Dietetics, Nutrition and Biological Sciences
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Item Transcriptional and physiological responses to chronic ACTH treatment by the mouse kidney(2009-11-17) Dunbar, D. R.; Khaled, H.; Evans, L. C.; Al-Dujaili, Emad A. S.; Mullins, L. J.; Mullins, J. J.; Kenyon, C. J.; Bailey, M. A.We investigated the effects on urinary steroid and electrolyte excretion and renal gene expression of chronic infusions of ACTH in the mouse. ACTH caused a sustained increase in corticosteroid excretion; aldosterone excretion was only transiently elevated. There was an increase in the excretion of deoxycorticosterone, a weak mineralocorticoid, to levels of physiological significance. Nevertheless, we observed neither antinatriuresis nor kaliuresis in ACTH-treated mice and plasma renin activity was not suppressed. We identified no changes in expression of mineralocorticoid target genes. Water turnover was increased in chronic ACTH-treated mice, as was hematocrit and hypertonicity: volume contraction is consistent with high levels of glucocorticoid. ACTH-treated mice exhibited other signs of glucocorticoid excess, such as enhanced weight gain and involution of the thymus. We identified novel ACTH-induced changes in i) genes involved in vitamin D (Cyp27b1, Cyp24a1, Gc) and calcium metabolism (Sgk, Calb1, Trpv5) associated with calciuria and phosphaturia; ii) genes that would be predicted to desensitize the kidney to glucocorticoid action (Nr3c1, Hsd11b1, Fkbp5) and iii) genes encoding transporters of enzymes systems associated with xenobiotic metabolism and oxidative stress. Although there is evidence that ACTH-induced hypertension is a function of physiological cross talk between glucocorticoids and mineralocorticoids, the present study suggest that the major changes in electrolyte and fluid homeostasis and renal function are attributable to glucocorticoids. The calcium and organic anion metabolism pathways that are affected by ACTH may explain some of the known adverse effects associated with glucocorticoid excess.Item Development and validation of a highly sensitive and specific enzyme immunosorbant assay for aldosterone: application to urine samples from cyp11b1 knockout mice(2008-04) Al-Dujaili, Emad A. S.; Kenyon, C. J.; Mullins, L. J.; Mullins, J. J.The majority of immunoassays used to measure aldosterone (most potent mineralocorticoid) levels are still based on radio-iodinated tracer, lack sensitivity and specificity and there is a definite need for their improvement. The aim of this project is to develop a highly sensitive and specific ELISA method for urinary aldosterone estimation in samples obtained from wild type and cyp11b1 knockout mice. 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 (Innova Biosciences, Cambridge) and used to develop the ELISA method. Urine samples obtained from wild type and null mice were first hydrolysed with Helix Pomatia (Sigma), extracted with dichloromethane and reconstituted in assay buffer. Aliquots were then assayed using the ELISA technique previously published following some modifications to sensitise the assay (Al-Dujaili 2006, Clinica Chimica Acta 364 172-179). The aldosterone ELISA was validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivity with major interfering steroids was minimal: corticosterone=0.018%, cortisol=0.0014%, DOC=0.013% except for 5-dihydro-aldosterone=1.65%. Minimum detection limit of this ELISA was 2.2 pg/ml (6.2 pmol/l). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC separation step (Y=1.048X+0.006, R2=0.998, n=42). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels in male wild type and null mice on normal sodium diet were 42.710.3 (S.E.M.) pmol/g per day and 16.12.7 pmol/g per day, and on low sodium diet were 132.524.2 and 37.610.3 pmol/g per day, respectively. In conclusion, a simple and highly sensitive ELISA has been developed to estimate urinary excretion of aldosterone and the assay can clearly confirm the animal's sodium intake status and distinguish between urinary aldosterone levels in wild type and null mice.Item Effect of green coffee bean extract and chlorogenic acid consumption on 11_HSD activity in humans and mice(2009-03) Almoosawi, Suzana; Dickinson, A.; Fyfe, Lorna; Kenyon, C. J.; Al-Dujaili, Emad A. S.Increased 11_ hydroxysteroid dehydrogenase type 1 (11_HSD1) activity is implicated in the