Dietetics, Nutrition and Biological Sciences
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Item Glucocorticoid receptor alters isovolumetric contraction and restrains cardiac fibrosis(Society for Endocrinology, 2017-01-05) Richardson, Rachel V.; Batchen, Emma J.; Thomson, Adrian J. W.; Darroch, Rowan; Pan, Xinlu; Rog-Zielinska, Eva A.; Wyrzykowska, Wiktoria; Scullion, Kathleen; Al-Dujaili, Emad A. S.; Diaz, Mary E.; Moran, Carmel M.; Kenyon, Christopher J.; Gray, Gillian A.; Chapman, Karen E.Corticosteroids directly affect the heart and vasculature and are implicated in the pathogenesis of heart failure. Attention is focussed upon the role of the mineralocorticoid receptor (MR) in mediating pro-fibrotic and other adverse effects of corticosteroids upon the heart. In contrast, the role of the glucocorticoid receptor (GR) in the heart and vasculature is less well understood. We addressed this in mice with cardiomyocyte and vascular smooth muscle deletion of GR (SMGRKO mice). Survival of SMGRKO mice to weaning was reduced compared with that of littermate controls. Doppler measurements of blood flow across the mitral valve showed an elongated isovolumetric contraction time in surviving adult SMGRKO mice, indicating impairment of the initial left ventricular contractile phase. Although heart weight was elevated in both genders, only male SMGRKO mice showed evidence of pathological cardiomyocyte hypertrophy, associated with increased myosin heavy chain-β expression. Left ventricular fibrosis, evident in both genders, was associated with elevated levels of mRNA encoding MR as well as proteins involved in cardiac remodelling and fibrosis. However, MR antagonism with spironolactone from birth only modestly attenuated the increase in pro-fibrotic gene expression in SMGRKO mice, suggesting that elevated MR signalling is not the primary driver of cardiac fibrosis in SMGRKO mice, and cardiac fibrosis can be dissociated from MR activation. Thus, GR contributes to systolic function and restrains normal cardiac growth, the latter through gender-specific mechanisms. Our findings suggest the GR:MR balance is critical in corticosteroid signalling in specific cardiac cell types.Item Introgressed chromosome 2 quantitative trait loci restores aldosterone regulation and reduces response to salt in the stroke-prone spontaneously hypertensive rat(Lippincott, Williams & Wilkins, 2014-10) Sampson, Amanda K.; Mohammed, Dashti; Beattie, Wendy; Graham, Delyth; Kenyon, Christopher J.; Al-Dujaili, Emad A. S.; Guryev, Victor; Mcbride, Martin W.; Dominiczak, Anna F.Paper adds to the growing body of evidence that children can acquire phonological systems before they are able to master the phonetic skills needed to convey the contrasts in that systemItem Evidence for the stress-linked immunocompetence handicap hypothesis in human male faces(2011-03-07) Moore, F. R.; Cornwell, R. E.; Law Smith, M. J.; Al-Dujaili, Emad A. S.; Sharp, M. A.; Perrett, D. I.The stress-linked immunocompetence handicap hypothesis (SL-ICHH) of sexual selection incorporates a role of the stress hormone corticosterone (C; cortisol in humans) in relationships between testosterone (T), immunity and secondary sexual trait expression. In support of this, C has been shown to mediate and moderate relationships between T and immune response and to be inversely related to attractiveness in some avian species. We predicted that female preferences for cues to T in human male faces would be contingent upon co-occurring cortisol levels. In study 1, we tested relationships between Tand cortisol and attractiveness, masculinity and health ratings of raw male faces. We found cortisol to be inversely related to attractiveness. In study 2, we tested female preferences for male faces that were parametrically manipulated on the basis of cues to naturally co-occurring levels of T and cortisol across the menstrual cycle. Women preferred cues to low cortisol in general and in the fertile phase of the cycle, and there was an interaction between Tand cortisol in general and in the non-fertile phase. Results were consistent with the SL-ICHH but not the original immunocompetence handicap model: females expressed preferences for cues to cortisol but not for cues to T, except in interaction with the stress hormone. Results inform the SL-ICHH by demonstrating female preferences for low cortisol and the nature of its interaction with T in humans, as well as indicating the traits that may be signalled by different combinations of the hormones including immune response, current health and resource acquisition characteristics. 