ORIGINAL
ARTICLE
25-Hydroxyvitamin D Can Interfere With a Common Assay for 1,25-Dihydroxyvitamin D in Vitamin D Intoxication Colin P. Hawkes, Sarah Schnellbacher, Ravinder J. Singh, and Michael A. Levine Division of Endocrinology and Diabetes (C.P.H., S.S., M.A.L.), The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; National Children’s Research Centre (C.P.H.), Dublin, Ireland; Department of Laboratory Medicine and Pathology (R.J.S.), The Mayo Clinic, Rochester, Minnesota 55905; and Department of Pediatrics (M.A.L.), Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Context: Vitamin D intoxication is characterized by elevated serum 25-hydroxyvitamin D (25(OH)D) and suppressed serum 1,25-dihydroxvitamin D (1,25(OH)2D). We evaluated two adolescents with hypercalcemia due to vitamin D intoxication; both had elevated serum 1,25(OH)2D by Diasorin RIA, but normal serum 1,25(OH)2D concentrations by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Objective: This study aimed to determine the effect of 25(OH)D2 and 25(OH)D3 on 1,25(OH)2D concentration determined using RIA and LC-MS/MS. Methods: Pools of normal serum and an artificial serum matrix were prepared and aliquots were spiked with ⬎99% pure 25(OH)D2 or 25(OH)D3 (50 –700 ng/mL). Samples were maintained at 4°C or heated to 56°C, and the concentrations of vitamin D metabolites were measured by LC-MS/MS and Diasorin RIA. Results: Median 1,25(OH)2D increased by 114% with RIA and 21% with LC-MS/MS with addition of 100 ng/mL 25(OH)D3, and 349% (RIA) and 117% (LC-MS/MS) with 700 ng/mL of 25(OH)D3. Each 1-ng/mL increase in 25(OH)D3 increased 1,25(OH)2D by 0.231 pg/mL (RIA) and 0.121 pg/mL (LCMS/MS). Spiking with 25(OH)D2 led to similar changes. Heat inactivation of serum, and using an artificial serum matrix, were associated with similar effects of 25(OH)D on 1,25(OH)2D assays. Conclusions: Vitamin D intoxication with high serum levels of 25(OH)D2 or 25(OH)D3 can be associated with elevated levels of 1,25(OH)2D due to interference in a commonly used RIA. A similar but attenuated effect also occurs when 1,25(OH)2D is measured using LC-MS/MS but does not seem to be clinically significant. The basis for this effect on the LC-MS/MS assay is presently uncertain. (J Clin Endocrinol Metab 100: 2883–2889, 2015)
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itamin D insufficiency has emerged as a worldwide public health problem (1, 2), particularly due to the high risk to the developing and growing skeleton during early infancy (3, 4) and adolescence (5). In response to these concerns, the American Academy of Pediatrics updated their guidelines in 2008 to recommend that all breastfed infants and all infants consuming less than 1 L
per day of vitamin D-fortified milk or formula receive 400 IU of supplemental vitamin D per day (6). More recently, the Institute of Medicine issued new dietary reference intakes for vitamin D that extended the American Academy of Pediatrics recommendations to recommend a daily intake of 600 IU of vitamin D per day for individuals greater than 1 year of age, and for 800 IU of vitamin D per day for
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA Copyright © 2015 by the Endocrine Society Received May 6, 2015. Accepted June 22, 2015. First Published Online June 29, 2015
Abbreviations: 1,25(OH)2D 1,25-dihydroxvitamin D; 25(OH)D 25 hydroxyvitamin D; D2 Ergocalcifierol; D3 Cholecalciferol; LC-MS/MS, liquid chromatography–tandem mass spectrometry; VDR, vitamin D receptor.
doi: 10.1210/jc.2015-2206
J Clin Endocrinol Metab, August 2015, 100(8):2883–2889
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individuals greater than 70 years of age (7). The revised dietary intake guidelines, in the context of continuing controversy regarding optimal plasma levels for 25(OH)D (8, 9), has resulted in increased interest in vitamin D intake and supplementation. Parental adherence with these guidelines of up to 75% has been reported (10), but adherence among adolescents remains a challenge (11). Moreover, easy access to high-potency preparations of vitamin D and growing but unsubstantiated claims regarding nonskeletal benefits of this vitamin (12) have encouraged higher intakes of vitamin D, with a consequent increased risk for inadvertent vitamin D intoxication. Clinically, this can result in hypercalcemia and hyperphosphatemia, calcifications, nephrocalcinosis, and lifethreatening renal failure (13–16). Vitamin D intoxication reflects a critical disturbance in normal vitamin D homeostasis, which involves a complex interplay of diet, sunlight exposure, and genetics (17). To become fully active, vitamin D, produced in the skin as cholecalciferol (vitamin D3) after exposure to UV light, or supplied in the diet as cholecalciferol or the related yeastand fungi-derived sterol ergocalciferol (vitamin D2) (18), must undergo two modifications by cytochrome P450 enzymes. The first step is 25-hydroxylation of parent vitamin D2 and D3 by hepatic microsomal CYP2R1, which generates the prohormones 25-hydroxyvitamin D3 (25(OH)D3) and 25-hydroxyvitamin D2 (25(OH)D2), respectively (19). A second hydroxylation occurs in the kidney where CYP27B1, a 1␣-hydroxylase located in the mitochondrion, converts 25(OH)D to the fully active form of the vitamin, 1,25-dihydroxyvitamin D (1,25(OH)2D). 1,25(OH)2D is a potent hormone, and binds with high affinity to the vitamin D receptor (VDR), which mediates most physiological actions of vitamin D via modulation of the transcription of target genes. The mechanism for hypercalcemia and hypercalciuria in vitamin D intoxication is thought to be activation of the VDR by 25(OH)D, which is markedly elevated, given that conversion of vitamin D to 25(OH)D by CYP2R1 seems to be substrate dependent and not highly regulated (20). By contrast, the conversion of 25(OH)D to 1,25(OH)2D by CYP27B1 is tightly regulated by calcium, PTH, and fibroblast growth factor 23. Hence, as serum levels of 25(OH)D increase excessively there is a parallel decrease in levels of serum 1,25(OH)2D, a phenomenon that reflects suppression of PTH by hypercalcemia and a corresponding loss of PTHdependent stimulation of CYP27B1 activity (17). Definitive proof that the cause of hypercalcemia in vitamin D intoxication is 25(OH)D rather than 1,25(OH)2D comes from studies by DeLuca et al (21), who showed that intoxication with vitamin D3 occurs equally well in the Cyp27b1 knockout mouse, which completely lacks 1␣-hydroxylase activity, as it does in the wild-type mouse. Thus, we were recently
surprised during the evaluation of two adolescents with vitamin D intoxication when we discovered that serum levels of 25(OH)D and 1,25(OH)2D were both markedly elevated. In this paper we characterize the false elevation of 1,25(OH)2D in vitamin D intoxication, and propose a possible mechanism.
