M. R. Hughes

Radioligand receptor assay for 25-hydroxyvitamin D2/D3 and 1 alpha, 25-dihydroxyvitamin D2/D3.

A competitive protein binding assay for measurement of the plasma concentration of 1 alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3] has been extended to include the immediate precursor of this hormone, 25-hydroxyvitamin D3 (25-OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely. 25-hydroxyvitamin D2 (25-OHD2) and 1alpha, 25-dihydroxyvitamin D2 [1alpha, 25-(OH)2D2]. The target tissue assay system consists of a high affinity cytosol receptor protein that binds the vitamin D metabolites and a limited number of acceptor sites on the nuclear chromatin. By utilizing a series of chromatographic purification steps, a single plasma sample can be assayed for any of the four vitamin D metabolites either individually or combined. Therefore, the assay procedure allows for both the quantitative and qualitative assessment of the total active vitamin D level in a given plasma sample. To show that the binding assay was capable of measuring 1alpha, 25-(OH)2D2 as well as 1alpha, 25 (OH)2D3, two groups of rats were raised. One group, supplemented with vitamin D3, produced assayable material that represented 1alpha, 25-(OH)2D3. The other group, fed only vitamin D2 in the diet, yielded plasma containing only 1alpha, 25-(OH)2D2 as the hormonal form of the vitamin. The circulating concentrations of the two active sterols were nearly identical (15 ng/100 ml) in both groups, indicating that the competitive binding assay can be used to measure both hormonal forms in plasma. In a separate experiment, 1alpha, 25-(OH)2D2 was generated in an in vitro kidney homogenate system using 25-OHD2 as substrate. Comparison of this sterol with 1alpha, 25-(OH)2D3 in the assay system showed very similar binding curves; the D2 form was slightly less efficient (77%). Comparison of the respective 25-hydroxy forms (25-OHD2 vs. 25-OHD3) at concentrations 500-fold that of 1alpha, 25-(OH)2D3, again suggested that the binding of the D2 metabolite was slightly less efficient (71%). Finally, the assay was employed to measure the total active vitamin D metabolite pools in the plasma of normal subjects and patients with varying degrees of hypervitaminosis D. The normal plasma levels of 25-OHD and 1alpha, 25-(OH)2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominately (greater than 90%) in the form of vitamin D3 metabolites in this environment. Typical cases of hypervitaminosis D exhibited approximately a 15-fold increase in the plasma 25-OHD concentration, and a dramatic changeover to virtually all metabolites existing in the form of D2 vitamins. In contrast, the circulating concentration of 1alpha, 25-(OH)2D was not substantially enhanced in vitamin D-intoxicated patients. We therefore conclude that hypervitaminosis D is not a result of abnormal plasma levels of 1alpha, 25-(OH)2D but may be cuased by an excessive circulating concentration of 25-OHD.

1,25-Dihydroxyvitamin D3-glycoside: Identification of a calcinogenic principle of solanum malacoxylon

Abstract The water soluble calcinogenic factor present in the plant Solanum malacoxylon is partially purified by selective extraction and chromatography on silicic acid and then hydrolyzed with a mixed preparation of glycosidases from the sea worm, Charonia lampus . Hydrolysis produces a chloroform soluble factor with biologic characteristics of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), the hormonal form of vitamin D. Purification of this factor is accomplished by chromatography on Sephadex LH-20, silicic acid, and Celite columns, yielding 3 μg of active material. During the isolation, bioactivity (as assessed by the ability of fractions to compete with labeled 1,25-(OH) 2 D 3 for binding to a specific intestinal receptor protein) migrates exactly with authentic tritiated 1,25-(OH) 2 D 3 . The purified factor has an ultraviolet absorption spectrum identical to that of 1,25-(OH) 2 D 3 and analysis via direct probe mass spectrometry yields a parent molecular ion of m/e 416 and a fragmentation pattern indistinguishable from synthetic 1,25-(OH) 2 D 3 hormone. We therefore conclude that the vitamin D-like principle in Solanum malacoxylon is a sterol-glycoside which contains the 1,25-(OH) 2 D 3 molecule as its active sterol component.

