Rapid and genomic biological responses are mediated by different shapes of the agonist steroid hormone, 1alpha,25(OH)2vitamin D3.

The hormone 1alpha,25(OH)2vitamin D3 (1,25-D) produces biological responses via both genomic and rapid mechanisms. The genomic responses are linked to a nuclear receptor, while the rapid responses are believed to utilize other signal transduction pathways that are likely linked to a putative cell membrane receptor for 1,25-D. The natural seco-steroid, 1,25-D, is capable of facile rotation about its 6,7 single carbon bond to permit generation of a continuum of potential ligand shapes extending from the 6-s-cis (6C) to the 6-s-trans (6T). To identify the shape of the conformer(s) that can serve as agonists for the genomic and rapid responses, we synthesized two families of analogs that were locked in either the 6T or 6C conformation. We found that 6T-locked analogs were inactive or significantly less active than 1,25-D in both rapid responses (transcaltachia or the rapid stimulation of intestinal Ca2+ absorption in perfused chick intestine, stimulation of whole cell chloride currents in osteoblastic ROS 17/2.8 cells, and stimulation of phosphorylation of mitogen-activated protein kinase in promyelocytic NB4 leukemic cells) and in genomic responses (induction of osteocalcin in human MG-63 osteoblastic cells). For genomic responses, the 6C-locked analogs bound poorly to the nuclear receptor and were much less potent than 1,25-D. In contrast, the 6C-locked analogs were potent agonists of the three rapid responses studied and had activities equivalent to 1,25-D. These results demonstrate that the signal transduction pathways that support rapid and genomic responses can discriminate between different shapes of the conformationally flexible 1,25-D.     

Side-chain modified vitamin D analogs require activation of both PI 3-K and erk1,2 signal transduction pathways to induce differentiation of human promyelocytic leukemia cells

Synthetic analogs of vitamin D for potential use in differentiation therapy should selectively regulate genes necessary for differentiation without inducing any perturbations in calcium homeostasis. PRI-1906, an analog of vitamin D2, and PRI-2191, an analog of vitamin D3 bind nuclear vitamin D receptor (nVDR) with substantially lower affinity than 1,25-dihydroxyvitamin D3 (1,25-D3), but have higher differentiation-inducing activity as estimated in HL-60 leukemia cellmodel. To examine how their increased differentiation-inducing activity is regulated we tested the hypothesis that membrane-mediated events, unrelated to nVDR, take part in the differentiation in response to PRI-1906 and PRI-2191. The induction of leukemia cell differentiation in response to the analogs of vitamin D was inhibited by LY294002 (phosphatidylinositol 3-kinase inhibitor), PD98059 (inhibitor of MEK1,2, an upstream regulator of extracellular-signal regulated kinase) and rapamycin (p70S6K inhibitor) pointing out that activation of signal transduction pathways unrelated to nVDR is necessary for differentiation. On the other hand, inhibition of cytosolic phospholipase A2 accelerated the differentiation of HL-60 cells induced by either 1,25-D3 or by the vitamin D analogs suggesting possible existence of a feedback loop between extracellular-signal regulated kinases and phospholipase A2.     

Vitamin D compounds in leukemia

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D3,] possess in vitro multiple anti-cancer activities including growth arrest, induction of apoptosis and differentiation of a variety of different types of malignant cells. However, its use as a therapeutic agent is hindered by its calcemic effects. Analogs of 1,25(OH)(2)D3 have enhanced anti-tumor activity, with reduced calcemic effects. However, limited clinical studies using vitamin D compounds have not yet achieved major clinical success. Nevertheless, pre-clinical studies suggest that the combination of either 1,25(OH)(2)D3 or its analogs with other agents can have additive or synergistic anti-cancer activities, suggesting future clinical studies.     

