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Dietetics, Nutrition and Biological Sciences

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End date: 2009Subject: search.filters.subject.ELISA

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    Development of a highly sensitive ELISA for aldosterone in mouse urine: Validation in physiological and pathophysiological states of aldosterone excess and depletion
    (2009-04) Al-Dujaili, Emad A. S.; Mullins, L. J.; Bailey, M. A.; Kenyon, C. J.
    Background: Clinical studies have established aldosterone as a critical physiological and pathophysiological factor in salt and water homeostasis, blood pressure control and in heart failure. Genetic and physiological studies of mice are used to model these processes. A sensitive and specific assay for aldosterone is therefore needed to monitor adrenocortical activity in murine studies of renal function and cardiovascular diseases. Methods: Antibodies against aldosterone were raised in sheep as previously described. HRP-Donkey-anti-sheep IgG enzyme tracer was produced in our laboratory using the Lightning-Link HRP technique. Aldosterone ELISA protocol was validated and optimised to achieve the best sensitivity. The assay was validated by analysing the urine of mice collected under various experimental conditions designed to stimulate or suppress aldosterone in the presence of other potentially interfering steroid hormones. Results: Cross-reactivity with the steroids most likely to interfere was minimal: corticosterone = 0.0028%, cortisol = 0.0006%, DOC = 0.0048% except for 5-dihydro-aldosterone = 1.65%. Minimum detection limit of this ELISA was 5.2 pmole/L (1.5 pg/mL). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between results obtained before and after solvent extraction and HPLC separation step (Y = 1.092X + 0.03, R2 = 0.995, n = 54). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels were (i) approximately 60-fold higher in females than males mice; (ii) increased 6-fold by dietary sodium restriction; (iii) increased 10-fold by ACTH infusion and (iv) reduced by >60% in Cyp11b1 null mice. Conclusion: We describe an ELISA for urinary aldosterone that is suitable for repeated non-invasive measurements in mice. Female aldosterone levels are higher than males. Unlike humans, most aldosterone in mouse urine is not conjugated. Increased levels were noted in response to dietary sodium restriction and ACTH treatment. The sensitivity of the assay is sufficient to detect suppressed levels in mouse models of congenital adrenal hyperplasia. 2009 Elsevier Inc. All rights reserved.
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    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.