4
Determination of vitamin D and its metabolites

https://doi.org/10.1016/j.beem.2013.06.001Get rights and content

The demand for analysis of 25-hydroxyvitamin D has increased dramatically throughout the world over the past decade. As a consequence, a number of new automated assays have been introduced for 25-hydroxyvitamin D measurement. Automated assays have shown variable ability to meet the technical challenges associated with 25-hydroxyvitamin D measurement. Assays are able to meet performance goals for precision at high concentrations but fail to do so at low concentrations of 25-hydroxyvitamin D. The overall accuracy of automated methods has improved over recent years and generally shows good overall agreement with reference methods; however, discrepancies persist for individual samples. Liquid chromatography-tandem mass spectrometry is used by some routine laboratories for 25-hydroxyvitamin D analysis but its widespread use is hampered by limited sample throughput. 1,25-Dihydroxyvitamin D is an important analyte in specific clinical situations, which remains in the hands of specialised laboratories using manual analytical methods.

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

References (91)

  • S. Messerlian et al.

    The 3-epi and 24-oxo-derivatives of 1a,25-dihydroxyvitamin D3 stimulate transcription through the vitamin D receptor

    Journal of Steroid Biochemistry and Molecular Biology

    (2000)
  • J.C. Fleet et al.

    1a,25-(OH)2-Vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelium calcium transport and mRNA expression in vitro

    Archives of Biochemistry and Biophysics

    (1996)
  • F.G. Strathmann et al.

    3-epi-25 hydroxyvitamin D concentrations are not correlated with age in a cohort of infants and adults

    Clinica Chimica Acta

    (2012)
  • T. Shinki et al.

    Parathyroid hormone inhibits 25-hydroxyvitmain D3-24-hydroxylase mRNA expression stimulation of 1 alpha25-dihydroxyvitamin D3 in rat kidney but not in intestine

    Journal of Biological Chemistry

    (1992)
  • C.R. Bosworth et al.

    The serum 24,25-dihydroxyvitamin D concentration, a marker of vitamin D catabolism, is reduced in chronic kidney disease

    Kidney International

    (2012)
  • R. Bouillon

    The vitamin D binding protein

  • R.P. Heaney et al.

    25-Hydroxylation of vitamin D3: relation to circulating vitamin D3 under various input conditions

    Amerian Journal of Clinical Nutrition

    (2008)
  • S. Epstein et al.

    Drug and hormone effects on vitamin D metabolism

  • A.B. Connell et al.

    Overreporting of vitamin D deficiency with the Roche Elecsys vitamin D3 (25-OH) method

    Pathology

    (2011)
  • B. Hollis

    Measuring 25-hydroxyvitamin D in a clinical environment: challenges and needs

    American Journal of Clinical Nutrition

    (2008)
  • Y. Chen et al.

    Performance evaluation of Siemens ADVIA centaur and Roche MODULAR analytics E170 total 25-OH vitamin D assays

    Clinical Biochemistry

    (2012)
  • J.A. Eisman et al.

    Determitamin D2 and 25-hydroxyvitamin D3 in human plasma using high-pressure liquid chromatography

    Analytical Biochemistry

    (1977)
  • G.D. Carter et al.

    Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation

    Journal Steroid Biochemistry and Molecular Biology

    (2004)
  • M.F. Holick

    Vitamin D status: measurement, interpretation and clinical application

    Annals of Epidemiology

    (2009)
  • A.M. Kissmeyer et al.

    Sensitive analysis of 1alpha,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography-tandem mass spectrometry

    Journal of Chromatography A

    (2001)
  • R. Singh

    Are clinical laboratories prepared for accurate testing of 25-hydroxyvitamin D?

    Clinical Chemistry

    (2008)
  • Vitamin D testing. Review of the funding arrangements for pathology services

    (2011)
  • P.R. von Hurst et al.

    Vitamin D supplementation reduces insulin resistance in South Asian women living in New Zealand who are insulin resistant and vitamin D deficient - a randomised, placebo-controlled trial

    British Journal of Nutrition

    (2010)
  • J.M. Lappe et al.

    Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial

    American Journal of Clinical Nutrition

    (2007)
  • T.J. Wang et al.

    Vitamin D deficiency and risk of cardiovascular disease

    Circulation

    (2008)
  • P. Autier et al.

    Vitamin D supplementation and total mortality: a meta-analysis of randomized controlled trials

    Archives of Internal Medicine

    (2007)
  • Pollack A. Quest acknowledges errors in vitamin D tests. New York Times. Available at:...
  • W.G. Tsiaras et al.

    Factors influencing vitamin D status

    Acta Dermato- Venereologica

    (2011)
  • M.F. Holick et al.

    Photosynthesis of previtamin D3 in human skin and the physiologic consequences

    Science

    (1980)
  • T.C. Chen et al.

    Photobiology of vitamin D

  • X.Q. Tian et al.

    Characterization of the translocation process of vitamin D3 from the skin into the circulation

    Endocrinology

    (1994)
  • A.R. Webb et al.

    Sunlight regulates the cutaneous production of vitamin D3 by causing its photodegradation

    Journal of Clinical Endocrinology and Metabolism

    (1989)
  • M.F. Holick et al.

    Regulation of cutaneous previtamin D3 photosynthesis in man: skin pigment is not an essential regulator

    Science

    (1981)
  • Institute of Medicine

    Food and nutrition board. Dietary reference intakes for calcium and vitamin D

    (2010)
  • National Institutes of Health, Office of Dietary Supplements. Dietary supplement fact sheet: vitamin D. Available at:...
  • B. Lehmann

    The vitamin D3 pathway in human skin and its role for regulation of biological processes

    Photochemisstry and Photobiology

    (2005)
  • D. Zehnder et al.

    Extrarenal expression of 25-hydroyxvitamin D3-1alpha-hydroxylase

    Journal of Clinical Endocrinology and Metabolism

    (2001)
  • A.J. Brown et al.

    1a,25-Dihydroxy-3-epi-vitamin D3, a natural metabolite of 1a-,25-dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion

    Journal of Cellular Biochemistry

    (1999)
  • R.J. Singh et al.

    C-3 Epimers can account for a significant proportion of total circulating 25-hdyroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status

    Journal of Clinical Endocrinology and Metabolism

    (2006)
  • G. Lensmeyer et al.

    The C-3 epimer of 25-hydroxyvitamin D3 is present in adult serum

    Journal of Clinical Endocrinology and Metabolism

    (2012)
  • Cited by (62)

    • Vitamin D<inf>3</inf> levels in women and factors contributing to explain metabolic variations

      2021, Journal of Steroid Biochemistry and Molecular Biology
      Citation Excerpt :

      The main reason to justify selection of 25(OH)D3 as the target vitamin D3 biomarker, despite it is not the active form of vitamin D3, is related to the lack of selective and sensitive methods for determination of dihydroxymetabolites [4,16]. Immunoassay based methods are affected by cross-reactivity with the rest of metabolites, which frequently provide inaccurate results [17]. On the other hand, mass-spectrometry methods have achieved low detection and quantitation limits for the active metabolite, 1,25(OH)2D3 (pmol/L) [18].

    View all citing articles on Scopus
    1

    Tel.: +61 2 9005 7000; Fax: +61 2 9770 1050.

    View full text