Chemistry and conformation of vitamin D molecules

1,25-Dihydroxyvitamin D₃ (1,25) is a structurally unique steroid hormone because it not only possesses the complete 25-hydroxycholesterol side chain, but most notably, it possesses a seco-B triene structure (it lacks a B-ring and is usually depicted in a non-steroidal, extended conformation). In contrast, the classical steroid hormones possess a truncated side chain (progesterone, cortisol, and aldosterone) or no side chain (estradiol and testosterone) and they all possess the fully intact ABCD steroid rings. These structural differences render the seco-B-steroid 1,25 considerably more conformationally flexible. Since 1,25 is now known to target a myriad of tissues where specific interactions occur to produce an array of biological responses, it is of interest to determine whether different topologies of 1,25 (resulting from different conformational orientations of 1,25) are necessary to interact effectively at the different target sites. The array of biological responses include both non-genomic and genomic effects and there is considerable promise for the efficacy of 1,25 analogs as chemotherapeutic agents in a variety of human disease states. For the non-genomic calcium transport response of transcaltachia, the finding that two 6-s-cis locked analogs, 1,25-dihydroxyprevitamin D₃ (pre-1,25) and 1,25-dihydroxylumisterol₃ (1,25-Lumi), are equipotent to 1,25, points strongly to the involvement of the 6-s-cis conformer of 1,25 as the biologically active conformer. Since there is a continuum of easily interconvertible 6,7-single bond conformers of the seco-B ring available to 1,25, conformational minima (either local or global) may have little to do with the manner in which 1,25 is bound to receptor. For the genomic calcium transport response, and for other genomic (or non-genomic) effects, there is no clear evidence whether the steroidal (s-cis) or non-steroidal (s-trans) conformer of 1,25 is involved. In order to address this matter further, efforts are underway to evaluate other conformationally locked analogs of 1,25 which might mimic either the planar 6-s-trans-1,25 or some intermediate conformer between it and the planar-6-s-cis form.     

Intestinal Na(+)/Ca(2+) exchanger protein and gene expression are regulated by 1,25(OH)(2)D(3) in vitamin D-deficient chicks

The role of 1,25(OH)(2)D(3) on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)(2)D(3) were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)(2)D(3) simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)(2)D(3). NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)(2)D(3). Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)(2)D(3) treatment. Rapid effects of 1,25(OH)(2)D(3) on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)(2)D(3) on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)(2)D(3) enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.     

Differential effects of Vitamin D analogs on calcium transport

It is well known that the efficiency of intestinal active calcium transport is regulated by the Vitamin D receptor pathway and Vitamin D analogs seem to exhibit differential effects on intestinal active calcium transport. To investigate the molecular basis for the difference among Vitamin D analogs, we tested three Vitamin D analogs: 1,25-dihydroxyvitamin D(3), 19-nor-1,25-dihydroxyvitamin D(2), and 1alpha-hydroxyvitamin D(2) ex vivo and in vitro. In 5/6 nephrectomized rat intestinal active calcium transport, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effects on intestinal active calcium transport at all the concentrations tested, while 1alpha-hydroxyvitamin D(2) at 0.33 and 0.67 microg/kg and 1,25-dihydroxyvitamin D(3) at 1microg/kg significantly stimulated calcium transport. In Caco-2 cells, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effect on calcium transport, while 1,25-dihydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(2) (the active form of 1alpha-hydroxyvitamin D(2)) stimulated calcium transport by 934 and 501% at 0.1microM, respectively. 1,25-Dihydroxyvitamin D(2) potently induced the expression of CALB3 and TRPV6 mRNA with an EC(50) of 0.3 and 1.0nM, whereas 19-nor-1,25-dihydroxyvitamin D(2) was 10-fold less potent than 1,25-dihydroxyvitamin D(2) in inducing CALB3 and TRPV6 mRNA. The three Vitamin D analogs had no significant effect on the expression of PMCA1 mRNA. These Vitamin D analogs did not change the expression of Vitamin D receptor (VDR) up to 10nM, but stimulated CYP24A1 expression in a dose-dependent manner with the potency in the order of 1,25-dihydroxyvitamin D(3)>1,25-dihydroxyvitamin D(2)=19-nor-1,25-dihydroxyvitamin D(2). These results suggest that the differential effect of Vitamin D analogs on stimulating intestinal and Caco-2 calcium transport may be in part due to its different effect on stimulating CALB3 and TRPV6 mRNA expression.     

Breast cancer cell regulation by high-dose Vitamin D compounds in the absence of nuclear vitamin D receptor

1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) inhibits growth and induces apoptosis in breast cancer cells in vivo and in vitro. To examine the role of the Vitamin D receptor (VDR) in mediating the actions of 1,25D(3) at nanomolar and micromolar concentrations, mammary epithelial tumor cell lines generated in wild type (WT) and VDR knockout (VDRKO) mice were utilized. WT cells express VDR and are growth inhibited by 1,25D(3) and synthetic analogs EB1089 and CB1093 at 1nM concentrations, while VDRKO cells do not express VDR and are insensitive to Vitamin D compounds at concentrations up to 100nM. In the current studies, we have confirmed and extended these previous observations. At nanomolar concentrations of 1,25D(3) and all analogs tested, including EB1089, CB1093, MC1288, and KH1230, WT cells are growth inhibited and exhibit apoptotic morphology, while VDRKO cells show no growth inhibition or apoptosis. At concentrations of 1-10microM, however, 1,25D(3) and synthetic analogs induce growth inhibition and apoptotic morphology in both WT and VDRKO cell lines. These data indicate that nanomolar concentrations of 1,25D(3) and analogs mediate growth regulatory effects via mechanisms requiring the nuclear VDR, but that micromolar concentrations of Vitamin D compounds can exert non VDR-mediated effects.     

