Sensitive measurement of serum 1α,25-dihydroxyvitamin D by liquid chromatography/tandem mass spectrometry after removing interference with immunoaffinity extraction.

Vitamin D plays important roles in bone health and a variety of other pathophysiological conditions. 1α,25-Dihydroxyvitamin D is the active form of vitamin D. Quantification of serum 1α,25-dihydroxyvitamin D is useful for evaluation of several diseases including chronic renal failure, hypoparathyroidism, sarcoidosis, and rickets. Measurement of 1α,25-dihydroxyvitamin D is very challenging due to its low circulating concentration and presence of interfering substances in serum. In this report, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantifying serum 1α,25-dihydroxyvitamin D is described. Lithium adducts of 1α,25-dihydroxyvitamin D were formed prior to mass spectrometry analysis to improve ionization efficiency. We tested a number of different sample preparation procedures and found that immunoaffinity extraction was the method of choice because it completely removed isobaric interferences and matrix effects present in patient serum. Extraction efficiency, expressed as absolute recovery, was greater than 60% in both patient serum and charcoal-stripped serum. This method was linear from 3.4 to 206.2 pg/mL for 1α,25-dihydroxyvitamin D(3) and 3.9 to 212.6 pg/mL for 1α,25-dihydroxyvitamin D(2) with an accuracy of 89.8-98.4% and 97.5-115.7%, respectively. Inter-assay and intra-assay coefficients of variance (CVs) for both analytes at two different concentration levels ranged from 2.5-7.0%. Comparison with a radioimmunoassay for measuring total 1α,25-dihydroxyvitamin D concentration using 40 patient samples showed a Deming regression slope of 0.751, a y-intercept of 0.84 pg/mL, an r value of 0.7909, and a mean percentage difference of -27.1%. Comparison with a reference LC/MS/MS method (n = 20) showed a Deming regression slope of 1.020, a y-intercept of 1.32 pg/mL, an r value of 0.9797, and a mean percentage difference of -2.9%. In conclusion, usage of immunoaffinity extraction enabled a sensitive LC/MS/MS method for quantification of 1α,25-dihydroxyvitamin D in serum.

Antagonistic action of novel 1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs on 25-hydroxyvitamin-D(3)-24-hydroxylase gene expression induced by 1alpha,25-dihydroxy-vitamin D(3) in human promyelocytic leukemia (HL-60) cells.

We have demonstrated that 1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs, (23S)- and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647, TEI-9648, respectively), inhibit HL-60 cell differentiation induced by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], but not differentiation caused by all-trans retinoic acid (D. Miura et al., 1999, J. Biol. Chem. 274, 16392). To assess whether the antagonistic actions of TEI-9647 and TEI-9648 in HL-60 cells are related to 1alpha,25(OH)(2)D(3) breakdown, we investigated their effects on catabolism of 1alpha,25(OH)(2)D(3). In HL-60 cells, the C-24 but not the C-23 side-chain oxidation pathway of 1alpha,25(OH)(2)D(3) has been reported. Here we demonstrate that 1alpha,25(OH)(2)D(3) was metabolized both to 24,25,26,27-tetranor-1alpha,23-(OH)(2)D(3) and 1alpha,25(OH)(2)D(3)-26,23-lactone; thus HL-60 cells constitutively possess both the 24- and the 23-hydroxylases. Metabolism of 1alpha, 25(OH)(2)D(3) was strongly suppressed by 10(-7) M TEI-9647 or 10(-6) M TEI-9648. 1alpha,25(OH)(2)D(3) alone slightly induced 24-hydroxylase gene expression by 8 h with full enhancement by 24-48 h; this induction was inhibited by 10(-6) M TEI-9647 and 10(-6) M TEI-9648 (86.2 and 31.9%, respectively) 24 h after treatment. However, analogs of TEI-9647 and TEI-9648 without the 25-dehydro functionality induced 24-hydroxylase gene expression. These results indicate that TEI-9647 and TEI-9648 clearly mediate their stereoselective antagonistic actions independent of their actions to block the catabolism of 1alpha,25(OH)(2)D(3). Therefore, TEI-9647 and TEI-9648 appear to be the first antagonists specific for the nuclear 1alpha,25(OH)(2)D(3) receptor-mediated genomic actions of 1alpha,25(OH)(2)D(3) in HL-60 cells.

Diastereoselective synthesis, binding affinity for vitamin D receptor, and chiral stationary phase chromatography of hydroxy analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol.

