Review
25-Hydroxyvitamin D assays: Potential interference from other circulating vitamin D metabolites

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Highlights

  • Both 3-epi-25-OHD3 and 24,25-dihydroxyvitamin D (24,25(OH)2D) show a strong correlation with 25-OHD3.

  • 3-epi-25-hydroxyvitamin D(3-epi-25-OHD) does not cross react in immunoassays for 25-hydroxyvitamin D (25-OHD).

  • Some HPLC/UV and LC–MS/MS methods may not resolve 3-epi-25-OHD3 from 25-OHD3.

  • Cross reaction of 24,25(OH)2D in 25-OHD immunoassays may contribute to the positive bias of some assays, particularly at high 25-OHD concentrations.

  • The use of exogenous 24,25(OH)2D gives misleading results in recovery experiments.

Abstract

The vitamin D External Quality Assessment Scheme (DEQAS) for 25-hydroxyvitamin D (25-OHD) has approximately 1100 participants in 53 countries using 26 different methods or variants of methods (October 2014). In April 2015, the scheme was extended to cover 24,25-dihydroxyvitamin D (24,25(OH)2D). Since 2013, the 25-OHD scheme has been accuracy-based with values assigned by the NIST reference measurement procedure (RMP). DEQAS is uniquely placed to assess the accuracy (bias) and specificity of 25-OHD methods in a routine laboratory setting. Other vitamin D metabolites are known to interfere in 25-OHD assays and DEQAS has distributed samples spiked with 3-epi-25-OHD3 (52.4 nmol/L), 24R,25(OH)2D3 (14.4 nmol/L) and 24S,25(OH)2D3 (57.9 nmol/L). The 3-epimer showed a cross reactivity of 56% in a competitive protein binding assay but was not detected in any antibody-based methods. Not all HPLC/UV or LC–MS/MS methods were able to resolve 3-epi-25-OHD3 from 25-OHD3 and thus overestimated total 25-OHD. The cross reactivity of 24R,25(OH)2D3 (24S,25(OH)2D3) ranged from <5% (<5%) to 548% (643%) in ligand binding assays. Both 24-hydroxylated metabolites were resolved by HPLC/UV and LC–MS/MS methods and thus caused no complications in the measurement of 25-OHD. Most antibodies to 25-OHD are known to cross-react with dihydroxylated metabolites but interference in some assays was far greater than expected. This may be related to the anomalous behaviour of exogenously added metabolites in these 25-OHD methods.

Introduction

The vitamin D External Quality Assessment Scheme (DEQAS) is an international external quality assessment scheme which has been assessing the performance of 25-hydroxyvitamin D (25-OHD) assays since 1989 and 1,25-dihydroxyvitamin D (1,25(OH)2D) assays since 1997. The scheme was extended in April 2015 to cover 24,25-dihydroxyvitamin D (24,25(OH)2D). Performance assessment is achieved by the quarterly distribution of 5 samples of human serum to the approximately 1100 laboratories participating in the 25-OHD scheme. DEQAS has participants in 53 countries (October 2014). Samples are prepared from blood donated locally by polycythaemic or haemochromatosis patients undergoing therapeutic venesection and, since April 2013, from serum purchased from the USA (Solomon Park, Kirkland, WA, USA).

Since April 2013, as a result of support from the NIH Office of Dietary Supplements, the 25-OHD scheme has been accuracy based with values assigned to each sample by the NIST reference measurement procedure (RMP) [1]. Thus DEQAS is well placed to assess the accuracy (bias) and specificity of 25-OHD methods in a routine laboratory setting.

The vitamin D metabolite 24,25(OH)2D, concentrations of which can range from 2% to 20% of total 25-OHD [2], is known to cross react in several ligand binding assays and might account for their variable bias, particularly in samples with high 25-OHD. Many routine HPLC/UV and LC–MS/MS assays fail to resolve 3-epi-25-OHD3 from 25-OHD3 which might contribute to the persistent positive bias seen in these methods [3].

The objectives of this study were twofold:

  • 1.

    To measure the concentration of 3-epi-25-OHD3 and 24R,25(OH)2D3 in a series of DEQAS samples and determine the relationship of each metabolite to 25-OHD3.

  • 2.

    To calculate the cross reactivity of 24R,25(OH)2D3 and 24S,25(OH)2D3 in commonly used ligand binding assays for 25-OHD and the degree of interference in HPLC/UV and LC–MS/MS methods.

Section snippets

Assignment of values to DEQAS samples

The concentrations of 25-OHD3 and 25-OHD2 were determined by NIST using their LC–MS/MS reference measurement procedure (RMP) [1]. A non-RMP, but similar method was employed for 3-epi-25OHD3 determination. The concentrations of 24,25(OH)2D3 were determined by LC–MS/MS using the method of Kaufmann et al. [4].

Spiking of pools with 3-epi-25-OHD3, 24S,25(OH)2D3 and 24R,25(OH)2D3

Synthetic vitamin D metabolites: 3-epi-25-OHD3, 24S,25(OH)2D3 and 24R,25(OH)2D3 were obtained from Sigma–Aldrich (Gillingham, Dorset, UK) and used without further purification. The

Results

3-epi-25-OHD3 and 24,25(OH)2D3 both showed strong positive correlations with 25-OHD3 (Fig. 1, Fig. 2) with the regression lines crossing the 25-OHD axis at approximately 20 and 15 nmol/L respectively.

The Roche total 25-OHD competitive protein binding assay showed a cross reactivity* of 56% with 3-epi-25-OHD3 but no significant interference was observed in any of the immunoassays. In general, HPLC/UV and LC–MS/MS methods did not resolve the 3-epimer from 25-OHD3 (Fig. 3).

In samples spiked with

Discussion

3-epi-25-OHD3 can be present in very high concentrations in neonates of up to one year of age(≈50% of 25-OHD3) and the metabolite is now known to be present in most adult sera [5], [6], [7]. In a study of 216 random clinical samples (211 from patients >20 years of age), mean (SD) 3-epi-25-OHD3 concentration was 3.8 (4.8) nmol/L (range 0.25–59.3 nmol/L); 92% of results were below 7.5 nmol/L [7]. Many routine HPLC/UV and LC–MS/MS assays fail to resolve the 3-epimer from 25-OHD3 in the chromatography

Acknowledgements

We are grateful to our NIST colleagues Carolyn Burdette, Johanna Camara, Lane Sander, Susan Tai and Stephen Wise for the provision of 25-OHD target values, without which DEQAS could not function as an accuracy based quality assessment scheme.

Our thanks to the patients and staff of the Haematology Clinic at Hammersmith Hospital for their invaluable assistance in providing blood donations.

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