Assessment and Interpretation of Circulating 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D in the Clinical Environment

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Methods of 25(OH)D quantitation

The assessment of circulating 25(OH)D started its journey approximately 4 decades ago with the advent of the competitive protein-binding assay (CPBA).3 From that early time to the present we have progressed to radioimmunoassay (RIA), high-performance liquid chromatography (HPLC), and liquid chromatography coupled with mass spectrometry (LC/MS). A brief description of each technique is given here.

Determining analytical recovery of 25(OH)D2 and 25(OH)D3 in human serum or plasma

Questions constantly arise regarding the various 25(OH)D assay procedures' ability to accurately measure total 25(OH)D (25(OH)D2 + 25 (OH)D3) levels in human samples.8 A brief study recently has described the ability of the DiaSorin Liaison Total-D 25(OH)D Assay System to perform this task as compared with the gold standard HPLC/UV quantitation of 25(OH)D2 and 25(OH)D3.18 Baseline serum samples that contained only 25(OH)D3 were obtained from 9 volunteers. All subjects then consumed 50,000 IU/d

Determining and defining a “normal” circulating 25(OH)D level

To define a “normal” circulating level of a given substance or nutrient, one usually obtains blood samples from a diverse population, measures the substance in question, plots the data by Gaussian distribution, and determines normality. This method works well for nutrients such as folate or vitamin E, and was precisely how normative circulating levels of 25(OH)D were defined in humans beginning about 40 years ago by Haddad and Chyu,3 who sampled a population of “normal” individuals whom were

Clinical reporting of circulating 25(OH)D concentrations

As highlighted earlier, all DiaSorin 25(OH)D assays are approved by the FDA for clinical utility. Thus, the diagnostic 25(OH)D tests sold by DiaSorin and IDS Diagnostics (Fountain Hills, AZ, USA) are under strict FDA control and monitoring for assay performance and reliability. In what is considered a distributing trend, many clinical reference laboratories are replacing these FDA-approved tests with “home-brew” LC/MS methods that are diverse and not under FDA scrutiny. The reasons for this

Methods of 1,25(OH)2D quantitation

Of all the steroid hormones, 1,25(OH)2D represented the most difficult challenge to the analytical biochemist with respect to quantitation. 1,25(OH)2D circulates at picomolar concentrations (too low for direct UV or MS quantitation), is highly lipophilic, and its precursor, 25(OH)D, circulates at nanomolar levels. The development of simple, rapid assay for this compound has proven to be a daunting task.

Determining and defining a “normal” circulating 1,25(OH)2D level

Unlike 25(OH)D, a normal circulating level of 1,25(OH)2D can be determined from a Gaussian distribution of subjects. Over the last 3 decades this has been accomplished, and a normal adult level has been defined as 16 to 56 pg/mL with a mean of 37.6 pg/mL.68 Circulating 1,25(OH)2D is diagnostic for several clinical conditions, including vitamin D–dependent rickets types I and II, hypercalcemia associated with sarcoidosis, and other hypercalcemic disorders causing increased 1,25(OH)2D levels.

Stability of 25(OH)D and 1,25(OH)2D in serum or plasma

Researchers have known for nearly 30 years that endogenous 25(OH)D and 1,25(OH)2D are extremely stable in serum or plasma.70 Lissner and colleagues70 showed that vitamin D metabolites in blood stored at 24°C for up to 72 hours remain intact. Recent studies on the stability of 25(OH)D in plasma or serum that has undergone many freeze-thaw cycles have reported the same stability.71 The author has used the same pooled human 25(OH)D and 1,25(OH)2D internal controls stored at −20°C for more than 10

Standardization of 25(OH)D and 1,25(OH)2D analysis

DEQAS (Internet: www.deqas.org) was founded in 1989 to compare the performance of then-available 25(OH)D tests. DEQAS has since become the largest vitamin D quality assessment program in the world, with approximately 600 participating laboratories worldwide. The organization's major aim today is to assess the analytical reliability of 25(OH)D and 1,25(OH)2D assays. The organization achieves this goal by:

  • Distributing serum pools at regular intervals

  • Conducting statistical analyses of submitted

Summary

The assessment of circulating 25(OH)D and, to a lesser degree, 1,25(OH)2D is rapidly becoming an important clinical tool in the diagnosis and management of many diverse pathologies. At present, the reference ranges for circulating 25(OH)D and 1,25(OH)2D are 32 to 100 ng/mL and 16 to 56 ng/mL, respectively, and are largely based on clinical data derived from the FDA-cleared DiaSorin assay procedures.

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    This article originally appeared in Endocrinology and Metabolism Clinics of North America Volume 39, Issue 2, June 2010.

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