Hydroxyvitamin D assays: An historical perspective from DEQAS.

Recent years have seen a substantial increase in demand for 25-hydroxyvitamin D (25-OHD) assays. DEQAS (the Vitamin D External Quality Assessment Scheme) has been monitoring the performance of these assays since 1989. The first DEQAS distribution was in June 1989 and results were submitted by 13 laboratories in the UK, two of which used HPLC/UV; the rest used ligand binding assays with a tritium tracer. Inter-laboratory CVs (ALTM) ranged from 29.3% (42.7nmol/L) to 53.7% (20.0nmol/L). Currently the scheme has participants in 56 countries using 30 methods or variants of methods. In January 2017, 918 participants returned results and inter-laboratory CVs (ALTM) ranged from 10.3% (73.1nmol/L) to 15.3% (29.4nmol/L). Over the last 27 years, there have been a number of significant milestones in assay development. The first major advance was the development of an iodinated 25-OHD tracer by Hollis and Napoli in 1992, subsequently used in an RIA kit marketed by DiaSorin. This and other commercial radioimmunoassays that followed brought 25-OHD assays within reach of many more non-specialist routine laboratories. With the introduction of fully automated non-isotopic assays without solvent extraction, measurement of 25-OHD became available to any clinical chemistry laboratory with an appropriate analytical platform. However, as the limitations of these non-extraction assays became apparent more laboratories started using LC-MS/MS methodology. Meanwhile the variable accuracy of 25-OHD methods has been addressed by the Vitamin D Standardization Program (VDSP) which encourages manufacturers to produce methods traceable to the reference measurement procedures (RMPs) of NIST, University of Ghent and the Centers for Disease Control and Prevention (CDC). DEQAS changed to an accuracy-based scheme in 2013 and now assesses assay accuracy against the NIST RMP. This review will use DEQAS results and statistics to chart the historical development in 25-OHD assay technology and highlight some of the problems encountered in obtaining reliable results for this most challenging of analytes.

Quality assessment of vitamin D metabolite assays used by clinical and research laboratories.

The Vitamin D External Quality Assessment Scheme (DEQAS) was launched in 1989 and monitors the performance of 25-hydroxyvitamin D (25-OHD) and 1,25- dihydroxyvitamin D (1,25(OH)2D) assays. In April 2015 a pilot scheme for 24,25-dihydroxyvitamin D (24,25(OH)2D) was introduced. The 25-OHD scheme is accuracy - based with target values assigned by the NIST Reference Measurement Procedure (RMP) for 25-OHD2 and 25-OHD3. A similar method is used to assign values for 3-epi-25-OHD. Five samples of human serum are distributed quarterly to over 1000 participants in 58 countries (April 2016) and clinical laboratories are expected to submit results within approximately 5 weeks. Research laboratories with assays run less frequently are not given a deadline. Archived samples with NIST- assigned values are also available. Performance is assessed on the first four samples with the fifth reserved for investigations e.g. recovery experiments or to assess the influence of other serum constituents such as lipids. DEQAS provides rapid feedback, with an on-line preliminary report available immediately after a participant submits results and a comprehensive report soon after the results deadline. In 2015, DEQAS investigations revealed that several 25-OHD immunoassays under-recovered 25-OHD2 and 25-OHD results were falsely low on a sample with a modestly raised triglyceride concentration. An RMP for 1,25 (OH)2D is not yet available and results are judged against the Method Mean. Free advice is available from the DEQAS Advisory Panel which includes experts on methodology and biostatistics. DEQAS collaborates closely with the Vitamin D Standardization Program (VDSP) and both organizations have successfully worked with participants and manufacturers to improve the accuracy of vitamin D assays.

Proficiency testing of 25-hydroxyvitamin D (25-OHD) assays.

The Vitamin D External Quality Assessment Scheme (DEQAS) has been monitoring 25-OHD assay performance since 1989. The scheme has expanded rapidly in recent years and has 670 participants in 35 countries (July 2009). Five samples of human serum are distributed quarterly and the results analyzed to give an All-Laboratory Trimmed Mean (ALTM) and SD. Each participant has internet access to a preliminary report after submission of results and, following the results deadline, a final report is e-mailed to designated staff in each laboratory. The last 15 years has seen an improvement in mean inter-laboratory imprecision (CV), from 32.0% (1994) to 15.3% (2009) and most major methods are now giving results within plus or minus 7.4% of the ALTM (2009). DEQAS has regularly conducted and reported on a number of investigations into the performance of 25-OHD methods. A gas chromatography-mass spectrometry (GC-MS) reference method for 25-OHD is under development and will be used to assess whether the ALTM remains the most appropriate target for DEQAS samples.

Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation.

The International Quality Assessment Scheme for Vitamin D metabolites (DEQAS) was introduced in 1989. Initially, the aim was to improve the reliability of 25-hydroxyvitamin D (25-OHD) assays but the scheme was extended in 1997 to include 1,25-dihydroxyvitamin D (1,25(OH)(2)D). DEQAS has 95 members in 18 countries (January 2003). Five serum samples are distributed quarterly and participants are given up to 6 weeks to return their results for statistical analysis. The majority of participants use commercial kits for both analytes. A performance target was set by an advisory panel in 1997 and, at present, requires participants to get 80% or more of their results within +/-30% of the All-Laboratory Trimmed Mean (ALTM). The performance targets are under continual review. In 2003, 59% of participants met the target (cf. 52% in 2000). A questionnaire, distributed in January 2003, requested information on methods and the interpretation of results. Reference ranges varied but there was reasonable agreement on the 25-OHD concentrations below which Vitamin D supplementation was advised. A minority (22%) of respondents was unsure whether Vitamin D(3) or Vitamin D(2) was used to treat patients in their locality. The majority (52%) of assays for 1,25(OH)(2)D were done 'on demand' and others for apparently spurious reasons. Most respondents thought participation in DEQAS extremely important and the planned introduction of on-line reporting should enhance its value.

In vitro metabolism of 19-nor-1alpha, 25-(OH)2D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites.

The active vitamin D analog, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25-(OH)2D2), has a similar structure to the natural vitamin D hormone, 1a,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), but lacks the C10-19 methylene group and possesses an ergosterol/ vitamin D2 rather than a cholesterol/vitamin D3 side chain. We have used this analog to investigate whether any of these structural features has any effect upon the type and rate of in vitro metabolism observed. Using a vitamin D-target cell, the human keratinocyte, HPK1A-ras, we observed formation of a number of metabolites, three of which were purified by extensive HPLC and conclusively identified by a combination of GC-MS and chemical derivatization as 19-nor-1alpha,24,25-(OH) 3D2, 19-nor-1alpha,24,25,26-(OH) 4D2, and 19-nor-1alpha,24,25,28-(OH)4,D2. The first metabolite is probably a product of the vitamin D-inducible cytochrome P450, P450cc24 (CYP24), while the latter two metabolites are likely to be further metabolic products of 19-nor-1alpha,24,25-(OH)3D2. These hydroxylated metabolites resemble those identified by other workers as products of the metabolism of 1alpha,25-(OH)2D2 in the perfused rat kidney. It therefore appears from the similar metabolic fate of 19-nor-1alpha,25-(OH)2D2 and 1alpha,25-(OH)2D2 that the lack of the C10-19 methylene group has little effect upon the nature of the lipid-soluble metabolic products and the rate of formation of these products seems to be comparable to that of products of 1alpha,25-(OH)2D3 in vitamin D-target cells. We also found extensive metabolism of 19-nor-1alpha,25(OH)2D2 to water-soluble metabolites in HPK1A-ras, metabolites which remain unidentified at this time. When we incubated 19-nor-1alpha,25-(OH)2D2 with the liver cell line HepG2, we obtained only 19-nor-1alpha,24,25-(OH)3D2. We conclude that 19-nor-1alpha,25-(OH)2D2 is efficiently metabolized by both vitamin D-target cells and liver cells.

Metabolism of a 20-methyl substituted series of vitamin D analogs by cultured human cells: apparent reduction of 23-hydroxylation of the side chain by the 20-methyl group.

