol/L) were found in 17 (11.8%), while folate deficiency (<7 nmol/L) in 10 (6.9%) participants. No one had vitamin B12 deficiency (<148 pmol/L). An elevated serum UMFA concentration was defined as > 1 nmol/L (90th percentile). UMFA concentrations were positively correlated with folic acid intake and negatively correlated to choline, methionine and vitamin B6 intakes. Participants in the lowest quartile of UMFA concentrations had lower dietary intake of total folate (DFEs) and folic acid, and higher dietary intake of methionine, choline and vitamin B6 than participants in the highest quartile of UMFA. Folic acid intake (OR [95% CI] = 1.02 [1.01-1.04)] and being a male (OR [95% CI] = 0.40 [0.19-0.87) were associated with increased and reduced odds for UMFA concentrations > 0.55 nmol/L (median values), respectively. CONCLUSION: UMFA concentrations were directly influenced by folic acid intake from fortified foods in a healthy convenience sample of adult Brazilians exposed to mandatory flour fortification with folic acid. |
Two international round-robin studies showed good comparability of 5-methyltetrahydrofolate but poor comparability of folic acid measured in serum by different high-performance liquid chromatography-tandem mass spectrometry methods
Fazili Z , Sternberg MR , Paladugula N , Pfeiffer CM . J Nutr 2017 147 (9) 1815-1825 Background: Serum folate methods produce different results. The comparability of HPLC-mass spectrometry (MS)/MS methods is not well documented.Objective: We conducted an international "round-robin" investigation to assess the comparability, precision, and accuracy of serum folate HPLC-MS/MS methods.Methods: The CDC laboratory, 7 laboratories with independently developed methods (group 1), and 6 laboratories with an adapted CDC method (group 2) analyzed folate forms in 6 serum pools and 6 calibrators from the CDC (duplicate analysis over 2 d) and in two 3-level reference materials (duplicate analysis).Results: All laboratories measured 5-methyltetrahydrofolate (5-methylTHF) and folic acid; some measured additional folate forms. The geometric mean (range) concentrations (nanomoles per liter) for 5-methylTHF in the 6 serum pools were 18.3 nmol/L (CDC), 13.8-28.9 nmol/L (group 1), and 16.8-18.6 nmol/L (group 2); for folic acid the concentrations were 3.42 nmol/L (CDC), 1.09-4.74 nmol/L (group 1), and 1.74-2.90 nmol/L (group 2). The median imprecision (CV) for 5-methylTHF was 4.1% (CDC), 4.6-11% (group 1), and 1.7-6.0% (group 2); for folic acid it was 6.9% (CDC), 4.9-20% (group 1), and 3.9-23% (group 2). The mean +/- SD (range) recovery of 5-methylTHF spiked into serum was 98% +/- 27% (59-138%) for group 1 and 98% +/- 10% (82-111%) for group 2; for folic acid it was 93% +/- 29% (67-198%) for group 1 and 81% +/- 16% (64-102%) for group 2. The mean relative bias for 5-methylTHF compared with the reference material certificate value was 12% (CDC), -24% to 30% (group 1), and -0.6% to 16% (group 2); for folic acid it was 73% (CDC), -47% to 578% (group 1), and -3.3% to 67% (group 2).Conclusions: For 5-methylTHF, group 2 laboratories demonstrated better agreement and precision, less variable spiking recovery, and less bias by using a reference material. Laboratory performance for folic acid was highly variable and needs improvement. Certified reference materials for serum folate forms and total folate are needed to improve method accuracy. |
A daily dose of 5 mg folic acid for 90 days is associated with increased serum unmetabolized folic acid and reduced natural killer cell cytotoxicity in healthy Brazilian adults