2010 The Royal Society.Item Validation and application of a highly specific and sensitive ELISA for the estimation of cortisone in saliva, urine and in vitro cell-culture media by using a novel antibody(Elsevier, 2012-05) Al-Dujaili, Emad A. S.; Baghdadi, Hussam H. S.; Howie, Forbes; Mason, IanIt is generally acknowledged that local tissue concentrations of cortisol and cortisone are modulated by site-specific actions of 11_-hydroxysteroid dehydrogenase (11_-HSD) isoenzymes 1 and 2. Cortisone, the inactive metabolite of cortisol is produced by 11_HSD type 2. To assess 11_-HSD types 1 and 2 activities, the cortisol/cortisone ratio has to be accurately determined. Immunoassays to measure cortisone levels are not widely available and tend to lack specificity. The aim of this project was to develop a highly specific and sensitive ELISA method for the estimation of free cortisone levels in urine, saliva and in vitro media samples without chromatographic separation. Antibodies against cortisone were raised in rabbits using cortisone-3-CMO-KLH as immunogen. HRP-goat anti-rabbit IgG conjugate was used as enzyme tracer. Cross-reactivities of the untreated cortisone antiserum with major interfering steroids were minimal except for cortisol (3.15%). However, following an immune-affinity purification of the antibodies using CNBr-activated sepharose-cortisol-3-CMO-BSA, cross-reactivity of the purified cortisone antibody with cortisol was reduced to 0.27%. The minimum detection limit of cortisone ELISA was 28 pg/mL (77.7 pM). The validity of the cortisone ELISA was confirmed by the excellent correlation obtained before and after an HPLC fractionation step (Y = 1.09X - 0.21, R2 = 0.98). Intra-assay and inter-assay imprecision were 5.5-11.7% and 8.7-12.8% CV, respectively. Using this assay, salivary cortisone levels showed a circadian rhythm in men and women (11.2 7.3 nM at 08.00 h and 5.1 3.6 nM at 18.00 h), and the levels were reduced following liquorice ingestion. In media of adrenocortical H295 cell line incubations, basal cortisone levels were 4.24 0.22 nM that increased to 8.6 1.2 nM post forskolin treatment. Urinary free cortisone excretion levels in healthy subjects were 56.66 36.9 nmol/day. In human volunteers following ingestion of green coffee bean extract for 2 weeks, urinary free cortisol excretion reduced significantly from 66.67 22.3 to 42.66 17.5 nmol/day (p = 0.02) and the cortisol/cortisone ratio from 2.04 1.33 to 1.49 1.13, p = 0.05. In conclusion, a simple and highly specific and sensitive ELISA has been developed and applied to estimate cortisone levels in biological fluids and culture media.Item Glucocorticoids Induce Nondipping Blood Pressure by Activating the Thiazide-Sensitive CotransporterNovelty and Significance(American Heart Association, 2016-03-07) Ivy, Jessica R.; Oosthuyzen, Wilna; Peltz, Theresa S.; Howarth, Amelia R.; Hunter, Robert W.; Dhaun, Neeraj; Al-Dujaili, Emad A. S.; Webb, David J.; Dear, James W.; Flatman, Peter W.; Bailey, Matthew A.Blood pressure (BP) normally dips during sleep, and nondipping increases cardiovascular risk. Hydrochlorothiazide restores the dipping BP profile in nondipping patients, suggesting that the NaCl cotransporter, NCC, is an important determinant of daily BP variation. NCC activity in cells is regulated by the circadian transcription factor per1. In vivo, circadian genes are entrained via the hypothalamic-pituitary-adrenal axis. Here, we test whether