Assay and properties of 18-hydroxylation of endogenous and exogenous corticosterone in rat adrenals. Evidence for heterogeneity of 18-hydroxylase activity.
A mass fragmentographic technique for assay of 18-hydroxylation of labeled (exogenous) and unlabeled (endogenous) corticosterone in adrenal mitochondria and in reconstituted cytochrome P-450 systems has been developed. An extract of an incubation of [14-14C]corticosterone is subjected both to thin-layer radiochromatography and to mass fragmentography (as O-methyloxime-trimethylsilyl ether derivative). In the latter procedure the ions at m/e 605 and 607 (specific for the derivatives of unlabeled and labeled 18-hydroxycorticosterone, respectively), at m/e 591 and 593 (specific for the derivatives of unlabeled labeled aldosterone, respectively) and at m/e 548 and 550 (specific for the derivatives of unlabeled and labeled corticosterone, respectively) were followed through the gas-liquid chromatography. From the ratio between the peaks obtained in the mass fragmentography and from the percentage conversion of [4-14C]corticosterone obtained in the thin-layer radiochromatography, the amount of endogenous and exogenous 18-hydroxycorticosterone and aldosterone could be calculated. The effects of time, enzyme, and substrate concentration of 18-hydroxylation were studied and optimal conditions for assay were determined. Under most conditions, the ratio between labeled and unlabeled 18-hydroxylated products was about constant, indicating that labeled and unlabeled corticosterone were not in equilibrium. It was ascertained that the 18-hydroxycorticosterone and aldosterone formed in the incubations were derived from corticosterone. [4-14C]18-Hydroxydeoxycorticosterone was not converted into aldosterone or 18-hydroxycorticosterone. In vitro studies with different 18-hydroxylase inhibitors (spironolactone, canrenone, and canrenoate-K) and studies with rats pretreated with KCl in drinking fluid suggest that 18-hydroxylation of corticosterone is catalyzed by an enzyme system different from that catalyzing 18-hydroxylation of deoxycorticosterone.
D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D002191:Canrenoic Acid / Q000494:pharmacology; D002192:Canrenone / Q000494:pharmacology; D003345:Corticosterone / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D013148:Spironolactone / Q000494:pharmacology; D013250:Steroid Hydroxylases / Q000378:metabolism*
D013148:Spironolactone; D011189:Potassium Chloride; D002192:Canrenone; D002191:Canrenoic Acid; D013250:Steroid Hydroxylases; D009251:NADPH-Ferrihemoprotein Reductase; D003345:Corticosterone