Inger Holmberg

25-Hydroxylase activity in subcellular fractions from human liver. Evidence for different rates of mitochondrial hydroxylation of vitamin D2 and D3

25-Hydroxylation of vitamin D2 and D3 was studied in subcellular fractions from human liver, using a technique based on isotope dilution-mass spectrometry. The mitochondrial fraction fortified with isocitrate catalysed 25-hydroxylation of vitamin D3 at a rate of about 10 pmol/mg protein X min. Under the same conditions, the rate of 25-hydroxylation of vitamin D2 was less than 2 pmol/mg protein X min. Crude microsomes fortified with NADPH catalysed 25-hydroxylation of vitamin D3 to a very low extent, and this activity was not linear with the amount of microsomal protein. A higher rate of conversion was obtained with a partially purified cytochrome P-450 fraction in the presence of NADPH-cytochrome P-450 reductase and NADPH. This fraction also catalysed 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. 25-Hydroxylation of vitamin D2 could not be detected, neither with crude microsomes, nor with the microsomal cytochrome P-450 fraction. Since the assay for 25-hydroxyvitamin D2 was less sensitive than that for 25-hydroxyvitamin D3, these experiments do not rule out the presence of some 25-hydroxylase activity towards vitamin D2 in the microsomes. The results are discussed in relation to previous work in which a lower toxicity has been reported for vitamin D2 than for vitamin D3 in some mammalian species.

A novel specific assay of 25-hydroxy vitamin D3

The synthesis of 25-hydroxy-[26-2H3]vitamin D3 is described. A fixed amount of this compound (usually 250 ng) is added to a fixed amount of serum (usually 2.5 ml) and the mixture is extracted with a chloroform/methanol mixture. The extract is chromatographed on a Sephadex LH-20 column together with a trace amount of 25-hydroxy-[263H3]vitamin D3. The chromatographic fraction corresponding to 25-hydroxy vitamin D3 is converted into trimethylsilyl ether and the amount of unlabeled 25hydroxy vitamin D3 is determined from the ratio between the mass fragmentographic recording at m/e 131 (base peak of unlabeled 25-hydroxy vitamin D3) and m/e 134 (base peak 25-hydroxy-[26-2H3]vitamin D3). The relative standard deviation of the method was about 5%.

Validation of a radioreceptor assay for 1,25-dihydroxyvitamin D using selected ion monitoring GC-MS.

This report describes two methods for the measurement of 1,25-dihydroxyvitamin D [1,25(OH)2D] in serum: A modified radio receptor assay (RRA), employing a 1,25(OH)2D receptor from calf thymus, and selected ion monitoring (SIM) with combined capillary gas chromatography (GC)-mass spectrometry (MS). The intra-assay coefficient of variation was close to 13% for both methods, and the inter-assay coefficients of variation were 14.0 and 6.5% for RRA and SIM (GC-MS), respectively. Aliquots of 2 ml (RRA) and 20 ml (SIM) serum were used, and the limits of detection were 10 and 6 pmol/l, respectively. The analytical recovery of each method was assessed, and a maximum deviation from the expected value of 10 and 2% was found for RRA and SIM, respectively. A correlation coefficient of 0.93 (slope 0.97) was obtained when 27 different serum samples were analyzed by both methods. Included in this study were serum samples from healthy subjects and patients with subnormal as well as supranormal 1,25(OH)2D levels. This result showed that the RRA accurately measured the serum levels of 1,25(OH)2D and therefore should be useful in the diagnosis and control of vitamin D dependent diseases.

[45] Mass fragmentographic assay of 25-hydroxyvitamin D3

Publisher Summary This chapter discusses mass fragmentographic assay of 25-hydroxyvitamin D 3 . The problems with respect to instability and lack of specificity can be overcome with the use of mass fragmentography and deuterium-labeled 25-hydroxyvitamin D 3 as internal standard. The internal standard is added to the biological fluid prior to purification and analysis, and it can be assumed that this standard is degraded or decomposed to the same extent as unlabeled 25-hydroxyvitamin D 3 . In the last step, the amount of unlabeled 25-hydroxyvitamin D 3 is determined by combined gas chromatography mass spectrometry with a multiple ion detector (MID). With the latter detector, a specific ion corresponding to the labeled and unlabeled derivative of 25-hydroxyvitamin D 3 , respectively, can be followed through a gas chromatogram, and the ratio between the two tracings can be used for the calculation of unlabeled 25-hydroxy vitamin D 3 . The specificity of the present assay is based on the fact that, to influence the results, a contaminating compound must have the same retention time in gas-liquid chromatography and contain the same ions in its mass spectrum as the derivative of 25-hydroxyvitamin D 3 . The very specific derivative used in the assay is expected to further increase the specificity.

Reconstitution of vitamin D3 25-hydroxylase activity with a cytochrome P-450 preparation from rat liver mitochondria

25-Hydroxylation of vitamin D3 has been shown catalyzed both by the microsomal [ 1,2] and the mitochondrial [3] fraction of rat liver. As yet, only the mitochondrial activity has been assayed under enzymological conditions involving substrate saturation [3], At present no definitive conclusions can be drawn concerning the relative physiologicai importance of these two hydroxylases. Both enzymes seem to be mixed function oxidases since the oxygen incorporated in the 25position is derived from molecular oxygen both when the mitochondrial [3] and the microsomal fraction [4] is used. In contrast to the mitochondrial activity [3], the microsomal activity seems to be affected by the vitamin D status of the animal [l]. Whether this is due to the hydroxylase per se or some other factor is unknown, however. In a study on the mitochondri~ vitamin Ds 25hydroxylase, it was shown that this enzyme had properties similar to a cytochrome P-450 monooxygenase [3]. Thus the enzyme required NADPH, wasinhibited by CO, and was stimulated by treatment with phenobarbital in vivo. Recently, cytochrome P-450 was solubilized from rat liver mitochondria [5-71. It is shown in the present work that this cytochrome P-450 preparation in the presence of NADPH, ferredoxin and ferredoxin reductase catalyzes the conversion of vitamin Da into a more polar product identified as 25hydroxyvitamin D3 by gas chromatograplly-rn~s spectrometry (%-MS).

