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Technical Briefs |
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia;
aaddress correspondence to this author at: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, NE, Mail Stop F55, Atlanta, GA 30345; fax 770-488-4139, e-mail CPfeiffer{at}cdc.gov
Abstract
Background: The Bio-Rad QuantaPhase II radioassay (BR), used for 25 years to measure total folate (TFOL) concentrations for the National Health and Nutrition Examination Survey (NHANES), will be discontinued in 2007. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) or a microbiologic assay (MA) will be used in the future.
Methods: We measured folate species by LC-MS/MS and TFOL by MA and BR in 327 serum samples.
Results: LC-MS/MS measured 5-methyltetrahydrofolic acid (5CH3THF; 82%), folic acid (FA; 8%), 5-formyltetrahydrofolic acid (5CHOTHF; 6%), tetrahydrofolic acid (THF; 4%), and 5,10-methenyltetrahydrofolic acid (5,10CH=THF; 0%). The sum of the folate species correlated well with TFOL measured by MA (R2 = 0.97) and BR (R2 = 0.91). Compared with LC-MS/MS results, MA and BR values were significantly lower (–6% and –29%, respectively); however, these differences were concentration dependent. The MA almost completely recovered folates added to serum samples except for FA [69% (3%)] and THF [36% (10%)]. The BR underrecovered 5CH3THF [61% (9%)] and 5CHOTHF [38% (14%)] and overrecovered 5,10CH=THF [234% (32%)]. Multiple linear regression models with log-transformed data yielded a good fit for converting BR data to MA or LC-MS/MS data and MA data to LC-MS/MS data.
Conclusions: The good correspondence between the sum of folate species determined by LC-MS/MS and TFOL determined by MA makes these 2 assays interchangeable. The BR produces much lower results, on average, probably because of 5CH3THF underrecovery. The conversion equations provided could be used for future NHANES time trend analyses.
The serum concentration of folate is an important marker of nutritional status and has been measured for >30 years as part of the National Health and Nutrition Examination Survey (NHANES). The Bio-Rad QuantaPhase II radioassay (BR) has been used since 1991 but will be discontinued in 2007. The introduction of mandatory folic acid (FA) fortification in 1998 has contributed to appreciable increases in serum folate concentrations in the US population (1); continued monitoring through NHANES is required. Two assays have been discussed for future surveys: the new isotope-dilution liquid chromatography–tandem mass spectrometry method (LC-MS/MS) (2)(3) and the traditional Lactobacillus casei microbiologic assay (MA) (4)(5). The LC-MS/MS measures 5 folate species: 5-methyltetrahydrofolic acid (5CH3THF), FA, 5-formyltetrahydrofolic acid (5CHOTHF), tetrahydrofolic acid (THF), and 5,10-methenyltetrahydrofolic acid (5,10CH=THF). The MA and BR measure total folate (TFOL). The 1st objective of this study was to compare the LC-MS/MS method with the traditional MA. The 2nd objective was to provide equations for converting results from the assay currently used for the NHANES (BR) to the new assay for future analyses (MA or LC-MS/MS).
Pristine serum samples (n = 237) collected as part of the NHANES from 1999 to 2004 and stored at –70 °C were analyzed between December 2005 and February 2006 for folate species by the LC-MS/MS and for TFOL by the MA. The BR has analyzed aliquots of the same samples for TFOL from 1999 to 2004. These samples are not representative of the US population. They were selected to cover a wide range of TFOL concentrations, as measured by the BR assay. The second set of samples consisted of 100 pristine serum samples obtained from a blood bank from January to March 2006. These samples were analyzed by all 3 assays in March and April 2006.
