Insertion/deletion polymorphism of the angiotensin converting enzyme gene and loss of the insertion allele in aldosterone-producing adenoma

The genetic mechanisms responsible for the formation of adrenocortical adenomas which autonomously produce aldosterone are largely unknown, The adrenal renin-angiotensin system has been implicated in the pathophysiology of these tumours, Angiotensin-converting enzyme (ACE) catalyses the generation of angiotensin II, and the insertion/deletion (I/D) polymorphism of the ACE gene regulates up to 50% of plasma and cellular ACE variability in humans. We therefore examined the genotypic and allelic frequency distributions of the ACE gene I/D polymorphism in 55 patients with aldosterone-producing adenoma, APA, (angiotensin-unresponsive APA n = 28, angiotensin-responsive APA n = 27), and 80 control subjects with no family history of hypertension, We also compared the ACE gene I/D polymorphism allelic pattern in matched tumour and peripheral blood DNA in the 55 patients with APA, The frequency of the D allele was 0.518 and 0.512 and the I allele was 0.482 and 0.488 in the APA and control subjects respectively, Genotypic and allelic frequency analysis found no significant differences between the groups, Examination of the matched tumour and peripheral blood DNA samples revealed the loss of the insertion allele in four of the 25 patients who were heterozygous for the ACE I/D genotype. The I/D polymorphism of the ACE gene does not appear to contribute to the biochemical and phenotypic characteristics of APA, however, the deletion of the insertion allele of the ACE gene I/D polymorphism in 16% of aldosterone-producing adenomas may represent the loss of a tumour suppressor gene/s or other genes on chromosome 17q which may contribute to tumorigenesis in APA.

Prepartum supplementation effects on growth and fertility in Bos indicus-cross cows

Three experiments were conducted in the dry tropics of north Australia using Bos indicus-cross cows. Cows in mid-late pregnancy were either unsupplemented during the late dry season or offered ad libitum (2 kg/day) molasses with 7.4% urea (w/w) (M8U) or cottonseed meal (1 kg/day) for up to 54 days commencing 2 months before the start of the calving season. Supplementation reduced weight loss in experiments 1 and 2 (P

Effect of creatine supplementation on measured glomerular filtration rate in postmenopausal women

We aimed to investigate whether creatine supplementation affects the measured glomerular filtration rate in postmenopausal women (age, 58 +/- 3 years). Subjects were randomly assigned to receive either creatine (20 g(.)day(-1) for 1 week and 5 g(.)day(-1) thereafter) or a placebo. Kidney function was assessed at baseline and after 12 weeks. [(51)Cr] EDTA clearance remained unchanged (CR-PRE: 86.16 +/- 14.36 mL(.)min(-1) per 1.73 m(2), POST: 87.25 +/- 17.60 mL(.)min(-1) per 1.73 m(2); PL-PRE: 85.15 +/- 8.54 mL(.)min(-1) per 1.73 m(2), POST: 87.18 +/- 9.64 mL(.)min(-1) per 1.73 m(2); p = 0.81). Thus, we concluded that creatine supplementation does not affect glomerular filtration rate in postmenopausal women.

Creatine supplementation does not impair kidney function in type 2 diabetic patients: a randomized, double-blind, placebo-controlled, clinical trial

Creatine supplementation may have a therapeutic role in diabetes, but it is uncertain whether this supplement is safe for kidney function. The aim of this study was to investigate the effects of creatine supplementation on kidney function in type 2 diabetic patients. A randomized, double-blind, placebo-controlled trial was performed. The patients were randomly allocated to receive either creatine or placebo for 12 weeks. All the patients underwent exercise training throughout the trial. Subjects were assessed at baseline and after the intervention. Blood samples and 24-h urine samples were obtained for kidney function assessments. Additionally, (51)Cr-EDTA clearance was performed. To ensure the compliance with creatine intake, we also assessed muscle phosphorylcreatine content. The creatine group presented higher muscle phosphorylcreatine content when compared to placebo group (CR Pre 44 +/- A 10, Post 70 +/- A 18 mmol/kg/wt; PL Pre 52 +/- A 13, Post 46 +/- A 13 mmol/kg/wt; p = 0.03; estimated difference between means 23.6; 95% confidence interval 1.42-45.8). No significant differences were observed for (51)Cr-EDTA clearance (CR Pre 90.4 +/- A 16.9, Post 96.1 +/- A 15.0 mL/min/1.73 m(2); PL Pre 97.9 +/- A 21.6, Post 96.4 +/- A 26.8 mL/min/1.73 m(2); p = 0.58; estimated difference between means -0.3; 95% confidence interval -24.9 to 24.2). Creatinine clearance, serum and urinary urea, electrolytes, proteinuria, and albuminuria were unchanged. CR supplementation does not affect kidney function in type 2 diabetic patients, opening a window of opportunities to explore its promising therapeutic role in this population. ClinicalTrials.gov registration number: NCT00992043.