High circulating levels of 25-hydroxyvitamin D3 in renal stone formers with hyperabsorptive hypercalciuria

Normocalcaemic male stone formers, 31-51 years old (n = 108) on a free diet, were divided into a hypercalciuric group (n = 47) with calcium excretion rates higher than 8.0 mmol/24 h, a normocalciuric group (n = 32) with calcium excretion rates below 6.1 mmol/24 h and an intermediate group (n = 29). There were no statistically significant differences between the hypercalciuric and the normocalciuric groups with respect to serum levels of calcium, phosphate, creatinine, urate, ALAT, albumin, PTH, 1,25-dihydroxyvitamin D or urinary excretion of cAMP. The group of patients with high calcium excretion had significantly higher serum levels of 25-hydroxyvitamin D3 (75 +/- 4 nmol/l) than the group with low calcium excretion (57 +/- 4 nmol/l) (p less than 0.002), while the group of patients with intermediate calcium excretion had 25-hydroxyvitamin D3 levels between the other two groups (69 +/- 4 nmol/l). A highly accurate method based on isotope dilution-mass spectrometry was used to assay 25-hydroxyvitamin D3. Of the patients with hypercalciuria (n = 47), seven were classified as hyperabsorbers on the basis of calcium load tests. These patients were found to have even higher serum levels of 25-hydroxyvitamin D3 (108 +/- 10 nmol/l)--significantly higher than that of the hypercalciuric patients as a whole. The above study was carried out in March 1983. In September, the group of patients with high urinary calcium excretion also had significantly higher levels of 25-hydroxyvitamin D3 than the group of patients with low calcium excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Relation Between Hypercalciuria and Vitamin D3-Status in Patients with Urolithiasis

Patients with urolithiasis were divided into two groups, one (n = 38) with a urinary excretion of calcium exceeding 6.0 mmol/24 h and one (n = 32) with a calcium excretion lower than 6.1 mmol/24 h. The group of patients with a high urinary excretion of calcium had a significantly higher level of 25-hydroxy vitamin D3 (26.2 +/- 1.6 ng/ml) than had the group of patients with a normal urinary excretion of calcium (17.6 +/- 0.9 ng/ml) (p less than 0.001). A highly specific and accurate method, based on isotope dilution--mass spectrometry was used in the assay of 25-hydroxy vitamin D3. There was no over-all correlation between level of 25-hydroxy vitamin D3 and serum level of calcium (r = 0.1). The results are in accordance with the contention that the vitamin D3-status might be of some importance for the development of hypercalciuria in these patients.

25-hydroxylation of vitamin D3 by a reconstituted system from rat liver microsomes

Abstract Using isotope dilution—mass fragmentography as assay technique, it was shown that highly purified preparations of cytochrome P-450 from rat liver microsomes catalyzed 25-hydroxylation of vitamin D 3 when combined with NADPH-cytochrome P-450 reductase and a phospholipid. The rate of conversion was approximately linear with the amount of cytochrome P-450, and was considerably higher than the rate of conversion obtained with crude liver microsomes. The possibility is discussed that the microsomal fraction contains inhibitors of 25-hydroxylase activity, which may be of regulatory importance in vitamin D 3 metabolism.

Assay of 25-hydroxy vitamin D3-1 α-hydroxylase in pig kidney mitochondria using isotope dilution-mass spectrometry

An assay of 1 alpha-hydroxylation of 25-hydroxy vitamin D3 in pig kidney mitochondria, based on selected ion monitoring, has been developed. Trideuterium-labeled 1,25-dihydroxy vitamin D3 was synthesized and used as internal standard. This standard was added immediately after incubation of 25-hydroxy vitamin D3 with the mitochondrial fraction. The incubation extracts were purified by high-performance liquid chromatography. After formation of the trimethylsilyl derivative, the product was quantitated by mass fragmentography using the ion at m/z 452 and m/z 455. With the use of this assay it was found that formation of 1,25-dihydroxy vitamin D3 was linear with the amount of mitochondrial protein and time of incubation. Substrate saturation was obtained at about 20 microM of 25-hydroxy vitamin D3. The maximal rate of conversion obtained under the conditions employed was about 0.1 pmol/mg protein X minute.

Differences in the metabolism of vitamin D2 and vitamin D3 by subcellular fractions from rat liver

The 25-hydroxylation of vitamin D2 and vitamin D3 was studied in the mitochondrial fraction from rat liver and in a reconstituted system containing cytochrome P-450 from rat liver microsomes. The mitochondrial fraction catalyzed the 25-hydroxylation of vitamin D3 at least two times more effectively than the 25-hydroxylation of vitamin D2. Microsomal cytochrome P-450 catalyzed an efficient 25-hydroxylation of vitamin D3, but no 25-hydroxylation of vitamin D2 could be detected. The present results show a difference in the 25-hydroxylation of vitamin D2 and vitamin D3 in rat liver in vitro.