Physiological and pathophysiological applications of sensitive ELISA methods for urinary deoxycorticosterone and corticosterone in rodents
ABSTRACT 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.
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- "Sensitivity The sensitivity (minimum detection limit) of the T and C measures derived from the IH E method was determined by 20 replicate measurements of the respective zero calibrators  . The concentration corresponding to the mean minus 2 * SD of the absorbance was calculated from its intercept with the displacement curve. "
ABSTRACT: OBJECTIVES: Salivary testosterone (T) and cortisol (C) concentrations were monitored across a sports competition. Data were compared using two enzyme-immunoassay (EIA) methods and two sample preparations to determine their influence on hormone concentrations. DESIGN AND METHODS: A group of male athletes (n = 19) provided a saliva sample the morning before and one day after (24 hours post) an international rugby union match. Following an extraction procedure, the samples were analysed for T and C concentrations using a commercial kit (CM(E)) and an in-house method (IH(E)). Raw samples (no extraction procedure) were also tested using the commercial kit (CM(R)). RESULTS: There were no significant changes in T and C levels from pre to post competition with each EIA method and sample preparation, but significant differences in T (IH(E) > CM(E) > CM(R)) and C (CM(R) > IH(E) and CM(E)) concentrations were seen when both samples were pooled. Bland-Altman analyses confirmed the presence of fixed and proportional bias. Strong and significant correlations were demonstrated between the IH(E) and CM(E) measures of salivary T (r = 0.93-0.97) and C (r = 0.95-0.97). The T and C values from the raw and extracted samples were also strongly correlated (r = 0.93-0.96). CONCLUSIONS: The measurement of salivary T and C concentrations across an international sports event was influenced by different EIA methods and sample preparations, but all measures were strongly correlated with some bias. Both T and C were unresponsive to the sports event, but within the group results large individual variation was seen.Clinical biochemistry 11/2012; 46(4-5). DOI:10.1016/j.clinbiochem.2012.11.019 · 2.23 Impact Factor
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ABSTRACT: The urocortin (Ucn) family of neuropeptides is suggested to be involved in homeostatic coping mechanisms of the central stress response through the activation of corticotropin-releasing factor receptor type 2 (CRFR2). The neuropeptides, Ucn1 and Ucn2, serve as endogenous ligands for the CRFR2, which is highly expressed by the dorsal raphe serotonergic neurons and is suggested to be involved in regulating major component of the central stress response. Here, we describe genetically modified mice in which both Ucn1 and Ucn2 are developmentally deleted. The double knockout mice showed a robust anxiolytic phenotype and altered hypothalamic–pituitary–adrenal axis activity compared with wild-type mice. The significant reduction in anxiety-like behavior observed in these mice was further enhanced after exposure to acute stress, and was correlated with the levels of serotonin and 5-hydroxyindoleacetic acid measured in brain regions associated with anxiety circuits. Thus, we propose that the Ucn/CRFR2 serotonergic system has an important role in regulating homeostatic equilibrium under challenge conditions.Molecular Psychiatry 04/2010; 15(4):442. DOI:10.1038/mp.2009.135 · 15.15 Impact Factor
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ABSTRACT: The mineralocorticoid effects of liquorice are mediated by the inhibitory effects of one of its active components glycyrrhetinic acid on 11β-hydroxysteroid dehydrogenase type 2. However, liquorice is reputed to have many medicinal properties and also contains a number of other potentially biologically active compounds. Here we have investigated the wider effects of oral liquorice on steroidogenesis focussing particularly on possible inhibitory effects of glycyrrhetinic acid on adrenal sulfotransferase activity.Molecular and Cellular Endocrinology 12/2010; 336(1-2):102-9. DOI:10.1016/j.mce.2010.12.011 · 4.24 Impact Factor