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The measurement of free 25-hydroxyvitamin D

Vitamin D status is currently assessed by measurements of total 25-hydroxyvitamin D [25(OH)D]. However, over 99% of circulating 25(OH)D is bound to protein, vitamin D binding protein in particular. The free hormone hypothesis stipulates that only the free form crosses the cell membrane to exert biologic action. Measurement of free 25(OH)D is now available.

by Professor Daniel D Bikle

Circulating levels of 25-hydroxyvitamin D [25(OH)D] are the most commonly used marker for the assessment of vitamin D nutritional status. This is because its concentration in blood is higher than all other vitamin D metabolites, making it easier to measure, and because its conversion from vitamin D is substrate dependent with minimal regulation. However, 25(OH)D is not the most biologically active metabolite of vitamin D. Instead 25(OH)D must be further metabolized to 1,25 dihydroxyvitamin D [1,25(OH)2D] for vitamin D to achieve its full biologic potential. 1,25(OH)2D is the ligand for a nuclear transcription factor, the vitamin D receptor (VDR), that mediates the genomic and at least some of the nongenomic actions of vitamin D within the cell. Nearly all, if not all, cells express the VDR at some stage in their development or activation. As the appreciation that vitamin D and its metabolites affect numerous physiologic processes and not just bone and mineral metabolism, and that these physiologic processes may have different requirements for these vitamin D metabolites, interest in determining optimal levels of the vitamin D metabolites to effect these different biologic processes has grown. Complicating this determination is the fact that all the vitamin D metabolites circulate in blood tightly bound to proteins, of which the vitamin D binding protein (DBP) plays the major role. For most cells, these binding proteins limit the flux of the vitamin D metabolites from blood into the cell where they exert their biologic activity. This raises the issue of what should we measure to determine vitamin D status: the total levels of these metabolites or their free levels?

The free hormone hypothesis: why measure free 25(OH)D
The free hormone hypothesis postulates that only the non-bound fraction (the free fraction) of hormones that otherwise circulate in blood bound to their carrier proteins is able to enter cells and exert their biologic effects. This hypothesis applies to steroid hormones, thyroid hormone and vitamin D. For the vitamin D metabolites this hypothesis needs to be qualified in that some tissues, kidney and parathyroid glands in particular, express a transport system, the megalin/cubilin complex, that enables 25(OH)D bound to DBP to be transported into these cells. However, for cells lacking this complex the free fraction is felt to be the fraction capable of entering these cells. In serum samples from normal individuals, ~85% of circulating vitamin D metabolites are bound to DBP, whereas albumin with its substantially lower binding affinity binds only ~15% of these metabolites despite its 10-fold higher concentration than DBP. Approximately 0.4% of total 1,25(OH)2D and 0.02–0.03% of total 25(OH)D is free in serum from normal non-pregnant individuals. The fraction of ‘bioavailable’ vitamin D metabolites is composed of the fraction of the free vitamin D and the fraction bound to albumin, thus measuring around 15% in normal individuals. At this point there is little evidence that the albumin fraction is truly bioavailable. A simple strategy might be to estimate the free concentration based on measurements of DBP and total 25(OH)D with known binding constants of DBP for 25(OH)D. This has in fact been done, but as subsequent research has documented, this relationship is affected by numerous clinical conditions and the different DBP variants with different affinities for 25(OH)D.

DBP is a 51–58 kDa multifunctional serum glycoprotein synthesized primarily in the liver. Initially, isoelectric focusing migration patterns identified phenotypic variants termed Group-Specific Component (Gc), the most common of which are Gc1f, Gcs and Gc2. Two common missense point mutations (SNPs) in exon 11 of the DBP gene, rs7041 (G/T single-nucleotide variation) and rs4588 (an A/C single-nucleotide variation), result in the three most common isoforms with amino acid changes at positions 416 and 420: Gc1f (Asp416, Thr420), Gc1s (Glu 416, Thr420), and Gc2 (Asp416, Lys420). Gc2 is the least abundant and Gc1f the most abundant. The distribution of the Gc alleles varies by race. Black and Asian populations are more likely to carry the Gc1f form, whereas the Gc2 form is rare, whereas Whites more frequently express the Gc1s and the Gc2 alleles. Although affinities of these DBP variants for 25(OH)D appear to vary, the rank order remains controversial, and their contribution of total 25(OH)D levels and the relationship between free and total 25(OH)D is modest in comparison to differences influenced by clinical condition. In the absence of disease or pregnancy, DBP levels are relatively constant over time in adults. That said, various substances in the blood such as polyunsaturated fatty acids may alter the affinity of DBP for the vitamin D metabolites, as can various clinical conditions. Liver disease leads to reduced levels of DBP, as do protein-losing nephropathies and acute illness (DBP is an acute phase reactant), whereas DBP levels are elevated during the latter stages of pregnancy. Moreover, various clinical conditions appear to shift the relationship between free and total 25(OH)D seemingly independent of DBP levels or DBP haplotypes. Thus, the measurement of total 25(OH)D may not provide the best assessment of vitamin D status. Calculation of free 25(OH)D from DBP and total 25(OH)D measurements using affinity constants obtained by measurements in normal sera may be inaccurate, at least in some clinical situations. Therefore, direct measurement of free 25(OH)D would appear to offer information about vitamin D nutritional status that at least complements that of total 25(OH)D.