Data were analyzed with SAS (version 9; SAS Institute) and Microsoft Excel with a clinical statistical analysis plug-in (Analyse-it; Analyse-it Software). Two NHANES samples were excluded as outliers because of an extremely high concentration of either 5CHOTHF or FA. The sample sets were analyzed first separately and then together. We used the sum of the folate species determined by the LC-MS/MS and TFOL determined by the MA or BR to compare the methods. Results are presented only for the combined set because we found no differences between the 2 data sets other than the concentration ranges (see Fig. 1
in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol53/issue4). Methods were compared by least-squares regression after log-transformation of the data to account for nongaussian distribution. The concentration-dependent relationship between these assays prompted us to develop multiple linear regression models that used a dummy binary variable (0, 1), IND, to account for intercept differences and an interaction variable (IND x log10 BR or IND x log10 MA) to account for differences in slope over 2 discrete concentration ranges (
45 vs >45 nmol/L for the BR and <50 vs 
50 nmol/L for the MA). The fit of the models was evaluated by comparing the sum of squared residuals (SSR) to the predicted residual sum of squares (PRESS). We tested the recoveries of the MA and BR methods (n = 2 days) by adding each of the 5 folate calibrators (Merck Eprova) at 10 nmol/L to a serum pool (21.8 nmol/L TFOL by LC-MS/MS).
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The mean concentrations for TFOL, 5CH3THF, and FA were higher in the NHANES set than in the blood bank set, whereas the mean 5CHOTHF concentration was higher in the blood bank set (Table 1
). In the combined set, the major folate forms were 5CH3THF (82%) and FA (8%). Most samples had only low FA (<2.5 nmol/L) and 5CHOTHF (<10 nmol/L) concentrations. Approximately half the samples had detectable THF concentrations; only 10% had THF concentrations >10 nmol/L.
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Mean and median TFOL concentrations measured by LC-MS/MS and MA were generally in agreement, but BR values were much lower. The sum of folate species and TFOL determined by the LC-MS/MS and the MA, respectively, were highly correlated (R2 = 0.97), but the latter produced slightly lower results, corresponding to a small but significant negative concentration-dependent difference of –5.94 nmol/L (95% confidence interval, –7.49 to –4.39 nmol/L) and a relative difference of –6%. The multiple linear regression model was developed for an MA cutoff of 50 nmol/L because of appreciably increased FA concentrations above this concentration. The FA concentration [mean (SD)] at <50 nmol/L was 0.8 (1.4) nmol/L, corresponding to 3% of the TFOL, whereas it was 38 (71) nmol/L at 50 nmol/L, corresponding to 20% of the TFOL (see Fig. 2A in the online Data Supplement). After log-transformation, R2 was 0.96 for the multiple linear regression equation: log10 LC-MS/MS = 0.1439 + (0.9193 x log10 MA) + (0.0048 x IND) + (0.029 x IND x log10 MA), with IND = 0 for MA results <50 nmol/L and IND = 1 for MA results 50 nmol/L (Fig. 1A
). The SSR (1.4042) agreed with the PRESS (1.4377).
We also obtained good correlation between TFOL obtained by LC-MS/MS or MA and TFOL obtained by the BR (R2 = 0.91 for both comparisons); the BR produced much lower results than either the LC-MS/MS or the MA. The mean negative concentration-dependent difference was 29%. The multiple linear regression model was developed for a 45 nmol/L cutoff for the BR, mainly because this value represents the upper end of the calibration curve, but also because of appreciably increased FA concentrations greater than 
50 nmol/L TFOL (see Fig. 2B in the online Data Supplement). After log-transformation, the R2 was 0.95 for both method comparisons. The conversion equations were as follows: log10 LC-MS/MS = 0.1436 + (1.0435 x log10 BR) + (0.51 x IND) – (0.3218 x IND x log10 BR), (SSR, 2.0119; PRESS, 2.0682); log10 MA = 0.0504 + (1.0958 x log10 BR) + (0.6358 x IND) – (0.4105 x IND x log10 BR), (SSR, 1.7889; PRESS, 1.8432). These equations were with IND = 0 for BR results 45 nmol/L and with IND = 1 for BR results >45 nmol/L (Fig. 1, B and C
).