Materials and Methods Patients We performed a retrospective chart review of two adolescents who presented to the Children’s Hospital of Philadelphia with vitamin D intoxication and hypercalcemia. We collected medical and demographic data from each subject, with particular emphasis on biochemical parameters of bone and mineral metabolism, and molecular analysis of the CYP24A1 gene was performed. This study was performed in accordance with the policies and procedures of the institutional review board of the Children’s Hospital of Philadelphia.
Biochemical and molecular studies We extracted genomic DNA from peripheral blood mononuclear cells using standard methods. All exons, exon-intron junctions, and the promoter of CYP24A1 (11p15.2) were amplified and sequenced by PCR using previously described primers and conditions (22). We performed a series of in vitro experiments to determine the effect of analytical grade (purity ⱖ 99%) 25(OH)D2 (Santa Cruz Biotechnology; Ref sc231277) and 25(OH)D3 (Santa Cruz Biotechnology; Ref sc288574) on the concentration of 1,25(OH)2D measured by RIA or LC-MS/MS. We collected deidentified excess serum that was to be discarded from the Clinical Chemistry laboratory and created separate serum pools. The serum samples were from a random set of inpatients and outpatients at the Children’s Hospital of Philadelphia and represented an unbiased spectrum of clinical diagnoses. Blood samples had been drawn into Vacutainer tubes (Becton Dickinson), either red top or serum separator tube. Once samples had arrived at the Clinical Chemistry laboratory the samples had been allowed to stand at room temperature for 30 minutes so serum tubes could clot, followed by centrifugation for 10 minutes at 2095⫻ g. Samples had been refrigerated (2– 8°C) for 2–3 days and then were frozen (⫺70°C) until used. Separate aliquots from each pool were spiked with the addition of either vehicle (final, 0.25% DMSO) or 25(OH)D2 or 25(OH)D3 at the following concentration: 0 ng/mL (control), 50, 100, 150, 250, and 750 ng/mL. Samples were maintained at 4°C or heat inactivated at 56°C for 30 minutes. Heat inactivation was performed to determine whether an observed increase was due to in vitro enzyme-mediated hydroxylation. We followed a similar protocol using aliquots of an artificial serum matrix that consisted of PBS, pH 7.4, containing BSA (4 g/dL). For each sample, we determined the concentration of 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, and 1,25(OH)2D3 by LC-MS/MS using an API 4000 and API 5000 instruments respectively (Applied Biosystems). Molecular-ion-specific fragments and transitions used for 1,25(OH)2D3 were 574.37 and 592.37 ⬎ 314.12 and for 1,25(OH)2D2 was 586.37 ⬎ 314.13 were measured, as previously published (23). We determined the
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concentration of total 1,25(OH)2D by RIA (DiaSorin); this assay is a competitive antibody RIA that involves a preliminary extraction and subsequent purification of vitamin D metabolites from serum or EDTA plasma using C18OH cartridges (24). The RIA method is based on a polyclonal antibody that is specific for both 1,25(OH)2D2 and 1,25(OH)2D3. According to the manufacturer’s product manual, a concentration of 25(OH)D3 greater than 1 mg/mL is required to achieve a 50% B/Bo in this assay, with less than 0.01% cross reactivity.
Statistical analysis Data were analyzed using SPSS 22 (IBM), and figures were generated using Prism 5 (GraphPad Software Inc.) and Adobe Illustrator 16.0 (Adobe Systems, Inc.). Linear regression analysis was performed to determine the influence of 25(OH)D2 or 25(OH)D3 on measured total 1,25(OH)2D by LC-MS/MS or RIA. A separate linear regression analysis was performed to determine the relative increase in measured 1,25(OH)2D by RIA and LC-MS/MS following the addition of either 25(OH)D2 or 25(OH)D3.
Results Case 1 A 15-year-old, previously healthy male presented with a 2-week history of postprandial vomiting, abdominal pain, and polyuria. He had taken a large dose of cholecalciferol numerous times daily over the previous month, but it was not possible to calculate the total intake. Initial laboratory evaluation revealed hypercalcemia, normal phosphorous, and suppressed PTH (Table 1) with a markedly elevated serum concentration of 25(OH)D3 (685 ng/mL by LC-MS/MS). At the same time, he had an elevated serum concentration of total 1,25(OH)2D (⬎230 pg/mL) as determined by RIA (ARUP Laboratories). He had an extensive evaluation to determine the basis for the combined elevation of 25(OH)D3 and 1,25(OH)2D, Table 1.
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which excluded granulomatous disorders and malignancy; the sequence of his CYP24A1 gene was normal. An aliquot of the serum sample that showed an elevated concentration of 1,25(OH)2D by RIA was subsequently retrieved and when analyzed by LC-MS/MS showed a normal concentration of 1,25(OH)2D of 45 pg/mL (ref, 25– 86 pg/mL). He was initially treated with saline hydration, calcitonin, and prednisone with no improvement. He received a single dose of zoledronic acid (0.025 mg/kg) on day 4 of admission, and his serum calcium rapidly declined to normal with a nadir of 7.2 mg/dL 3 days later, and his serum creatinine returned to normal. Three months after discharge from hospital his serum calcium concentration remained normal, and his serum 25(OH)D3 remained elevated at 171.6 ng/mL. Case 2 A 17-year-old female with a background of ocular albinism and autism presented with a history of weight loss and hypercalcemia. She denied a history of excessive ingestion of vitamin D or calcium. Her biochemical studies showed hypercalcemia, normal serum level of phosphorus, and suppressed PTH (Table 1). Measurement of vitamin D metabolites showed elevated serum concentrations of 25(OH)D3 (143 ng/mL) and 1,25(OH)2D (⬎190 pg/mL by RIA). Reanalysis of the serum by LC-MS/MS showed a normal concentration of 1,25(OH)2D of 68 pg/ mL. Her serum concentration of 24,25(OH)2D3 was normal at 17.1 ng/mL (Heartland Assays), and analysis of the CYP24A1 gene was normal. The serum calcium normalized after dietary intervention to reduce vitamin D intake. Vitamin D experiments Concentrations of 25(OH)D2 or 25(OH)D3 increased as expected based on the amount of vitamin D metabolite
Clinical Characteristics and Laboratory Findings in Two Adolescents Presenting With 25(OH)D Intoxication
Clinical Characteristic Demographics Background Diagnoses Calcium, mg/dL Phosphorous, mg/dL Albumin, g/dL PTH, pg/mL Creatinine, mg/dL PTHrP, pmol/L CYP24A1 gene 25(OH)D3, ng/mL LC-MS/MS 1,25(OH)D3, pg/mL RIA 1,25(OH)D3, pg/mL LC-MS/MS
Reference Range
Case 1
Case 2
15-year-old male Well child 13.8 4.2 4.3 ⬍3 3.1 ⬍2 Normal
17-year-old female Ocular albinism, autism 11.3 4 4.1 ⬍5.5 0.48 ⬍2 Normal
30 – 80
685
143
15–75
⬎230
⬎190
25– 86
45
68
8.8 –10.1 2.9 –5.4 3.7–5.6 9 –52 0.6 –1.3 ⬍2
Abbreviation: CYP, cytochrome P450.
