is likely to provide a better assessment of vitamin D status than total level [76–78]. Prior to the development of a commercially available direct measure of free 25(OH)D, investigators have calculated the free level using the published affinity constants of 25(OH)D (and 1,25[OH]2D) for DBP and albumin. This method has several drawbacks in that it assumes that these affinity constants do not change with different physiologic states and are identical from one individual to the next. Neither assumption is valid [79–81]. Moreover, these calculations rely on accurate measurement of the levels of DBP and albumin. Although the measurement of albumin is standard from laboratory to laboratory, the measurement of DBP has been more problematic. DBP has a number of polymorphisms, which affect not only its affinity for the vitamin D metabolites [81], but also its measurement. A commercially available monoclonal antibody used in an ELISA from R&D Systems was found to underestimate the levels of the 1F allele of DBP, an allele common in Africans (Gambians) and African Americans, but seldom found in whites of European descent [82]. Using this ELISA resulted in substantially lower measurements of DBP levels in African Americans compared to Caucasians [83], results that were not substantiated when polyclonal antibodies were used either as ELISAs or with radial immunodiffusion [82]. When mass spectrometry was used to measure DBP, the results confirmed that DBP levels differed little on the basic of allelic differences, but measurements were substantially below that obtained with the polyclonal assay [84]. This disparity between the polyclonal immunoassays and mass spectrometry has not been resolved, but this disparity further points to the difficulty in calculating free vitamin D metabolite levels rather than measuring these levels directly. Centrifugal ultrafiltration was the first of the direct assays for the free metabolites, and provided much of the initial data on affinity constants and levels in different groups varying in DBP levels, but because of its labor intensive requirements it is no longer used. Instead, an immunoassay has been developed by Future Diagnostics that is now commercially available, and measurement of free 25(OH)D by LC-MS may soon be available.
Centrifugal Ultrafiltration. The centrifugal ultrafiltration assay consists of an inner vial capped on one end with dialysis membrane resting on filter pads at the bottom of an outer vial. The serum sample following incubation with freshly purified 3H-labeled vitamin D metabolite and 14C-labeled glucose as a marker of free water was placed in the inner vial and centrifuged at 37°C for 45 min. The ratio of 3H/14C in the ultrafiltrate to that in the sample determined the % free. The free concentration was then calculated by multiplying the % free times the total metabolite concentration [77]. This method is dependent on the purity of the labeled vitamin D metabolite requiring purification by HPLC before each assay, as degradation products could increase the fraction filtered. Also, the assembly of the ultrafiltration apparatus is labor intensive, as no suitable commercial equipment is available that does not bind to the filtered vitamin D metabolite. This assay is not commercially available.
Immunoassay. In this assay, developed by Future Diagnostics B.V., Wijchen, The Netherlands, antibodies reactive to 25(OH)D are immobilized in a microtiter well (solid phase). Standards, controls, and patient samples are added to the wells, binding to the solid-phase antibodies. The solid phase is then washed and a biotin-labeled analog of 25(OH)D is added to react with the remaining antibody in a second incubation. After washing, the wells are incubated with a streptavidin-peroxidase conjugate and bound enzyme is quantitated using a colorimetric reaction. Intensity of the signal is inversely proportional to the level of free 25(OH)D in the sample. This assay like all immunoassays is dependent on the specificity of the antibody. It is reported to modestly (70–90%) underestimate 25(OH)D2 relative to 25(OH)D3 (http://www.future-diagnostics.nl/).
The measurements of free 25(OH)D using this immunoassay are in agreement with the measurements obtained with centrifugal ultrafiltration in subjects with a large range of DBP and albumin concentrations [76, 85].
LC-MS. As noted earlier, LC-MS has been used to detect 25(OH)D in saliva, which is expected to be free of DBP and albumin and so represents free 25(OH)D [54]. In this method, 1 mL of saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with PTAD, ionized by ESI and subjected to LC-MS. The limits of detection were reported as 2 pg/mL. The range of values obtained in normal controls was between 3 and 15 pg/mL, correlating well with total serum 25(OH)D (10–30 ng/mL). The intercept was positive, but the free fraction in the mid range of the assay was approximately 0.04%, in line with the results from centrifugal ultrafiltration and the Future Diagnostics immunoassay.
Conclusions
The request for vitamin D metabolite measurements has exploded over the last several years due to the growing appreciation of the role of vitamin D in health maintenance. Much interest is shown in the measurement of 25(OH)D and to a lesser extent in the measurement of 1,25(OH)2D, but as the assays develop, requests for multiple metabolites in a single sample are increasing. Moreover, there is increased interest in the possibility that free vitamin D metabolite levels might be better markers of vitamin D status than total levels especially in individuals with altered DBP concentrations. Although immunoassays have been and remain the most prevalent assay in use today, LC-MS measurements are becoming more widespread. Each method has its advantages and disadvantages. However, as methods improve, especially for LC-MS, it is expected that LC-MS will become the more widely used method for most applications because it offers precision without the variability intrinsic to immunoassays with different antibodies. A major advance in reducing the variability between laboratories is the introduction of standards for many of the vitamin D metabolites provided by the NIST. Thus, we are approaching a time that the physician requesting these measurements from a certified laboratory can have confidence that the results are reliable in guiding clinical decision making.
References
1Bikle DD: Vitamin D: newly discovered actions require reconsideration of physiologic requirements. Trends Endocrinol Metab 2010;21:375–384.
2Wagner D, Hanwell HE, Schnabl K, Yazdanpanah M, Kimball S, Fu L, et al: The ratio of serum 24,25-dihydroxyvitamin D(3) to 25-hydroxyvitamin D(3) is predictive of 25-hydroxyvitamin D(3) response to vitamin D(3) supplementation. J Steroid Biochem Mol Biol 2011;126:72–77.
3Bikle DD, Gee E: Free, and not total, 1,25-dihydroxyvitamin D regulates 25-hydroxyvitamin D metabolism by keratinocytes. Endocrinology 1989;124:649–654.
4Holick MF, MacLaughlin JA, Clark MB, Holick SA, Potts JT Jr, Anderson RR, et al: Photosynthesis of previtamin D3 in human skin and the physiologic consequences. Science 1980;210:203–205.
5Webb AR, DeCosta BR, Holick MF: Sunlight regulates the cutaneous production of vitamin D3 by causing its photodegradation. J Clin Endocrinol Metab 1989;68:882–887.