We describe here for the first time the effect of introducing a 20-methyl group on the side-chain metabolism of the vitamin D molecule. Using a series of 20-methyl-derivatives of 1alpha,25-(OH)2D3 incubated with two different cultured human cell lines, HPK1A-ras and HepG2, previously shown to metabolize vitamin D compounds, we obtained a series of metabolic products that were identified by comparison to chemically synthesized standards on HPLC and GC-MS. 24-Hydroxylated-, 24-oxo-hydroxylated-, and 24-oxo-23-hydroxylated products of 20-methyl-1alpha,25-(OH)2D3 were observed, but the efficiency of 23-hydroxylation was low as compared with that of the natural hormone and, in contrast to 1alpha,25-(OH)2D3, no truncated 23-alcohol was formed from the 20-methyl analog. These data, taken together with results from other analogs with changes in the vicinity of the C17-C20 positions, lead us to speculate that such changes must alter the accessibility of the C-23 position to the cytochrome P450 involved. Using the HepG2 cell line, we found evidence that the 24S-hydroxylated product of 20-methyl-1alpha,25-(OH)2D3 predominates, implying that the liver cytochrome involved in metabolism is a different isoform. Studies with a more metabolically resistant analog of the series, 20-methyl-Delta(23)-1alpha,25-(OH)2D3, gave the expected block in 23- and 24-hydroxylation, and evidence of an alternative pathway, namely 26-hydroxylation. 20-Methyl-Delta(23)-1alpha,25-(OH)2D3 was also more potent in biological assays, and the metabolic studies reported here help us to suggest explanations for this increased potency. We conclude that the 20-methyl series of vitamin D analogs offers new perspectives into vitamin D analog action, as well as insights into the substrate preferences of the cytochrome(s) P450 involved in vitamin D catabolism.

Potent suppression of the parathyroid glands by hydroxylated metabolites of dihydrotachysterol(2).

BACKGROUND: Dihydrotachysterol(2), a licensed pharmaceutical, is hydroxylated to 25-hydroxydihydrotachysterol(2) (25(OH)DHT(2)) and 1 alpha,25-dihydroxydihydrotachysterol(2) (1 alpha,25(OH)(2)DHT(2)) in man. We have compared the biological activity of these metabolites with calcitriol and the 'non-calcaemic' analogue, 22-oxacalcitriol (OCT) in bovine parathyroid cell cultures and in rats. METHODS: The effect of each sterol on parathyroid hormone (PTH) secreted by primary bovine parathyroid cells was measured. High-performance liquid chromotography and gas chromotography-mass spectrometry were used to investigate in vitro 25(OH)DHT(2) metabolism. Rats were given a single intraperitoneal injection or five daily injections of each sterol, and changes in ionized calcium and PTH were measured. RESULTS: In vitro, all sterols suppressed PTH significantly. Calcitriol and OCT were of similar potency, but 1 alpha, 25(OH)(2)DHT(2) and 25(OH)DHT(2) required higher concentrations to suppress PTH equally. We were unable to detect metabolism of 25(OH)DHT(2) to 1 alpha,25(OH)(2)DHT(2) in vitro. In rats, a single dose of 0.5 microg/rat of calcitriol increased ionized calcium at 30 and 40 h (statistically significant at 48 h). 50 microg of OCT and 1 alpha,25(OH)(2)DHT(2) did not cause significant hypercalcaemia at 48 h, although 1 alpha,25(OH)(2)DHT(2) caused hypercalcaemia at 30 h. In contrast, 50 microg of 25(OH)DHT(2) caused hypercalcaemia at 48 h but not at 30 h. Five daily doses of 0.001 microg/rat of calcitriol caused a significant rise in calcium and a 50% fall in PTH. OCT and 1 alpha,25(OH)(2)DHT(2) at 0.025 and 0.5 microg/rat respectively caused similar suppression of PTH but without hypercalcaemia. CONCLUSION: 1 alpha,25(OH)(2)DHT(2) and 25(OH)DHT(2) are potent suppressors of PTH in vitro and in vivo. 25(OH)DHT(2) may be active by virtue of its pseudo-1 alpha-hydroxyl group. Hypercalcaemia caused by a single dose of 1 alpha,25(OH)(2)DHT(2) appeared to be more transient than calcitriol. Five daily doses of 1 alpha, 25(OH)(2)DHT(2) and OCT could achieve 50% suppression of PTH without significant increments in ionized calcium. In contrast, suppression of PTH by calcitriol was associated with significant increments in ionized calcium. These data suggest that like OCT, 1 alpha, 25(OH)(2)DHT(2) can dissociate calcaemic actions from parathyroid-suppressing actions in a manner that may be therapeutically useful.

Characterization of 3-epi-1alpha,25-dihydroxyvitamin D3 involved in 1alpha,25-dihydroxyvitamin D3 metabolic pathway in cultured cell lines.