A series of analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol were obtained with an additional hydroxyl in the aliphatic side chain at carbon atom C-24. These analogs were synthesized by direct and diastereo-selective alpha-hydroxylation of enolates derived from respective vitamin D esters using Davies chiral oxaziridines. The use of (+)-(2R,8aS)-(8, 8-dichlorocamphoryl)sulfonyl oxaziridine resulted in (R) stereochemistry of the new asymmetric center for both series of analogs. Similarly, (-)-(2S,8aR) oxaziridine gave (S) analogs. The diastereomeric purity of hydroxy analogs was determined by high-performance liquid chromatography on a chiral stationary phase. High diastereopurity of hydroxylation of vitamin D esters was obtained without the use of any chiral auxiliary. The binding affinity of (24R)-1,24,25-trihydroxycholecalciferol for the calf thymus intracellular vitamin D receptor was one order of magnitude higher than that of the respective (24S)-diastereomer.

Separation from related compounds and assay of calcipotriol by high-performance liquid chromatography.

In this paper, two methods are presented. One involves the separation of calcipotriol, a new synthetic analogue, from two related compounds, specifically cholecalciferol (Vitamin D3) and calcitriol (1,25-dihydroxyvitamin D3). The other involves the isolation and assay of calcipotriol from a topical ointment. The study was performed with reversed-phase high-performance liquid chromatography using an RP18 column and ultraviolet detection. Applying the method of Snyder, a mobile phase mixture containing methanol-acetonitrile-water (67:23:10, v/v) was found which achieved a total separation within 18 min. A mobile phase of methanol-water (80:20, v/v) attained a slower elution of calcipotriol. For isolation and assay of calcipotriol from an ointment (Daivonex), dissolution in chloroform gave the highest recovery (> 98%). The isolation and assay process can be performed within 2 h.

Fluorimetric assay of 1 alpha,25-dihydroxyvitamin D3 in human plasma.

The first fluorimetric method for assaying plasma 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is described. Lipid extracts from human plasma were successively purified by a Bond Elut NH2 column and a normal-phase HPLC column, and the 1,25-(OH)2D3 fractions obtained were fluorescence-labelled with 4-[2-(6,7-dimethoxy-4-methtyl-3-oxo-3,4-dihydroquinoxalyl)et hyl]-1, 2,4-triazoline-3,5-dione (DMEQ-TAD). The fluorescent adducts of 1,25-(OH)2D3 were analyzed by HPLC on a reversed-phase column, after extensive elimination of the degradation products of excess reagents, by a Bond Elut PSA column followed by a normal-phase HPLC column. The intra- and inter-assay coefficients of variation were within 10% in both assays. The analytical recoveries of standard 1,25-(OH)2D3 added to plasma were quantitative. The present fluorimetric assay was compared with the established radioreceptor assay (RRA) used in measuring plasma 1,25-(OH)2D3 concentration. The regression analysis of the data afforded a relationship y = 1.049x - 2.657 and a correlation coefficient (r) of 0.900.

Tandem immunoaffinity chromatography for plasma 1 alpha,25-dihydroxyvitamin D3 utilizing two antibodies having different specificities: a novel and powerful pretreatment tool for 1 alpha,25-dihydroxyvitamin D3 radioreceptor assays.

We report here a novel and powerful pretreatment method for radioreceptor assays (RRAs) for human plasma 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) based on "Tandem" immunoaffinity chromatography (Tandem IAC). Two antibodies having different specificities were each immobilized on agarose gel with cyanogen bromide to produce immunosorbents which were stable and repeatedly usable. An ethyl ether extract of plasma was applied to the first affinity column, from which 1,25(OH)2D3 could be preferentially eluted and separated from 1 alpha-deoxy type metabolites. The effluent was then submitted to the second column, and the 1,25(OH)2D3 retained was eluted after non- or weakly-absorbed interfering substances were washed out. This procedure allowed efficient purification without careful handling or strict time-management in the entire operation and enabled avoiding preparative high-performance liquid chromatography (HPLC) from RRA even with a conventional chick intestinal vitamin D receptor. Mean (+/- SD) plasma 1,25(OH)2D3 values of 56 normal subjects and 10 patients with chronic renal failure, obtained with this Tandem IAC/RRA system, were 36.4 (8.7) and 11.2 (4.0) pg/ml, respectively. The Tandem IAC will also be useful for developing immunoassays or gas chromatography-mass spectrometry of 1,25(OH)2D3.

Further oxidation of hydroxycalcidiol by calcidiol 24-hydroxylase. A study with the mature enzyme expressed in Escherichia coli.

