A randomised controlled trial evaluating the effect of potassium supplementation on vascular function and the renin-angiotensin-aldosterone system.

There is limited evidence on the effect of potassium supplementation on the vasculature in patients at increased cardiovascular risk. Potassium increases aldosterone and there is a strong association of hyperaldosteronism with poor cardiac outcomes. We aimed to determine whether potassium supplementation has a significant medium-term effect on aldosterone levels and, if so, what the overall effect of this is on vascular function in patients at moderate cardiovascular disease risk. Forty patients at moderate cardiovascular disease risk were included in a randomised placebo-controlled crossover study. Patients were assigned to 64 mmol potassium chloride or placebo for 6 weeks. Vascular function was assessed using pulse-wave analysis including the detection of a change in augmentation index to salbutamol and nitroglycerine-induced vasodilation. There was no change in augmentation index with potassium vs placebo (25.2±1.4 vs. 26.0±1.3%, respectively). Potassium improved brachial systolic blood pressure (131.8±2.2 vs. 137.1±2.4 mm Hg; P=0.013), central systolic blood pressure (123.2±2.3 vs. 128.4±2.3 mm Hg; P=0.011) and central diastolic blood pressure (80.3±1.3 vs. 83.7±1.4 mm Hg; P=0.019). Plasma renin activity and serum aldosterone both increased with potassium (P=0.001 and P=0.048 respectively). We found that potassium supplementation had no effect on endothelial function or pulse-wave analysis. It lowered brachial systolic and central blood pressure. It was associated with increased plasma renin activity and serum aldosterone.

Effect of hydration on whole blood viscosity in firefighters.

CONTEXT: Cardiovascular disease (CVD) is the leading cause of on-duty death among firefighters, totaling 45% of on-duty fatalities. Heat stress and fluid losses can result in decreases in cardiac output of firefighters, despite sustained tachycardia and maximally elevated heart rate during emergencies. Measurements of whole blood viscosity (WBV) may serve as an independent biomarker of the hydration and dehydration states of on-duty firefighters. OBJECTIVE: The current pilot study investigates the effects of a strenuous firefighting simulation and subsequent rehydration on WBV and other biological metrics in nine healthy, nonsmoking firefighters to (1) determine whether dehydration and rehydration result in detectable changes in WBV and (2) compare WBV with the results from a range of conventional medical tests. DESIGN: The research team designed a single-center, unblinded pilot study. SETTING: Fire Training Division, 1900 Lind Ave SW, Renton, WA, 98057. PARTICIPANTS: Participants were 9 healthy, nonsmoking firefighters who were volunteers. OUTCOME MEASURE(S): Vital signs, traditional medical blood tests, and WBV were measured for each firefighter (1) at baseline, (2) after exercise but before rehydration with alkaline water, and (3) postexercise and after rehydration. Hematocrit (HCT), hemoglobin (Hb), and WBV increased after exercise and before rehydration. RESULTS: Dehydration during the mock fire drill resulted in elevated WBV at both low- and high-shear rates. HCT and Hb increased due to dehydration and hemoconcentration. Hb and HCT returned to baseline values after exercise and rehydration, and while WBV improved, baseline values were not restored. After exercise but before rehydration, WBV changes were significantly larger than HCT and Hb changes, suggesting the profound influence of hydration states on WBV. CONCLUSIONS: WBV measurements were better determinants of hydration states than HCT or Hb and should be performed to monitor the cardiovascular health of at-risk firefighters.

Haptoglobin phenotype, pre-eclampsia, and response to supplementation with vitamins C and E in pregnant women with type-1 diabetes.