Vitamin D analogs: therapeutic applications and mechanisms for selectivity

The vitamin D endocrine system plays a central role in mineral ion homeostasis through the actions of the vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], on the intestine, bone, parathyroid gland, and kidney. The main function of 1,25(OH)(2)D(3) is to promote the dietary absorption of calcium and phosphate, but effects on bone, kidney and the parathyroids fine-tune the mineral levels. In addition to these classical actions, 1,25(OH)(2)D(3) exerts pleiotropic effects in a wide variety of target tissues and cell types, often in an autocrine/paracrine fashion. These biological activities of 1,25(OH)(2)D(3) have suggested a multitude of potential therapeutic applications of the vitamin D hormone for the treatment of hyperproliferative disorders (e.g. cancer and psoriasis), immune dysfunction (autoimmune diseases), and endocrine disorders (e.g. hyperparathyroidism). Unfortunately, the effective therapeutic doses required to treat these disorders can produce substantial hypercalcemia. This limitation of 1,25(OH)(2)D(3) therapy has spurred the development of vitamin D analogs that retain the therapeutically important properties of 1,25(OH)(2)D(3), but with reduced calcemic activity. Analogs with improved therapeutic indices are now available for treatment of psoriasis and secondary hyperparathyroidism in chronic kidney disease, and research on newer analogs for these indications continues. Other analogs are under development and in clinical trials for treatment of various types of cancer, autoimmune disorders, and many other diseases. Although many new analogs show tremendous promise in cell-based models, this article will limit it focus on the development of analogs currently in use and those that have demonstrated efficacy in animal models or in clinical trials.     

Differential effects of 1,25-dihydroxyvitamin D3 upon intestinal vitamin D3-dependent calbindin (a 28,000-dalton calcium binding protein) and its mRNA in D-replete and D-deficient chickens

The effect of vitamin D3 status upon the responsiveness of chick intestinal epithelium to exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was studied. Intestinal calbindin [A recent consensus decision was made to redesignate the vitamin D-dependent calcium binding protein as "calbindin-D28K" (R.H. Wasserman (1985) in Vitamin D: Chemical, Biochemical, and Clinical Update (Norman, A.W., Schaefer, K., Grigoleit, H.-G., and Herrath, D.V., Eds.), pp. 321-322, de Gruyter, Berlin/New York).] protein and intestinal calbindin mRNA were quantitated in birds which had been raised on a vitamin D3-deplete (-D) or on a vitamin D3-replete (+D) diet. 1,25(OH)2D3 stimulated intestinal calbindin mRNA levels in -D chickens in a proportional dose-dependent manner, when measured at both 12 and 48 h after administration of the hormone. A first increase was observed with 1,25(OH)2D3 concentrations between 0.065 and 0.65 nmol. The maximal stimulation achieved by 1,25(OH)2D3 (6.5-18 nmol) in -D tissue was approximately 10-fold over the calbindin mRNA levels present in vehicle-treated birds. The increase of calbindin mRNA in -D birds was associated with a similar dose-dependent increase in calbindin protein in 1,25(OH)2D3-treated -D birds after 12 or 48 h. In +D intestine, while exogenous 1,25(OH)2D3 also increased calbindin mRNA levels in a dose-dependent fashion, the maximal stimulation observed after 5 h (1.2- to 2-fold) was clearly less than that observed in -D intestine. In contrast to -D birds, intestinal calbindin levels in +D birds were decreased by administration of exogenous 1,25(OH)2D3. Administration of 32.5 to 65 nmol 1,25(OH)2D3 resulted in an approximately 1.8-fold repression compared to vehicle-treated birds. This differential responsiveness between +D and -D intestines with respect to 1,25(OH)2D3 was not explained either by differences in the uptake in the chromatin fractions of these tissues or by metabolism of radiolabeled 1,25(OH)2D3. Dietary withdrawal of vitamin D3 led to a gradual decline in ambient intestinal calbindin levels, while intestinal sensitivity to 1,25(OH)2D3 was restored. These findings suggest that vitamin D3 status regulates intestinal responsiveness to the seco-steroid 1,25(OH)2D3.     