Paniz C , Bertinato JF , Lucena MR , De Carli E , Amorim Pmds , Gomes GW , Palchetti CZ , Figueiredo MS , Pfeiffer CM , Fazili Z , Green R , Guerra-Shinohara EM . J Nutr 2017 147 (9) 1677-1685 Background: The effects of high-dose folic acid (FA) supplementation in healthy individuals on blood folate concentrations and immune response are unknown.Objective: The aim of the study was to evaluate the effects of daily consumption of a tablet containing 5 mg FA on serum folate; number and cytotoxicity of natural killer (NK) cells; mRNA expression of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR), interferon gamma (IFNG), tumor necrosis factor alpha (TNFA), and interleukin 8 (IL8) genes; and concentrations of serum inflammatory markers.Methods: This prospective clinical trial was conducted in 30 healthy Brazilian adults (15 women), aged 27.7 y (95% CI: 26.4, 29.1 y), with a body mass index (in kg/m2) of 23.1 (95% CI: 22.0, 24.3). Blood was collected at baseline and after 45 and 90 d of the intervention. Serum folate concentrations were measured by microbiological assay and HPLC-tandem mass spectrometry [folate forms, including unmetabolized folic acid (UMFA)]. We used real-time polymerase chain reaction to assess mononuclear leukocyte mRNA expression and flow cytometry to measure the number and cytotoxicity of NK cells.Results: Serum folate concentrations increased by approximately 5-fold after the intervention (P < 0.001), and UMFA concentrations increased by 11.9- and 5.9-fold at 45 and 90 d, respectively, when compared with baseline (P < 0.001). UMFA concentrations increased (>1.12 nmol/L) in 29 (96.6%) participants at day 45 and in 26 (86.7%) participants at day 90. We observed significant reductions in the number (P < 0.001) and cytotoxicity (P = 0.003) of NK cells after 45 and 90 d. Compared with baseline, DHFR mRNA expression was higher at 90 d (P = 0.006) and IL8 and TNFA mRNA expressions were higher at 45 and 90 d (P = 0.001 for both).Conclusion: This noncontrolled intervention showed that healthy adults responded to a high-dose FA supplement with increased UMFA concentrations, changes in cytokine mRNA expression, and reduced number and cytotoxicity of NK cells. This trial was registered at www.ensaiosclinicos.govbr as RBR-2pr7zp. |
Biomarkers of nutrition for development - folate review
Bailey LB , Stover PJ , McNulty H , Fenech MF , Gregory JF 3rd , Mills JL , Pfeiffer CM , Fazili Z , Zhang M , Ueland PM , Molloy AM , Caudill MA , Shane B , Berry RJ , Bailey RL , Hausman DB , Raghavan R , Raiten DJ . J Nutr 2015 145 (7) 1636S-80S 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 status and concentrations of serum folate forms in the US population: National Health and Nutrition Examination Survey 2011-2
Pfeiffer CM , Sternberg MR , Fazili Z , Lacher DA , Zhang M , Johnson CL , Hamner HC , Bailey RL , Rader JI , Yamini S , Berry RJ , Yetley EA . Br J Nutr 2015 113 (12) 1-13 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 (37.5 nmol/l; 100 %), UMFA (1.21 nmol/l; 99.9 %), MeFox (1.53 nmol/l; 98.8 %), and THF (1.01 nmol/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. |
The association between circulating total folate and folate vitamers with overall survival after postmenopausal breast cancer diagnosis
McEligot AJ , Ziogas A , Pfeiffer CM , Fazili Z , Anton-Culver H . Nutr Cancer 2015 67 (3) 442-8 We studied the relationship between plasma total folate and folate vitamer concentrations [5-methyltetrahydrofolic acid, pteroylglutamic acid (folic acid) and tetrahydrofolic acid] with overall survival after breast cancer diagnosis. A secondary aim was to assess the relationship between folic acid supplement use with circulating total folate and folate vitamer concentrations. Participants were postmenopausal women diagnosed with breast cancer (n = 498) with an average follow-up of 6.7 yr. Plasma total folate and folate vitamers were measured by isotope-dilution LC-MS/MS in samples collected at or postdiagnosis. Cox proportional multivariate hazards models (controlled for stage, age at diagnosis, body mass index, parity, hormone replacement therapy use, treatment, alcohol use, folic acid use, and energy intake), were used to assess overall survival after breast cancer diagnosis. We found that the relative risk of dying for women with plasma total folate concentrations in the highest quartile was 59% lower (hazard ratio: 0.41, 95% confidence interval: 0.19-0.90) compared with the lowest quartile. Data on supplement use showed that women taking folic acid supplements had significantly higher circulating total folate and folate vitamer concentrations (P < 0.0001), suggesting that increased folate consumption through diet and/or supplementation may improve prognosis after breast cancer diagnosis. |
