Rats were dosed orally for 14 days with 0.65, 6.5, 65, 650, or 6500 nmol/day vitamin D3 or 0.46, 4.6, 46, 460, or 4600 nmol/day 25-hydroxyvitamin D3 and examined for morbidity, weight loss, plasma phosphorus, and plasma calcium concentration. Vitamin D intoxication was produced, as indicated by hypercalcemia, by 650 nmol/day vitamin D3 and 4600 nmol/day 25-hydroxyvitamin D3; 6500 nmol/day vitamin D3 caused hypercalcemia and death. For all groups of rats, plasma concentrations of vitamin D3, 25-hydroxyvitamin D3, 25-hydroxyvitamin D3-26,23-lactone, 24,25-dihydroxyvitamin D3, 25,26-dihydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 were determined by a multiple assay procedure. The following changes in plasma concentrations of these metabolites occurred as the dose level of vitamin D3 was increased from 0.65 to 650 nmol/day: vitamin D3, 11.3 to 1339 ng/ml; 25-hydroxyvitamin D3, 2.3 to 643 ng/ml; 25-hydroxyvitamin D3-26,23-lactone, <0.06 to 64.5 ng/ml; 24,25-dihydroxyvitamin D3, 0.56 to 73.5 ng/ml; 25,26-dihydroxyvitamin D3, <0.2 to 16.4 ng/ml; 1,25-dihydroxyvitamin D3, 80 to 51 pg/ml. The following changes in plasma concentrations of these metabolites occurred as the dose level of 25-hydroxyvitamin D3 increased from 0.46 to 4600 nmol/day: vitamin D3, undetectable; 25-hydroxyvitamin D3, 6.2 to 688 ng/ml; 25-hydroxyvitamin D3-26,23-lactone, 0.31 to 110 ng/ml; 24,25-dihydroxyvitamin D3, 2.29 to 214 ng/ml; 25,26-dihydroxyvitamin D3, <0.2 to 6.31 ng/ml; 1,25-dihydroxyvitamin D3, 187 to 22 pg/ml. Based on plasma concentrations and relative effectiveness of interaction with the vitamin D receptor protein of in vitro bone and intestinal systems, 25-hydroxyvitamin D3 could be the cause of the vitamin D toxicity, although other unmeasured metabolites of vitamin D3 cannot be ruled out. 25-Hydroxyvitamin D3-26,23-lactone is of interest because it shows the greatest relative increase of all the vitamin D3 metabolites in plasma with increasing dose of vitamin D. Based on relative ultraviolet absorption and rat plasma transport protein binding assay, the lactone has about fourfold the affinity of 24,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 for the rat plasma binding protein.

This chapter discusses a method for the determination of vitamin D and its metabolites in plasma. It has been found that vitamin D must be metabolized before its biological activity can be expressed. Vitamin D is hydroxylated to 25-hydroxyvitamin D (25-OH-D) in the liver and then further hydroxylated in the kidney to either 1,25-dihydroxyvitamin D [1,25-(OH)2D] or 24R,25-dihydroxyvitamin D (24R,25-(OH)2D). The 1,25-(OH)2D is now recognized as the active form of vitamin D in bone mineral mobilization and is exclusively responsible for the initiation of active intestinal absorption of calcium and phosphorus. The methodology outlined in this chapter describes a multiple assay capable of quantitating vitamin D and its metabolites in a single small sample of plasma by means of high-pressure liquid chromatography (HPLC) with an ultraviolet absorbance detector or competitive protein-binding detection. The extraction of vitamin D metabolites from plasma, the purification and separation of the metabolites by conventional column chromatography and HPLC, and their detection by optical absorbance or competitive protein binding are discussed. This method has been put to routine use in author's laboratory to measure metabolite levels in plasma samples from humans, rats, chicks, and cows.

A major vitamin D metabolite was isolated in pure form from the blood plasma of chicks either maintenance levels or large doses of vitamin D3. The isolation involved methanol-chloroform extraction and five column chromatographic procedures. The metabolite purification and elution position on these columns were followed by a competitive protein binding assay. The metabolite was identified, using high- and low-resolution mass spectrometry, 270-MHz proton nuclear magnetic resonance spectrometry, ultraviolet absorption spectrophotometry, Fourier transform infrared spectrophotometry, and specific chemical reactions, as 3 beta,-25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-lactone. The trivial names 25-hydroxyvitamin D3 26,23-lactone or calcidiol 26,23-lactone are suggested for this compound.