The effect of vitamin D analogs and of vitamin D-binding protein on lymphocyte proliferation

In the absence of vitamin D-binding protein (DBP), 1,25-(OH)2D3 at 10(-12) M significantly inhibited the [3H]thymidine incorporation in human lymphocytes during mixed lymphocyte cultures (MLC) or after phyto-hemaglutinin (PHA) stimulation. In the presence of a physiological concentration of DBP (5 x 10(-6) M), the concentration of 1,25-(OH)2D3 required for inhibition was 10(-10) M (for PHA-cultures) and 10(-9) M (for MLC). Several vitamin D analogs were compared for their inhibitory action on PHA stimulation. In the absence of DBP, the concentration necessary for 50% inhibition of [3H]thymidine incorporation ranged from 10(-12) M [1,25-(OH)2D3 and 24,24-F2-1,25-(OH)2D3], over 10(-10) M [1,24R, 25-(OH)3D3; 1,25S, 26-(OH)3D3 and 26,27-F6-1,25-(OH)2D3] and 10(-8) M [25 OHD3 and 24,25-(OH)2D3] to 10(-6) M [calcitriol-lactone]. This rank order correlates with the binding affinity of the various analogs to the cytoplasmic 1,25-(OH)2D3-receptor. DBP counteracted the inhibitory effect of all analogs and the degree of counteraction was directly proportional to the binding affinity between DBP and the vitamin D analog. DBP thus decreased the in vitro inhibitory action of 1,25-(OH)2D3 and its analogs on lymphocyte proliferation. Of all analogs tested, only 1,25-(OH)2D3 had a significant effect at a physiological concentration.     

Novel Gemini vitamin D(3) analogs have potent antitumor activity

The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D(3) has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose-response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH)(2)D(3). Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH)(2)D(3) in causing 50% clonal growth inhibition (ED(50)) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10(-9)M, 48h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10(-11)M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH)(2)D(3). Annexin V assay showed that BXL-01-0120 (10(-10)M, 4 days) induced significantly (p<0.05) more apoptosis than 1,25(OH)(2)D(3). In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.     

Probing the vitamin D sterol-binding pocket of human vitamin D-binding protein with bromoacetate affinity labeling reagents containing the affinity probe at C-3, C-6, C-11, and C-19 positions of parent vitamin D sterols

The multiple physiological properties of vitamin D-binding protein (DBP) include organ-specific transportation of vitamin D(3) and its metabolites, manifested by its ability to bind vitamin D sterols with high affinity. In the present investigation we probed the vitamin D sterol-binding pocket of human DBP with affinity labeling analogs of 25-hydroxyvitamin D(3) ?25-OH-D(3) and 1, 25-dihydroxyvitamin D(3) ?1,25(OH)(2)D(3) containing bromoacetate alkylating probe at C-3 (A-ring), C-6 (triene), C-11 (C-ring), and C-19 (exocyclic methylene) of the parent sterol. Competitive binding assays with DBP showed approximately 22-, 68-, and 2000-fold decrease in the binding of 1,25(OH)(2)-D(3)-11-BE, 25-OH-D(3)-3-BE, and 25-OH-D(3)-6-BE, respectively, compared to that seen with 25-OH-D(3), while there was no significant difference in the DBP-binding affinity of 25-OH-D(3)-19-BE and 25-OH-D(3). Surprisingly, ?(14)C25-OH-D(3)-11-BE and ?(14)C1, 25(OH)(2)-D(3)-19-BE failed to label DBP despite high-affinity DBP-binding, indicating the absence of any nucleophilic amino acid in the vicinity of their bromoacetate moiety to form a covalent bond, while these analogs are inside the binding pocket. In contrast, ?(14)C25-OH-D(3)-6-BE and ?(14)C25-OH-D(3)-3-BE specifically labeled DBP. BNPS-skatole digestion of DBP labeled with ?(14)C25-OH-D(3)-3-BE or ?(14)C25-OH-D(3)-6-BE produced two peptides (M(r) 17,400 and 33,840), with radioactivity associated with the N- and C-terminal peptides, respectively, raising the possibility that either different areas of the same vitamin D sterol-binding pocket, or different domains of DBP might be labeled by these analogs. Successful affinity labeling of recombinant domain I (1-203) of DBP with both reagents indicated that different areas of the same vitamin D-binding pocket (domain I) were labeled. These affinity analogs are potentially useful for "mapping" the vitamin D sterol-binding pocket and developing a functional model.     