OBJECTIVE: The phenotype of the antioxidant and pro-angiogenic protein haptoglobin (Hp) predicts cardiovascular disease risk and treatment response to antioxidant vitamins in individuals with diabetes. Our objective was to determine whether Hp phenotype influences pre-eclampsia risk, or the efficacy of vitamins C and E in preventing pre-eclampsia, in women with type-1 diabetes. DESIGN: This is a secondary analysis of a randomised controlled trial in which women with diabetes received daily vitamins C and E, or placebo, from 8 to 22 weeks of gestation until delivery. SETTING: Twenty-five antenatal metabolic clinics across the UK (in north-west England, Scotland, and Northern Ireland). POPULATION: Pregnant women with type-1 diabetes. METHODS: Hp phenotype was determined in white women who completed the study and had plasma samples available (n = 685). MAIN OUTCOME MEASURE: Pre-eclampsia. RESULTS: Compared with Hp 2-1, Hp 1-1 (OR 0.59, 95% CI 0.30-1.16) and Hp 2-2 (OR 0.93, 95% CI 0.60-1.45) were not associated with significantly decreased pre-eclampsia risk after adjusting for treatment group and HbA1c at randomisation. Our study was not powered to detect an interaction between Hp phenotype and treatment response; however, our preliminary analysis suggests that vitamins C and E did not prevent pre-eclampsia in women of any Hp phenotype (Hp 1-1, OR 0.77, 95% CI 0.22-2.71; Hp 2-1, OR 0.81, 95% CI 0.46-1.43; Hp 2-2, 0.67, 95% CI 0.34-1.33), after adjusting for HbA1c at randomisation. CONCLUSIONS: The Hp phenotype did not significantly affect pre-eclampsia risk in women with type-1 diabetes.

Placental protein tyrosine nitration and MAPK in type 1 diabetic pre-eclampsia: Impact of antioxidant vitamin supplementation.

AIM: To examine the role of placental protein tyrosine nitration and p38-Mitogen-Activated Protein Kinase α (p38-MAPKα), Extra Cellular-Signal Regulated Kinase (ERK) and c-Jun NH2-Terminal Kinase (JNK) activity, in the pathogenesis of type 1 diabetic pre-eclampsia, and the putative modulation of these indices by maternal vitamin C and E supplementation. METHODS: Placental samples were obtained from a sub-cohort of the DAPIT trial: a randomised placebo-controlled trial of antioxidant supplementation to reduce pre-eclampsia in type 1 diabetic pregnancy. Placenta from placebo-treated: normotensive (NT) [n=17], gestational hypertension (GH) [n=7] and pre-eclampsia (PE) [n=6] and vitamin-treated: NT (n=20), GH (n=4) and PE (n=3) was analysed. Protein tyrosine nitration was assessed by immunohistochemistry in paraffin-embedded tissue. Catalytic activities of placental p38-MAPKα, ERK and JNK were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Nitrotyrosine immunostaining was present in placebo-treated NT, GH and PE placentae, with no significant difference observed between the groups. There was a non-significant trend towards decreased p38-MAPKα activity in PE vs NT control placentae. ERK and JNK were similar among the three outcome placebo groups and vitamin supplementation did not significantly alter their activity. CONCLUSION: Nitrotyrosine immunopositivity in normotensive diabetic placentae indicates some degree of tyrosine nitration in uncomplicated diabetic pregnancy, possibly due to inherent oxidative stress and peroxynitrite production. Our results suggest that p38-MAPKα, ERK and JNK are not directly involved in the pathogenesis of type 1 diabetic pre-eclampsia and are not modulated by vitamin-supplementation.

Factors associated with serum/plasma concentrations of vitamins A, C, E and carotenoids in older people throughout Europe: the EUREYE study.

PURPOSE: To report on plasma/serum levels of antioxidant vitamin and carotenoids in older adults resident in multiple countries in Europe and examine relationships with potential modifiers. METHODS: Population-based cross-sectional European Eye Study in 7 centres from northern to southern Europe. In total, 4,133 participants aged 65 years or over, collected by random sampling, were recruited. Questionnaires relating to diet, lifestyle and medical history were administered. Non-fasting blood samples were analysed in a single laboratory for vitamins A, C and E and a panel of carotenoids. Associations were analysed by bootstrapped multivariable regression analysis. RESULTS: Centre and season influenced the serum and plasma concentrations of all antioxidant vitamins and carotenoids. Gender, BMI, smoking, age, education, alcohol consumption and supplement use were also significantly associated with some, but not all, of the antioxidant vitamins and carotenoids examined. The proportion of variance explained ranged from 4.8 % for retinol to 25.2 % for zeaxanthin. CONCLUSIONS: In older people, antioxidant vitamin and carotenoid status varies by centre and season, but is also associated with other behavioural and lifestyle variables. Studies aiming to demonstrate an association between antioxidant vitamins and carotenoid status and chronic disease risk should consider these potential confounders.

