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Assay Variation Confounds the Diagnosis of Hypovitaminosis D: A Call for Standardization

Department of Medicine Osteoporosis Clinical Center and Research Program (N.B., D.K., C.S.C., K.E.H., M.K.D.); Institute on Aging (N.B., D.K., C.S.C., K.E.H., M.K.D.); Geriatric Research Education and Clinical Center, William S. Middleton Veterans Affairs Medical Center (M.K.D.); and Department of Biochemistry (L.P., E.L., H.F.D.), University of Wisconsin-Madison, Madison, Wisconsin 53705

Address all correspondence and requests for reprints to: Neil Binkley, M.D., 2870 University Avenue, Suite 100, Madison, Wisconsin 53705. E-mail: nbinkley{at}facstaff.wisc.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Endemic hypovitaminosis D contributes to osteoporosis development. However, variation in 25-hydroxyvitamin D (25OHD) measurement is reported and confounds the diagnosis of vitamin D insufficiency/deficiency. This report emphasizes the marked variability observed in serum 25OHD measurements between laboratories.

Initially, postmenopausal women had serum 25OHD determinations: 42 in laboratory A, 20 in laboratory B. Their mean (SEM) serum 25OHD concentrations were 46 (2.1) and 21 (2.3) ng/ml in laboratories A and B, respectively. Furthermore, there was little overlap in serum 25OHD among these clinically similar individuals. Specifically, 17% of those measured in laboratory A but 90% in laboratory B were below an arbitrary threshold value of 32 ng/ml.

Subsequently, serum was obtained from 10 healthy adults. Two aliquots from each individual, one of which was spiked with 20 ng/ml 25OHD, were sent to six laboratories. Substantial variability was noted between these six laboratories. The mean serum 25OHD concentration ranged from 17.1–35.6 ng/ml. Similarly, the mean increase produced by spiking with 20 ng/ml ranged from 7.7–18.0 ng/ml.

In conclusion, 25OHD assays yield markedly differing results; whether an individual is found to have low or normal vitamin D status is a function of the laboratory used. If the medical community is to make progress in correcting widespread hypovitaminosis D, 25OHD measurement must be standardized.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
HYPOVITAMINOSIS D is common due to low dietary vitamin D intake coupled with sun avoidance or minimal exposure (1, 2, 3). As a result, vitamin D inadequacy is a relatively recently recognized, but common, cause of secondary hyperparathyroidism with consequent bone loss and osteoporosis (4, 5). Furthermore, vitamin D insufficiency is associated with muscle weakness, and its correction reduces the risk for falls (6). Consequently, simply correcting this vitamin inadequacy should lead to improved skeletal health. Consistent with this, provision of supplemental vitamin D (7) or vitamin D plus calcium (8, 9) reduces fracture risk. Moreover, evidence is accumulating that vitamin D inadequacy may have other adverse health consequences, including increased risk of malignancies, hypertension, and diabetes mellitus (10, 11). Not surprisingly, much work has focused on defining the optimal circulating concentration of 25-hydroxyvitamin D (25OHD) and determination of the required intake to achieve this concentration. In this regard, expert opinion suggests that this serum concentration is approximately 80 nmol/liter (32 ng/ml) (12, 13). Thus, it is often recommended that clinicians strive to maintain 25OHD concentrations above 32 ng/ml in their patients to maximize bone health. Although previously noted (14, 15, 16, 17), it is perhaps less widely appreciated that the assays used to measure 25OHD may yield discrepant results. This study highlights variability in 25OHD measurements obtained using current assays in different laboratories.

	Subjects and Methods

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Initial observation

Fifty-nine postmenopausal women were screened for potential participation in two osteoporosis clinical trials. A serum specimen from each woman was collected for 25OHD measurement; 42 samples were sent to laboratory A, and 20 were sent to laboratory B for analysis. Because three volunteers did not meet bone density criteria for the first study, they were subsequently screened for the second study.

Subsequent evaluation in healthy adults

Venous blood was obtained on April 8, 2003, from 10 healthy adult research staff and clinicians living in Madison, Wisconsin (43° north latitude). Serum aliquots were prepared, and 20 ng/ml 25OHD (Amersham Searle, Arlington Heights, IL) was added to half of the aliquots (spiked specimen) from each volunteer. All aliquots were promptly frozen and maintained at –80 C until shipped for analysis. Spiked and unspiked aliquots from all individuals were sent to two clinical reference laboratories (C and D), two national central research laboratories (F and G), and two academic research laboratories (E and H) for 25OHD measurement. All laboratories were blinded to sample status, i.e. basal or spiked. Additionally, a local clinical laboratory measured serum calcium using a Cobas Integra 800 Autoanalyzer (Roche, Indianapolis, IN) and intact PTH with an immunochemiluminometric assay.