Using six different cultured cell models representing osteoblast, intestine, kidney and keratinocyte, we have demonstrated that 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) is metabolized into 3-epi-1alpha,25(OH)2D3 in vitamin D-target cells. Although differences existed in the amount of 3-epi-1alpha,25(OH)2D3 formed with different cell types, it was apparent that 1alpha,25(OH)2D3 was subjected to metabolism both through the C24-oxidation and 3-epimerization pathways. Time course and dose response studies showed that the production of 3-epi-1alpha,25(OH)2D3 was enzymatic. It is interesting to note that this epimerization proceeded from 3beta towards 3alpha unidirectionally, and this conversion was not inhibited by ketoconazole. These data suggest that cytochrome P450 related enzymes including the 24-hydroxylase would not affect this reaction. The biological activity of 3-epi-1alpha,25(OH)2D3 was found to be lower than the native 1alpha,25(OH)2D3 in suppressing of proliferation of HL-60 cells, while the affinity of 3-epi-1alpha,25(OH)2D3 for vitamin D-binding protein was 2.5-fold higher than that of 1alpha,25(OH)2D3. The results indicate that 3-epimerization may change the pharmacokinetics and catabolism of 1alpha,25(OH)2D3 in vitamin D-target cells.

Contribution of several metabolites of the vitamin D analog 20-epi-22-oxa-24a,26a,27a-tri-homo-1,25-(OH)(2) vitamin D(3) (KH 1060) to the overall biological activity of KH1060 by a shared mechanism of action.

The synthetic 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) analog 20-epi-22-oxa-24a,26a,27a-tri-homo-1,25-(OH)(2)vitamin D(3) (KH1060) is considerably more potent than its cognate hormone. The mechanism of action of KH1060 includes interaction with the vitamin D receptor (VDR). We previously showed that KH1060 increases VDR stability in ROS 17/2.8 osteoblastic cells by inducing a specific conformational change in the VDR. KH1060 is metabolized, both in vivo and in vitro, into several stable products. In the present study, we investigated whether these metabolites might contribute to the increased biological activity of KH1060. We found that the potencies of two of these metabolites, 24a-OH-KH1060 and 26-OH-KH1060, were similar to that of 1,25-(OH)(2)D(3) in inducing osteocalcin production by the osteoblast cell line ROS 17/2.8. This report further showed that these metabolites had the same effects as KH1060 on VDR: they increased VDR stability in ROS 17/2.8 cells, while limited proteolytic analysis revealed that they caused a conformational change in the VDR, resulting in an increased resistance against proteolytic cleavage. Furthermore, as shown in gel mobility shift assays, both compounds clearly induced VDR binding to vitamin D response elements. Together, these results show that the potent in vitro activity of KH1060 is not only directed by the effects on the VDR conformation/stabilization of the analog itself, but also by certain of its long-lived metabolites, and emphasizes the importance of detailed knowledge of the metabolism of synthetic hormonal analogs.

Unique 24-hydroxylated metabolites represent a significant pathway of metabolism of vitamin D2 in humans: 24-hydroxyvitamin D2 and 1,24-dihydroxyvitamin D2 detectable in human serum.

We have produced evidence for a new metabolic pathway for vitamin D2 in humans involving the production of 24-hydroxyvitamin D2 (24OHD2) and 1,24-dihydroxyvitamin D2 [1,24-(OH)2D2]. These metabolites were produced after either a single large dose (10(6) IU) of vitamin D2 or repeated daily doses between 10(3) and 5 x 10(4) IU. We developed assay systems for the metabolites in human serum and showed that in some chronically treated patients, the concentration of 1,24-(OH)2D2 equalled that of 1,25-(OH)2D2 at about 100 pmol/L. The metabolites were identified by high performance liquid chromatography with diode array spectrophotometry for 24OHD2 and by high resolution gas chromatography-mass spectrometry for 1,24-(OH)2D2. We show that 1,24-(OH)2D2 synthesis can be stimulated by PTH, indicating a renal origin for this metabolite and postulate that it is formed from 24OHD2, which may be synthesized in liver. We conclude from this study that vitamin D2 gives rise to two biologically active products, 1,24-(OH)2D2 and 1,25-(OH)2D2, and that 1,24-(OH)2D2 could be an attractive naturally occurring analog of 1,25-(OH)2D3 for clinical use.

