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Properties of guinea-pig kidney 25-hydroxyvitamin D3 1 alpha-hydroxylase assayed by isotope dilution-mass spectrometry.

ABSTRACT: 1. A highly specific and accurate method based on isotope dilution-mass spectrometry was used for characterization of the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase in untreated guinea pigs with a normal vitamin D status. In previous work, the properties of the enzyme had been determined in rachitic animals only. 2. With intact mitochondria, the reaction required the presence of citric acid-cycle intermediates. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had an inhibitory effect on the isocitrate-supported reaction, indicating that energy-dependent transhydrogenation is of importance. Mitochondrial respiratory-chain inhibitors (cyanide, rotenone, antimycin A) had no effect on the hydroxylation. CO had an inhibitory effect, suggesting participation of a species of cytochrome P-450 in the reaction. A fraction solubilized from mitochondria by cholate became catalytically active in 1 alpha-hydroxylation of 25-hydroxyvitamin D3 after addition of ferredoxin and ferredoxin reductase. The isocitrate-supported reaction catalysed by crude mitochondria had an apparent Km of about 1 microM. 3. An atmosphere containing 50% O2 was found to be necessary for optimal activity. It is thus possible that O2 may be a limiting factor under normal conditions in vivo. 4. The results demonstrate that the mammalian renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is a cytochrome P-450-dependent mixed-function oxidase with properties similar to those previously reported for the same enzyme system in chicken. The present assay and animal system seem to be suitable for further studies on the mechanism of regulation of the mammalian renal 25-hydroxyvitamin D3 1 alpha-hydroxylase under conditions when the vitamin D status is normal.

SUBMITTER: Hagenfeldt Y

PROVIDER: S-EPMC1148886 | biostudies-other | 1988 Mar

REPOSITORIES: biostudies-other

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Project description:The inaccuracy of routine serum 25-hydroxyvitamin D measurements hampers the interpretation of data in patient care and public health research. We developed and validated a candidate reference measurement procedure (RMP) for highly accurate quantitation of two clinically important 25-hydroxyvitamin D metabolites in serum, 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3]. The two compounds of interest together with spiked deuterium-labeled internal standards [d 3-25(OH)D2 and d 6-25(OH)D3] were extracted from serum via liquid-liquid extraction. The featured isotope-dilution LC-MS/MS method used reversed-phase chromatography and atmospheric pressure chemical ionization in positive ion mode. A pentafluorophenylpropyl-packed UHPLC column together with isocratic elution allowed for complete baseline resolution of 25(OH)D2 and 25(OH)D3 from their structural C-3 isomers within 12 min. We evaluated method trueness, precision, potential interferences, matrix effects, limits of quantitation, and measurement uncertainty. Calibration materials were, or were traceable to, NIST Standard Reference Materials 2972. Within-day and total imprecision (CV) averaged 1.9 and 2.0% for 25(OH)D3, respectively, and 2.4 and 3.5% for 25(OH)D2, respectively. Mean trueness was 100.3% for 25(OH)D3 and 25(OH)D2. The limits of quantitation/limits of detection were 4.61/1.38 nmol/L for 25(OH)D3 and 1.46/0.13 nmol/L for 25(OH)D2. When we compared our RMP results to an established RMP using 40 serum samples, we found a nonsignificant mean bias of 0.2% for total 25(OH)D. This candidate RMP for 25(OH)D metabolites meets predefined method performance specifications (?5% total CV and ?1.7% bias) and provides sufficient sample throughput to meet the needs of the Centers for Disease Control and Prevention Vitamin D Standardization Certification Program. Graphical abstract Bias assessment using NIST standard reference materials. Legend CDC mean mass fractions (ng/g) ± U 95 (6 replicates per mean). NIST-certified mass fractions (ng/g) ± U 95 from the Certificates of Analysis.

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Properties of guinea-pig kidney 25-hydroxyvitamin D3 1 alpha-hydroxylase assayed by isotope dilution-mass spectrometry.

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