These studies were reviewed and approved by the University of Wisconsin Health Sciences Human Subjects Committee. Informed consent was obtained from all participants before any study-related procedure.

Vitamin D assay methodology

Table 1 lists the methods and normal range for 25OHD measurement in these laboratories.

For the purpose of analysis, HPLC measurement of 25OHD (laboratory H) was established as the gold standard to which the results from other laboratories/assays were compared. Serum 25OHD measured in laboratories C–H was compared in basal and spiked specimens using factorial ANOVA with subsequent Fisher’s protected least significant difference testing using Statview software (Abacus Concepts, Cary, NC). Additionally, results of laboratories C–G were compared with laboratory H using Bland-Altman plots and Passing/Bablok regression analysis using Analyse-it software (Analyse-it Software Ltd., Leeds, UK).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Demographics

Initial observation group. All of the subjects in both groups were Caucasian, postmenopausal women. Overall, their mean age was 65.0 ± 1.1 (SEM) yr (range, 49–86 yr). Osteoporosis was present in all of these women based on bone mineral density measurement at the lumbar spine and/or proximal femur using dual-energy x-ray absorptiometry. Their mean T score (lowest of the L1–L4 spine, total femur, femoral neck, or trochanter) was –2.7 ± 0.1. No differences were present in age, weight, or bone density between groups (Table 2).

25OHD concentration/postmenopausal women

Despite the subjects in groups A and B being of similar age, geographic residence, and bone mass, there was almost no overlap in measured serum 25OHD concentration at these two national central laboratories (Fig. 1A). Additionally, if optimal serum 25OHD concentration is defined as being greater than 32 ng/ml, 90% of these women were insufficient at laboratory B, but only 17% in laboratory A. Furthermore, serum 25OHD concentration was measured in three women at both laboratories A and B with markedly differing results (Fig. 1B).

View larger version (26K): [in this window] [in a new window]   FIG. 1. Initial observation. A, Although these postmenopausal women were clinically similar, there was almost no overlap in measured serum 25OHD concentration at reference laboratories. Additionally, if optimal serum 25OHD concentration is defined as being greater than 32 ng/ml (dashed line), 90% of these women were insufficient at laboratory B, but only 17% were insufficient in laboratory A. B, Three women were measured in both laboratories A and B with markedly differing results.

In these 10 healthy adults, the mean serum 25OHD varied widely from 17.1 (4.6) to 35.6 (5.2) ng/ml (P < 0.005) between laboratories (Fig. 2A). Similarly, for a given subject, the 25OHD result was substantially different depending on the laboratory used (Fig. 2B). Interestingly, laboratories C and D are the sites to which clinical specimens are routinely sent in our area. As evident from these observations, whether a patient is considered as being vitamin D insufficient (using 32 ng/ml as a threshold) depends, in large measure, on the laboratory used. For example, subjects 1, 4, 6, 7, and 9 (Fig. 2B) would be classified as insufficient in some laboratories and normal in others. Thus, 50% of these individuals might be classified as having a different vitamin D status (insufficient or normal) depending solely on the laboratory used.

View larger version (31K): [in this window] [in a new window]   FIG. 2. Laboratory personnel basal serum 25OHD concentration. A, In these 10 individuals, the mean serum 25OHD varied widely [from 17.1 (4.6) to 35.6 (5.2) ng/ml; P < 0.005] between laboratories (error bars represent SEM). B, Similarly, marked within-individual variation was observed in 25OHD measurement in different laboratories. Whether an individual has hypovitaminosis D (arbitrary threshold, 32 ng/ml shown as dashed line) depends on which laboratory was used. Note that laboratory H used HPLC and was considered to be the "gold standard" against which others were compared.

In the spiked specimens, the mean serum 25OHD also varied (P < 0.05) between laboratories from 27.4 (4.5) to 44.6 (6.1) ng/ml (Fig. 3). Furthermore, the increase observed upon spiking with 20 ng/ml 25OHD was less than anticipated, e.g. from 7.7–18.0 ng/ml with the greatest increment detected by HPLC (Fig. 3).

View larger version (42K): [in this window] [in a new window]   FIG. 3. Laboratory personnel effect of spiking with 25OHD on the measured concentration. In the spiked specimens, the mean serum 25OHD varied (P < 0.05) between laboratories from 27.4 (4.5) to 44.6 (6.1) ng/ml. Furthermore, the increment was less than anticipated and varied between laboratories (P = 0.005) from 7.7–18 ng/ml, with the greatest increment detected by HPLC. The solid portion of the bars depicts mean basal 25OHD concentration, and the hatched portion illustrates the measured increase from spiking with 20 ng/ml 25OHD. Error bars represent SEM.