Evaluation of the status of canine hydrotherapy in the UK.

To establish the current status of canine hydrotherapy in the UK and to ascertain information regarding the current use of hydrotherapy, a questionnaire was sent to 152 hydrotherapy centres throughout the UK, from which 89 responded. Hydrotherapy was found to be a rapidly growing business. Stand-alone centres were in existence; however, many centres were connected to other businesses, including boarding kennels and general practice veterinary surgeries. The dogs using the facility were mainly pedigree breeds, particularly labrador retrievers (30 per cent), and the most commonly encountered conditions were rupture of the cranial cruciate ligament (25 per cent), hip dysplasia (24 per cent) and osteoarthritis (18 per cent). The proportion of qualified versus unqualified staff varied between centres, highlighting a need for improved regulation of this aspect of the industry. However, all the dogs treated by the hydrotherapy centres surveyed were direct veterinary referrals, suggesting a good degree of professionalism in the field and a high regard for the benefits of hydrotherapy.

Lipid content and fatty acid composition of the digital cushion of bulls offered different amounts of linseed.

Previous research suggests that the digital cushion, a shock-absorbing structure in the claw, plays an important role in protecting cattle from lameness. This study aimed to assess the degree to which nutritional factors influence the composition of the digital cushion. This involved quantifying lipid content and fatty acid composition differences in digital cushion tissue from cattle offered diets with different amounts of linseed. Forty-six bulls were allocated to 1 of 4 treatments, which were applied for an average of 140 +/- 27 d during the finishing period. The treatments consisted of a linseed supplement offered once daily on top of the basal diet (grass silage:concentrate) at 0, 400, 800, or 1,200 g of supplement/animal per day. For each treatment, the concentrate offered was adjusted to ensure that total estimated ME intake was constant across treatments. Target BW at slaughter was 540 kg. Legs were collected in 3 batches after 120, 147 and 185 d on experiment. Six samples of the digital cushion were dissected from the right lateral hind claw of each animal. Lipids were extracted and expressed as a proportion of fresh tissue, and fatty acid composition of the digital cushion was determined by gas chromatography. Data were analyzed by ANOVA, with diet, location within the digital cushion, and their interactions as fixed effects and fat content (grams per 100 g of tissue) as a covariate. Linear or quadratic contrasts were examined. The lipid content of digital cushion tissue differed between sampling locations (P < 0.001) but did not vary by treatment. There were quadratic responses to increasing linseed supplementation for several fatty acids. Although the overall proportion of C18:3n-3 (the most abundant fatty acid in linseed) did not differ (P < 0.33) by treatment, there was a quadratic influence of diet on total PUFA concentration (P = 0.003) and a linear increase in C18:3n-3 as a proportion of total PUFA (P = 0.01) in the digital cushion. This work demonstrates that dietary fatty acid composition influences the concentration of fatty acids incorporated in the digital cushion of cattle. Based on the large number of quadratic responses among the fatty acids, it appears there is a threshold amount of fatty acid incorporation in the digital cushion.

Molecular detection of exercise-induced free radicals following ascorbate prophylaxis in type 1 diabetes mellitus: a randomised controlled trial.