Unmetabolized folic acid is detected in nearly all serum samples from US children, adolescents, and adults
Pfeiffer CM , Sternberg MR , Fazili Z , Yetley EA , Lacher DA , Bailey RL , Johnson CL . J Nutr 2015 145 (3) 520-31 BACKGROUND: Serum total folate consists mainly of 5-methyltetrahydrofolate (5-methylTHF). Unmetabolized folic acid (UMFA) may occur in persons consuming folic acid-fortified foods or supplements. OBJECTIVES: We describe serum 5-methylTHF and UMFA concentrations in the US population ≥1 y of age by demographic variables and fasting time, stratified by folic acid-containing dietary supplement use. We also evaluate factors associated with UMFA concentrations >1 nmol/L. METHODS: Serum samples from the cross-sectional NHANES 2007-2008 were measured for 5-methylTHF (n = 2734) and UMFA (n = 2707) by HPLC-tandem mass spectrometry. RESULTS: In supplement users compared with nonusers, we found significantly higher geometric mean concentrations of 5-methylTHF (48.4 and 30.7 nmol/L, respectively) and UMFA (1.54 and 0.794 nmol/L, respectively). UMFA concentrations were detectable (>0.3 nmol/L) in >95% of supplement users and nonusers, regardless of demographic or fasting characteristics; concentrations differed significantly by age and fasting time, but not by sex and race-ethnicity, both in supplement users and nonusers. The prevalence of UMFA concentrations >1 nmol/L was 33.2% overall and 21.0% in fasting (≥8 h) adults (≥20 y of age). Using multiple logistic regression analysis, UMFA concentrations >1 nmol/L were associated with being older, non-Hispanic black, nonfasting (<8 h), having smaller body surface area, higher total folic acid intake (diet and supplements), and higher red blood cell folate concentrations. In fasting adults, a decrease in the mean daily alcohol consumption was also associated with increased odds of UMFA concentrations >1 nmol/L. CONCLUSIONS: UMFA detection was nearly ubiquitous, and concentrations >1 nmol/L were largely but not entirely explained by fasting status and by total folic acid intake from diet and supplements. These new UMFA data in US persons ≥1 y of age provide much-needed information on this vitamer in a fortified population with relatively high use of dietary supplements. |
The loss of 5-methyltetrahydrofolate in human serum under suboptimal preanalytical conditions can only partially be recovered by an oxidation product
Fazili Z , Sternberg MR , Paladugula N , Whitehead RD Jr , Chen H , Pfeiffer CM . J Nutr 2014 144 (11) 1873-9 BACKGROUND: Maintaining folate stability during sample handling is important, yet challenging. OBJECTIVE: We investigated the effects of suboptimal preanalytical conditions on serum folate stability. METHODS: By using an HPLC-tandem MS method we measured folates [5-methyltetrahydrofolate (5-methylTHF), folic acid, MeFox (5-methylTHF oxidation product, pyrazino-s-triazine derivative of 4alpha-hydroxy-5-methylTHF), and other minor folate forms at or below the limit of detection] in human serum exposed to suboptimal conditions. RESULTS: Whole blood samples (n = 21) stored at 32 degrees C for ≤3 d (Expt. 1: delayed processing) showed significant decreases in serum total folate (tFOL; sum of folate forms: 11-32%, 5.5-15.9 nmol/L) and 5-methylTHF (36-62%, 14.5-25.1 nmol/L) and a significant increase in MeFox (346-415%, 7.17-8.63 