The anomalous behaviour of exogenous 25-hydroxyvitamin D in competitive binding assays

doi:10.1016/j.jsbmb.2006.11.007

The Journal of Steroid Biochemistry and Molecular Biology

Volume 103, Issues 3–5, March 2007, Pages 480-482

https://doi.org/10.1016/j.jsbmb.2006.11.007 Get rights and content

Abstract

The Vitamin D International External Quality Assessment Scheme (DEQAS) was established in 1989 to monitor the performance of assays for 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D (I,25(OH)₂D). This is achieved through the quarterly distribution of five samples of human serum. Results are used to calculate an All-Laboratory Trimmed Mean and a Method Mean for each of the methods used by participants. In July 2005, participants were asked to assay serum to which 50.9 nmol of either 25-OHD₃ or 25-OHD₂ had been added as ethanolic solutions. The final concentration of ethanol in the serum was 0.7%. The distribution also included a sample of the original serum (OS) containing 0.7% pure ethanol. The percentage recoveries of exogenous 25-OHD₃ (R1) and 25-OHD₂ (R2) were calculated for each method. Results (OS nM, R1 and R2) were as follows: DiaSorin RIA (n = 53); 39.2, 82.1%, 83.3%, DiaSorin Liason (n = 16); 36.8, 81.4%, 88.6%, IDS RIA (n = 21); 36.4, 54.2%, 29.1%, IDS OCTEIA (n = 16); 47.3, 78.8%, 56.4%, Nichols Advantage (n = 21); 58.9, 46.4%, 43.2%, HPLC (n = 9); 42.6, 112.2%, 97.1%, LC–MS (n = 4); 34.0, 111.5%, 118.1%. The IDS RIA and Nichols assays gave unexpectedly low recoveries. This does not appear to be a calibration problem or the effect of ethanol.

Introduction

Difficulties in measuring Vitamin D metabolites are compounded by frequent misunderstanding among clinicians and even laboratory staff of the terminology used. Assays for 25-OHD are often collectively referred to as ‘Vitamin D assays’ and no distinction made between the metabolites of cholecalciferol (Vitamin D₃) and ergocalciferol (Vitamin D₂). The latter is exclusively used in treating Vitamin D deficiency in the US and widely used elsewhere [1].

Most, but not all, HPLC and LC–MS methods for 25-OHD are capable of separating and measuring 25-hydroxyvitamin D₃ (25-OHD₃) and 25-hydroxyvitamin D₂ (25-OHD₂), whereas most competitive binding assays are designed to measure total 25-OHD. However, all competitive assays are calibrated against 25-OHD₃ and some give falsely low results for 25-OHD₂[2]. For the correct interpretation of 25-OHD assays, it is important that, both the analyst, and the clinician understands the characteristics and limitations of the assay used.

The international Vitamin D External Quality Assessment Scheme (DEQAS) was established in 1989 after several surveys reported poor performance among laboratories measuring 25-OHD [3], [4], [5] and 1,25-dihydroxyvitamin D (1,25(OH)₂D) [4]. The scheme has over 180 participants in 23 countries.

The primary aim of DEQAS is to monitor the performance of individual laboratories. The scheme also provides a unique opportunity to assess and compare the performance characteristics of the methods used by the participants.

Details of how the scheme operates have been given previously [6] and are available on the DEQAS website (http://www.deqas.org/). Briefly, five liquid samples of human serum are distributed quarterly at ambient temperature. Results are analysed by the method of Healey [7] to produce an All-Laboratory Trimmed Mean (ALTM) and standard deviation (S.D.). The accuracy of individual results is measured by calculating the percentage bias from the ALTM, the current target value. The mean and S.D. is also calculated for each of the major methods used by participants. The accuracy of each method can be assessed from the percentage bias of the method mean (MM) from the ALTM ({MM − ALTM}100/ALTM).

Periodically, distributions have been designed to test linearity, specificity and recovery of 25-OHD and 1,25-(OH)₂D methods.

In a recovery experiment performed in July 2005, participants were asked to analyse samples to which known amounts of either 25-OHD₃ or 25-OHD₂ had been added.

Section snippets

Serum pools

Serum was harvested from blood donated by patients undergoing venesection for haemochromatosis or polycythaemia. Its use was approved by the local ethics committee and each patient gave their informed consent. Serum was screened for hepatitis B and C and HIV, stored at −30 °C and pooled before use.

Vitamin D metabolites

25-OHD₃ and 25-OHD₂ were purchased from Sigma (Sigma–Aldrich Company Ltd., Gillingham, UK) and purified by straight phase HPLC (Waters Associates, Milford, MA) using a Hewlett Packard Zorbax-Sil column

Results

Results for the recovery of exogenous 25-OHD are shown in Fig. 1 and should be compared with the results for the sample containing only endogenous 25-OHD (Fig. 2).