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is measured in nanograms per milliliter whereas 1,25(OH)2D is 10001,25(OH) D RIA 1,25(OH) D LC-MS/MS fold lower concentrations and is 25(OH)D measured in picograms per milliliter. When adjusted for these different concentrations, there is 0.013% (RIA) or 0.011% (LC-MS/MS) cross reactivity of 25(OH)D2 with 1,25(OH)2D, and 0.023% (RIA) or Concentration of 25(OH)D2 Added, ng/ml 0.012% (LC-MS/MS) cross reactivity of 25(OH)D3 with 1,25(OH)2D. Heat Inactivated (56 C) Artificial Serum Matrix B Standard Temperature (4 C) These cross reactivities are greater 1,25(OH) D RIA than the ⬍ 0.01% cross reactivity de1,25(OH) D LC-MS/MS scribed by Diasorin for their RIA. 25(OH)D To investigate whether the increase in measured 1,25(OH)2D that we observed in vitro might be due to a previously unidentified biological or chemical conversion of 25(OH)D to 1,25(OH)2D, we repeated the Concentration of 25(OH)D3 Added, ng/ml spiking experiments under two adFigure 1. The concentrations of 1,25(OH)2D measured by RIA and LC-MS/MS in response to ditional conditions. We heated seincreasing concentrations of 25(OH)D2 (A) or 25(OH)D3 (B) at 4°C, 56°C, and using a serum rum to 56°C to inactivate potential matrix. Mean and ranges of measured 1,25(OH)2D from 3 pools for each concentration of enzymes that might be present in se25(OH)D2 or 25(OH)D3 are shown. rum, and used an artificial serum matrix to exclude both biological and added to pooled serum samples or artificial serum matrix chemical processing. Using both of these experimental in all experiments (Figure 1). The addition of 100 ng/mL conditions, we found similar increases in measured of 25(OH)D3 to pooled patient serum resulted in a median 1,25(OH)2D with both RIA and LC-MS/MS (Figure 1) (range) increase of 114% (33–385%) in measured regardless of whether 25(OH)D2 or 25(OH)D3 was 1,25(OH)D2 via RIA and a 21% (21–23%) increase via added. LC-MS/MS. At 700 ng/mL, the increase was 349% (150 – Using LC-MS/MS, we separated 1,25(OH)2D2 from 654%) with RIA and 117% (112–122%) with LC-MS/ 1,25(OH)2D3 to determine which of the two metabolites MS. Similar increases were seen with the addition of accounted for the increases. We found that most increased 25(OH)D2. When 100 ng/mL was added, a 94% (6 – total 1,25(OH)2D was due to an increase in 1,25(OH)2D3 227%) increase was seen with RIA and 20% (19 –22%) regardless of whether 25(OH)D2 or 25(OH)D3 was added with LC-MS/MS. Following a 700-ng/mL spike, this in- (Figure 4). creased the measured 1,25(OH)2D by 249% (77–554%) with RIA and 140% (126 –145%) with LC-MS/MS (Figure 2). The increase in measured 1,25(OH)2D was greater Conclusion with RIA than LC-MS/MS, as demonstrated in Figure 3. Linear regression analysis was performed to determine We report here two cases of hypercalcemia due to vitamin the influence of 25(OH)D on the measured total D intoxication in which both patients were found to have 1,25(OH)2D either by RIA or LC-MS/MS. For every in- elevated serum levels of both 25(OH)D and 1,25(OH)2D. crease in measured 25(OH)D2 by 1 ng/mL, 1,25(OH)2D A similar case of vitamin D intoxication with markedly increased by 0.131 pg/mL (R2 ⫽ 0.8) by RIA. This increase elevated levels of both 25(OH)D and 1,25(OH)2D was was lower at 0.109 for LC-MS/MS (R2 ⫽ 0.99). The ad- recently reported by one of us (16). A large body of literdition of 25(OH)D3 increased the measured 1,25(OH)2D ature [reviewed in the recent Institute of Medicine report more than 25(OH)D2. The increase in 1,25(OH)2D was (7)] suggests that 25(OH)D and not 1,25(OH)2D is the greater when measured by RIA than LC-MS/MS. For ev- basis for hypercalcemia in vitamin D intoxication, and this ery increase in 25(OH)D3 by 1 ng/mL, an increase of is supported by the finding that vitamin D intoxication is 1,25(OH)2D by 0.231 was seen by RIA (R2 ⫽ 0.84) or accompanied by a decrease in plasma levels of 0.121 by LC-MS/MS (R2 ⫽ 0.98). Note that 25(OH)D 1,25(OH)2D in multiple species (20, 25, 26). Because of
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Figure 4. The increase in total 1,25(OH)2D concentration measured by LC-MS/MS was due mainly to an increase in 1,25(OH)2D3, even when 25(OH)D2 was added to serum. Median and ranges for three samples at each concentration are shown.