nmol/L). Serum samples (n = 21) stored at 11 degrees C for 7-14 d (Expt. 2: delayed freezing) also showed significant decreases in tFOL (4.6-10.4%, 2.3-5.1 nmol/L) and 5-methylTHF (8.4-29%, 3.4-11.6 nmol/L) and significant increases in MeFox (88-320%, 1.82-6.62 nmol/L). The molar loss in 5-methylTHF exceeded the gain in MeFox in these 2 experiments. When we exposed 3 serum pools (tFOL: 16.7-58.3 nmol/L) for 24 h to an elevated temperature of 37 degrees C (Expt. 3), the significant decrease in 5-methylTHF (33% on average) was compensated for by an equimolar gain in MeFox. Repeated freeze/thaw cycles (≤3 cycles) of serum [closed (Expt. 4) and open (Expt. 5) vials] showed generally stable folates with small (<1 nmol/L) changes. Long-term (≤12 mo) exposure of 3 serum pools (tFOL: 17.5-63.7 nmol/L) to a suboptimal (-20 degrees C) freezing temperature (Expt. 6) showed significant decreases in tFOL (5% on average) already after 3 mo. The molar loss in 5-methylTHF exceeded the gain in MeFox. Folic acid generally showed good stability. CONCLUSIONS: To avoid folate losses, unprocessed whole blood should be protected from elevated temperatures and serum should not be refrigerated for >2 d or for a long term stored at -20 degrees C. |
Folate is absorbed across the human colon: evidence by using enteric-coated caplets containing 13C-labeled [6S]-5-formyltetrahydrofolate
Lakoff A , Fazili Z , Aufreiter S , Pfeiffer CM , Connolly B , Gregory JF 3rd , Pencharz PB , O'Connor DL . Am J Clin Nutr 2014 100 (5) 1278-86 BACKGROUND: Folate intakes that do not meet or greatly exceed requirements may be associated with negative health outcomes. A better understanding of contributors that influence the input side will help establish dietary guidance that ensures health benefits without associated risks. Colonic microbiota produce large quantities of folate, and [(13)C5]5-formyltetrahydrofolate infused during colonoscopy is absorbed. However, it is unclear if significant quantities of folate are absorbed in an intact microbiome. OBJECTIVE: We determined whether and how much of a physiologic dose of [(13)C5]5-formyltetrahydrofolate delivered in a pH-sensitive enteric caplet to an intact colonic microbiome is absorbed. DESIGN: Healthy adults ingested a specially designed pH-sensitive acrylic copolymer-coated barium sulfate caplet that contained 855 nmol (400 mug) [(13)C5]5-formyltetrahydrofolate. After a washout period ≥4 wk, subjects received an intravenous injection of the same compound (214 nmol). Serially collected blood samples before and after each test dose were analyzed by using a microbiological assay and liquid chromatography-tandem mass spectrometry. RESULTS: Caplet disintegration in the colon was observed by fluoroscopic imaging for 6 subjects with a mean (+/-SD) complete disintegration time of 284 +/- 155 min. The mean (+/-SEM) rate of appearance of [(13)C5]5-methyltetrahydrofolate in plasma was 0.33 +/- 0.09 (caplet) and 5.8 +/- 1.2 (intravenous) nmol/h. Likely because of the significant time in the colon, the mean apparent absorption across the colon was 46%. CONCLUSIONS: Folate is absorbed across the colon in humans with an undisturbed microbiome. This finding and previous observations of the size of the colonic depot of folate and its potential for manipulation by diet (eg, dietary fiber, oligosaccharides, and probiotics) suggest that an individual's dietary folate requirement may differ depending on the consumption of dietary constituents that affect the size and composition of their gastrointestinal microbiota. In addition, a systematic investigation of the role of colonic folate on gastrointestinal development and the prevention of colorectal cancer is warranted. This trial was registered at clinicaltrials.gov as NCT00941174. |
Brief report: are autistic-behaviors in children related to prenatal vitamin use and maternal whole blood folate concentrations?
