Comparison of Two ELISA Methods and Mass Spectrometry for Measurement of Vitamin D-Binding Protein: Implications for the Assessment of Bioavailable Vitamin D Concentrations Across Genotypes

Authors

  • Michelle R Denburg,

    Corresponding author
    1. Department of Pediatrics, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
    2. Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
    • Address correspondence to: Michelle R. Denburg, MD, MSCE, Assistant Professor of Pediatrics, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, 34th Street and Civic Center Blvd, Philadelphia, PA 19104, USA., E-mail: denburgm@email.chop.edu

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  • Andrew N Hoofnagle,

    1. Department of Laboratory Medicine, Washingon University School of Medicine, Seattle, WA, USA
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  • Samir Sayed,

    1. Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, USA
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  • Jayanta Gupta,

    1. Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA
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  • Ian H de Boer,

    1. Department of Medicine, Washingon University School of Medicine, Seattle, WA, USA
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  • Lawrence J Appel,

    1. Departments of Medicine, Epidemiology and International Health, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Ramon Durazo-Arvizu,

    1. Department of Public Health Sciences, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA
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  • Krista Whitehead,

    1. Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
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  • Harold I Feldman,

    1. Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
    2. Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
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  • Mary B Leonard,

    1. Departments of Pediatrics and Medicine, Stanford University School of Medicine, Stanford, CA, USA
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  • on behalf of the Chronic Renal Insufficiency Cohort study investigators


  • For a Commentary on this article, please see Bouillon (J Bone Miner Res. 2016;31(6):1124–1127. DOI: 10.1002/jbmr.2871).

ABSTRACT

Studies using vitamin D-binding protein (DBP) concentrations to estimate free and bioavailable vitamin D have increased dramatically in recent years. Combinations of two single-nucleotide polymorphisms (SNPs) produce three major DBP isoforms (Gc1f, Gc1s, and Gc2). A recent study showed that DBP concentrations quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) did not differ by race, whereas a widely used monoclonal enzyme-linked immunosorbent assay (ELISA) quantified DBP differentially by isoform, yielding significantly lower DBP concentrations in black versus white individuals. We compared measurements of serum DBP using a monoclonal ELISA, a polyclonal ELISA, and LC-MS/MS in 125 participants in the Chronic Renal Insufficiency Cohort (CRIC). Serum free and bioavailable 25OHD were calculated based on DBP concentrations from these three assays in homozygous participants, and race differences were compared. We confirmed that the monoclonal ELISA quantifies DBP differentially by isoform and showed that the polyclonal ELISA is not subject to this bias. Whereas ≤9% of the variability in DBP concentrations quantified using either LC-MS/MS or the polyclonal ELISA was explained by genotype, 85% of the variability in the monoclonal ELISA-based measures was explained by genotype. DBP concentrations measured by the monoclonal ELISA were disproportionately lower than LC-MS/MS-based results for Gc1f homozygotes (median difference –67%; interquartile range [IQR] –71%, –64%), 95% of whom were black. In contrast, the polyclonal ELISA yielded consistently and similarly higher measurements of DBP than LC-MS/MS, irrespective of genotype, with a median percent difference of +50% (IQR +33%, +65%). Contrary to findings using the monoclonal ELISA, DBP concentrations did not differ by race, and free and bioavailable 25OHD were significantly lower in black versus white participants based on both the polyclonal ELISA and LC-MS/MS, consistent with their lower total 25OHD. Future studies of DBP and free or bioavailable vitamin D metabolites should employ DBP assays that are not biased by DBP genotype. © 2016 American Society for Bone and Mineral Research.

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