development of the metabolic syndrome. Identifying natural compounds that influence 11_HSD1 activity could lead to novel methods of treating obesity, cardiovascular disease and diabetes. In the present study, we tested the effect of green coffee bean extract (GCBE), rich in chlorogenic acid (CGA), in human volunteers and of CGA in mice on blood pressure (BP), lipid and glucose metabolism. Our hypothesis was that CGA would improve these parameters, by blocking the uptake of microsomal glucose-6-phosphate which in turn would limit the production of co-factor for 11_HSD1 reductase activity. With local Ethics Committee approval, 13 healthy overweight subjects were given GCBE containing 90 mg CGA twice daily for 2 weeks. Urinary 24 h free cortisol was reduced from 1.05230.45 to 0.7630.40 nmol/kg (P=0.07). Free cortisone excretion was reduced from 0.7120.38 to 0.4320.24 nmol/kg (P=0.007). Systolic BP decreased from 119.410.5 to 113.89.1 mmHg (P=0.05). Fasting plasma glucose (P=0.101), diastolic BP (P=0.114), free cortisol:cortisone ratio (P=0.216) and anthropometrical measurement were not affected. In vitro, 11_HSD1 activity (conversion of added cortisone to cortisol) in isolated mouse microsomes was inhibited dose-dependently by CGA. The effects of feeding diet containing 0.15% CGA for 17 days was tested in male C57BL6 mice. Adiposity was unaffected but liver (27.74.9 vs 15.52.2 mg/g, P<0.04) and plasma (1.240.18 vs 0.860.08 mg/ml, P<0.08) triglycerides tended to be reduced. Urinary 24 h cortisol excretion following IP injection of 20 mg/kg cortisone was 30.14.1 vs 24.25.3 nmol/kg (P<0.4) for control and CGA-treated mice. Peak plasma glucose levels in tolerance tests were earlier with CGA treatment although, over a 2 h period, glucose clearance was not affected.Item Development and validation of highly sensitive and specific enzyme immunosorbant assays for deoxycorticosterone and corticosterone: application to urine samples from cyp11b1 knockout mice(2008-04) Al-Dujaili, Emad A. S.; Kenyon, C. J.; Mullins, L. J.; Mullins, J. J.Deoxycorticosterone (DOC: a weak mineralocorticoid) is the precursor to corticosterone (B: the major glucocorticoid in rodents) and aldosterone. Cyp11b1 encodes 11_-hydroxylase which catalyses the conversion of DOC to B in rodents. The aim of this study is to develop sensitive and specific ELISA methods to estimate urinary DOC and B levels in mice. Antibodies against DOC and B were raised in rabbits by our laboratories as previously described and HRP-Goat anti-Rabbit IgG enzyme tracer (Upstate, UK) were used to develop the ELISA methods. Urine samples obtained from wild type and null mice were first hydrolysed with Helix Pomatia, extracted with dichloromethane and reconstituted in assay buffer. Aliquots were then assayed using the ELISA technique previously published (Al-Dujaili 2006, Clinica Chimica Acta. 364: 172-179). The assays were validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivity with major interfering steroids was minimal: DOC assay (progesterone=0.735% and corticosterone=0.045%), and for B assay (11-dehydro-B=0.006%, cortisol=0.016%, DOC=0.04% and aldosterone=0.14%). Minimum detection limit for DOC ELISA was 2.8 pg/ml (8.5 pmol/l), and for B ELISA was 12.2 pg/ml (0.035 nmol/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 separation step (DOC ELISA: Y=1.092X-0.012, R2=0.988, n=42; B ELISA: Y=1.047X-0.226, R2=0.996, n=42). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using these assays: mean urinary DOC and B levels in female wild type mice were 0.1960.033 (S.E.M.) and 65.3813.04 nmol/g per day and in null mice were 8.561.27 and 3.6610.48 nmol/g per day respectively. In male mice, DOC and B levels in wild type were 0.0480.14 and 6.060.87 nmol/g per day versus 1.280.298 and 0.6410.112 nmol/g per day in null animals. In conclusion, simple, rapid and sensitive ELISAs have been developed to estimate urinary excretion of DOC and B and the assays can clearly distinguish between urinary steroid excretion of wild type and null mice.