abnormalities in the day:night variation of circulating glucocorticoid influence NCC activity and BP control. C57BL6/J mice were culled at the peak (1:00 AM) and trough (1:00 PM) of BP. We found no day:night variation in NCC mRNA or protein but NCC phosphorylation on threonine53 (pNCC), required for NCC activation, was higher when mice were awake, as was excretion of NCC in urinary exosomes. Peak NCC activity correlated with peak expression of per2 and bmal1 (clock genes) and sgk1 and tsc22d3 (glucocorticoid-responsive kinases). Adrenalectomy reduced NCC abundance and blunted the daily variation in pNCC levels without affecting variation in clock gene transcription. Chronic corticosterone infusion increased bmal1, per1, sgk1, and tsc22d3 expression during the inactive phase. Inactive phase pNCC was also elevated by corticosterone, and a nondipping BP profile was induced. Hydrochlorothiazide restored rhythmicity of BP in corticosterone-treated mice without affecting BP in controls. Glucocorticoids influence the day:night variation in NCC activity via kinases that control phosphorylation. Abnormal glucocorticoid rhythms impair NCC and induce nondipping. Night-time dosing of thiazides may be particularly beneficial in patients with modest glucocorticoid excess.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 Effect of meal fat content on salivary testosterone and cortisol levels in healthy female volunteers(2005) Al-Dujaili, Emad A. S.; Bryant, M. L.The aim of this study was to determine if a change in the amount of fat consumed in the diet influenced female salivary postprandial testosterone and cortisol concentrations and whether any changes affected circadian rhythm. The study was conducted on 9 healthy female subjects aged 21-45 years (BMI mean=22.51.61) and has been approved by the University College Ethical Committee. Over 3 non-consecutive days, each subject consumed 2 meals, lunch and evening dinner, containing either, high fat (40-45% of total energy), low fat (20-25% of total energy), or the subjects usual daily meals. Saliva samples were collected at 11 predetermined times during the challenge days. Testosterone and cortisol levels in each sample were measured by specific and sensitive ELISA methods following ether extraction of saliva samples. Paired t-tests and repeated measures ANOVA were used for statistical analyses. On high fat diet, testosterone levels post lunch rose from a mean of 155 to 307 pg/ml.Testosterone results were significantly higher on the high fat diet compared to usual daily diet (p=0.018 at 30 minutes post-lunch, and p=0.006 at 1 hour post-lunch). Testosterone levels on low and high fat diets differed significantly 30 minutes post-lunch (p <0.05) and 5 minutes pre-dinner (p=0.03). There was a slight rise, though not significant, in testosterone level post dinner (From a mean of 136 to 189 pg/ml, p=0.22) on high fat content compared with a drop on low fat meal (from a mean of 212 pg/ml to 78 pg/ml). Cortisol levels on the low fat meal differed significantly from the high fat meal at1 hour and 2 hours post lunch (p<0.05 and p<0.01 respectively) and 2 hours post-dinner (p=0.041). Mean cortisol level on high fat meal rose from a mean of 3.34 to 4.935 ng/ml post lunch, with a smaller increase after evening meal (from 2.402 to 3.641 ng/ml). The findings of this study indicate that the amount of fat consumed in a meal, can influence postprandial levels of salivary testosterone and cortisol and their circadian rhythm profile. Such an effect on steroid hormones might have an impact on the person's daily activities and general health and wellbeing.Item Effects of green tea consumption on blood pressure, total cholesterol, body weight and fat in healthy volunteers(2009) Al-Dujaili, Emad A. S.; Bradley, Jon-Paul; Almoosawi, Suzana; Fyfe, LornaBackground: Hypertension, obesity and hyperlipidemia are key interlinked features of both metabolic syndrome and cardiovascular disease. Numerous studies have suggested that green tea may reduce blood pressure