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Added 25(OH)D, ng/ml Figure 2. The median and upper range for the percentage increase in measured 1,25(OH)2D from baseline following the addition of increasing concentrations of 25(OH)D2 or 25(OH)D3. This increase was greater when measured by RIA, both for 25(OH)D2 and 25(OH)D3.
this, the two cases reported here and previously (16) underwent extensive investigations for alternative or confounding disorders, including analyses of the CYP24A1 gene, which failed to disclose the basis for the unusual vitamin D metabolites in what seemed to be simple vitamin D intoxication. Remarkably, when serum samples that showed markedly elevated levels of 1,25(OH)2D by RIA were reanalyzed by LC-MS/MS the levels of 1,25(OH)2D were normal; nevertheless, these normal levels can still be considered to be inappropriate given that corresponding serum levels of PTH were undetectable. In contrast, our results suggest that when levels of substrate 25(OH)D are very high CYP27B1 can produce 1,25(OH)2D even in the absence of PTH action, which may explain in part the 25(OH)D2 Added 25(OH)D3 Added Increase in 1,25(OH)2D (pg/ml) by LC-MS/MS
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Figure 3. There is an increase in total 1,25(OH)2D measured by RIA and LC-MS/MS when 25(OH)D2 or 25(OH)D3 was added. Note that this increase was greater with RIA than LC-MS/MS.
effectiveness of therapy with parent vitamin D2 in patients with hypoparathyroidism (Elizabeth Streeten, MD, University of Maryland, Personal Communication). We performed in vitro experiments to understand the basis for the elevation in 1,25(OH)2D in these two patients with vitamin D intoxication, and found that addition of high concentrations of 25(OH)D in vitro to serum or an artificial serum matrix can reproduce this artifact even when serum is denatured at high temperatures, thus excluding a simple biological or chemical transformation of 25(OH)D to 1,25(OH)2D. In healthy individuals, 25(OH)D levels are normally between 32 and 70 ng/mL (80 and 175 nmol/L), but these levels may be as much as 15-fold greater in cases of vitamin D toxicity. Given that normal circulating concentrations of 25(OH)D are 1000fold greater than serum concentrations of 1,25(OH)2D, it is understandable how even only moderately increased concentrations of 25(OH)D, as occurred in Case 2, can lead to falsely high signals in a 1,25(OH)2D RIA with a 0.01– 0.02% cross reactivity for 25(OH)D. Although the LC-MS/MS assay provided reassurance in both cases of vitamin D intoxication that we report here that levels of serum 1,25(OH)2D were not elevated, our in vitro studies did show that addition of 25(OH)D to samples led to a small but significant increases in measured 1,25(OH)2D concentrations. Our observation that serum levels of 1,25(OH)2D may be elevated in patients with vitamin D intoxication draws attention to a previously unaddressed effect. In addition to the cases we report here, there have been many other prior reports of vitamin D intoxication in which patients also had elevated serum concentrations of 1,25(OH)2D (16, 27–29), but little attention was paid to this unexpected finding. Two of eight (25%) reported cases of vitamin D intoxication reported by Jacobus et al (28) had elevated 1,25(OH)2D levels, as did three of eleven (27%) of cases reported by Pettifor et al (27). Although it is possible that
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these unusual cases represent examples of vitamin D hypersensitivity due to the unrecognized contribution of loss-of-function mutations in CYP24A1 (22, 30), as has been at least suggested (31) as a basis for the cases of hypercalcemia that followed vitamin D fortification of foods in Great Britain in the 1950s reported by Lightwood and others (32–34), it is more likely given the rarity of these gene defects that assay artifact and not genetic mutation provides a more realistic explanation for the elevated 1,25(OH)2D levels. The significance of our observations is based on the critical role that reliable laboratory testing plays in the process of clinical diagnosis. Because 1,25(OH)2D is irrelevant to the pathophysiology of vitamin D intoxication, the astute clinician will recognize an elevated serum level of 1,25(OH)2D as a justification to pursue alternative or confounding explanations for hypercalcemia. In some cases this will result in the discovery of a granulomatous disease (35) or lymphoma (36), whereas in other cases a comprehensive analysis will disclose a mutation in CYP24A1 (22, 31), in which case a low serum level of 24,25(OH)2D can provide early evidence of the decreased clearance of vitamin D metabolites. This presentation may not be limited to infancy, given that CYP24A1 mutations have been reported as a cause of adult-onset hypercalcemia and/or nephrolithiasis (37–39). Our results suggest that 25(OH)D can interfere with both with RIA and LC-MS/MS assays for 1,25(OH)2D. Denaturing and serum replacement experiments have demonstrated that this effect is not due to biological or chemical conversion of 25(OH)D to 1,25(OH)2D. It is unlikely but possible that the vitamin D supplements taken by the two patients we report here and the analytical grade 25(OH)D metabolites that we used are all contaminated with 1,25(OH)2D, but we suspect that the effect is most likely due to assay interference. Although it is understandable how an antibody used in an RIA for 1,25(OH)2D can have cross reactivity with 25(OH)D, it is less obvious how 25(OH)D can be misread as 1,25(OH)2D in LC-MS/MS. Nevertheless, it is reassuring that in both clinical cases described here, reanalysis of 1,25(OH)2D using LCMS/MS demonstrated normal concentrations and led to the proper diagnosis. Thus, whereas interference was seen with both assays in vitro, it is possible that interference is more relevant clinically in the conventional Diasorin RIA. Our study has several limitations. We did not evaluate, and hence cannot describe the corresponding performance, of RIAs for 1,25(OH)2D that are manufactured by other companies. In addition, we did not evaluate the performance of the new Liason XL chemiluminescent immunoassay for 1,25(OH)2D that has recently been introduced by Diasorin, and which employs a recombinant
VDR fusion protein for capture of the 1,25(OH)2D molecule and a murine monoclonal antibody that specifically recognizes the complex formed by the recombinant fusion protein with the 1,25(OH)2D molecule (40). We conclude that at least some RIAs for 1,25(OH)2D may produce falsely elevated signals when serum levels of 25(OH)D are elevated, and that this potential artifact should be considered along with potential mutations in CYP24A1 in patients with suspected vitamin D intoxication. We recommend measurement of serum 24,25(OH)2D and use of LC-MS/MS, which seems less susceptible to this interference, to reassess serum levels of 1,25(OH)2D when the clinical scenario is confusing.
Acknowledgments We thank Dr Ryan A. Housam and Dr Dean Carlow for their assistance with early collection of data and preliminary experiments, respectively, and Mr. Evan Opas for his technical assistance. Address all correspondence and requests for reprints to: Dr Michael A. Levine, Division of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104. E-mail: [email protected]. C.P.H. supported by a PhD grant by the National Children’s Research Centre, Dublin, Ireland. This work was also supported in part by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK079970) and by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1TR000003. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosure Summary: The authors have nothing to disclose.