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.Item Targeting Cyp11b1 expression in mice to model sequelae of congenital adrenal hyperplasia(2008) Mullins, L. J.; Peter, A.; Wrobel, N.; Al-Dujaili, Emad A. S.; McNeilly, J. R.; Brownstein, D. G.; McNeilly, J. R.; Mullins, J. J.; Kenyon, C. J.We have created transgenic mice in which Cyp11b1, the gene encoding 11_-hydroxylase, has been knocked out. Since mice do not secrete adrenal androgens, this knockout line allows a more detailed investigation of phenotypes associated with congenital adrenal hyperplasia (CAH) without the overwhelming virilisation that characterises patients with CAH. Starting with a BAC containing the mouse Cyp11b1/b2 locus and including flanking up- and downstream sequences, a construct was engineered in which exons 3-7 of Cyp11b1 were substituted with DNA encoding the fluorescent reporter protein ECFP. An IRES site preceding and a farnesylation signal following the ECFP gene were added to the construct which was then homologously recombined in ES cells before injection into blastocysts. Successful targeting of the Cyp11b1 gene was confirmed in ES cells by FISH analysis and in homozygous transgenic mice by the absence of adrenal Cyp11b1 mRNA (RT-PCR) and 11beta-hydroxylase (immunocytochemistry) expression. The expected increase in adrenal mass (3-fold, P<0.001) was observed with changes due to cell hypertrophy rather than hyperplasia. Urinary steroid profiles showed marked reductions in corticosterone (9.5-fold, P<0.001) and concomitant increases of earlier intermediates in the glucocorticoid biosynthetic pathway (deoxycorticosterone 25-fold, P<0.001; progesterone 3-fold, P<0.001). Urinary DHEA and testosterone were higher in both males and females although not to the extent seen in patients with CAH. Estradiol in females was unaffected. Several unexpected phenotypes relating to reproduction were observed. In initial crosses of heterozgotes, offspring carrying the Cyp11b1 null allele were underrepresented. Also female homozygote mice were infertile with poorly defined corpora lutea, endometrial hyperplasia, and late-onset adenomyosis. We conclude that Cyp11b1 null mice exhibit signs of glucocorticoid deficiency, mineralocorticoid and progesterone excess and mild hyperandrogenism. Infertility which is known to be a problem of CAH patients, appears to be caused by abnormalities in uterine and ovarian tissues.Item Investigating the effects of Liquorice consumption on Salivary Steroid hormones profile and blood pressure in healthy volunteers(2008) Al-Dujaili, Emad A. S.; Macdonald, Claire; Kenyon, C. J.; Nicol, MoiraGlycyrrhetinic acid (GA) has diverse in vitro effects as an inhibitor of 11beta hydroxysteroid dehydrogenase (11HSD), 5alpha reductase and hormone receptor binding. However, in vivo GA studies have focussed on the hypertensive effects associated with the syndrome of apparent mineralocorticoid excess (SAME) in which 11HSD inhibition allows glucocorticoid hormones to bind inappropriately to MR and subsequent decreased aldosterone synthesis. Here we consider whether GA's other in vitro effects are reflected in altered steroid hormone profiles in vivo. The effect of liquorice (containing GA) has been assessed by measuring steroid hormone levels in healthy individuals. Ten men and 10 women (18-30 years) were given 100 g liquorice sweets (3% liquorice extract) or non-liquorice containing confectionary for 7 days in a crossover study. Saliva was collected 30 min after waking, at 1100-1300 h and at 1800-2100 h for cortisol, cortisone, aldosterone (Aldo), deoxycorticosterone (DOC), DHEA and testosterone measurements by in-house, sensitive and specific ELISA methods. Systolic blood pressure measured at the end of the two periods of treatment showed non-significant increases (P=0.127) in response to liquorice consumption. Cortisol was significantly higher (P=0.003) and cortisone and aldo were reduced by liquorice (P<0.001) consistent with the SAME. DHEA, testosterone and DOC were increased (P<0.001) possibly because of reduced hepatic clearance. However, these steroids are not 11HSD type 2 substrates indicating liquorice might also have inhibited hepatic 5alpha reductase. Recent studies also implicate the bi-directional 11HSD type1 enzyme in DHEA metabolism. GA is equipotent as an inhibitor of 11HSD type 1 and type 2 oxidase activity. 7-Hydroxy-DHEA, a major metabolite of DHEA is inter-converted with 7-keto-DHEA by 11HSD type 1 and could, therefore, secondarily interfere with DHEA metabolism. We conclude that in addition to cortisol, other biologically active steroid hormones are affected by liquorice. Increased salivary levels of DHEA and testosterone suggest that liquorice modulates their bioavailability.