Braun JM , Froehlich T , Kalkbrenner A , Pfeiffer CM , Fazili Z , Yolton K , Lanphear BP . J Autism Dev Disord 2014 44 (10) 2602-7 Prenatal multivitamin/folic acid supplement use may reduce the risk of autism spectrum disorders. We investigated whether 2nd trimester prenatal vitamin use and maternal whole blood folate (WBF) concentrations were associated with Social Responsiveness Scale (SRS) scores at 4-5 years of age in a prospective cohort of 209 mother-child pairs. After confounder adjustment, children born to women taking prenatal vitamins weekly/daily (n = 179) had lower odds of clinically elevated SRS scores (odds ratio 0.26; 95 % confidence interval 0.08, 0.89) than those who rarely/never took them (n = 30). WBF concentrations were not associated with SRS scores. The lack of association between WBF and autistic-behaviors may be due to the timing of biomarker measures relative to critical periods of brain development, confounding, or other modifying factors. |
Neither folic acid supplementation nor pregnancy affects the distribution of folate forms in the red blood cells of women
Hartman BA , Fazili Z , Pfeiffer CM , O'Connor DL . J Nutr 2014 144 (9) 1364-9 It is not known whether folate metabolism is altered during pregnancy to support increased DNA and RNA biosynthesis. By using a state-of-the-art LC tandem mass spectrometry technique, the aim of this study was to investigate differences in RBC folate forms between pregnant and nonpregnant women and between nonpregnant women consuming different concentrations of supplemental folic acid. Forms of folate in RBCs were used to explore potential shifts in folate metabolism during early erythropoiesis. Total RBC folate and folate forms [tetrahydrofolate (THF); 5-methyltetrahydrofolate (5-methyl-THF); 4alpha-hydroxy-5-methyl-THF (an oxidation product of 5-methyl-THF); 5-formyl-THF; and 5,10-methenyl-THF] were measured in 4 groups of women (n = 26): pregnant women (PW) (30-36 wk of gestation) consuming 1 mg/d of folic acid, and nonpregnant women consuming 0 mg/d (NPW-0), 1 mg/d (NPW-1), and 5 mg/d (NPW-5) folic acid. Mean (+/- SD) RBC folate concentration of the NPW-0 group (890 +/- 530 nmol/L) was lower than the NPW-1 (1660 +/- 350 nmol/L) and NPW-5 (1980 +/- 570 nmol/L) groups as assessed by microbiologic assay (n = 26, P < 0.0022). No difference was found between the NPW-1 and NPW-5 groups. We detected 5-methyl-THF [limit of detection (LOD) = 0.06 nmol/L] in all groups and THF (LOD = 0.2 nmol/L) in most women regardless of methylenetetrahydrofolate reductase genotype. Most women consuming folic acid supplements had detectable concentrations of 5,10-methenyl-THF (LOD = 0.31 nmol/L). However, there was no difference in the relative distribution of 5-methyl-THF (83-84%), sum of non-methyl folates (0.6-3%), or individual non-methyl folate forms in RBCs across groups. We conclude that although folic acid supplementation in nonpregnant women increases RBC total folate and the concentration of individual folate forms, it does not alter the relative distribution of folate forms. Similarly, distribution of RBC folate forms did not differ between pregnant and nonpregnant women. This trial was registered at clinicaltrials.gov as NCT01741077. |
Serum cotinine and whole blood folate concentrations in pregnancy
Prasodjo A , Pfeiffer CM , Fazili Z , Xu Y , Liddy S , Yolton K , Savitz DA , Lanphear BP , Braun JM . Ann Epidemiol 2014 24 (7) 498-503 e1 PURPOSE: Prenatal tobacco smoke exposure may be associated with low maternal folate levels that increase the risk of adverse infant and child health outcomes by reducing folate availability during fetal development. METHODS: Using data from the Health Outcomes and Measures of the Environment Study, we examined the relationship between secondhand or active tobacco smoke exposure and whole blood folate concentrations in pregnant women from Cincinnati, Ohio (n = 362) at approximately 16-week gestation. We used multivariable linear regression to examine the association between continuous or categorical serum cotinine levels and whole blood folate levels, adjusting for sociodemographic, dietary, and perinatal variables. RESULTS: After adjustment for potential confounders, an interquartile range increases in serum cotinine concentration (0.012-0.224 ng/mL) was suggestively associated with decreased whole blood folate levels (beta, -23 nmol/L; 95% confidence interval (CI), -49, 3; P value = .08). Compared with unexposed women, reductions in mean whole blood folate were observed among active smokers (beta, -94, 95% CI, 195, 6 nmol/L; P value = .40); smaller reductions were observed among women with secondhand exposure (beta, 26; CI, 84, 32 nmol/L; P value = .07). CONCLUSIONS: Consistent with prior studies, active smoking was associated with reduced whole blood folate levels among these pregnant women. Secondhand tobacco smoke exposures were associated with small and imprecise reductions in whole blood folate levels. |