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ARTICLE
25-Hydroxyvitamin D Can Interfere With a Common Assay for 1,25-Dihydroxyvitamin D in Vitamin D Intoxication Colin P. Hawkes, Sarah Schnellbacher, Ravinder J. Singh, and Michael A. Levine Division of Endocrinology and Diabetes (C.P.H., S.S., M.A.L.), The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; National Children’s Research Centre (C.P.H.), Dublin, Ireland; Department of Laboratory Medicine and Pathology (R.J.S.), The Mayo Clinic, Rochester, Minnesota 55905; and Department of Pediatrics (M.A.L.), Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Context: Vitamin D intoxication is characterized by elevated serum 25-hydroxyvitamin D (25(OH)D) and suppressed serum 1,25-dihydroxvitamin D (1,25(OH)2D). We evaluated two adolescents with hypercalcemia due to vitamin D intoxication; both had elevated serum 1,25(OH)2D by Diasorin RIA, but normal serum 1,25(OH)2D concentrations by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Objective: This study aimed to determine the effect of 25(OH)D2 and 25(OH)D3 on 1,25(OH)2D concentration determined using RIA and LC-MS/MS. Methods: Pools of normal serum and an artificial serum matrix were prepared and aliquots were spiked with ⬎99% pure 25(OH)D2 or 25(OH)D3 (50 –700 ng/mL). Samples were maintained at 4°C or heated to 56°C, and the concentrations of vitamin D metabolites were measured by LC-MS/MS and Diasorin RIA. Results: Median 1,25(OH)2D increased by 114% with RIA and 21% with LC-MS/MS with addition of 100 ng/mL 25(OH)D3, and 349% (RIA) and 117% (LC-MS/MS) with 700 ng/mL of 25(OH)D3. Each 1-ng/mL increase in 25(OH)D3 increased 1,25(OH)2D by 0.231 pg/mL (RIA) and 0.121 pg/mL (LCMS/MS). Spiking with 25(OH)D2 led to similar changes. Heat inactivation of serum, and using an artificial serum matrix, were associated with similar effects of 25(OH)D on 1,25(OH)2D assays. Conclusions: Vitamin D intoxication with high serum levels of 25(OH)D2 or 25(OH)D3 can be associated with elevated levels of 1,25(OH)2D due to interference in a commonly used RIA. A similar but attenuated effect also occurs when 1,25(OH)2D is measured using LC-MS/MS but does not seem to be clinically significant. The basis for this effect on the LC-MS/MS assay is presently uncertain. (J Clin Endocrinol Metab 100: 2883–2889, 2015)
V
itamin D insufficiency has emerged as a worldwide public health problem (1, 2), particularly due to the high risk to the developing and growing skeleton during early infancy (3, 4) and adolescence (5). In response to these concerns, the American Academy of Pediatrics updated their guidelines in 2008 to recommend that all breastfed infants and all infants consuming less than 1 L
per day of vitamin D-fortified milk or formula receive 400 IU of supplemental vitamin D per day (6). More recently, the Institute of Medicine issued new dietary reference intakes for vitamin D that extended the American Academy of Pediatrics recommendations to recommend a daily intake of 600 IU of vitamin D per day for individuals greater than 1 year of age, and for 800 IU of vitamin D per day for
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA Copyright © 2015 by the Endocrine Society Received May 6, 2015. Accepted June 22, 2015. First Published Online June 29, 2015
Abbreviations: 1,25(OH)2D 1,25-dihydroxvitamin D; 25(OH)D 25 hydroxyvitamin D; D2 Ergocalcifierol; D3 Cholecalciferol; LC-MS/MS, liquid chromatography–tandem mass spectrometry; VDR, vitamin D receptor.
doi: 10.1210/jc.2015-2206
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individuals greater than 70 years of age (7). The revised dietary intake guidelines, in the context of continuing controversy regarding optimal plasma levels for 25(OH)D (8, 9), has resulted in increased interest in vitamin D intake and supplementation. Parental adherence with these guidelines of up to 75% has been reported (10), but adherence among adolescents remains a challenge (11). Moreover, easy access to high-potency preparations of vitamin D and growing but unsubstantiated claims regarding nonskeletal benefits of this vitamin (12) have encouraged higher intakes of vitamin D, with a consequent increased risk for inadvertent vitamin D intoxication. Clinically, this can result in hypercalcemia and hyperphosphatemia, calcifications, nephrocalcinosis, and lifethreatening renal failure (13–16). Vitamin D intoxication reflects a critical disturbance in normal vitamin D homeostasis, which involves a complex interplay of diet, sunlight exposure, and genetics (17). To become fully active, vitamin D, produced in the skin as cholecalciferol (vitamin D3) after exposure to UV light, or supplied in the diet as cholecalciferol or the related yeastand fungi-derived sterol ergocalciferol (vitamin D2) (18), must undergo two modifications by cytochrome P450 enzymes. The first step is 25-hydroxylation of parent vitamin D2 and D3 by hepatic microsomal CYP2R1, which generates the prohormones 25-hydroxyvitamin D3 (25(OH)D3) and 25-hydroxyvitamin D2 (25(OH)D2), respectively (19). A second hydroxylation occurs in the kidney where CYP27B1, a 1␣-hydroxylase located in the mitochondrion, converts 25(OH)D to the fully active form of the vitamin, 1,25-dihydroxyvitamin D (1,25(OH)2D). 1,25(OH)2D is a potent hormone, and binds with high affinity to the vitamin D receptor (VDR), which mediates most physiological actions of vitamin D via modulation of the transcription of target genes. The mechanism for hypercalcemia and hypercalciuria in vitamin D intoxication is thought to be activation of the VDR by 25(OH)D, which is markedly elevated, given that conversion of vitamin D to 25(OH)D by CYP2R1 seems to be substrate dependent and not highly regulated (20). By contrast, the conversion of 25(OH)D to 1,25(OH)2D by CYP27B1 is tightly regulated by calcium, PTH, and fibroblast growth factor 23. Hence, as serum levels of 25(OH)D increase excessively there is a parallel decrease in levels of serum 1,25(OH)2D, a phenomenon that reflects suppression of PTH by hypercalcemia and a corresponding loss of PTHdependent stimulation of CYP27B1 activity (17). Definitive proof that the cause of hypercalcemia in vitamin D intoxication is 25(OH)D rather than 1,25(OH)2D comes from studies by DeLuca et al (21), who showed that intoxication with vitamin D3 occurs equally well in the Cyp27b1 knockout mouse, which completely lacks 1␣-hydroxylase activity, as it does in the wild-type mouse. Thus, we were recently
surprised during the evaluation of two adolescents with vitamin D intoxication when we discovered that serum levels of 25(OH)D and 1,25(OH)2D were both markedly elevated. In this paper we characterize the false elevation of 1,25(OH)2D in vitamin D intoxication, and propose a possible mechanism.