AIMS/HYPOTHESIS: Patients with type 1 diabetes mellitus are more susceptible than healthy individuals to exercise-induced oxidative stress and vascular endothelial dysfunction, which has important implications for the progression of disease. Thus, in the present study, we designed a randomised double-blind, placebo-controlled trial to test the original hypothesis that oral prophylaxis with vitamin C attenuates rest and exercise-induced free radical-mediated lipid peroxidation in type 1 diabetes mellitus. METHODS: All data were collected from hospitalised diabetic patients. The electron paramagnetic resonance spectroscopic detection of spin-trapped alpha-phenyl-tert-butylnitrone (PBN) adducts was combined with the use of supporting markers of lipid peroxidation and non-enzymatic antioxidants to assess exercise-induced oxidative stress in male patients with type 1 diabetes (HbA(1c) 7.9 +/- 1%, n = 12) and healthy controls (HbA(1c) 4.6 +/- 0.5%, n = 14). Following participant randomisation using numbers in a sealed envelope, venous blood samples were obtained at rest, after a maximal exercise challenge and before and 2 h after oral ingestion of 1 g ascorbate or placebo. Participants and lead investigators were blinded to the administration of either placebo or ascorbate treatments. Primary outcome was the difference in changes in free radicals following ascorbate ingestion. RESULTS: Six diabetic patients and seven healthy control participants were randomised to each of the placebo and ascorbate groups. Diabetic patients (n = 12) exhibited an elevated concentration of PBN adducts (p < 0.05 vs healthy, n = 14), which were confirmed as secondary, lipid-derived oxygen-centred alkoxyl (RO.) radicals (a(nitrogen) = 1.37 mT and abeta(hydrogen) = 0.18 mT). Lipid hydroperoxides were also selectively elevated and associated with a depression of retinol and lycopene (p < 0.05 vs healthy). Vitamin C supplementation increased plasma vitamin C concentration to a similar degree in both groups (p < 0.05 vs pre-supplementation) and attenuated the exercise-induced oxidative stress response (p < 0.05 vs healthy). There were no selective treatment differences between groups in the primary outcome variable. CONCLUSIONS/INTERPRETATION: These findings are the first to suggest that oral vitamin C supplementation provides an effective prophylaxis against exercise-induced free radical-mediated lipid peroxidation in human diabetic blood. CLINICAL TRIALS REGISTRATION NUMBER: ISRCTN96164937.

Hypothalamic input is required for development of normal numbers of thyrotrophs and gonadotrophs, but not other anterior pituitary cells in late gestation sheep.

To evaluate the hypothalamic contribution to the development of anterior pituitary (AP) cells we surgically disconnected the hypothalamus from the pituitary (hypothalamo-pituitary disconnection, HPD) in fetal sheep and collected pituitaries 31 days later. Pituitaries (n = 6 per group) were obtained from fetal sheep (term = 147 +/- 3 days) at 110 days (unoperated group) of gestation and at 141 days from animals that had undergone HPD or sham surgery at 110 days. Cells were identified by labelling pituitary sections with antisera against the six AP hormones. Additionally, we investigated the colocalization of glycoprotein hormones. The proportions of somatotrophs and corticotrophs were unchanged by age or HPD. Lactotrophs increased 80% over time, but the proportion was unaffected by HPD. Thyrotrophs, which were unaffected by age, increased 70% following HPD. Gonadotrophs increased with gestational age (LH+ cells 55%; FSH+ cells 19-fold), but this was severely attenuated by HPD. We investigated the possible existence of a reciprocal effect of HPD on multipotential glycoprotein-expressing cells. Co-expression of LH and TSH was extremely rare (< 1%) and unchanged over the last month of gestation or HPD. The increase of gonadotrophs expressing FSH only or LH and FSH was attenuated by HPD. Therefore, the proportions of somatotrophs, lactotrophs and corticotrophs are regulated independently of hypothalamic input in the late gestation fetal pituitary. In marked contrast, the determination of the thyrotroph and gonadotroph lineages over the same time period is subject to complex mechanisms involving hypothalamic factors, which inhibit differentiation and/or proliferation of thyrotrophs, but stimulate gonadotrophs down the FSH lineage. Development of a distinct population of gonadotrophs, expressing only LH, appears to be subject to alternative mechanisms.

Effect of lycopene supplementation on insulin-like growth factor-1 and insulin-like growth factor binding protein-3: a double-blind, placebo-controlled trial.

