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

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 1α-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 µM. Ketoconazole, a cytochrome P450 inhibitor, blocked calcitriol production in a dose dependent fashion. Total inhibition of the liver 1α-hydroxylase was achieved with 180 µM ketoconazole. We next compared the kinetics of the 1α-hydroxylases of normal and uremic rat livers. Maximal velocities were not statistically different (139.6 ± 22.3 pg/mg/min for normals and 217.1 ± 73.3 pg/mg/min for uremic rats). However, the apparent Km was 35.9 ± 3.2 µM for uremic animals, significantly higher (P ≤ 0.001) than that of normal rats (16.6 ± 0.7 µM). These results demonstrate that: (1) 1α-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