The free 25(OH)D assay
The original free 25(OH)D assay employed centrifugal ultrafiltration. This was a labour- and reagent-intensive assay suitable only for a dedicated research laboratory. However, it sufficed to determine free 25(OH)D levels in a number of patient groups including cirrhotics and pregnant women, providing proof of concept that the free 25(OH)D measurement would add to the assessment of vitamin D nutritional status. This assay has subsequently been superseded by a much simpler method capable of high throughput.

A two-step ELISA that directly measures free 25(OH)D levels was recently developed by Future Diagnostics Solutions using monoclonal antibodies from DIAsource Immunoassays. In the first incubation step, an anti-25(OH)D monoclonal antibody immobilized on a microtitre plate binds the free 25(OH)D in the serum sample. The serum is removed and biotinylated 25(OH)D in a known amount is added to react with the unoccupied binding sites on the monoclonal antibody attached to the plate. The non-bound biotinylated 25(OH)D is then removed followed by the addition of streptavidin peroxidase conjugate and the substrate 3,3ʹ,5,5ʹ-Tetramethylbenzidine (TMB). The bound streptavidin peroxidase can be quantified by measuring the absorbance at 450 nm generated in the reaction. The intensity is inversely proportional to the level of free 25(OH)D. The limit of detection is 2.8 pg/mL. The antibody in the current assay does not recognize 25(OH)D2 as well as 25(OH)D3 (77% of the 25(OH)D3 value), and so it underestimates the free 25(OH)D2. However, under most situations where the predominant vitamin D metabolite is 25(OH)D3 this issue is not a major concern. The data for both normal subjects and those with different DBP levels (cirrhotics, pregnant women) compare quite well to those obtained from similar populations using the centrifugal ultrafiltration assay.

Clinical implications
In a study currently under review for publication we compiled data from over 1600 individuals in whom free 25(OH)D had been measured by this ELISA. The samples included sera from both normal subjects and those with a variety of clinical conditions and a variety of DBP alleles. In the nearly 1000 normal and community dwelling outpatient subjects the normal range for free 25(OH)D was established at 4.3±1.9 pg/mL with a mean total 25(OH)D of 21.9±9.9 ng/mL, providing a percent free 25(OH)D of 0.02%. These results are essentially identical to those reported by the author using centrifugal ultrafiltration 30 years ago. As expected, clinical conditions affecting DBP values made a big difference. Liver disease resulted in lower DBP levels and higher percentage free 25(OH)D resulting in the population of cirrhotics studied having among the highest free 25(OH)D despite the lowest total 25(OH)D. Nursing home patients also had unexpectedly high free 25(OH)D, higher than that of the cirrhotics, with only modest reductions in DBP levels. Pregnancy (third trimester), however, resulted in increased DBP levels and the lowest free 25(OH)D levels, although the free fraction was not lower than that of the normal subjects. Overall, these results indicate that the free fraction is altered by the clinical situation not only in terms of altered DBP levels but in the relationship between total and free 25(OH)D for any given DBP level. Therefore, it is recommended that the free 25(OH)D level needs to be measured directly if the free level is thought to have particular relevance to the clinical situation that cannot be captured by measuring total 25(OH)D.

At this point it is not yet clear whether the determination of free 25(OH)D is a better marker of vitamin D nutritional status and biologic action than the determination of total 25(OH)D. Using a convenient marker such as parathyroid hormone (PTH), much as we use thyroid-stimulating hormone (TSH) as a marker of thyroid status, is problematic. First of all PTH levels are controlled by calcium as well as
vitamin D. Second, regulation of PTH secretion is mediated primarily by the 1,25(OH)2D produced within the gland itself (much as TSH secretion is controlled by triiodothyronine (T3) produced within the pituitary). Third, the parathyroid gland has the megalin/cubilin transport system to enable 25(OH)D bound to DBP to enter the cells, obviating any advantage free 25(OH)D might have in cell uptake. However, several studies have demonstrated a stronger correlation between free 25(OH)D and bone markers than that observed with total 25(OH)D. But at this point, determining the role that free 25(OH)D measurements play in the assessment of vitamin D nutrition and action requires further investigation.

1. Bikle DD. Vitamin D Assays. Front Horm Res 2018; 50: 14–30.
2. Malstroem S, Rejmark L, et al. Current assays to determine free 25-hydroxyvitamin D in serum. J AOAC Internl 2017; 100: 1323–1327.
3. Bikle D, Bouillon R, et al. Vitamin D metabolites in captivity? Should we measure free or total 25(OH)D to assess vitamin D status? J Steroid Biochem Mol Biol 2017; 173: 1054–1116.
4. Bikle DD, Malmstroem S, Schwartz J. Current controversies: are free vitamin metabolite levels a more accurate assessment of vitamin D status than total levels? Endo Clinics NA 2017; 46: 901–918.
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6. Schwartz JB,  Lai J, et al. A comparison of direct and calculated free 25-OH vitamin D levels in clinical populations. J Clin Endocrinol Metab 2014; 99: 1631–1637.

The author
Daniel D Bikle MD, PhD
VA Medical Center and University of
California San Francisco, San Francisco,
CA 94158, USA

E-mail: Daniel.bikle@ucsf.edu