OBJECTIVE: Studies have suggested a link between lycopene and insulin-like growth factor-1 (IGF-1). The aim of this study was to test the effect of lycopene supplementation on IGF-1 and binding protein-3 (IGFBP-3) status in healthy male volunteers. DESIGN, SETTING, SUBJECTS AND INTERVENTION: This was a 4 week randomized, double-blind, placebo-controlled study of lycopene supplementation (15 mg/day) in healthy male volunteers (n=20). Fasting blood samples were collected at baseline and after 4 weeks. Samples were analysed for lycopene by high-performance liquid chromatography (HPLC) and IGF-1 and IGFBP-3 by enzyme-linked immunosorbent assay (ELISA). Changes in end points from baseline were compared in those who received placebo versus those who received the lycopene supplement. RESULTS: Median change in lycopene from baseline (post-supplement - baseline) was higher in subjects in the intervention than those on placebo (lycopene group 0.29 (0.09, 0.46); placebo group 0.03 (-0.11, 0.08) micromol/l; median (25th, 75th percentiles), P<0.01). There was no difference in median change in IGF-1 concentrations (lycopene group -0.6 (-2.6, 1.9); placebo group -1.15 (-2.88, 0.95) nmol/l, P=0.52), or median change in IGFBP-3 concentrations (lycopene group 245 (-109, 484); placebo group 101 (-34, 234) nmol/l, P=0.55) between intervention and control groups. Change in lycopene concentration was associated with the change in IGFBP-3 in the intervention group (r=0.78; P=0.008; n=10). CONCLUSIONS: Lycopene supplementation in healthy male subjects has no effect on IGF-1 or IGFBP-3 concentrations in a healthy male population. However, the association between change in lycopene concentration and change in IGFBP-3 in the intervention group suggests a potential effect of lycopene supplementation on IGFBP-3.

The Diabetes and Pre-eclampsia Intervention Trial.

OBJECTIVE: Rates of pre-eclampsia in women with type 1 diabetes are two to four times higher than in normal pregnancies. Diabetes is associated with antioxidant depletion and increased free radical production, and an increasing body of evidence suggests that oxidative stress and endothelial cell activation may be relevant to disease pathogenesis in pre-eclampsia. The Diabetes and Pre-eclampsia Intervention Trial (DAPIT) aims to establish if pregnant women with type 1 diabetes supplemented with vitamins C and E have lower rates of pre-eclampsia and endothelial activation compared with placebo treatment. METHODS: DAPIT is a randomised multicentre double-blind placebo-controlled trial that will recruit 756 pregnant women with type 1 diabetes from 20 metabolic-antenatal clinics in the UK over 4 years. Women are randomised to daily vitamin C (1000 mg) and vitamin E (400 IU) or placebo at 8-22 weeks of gestation until delivery. Maternal venous blood is obtained at randomisation, 26 and 34 weeks, for markers of endothelial activation and oxidative stress and to assess glycaemic control. The primary outcome of DAPIT is pre-eclampsia. Secondary outcomes include endothelial activation (PAI-1/PAI-2) and birthweight centile.

Impact of video colonoscopy on stage and outcome of patients with symptomatic colorectal cancer.

BACKGROUND: Double-contrast barium enema still is regarded by many as the investigation of choice for patients with large bowel symptoms. The aim of this study was to compare the stage and outcome of patients with colorectal cancer diagnosed by video colonoscopy (VC) and barium enema (BE) in a single institution. METHODS: Between July 1997 and December 2001, data were gathered prospectively in a series of 489 patients presenting consecutively with symptomatic colorectal cancer. Selection of patients for either VC or BE investigation was made by the clinician who examined the patient in the clinic. Of the 489 patients, 82 were excluded because they presented acutely or other methods were used for the diagnosis. RESULTS: A diagnosis of colorectal cancer was determined by VC for 292 patients and by BE for 115 patients. The patients in both groups were similar in terms of age, gender, and site of disease. Stage 1 disease (T1/2NO) was diagnosed for 87 (29.8%) patients in the VC group, as compared with 10 (8.7%) in the BE group (p < 0.0001). Early colorectal cancer (T1) was diagnosed for 43 patients in the VC group as compared to 1 patient in the BE group (p < 0.0001). During a median follow-up period of 33 months, 8.2% of the patients in the VC group had experienced recurrence after curative resection, as compared with 17.4% of the patients in the BE group p = 0.018). Freedom from disease (p = 0.02) and overall survival (p = 0.03) were significantly increased in the VC group. CONCLUSIONS: Videocolonoscopy used as the investigation of choice for patients with large bowel symptoms detects colorectal cancer at an earlier stage and has a significant impact on the outcome for this condition.

Rate of optic disc cup progression in treated primary open-angle glaucoma.

