title: In-vitro evidence for the regulation of 17,20-lyase activity by cytochrome b5 in adrenocortical adenomas from patients with Cushing's syndrome.
abstract: OBJECTIVE
  The electron transfer system molecules, NADPH-cytochrome P450 reductase (Red) and cytochrome b5 (b5) are known to increase the relative activity of 17,20-lyase to 17 alpha-hydroxylase in vitro. Consistent with this hypothesis, we have reported recently that adrenocortical adenomas from patients with Cushing's syndrome that produced exceptionally high concentrations of androgens also contained more b5 mRNA as well as greater 17,20-lyase activity than adenomas that produced low concentrations of androgens. This finding was suggestive but inconclusive in linking b5 functionally to this difference in adenoma 17,20-lyase activity. In the present study, we have extended this finding by examining the effect of b5 on microsomal 17,20-lyase activity using an antibody against cytochrome b5.
  
  
  METHODS
  Biochemical quantitation of the content of b5 and Red activity in the microsomal fraction of the tumours and determination of 17,20-lyase activity in the microsomes in the presence or absence of an antibody against b5.
  
  
  METHODS
  Seven patients with a clinical diagnosis of Cushing's syndrome secondary to benign adrenocortical adenoma were studied.
  
  
  METHODS
  The microsomal activities of 17 alpha-hydroxylase, 17,20-lyase and 3 beta-hydroxysteroid dehydrogenase were measured by in-vitro enzyme assay with thin layer chromatography. Microsomal Red activity was assayed by measuring NADPH-dependent cytochrome c reduction reaction. Cytochrome b5 concentration was determined by spectrophotometric analysis.
  
  
  RESULTS
  An in-vitro enzyme assay of microsomal fractions from the adenoma showed that the 17,20-lyase activities of two adenomas that produced high concentrations of adrenal androgen were threefold greater than those of five other adenomas that produced low concentrations of androgens. Cytochrome b5 concentrations were greater in the two adenomas with high 17,20-lyase activity than in the other adenomas, while no significant difference in Red activity was observed among all the adenomas. The increased 17,20-lyase activity in the two adenomas was partially but significantly antagonized by an antibody against b5.
  
  
  CONCLUSIONS
  These results suggest that differences in tissue b5 are functionally associated with differences in 17,20-lyase activity in adrenocortical adenomas in Cushing's syndrome, resulting in a dissociated secretion of cortisol and androgens in some patients.
authors.affiliation: Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Sakai
authors.forename: T
authors.initials: T
authors.lastname: Yanase
authors.forename: T
authors.initials: T
authors.lastname: Hara
authors.forename: R
authors.initials: R
authors.lastname: Takayanagi
authors.forename: M
authors.initials: M
authors.lastname: Haji
authors.forename: H
authors.initials: H
authors.lastname: Nawata
mesh_terms: D000236:Adenoma / Q000201:enzymology*; D000306:Adrenal Cortex Neoplasms / Q000201:enzymology*; D000446:Aldehyde-Lyases / Q000378:metabolism*; D000906:Antibodies / Q000378:metabolism; D003480:Cushing Syndrome / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015786:Cytochromes b5 / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008861:Microsomes / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase
chemical_list: D000906:Antibodies; D009249:NADP; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D000446:Aldehyde-Lyases