IntroductionTransport of vitamin D3 from its sites of cutaneous synthesis into the circulation has been assumed to be via the plasma vitamin D binding protein (DBP). We studied vitamin D transport from the skin in seven healthy volunteers who received whole body irradiation with 27 mJ /cm2 dosage of ultraviolet B light (290-320 nm). Samples of venous blood were collected serially in EDTA and immediately chilled. In KBr, plasma samples were ultracentrifuged to provide a rapid separation of proteins of density < and > 1.3 g/ml. Upper and lower phases and serial fractions were analyzed for vitamin D3 (extraction, HPLC), cholesterol (enzyme assay), and human DBP (hDBP) (radial immunodiffusion). Total plasma vitamin D (basal level < 1 ng/ml) increased by 10 h and peaked at 24 h (9 ± 1 ng/ml). 98% of the D3 remained at the density > 1.3 layers for up to 7 d, whereas cholesterol (> 85%) was detected at density < 1.3 and all of the hDBP was at density > 1. Vitamin D is a precursor ofa renal steroid hormone, 1 ,25-dihydroxyvitamin D (1). This precursor, however, cannot be provided by enzymatic synthesis. Its supply depends on ultraviolet (UV) irradiation ofthe skin or absorption from the diet. Since few natural foodstuffs contain much vitamin D (2), observers consider the endogenous, cutaneous production of cholecalciferol to be the physiological mechanism of precursor supply (2-5).Distinct differences are recognized for the cutaneous synthesis vs. diet-derived supply of vitamin D (2, 6). A more efficient and sustained supply of vitamin D is associated with UV irradiation of the skin or parenteral administration of vitamin D (2, 6). In contrast, oral vitamin D consumption leads to rapid but less sustained availability of sterol and a similar pattern of increase in the hepatic metabolite, 25-hydroxycholecalciferol (2, 6-9).Many studies have addressed the mode of vitamin D transport after oral consumption (6), but no direct studies of the plasma transport of cutaneously-derived vitamin D are reported. It has been assumed that vitamin D synthesized in skin enters the blood stream on the plasma binding protein for vitamin D and its metabolites (DBP)' and this assumption is based on in vitro studies ofthe relative potency of vitamin D in binding to DBP as compared with 7-dehydroxycholesterol, lumisterol, tachysterols3 and pre-D3 (10-12). However, other plasma carriers are recognized and some facilitate hepatic ( 13-15) and hepatocyte (16) Cutaneous irradiation. Seven subjects were exposed to 27 mJ/cm2 of . Venous blood samples were taken at baseline, 10,24, 72, and 168 h after the UV-B exposure, immediately 1. Abbreviation used in this paper: DBP, vitamin D binding protein.

The mammalian plasma vitamin D binding protein (DBP), or Gc-globulin, is recognized to have at least two functional properties: sterol binding and G-actin sequestration. Affinity labeling of the sterol binding site with the radioactive electrophilic ligand, 3 beta-(bromoacetoxy)-25-hydroxycholecalciferol, followed by limited proteolysis, permitted the isolation and identification of three overlapping peptides in the amino terminus of the molecule. When G-actin affinity chromatography was applied to other proteolytic fragments, two fragments from the carboxy terminus of the molecule were isolated and identified. Another, large, tryptic fragment displayed both sterol- and actin-binding properties. The amino-terminal assignment of the sterol-binding domain was confirmed by demonstrating sterol-specific binding by an in vitro transcribed and translated product of a mutated rat DBP cDNA encoding a protein truncated in its carboxy terminus. The sterol-binding domain was localized to the region between the first-amino-terminal disulfide bond, and the actin-binding domain was found between residues 350 and 403. A high degree of sequence conservation in these regions was found among human, rat, and mouse DBP's. These functional domain assignments confirm the apparent independence of these two binding activities and help to explain the observed triprotein complex of DBP-actin-DNase I and the competition between DBP and profilin for G-actin binding. Our findings should facilitate more precise delineation of the binding domains by site-directed mutagenesis experiments.