Materials and Methods Patients We performed a retrospective chart review of two adolescents who presented to the Children’s Hospital of Philadelphia with vitamin D intoxication and hypercalcemia. We collected medical and demographic data from each subject, with particular emphasis on biochemical parameters of bone and mineral metabolism, and molecular analysis of the CYP24A1 gene was performed. This study was performed in accordance with the policies and procedures of the institutional review board of the Children’s Hospital of Philadelphia.
Biochemical and molecular studies We extracted genomic DNA from peripheral blood mononuclear cells using standard methods. All exons, exon-intron junctions, and the promoter of CYP24A1 (11p15.2) were amplified and sequenced by PCR using previously described primers and conditions (22). We performed a series of in vitro experiments to determine the effect of analytical grade (purity ⱖ 99%) 25(OH)D2 (Santa Cruz Biotechnology; Ref sc231277) and 25(OH)D3 (Santa Cruz Biotechnology; Ref sc288574) on the concentration of 1,25(OH)2D measured by RIA or LC-MS/MS. We collected deidentified excess serum that was to be discarded from the Clinical Chemistry laboratory and created separate serum pools. The serum samples were from a random set of inpatients and outpatients at the Children’s Hospital of Philadelphia and represented an unbiased spectrum of clinical diagnoses. Blood samples had been drawn into Vacutainer tubes (Becton Dickinson), either red top or serum separator tube. Once samples had arrived at the Clinical Chemistry laboratory the samples had been allowed to stand at room temperature for 30 minutes so serum tubes could clot, followed by centrifugation for 10 minutes at 2095⫻ g. Samples had been refrigerated (2– 8°C) for 2–3 days and then were frozen (⫺70°C) until used. Separate aliquots from each pool were spiked with the addition of either vehicle (final, 0.25% DMSO) or 25(OH)D2 or 25(OH)D3 at the following concentration: 0 ng/mL (control), 50, 100, 150, 250, and 750 ng/mL. Samples were maintained at 4°C or heat inactivated at 56°C for 30 minutes. Heat inactivation was performed to determine whether an observed increase was due to in vitro enzyme-mediated hydroxylation. We followed a similar protocol using aliquots of an artificial serum matrix that consisted of PBS, pH 7.4, containing BSA (4 g/dL). For each sample, we determined the concentration of 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, and 1,25(OH)2D3 by LC-MS/MS using an API 4000 and API 5000 instruments respectively (Applied Biosystems). Molecular-ion-specific fragments and transitions used for 1,25(OH)2D3 were 574.37 and 592.37 ⬎ 314.12 and for 1,25(OH)2D2 was 586.37 ⬎ 314.13 were measured, as previously published (23). We determined the
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concentration of total 1,25(OH)2D by RIA (DiaSorin); this assay is a competitive antibody RIA that involves a preliminary extraction and subsequent purification of vitamin D metabolites from serum or EDTA plasma using C18OH cartridges (24). The RIA method is based on a polyclonal antibody that is specific for both 1,25(OH)2D2 and 1,25(OH)2D3. According to the manufacturer’s product manual, a concentration of 25(OH)D3 greater than 1 mg/mL is required to achieve a 50% B/Bo in this assay, with less than 0.01% cross reactivity.
Statistical analysis Data were analyzed using SPSS 22 (IBM), and figures were generated using Prism 5 (GraphPad Software Inc.) and Adobe Illustrator 16.0 (Adobe Systems, Inc.). Linear regression analysis was performed to determine the influence of 25(OH)D2 or 25(OH)D3 on measured total 1,25(OH)2D by LC-MS/MS or RIA. A separate linear regression analysis was performed to determine the relative increase in measured 1,25(OH)2D by RIA and LC-MS/MS following the addition of either 25(OH)D2 or 25(OH)D3.
Results Case 1 A 15-year-old, previously healthy male presented with a 2-week history of postprandial vomiting, abdominal pain, and polyuria. He had taken a large dose of cholecalciferol numerous times daily over the previous month, but it was not possible to calculate the total intake. Initial laboratory evaluation revealed hypercalcemia, normal phosphorous, and suppressed PTH (Table 1) with a markedly elevated serum concentration of 25(OH)D3 (685 ng/mL by LC-MS/MS). At the same time, he had an elevated serum concentration of total 1,25(OH)2D (⬎230 pg/mL) as determined by RIA (ARUP Laboratories). He had an extensive evaluation to determine the basis for the combined elevation of 25(OH)D3 and 1,25(OH)2D, Table 1.
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which excluded granulomatous disorders and malignancy; the sequence of his CYP24A1 gene was normal. An aliquot of the serum sample that showed an elevated concentration of 1,25(OH)2D by RIA was subsequently retrieved and when analyzed by LC-MS/MS showed a normal concentration of 1,25(OH)2D of 45 pg/mL (ref, 25– 86 pg/mL). He was initially treated with saline hydration, calcitonin, and prednisone with no improvement. He received a single dose of zoledronic acid (0.025 mg/kg) on day 4 of admission, and his serum calcium rapidly declined to normal with a nadir of 7.2 mg/dL 3 days later, and his serum creatinine returned to normal. Three months after discharge from hospital his serum calcium concentration remained normal, and his serum 25(OH)D3 remained elevated at 171.6 ng/mL. Case 2 A 17-year-old female with a background of ocular albinism and autism presented with a history of weight loss and hypercalcemia. She denied a history of excessive ingestion of vitamin D or calcium. Her biochemical studies showed hypercalcemia, normal serum level of phosphorus, and suppressed PTH (Table 1). Measurement of vitamin D metabolites showed elevated serum concentrations of 25(OH)D3 (143 ng/mL) and 1,25(OH)2D (⬎190 pg/mL by RIA). Reanalysis of the serum by LC-MS/MS showed a normal concentration of 1,25(OH)2D of 68 pg/ mL. Her serum concentration of 24,25(OH)2D3 was normal at 17.1 ng/mL (Heartland Assays), and analysis of the CYP24A1 gene was normal. The serum calcium normalized after dietary intervention to reduce vitamin D intake. Vitamin D experiments Concentrations of 25(OH)D2 or 25(OH)D3 increased as expected based on the amount of vitamin D metabolite
Clinical Characteristics and Laboratory Findings in Two Adolescents Presenting With 25(OH)D Intoxication
Clinical Characteristic Demographics Background Diagnoses Calcium, mg/dL Phosphorous, mg/dL Albumin, g/dL PTH, pg/mL Creatinine, mg/dL PTHrP, pmol/L CYP24A1 gene 25(OH)D3, ng/mL LC-MS/MS 1,25(OH)D3, pg/mL RIA 1,25(OH)D3, pg/mL LC-MS/MS
Reference Range
Case 1
Case 2
15-year-old male Well child 13.8 4.2 4.3 ⬍3 3.1 ⬍2 Normal
17-year-old female Ocular albinism, autism 11.3 4 4.1 ⬍5.5 0.48 ⬍2 Normal
30 – 80
685
143
15–75
⬎230
⬎190
25– 86
45
68
8.8 –10.1 2.9 –5.4 3.7–5.6 9 –52 0.6 –1.3 ⬍2
Abbreviation: CYP, cytochrome P450.