The vitamin D analog, KH1060, is rapidly degraded both in vivo and in vitro via several pathways: principal metabolites generated retain significant biological activity.

Vitamin D analogs are valuable drugs with established and potential uses in hyperproliferative disorders. Lexacalcitol (KH1060) is over 100 times more active than 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3], as judged by in vitro antiproliferative and cell differentiating assays. The underlying biochemical reasons for the increased biological activity of KH1060 are unknown, but are thought to include 1) metabolic considerations in addition to explanations based upon 2) enhanced stability of KH1060-liganded transcriptional complexes. In this study we explored the in vivo and in vitro metabolism of KH1060. We established by physicochemical techniques the existence of multiple side-chain hydroxylated metabolites of KH1060, including 24-, 24a-, 26-, and 26a-hydroxylated derivatives as well as side-chain truncated forms. KH1060 metabolism could be blocked by the cytochrome P450 inhibitor, ketoconazole. KH1060 was not an effective competitor of C24 oxidation of 1alpha,25-(OH)2D3. Certain hydroxylated metabolites of KH1060 retained significant biological activity in vitamin D-dependent reporter gene systems (chloramphenicol acetyltransferase). Likewise, those metabolites accumulating in the target cell culture models in metabolism studies, particularly 24a-hydroxy-KH1060 and 26-hydroxy-KH1060, retained biological activities superior to those of 1alpha,25-(OH)2D3 in native gene expression systems in vitamin D target cells (osteopontin and P450cc24). We conclude that KH1060 is rapidly metabolized by a variety of cytochrome P450-mediated enzyme systems to products, many of which retain significant biological activity in vitamin D-dependent assay systems. These results provide an explanation for the considerable biological activity advantage displayed by KH1060 compared with 1alpha,25-(OH)2D3 in various in vitro assay systems.

Metabolism of the vitamin D analog EB 1089: identification of in vivo and in vitro liver metabolites and their biological activities.

1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3'(E)-dien -1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB 1089) is a novel analog of the vitamin D hormone, calcitriol that has been modified in the side-chain resulting in an increased metabolic stability relative to other side-chain modified analogs (e.g. calcipotriol and 22-oxacalcitriol). To further investigate the metabolism of EB 1089, we set out to study this metabolism both in the rat in vivo as well as in the postmitochondrial liver fractions from rat, man, and minipig in vitro. The same pattern of metabolism was observed in all biological systems employed, both in vivo and in vitro, namely 26- and 26a-hydroxylation of EB 1089. The same metabolites were produced using cultured cell systems (Shankar et al., see this issue). All the possible isomers of 26- and 26a-hydroxy EB 1089 were synthesised and these were compared to biologically generated material using HPLC, NMR, and GC-MS techniques. The predominant natural isomer observed in vitro and in vivo in rats as well as in vitro in humans was identified to be (25S),26R-hydroxy EB 1089. The biological activities of the EB 1089 metabolites on cell growth regulation were 10- to 100-fold lower than that of EB 1089. The effects of the metabolites on calcium metabolism in vivo were comparable to the effect of EB 1089; however, these effects were reduced for the major metabolite in rat and man and for the isomers of 26a-hydroxy EB 1089. We conclude that EB 1089 is metabolised by a different route of side-chain metabolism than calcitriol and that this may explain its relative metabolic stability in pharmacokinetic experiments in vivo compared to that of other vitamin D analogs.

Metabolism of the vitamin D analog EB1089 by cultured human cells: redirection of hydroxylation site to distal carbons of the side-chain.

