Physiological and pathophysiological applications of sensitive ELISA methods for urinary deoxycorticosterone and corticosterone in rodents
Queen Margaret University, Edinburgh, Scotland, UK.Steroids (Impact Factor: 2.64). 08/2009; 74(12):938-44. DOI: 10.1016/j.steroids.2009.06.009
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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- "Samples were then reconstituted in 250 ml of assay buffer (PBS (Sigma) + 0.1% Bovine serum Albumin, Sigma), vortexed and assayed. An indirect ELISA using an in-house protocol developed by co-author Al-Dujaili (Al-Dujaili et al. 2009; 2012) determined cortisol concentrations using a spectrophotometer (Thermo Scientific Multiskan FC Microplate Photometer) with a filter of 595nm. Placental cotyledon cortisol was expressed as ng of cortisol per g of original tissue used. "
ABSTRACT: Dairy animals are subjected to a number of potential stressors throughout their lives, including daily interactions with humans. The quality of these interactions may have direct consequences for the animal undergoing the experience, but if such events occur during gestation it may also affect the developing fetus. This study examined the effects of differential handling during mid-gestation in 40 twin-bearing Saanen × Toggenburg primiparous goats. Between days 80 and 115 of gestation (gestation = 150 days), goats were subjected to aversive (AVS, n = 13), gentle (GEN, n = 13) or minimal (M, n = 14) handling protocols for 10 minute periods twice daily. The control (M) group did not receive handling treatments and all goats received normal husbandry procedures outside treatment periods. Salivary cortisol measured during the treatment period was higher in AVS goats (mean cortisol (sem) in pg/μl: AVS: 176.7 (18.2), GEN: 119.6 (11.1), M: 126.5 (13.7); P = 0.007). Data collection was focussed on mother-offspring behaviors 2 h post-partum, placental morphology and colostrum quality. AVS goats were the only treatment group to suffer fetal loss (16% loss vs 0% in GEN and M, P = 0.05). Treatment also influenced placental morphology with a tendency for fewer cotyledons evident in placentae from the aversive treatment (AVS: 87.9 (7.8), GEN: 107.1 (7.9), M: 112.1 (9.3), P = 0.093), and significantly fewer medium sized cotyledons (AVS: 67.6 (7.8), GEN: 89.3 (6.4), M: 84.3 (5.4), P = 0.042). GEN goats displayed more grooming and nosing behaviors towards their young during the first 2 h post-partum (grooming: GEN: 89.3% (7.1), AVS: 72.6% (7.7), M: 63.4% (9.0), P = 0.045; nosing frequency: GEN: 58.8 (12.5), AVS: 28.6 (11.1), M: 34.7 (6.5), P = 0.021). There was an overall trend for kids from mothers experiencing the AVS treatment to take longer to stand, reach the udder and suck compared to kids from GEN and M treatment groups. Treatment significantly affected latency to perform play behavior, with kids from AVS goats taking on average 25 min longer to play for the first time than kids from GEN and M treatment groups (P < 0.001). The results show that handling during gestation affects placental morphology, fetal survival and post-partum maternal behaviors, and influences kid behavioral development. Such results have important animal welfare implications, demonstrating that negative handling of pregnant females results in poorer placental quality with potential for fetal loss. It also demonstrates the beneficial effects of positive handling on enhancement of maternal behaviors.
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- "Plasma and urinary corticosterone and aldosterone were measured by ELISA as described by , . Plasma arginine vasopressin (AVP) was measured by ELISA following the kit instructions (arg8-vasopressin EIA; Enzo Life sciences, Exeter, UK). "
ABSTRACT: Hypertension is common and contributes, via cardiovascular disease, towards a large proportion of adult deaths in the Western World. High salt intake leads to high blood pressure, even when occurring prior to birth - a mechanism purported to reside in altered kidney development and later function. Using a combination of in vitro and in vivo approaches we tested whether increased maternal salt intake influences fetal kidney development to render the adult individual more susceptible to salt retention and hypertension. We found that salt-loaded pregnant rat dams were hypernatraemic at day 20 gestation (147±5 vs. 128±5 mmoles/L). Increased extracellular salt impeded murine kidney development in vitro, but had little effect in vivo. Kidneys of the adult offspring had few structural or functional abnormalities, but male and female offspring were hypernatraemic (166±4 vs. 149±2 mmoles/L), with a marked increase in plasma corticosterone (e.g. male offspring; 11.9 [9.3-14.8] vs. 2.8 [2.0-8.3] nmol/L median [IQR]). Furthermore, adult male, but not female, offspring had higher mean arterial blood pressure (effect size, +16 [9-21] mm Hg; mean [95% C.I.]. With no clear indication that the kidneys of salt-exposed offspring retained more sodium per se, we conducted a preliminary investigation of their gastrointestinal electrolyte handling and found increased expression of proximal colon solute carrier family 9 (sodium/hydrogen exchanger), member 3 (SLC9A3) together with altered faecal characteristics and electrolyte handling, relative to control offspring. On the basis of these data we suggest that excess salt exposure, via maternal diet, at a vulnerable period of brain and gut development in the rat neonate lays the foundation for sustained increases in blood pressure later in life. Hence, our evidence further supports the argument that excess dietary salt should be avoided per se, particularly in the range of foods consumed by physiologically immature young.
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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 (24h 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.
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