Development of a Standard Reference Material for metabolomics research
Phinney KW , Ballihaut G , Bedner M , Benford BS , Camara JE , Christopher SJ , Davis WC , Dodder NG , Eppe G , Lang BE , Long SE , Lowenthal MS , McGaw EA , Murphy KE , Nelson BC , Prendergast JL , Reiner JL , Rimmer CA , Sander LC , Schantz MM , Sharpless KE , Sniegoski LT , Tai SS , Thomas JB , Vetter TW , Welch MJ , Wise SA , Wood LJ , Guthrie WF , Hagwood CR , Leigh SD , Yen JH , Zhang NF , Chaudhary-Webb M , Chen H , Fazili Z , Lavoie DJ , McCoy LF , Momin SS , Paladugula N , Pendergrast EC , Pfeiffer CM , Powers CD , Rabinowitz D , Rybak ME , Schleicher RL , Toombs BM , Xu M , Zhang M , Castle AL . Anal Chem 2013 85 (24) 11732-8 The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research. |
A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product
Fazili Z , Whitehead RD Jr , Paladugula N , Pfeiffer CM . Anal Bioanal Chem 2013 405 (13) 4549-60 Small specimen volume and high sample throughput are key features needed for routine methods used for population biomonitoring. We modified our routine eight-probe solid phase extraction (SPE) LC-MS/MS method for the measurement of five folate vitamers [5-methyltetrahydrofolate (5-methylTHF), folic acid (FA), plus three minor forms: THF, 5-formylTHF, 5,10-methenylTHF] and one oxidation product of 5-methylTHF (MeFox) to require less serum volume (150 muL instead of 275 muL) by using 96-well SPE plates with 50 mg instead of 100 mg phenyl sorbent and to provide faster throughput by using a 96-probe SPE system. Total imprecision (10 days, two replicates/day) for three serum quality control pools was 2.8-3.6% for 5-methylTHF (19.5-51.1 nmol/L), 6.6-8.7% for FA (0.72-11.4 nmol/L), and ≤11.4% for the minor folate forms (<1-5 nmol/L). The mean (+/-SE) recoveries of folates spiked into serum (3 days, four levels, two replicates/level) were: 5-methylTHF, 99.4 +/- 3.6%; FA, 100 +/- 1.8%; minor folates, 91.7-108%. SPE extraction efficiencies were ≥85%, except for THF (78%). Limits of detection were ≤0.3 nmol/L. The new method correlated well with our routine method [n = 150, r = 0.99 for 5-methylTHF, FA, and total folate (tFOL, sum of folate forms)] and produced slightly higher tFOL (5.6%) and 5-methylTHF (7.3%) concentrations, likely due to the faster 96-probe SPE process (1 vs. 5 h), resulting in improved SPE efficiency and recovery compared to the eight-probe SPE method. With this improved LC-MS/MS method, 96 samples can be processed in ~2 h, and all relevant folate forms can be accurately measured using a small serum volume. |
Accounting for an isobaric interference allows correct determination of folate vitamers in serum by isotope dilution-liquid chromatography-tandem MS
Fazili Z , Pfeiffer CM . J Nutr 2012 143 (1) 108-13 Mild and prolonged oxidative degradation of 5-methyltetrahydrofolate (5-methylTHF) leads to the biologically inactive pyrazino-s-triazine derivative of 4alpha-hydroxy-5-methylTHF (MeFox). MeFox and the biologically active 5-formyltetrahydrofolate (5-formylTHF) are isobaric compounds that behave similarly during chromatographic and mass separation, making coelution and misidentification likely. Our published routine liquid chromatography-tandem MS (LC-MS/MS) method did not discern between 5-formylTHF and MeFox, measuring the sum of these compounds at a mass to charge ratio (m/z) of 474-->327 as 5-formylTHF. We modified this method to separate MeFox and 5-formylTHF by either chromatography or unique mass transitions and then applied the 2 methods to serum specimens to determine typical concentrations of these compounds. The 2 unique transitions (m/z: 5-formylTHF, 474-->299; MeFox, 474-->284) showed good sensitivity [limit of detection (nmol/L): 5-formylTHF, 0.21; MeFox, 0.34], selectivity (no interfering peaks), spiking recovery (mean +/- SD: 5-formylTHF, 103 +/- 3.4%; MeFox, 94 +/- 10%), and low imprecision (CV: 5-formylTHF, 3.9% at 2.4 nmol/L; MeFox, 5.1% at 2.9 nmol/L). The mass separation method detected 5-formylTHF in the same specimens as the chromatographic separation method. Analysis of several thousand serum specimens showed that the majority ( approximately 85%) contained MeFox at <3 nmol/L but no detectable 5-formylTHF concentrations, some ( approximately 14%) contained 5-formylTHF at <0.5 nmol/L, and a few specimens contained 5-formylTHF at >1 nmol/L and MeFox at >10 nmol/L. In summary, serum can contain 5-formylTHF high enough to contribute to total folate and contains MeFox that will bias total folate if not appropriately separated. Including measurements of MeFox and 5-formylTHF along with the other folate vitamers will enhance assessments of the association between biologically active folate and health effects. |
Assessing the influence of 5,10-methylenetetrahydrofolate reductase polymorphism on folate stability during long-term frozen storage, thawing, and repeated freeze/thawing of whole blood.