PURPOSE: To quantitatively evaluate the rate of cup-to-disc ratio progression in treated patients with primary open-angle glaucoma and to identify clinical factors associated with cup progression. PATIENTS AND METHODS: Fifty one eyes of 51 treated primary open-angle glaucoma patients with a minimum of 9-year longitudinal series of stereoscopic optic disc photographs were studied. Eyes with any other ocular disease except for mild cataract were excluded. Each set of stereoscopic photographs was digitized and viewed stereoscopically on a computer screen using a hand-held stereoscope. Computer-aided planimetry was performed on each set of photographs with examiner-defined cup and disc margins using custom-made software. The software computed linear cup-to-disc ratios as well as peripapillary atrophy area. Both inter-observer and intra-observer reliabilities were evaluated in a masked, random fashion using intra-class correlation. Changes in linear cup-to-disc ratios and peripapillary atrophy were estimated using linear regression over time. All available clinical factors were evaluated for association with the rate of cup progression using a multiple regression model. RESULTS: All patients studied were Caucasian; 31(61%) were females. The mean age at the beginning of the study was 61.6 +/- 7.8 years (range 46-81). The mean follow-up period was 14.3 +/- 3.5 years (median 14.0, range 9.6-22.3). A total of 173 sets of stereo disc photographs were analyzed (3.4 +/- 1.3 per patient). The initial and final linear cup-to-disc ratios were 0.64 +/- 0.15 and 0.73 +/- 0.14 respectively. The inter-rater and intra-rater intraclass correlations were 0.76 (95% CI 0.61-0.87) and 0.97 (95% CI 0.93-0.98) respectively. Using linear regression the rate of linear cup-to-disc ratio change was 0.0068 +/- 0.0062 per year (range -0.0025- 0.0269). Three eyes had an increase in the peripapillary atrophy area. The higher yearly average intraocular pressure was significantly associated with faster rate of linear cup-to-disc ratios progression (P = 0.03). CONCLUSIONS: In treated patients with primary open-angle glaucoma, the rate of progressive optic disc cupping was slow (LCDR progression of 0.0068 per year). The higher yearly average intraocular pressure was significantly associated with a faster rate of cup progression.

Dietary supplements of soya flour lower serum testosterone concentrations and improve markers of oxidative stress in men.

OBJECTIVE: We examined the effects on serum sex steroids, lipids and markers of oxidative stress of supplementing the diets of healthy male volunteers with scones made with soya flour. DESIGN: A randomized placebo controlled cross-over trial. SETTING: University Hospital of Wales. SUBJECTS: Twenty volunteers recruited by advertisement. INTERVENTIONS: Male volunteers ate three scones a day in addition to their normal diet for a period of 6 weeks. The scones were made with either wheat or soya flour (containing 120 mg/day of isoflavones). Blood was analysed for sex steroids (testosterone, dihydro-testosterone, oestradiol, oestrone, sex hormone binding globulin, albumin and the concentration of non-protein bound sex steroids were calculated), lipid profile (total cholesterol, high density lipoprotein cholesterol and triglycerides) and measures of oxidative stress (hydroperoxides, susceptibility of LDL to oxidation with copper and myeloperoxidase). RESULTS: The volunteers' mean age was 35.6 (s.d. 11.2) y. Total serum testosterone fell in volunteers taking the soya scones (19.3-18.2 nmol/l; 95% CI 1.01, 1.12; P=0.03). No significant changes were seen in the concentrations of the other serum sex steroids, albumin or sex hormone binding globulin throughout the study. Significant improvements in two of the three markers of oxidative stress were seen in volunteers taking soya scones. Lag time for myeloperoxidase rose from 55.0 to 68.0 min (95% CI -16.0, -3.5; P=0.009) and the presence of hydroperoxides decreased from 2.69 to 2.34 micro mol/l (95% CI 0.12, 0.71; P=0.009). There were no changes seen in serum triglycerides or cholesterol. CONCLUSIONS: We have shown that soya supplements reduce serum testosterone and improve markers of oxidative stress. These findings provide a putative mechanism by which soya supplements could protect against prostatic disease and atherosclerosis. Further dietary studies with clinical end points are warranted.

