Browsing by Person "Mullins, J. J."

Now showing 1 - 5 of 5

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.
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.
Hsd11b2 haploinsufficiency in mice causes salt sensitivity of blood pressure
(2011-03) Bailey, M. A.; Craigie, E.; Livingstone, D. E. W.; Kotelevtsev, K. V.; Al-Dujaili, Emad A. S.; Kenyon, C. J.; Mullins, J. J.
Salt sensitivity of blood pressure is an independent risk factor for cardiovascular morbidity. Mechanistically, abnormal mineralocorticoid action and subclinical renal impairment may blunt the natriuretic response to high sodium intake, causing blood pressure to rise. 11_-Hydroxysteroid dehydrogenase type 2 (11_HSD2) controls ligand access to the mineralocorticoid receptor, and ablation of the enzyme causes severe hypertension. Polymorphisms in HSD11B2 are associated with salt sensitivity of blood pressure in normotensives. In this study, we used mice heterozygote for a null mutation in Hsd11b2 (Hsd11b2) to define the mechanisms linking reduced enzyme activity to salt sensitivity of blood pressure. A high-sodium diet caused a rapid and sustained increase in blood pressure in Hsd11b2 mice but not in wild-type littermates. During the adaptation to high-sodium diet, heterozygotes displayed impaired sodium excretion, a transient positive sodium balance, and hypokalemia. After 21 days of high-sodium feeding, Hsd11b2 mice had an increased heart weight. Mineralocorticoid receptor antagonism partially prevented the increase in heart weight but not the increase in blood pressure. Glucocorticoid receptor antagonism prevented the rise in blood pressure. In Hsd11b2 mice, high-sodium feeding caused suppression of aldosterone and a moderate but sustained increase in corticosterone. This study demonstrates an inverse relationship among 11_HSD2 activity, heart weight, and blood pressure in a clinically important context. Reduced activity causes salt sensitivity of blood pressure, but this does not reflect illicit activation of mineralocorticoid receptors by glucocorticoids. Instead, we have identified a novel interaction among 11_HSD2, dietary salt, and circulating glucocorticoids. 2011 American Heart Association, Inc.
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.
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.