25-HYDROXYVITAMIN D: METHOLOGICAL ASPECTS AND OPTIMAL SERUM LEVELS

Abstract

In biochemical practice problems such as standardization, specificity, sensitivity, functional sensitivity,

matrix, uncertainty, etc., are relatively common. Assay of 25-hydroxyvitamin D(25D) is not free of these

problems.Assay systems without prior chromatography give higher values, due to lack of separation of

interfering metabolites. With prior chromatography –either normal or in reverse phase– this problem is

obviated. Radioimmunoassays, enzymoimmunoassays and automated assays have simplified the process. Both

HPLC and gas chromatography, followed by mass spectrometry, have been proposed as reference methods,

but they are difficult to perform in conventional laboratories. The variety of methods used along the last 20

years and the absence of an international 25D reference standard makes it necessary to evaluate, in every

publication, which method has been employed; also, comparisons between papers should be made with

caution, specially if no correction factor is known. Assay specificity should be always considered carefully.

Although kit inserts claim that both 25D3 and 25D2 are recognized, this is not so in every case. There is a

difference between the reference range and the “healthy” or “optimal” level of 25D. The distribution of 25D

values in a population, although typical, cannot be considered normal. Because of this, different criteria

have been described to define which is the optimal 25D level. One of them is to evaluate the relation between PTH and 25D, and to define at which 25D level PTH is maximally suppressed (although some studies have

not found such suppression). Using this criterion, and based on some of the most recent papers examining the

decrease in fracture rates with vitamin D supplements, as well a other elements, there is consensus nowadays

that the optimal level of 25D is well above the traditional 10-12 ng/ml cut-off, and averages 30 ng/ml.