Effect of antalarmin, a novel corticotropin-releasing hormone antagonist, on the dynamic function of the preterm ovine fetal hypothalamo-pituitary-adrenal axis.

This study describes the effect of antalarmin on basal and stimulated activity of the hypothalamo-pituitary-adrenal (HPA) axis function in the late gestation ovine fetus. Fetuses received antalarmin (15 mg/h i.v.) or vehicle (cremophor El 50% in ethanol) from day 130 gestational age. Antalarmin infusion did not significantly affect immunoreactive corticotropin (ir-ACTH) concentrations, although there was a tendency for ir-ACTH to be lower and cortisol concentrations were lower in the antalarmin-treated fetuses (p < 0.01). The ir-ACTH response to corticotropin-releasing hormone (CRH) challenge was attenuated (p < 0.05) in the antalarmin-treated fetuses, but neither antalarmin- nor vehicle-treated fetuses had significant cortisol responses to CRH. The ir-ACTH response to hypoxia was diminished (p < 0.05) in the antalarmin-treated fetuses while the cortisol responses of antalarmin- and vehicle-treated fetuses were indistinguishable. Deconvolution analysis revealed no effect of antalarmin treatment on ir-ACTH secretory dynamics. In contrast, antalarmin decreased (p < 0.05) basal, mean and integrated cortisol. The plasma cortisol responses of antalarmin- and vehicle-treated fetuses to exogenous ACTH(1-24) were indistinguishable. These data indicate that, while antalarmin inhibits CRH- and stress-induced ir-ACTH secretion, basal ir-ACTH secretion may be less affected by antalarmin treatment. Paradoxically, cortisol secretion is impaired by antalarmin infusion, although adrenal responsiveness to ACTH is not impaired. These results confirm a role for CRH in stress-induced ACTH secretion in the ovine fetus, though its role in the regulation of basal ACTH and cortisol secretion is unclear.

Site and mechanism of action of dynorphin A-(1-13) and N-methyl-D-aspartate on ACTH release in fetal sheep.

Dynorphin A (Dyn A) stimulates the release of ACTH in fetal sheep, a response that involves N-methyl-D-aspartate (NMDA) receptors but not the secretogogues corticotropin-releasing hormone or arginine vasopressin. We now find that neither Dyn A-(1-13) (0.5 mg/kg, i.v.) nor NMDA (4 mg/kg, i.v.) elicits ACTH release in postnatal lambs. This led us to hypothesize that Dyn A-(1-13) and NMDA might act to release placental ACTH. However, the ability of Dyn A-(1-13), NMDA, and the kappa-opioid receptor agonist U-50488H (1 mg/kg, i.v.) to release ACTH was lost after either fetal hypophysectomy (n = 4) or hypothalamo-pituitary disconnection (n = 4). These results indicate that neither the placenta nor the fetal pituitary is the site of action for these agonists and suggest a hypothalamic or suprahypothalamic site of action. Furthermore, the release of ACTH by Dyn A-(1-13) and NMDA was abolished after pretreatment with indomethacin, suggesting that they might cause the release of a prostanoid, possibly from the placenta, that subsequently acts at the hypothalamus or serves as a permissive factor in the action of Dyn A-(1-13) and NMDA at the hypothalamus.

Expression, crystallization and derivatization of the complete extracellular domain of the beta(c) subunit of the human IL-5, IL-3 and GM-CSF receptors.

The major signalling entity of the receptors for the haemopoietic cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is the shared beta(c) receptor, which is activated by ligand-specific alpha receptors. The beta(c) subunit is a stable homodimer whose extracellular region consists of four fibronectin domains and appears to be a duplication of the cytokine receptor homology module. No four domain structure has been determined for this receptor family and the structure of the beta(c) subunit remains unknown. We have expressed the extracellular domain in insect cells using the baculovirus system, purified it to homogeneity and determined its N-terminal sequence. N-glycosylation at two sites was demonstrated. Crystals of the complete domain have been obtained that are suitable for X-ray crystallographic studies, following mutagenesis to remove one of the N-glycosylation sites. The rhombohedral crystals of space group R3, with unit cell dimensions 186.1 A and 103.5 A, diffracted to a resolution of 2.9 A using synchrotron radiation. Mutagenesis was also used to engineer cysteine substitution mutants which formed isomorphous Hg derivatives in order to solve the crystallographic phase problem. The crystal structure will help to elucidate how the beta(c) receptor is activated by heterodimerization with the respective alpha/ligand complexes.