Vitamin D status and its relation to age and body mass index

BACKGROUND/AIMS: While numerous studies have examined 25(OH)-vitamin D(3) (25-D) concentrations and their relation to parathyroid hormone (PTH) levels there is only limited information on the interrelation between 25-D, 1,25(OH)(2)-vitamin D(3) (1,25-D) and PTH. It was the aim of this study to assess the vitamin D endocrine system and its relation to age and body mass index (BMI). METHODS: This cross-sectional study comprised a convenience sample of 483 adults which attended the endocrinology outpatient service of a university hospital in the years 2002-2004. RESULTS: The mean concentrations of 25-D, 1,25-D, calcium and PTH were 21.0 +/- 10.6 ng/ml, 47.9 +/- 21.7 pg/ml, 9.48 +/- 0.48 mg/dl and 51.0 +/- 27.2 pg/ml, respectively. 25-D was related (p < 0.01) to BMI, age, PTH and 1,25-D. After correction for 25-D, we found no relation between BMI and 1,25-D. PTH was related (p < 0.01) to serum calcium, BMI, age and 1,25-D (p = 0015). There was a seasonal variation in both, 25-D and 1,25-D serum concentrations: 25-D levels were lowest in January and increased until July while the nadir and zenith of 1,25-D were found in April and October, respectively. CONCLUSION: Since BMI was negatively related to 25-D the prevalence of 25-D deficiency (<8.8 ng/ml) increased from 8.8% in subjects with BMI <30 kg/m(2) to 15.0% in subjects with BMI >30 kg/m(2). BMI, age and season should be taken into account when assessing a patients vitamin D status and more aggressive vitamin D supplementation should be considered for obese subjects.     

Immunochemical studies on the putative plasmalemmal receptor for 1, 25-dihydroxyvitamin D(3). III. Vitamin D status

The effect of vitamin D status on levels of the putative 1, 25(OH)(2)D(3) membrane receptor (pmVDR) was studied in chick intestine, kidney, and brain. Western analyses and assays for specific [(3)H]1,25(OH)(2)D(3) binding indicated that, in intestine, pmVDR levels were greatest in -D chicks relative to +1,25D and +D animals (P < 0.05). In kidney, protein levels and specific binding followed the order +D > +1,25D, -D. In brain, vitamin D status did not affect protein levels or specific binding levels. In tissue from normal chicks, both protein and specific binding followed the order of intestine > kidney > brain membranes. Intestinal cells were further evaluated for the effect of 1,25(OH)(2)D(3) on selected "rapid responses." Extrusion of (45)Ca in response to 130 pM 1, 25(OH)(2)D(3) in vitro was greater in cells from -D chicks than from +1,25D or normal birds. Analyses of signal transduction events revealed diminished hormone-induced intracellular calcium oscillations (as assessed by fura-2 fluorescence), and lack of steroid-enhanced protein kinase (PK) A activity in intestinal epithelial cells from -D chicks relative to +D chicks. PK C activation by 130 pM 1,25(OH)(2)D(3) was approximately twofold in cells from +D or -D chicks. The combined results indicate that vitamin D status differentially affects the pmVDR in intestine, kidney, and brain. In intestine, vitamin D deficiency differentially affects (45)Ca handling, intracellular calcium oscillations, PK A and PK C activities in response to 1,25(OH)(2)D(3).     

Production of 10-oxo-19-nor-25-hydroxyvitamin D3 by solubilized kidney mitochondria from chick and rat

Cholate-solubilized chick kidney mitochondria that 1-hydroxylated 25-hydroxyvitamin-D3 (25-OH-D3) upon reconstitution also produced 10-oxo-19-nor-25-OH-D3, which co-eluted with 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) on normal phase high performance liquid chromatography (HPLC) with hexane:propanol-2 (9:1), the traditional chromatographic system for isolating 1,25-(OH)2-D3. The 10-oxo derivative was separated from 1,25-(OH)2-D3 by normal phase HPLC with dichloromethane:propanol-2 (19:1) or by reverse phase HPLC with methanol:water (4:1). Unlike 1,25-(OH)2-D3 production, formation of 10-oxo-19-nor-25-OH-D3 did not require a source of reducing equivalents and was blocked by the antioxidants, diphenyl-rho-phenylenediamine, and butylated hydroxytoluene, implicating a free radical or peroxidative synthetic mechanism. Rat kidney mitochondria solubilized with cholate or with cholate and Emulgen 911 produced 10-oxo-19-nor-25-OH-D3 but no detectable 1 alpha,25-(OH)2-D3. These results stress the importance of careful identification of vitamin D metabolites produced in vitro and suggest the use of alternate chromatographic conditions for isolating 1,25-(OH)2-D3 or inclusion of antioxidants in the assay of solubilized 1 alpha-hydroxylase to eliminate contamination of 1,25-dihydroxyvitamin D3 with 10-oxo-19-nor-25-OH-D3.