1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3' (E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089) is a novel synthetic analog of 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D3] with potential for use in the treatment of hyperproliferative disorders. It has an altered side-chain structure compared to 1,25-(OH)2D3, featuring 26,27 dimethyl groups, insertion of an extra carbon atom (24a) at C-24, and two double bonds at C-22,23 and C-24,24a. In vitro metabolism of EB1089 was studied in a human keratinocyte cell model, HPK1A-ras, previously shown to metabolize 1,25-(OH)2D3. Four metabolites were formed, all of which possessed the same UV chromophore as EB1089, indicating the retention of the side-chain conjugated double bond system. Two metabolites were present in sufficient quantities to identify them as 26-hydroxy EB1089 (major product) and 26a-hydroxy EB1089 (minor product), based on mass spectral analysis and cochromatography with synthetic standards. Similar metabolites were generated in vivo and using a liver postmitochondrial fraction in vitro (Kissmeyer et al., companion paper). Studies with the human hepatoma Hep G2 gave rise to 2 isomers of 26-hydroxy EB1089. Studies using ketoconazole, a general cytochrome P450 inhibitor, implicated cytochrome P450s in the formation of the EB1089 metabolites. COS-1 transfection cell experiments using vectors containing CYP27 and CYP24 suggest that these cytochrome P450s are probably not involved in 26- or 26a-hydroxylation of EB1089. Other experiments that examined the HPK1A-ras metabolism of related analogs containing only a single side-chain double bond: 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1' (E)-en-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (MC1473; double bond at C-22,23) and 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-3'(E)-en-1'-yl)-9, 10-secopregna-5(Z),7(E),10(19)-triene (MC1611; double bond at C-24,24a) revealed that the former compound was subject to 24-hydroxylation and the latter compound was mainly 23-hydroxylated. Metabolism experiments involving EB1089, MC1473, and MC1611 in competition with [1 beta-3H]1,25-(OH)2D3 in HPK1A-ras confirmed that CYP24 is probably not involved in the metabolism of EB1089 whereas, in the case of MC1473 and MC1611, it does appear to carry out side-chain hydroxylation. Our interpretation is that the conjugated double bond system in the side-chain of EB1089 is responsible for directing the target cell hydroxylation to the distal positions, C-26 and C-26a. We conclude that EB1089 is slowly metabolized via unique in vitro metabolic pathways, and that these features may explain the relative stability of EB1089 compared to other analogs in vivo.

Use of gas chromatographic-mass spectrometric techniques in studies of androst-16-ene and androgen biosynthesis in human testis; cytosolic specific binding of 5alpha-androst-16-en-3-one.

Homogenates of histologically normal human testis from three men were incubated separately with pregnenolone, 16-dehydropregnenolone, 5alpha-pregnane-3,20-dione, 3beta-hydroxy-5alpha-pregnan-20-one and androsta-5,16-dien-3beta-ol (androstadienol) in the presence of NADPH in a study of androst-16-ene and androgen biosynthesis. After the addition of internal standards and initial extraction and purification, metabolites were identified using gas chromatography-mass spectrometry (GC-MS) and monitoring selectively for three principal ions in each case at the appropriate GC retention time. Quantification was achieved by comparison with calibration lines for authentic steroids, together with the appropriate internal standards, prepared by monitoring three ion fragments for each analyte. In all experiments, androstadienol was found to be the major androst-16-ene metabolite of pregnenolone (seven times the control, i.e. endogenous, quantity; 19.8 +/- 3 ng/100 mg homogenate protein, mean +/- SEM, n = 9). Pregnenolone was also converted to androsta-4,16-dien-3-one (androstadienone) with three times the endogenous quantity (44 +/- 10 ng/100 mg homogenate protein, mean +/- SEM, n = 9) being formed. The formation of testosterone occurred only in trace amounts in the incubations of testis taken from one man (a 69-yr-old) but appreciable yields (six times endogenous levels 90 +/- 7 ng/100 mg homogenate protein, mean +/- SEM, n = 9) were found with testes from two younger men. Only traces of 5alpha-dihydrotestosterone were detected. Using androstadienol as the substrate, androstadienone was shown to be the major metabolite (approximately 10 times greater than control incubations) together with 5alpha-androst-16-en-3alpha- and 3beta-ols at approximately twice the endogenous quantities (5 ng/100 mg homogenate protein). In some incubations with androstadienol, 5alpha-androst-16-en-3-one (5alpha-androstenone) was formed (32 +/- 1 ng/100 mg homogenate protein/h; mean +/- SEM, n = 3); surprisingly, no endogenous 5alpha-androstenone could be detected. No evidence was obtained for the production of testosterone or 5alpha-DHT from androstadienol. Using cytosolic fractions of human testis, specific (displaceable) binding of 5alpha-androstenone was determined, with binding sites of approximately 200 fmol/mg tissue and a Ka of approximately 8 nmol/l.

Anti-proliferative activity and target cell catabolism of the vitamin D analog 1 alpha,24(S)-(OH)2D2 in normal and immortalized human epidermal cells.