Beneficial Effect of Creatine Supplementation in Knee Osteoarthritis

NEVES JR., M., B. GUALANO, H. ROSCHEL, R. FULLER, F. B. BENATTI, A. L. DE SA PINTO, F. R. LIMA, R. M. PEREIRA, A. H. LANCHA JR., E. BONFA. Beneficial Effect of Creatine Supplementation in Knee Osteoarthritis. Med. Sci. Sports Exerc., Vol. 43, No. 8, pp. 1538-1543, 2011. Introduction: The aim of this study was to investigate the efficacy of creatine (CR) supplementation combined with strengthening exercises in knee osteoarthritis (OA). Methods: A randomized, double-blind, placebo-controlled trial was performed. Postmenopausal women with knee OA were allocated to receive either CR (20 g.d(-1) for 1 wk and 5 g.d(-1) thereafter) or placebo (PL) and were enrolled in a lower limb resistance training program. They were assessed at baseline (PRE) and after 12 wk (POST). The primary outcome was the physical function as measured by the timed-stands test. Secondary outcomes included lean mass, quality of life, pain, stiffness, and muscle strength. Results: Physical function was significantly improved only in the CR group (P = 0.006). In addition, a significant between-group difference was observed (CR: PRE = 15.7 +/- 1.4, POST = 18.1 +/- 1.8; PL: PRE = 15.0 +/- 1.8, POST = 15.2 +/- 1.2; P = 0.004). The CR group also presented improvements in physical function and stiffness subscales as evaluated by the Western Ontario and McMaster Universities Osteoarthritis Index (P = 0.005 and P = 0.024, respectively), whereas the PL group did not show any significant changes in these parameters (P > 0.05). In addition, only the CR group presented a significant improvement in lower limb lean mass (P = 0.04) as well as in quality of life (P = 0.01). Both CR and PL groups demonstrated significant reductions in pain (P G 0.05). Similarly, a main effect for time revealed an increase in leg-press one-repetition maximum (P = 0.005) with no significant differences between groups (P = 0.81). Conclusions: CR supplementation improves physical function, lower limb lean mass, and quality of life in postmenopausal women with knee OA undergoing strengthening exercises.

Effects of carbohydrate supplementation on competitive runners undergoing overload training followed by a session of intermittent exercise

This study evaluated the effects of a micro cycle of overload training (1st-8th day) on metabolic and hormonal responses in male runners with or without carbohydrate supplementation and investigated the cumulative effects of this period on a session of intermittent high-intensity running and maximum-performance-test (9th day). The participants were 24 male runners divided into two groups, receiving 61% of their energy intake as CHO (carbohydrate-group) and 54% in the control-group (CON). The testosterone was higher for the CHO than the CON group after the overload training (694.0 +/- A 54.6 vs. CON 610.8 +/- A 47.9 pmol/l). On the ninth day participants performed 10 x 800 m at mean 3 km velocity. An all-out 1000 m running was performed before and after the 10 x 800 m. Before, during, and after this protocol, the runners received solution containing CHO or the CON equivalent. The performance on 800 m series did not differ in either group between the first and last series of 800 m, but for the all-out 1000 m test the performance decrement was lower for CHO group (5.3 +/- A 1.0 vs. 10.6 +/- A 1.3%). The cortisol concentrations were lower in the CHO group in relation to CON group (22.4 +/- A 0.9 vs. 27.6 +/- A 1.4 pmol/l) and the IGF1/IGFBP3 ratio increased 12.7% in the CHO group. During recovery, blood glucose concentrations remained higher in the CHO group in comparison with the CON group. It was concluded that CHO supplementation possibly attenuated the suppression of the hypothalamic-pituitary-gonadal axis and resulted in less catabolic stress, and thus improved running performance.

Aerobic Exercise Training-Induced Left Ventricular Hypertrophy Involves Regulatory MicroRNAs, Decreased Angiotensin-Converting Enzyme-Angiotensin II, and Synergistic Regulation of Angiotensin-Converting Enzyme 2-Angiotensin (1-7)

