Читать книгу Vitamin D in Clinical Medicine - Группа авторов - Страница 33

The measurement of 1,25(OH)₂D is more challenging than that of 25(OH)D because it circulates in blood at levels that are nearly 1/1,000 that of 25(OH)D. As for the measurement of 25(OH)D, there are 3 basic methods: CPBA, immunoassay, and LC-MS. LC-UV does not have the sensitivity to measure 1,25(OH)₂D so is not included in this discussion.

CPBA. The first assay for 1,25(OH)₂D was a CPBA using a chicken intestinal extract containing the VDR and ³H-1,25(OH)₂D as tracer [58]. The intestinal extract was subsequently replaced by calf thymus VDR [59]. These assays required substantial sample preparation by HPLC to avoid interfering substances, and have subsequently been replaced by immunoassays.

Immunoassays. The most common immunoassay in use today is the RIA available in kit form from DiaSorin and IDS among others with antibody (e.g., sheep polyclonal) and ¹²⁵I-1,25(OH)₂D as tracer. This assay does not require HPLC preparation of the sample and uses serum samples as standards without an internal standard to monitor recovery [60]. However, the IDS kit has been reported to have less than 100% recovery for 1,25(OH)₂D₂[61]. ELISA kits are also available from IDS and Immunodiagnostik. The IDS assay uses solid phase immunoextraction in terms of their RIA and colorimetric detection as described for 25(OH)D, but again may underestimate 1,25(OH)₂D₂. Less is known about the performance of the Immunodiagnostik kit. Recently, a fully automated chemiluminescent assay for 1,25(OH)₂D has been introduced (DiaSorin Liason XL) [62]. This method uses the ligand binding domain of VDR as the capture molecule, reaction conditions favoring the binding of 1,25(OH)₂D to VDR versus DBP in the sample but retaining the preferential binding of 25(OH)D, 24,25(OH)₂D, and 25,26(OH)₂D to DBP, and using a monoclonal antibody that selectively detects the VDR conformation induced by ligand binding. This antibody is attached to magnetic beads enabling nonbound materials to be washed away and then followed by a monoclonal antibody conjugated with a chemiluminescent label and specific for an epitope in the ligand binding domain. After washing, the chemiluminescent signal is triggered and quantitated, the strength of which is directly proportional to the amount of 1,25(OH)₂D in the sample. This assay was compared to 2 LCMS assays using immuno enrichment as a preliminary step and found to have a correlation coefficient around 0.92–0.94, a slope approximately of 1, a mean bias of 2.4–15.5%, and an intercept of approximately 2–4. In this regard, it outperformed the earlier DiaSorin immunoassay. Its LOQ was reported as 2 pg/mL using 75 microliter samples.

An important problem for all immunoassays for 1,25(OH)₂D with the possible exception of the above-described chemiluminescent assay is that the polyclonal antibodies generally employed show some cross reactivity with 25(OH)D and 24,25(OH)₂D, which circulate at much higher concentrations in blood than does 1,25(OH)₂D [63]. This can be prevented by careful preparation of the sample to separate these metabolites from 1,25(OH)₂D, but this is not always done.

LC-MS. The major problem to be overcome using LC-MS to measure 1,25(OH)₂D is the limited sensitivity caused by the low circulating levels of 1,25(OH)₂D and the poor ionization efficiency. The sensitivity problem can be addressed by using immunoaffinity extraction with an antibody to 1,25(OH)₂D prior to HPLC and tandem mass spectrometry. This would eliminate the use of LC-MS to measure 1,25(OH)₂D along with other vitamin D metabolites and does not lend itself to making this measurement in small samples. The second method is to use derivatization with polar groups such as PTAD [64] or adduct formation with ammonia [65] or lithium [66]. In these measurements, deuterated 1,25(OH)₂D is used as an internal standard to calculate recovery. Combining immunoaffinity extraction with derivitazation has achieved an LOQ for 1,25(OH)₂D₃ of 3 pM (1.2 pg/mL) and for 1,25(OH)₂D₂ of 1.5 pM (0.6 pg/mL) [63]. The advantages and limitations of LC-MS over and above the sensitivity issue are similar to those described for the LC-MS measurement of 25(OH)D. However, at this point, there is no universally accepted NIST standard for 1,25(OH)₂D by which labs can compare their results, or investigators and clinicians can compare one assay to another. Moreover, like 25(OH)D, the C3-beta epimer of 1,25(OH)₂D is found in serum [67], and other dihydroxy vitamin D metabolites such as 23,25(OH)₂D, 24,25(OH)₂D, 25,26(OH)₂D, and 4β,25(OH)₂D (the product of CYP3A4 hydroxylation of 25[OH]D) need to be separated from 1,25(OH)₂D prior to MS, as all exhibit the same molecular weight and m/z ratios. 4β,25(OH)₂D was found in similar concentrations as 1,25(OH)₂D in one study [68].