The coding region of the cDNA for rat kidney calcidiol 24-hydroxylase (P450cc24), which is involved in calcium homeostasis in animals, was inserted into an expression vector pKK223-3. The recombinant plasmid was formed in a specific manner without deletion or substitution of any parts of the coding region of the cDNA. When the resulting plasmid was introduced into Escherichia coli JM109, the recombinant cells produced a protein which was immunoreactive to an antibody against P450cc24. When the cell-free extract of the transformed cells was incubated with calcidiol together with bovine adrenodoxin and NADPH-adrenodoxin reductase, not only hydroxycalcidiol but also other metabolites such as oxocalcidiol and oxohydroxycalcidiol were produced. Similarly, calcitriol was converted not only to calcitetrol but also to oxocalcitriol and oxohydroxycalcitriol. These results indicate that a single enzyme expressed in the bacteria is responsible for all these successive reactions.

Modification of 1 alpha,25-dihydroxyvitamin D3 metabolism by introduction of 26,26,26,27,27,27-hexafluoro atoms in human promyelocytic leukaemia (HL-60) cells: isolation and identification of a novel bioactive metabolite, 26,26,26,27,27,27-hexafluoro-1 alpha,23(S),25-trihydroxyvitamin D3.

To study whether the introduction of fluoro atoms into C-26 and C-27 positions on the 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] molecule could affect metabolism in human promyelocytic leukaemia (HL-60) cells, we compared the metabolism of 26,26,26,27,27,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 [26,27-F6-1 alpha,25(OH)2D3] and 1 alpha,25(OH)2D3 in HL-60 cells. 26,27-F6-1 alpha,25(OH)2D3 was mainly converted into a new bioactive metabolite, 26,26,26,27,27,27-hexafluoro-1 alpha,23(S),25- trihydroxyvitamin D3 [26,27-F6-1 alpha,23(S),25(OH)3D3], but not into 26,26,26,27,27,27-hexafluoro-1 alpha,24(R),25-trihydroxyvitamin D3 [26,27-F6-1 alpha,24(R),25(OH)3D3] in HL-60 cells. 26,27-F6-1 alpha,23(S),25(OH)3D3 was identified by combinations of h.p.l.c., u.v. spectroscopy and g.c.-mass spectrometry. Evidence is presented that 26,27-F6-1 alpha,25(OH)2D2 was metabolized to 26,27-F6-1 alpha,23(S),25(OH)3D3 by C-23 hydroxylation as a first step of the metabolism, and the 23-hydroxylated bioactive metabolite was accumulated in the cells, whereas 1 alpha,25(OH)2D3 was initially deactivated and metabolized to 1 alpha,24(R),25(OH)3D3 by C-24 hydroxylation through a side-chain oxidation pathway resulting in C23-C24 cleavage, yielding 24,25,26,27-tetranor-1 alpha,23(OH)2D3 in HL-60 cells. These results show that 26,27-F6-1 alpha,25(OH)2D3 and 1 alpha,25(OH)2D3 are metabolized by different metabolic pathways in HL-60 cells.

Gross and separative determination of 1 alpha,25-dihydroxyvitamin D2 and D3 in plasma using calf thymus receptor.

Determination of 1 alpha,25-dihydroxyvitamin D [1,25(OH)2D, gross amounts of 1,25(OH)2D2 and 1,25(OH)2D3] and separative determination of 1,25(OH)2D2 and 1,25(OH)2D3 in plasma using calf thymus receptor have been investigated. A lipid extract from 1 ml of plasma is applied to a Bond Elut C18OH column and an eluate corresponding to 1,25-(OH)2D including both 1,25(OH)2D2 and 1,25(OH)2D3 is applied to calf thymus receptor to assay a gross amount of the two compounds. On the other hand, when separative assay of the two compounds is performed, the 1,25(OH)2D eluate obtained from the Bond Elut C18OH column is further applied to HPLC using a Zorbax SIL column with 5% isopropanol in methylene chloride as a developing solvent to separate the two compounds from one another. The separated eluates are independently applied to the receptor to assay the two compounds. Since less amounts of unknown components non-specifically bound to interfering concomitants besides 1,25(OH)2D exist in the calf thymus receptor, complicated purification steps to eliminate the concomitants are unnecessary. The detection limit by this method is 1.25 pg/tube which is sensitive enough for a routine method to assay 1,25(OH)2D in plasma.

Subcellular localization and partial purification of the 25-hydroxyvitamin D3 1-hydroxylation reaction in the chick myelomonocytic cell line HD-11.