Of the competitive binding assays, the IDS radioimmunoassay and Nichols Advantage methods gave very low recoveries for both metabolites. The two DiaSorin assays and the IDS OCTEIA method (for 25-OHD₃) gave higher recoveries (approximately 80%). The chromatographic methods generally gave high recoveries, the exception being HPLC, for

Discussion

Techniques for measuring recovery are inevitably a compromise and spiking serum with lipophilic molecules, such as 25-OHD, poses particular difficulties. To overcome potential solubility problems, the metabolite was added in ethanol; this runs the risk of introducing a matrix problem, which may affect some assays more than others. However, ethanol did not have a significant effect on the results for sample C, which contained only endogenous 25-OHD. Ethanolic solutions of steroids have

References (9)

G.D. Carter et al.
Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation
J. Steroid Biochem. Mol. Biol.
(2004)
B.W. Hollis
Editorial: the determination of circulating 25-hydroxyvitamin D: no easy task
J. Clin. Endocrinol. Metabol.
(2004)
G.D. Carter et al.
How accurate are assays for 25-hydroxyvitamin D? Data from the international Vitamin D external quality assessment scheme
Clin. Chem.
(2004)
G.D. Carter et al.
25-hydroxyvitamin D: results of a national quality assessment
J. Endocrinol.
(1988)

There are more references available in the full text version of this article.

Cited by (71)