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is measured in nanograms per milliliter whereas 1,25(OH)2D is 10001,25(OH) D RIA 1,25(OH) D LC-MS/MS fold lower concentrations and is 25(OH)D measured in picograms per milliliter. When adjusted for these different concentrations, there is 0.013% (RIA) or 0.011% (LC-MS/MS) cross reactivity of 25(OH)D2 with 1,25(OH)2D, and 0.023% (RIA) or Concentration of 25(OH)D2 Added, ng/ml 0.012% (LC-MS/MS) cross reactivity of 25(OH)D3 with 1,25(OH)2D. Heat Inactivated (56 C) Artificial Serum Matrix B Standard Temperature (4 C) These cross reactivities are greater 1,25(OH) D RIA than the ⬍ 0.01% cross reactivity de1,25(OH) D LC-MS/MS scribed by Diasorin for their RIA. 25(OH)D To investigate whether the increase in measured 1,25(OH)2D that we observed in vitro might be due to a previously unidentified biological or chemical conversion of 25(OH)D to 1,25(OH)2D, we repeated the Concentration of 25(OH)D3 Added, ng/ml spiking experiments under two adFigure 1. The concentrations of 1,25(OH)2D measured by RIA and LC-MS/MS in response to ditional conditions. We heated seincreasing concentrations of 25(OH)D2 (A) or 25(OH)D3 (B) at 4°C, 56°C, and using a serum rum to 56°C to inactivate potential matrix. Mean and ranges of measured 1,25(OH)2D from 3 pools for each concentration of enzymes that might be present in se25(OH)D2 or 25(OH)D3 are shown. rum, and used an artificial serum matrix to exclude both biological and added to pooled serum samples or artificial serum matrix chemical processing. Using both of these experimental in all experiments (Figure 1). The addition of 100 ng/mL conditions, we found similar increases in measured of 25(OH)D3 to pooled patient serum resulted in a median 1,25(OH)2D with both RIA and LC-MS/MS (Figure 1) (range) increase of 114% (33–385%) in measured regardless of whether 25(OH)D2 or 25(OH)D3 was 1,25(OH)D2 via RIA and a 21% (21–23%) increase via added. LC-MS/MS. At 700 ng/mL, the increase was 349% (150 – Using LC-MS/MS, we separated 1,25(OH)2D2 from 654%) with RIA and 117% (112–122%) with LC-MS/ 1,25(OH)2D3 to determine which of the two metabolites MS. Similar increases were seen with the addition of accounted for the increases. We found that most increased 25(OH)D2. When 100 ng/mL was added, a 94% (6 – total 1,25(OH)2D was due to an increase in 1,25(OH)2D3 227%) increase was seen with RIA and 20% (19 –22%) regardless of whether 25(OH)D2 or 25(OH)D3 was added with LC-MS/MS. Following a 700-ng/mL spike, this in- (Figure 4). creased the measured 1,25(OH)2D by 249% (77–554%) with RIA and 140% (126 –145%) with LC-MS/MS (Figure 2). The increase in measured 1,25(OH)2D was greater Conclusion with RIA than LC-MS/MS, as demonstrated in Figure 3. Linear regression analysis was performed to determine We report here two cases of hypercalcemia due to vitamin the influence of 25(OH)D on the measured total D intoxication in which both patients were found to have 1,25(OH)2D either by RIA or LC-MS/MS. For every in- elevated serum levels of both 25(OH)D and 1,25(OH)2D. crease in measured 25(OH)D2 by 1 ng/mL, 1,25(OH)2D A similar case of vitamin D intoxication with markedly increased by 0.131 pg/mL (R2 ⫽ 0.8) by RIA. This increase elevated levels of both 25(OH)D and 1,25(OH)2D was was lower at 0.109 for LC-MS/MS (R2 ⫽ 0.99). The ad- recently reported by one of us (16). A large body of literdition of 25(OH)D3 increased the measured 1,25(OH)2D ature [reviewed in the recent Institute of Medicine report more than 25(OH)D2. The increase in 1,25(OH)2D was (7)] suggests that 25(OH)D and not 1,25(OH)2D is the greater when measured by RIA than LC-MS/MS. For ev- basis for hypercalcemia in vitamin D intoxication, and this ery increase in 25(OH)D3 by 1 ng/mL, an increase of is supported by the finding that vitamin D intoxication is 1,25(OH)2D by 0.231 was seen by RIA (R2 ⫽ 0.84) or accompanied by a decrease in plasma levels of 0.121 by LC-MS/MS (R2 ⫽ 0.98). Note that 25(OH)D 1,25(OH)2D in multiple species (20, 25, 26). Because of
A
300
Standard Temperature (4oC)
Heat Inactivated (56oC)
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25(OH)D2, ng/ml
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1,25(OH)2D, pg/ml
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LC-MS/MS
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25(OH)D2 Added
25(OH)D3 Added
25(OH)D3 Added
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1,25(OH)2D3 150
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Concentration of 25(OH)D3 Added, ng/ml
Figure 4. The increase in total 1,25(OH)2D concentration measured by LC-MS/MS was due mainly to an increase in 1,25(OH)2D3, even when 25(OH)D2 was added to serum. Median and ranges for three samples at each concentration are shown.