Hypercalcemia in human granuloma-forming diseases like sarcoidosis results from the endogenous overproduction of 1,25-dihydroxyvitamin D [1,25-(OH)2D] by disease-activated tissue macrophages. The recent identification of an immortalized chick myelomonocytic cell line, HD-11, that constitutively expresses a 25-hydroxyvitamin D (25-OHD) 1-hydroxylation reaction has alleviated dependence on studying primary macrophage cultures with no replicative potential in vitro. In these experiments we established conditions for the maximal expression of the HD-11 cell 25-OHD3-1-hydroxylation reaction and localized this activity to the mitochondrial fraction. On a per cell basis, the activity of HD-11 cell 25-OHD3 1-hydroxylation reaction was comparable to that in primary cultures of chick renal tubular epithelial cells, which express the authentic renal 25-OHD3 1-hydroxylase. Maximal product yield was achieved after incubation of HD-11 cells with 200 nM 25-OHD3 for 3 h. Although adherent monolayers possessed 3- to 4-fold more capacity for hormone production than cells in suspension, suspended cells exhibited easily detectable 25-OHD3 catalytic activity (0.58 +/- 0.08 pmol per 10(6) cells per h; +/- SEM), 50% of which remained solubilized in a sonicate of suspended cells cleared of nuclei and plasma membrane. Subcellular localization disclosed 91% of the residual activity to be concentrated in the mitochondrial subfraction. A detergent-solubilized extract of this mitochondrial subfraction contained 1.9 +/- 0.3 pmol 1,25-(OH)2D3 synthetic capacity per mg protein. The catalytic activity (1-hydroxylase activity) was concentrated 20.2-fold after chromatography on octyl-amino agarose and was associated with 0.054 nmol cytochrome P450 per mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and identification of vitamin D3, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 in Solanum malacoxylon incubated with ruminal fluid.

It has been shown that Solanum malacoxylon contains 1 alpha,25-dihydroxyvitamin D3-glycoside. The presence of vitamin D3 and 25-hydroxyvitamin D3 has also been suggested. In the present study vitamin D3 and three of its metabolites, including 1 alpha,25-dihydroxyvitamin D3, were detected in plant leaf extracts preincubated with ruminal fluid (SMRF). Extraction of SMRF with non-polar organic solvents and purification of the lipid extract by TLC followed by HPLC yielded nine ultraviolet-absorbing (264 nm) peaks. Four of them comigrated on a Zorbax-Sil HPLC column with synthetic standards of vitamin D3, 25-hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3, respectively. These compounds were unequivocally identified by means of mass spectrometry. The results confirm that Solanum malacoxylon contains, in addition to 1 alpha,25-dihydroxyvitamin D3, vitamin D3, 25-hydroxyvitamin D3 and possibly other as yet unidentified derivatives. As 1,24,25-trihydroxyvitamin D3 is absent in plant extracts not incubated with ruminal fluid, the data also indicate that rumen microbes may convert 1 alpha,25-dihydroxyvitamin D3 into 1,24,25-trihydroxyvitamin D3.

The use of octadecyl-bonded microparticulate silica in the separation of free and bound fractions during saturation analysis of vitamin D metabolites.

The use of octadecyl-bonded microparticulate silica to separate free and bound fractions during the saturation analysis of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D has been investigated. A slurry of octadecyl-bonded silica in an appropriate incubation buffer was prepared and used in parallel with a conventional dextran-coated charcoal suspension in several assay procedures. Standard curves, non-specific binding and plasma values were compared. A competitive protein binding assay for 25-hydroxyvitamin D and two radioreceptor assays and one radioimmunoassay for 1,25-dihydroxyvitamin D were investigated. In most cases the octadecyl-bonded silica preparation gave the more favourable results; its action was rapid, time- and temperature-independent, and it produced low non-specific binding and higher B0 values in all the assays examined. It was in our hands easier to use than dextran-coated charcoal. The use of octadecyl-bonded silica is recommended as an efficient agent for the separation of free and bound fractions in the saturation analysis of vitamin D metabolites.

Variations of vitamin D-like reactivity in the crustacean Orchestia cavimana during the molt cycle.

An investigation into vitamin D-like molecules has been performed on whole extracts of the terrestrial amphipod Orchestia cavimana, using a sensitive nonequilibrium assay employing 1,25-(OH)2 D receptor from calf thymus. Relatively large amounts of these secosteroid-like molecules were observed and they varied in concentration according to the stages of the molt cycle. The amplitude of these variations reaches a ratio of about 40 from the minimum in premolt to the intermolt sharp peak.

Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway.

