Repository logo
 

Dietetics, Nutrition and Biological Sciences

Permanent URI for this collectionhttps://eresearch.qmu.ac.uk/handle/20.500.12289/23

Browse

Filters

20092

Settings

Author: search.filters.author.Andrew, R.

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Physiological and pathophysiological applications of sensitive ELISA methods for urinary deoxycorticosterone and corticosterone in rodents
    (Elsevier, 2009-11-04) Al-Dujaili, Emad A. S.; Mullins, L. J.; Bailey, M.; Andrew, R.; Kenyon, C. J.
    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 (11_-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 were confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC (DOC ELISA: Y = 1.092X - 0.012, R2 = 0.988; B ELISA: Y= 1.047X - 0.226, R2=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.
  • No Thumbnail Available
    Item
    Increased mineralocorticoid activity in obese Zucker rats is independent of the renin-angiotensin system and hepatic steroid metabolism.
    (2009) Kenyon, C. J.; Livingstone, D.; Ingram, M.; Al-Dujaili, Emad A. S.; Rutter, A.; Fraser, R.; Andrew, R.
    Hypertension in obese Zucker rats is associated with raised plasma aldosterone, suppressed renin and increased hypothalamo-pituitary-adrenal activity. We investigated the possible causes and consequences of these associations by comparing adrenal gene expression and urinary electrolyte and steroid excretion patterns in lean and obese rats. Groups (n=11) of adult male lean and obese Zucker rats were held in metabolism cages for 7 days. Urine collected over the last 2 days was analysed for electrolytes and steroid content by flame photometry, ELISA and LCMS. Urine volumes, sodium, potassium and creatinine were higher in obese vs lean rats (P<0.05): ratios of Na/creatinine and K/creatinine were lower (P<0.01) but Na/K was unaffected. Urinary aldosterone and 18OH deoxycorticosterone were not significantly different. Urinary corticosterone was slightly higher (1.5 fold, P<0.001) in obese vs lean rats in agreement with previous evidence of adrenocortical cell hypertrophy. Deoxycorticosterone (11_ hydroxylase precursor) and 11 dehydrocorticosterone (11_ hydroxysteroid dehydrogenase product) were markedly higher (3.3 and 2.6 fold respectively, P<0.001). We considered whether these changes could be attributed to intra-adrenal differences in the expression of enzymes involved in the synthesis and metabolism of corticosterone. Cyp11b1 (11_ hydroxylase) was increased and 5alpha reductase type 1, 11_HSD1 and 2 mRNA differences were not statistically significant. We conclude that extra-adrenal metabolism of corticosterone is compensated by HPA-mediated adrenal hypertrophy. Increased antinatriuretic activity and suppressed renin can be attributed to higher aldosterone and/or deoxycorticosterone levels or impaired renal 11_HSD2 activity. These differences are not caused by changes in the pattern of adrenal steroidogenesis nor are they likely to be due reduced hepatic metabolism of mineralocorticoid hormones. Raised plasma aldosterone:renin ratios in obese Zucker rats could be explained by a novel adipose factor(s) that has recently been shown to stimulate aldosterone synthesis by Wnt signalling effects on StAR protein.