Physiological and pathophysiological applications of sensitive ELISA methods for urinary deoxycorticosterone and corticosterone in rodents.
Deoxycorticosterone (DOC: a weak mineralocorticoid) is the precursor to corticosterone (B: the major glucocorticoid in rodents) and aldosterone (the major mineralocorticoid). The genes Cyp11b1 and Cyp11b2 that encode the enzymes responsible for DOC to B (11beta-hydroxylase) and DOC to aldosterone (aldosterone synthase) conversions are located on the same chromosome. The aim of this study was to develop sensitive and specific ELISA methods to quantify urinary DOC and B concentrations to assess the physiological and genetic control of the Cyp11b1/b2 locus. Antibodies raised in rabbits against DOC and B and horse radish peroxidase-goat anti-rabbit IgG enzyme tracer were used to develop the assays. Urine samples collected from mice held in metabolic cages were extracted with dichloromethane and reconstituted in assay buffer. The assays were validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivities with major interfering steroids were minimal: DOC assay (progesterone=0.735% and corticosterone=0.045%), and for B assay (aldosterone=0.14%, 11-dehydro-B=0.006%, cortisol=0.016% and DOC=0.04%) and minimum detection limit for DOC ELISA was 2.2 pg/mL (6.6 pmol/L), and for B ELISA was 6.2 pg/mL (17.9 pmol/L). The validity of urinary DOC and B ELISAs was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC (DOC ELISA: Y=1.092X-0.054, R(2)=0.988; B ELISA: Y=1.047X-0.226, R(2)=0.996). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. The methods were used in a series of metabolic cage studies which demonstrated that (i) females produce more DOC and corticosterone than males; (ii) DOC and corticosterone respond to ACTH treatment but not dietary sodium restriction; (iii) DOC:B ratios in Cyp11b1 null mice were >200-fold greater than wild type.
Al-Dujaili
Emad A S
EA
Queen Margaret University, Edinburgh, Scotland, UK. ealdujaili@qmu.ac.uk
Mullins
Linda J
LJ
Bailey
Matthew A
MA
Andrew
Ruth
R
Kenyon
Christopher J
CJ
D000818:Animals; D002138:Calibration; D002417:Cattle; D003345:Corticosterone / Q000652:urine*; D019405:Cytochrome P-450 CYP11B2 / Q000235:genetics / Q000502:physiology; D003900:Desoxycorticosterone / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008297:Male; D051379:Mice; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics / Q000502:physiology; D016482:Urinalysis / Q000379:methods* / Q000592:standards
D003900:Desoxycorticosterone; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase; D003345:Corticosterone