Immunological and immunoassay studies of the binding protein for vitamin D and its metabolites in human serum.

Abstract

This study reports the development of a specific and sensitive radioimmunoassay and a simple and accurate radial immunodiffusion (RID) assay for the human serum-binding protein for vitamin D and its metabolites (DBP). These immunoassays employed a monospecific antiserum that was prepared in rabbits against human DBP. The radioimmunoassay effectively measured DBP in amounts of 1-10 ng, whereas the RID assay measured DBP accurately in amounts of 0.2-0.8 mug. The results obtained with the two immunoassays on the same samples of serum agreed well with each other. Using the RID assay, the mean (+/- SD) serum DBP concentration observed in 35 normal persons was 422 +/- 27 micrograms/ml. Generally similar levels were observed in 66 hyperlipidemic subjects. In molar terms, the mean DBP concentration (approximately 8 microgramsM) was of the order of 50 times the usual serum level of 25-hydroxyvitamin D (25-OH-D) plus vitamin D. Thus, most of plasma DBP circulates as apo-DBP, not containing a bound molecule of 25-OH-D or of vitamin D. DBP and 25-OH-D concentrations were measured in a limited number of patients with hypercalcemia, mild hypocalcemia, and markedly elevated serum 25-OH-D levels due to oral vitamin D supplementation. It was found that major changes can occur in the serum levels of 25-OH-D and of calcium with very little or no associated changes occurring in the serum concentration of DBP, The results suggest that neither serum 25-OH-D nor serum calcium plays an important role in the regulation of the metabolism of DBP. Data were obtained that confirmed and extended an earlier report on the identity of the group-specific component (Gc) protein in plasma with the plasma vitamin D-binding protein. On immunodiffusion against whole serum, the line formed with the anti-DBP antiserum showed a complete reaction-of-identity with the line formed with commercial antiserum against Gc protein. Furthermore, serum that had been depleted of DBP by treatment with Sepharose containing covalently coupled antibodies against DBP was found to be depleted also of immunoreactivity against anti-GC protein antiserum. In addition, the properties of the purified DBP preparation agreed closely with those previously reported by others for Gc protein. Finally, a comparative immunology study showed that sera from several different mammalian orders showed some immunoreactivity against the antihuman DBP antiserum. Thus, proteins immunologically similar to human DBP are present in sera from a number of mammalian species and orders.