Fazili Z , Sternberg MR , Pfeiffer CM . Clin Chim Acta 2012 413 966-72 BACKGROUND: Limited information is available on folate stability, particularly vitamer stability by 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T genotype, during frozen storage, thawing, and repeated freeze/thawing (F/T) of whole blood (WB). METHODS: We assessed folate stability after storing undiluted WB for up to 30mo at -70 degrees C and measuring folate vitamers by LC-MS/MS at 6, 14, 20 and 30mo in samples with C/C and T/T genotype (n=13 each). We investigated folate stability during 3-h thawing of WB (n=2 each/genotype) and during repeated F/T of WB (n=4 each/genotype). RESULTS: We found significant decreases in total folate (TFOL) (median decrease: 8.8% for C/C and 16% for T/T), methyl folate (7.9% for C/C and 10% for T/T), and non-methyl folate (19% for C/C and 24% for T/T) concentrations from 6 to 30mo WB frozen storage. During thawing of WB at room temperature and repeated F/T, samples with T/T genotype were susceptible to greater folate losses than samples with C/C genotype. CONCLUSIONS: Long-term frozen storage of WB resulted in significant folate losses of ~10-25% that are clinically unacceptable. Frozen WB should not be exposed to more than 1h of thawing time and repeated F/T of WB should be avoided. |
Isotope dilution-LC-MS/MS reference method assessment of serum folate assay accuracy and proficiency testing consensus mean
Blackmore S , Pfeiffer CM , Lee A , Fazili Z , Hamilton MS . Clin Chem 2011 57 (7) 986-94 BACKGROUND: Current methods for measuring folates in clinical laboratories are competitive folate binding protein assays. These assays show a considerable lack of agreement that has implications for the comparability of data across studies as well as for long-term population studies. The development of isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference methods permitted the evaluation of method accuracy and consistency over time. METHODS: We measured 3 pools of human serum by ID-LC-MS/MS, calculated values for total folate, and distributed the same pools to participants in a national External Quality Assessment scheme. We used linear regression to compare the all-laboratory and method data with reference method values. The exercise was repeated after 18 months to assess the stability of the all-laboratory and method data. RESULTS: The distributed serum pools had mass spectrometry values for folate species typical of those found in healthy individuals from populations not receiving dietary folic acid fortification. There was good agreement of the all-laboratory data set with the reference method (y =0.86x + 0.91 mcg/L) at both time points. Linear regression demonstrated that the Abbott Architect showed the closest agreement with the reference method. The Roche Elecsys method was nonlinear and showed a calibration offset of 2.6 mcg/L (4.57 nmol/L). CONCLUSIONS: Calibration of serum folate assays traceable to higher-order reference methods increases method accuracy and improves consistency. The all-laboratory consensus mean proved sufficiently accurate and stable to be used as the target for monitoring laboratory performance. |
Biomarkers of folate status in NHANES: a roundtable summary
Yetley EA , Pfeiffer CM , Phinney KW , Fazili Z , Lacher DA , Bailey RL , Blackmore S , Bock JL , Brody LC , Carmel R , Curtin LR , Durazo-Arvizu RA , Eckfeldt JH , Green R , Gregory JF 3rd , Hoofnagle AN , Jacobsen DW , Jacques PF , Molloy AM , Massaro J , Mills JL , Nexo E , Rader JI , Selhub J , Sempos C , Shane B , Stabler S , Stover P , Tamura T , Tedstone A , Thorpe SJ , Coates PM , Johnson CL , Picciano MF . Am J Clin Nutr 2011 94 (1) 303S-312S A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999-2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991-1994 and NHANES 1999-2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007-2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA. |
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