by activating endothelial nitric oxide synthase and reducing total cholesterol by disrupting the production of apo B and synthesis of chylomicrons and thus have cardio-protective effects. The aim of this pilot study was to investigate the effects of increasing the consumption of green tea-rich catechins on blood pressure (BP), total cholesterol and other body composition parameters in healthy volunteers living in Scotland. Methods: Following a 2 day green tea free period, participants (n=12; 9 females and 3 males) were asked to drink 4 cups of green tea (organic Mao Jian Green Tea from the Zhejiang region of China) for 14 days (~800 ml green tea infusion containing 600-800 mg total catechins). Fasting total plasma cholesterol, BP, weight, BMI and %body fat were measured at day 0 (baseline), day 7 and day 14. Results: Mean systolic BP was reduced significantly by 7.1 mmHg (P<0.0001) and mean diastolic BP reduced by 7.8 mmHg over 14 days (P<0.0001). Mean fasting total cholesterol was reduced significantly by 0.556 mmol/l (P<0.008), BMI by 0.34 kg/m2 (P<0.001), body weight by 0.96 kg (P<0.001) and body fat by 2.36% (P<0.005). Conclusion: Our results has shown that short term consumption of commercial green tea reduces systolic and diastolic BP, fasting total cholesterol, %body fat and body weight suggesting a role for green tea in decreasing established potential cardiovascular risk factors. This study also suggests that reductions may be more pronounced in the overweight population where a significant proportion are obese and have a high risk of cardiovascular disease. Green tea consumption is cost effective, accepted by patients and has no reported side effects.Item Detection of endogenous nandrolone in the urine of healthy volunteers by utilising a sensitive ELISA(2005) Al-Dujaili, Emad A. S.; Mason, J. I.; Swart, P.Recent studies report that nandrolone, a widely used anabolic steroid by athletes to enhance their performance, is produced endogenously in the humans. However, the detection of nandrolone has mainly been indicated by the measurement of its urinary metabolites (19-norandrosterone and 19-noretiocholanone). The aim of this study was to develop a sensitive and specific ELISA for nandrolone to investigate whether the parent steroid can be detected in the urine of healthy volunteers. Nandrolone antibodies were produced by immunising sheep with nandrolone-3-CMO-KLH immunogen and used with HRP-donkey anti-sheep IgG conjugate as tracer to develop the ELISA method. Cross-reactivity values of anti-nandrolone antibody with major interfering steroids including nandrolone metabolites were minimal except for testosterone (1.8%) and dihydrotestosterone (3.98%). A sensitive standard curve for nandrolone ELISA has been constructed with good reproducibility and a minimum detection limit of 12.75 pmole/L (3.5 pg/mL). The assay was evaluated for specificity, sensitivity, parallelism, accuracy and imprecision and all found to be satisfactory. The validity of the urinary nandrolone assay was confirmed by the excellent correlation between the ELISA results and those obtained by LC/MS/MS (ELISANand=1.06 LC/MS/MS Nand+0.032, R2=0.98, p<0.001). Urinary nandrolone excretion in healthy volunteers who were known not to have taken any anabolic steroids was assessed in exercising and non-exercising individuals. In non-exercising females, endogenous urinary nandrolone levels were found to range from 0.014-2.122 ng/mL (0.069-8.98 nmol/ day), and 0.017-1.291 nmol/day for exercising females. In non-exercising males, the levels ranged from 0.018-0.486 ng/mL (0.078-2.341 nmol/day), and 0.041-2.44 nmol/day for exercising males. In conclusion, a simple, rapid and sensitive ELISA method has been developed to estimate urinary excretion of nandrolone, and used for the detection of this steroid in urine. It seems likely that nandrolone as such, though at parts per billion, is excreted in the urine of healthy subjects not knowingly ingesting steroids.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.