A multiple assay capable of reliably determining vitamins D(2) and D(3) (ergocalciferol and cholecalciferol), 25(OH)D(2) (25-hydroxyvitamin D(2)) and 25(OH)D(3) (25-hydroxyvitamin D(3)), 24,25(OH)(2)D (24,25-dihydroxyvitamin D), 25,26(OH)(2)D (25,26-dihydroxyvitamin D) and 1,25(OH)(2)D (1,25-dihydroxyvitamin D) in a single 3-5ml sample of human plasma was developed. The procedure involves methanol/methylene chloride extraction of plasma lipids followed by separation of the metabolites and purification from interfering contaminants by batch elution chromatography on Sephadex LH-20 and Lipidex 5000 and by h.p.l.c. (high-pressure liquid chromatography). Vitamins D(2) and D(3) and 25(OH)D(2) and 25(OH)D(3) are quantified by h.p.l.c. by using u.v. detection, comparing their peak heights with those of standards. 24,25(OH)(2)D and 25,26(OH)(2)D are measured by competitive protein-binding assay with diluted plasma from vitamin D-deficient rats. 1,25(OH)(2)D is measured by competitive protein-binding assay with diluted cytosol from vitamin D-deficient chick intestine. Values in normal human plasma samples taken in February are: vitamin D 3.5+/-2.5ng/ml; 25(OH)D 31.6+/-9.3ng/ml; 24,25(OH)(2)D 3.5+/-1.4ng/ml; 25,26(OH)(2)D 0.7+/-0.5ng/ml; 1,25(OH)(2)D 31+/-9pg/ml (means+/-s.d.). Values in two normal human plasma samples taken in February after 1 week of high sun exposure are: vitamin D 27.1+/-7.9ng/ml; 25(OH)D 56.8+/-4.2ng/ml; 24,25(OH)(2)D 4.3+/-1.6ng/ml; 25,26(OH)(2)D 0.5+/-0.2ng/ml. Values in anephric-human plasma are: vitamin D 2.7+/-0.8ng/ml; 25(OH)D 36.4+/-16.5ng/ml; 24,25(OH)(2)D 1.9+/-1.3ng/ml; 25,26(OH)(2)D 0.6+/-0.3ng/ml; 1,25(OH)(2)D was undetectable.

Methods have been developed for the precise measurement of the major known vitamin D metabolites in a single sample of cow plasma (~5 ml). The procedure involves initial extraction with methylene chloride-methanol followed by chromatography on Sephadex LH-20. 25-Hydroxyvitamin D2 and 25-hydroxyvitamin D3 were determined using high-pressure liquid chromatography and comparing ultraviolet absorption peak height with absorption peak heights of standards. The dihydroxylated metabolites were further purified and resolved by high-pressure liquid chromatography and determined by radioligand binding assays. The assays were employed to measure the total vitamin D metabolite levels in the plasma of paretic and normal dairy cows at parturition. Parturition had no effect on 25-hydroxyvitamin D levels in either group of cows (paretic, 37–44 ng/ml; normal, 35–38 ng/ml). However, normal cows did show lower mean 25-hydroxyvitamin D levels at every sampling period with the lowest levels in both groups occurring at 7 days postpartum. Plasma 25, 26-dihydroxyvitamin D was higher in paretic animals prepartum and at parturition (0.7–1.0 ng/ml) when compared to nonparetic animals (0.4–0.45 ng/ml). Similar levels (0.6 ng/ml) were observed in both groups postpartum. Cows developing parturient paresis showed a significant (P < 0.05) elevation of 1,25-dihydroxyvitamin D at parturition with a maximum level of 350 pg/ml attained at 1 day postpartum compared to prepartum levels of 60 pg/ml. Normal animals also showed a rise in plasma levels of 1,25-dihydroxyvitamin D with a maximum level of 185 pg/ml observed at 1 day postpartum. Plasma 24,25-dihydroxyvitamin D was initially higher in paretic cows (1.9 ng/ml) with a significant (P < 0.05) drop to 1.05 ng/ml occurring at parturition. This level was maintained for 7 days postpartum. The levels of this steroid were maintained at 1.3–1.4 ng/ml in the normal cows throughout the entire sampling period.

A rapid, relatively sensitive and highly accurate method of determining 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 levels in plasma has been devised. The method involves methanol-chloroform extraction of 4 ml of serum, prepurification of 25-hydroxyvitamin D on batch columns of Sephadex LH-20, and detection by absorption at 254 nm coincident with high-pressure liquid chromatography. Recoveries are monitored by adding known amounts of 25-hydroxy-[3H]vitamin D3 to the serum initially and counting the 3H recovered in the 25-hydroxyvitamin D3 peak on high-pressure liquid chromatography. Normal human serum collected during the summer contains 25-hydroxyvitamin D levels of 31.9 ± 1.7 ng/ml (mean ± SE, n = 19).