When compared with HPLC, all laboratories (with the exception of laboratory E) demonstrated a highly significant correlation when evaluated by Bablock regression analysis (Fig. 4). However, significant positive bias was observed in the basal samples measured in laboratories D, F, and G (Table 3 and Fig. 4). In the spiked specimens, significant positive bias was observed only in laboratory D (Table 3 and Fig. 4).

View larger version (30K): [in this window] [in a new window]   FIG. 4. Comparison with HPLC. Significant bias was observed in 25OHD measurement in laboratories D, F, and G compared with HPLC. This positive bias ranged from 9.8–18.5 ng/ml. Positive bias was present only in laboratory D in the spiked samples. Overall excellent correlation without significant bias was demonstrated in laboratory C.

View larger version (26K): [in this window] [in a new window]   FIG. 4A. Continued.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Vitamin D deficiency classically causes impairment of skeletal mineralization, resulting in rickets and osteomalacia in children or osteomalacia alone in adults. However, less severe degrees of vitamin D insufficiency contribute to bone loss without necessarily impairing bone mineralization. Specifically, vitamin D inadequacy leads to calcium malabsorption with resultant secondary hyperparathyroidism leading, over time, to bone loss and osteoporosis (5). In this context, optimal vitamin D status is defined as the serum 25OHD concentration above which PTH is not further suppressed. Applying this definition, hypovitaminosis D is extremely common due to low dietary intake and minimal cutaneous sunlight exposure (4, 13, 20, 21, 22, 23, 24). Additionally, vitamin D supplementation increases calcium absorption, improves bone density, reduces risk of falls, and decreases the likelihood of osteoporotic fracture (6, 8, 25, 26, 27, 28). Finally, nonskeletal adverse effects related to inadequate vitamin D status, including such diverse consequences as hypertension, diabetes mellitus, cancer, and autoimmune disease, are receiving increased attention (29, 30, 31, 32). As a result, there is an appropriately increasing emphasis on the measurement of 25OHD in the clinic and subsequent supplementation to maintain or enhance health status by attaining an optimal vitamin D status. In this regard, Chapuy et al. (13) and Heaney (12) have suggested that the optimal 25OHD serum concentration is above approximately 31–32 ng/ml (78–80 nmol/liter). However, our data indicate that widespread clinical application of a single target value, e.g. 32 ng/ml, is not appropriate at this time due to marked interlaboratory variability. Thus, practicing clinicians do not have a reliable tool to detect hypovitaminosis D in their patients.

It is not surprising that discordant results are obtained because these laboratories use substantially different methodology for measurement of 25OHD, which, to this time, has not been standardized. For example, the chemiluminescent assay is, in essence, a protein binding assay that uses a reagent to separate vitamin D from its binding proteins without preparative chromatography (33). Thus, this procedure has similarities to protein binding assays that have been used for some time (34, 35, 36) and usually (14, 15, 37), but not always (17, 38), produce higher 25OHD results than other methodologies, perhaps reflecting the absence of a prior chromatographic separation (39). It is important to recognize that the competitive protein binding assays may use different sources of vitamin D binding proteins and modify the extraction or preliminary purification procedures and/or incubation conditions (33, 40). Thus, not all competitive protein binding assays are identical. Subsequently, RIA methodology using antibodies was developed (41) and documented to compare very well with an HPLC gold standard (42, 43). However, it is important to recognize that not all RIA assays are the same; some antibodies recognize both 25OHD₂ and 25OHD₃, whereas others underestimate 25OHD₂ (16). Furthermore, the fact that an RIA, which has previously been reported to correlate well with HPLC, performed variably in this study likely indicates that this procedure is user-dependent and requires rigorous quality control to ensure reliable results. Finally, HPLC has long been recognized as the gold standard (44, 45, 46); however, despite recent advances to improve its clinical applicability (47), it is not widely available to clinicians.

We recognize that assay standardization is not a simple or inexpensive endeavor (48). However, given what appears to be an epidemic of hypovitaminosis D, the simplicity and low cost of its correction, and the potential beneficial skeletal and nonskeletal consequences of doing so, it is essential that the medical community define a threshold for optimal vitamin D status using accurate, reproducible assays. These assays must subsequently be internationally standardized and made available to practicing clinicians.

Limitations of this study include the small sample size and the fact that not every available methodology for 25OHD measurement was evaluated. However, these limitations do not negate the conclusion that widespread clinical application of a single threshold value to define optimal vitamin D status is impossible at this time.