Consumption of flavonoids in onions and black tea: lack of effect on F2-isoprostanes and autoantibodies to oxidized LDL in healthy humans.

BACKGROUND: Oxidative damage to lipids in vivo may be involved in the development of atherosclerosis and cancer. Onions and black tea are foods rich in flavonoids, predominantly the flavonoid quercetin, which is a potent in vitro inhibitor of membrane lipid peroxidation and LDL oxidation. OBJECTIVE: Our objective was to investigate the effects of consuming a high-flavonoid (HF) diet enriched with onions and black tea on indexes of oxidative damage in vivo compared with a low-flavonoid (LF) diet. DESIGN: Thirty-two healthy humans were studied in a randomized crossover design. Indexes of oxidative damage used were plasma F2-isoprostanes (a biomarker of lipid peroxidation in vivo) and the titer of antibodies to malondialdehyde (MDA)-modified LDL. RESULTS: There were no significant differences in the intake of macronutrients or assessed micronutrients, plasma F2-isoprostane concentrations, and MDA-LDL autoantibody titer between the HF and LF dietary treatments. In the men, however, plasma concentrations of the F2-isoprostane 8-epi-prostaglandin F2alpha were slightly higher after the HF treatment phase than after the LF treatment [0.31 +/- 0.029 nmol/L (111 +/- 10.4 ng/L) compared with 0.26 +/- 0.022 nmol/L (92 +/- 7.8 ng/L); P = 0.041]. In all subjects, plasma quercetin concentrations were significantly higher after the HF treatment phase than after the LF treatment: 221.6 +/- 37.4 nmol/L compared with less than the limit of detection of 66.2 nmol/L. CONCLUSION: Flavonoid consumption in onions and tea had no significant effect on plasma F2-isoprostane concentrations and MDA-LDL autoantibody titer in this study and thus does not seem to inhibit lipid peroxidation in humans.

Antioxidants and protein carbonyls in bronchoalveolar lavage fluid of children: normal data.

Antioxidant-oxidant imbalances in bronchoalveolar lavage fluid (BAL) are thought to contribute to oxidative stress in respiratory disease. However, normal reference ranges for BAL antioxidants and oxidized proteins in children are not available. In this study, we recruited 124 children attending for elective surgery for a noninflammatory condition; 83 were nonasthmatic, nonatopic (N) and 41 were nonasthmatic, atopic (NA). A nonbronchoscopic lavage was performed and ascorbate, uric acid, alpha-tocopherol, and protein carbonyl (as a measure of oxidative damage) concentrations were determined in BAL fluid. The 95% reference range was 0.112-1.897 micromol/L for ascorbate, 0.149-2.163 micromol/L for urate, 0.0029-0.066 micromol/L for alpha-tocopherol, and 0.280-4.529 nmol/mg for protein carbonyls in BAL fluid. Age, gender, and exposure to environmental tobacco smoke did not affect the concentration of ascorbate, urate, alpha-tocopherol, or protein carbonyls. However, in multiple linear regression analyses, the type of home heating (glass-fronted fires or oil-fired central heating) was found to influence ascorbate and urate concentrations in the BAL fluid (ss-coefficient for ascorbate: 0.445, p = 0.031; for urate: 0.114, p = 0.001). There was no significant difference between the N and NA group in BAL fluid concentrations of ascorbate, urate, or protein carbonyls. The alpha-tocopherol concentration was significantly increased in the NA group (p = 0.037). Uric acid and alpha-tocopherol concentrations in BAL fluid and serum were not correlated. Intriguingly, serum and BAL ascorbate concentrations were significantly correlated (r = 0.297, p = 0.018, n = 63), which may offer an explanation for why supplementing the diet with vitamin C can improve asthma symptoms. Further studies will investigate the role of BAL antioxidant concentrations in children with inflammatory respiratory diseases.