Vitamin D analogs represent valuable new agents for the suppression of proliferation of a variety of cell types, including those of the skin. One such analog is the vitamin D2 metabolite, 1 alpha,24(S)-dihydroxyvitamin D2, which binds strongly to the vitamin D receptor and induces vitamin D-dependent gene expression in vitro. In the work described here, we studied the anti-proliferative activity and target cell metabolism of 1 alpha,24(S)-dihydroxyvitamin D2 in cells of human epidermal origin. We found this analog to be equally potent in its anti-proliferative effect to the hormone 1 alpha,25-dihydroxyvitamin D3. Furthermore, 1 alpha,24(S)-dihydroxyvitamin D2 was metabolized by the human keratinocyte cell line HPK1A-ras at a slower rate than either 1 alpha,25-dihydroxyvitamin D3 or calcipotriol, a drug used effectively in the treatment of psoriasis. We characterized the metabolic products of 1 alpha,24(S)-dihydroxyvitamin D2 as a mixture of side-chain truncated and hydroxylated products. The main product was identified by GC-MS and NMR techniques as 1 alpha,24(S),26-trihydroxyvitamin D2. The biological activity of this main product was determined in a vitamin D-dependent, growth-hormone reporter gene expression system to be lower than that of the parent molecule. We conclude from these data that 1 alpha,24(S)-dihydroxyvitamin D2 is a valuable new anti-proliferative agent with a slower rate of catabolism by cells of epidermal origin. Preliminary evidence suggests that the parent molecule, and not its products, is responsible for this biological activity in vitro.

In vitro metabolism of the vitamin D analog, 22-oxacalcitriol, using cultured osteosarcoma, hepatoma, and keratinocyte cell lines.

Using four cultured cell models representing liver, keratinocyte, and osteoblast, we have demonstrated that the vitamin D analog, 22-oxacalcitriol is degraded into a variety of hydroxylated and side chain truncated metabolites. Four of these metabolic products have been rigorously identified by high pressure liquid chromatography, diode array spectrophotometry, and gas chromatography-mass spectrometry analysis as 24-hydroxylated and 26-hydroxylated derivatives as well as the cleaved molecules, hexanor-1alpha,20-dihydroxyvitamin D3 and hexanor-20-oxo-1alpha-hydroxyvitamin D3. Comparison with chemically synthesized standards has revealed the stereochemistry of the biological products. Although differences exist in the amounts of products formed with the different cell types, it is apparent that 22-oxacalcitriol is subject to metabolism by both vitamin D-inducible and noninducible enzymes. Time course studies suggest that the truncated 20-alcohol is derived from a side chain hydroxylated molecule via a hemiacetal intermediate and the 20-oxo derivative is likely formed from the 20-alcohol. Biological activity measurements of the metabolites identified in our studies are consistent with the view that these are catabolites and that the biological activity of 22-oxacalcitriol is due to the parent compound. These results are also consistent with recent findings of others that the biliary excretory form of 22-oxacalcitriol is a glucuronide ester of the truncated 20-alcohol.

1 alpha,24(S)-dihydroxyvitamin D2: a biologically active product of 1 alpha-hydroxyvitamin D2 made in the human hepatoma, Hep3B.

A major metabolite of the vitamin D analogue 1 alpha-hydroxyvitamin D2 in human liver cells in culture has been identified as 1 alpha,24(S)-dihydroxyvitamin D2 [1 alpha,24(S)-(OH)2D2]. 1 alpha-Hydroxyvitamin D3 incubated with the same cells gives rise to predominantly 25- and 27-hydroxylated products. Our identification of 1 alpha,24(S)-dihydroxyvitamin D2 is based on comparisons of the liver cell metabolite with chemically synthesized 1 alpha,24(S)-(OH)2D2 and 1 alpha,24(R)-(OH)2D2 by using HPLC, GC and GC-MS techniques. The stereochemical orientation of the 24-hydroxyl group was inferred after X-ray-crystallographic analysis of the 24(R)-OH epimer. 1 alpha,24(S)-Dihydroxyvitamin D2 binds strongly to the vitamin D receptor and is biologically active in growth hormone and chloramphenicol acetyltransferase reporter gene expression systems in vitro, but binds poorly to rat vitamin D-binding globulin, DBP. We suggest that this metabolite, 1 alpha,24(S)-(OH)2D2, possesses the spectrum of biological properties to be useful as a drug in the treatment of psoriasis, metabolic bone disease and cancer.