About a decade ago calcitroic acid was isolated as a major side chain cleaved water-soluble metabolite of 1,25-dihydroxyvitamin D3 [Esvelt, R. P., Schnoes, H. K., & Decula, H. F. (1979) Biochemistry 18, 3977]. Presently, calcitroic acid is being considered as the major excretory form of 1,25-dihydroxyvitamin D3. However, the exact site or sites of calcitroic acid production and the possible side chain modified intermediary metabolites that may be formed during the conversion of 1,25-dihydroxyvitamin D3 into calcitroic acid are not fully understood. In the mean time there have been many advances in our understanding of the side-chain metabolism of 1,25-dihydroxyvitamin D3. It is now well established that both the kidney and the intestine metabolize 1,25-dihydroxyvitamin D3 through the C-24 oxidation pathway according to the following steps: 1,25-dihydroxyvitamin D3----1,24,25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3-----1,23,25-trihydroxy-24-oxovitamin D3. Recently, we identified 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-23 alcohol) as a major side chain cleaved lipid-soluble metabolite of 1,25-dihydroxyvitamin D3 and further extended the aforementioned C-24 oxidation pathway in the kidney by demonstrating 1,23,25-trihydroxy-24-oxovitamin D3 as the precursor of C-23 alcohol [Reddy, G. S., Tserng, K. Y., Thomas, B. R., Dayal, R., & Norman, A. W. (1987) Biochemistry 26, 324]. In this present study, we investigated the metabolic fate of 1,25-dihydroxyvitamin D3 (3 X 10(-10) M) in the perfused rat kidney and identified calcitroic acid as the major water-soluble metabolite of 1,25-dihydroxyvitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of 25-hydroxyvitamin D3 metabolism in chick embryo.

We investigated the time course of the development of renal 25-hydroxyvitamin D-1-hydroxylase and 25-hydroxyvitamin D-24-hydroxylase in chick embryos grown in the presence and absence of the eggshell. In embryos with the eggshell, the specific activity (SA) of 25-hydroxyvitamin D-1-hydroxylase in kidney homogenates increased from 0.68 fmol X min-1 X mg protein-1 at 12 days of gestation to a peak of 2.55 +/- 0.50 fmol X min-1 X mg-1 protein-1 at 17 days. In contrast, the SA of 25-hydroxyvitamin D-24-hydroxylase decreased from 2.5 fmol X min-1 X mg protein-1 to 0.90 +/- 0.25 fmol X min-1 X mg protein-1 during the interval. The total plasma calcium was significantly reduced in embryos without shells at 14 to 15 days of gestation (1.1 +/- 0.1 mM, mean +/- SE) compared with normal embryos of the same gestation (2.3 +/- 0.3 mM, P less than 0.002). In embryos without the eggshell, renal 25-hydroxyvitamin D-1-hydroxylase increased from 6.0 to 8.2 +/- 0.6 fmol X min-1 X mg protein-1 at 17 days of gestation and was from four- to sixfold higher than corresponding enzymatic activities for intact embryos. The increased enzyme activity resulting from loss of the eggshell was due to an increase in Vmax. The findings indicate that renal 25-hydroxyvitamin D-1-hydroxylase and 25-hydroxyvitamin D-24-hydroxylase in the chick embryo exhibit activity and show a large capacity for regulation in response to changes in calcium metabolism.

Isolation and identification of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, a new metabolite of 1,25-dihydroxyvitamin D3 produced in rat kidney.

A new metabolite of vitamin D3 was produced in vitro by perfusing rat kidneys with 1,25-dihydroxyvitamin D3 (4 X 10(-6) M). It was isolated and purified from the lipid extract of the kidney perfusate by high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, chemical derivatization, and chemical synthesis, the new metabolite was identified as 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Along with the new metabolite, three other previously identified metabolites, namely, 1,24,25-trihydroxyvitamin D3, 1,25-dihydroxy-24-oxovitamin D3, and 1,23,25-trihydroxy-24-oxovitamin D3, were also isolated. The new metabolite was also formed when 1,23,25-trihydroxy-24-oxovitamin D3 was used as the substrate. Thus, the new metabolite fits into the following metabolic pathway: 1,25-dihydroxyvitamin D3----1,24(R),25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3----1,23,25-trihydroxy-24-oxovitamin D3----1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Further, we used 1 alpha,25-dihydroxy[1 beta-3H]vitamin D3 in the kidney perfusion system and demonstrated 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 as the major further metabolite of 1,25-dihydroxyvitamin D3, circulating in the final perfusate when kidneys were perfused with 1,25-dihydroxyvitamin D3 (6 X 10(-10) M) for 4 h. The biological activity of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-3 alcohol) and its metabolic relationship to 1-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D3 (calcitroic acid or C-23 acid), the other previously identified side-chain cleavage metabolite of 1,25-dihydroxyvitamin D3, are unknown and are presently undergoing investigation.