Best Practice & Research Clinical Endocrinology & Metabolism
4Determination of vitamin D and its metabolites
Introduction
Testing for vitamin D has increased exponentially in the past decade. In the United States, requests to clinical laboratories have been increasing at a rate of 80–90% per year [1]. Similarly, in Australia there was a 100-fold increase in vitamin D tests between 2000 and 2010 [2]. The demand is a consequence of the recognition of a high prevalence of deficiency in diverse populations [3] and research uncovering the importance of vitamin D in multiple physiological functions. The role of vitamin D in promoting absorption of dietary calcium and phosphate and increasing bone mineral density is well-recognised. However, vitamin D deficiency incurs an increased risk of conditions as disparate as insulin resistance and diabetes, cancer, autoimmune disease, cardiovascular disease and all-cause mortality [4], [5], [6], [7], [8], [9].
The increase in requests for vitamin D analyses has placed pressure on clinical laboratories to offer testing procedures capable of providing results for large numbers of samples in a timely fashion. Multiple in vitro diagnostic companies have therefore been motivated to offer a vitamin D assay on their automated immunoassay platforms. Consequently, there has been an influx of new vitamin D assays onto the market and clinical laboratories can now select from a range of possible assays. However, due to the highly lipophilic nature of vitamin D, high affinity for vitamin D binding protein (DBP) as well as presence of multiple vitamin D metabolites in the circulation, vitamin D is a challenging analyte to measure accurately. These challenges are most easily met in specialist laboratories using time-consuming methods and manufacturers have had difficulty in producing high-throughput assays capable of producing results of satisfactory accuracy. In fact, a number of automated immunoassays have been withdrawn from the market because of poor analytical accuracy. Even higher-order methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) have had well-publicised problems with accuracy in the routine setting [10]. Furthermore, many other assays have been re-formulated as manufacturers seek to improve the performance of sub-optimal assays.
This article reviews the physiology of vitamin D metabolites and evaluates the performance of automated and chromatographic assays for 25-hydroxyvitamin D (25-OHD), the best general marker of vitamin D status. It also addresses the indications for measurement and methodologies available for measurement of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-OH2D).
Section snippets
Vitamin D3 metabolism
The primary source of vitamin D in humans is via production in the skin. This is achieved through the action of UV light on 7-dehydrocholesterol, an intermediate in the cholesterol biosynthetic pathway, also referred to as ‘provitamin D’. Cholesterol is an important component in the lipid barrier of the skin and the epidermis is an active site of cholesterol synthesis. Local synthesis thus provides a ready source of provitamin D where it is incorporated into the plasma membrane lipid bilayers
Measurement of 25-hydroxyvitamin D
The total serum 25-OHD concentration (i.e. sum of D3 and D2 forms) is regarded as the best single marker of vitamin D status [26]. There is essentially complete conversion of vitamin D to 25-OHD when vitamin D production and/or ingestion is below 2000 IU per day [38]. Furthermore, 25-OHD has a long half-life in the circulation, approximately 3 weeks. The circulating 25-OHD concentration also indicates the availability of substrate for local tissue production and autocrine/paracrine action of
Indications for measurement
Although 25-OHD is generally the preferred marker of vitamin D status, 1,25-OH2D measurement is important in a limited number of circumstances. In particular, the 1,25-OH2D concentration is clinically relevant in circumstances in which there may be a disorder of 1α-hydroxylation. This most commonly occurs in the context of chronic kidney disease (CKD). A number of mechanisms may contribute to impaired 1α-hydroxylation in CKD: decreased quantity of 1α-hydroxylase due to the decreased kidney
Conclusions
The analysis of vitamin D and its metabolites is a rapidly evolving field. The recognition of the importance of vitamin D beyond simply skeletal health and a high prevalence of deficiency in diverse populations has created a rapid increase in demand for vitamin D testing. Consequently, vitamin D analysis has moved from specialist laboratories using highly manual assays to routine laboratories using automated assays on high-throughput analysers. The difficulty of providing accurate automated
Summary
There has been a dramatic worldwide increase in demand for the laboratory assessment of patients' vitamin D status. Although laboratories are able to measure a number of vitamin D metabolites, it is total serum 25-hydroxyvitamin D (i.e. sum of D3 and D2 forms) which is considered the best marker of status in most patients. To cope with the increased demand for testing laboratories have increasingly be using automated 25-OHD assays and a number of new automated assays have been recently
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