title: 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.
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.
authors.affiliation: Department of Pediatrics, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Denburg
authors.affiliation: Department of Laboratory Medicine, Washingon University School of Medicine, Seattle, WA, USA.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Hoofnagle
authors.affiliation: Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Samir
authors.initials: S
authors.lastname: Sayed
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA.
authors.forename: Jayanta
authors.initials: J
authors.lastname: Gupta
authors.affiliation: Department of Medicine, Washingon University School of Medicine, Seattle, WA, USA.
authors.forename: Ian H
authors.initials: IH
authors.lastname: de Boer
authors.affiliation: Departments of Medicine, Epidemiology and International Health, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Appel
authors.affiliation: Department of Public Health Sciences, Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA.
authors.forename: Ramon
authors.initials: R
authors.lastname: Durazo-Arvizu
authors.affiliation: Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Krista
authors.initials: K
authors.lastname: Whitehead
authors.affiliation: Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Harold I
authors.initials: HI
authors.lastname: Feldman
authors.affiliation: Departments of Pediatrics and Medicine, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Mary B
authors.initials: MB
authors.lastname: Leonard
mesh_terms: D000328:Adult; D000368:Aged; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006720:Homozygote; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000097:blood; D051436:Renal Insufficiency, Chronic / Q000097:blood*; D014807:Vitamin D / Q000097:blood*; D014809:Vitamin D-Binding Protein / Q000097:blood / Q000235:genetics
chemical_list: D020033:Protein Isoforms; D014809:Vitamin D-Binding Protein; D014807:Vitamin D
keywords: BIOAVAILABLE VITAMIN D; GENOTYPE; Gc; LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY; VITAMIN D-BINDING PROTEIN