Whole-Blood Folate Values in Subjects with Different Methylenetetrahydrofolate Reductase Genotypes: Differences Between the Radioassay and Microbiological Assays

¹ Dept. of Clin. Med. ,
² Dept. of Genetics,
³ Dept. of Biochem., Trinity College Dublin, ,
⁴ Health Research Board, Dublin, Ireland, ,
⁵ Natl. Inst. of Child Health and Human Devel., Bethesda, MD 20892-7510
^a Author for correspondence: Dept. of Biochemistry, Trinity College Dublin, Dublin 2, Ireland. Fax 353 1 6772400.

To the Editor:

Traditionally, serum and erythrocyte folate were analyzed by microbiological assay (1). Because of the original technical difficulty of such assays, more and more laboratories switched over to the radiometric competitive binding assays (radioassays) as they became available. However, it was subsequently found that these newer assays were problematic, particularly with respect to erythrocyte folate estimates (2). These difficulties are said to have been corrected in the newer versions of the radioassay, and it is now believed that both assays are measuring similar folates, albeit by a different principle, and that the assays give equivalent results (3).

The C677T genetic variant results in a thermolabile and less active form of the folate-dependent enzyme 5,10-methylenetetrahydrofolate reductase and has a homozygous prevalence of between 5% and 15% depending on the population studied (4). This variant is of interest clinically because it has been found to be more prevalent in people with neural tube defects (4)(5)(6) and also in those with increased plasma homocysteine, a new risk factor, or at least marker, for cardiovascular disease (7). One group has published (8) and to our knowledge two other groups have found that subjects homozygous for the variant (TT) have higher erythrocyte folate values compared with those heterozygous (CT) or those without the variant wild-type (CC). All three groups have used radioassays for these determinations. It has been our consistent finding with the microbiological assay, however, that the converse is true; we find lower erythrocyte folates in those homozygous for the variant compared with those who are heterozygous or wild-type (9). Although the populations studied by us and others are different, it is difficult to understand why those with the thermolabile variant should have higher concentrations of folate in some populations (8) and lower in other populations (9). An obvious explanation is that the method of folate assay used in the former case was radioassay (8) and in the latter (9), microbiological assay.

To resolve this issue, we carried out comparative assays on the three different genotypes for samples selected at random from a normal group of women (group I). Because van der Put et al. (8) had used children with neural tube defects and their parents in their studies, we also selected a group of the three genotypes from such subjects (group II). All procedures were approved by the relevant ethics committees. The microbiological assay was carried out on microtiter plates with the chloramphenicol-resistant strain of Lactobacillus casei (10), and the radioassay was performed with the Dualcount Solid Phase Boil assay (Diagnostic Products Corp.), which was that used by van der Put et al. (8). We have expressed the results as whole- blood folate for reasons of clarity because these values are converted in the same way to erythrocyte folate values by using the packed cell volume. Serum or plasma folates were not analyzed because the radioassay (8) and microbiological assay (9) give similar results: lower folate values for TT homozygotes.

Table 1 shows that, overall, the microbiological assay gives higher values than the radioassay, as reported in the literature (2)(11). After subdivision of the data by genotype and reanalyzing either as individual groups or combined, there was a decrease in whole-blood folate for the wild-type (CC) and heterozygous (CT) groups [statistically significant for group I (P = 0.0005 and 0.017, respectively) and for the combined groups (P = 0.0001 and 0.006, respectively)] when carried out by the radioassay compared with the microbiological assay. However, in the homozygous (TT) groups, the whole- blood folate concentrations measured by radioassay were higher, and the difference was statistically significant (P = 0.026) in the combined analysis. Thus, in two different populations, subdivided only with respect to this genotype, the assays are measuring components that change with respect to genotype.

Explanations could be that in those with the homozygous (TT) variant, a species of folate or a degradation product accumulates because the enzyme is compromised. This form could be bound in the radioassay and thus appear active and measurable but might not be microbiologically active. It is also possible, but less likely, that the microbiological assay is underestimating the concentration of some active folate. Alternatively, a folate may accumulate that is estimated in both assays but gives values quantitatively different from the forms of folate normally present. We are at present searching for such putative derivatives. The nature of such derivatives showing either accumulation of known folates or the existence of a known or as yet unknown naturally occurring degradation product may be of interest with respect to the etiology of neural tube defects or the homocysteinemia that is associated with this variant. At a more practical level, the comparative evaluation of the two assays reported here indicates that folate concentrations are not being measured accurately by one of the assays in the 5% to 15% of people who are homozygous for the thermolabile variant.

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