Further evidence for the 1,25-dihydroxyvitamin D-like activity of Solanum malacoxylon

Abstract Administration of an aqueous extract of the calcinogenic plant Solanum malacoxylon (S.m.) to vitamin D-deficient or strontium fed chicks produces significant plasma 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) activity within 6 hr. (via radioreceptor assay) and subsequently elicits the appearance of immunoreactive intestinal calcium binding protein. Studies of a purified aqueous extract of S.m. show that it does not compete effectively with radioactive 1,25-(OH)2D3 for binding to the sterols intestinal receptor. However, treatment of the extract with β-glucosidase releases a biologically active substance which is soluble in organic solvents and efficiently competes with labeled sterol for the receptor. This factor migrates exactly with tritiated 1,25-(OH)2D3 on high resolution Celite liquid-liquid partition columns. Thus, S.m. contains a molecule very similar or identical to 1,25-(OH)2D3 which is combined with one or more carbohydrate moieties in the native plant. This glycoside is probably cleaved in vivo before biological activity is attained.

Effect of oral contraceptives on plasma clearance

The effects of chronic steroid contraceptive therapy on drug clearance from plasma were studied by using plasma antipyrine, phenylbutazone, and cholecalciferol half‐lives in women. After 3 mo of oral steroid therapy (Norinyl, 2 mg; norethindrone + mestranol), the antipyrine half‐life was increased in 3 of 6 subjects, phenylbutazone half‐life was not consistently altered, and vitamin Da half‐life was increased in 3 of 4 patients. After 1 to 7 yr of oral steroid therapy, the antipyrine half‐life was longer while taking the contraceptive than when the contraceptive treatment was discontinued in 4 of 6 subjects, whereas that of phenylbutazone was not consistently altered.

Partial purification of the chick intestinal receptor for 1,25-dihydroxy vitamin D by ion exchange and blue dextran–sepharose chromatography

1,25Dihydroxyvitamin Ds (1,25(OH)zDs) is now considered to function analogously to classic steroid hormones. The ultimate site of action of 1,25(OH)2D, is the intestinal mucosa cell nucleus [ 1,2] , where it apparently induces the formation of new mRNAs which code for proteins functional in calcium and phosphate absorption [3-63. The 1,2.5(OH)2D3 sterol is transported to the intestinal chromatin via a specific, high affinity cytoplasmic receptor protein which migrates at 3.7 S in high saltsucrose gradients [7-l 1] . Other than agarose gel filtration of analytical amounts [9], no chromatographic purification of the 1 ,25-(OH)2D, receptor has been reported to date. Other steroid, hormone receptors, such as those for progesterone, have been purified to homogeneity, either by ion-exchange [ 121 or affinity [ 131 chromatography, However, this has proven to be difficult because of the extremely low percentage of cytosolic proteins representing receptor molecules and the high lability of these receptors, especially in more purified states. In the present study, we report that the intestinal cytosol receptor for 1 ,25-(OH)zDs from the chick can be purified approx. 800-fold by ammonium sulfate precipitation, ion-exchange chromatography

1,25-Dihydroxyvitamin D3: mode of action in intestine and parathyroid glands, assay in humans and isolation of its glycoside from Solanum malacoxylon.

Vitamin D3 is metabolized first to 25-hydroxyvitamin D 3 (25-OH-D3) and then to several dihydroxylated products (Fig. 1). The most biologically active of these metabolites (Haussler et al., 1971), la,25-dihydroxyvitamin D3 (1,25-(OH)2D3), is formed in the kidney (Fraser and Kodicek, 1970). The action of vitamin D 3 to mobilize calcium and phosphate from sites such as intestine and bone is thought to be mediated by the 1,25-(OH)2D 3 sterol and, therefore, this metabolite is considered to be the hormonal form of vitamin D (DeLuca, 1974). Furthermore 1,25-(OH)2D3 qualifies as a renal hormone controlling calcium and phosphate metabolism since its biosynthesis is regulated by the calcium and phosphorus status of animals (DeLuca, 1974) and its biochemical action on the target intestine resembles the molecular functioning of classic steroid hormones in their respective target organs (Haussler, 1974). The primary regulated step in the formation of 1,25-(OH)2D3 from the parent vitamin is the la-hydroxylation of 25-OH-Da (Tucker et al., 1973), and the renal la-hydroxylase (la-OHase) enzyme has become the focal point of investigations of the dietary, physiological, and pathological conditions that affect the biosynthesis of 1,25-(OH)2D a. Fig. 1 illustrates that the formation of 1,25-(OH)2Da from 25OH-Da is controlled by calcium (Boyle et al., 1971), parathyroid hormone (PTH) (Garabedian et al., 1972), and phosphate (Tanaka and DeLuca, 1973). Evidence suggests that hypocalcemia stimulates PTH secretion which, in turn, increases the production of 1,25-(OH)zD a (Hughes et al., 1975). Thus, PTH, rather than calcium, may be the dominant modulator of the la-OHase with respect to calcium