The Power of Mass Spectroscopy as Arbiter for Immunoassays

Abstract

In this issue of Clinical Chemistry, Henderson et al. present a new, elaborate method that uses mass spectrometry to analyze and quantify serum vitamin D binding protein (DBP) 2 (1 ). This protein, initially discovered by its different electrophoretic mobility in various populations and therefore called group-specific component (GC), is used in population genetics and forensic medicine (2, 3 ). The genetic basis of the polymorphism is known to be 2 amino acid differences (aa 432 and 436) in the C domain of the protein, thereby generating 3 isoforms, GC1f, GC1s, and GC2, detectable by genetic analysis (single nucleotide polymorphisms RS7041 and RS4588). Because both alleles are codominant, this genetic diversity generates 3 homozygous and 3 heterozygous possibilities. A remarkably strange south-to-north gradient is evident: members of the original African population (and their descendants around the world) mainly express GC1f, whereas populations living further from the equator have gradually higher percentages of GC1s or GC2 alleles. Moreover, based on isoelectric focusing, Ͼ120 different types of DBP GCs have been identified, although the genetic or posttranslational origin of these isoforms remains largely unexplained.DBP is a glycoprotein with either 3 (GC1 isoforms) or 2 carbohydrate chains on some (2%-20%) of the circulating DBP molecules (4 ); it is a multifunctional protein probably belonging to the oldest member of the albumin gene family (1, 2 ). It has a very high affinity for actin and helps to depolymerize filamentous actin into globular actin when released in the bloodstream after cell damage (5 ). It is also the major binding protein for all vitamin D metabolites, with higher affinity for 25-. This is very similar to the situation for thyroid hormones, in which the T 4 binds thyroxinebinding globulin much tighter than T 3 . Inversely, these prohormones have a much lower affinity for their intracellular receptors, VDR and TR, both members of the same nuclear receptor transcription family.For thyroid hormones, as for sex steroid hormones and cortisol, it is generally accepted that functional activity is better reflected by measurement of free rather than total hormone concentrations. Therefore, accurate and reliable measurements of these free hormones were developed, initially by measuring the total hormone concentrations and that of their binding proteins, followed by mathematical calculation to obtain the free hormone concentrations. Increasingly, over the last 2 decades, this approach has been replaced by direct measurements of free hormones with improving accuracy (6 ). In view of the many similarities between thyroid and vitamin D metabolism, transport, and action, one can presume that free vitamin D metabolites would behave as free thyroid hormones and that measurements of free metabolites would reflect physiologic or disease outcomes better than their total concentrations (7,8 ). Therefore, it seems logical to develop methods for direct measurement of free vitamin D metabolites as well as me...

Current vitamin D status in European and Middle East countries and strategies to prevent vitamin D deficiency: a position statement of the European Calcified Tissue Society

“…As the free concentration of 25(OH)D is dependent on both DBP concentration and affinity it is yet not possible to conclude whether clinical correlates and thresholds for vitamin D deficiency depend on genetic polymorphisms of DBP. An assay to measure the free 25(OH)D concentration is available, but currently it is uncertain whether the measurement of this metabolite in its free state has clinical implications (189,190).…”

Free or Total 25OHD as Marker for Vitamin D Status?

“…Mass spectroscopy can be used as an arbiter to interpret immunoassays because such method allows measuring peptides from the region of DBP common to all genetic variants separately from genotype‐specific sequences . Based on such techniques, at least two laboratories independently confirmed the absence of racial or genetic differences in DBP concentrations.…”