200 150 100 50 0 50
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Added 25(OH)D, ng/ml Figure 2. The median and upper range for the percentage increase in measured 1,25(OH)2D from baseline following the addition of increasing concentrations of 25(OH)D2 or 25(OH)D3. This increase was greater when measured by RIA, both for 25(OH)D2 and 25(OH)D3.
this, the two cases reported here and previously (16) underwent extensive investigations for alternative or confounding disorders, including analyses of the CYP24A1 gene, which failed to disclose the basis for the unusual vitamin D metabolites in what seemed to be simple vitamin D intoxication. Remarkably, when serum samples that showed markedly elevated levels of 1,25(OH)2D by RIA were reanalyzed by LC-MS/MS the levels of 1,25(OH)2D were normal; nevertheless, these normal levels can still be considered to be inappropriate given that corresponding serum levels of PTH were undetectable. In contrast, our results suggest that when levels of substrate 25(OH)D are very high CYP27B1 can produce 1,25(OH)2D even in the absence of PTH action, which may explain in part the 25(OH)D2 Added 25(OH)D3 Added Increase in 1,25(OH)2D (pg/ml) by LC-MS/MS
100
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Increase in 1,25(OH)2D (pg/ml) by Radioimmunoassay -50
Figure 3. There is an increase in total 1,25(OH)2D measured by RIA and LC-MS/MS when 25(OH)D2 or 25(OH)D3 was added. Note that this increase was greater with RIA than LC-MS/MS.
effectiveness of therapy with parent vitamin D2 in patients with hypoparathyroidism (Elizabeth Streeten, MD, University of Maryland, Personal Communication). We performed in vitro experiments to understand the basis for the elevation in 1,25(OH)2D in these two patients with vitamin D intoxication, and found that addition of high concentrations of 25(OH)D in vitro to serum or an artificial serum matrix can reproduce this artifact even when serum is denatured at high temperatures, thus excluding a simple biological or chemical transformation of 25(OH)D to 1,25(OH)2D. In healthy individuals, 25(OH)D levels are normally between 32 and 70 ng/mL (80 and 175 nmol/L), but these levels may be as much as 15-fold greater in cases of vitamin D toxicity. Given that normal circulating concentrations of 25(OH)D are 1000fold greater than serum concentrations of 1,25(OH)2D, it is understandable how even only moderately increased concentrations of 25(OH)D, as occurred in Case 2, can lead to falsely high signals in a 1,25(OH)2D RIA with a 0.01– 0.02% cross reactivity for 25(OH)D. Although the LC-MS/MS assay provided reassurance in both cases of vitamin D intoxication that we report here that levels of serum 1,25(OH)2D were not elevated, our in vitro studies did show that addition of 25(OH)D to samples led to a small but significant increases in measured 1,25(OH)2D concentrations. Our observation that serum levels of 1,25(OH)2D may be elevated in patients with vitamin D intoxication draws attention to a previously unaddressed effect. In addition to the cases we report here, there have been many other prior reports of vitamin D intoxication in which patients also had elevated serum concentrations of 1,25(OH)2D (16, 27–29), but little attention was paid to this unexpected finding. Two of eight (25%) reported cases of vitamin D intoxication reported by Jacobus et al (28) had elevated 1,25(OH)2D levels, as did three of eleven (27%) of cases reported by Pettifor et al (27). Although it is possible that
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2888
Hawkes et al
Assay Interference in Vitamin D Intoxication
J Clin Endocrinol Metab, August 2015, 100(8):2883–2889
these unusual cases represent examples of vitamin D hypersensitivity due to the unrecognized contribution of loss-of-function mutations in CYP24A1 (22, 30), as has been at least suggested (31) as a basis for the cases of hypercalcemia that followed vitamin D fortification of foods in Great Britain in the 1950s reported by Lightwood and others (32–34), it is more likely given the rarity of these gene defects that assay artifact and not genetic mutation provides a more realistic explanation for the elevated 1,25(OH)2D levels. The significance of our observations is based on the critical role that reliable laboratory testing plays in the process of clinical diagnosis. Because 1,25(OH)2D is irrelevant to the pathophysiology of vitamin D intoxication, the astute clinician will recognize an elevated serum level of 1,25(OH)2D as a justification to pursue alternative or confounding explanations for hypercalcemia. In some cases this will result in the discovery of a granulomatous disease (35) or lymphoma (36), whereas in other cases a comprehensive analysis will disclose a mutation in CYP24A1 (22, 31), in which case a low serum level of 24,25(OH)2D can provide early evidence of the decreased clearance of vitamin D metabolites. This presentation may not be limited to infancy, given that CYP24A1 mutations have been reported as a cause of adult-onset hypercalcemia and/or nephrolithiasis (37–39). Our results suggest that 25(OH)D can interfere with both with RIA and LC-MS/MS assays for 1,25(OH)2D. Denaturing and serum replacement experiments have demonstrated that this effect is not due to biological or chemical conversion of 25(OH)D to 1,25(OH)2D. It is unlikely but possible that the vitamin D supplements taken by the two patients we report here and the analytical grade 25(OH)D metabolites that we used are all contaminated with 1,25(OH)2D, but we suspect that the effect is most likely due to assay interference. Although it is understandable how an antibody used in an RIA for 1,25(OH)2D can have cross reactivity with 25(OH)D, it is less obvious how 25(OH)D can be misread as 1,25(OH)2D in LC-MS/MS. Nevertheless, it is reassuring that in both clinical cases described here, reanalysis of 1,25(OH)2D using LCMS/MS demonstrated normal concentrations and led to the proper diagnosis. Thus, whereas interference was seen with both assays in vitro, it is possible that interference is more relevant clinically in the conventional Diasorin RIA. Our study has several limitations. We did not evaluate, and hence cannot describe the corresponding performance, of RIAs for 1,25(OH)2D that are manufactured by other companies. In addition, we did not evaluate the performance of the new Liason XL chemiluminescent immunoassay for 1,25(OH)2D that has recently been introduced by Diasorin, and which employs a recombinant
VDR fusion protein for capture of the 1,25(OH)2D molecule and a murine monoclonal antibody that specifically recognizes the complex formed by the recombinant fusion protein with the 1,25(OH)2D molecule (40). We conclude that at least some RIAs for 1,25(OH)2D may produce falsely elevated signals when serum levels of 25(OH)D are elevated, and that this potential artifact should be considered along with potential mutations in CYP24A1 in patients with suspected vitamin D intoxication. We recommend measurement of serum 24,25(OH)2D and use of LC-MS/MS, which seems less susceptible to this interference, to reassess serum levels of 1,25(OH)2D when the clinical scenario is confusing.
Acknowledgments We thank Dr Ryan A. Housam and Dr Dean Carlow for their assistance with early collection of data and preliminary experiments, respectively, and Mr. Evan Opas for his technical assistance. Address all correspondence and requests for reprints to: Dr Michael A. Levine, Division of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104. E-mail: [email protected]. C.P.H. supported by a PhD grant by the National Children’s Research Centre, Dublin, Ireland. This work was also supported in part by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK079970) and by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1TR000003. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosure Summary: The authors have nothing to disclose.
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![[Fatal vitamin D intoxication].](/assets/img/hold.png)
