Clinical Investigation

Kidney International (1993) 44, 134–138; doi:10.1038/ki.1993.223

Lower affinity for substrate for extrarenal synthesis of calcitriol in chronic uremia

Lavinia Negrea1, Eduardo Slatopolsky1 and Adriana Dusso1

1Department of Internal Medicine, Renal Division, Washington University School of Medicine, St. Louis, Missouri, USA

Correspondence: Adriana Dusso PhD, Washington University School of Medicine, Department of Medicine, Renal Division, 660 South Euclid Avenue, Box 8129, St. Louis, Missouri 63110, USA.

Received 3 August 1992; Revised 21 January 1993; Accepted 8 February 1993.

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Abstract

Lower affinity for substrate for extrarenal synthesis of calcitriol in chronic uremia. Previous studies from our laboratory have shown that anephric patients have very low, but detectable, levels of 1,25(OH)2D3 (calcitriol) that can be increased to normal by administration of large doses of 25(OH)D3. The report of 1alpha-hydroxylase activity in pig liver with an affinity for substrate significantly lower than that of the renal enzyme, led us to use the rat as an experimental model to further clarify the need of supraphysiological levels of 25(OH)D3 to correct calcitriol deficiency in chronic uremia. We have measured 1,25(OH)2D3 production by rat liver. Cytosol free liver homogenates (CFH) from normal rats were incubated with 25(OH)D3 and the production of 1,25(OH)2D3 was measured using the thymus radioreceptor assay after solid phase C18 extraction and HPLC purification of the samples. 1,25(OH)2D3 production was linear up to 30 minutes and a CFH protein concentration up to 20 mg. Saturability was attained for a substrate concentration of approximately 60 microM. Ketoconazole, a cytochrome P450 inhibitor, blocked calcitriol production in a dose dependent fashion. Total inhibition of the liver 1alpha-hydroxylase was achieved with 180 microM ketoconazole. We next compared the kinetics of the 1alpha-hydroxylases of normal and uremic rat livers. Maximal velocities were not statistically different (139.6 plusminus 22.3 pg/mg/min for normals and 217.1 plusminus 73.3 pg/mg/min for uremic rats). However, the apparent Km was 35.9 plusminus 3.2 microM for uremic animals, significantly higher (P less than or equal to 0.001) than that of normal rats (16.6 plusminus 0.7 microM). These results demonstrate that: (1) 1alpha-hydroxylase is expressed in rat livers; (2) the liver enzyme appears to be a cytochrome P450 mixed function oxidase; (3) the affinity of the liver enzyme for 25(OH)D3 is significantly reduced in uremia, a finding which may explain the need of supraphysiological levels of substrate to normalize serum calcitriol in anephric humans.

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