In conclusion, marked between-laboratory variability exists in serum 25OHD measurement. As a result of this variability, an arbitrary definition of optimal serum 25OHD concentration is neither possible nor widely applicable. International standardization of 25OHD measurement is essential before elimination of endemic hypovitaminosis D is achievable. Given increased recognition of this situation by the National Institutes of Health (49), such standardization, perhaps through agencies such as the National Institute of Standards and Technology, seems attainable in the near future. In the interim, it is prudent that clinicians recommend routine vitamin D supplementation but, because of the difficulties inherent in measuring 25OHD, refrain from using a single 25OHD value to define optimal vitamin D status. Furthermore, the amount of supplemental vitamin D required remains controversial and likely varies with several factors, including age and severity of extant vitamin D deficiency. Although the recommended daily intake remains at 400–600 IU in the United States, recent data and, consequently, expert opinion suggest that substantially more (1000 IU) may be required. Ideally, these higher doses should be monitored using an accurate 25OHD assay. Based on our study and those of others, clinicians should consider using RIA for 25OHD measurement to assess vitamin D status and response to supplementation until other methodologies are further validated.

	Footnotes

 
Abbreviation: 25OHD, 25-Hydroxyvitamin D.

Received November 14, 2003.

Accepted February 24, 2004.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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				J. A. Schmidt Measurement of 25-Hydroxyvitamin D Revisited Clin. Chem., December 1, 2006; 52(12): 2304 - 2305. [Full Text] [PDF]

				G. Lensmeyer, D. Wiebe, N. Binkley, and M. Drezner The authors of the article cited above respond: Clin. Chem., December 1, 2006; 52(12): 2305 - 2306. [Full Text] [PDF]

				R. J. Singh, R. L. Taylor, G. S. Reddy, and S. K. G. Grebe C-3 Epimers Can Account for a Significant Proportion of Total Circulating 25-Hydroxyvitamin D in Infants, Complicating Accurate Measurement and Interpretation of Vitamin D Status J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 3055 - 3061. [Abstract] [Full Text] [PDF]

				M. F. Holick, N. Binkley, E. Chen, and A. E. de Papp Authors' Response: 25-OH-Vitamin D Assays J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6338 - 6338. [Full Text] [PDF]

				E. E. Fradinger Letter re: 25-OH-Vitamin D Assays J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6337 - 6338. [Full Text] [PDF]

				P. G. Masse, J. Dosy, J.-L. Jougleux, M. Caissie, and D. S. Howell Bone Mineral Density and Metabolism at an Early Stage of Menopause When Estrogen and Calcium Supplement Are Not Used and without the Interference of Major Confounding Variables J. Am. Coll. Nutr., October 1, 2005; 24(5): 354 - 360. [Abstract] [Full Text] [PDF]

				S. Gaugris, R.P. Heaney, S. Boonen, H. Kurth, J.D. Bentkover, and S.S. Sen Vitamin D inadequacy among post-menopausal women: a systematic review QJM, September 1, 2005; 98(9): 667 - 676. [Abstract] [Full Text] [PDF]

				Z. Maunsell, D. J. Wright, and S. J. Rainbow Routine Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Measurement of the 25-Hydroxy Metabolites of Vitamins D2 and D3 Clin. Chem., September 1, 2005; 51(9): 1683 - 1690. [Abstract] [Full Text] [PDF]

				D. M. Antoniucci, E. Vittinghoff, T. Blackwell, D. M. Black, and D. E. Sellmeyer Vitamin D Insufficiency Does Not Affect Bone Mineral Density Response to Raloxifene J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4566 - 4572. [Abstract] [Full Text] [PDF]

				M. J. Favus Postmenopausal Osteoporosis and the Detection of So-Called Secondary Causes of Low Bone Density J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3800 - 3801. [Full Text] [PDF]

				M. F. Holick, E. S. Siris, N. Binkley, M. K. Beard, A. Khan, J. T. Katzer, R. A. Petruschke, E. Chen, and A. E. de Papp Prevalence of Vitamin D Inadequacy among Postmenopausal North American Women Receiving Osteoporosis Therapy J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3215 - 3224. [Abstract] [Full Text] [PDF]

				P. Leventis, L. Garrison, M. Sibley, P. Peterson, M. Egerton, G. Levin, and P. Kiely Underestimation of Serum 25-Hydroxyvitamin D by the Nichols Advantage Assay in Patients Receiving Vitamin D Replacement Therapy Clin. Chem., June 1, 2005; 51(6): 1072 - 1074. [Full Text] [PDF]

P. Glendenning and W. D. Fraser 25-OH-Vitamin D Assays J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3129 - 3129.