In 2005 the NIST released a new standard reference material for homocysteine and folate in human serum, SRM 1955 (6). We found similar good agreement for TFOL between our LC-MS/MS and MA methods (level 1, 6.0 vs 5.6 nmol/L; level 2, 13 vs 14 nmol/L; level 3, 41 vs 44 nmol/L). TFOL concentrations obtained by the BR were 25%–40% lower (level 1, 4.5 nmol/L; level 2, 10 nmol/L; level 3, 25 nmol/L).
The lower response of the BR compared with the MA has been known for many years (7). The BR was initially marketed (QuantaPhase I) with calibrator concentrations to match MA performance, but questions raised by Levine (8) prompted introduction of the QuantaPhase II assay in 1993 with spectrophotometrically verified FA calibrator concentrations, resulting in a 30% downward shift in TFOL concentrations. One potential cause for lower TFOL concentrations by the BR is underrecovery of certain folate forms. Our recovery experiments with serum samples showed satisfactory recovery of FA [91% (10%)] and THF [106% (27%)], underrecovery of 5CH3THF [61% (9%)] and 5CHOTHF [38% (14%)], and overrecovery of 5,10CH=THF [234% (32%)]. Others have also reported lower BR recoveries of 5CH3THF (60%) (9). We obtained satisfactory MA recoveries for 5CH3THF [88% (9%)], 5CHOTHF [120% (9%)], and 5,10CH=THF [101% (7%)]. FA and THF recoveries were 69% (3%) and 36% (10%), respectively. Because we calibrate the MA with 5CH3THF, which produces a slightly higher response curve than FA, we expect to underrecover FA. The lower recovery of THF, probably due to oxidative loss of THF during the 42-h incubation, might be improved by increasing the ascorbic acid concentration of the medium. Differential recovery of different folate forms by the BR is likely the reason for the concentration-dependent relationship between this assay and the LC-MS/MS or MA. The lower MA recovery of FA is probably the reason for the increased difference between LC-MS/MS and MA results at higher TFOL concentrations.
We conclude that the new LC-MS/MS method agrees well with the traditional MA method. The small difference in results between these 2 methods is not clinically relevant. Irrespective of the assay used for future NHANES monitoring, population reference ranges will change to higher values; however, MA-determined cutoff values for deficiency (10) could be directly applied to the LC-MS/MS because of its excellent agreement with the MA. Some advantages of the LC-MS/MS compared with the MA are that it provides information on the different folate species in addition to TFOL and it is less prone to interferences such as antibiotics. BR underrecovery of 5CH3THF, the main circulating form of folate, is likely the major reason for its lower results. A model will be required to convert results from the old or the new NHANES method for time trend analysis. Our model provides an excellent fit over a wide concentration range. This information may also be useful to the international community in that national data generated with the MA can now potentially be compared with US reference ranges.
Acknowledgments
We thank Joseph Jacobson (Battelle, Columbus, OH) and Irene Williams (Centers for Disease Control and Prevention, Atlanta, GA) for technical assistance with the LC-MS/MS and BR methods.
References
The following articles in journals at HighWire Press have cited this article:
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  R. Hannisdal, P. M. Ueland, and A. Svardal Liquid Chromatography-Tandem Mass Spectrometry Analysis of Folate and Folate Catabolites in Human Serum Clin. Chem., June 1, 2009; 55(6): 1147 - 1154. [Abstract] [Full Text] [PDF]
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 L. A Houghton, J. Yang, and D. L O'Connor Unmetabolized folic acid and total folate concentrations in breast milk are unaffected by low-dose folate supplements Am. J. Clinical Nutrition, January 1, 2009; 89(1): 216 - 220. [Abstract] [Full Text] [PDF]
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Z. Fazili, C. M. Pfeiffer, M. Zhang, R. B. Jain, and D. Koontz Influence of 5,10-Methylenetetrahydrofolate Reductase Polymorphism on Whole-Blood Folate Concentrations Measured by LC-MS/MS, Microbiologic Assay, and Bio-Rad Radioassay Clin. Chem., January 1, 2008; 54(1): 197 - 201. [Abstract] [Full Text] [PDF]
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