Biotin supplementation causes erroneous elevations of results in some commercial serum 25-hydroxyvitamin d (25OHD) assays
2020, Journal of Steroid Biochemistry and Molecular Biology
The Vitamin D External Quality Assessment Scheme (DEQAS) distributes serum samples globally, on a quarterly basis, to assess participants’ performance of specific methods for 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25-(OH)₂D). DEQAS occasionally circulates samples containing high levels of substances found in certain clinical situations e.g. 25-OHD₂, 24,25-(OH)₂D₃, hypertriglyceridemia. The increased availability and use of health supplements containing biotin has led to case reports of assay interference in methods utilizing a biotin-streptavidin detection system. In October 2018, DEQAS included a serum sample (545) containing exogenous biotin (concentration =586 μg/L) which was analyzed by a total of 683 laboratories using 35 different methods. The same serum sample (544) without exogenous biotin was also included in the 5-sample set. All methods (760 laboratories) performed satisfactorily on sample 544 giving an All-Laboratory Trimmed Mean = 50.2 ± 6.5 nmol/L (±SD, CV = 12.9 %). The target value for this sample 544 (& 555) was 47.4 nmol/L as determined by Centers for Disease Control and Prevention (CDC) Atlanta, Georgia using their LC–MS/MS reference method. In contrast, #545 containing the exogenous biotin was reported by only 683 laboratories and gave an All-Laboratory Trimmed Mean = 66.8 ± 37.6 nmol/L (±SD, CV = 56.3 %). As expected, LC-MS/MS methods (143 labs) reported similar results for both 544 = 48.9 ± 4.4 nmol/L (±SD) and 545 = 48.3 ± 4.5 nmol/L (±SD) showing that assays involving chromatographic steps are unaffected by the presence of biotin. Several of the antibody-based assays including Abbott Architect, DiaSorin Liaison, Beckman Unicel and Siemens Centaur are also unaffected by the addition of biotin. Two assays, IDS-iSYS and Roche Total 25OHD, both of which use biotin-streptavidin, exhibit biotin interference yielding values with a significant positive bias for 545 of 102.6 nmol/L ± 78.7 nmol/L (±SD) and 517.8 nmol/L ± 209.8 nmol/L (±SD) respectively. Interestingly, the failure to report sample 545 data from 77 laboratories is due solely to those running Roche Total 25OHD or Roche Vitamin D Total II assays. Given the prevalence of the adversely affected assays (25 % of DEQAS users) and the high volume of 25OHD testing, clinicians using these assays should, where possible, only measure 25OHD when patients are off biotin.
Efficacy of two vitamin D immunoassays to detect 25-OH vitamin D2 and D3
2019, Practical Laboratory Medicine
Assessment of Vitamin D status by measurement of 25-Hydroxyvitamin D (25-OH-D) is widely performed by immunoassay. Yet, the ability of these assays to detect Vitamin D2 (as 25-OH-D2) or Vitamin D3 (as 25-OH-D3) varies. It is important to recognize the ability of an assay to quantitate either form of 25-OH-D to evaluate Vitamin D status of supplemented patients. We evaluated detection of 25-OH-D2 and 25-OH-D3 by two assays in our medical center.
The Abbott Architect i1000 SR 25-OH Vitamin D assay and Roche Cobas 8000 Vitamin D assay were compared for their recovery of 25-OH-D2 or D3 from spiked serum samples. Samples with known endogenous concentrations of 25-OH-D2 or D3 by LC-MS/MS were also measured to calculate bias between our assays and LC-MS/MS.
Recovery of 25-OH-D3 in spiked samples was similar by Architect (84–87%) and Cobas (90%). Recovery of 25-OH-D2 was lower than 25-OH-D3, and was poorer by Architect (37–40%) than by Cobas (69–71%). In measurement of samples with known 25-OH-D concentrations, performance of Architect and Cobas assays was similar for 25-OH-D3. However, at concentrations >50 nmol/L 25-OH-D2, the Architect assay exhibited large average negative bias (−27%).
While the Architect and Cobas assays performed similarly in detection of 25-OH-D3, the Architect assay was significantly poorer at detecting 25-OH-D2 than Cobas, with poorer recovery and significant negative bias at higher concentrations of 25-OH-D2. This agrees with other studies, and indicates that caution should be used in interpreting Architect 25-OH-D results in patients supplemented with Vitamin D2.
External Quality Assessment of 24,25-dihydroxyvitamin D <inf>3</inf> (24,25(OH) <inf>2</inf> D <inf>3</inf> ) assays
2019, Journal of Steroid Biochemistry and Molecular Biology
The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)₂D₃. Several studies have also suggested that a high 25-hydroxyvitamin D₃(25-OHD₃):24,25(OH)₂D₃ ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)₂D₃ is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with ‘target values’ assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)₂D₃ was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)₂D₃ RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.
Methods for assessment of vitamin D
2018, Laboratory Assessment of Vitamin Status
The vitamins D belong to a group of antirachitic compounds of which the most important in humans is cholecalciferol (vitamin D₃), formed by the action of sunlight on the skin. Ergocalciferol (vitamin D₂), a structurally similar compound, is found in invertebrates, plants, and fungi and is sometimes used as a supplement in vitamin D deficiency. Both molecules are biologically inactive and are activated by two hydroxylation steps, the first in the liver to produce 25-hydroxyvitamin D₃ (25-OHD₃) and D2 (25-OHD₂), the second in the kidneys and other tissues to produce the bioactive form 1,25-dihdroxyvitamin D₃ (1,25(OH)₂D₃) and D₂ (1,25(OH)₂D₂). Serum total 25-OHD (the sum of 25-OHD₃ and 25-OHD₂) is universally used in the laboratory assessment of vitamin D status. Increasing workloads have led many laboratories to use commercial automated nonextraction ligand-binding assays but results from these methods can be affected by the sample matrix or presence of other hydroxylated metabolites. More rigorous liquid chromatography-tandem mass spectrometry methods are increasingly used in specialist laboratories and are the basis of Reference Measurement Procedures against which many of the commercially available immunoassays are now calibrated.
25-Hydroxyvitamin D Assays: Standardization Guidelines, Problems, and Interpretation
2018, Vitamin D: Fourth Edition
Widespread variation in assay measurement of total 25-hydroxyvitamin D (25(OH)D) confounds international efforts to develop evidence-based clinical and public health guidelines. This chapter's subject is the Vitamin D Standardization Program (VDSP), its tools, and methods for measuring 25(OH)D in both commercially and laboratory developed assays worldwide. Two distinct VDSP protocols exist for standardizing 25(OH)D measurements: one for current/future measurements, i.e., Prospective Standardization and another for Retrospective Standardization of 25(OH)D measurements from past studies; both are described in detail. Special attention is given to the role that accuracy-based Performance Testing/External Quality Assessment schemes play in standardizing measurements in routine laboratories. The steps involved in retrospective standardization and its potential impact on interpretation of vitamin D research data are discussed in detail. Given the impact of assay standardization on research data interpretation, publication of meta-analyses based on unstandardized 25(OH)D data should be suspended. As discussed, both prospective and retrospective standardization are the way forward to developing consensus vitamin D guidelines.
Toward Clarity in Clinical Vitamin D Status Assessment: 25(OH)D Assay Standardization
2017, Endocrinology and Metabolism Clinics of North America

View all citing articles on Scopus

View full text

The anomalous behaviour of exogenous 25-hydroxyvitamin D in competitive binding assays

Abstract

Introduction

Section snippets

Serum pools

Vitamin D metabolites

Results

Discussion

J. Steroid Biochem. Mol. Biol.

Editorial: the determination of circulating 25-hydroxyvitamin D: no easy task

J. Clin. Endocrinol. Metabol.

How accurate are assays for 25-hydroxyvitamin D? Data from the international Vitamin D external quality assessment scheme

Clin. Chem.

25-hydroxyvitamin D: results of a national quality assessment

J. Endocrinol.