25-hydroxyvitamin D can interfere with a common assay for 1,25-dihydroxyvitamin D in vitamin D intoxication

Colin P. Hawkes, Sarah Schnellbacher, Ravinder Jit Singh, Michael A. Levine

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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)<inf>2</inf>D). We evaluated two adolescents with hypercalcemia due to vitamin D intoxication; both had elevated serum 1,25(OH)<inf>2</inf>D by Diasorin RIA, but normal serum 1,25(OH)<inf>2</inf>D concentrations by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Objective: This study aimed to determine the effect of 25(OH)D<inf>2</inf> and 25(OH)D<inf>3</inf> on 1,25(OH)<inf>2</inf>D 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)D<inf>2</inf> or 25(OH)D<inf>3</inf> (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)2 D increased by 114% with RIA and 21% with LC-MS/MS with addition of 100 ng/mL 25(OH)D<inf>3</inf>, and 349% (RIA) and 117% (LC-MS/MS) with 700 ng/mL of 25(OH)D<inf>3</inf>. Each 1-ng/mL increase in 25(OH)D<inf>3</inf> increased 1,25(OH)<inf>2</inf>D by 0.231 pg/mL (RIA) and 0.121 pg/mL (LC-MS/MS). Spiking with 25(OH)D<inf>2</inf> 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)<inf>2</inf>D assays. Conclusions: Vitamin D intoxication with high serum levels of 25(OH)D<inf>2</inf> or 25(OH)D<inf>3</inf> can be associated with elevated levels of 1,25(OH)<inf>2</inf>D due to interference in a commonly used RIA. A similar but attenuated effect also occurs when 1,25(OH)2 D 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.

Original languageEnglish (US)
Pages (from-to)2883-2889
Number of pages7
JournalJournal of Clinical Endocrinology and Metabolism
Volume100
Issue number8
DOIs
StatePublished - Aug 1 2015

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Vitamin D
Assays
Serum
Liquid chromatography
Metabolites
Mass spectrometry
1,25-dihydroxyvitamin D
25-hydroxyvitamin D
Hypercalcemia
Tandem Mass Spectrometry
Liquid Chromatography
Hot Temperature

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Endocrinology
  • Biochemistry, medical
  • Endocrinology, Diabetes and Metabolism

Cite this

25-hydroxyvitamin D can interfere with a common assay for 1,25-dihydroxyvitamin D in vitamin D intoxication. / Hawkes, Colin P.; Schnellbacher, Sarah; Singh, Ravinder Jit; Levine, Michael A.

In: Journal of Clinical Endocrinology and Metabolism, Vol. 100, No. 8, 01.08.2015, p. 2883-2889.

Research output: Contribution to journalArticle

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title = "25-hydroxyvitamin D can interfere with a common assay for 1,25-dihydroxyvitamin D in vitamin D intoxication",
abstract = "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)2 D 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 (LC-MS/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)2 D 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.",
author = "Hawkes, {Colin P.} and Sarah Schnellbacher and Singh, {Ravinder Jit} and Levine, {Michael A.}",
year = "2015",
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volume = "100",
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T1 - 25-hydroxyvitamin D can interfere with a common assay for 1,25-dihydroxyvitamin D in vitamin D intoxication

AU - Hawkes, Colin P.

AU - Schnellbacher, Sarah

AU - Singh, Ravinder Jit

AU - Levine, Michael A.

PY - 2015/8/1

Y1 - 2015/8/1

N2 - 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)2 D 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 (LC-MS/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)2 D 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.

AB - 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)2 D 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 (LC-MS/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)2 D 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.

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