Aerobic exercise training leads to a physiological, nonpathological left ventricular hypertrophy; however, the underlying biochemical and molecular mechanisms of physiological left ventricular hypertrophy are unknown. The role of microRNAs regulating the classic and the novel cardiac renin-angiotensin (Ang) system was studied in trained rats assigned to 3 groups: (1) sedentary; (2) swimming trained with protocol 1 (T1, moderate-volume training); and (3) protocol 2 (T2, high-volume training). Cardiac Ang I levels, Ang-converting enzyme (ACE) activity, and protein expression, as well as Ang II levels, were lower in T1 and T2; however, Ang II type 1 receptor mRNA levels (69% in T1 and 99% in T2) and protein expression (240% in T1 and 300% in T2) increased after training. Ang II type 2 receptor mRNA levels (220%) and protein expression (332%) were shown to be increased in T2. In addition, T1 and T2 were shown to increase ACE2 activity and protein expression and Ang (1-7) levels in the heart. Exercise increased microRNA-27a and 27b, targeting ACE and decreasing microRNA-143 targeting ACE2 in the heart. Left ventricular hypertrophy induced by aerobic training involves microRNA regulation and an increase in cardiac Ang II type 1 receptor without the participation of Ang II. Parallel to this, an increase in ACE2, Ang (1-7), and Ang II type 2 receptor in the heart by exercise suggests that this nonclassic cardiac renin-angiotensin system counteracts the classic cardiac renin-angiotensin system. These findings are consistent with a model in which exercise may induce left ventricular hypertrophy, at least in part, altering the expression of specific microRNAs targeting renin-angiotensin system genes. Together these effects might provide the additional aerobic capacity required by the exercised heart. (Hypertension. 2011;58:182-189.).

Supplementation of Vitamin E, Vitamin C, and Zinc Attenuates Oxidative Stress in Burned Children: A Randomized, Double-Blind, Placebo-Controlled Pilot Study

The aim of this study was to investigate the effect of supplementation of vitamin E, vitamin C, and zinc on the oxidative stress in burned children. In a prospective double-blind placebo-controlled pilot study, 32 patients were randomized as no supplementation (n = 15) or antioxidant supplementation (n = 17) groups. Supplementation consisted of the antioxidant mixture of vitamin C (1.5 times upper intake level), vitamin E (1.35 times upper intake level), and zinc (2.0 times recommended dietary allowance) administered during 7 days starting on the second day of admittance into the hospital. Energy requirement was calculated by the Curreri equation, and protein input was 3.0 g/kg of ideal body mass index (percentile 50 degrees). Total antioxidant capacity of plasma and malondialdehyde were used to monitor oxidative stress. The time of wound healing was evaluated as the main clinical feature. Patients (age 54.2 +/- 48.9 months, 65.6% males), who exhibited 15.5 +/- 6.7% of total burn area, showed no differences in age and sex, when compared with controls. Intake of the administered antioxidants was obviously higher in treated subjects (P = .005), and serum differences were confirmed for vitamin E and C, but not for zinc (P = .180). There was a decrease in lipid peroxidation (malondialdehyde level) (P = .006) and an increase in vitamin E concentrations in the antioxidant supplementation group (P = .016). The time of wound healing was lower in the supplemented group (P < .001). The antioxidant supplementation through vitamin E and C and the mineral zinc apparently enhanced antioxidant protection against oxidative stress and allowed less time for wound healing. (J Burn Care Res 2009;30:859-866)

Kidney Function and 24-Hour Proteinuria in Patients with Fabry Disease during 36 Months of Agalsidase Alfa Enzyme Replacement Therapy: A Brazilian Experience

Background. Prior to the introduction of enzyme replacement therapy (ERT), management of Fabry disease (FD) consisted of symptomatic and palliative measures. ERT has been available for several years using recombinant human agalsidase alfa, an analogue of alpha-galactosidase A (GALA). However, the limitations of ERT in improving kidney function have not been established. This study evaluates the safety and therapeutic effect of agalsidase alfa replacement in terms of kidney function and reduction in 24-hour proteinuria. Methods. During the period between January 1, 2002, and August 1, 2005, nine Fabry patients (7 male, 2 female) were treated according to protocol, receiving 0.2 mg/kg agalsidase alfa IV every two weeks. Kidney function was evaluated by measuring the glomerular filtration rate (GFR) using chromium ethylene diamine tetra-acetate clearance ((51)Cr-EDTA mL/min/1.73 m(2)) at baseline, 12, 24, and 36 months. 24-hour proteinuria was measured at baseline, 3, 6, 12, 18, 24, and 36 months of ERT. Kidney disease was classified according to National Kidney Foundation Disease Outcome Quality Initiative (NKF/DOQI) Advisory Board criteria, which define stage I chronic kidney disease (CKD) as GFR >= 90mL/min/1.73 m(2), stage II as 60-89 mL/min/1.73m(2), stage III as 30-59 mL/min/1.73 m(2), stage IV as 15-29 mL/min/1.73m(2), and stage V as < 15 mL/min/1.73m(2). Results. Six patients completed 36 months of therapy, 2 patients completed 18 months, and 1 patient completed 12 months. Mean patient age at baseline was 34.6 +/- 11.3 years. During the study period, kidney function remained stable in patients with stages I, II, or III CKD. One patient, who entered the study with stage IV CKD, progressed to end-stage chronic kidney disease, beginning hemodialysis after 7 months and receiving a kidney transplant after 12 months of ERT. Proteinuria also remained stable in the group of patients with pathologic proteinuria. The use of agalsidase alfa was well tolerated in 99.5% of the infusions administered. Conclusion. Over the course of 36 months of ERT, there was no change in kidney function and 24-hour proteinuria. This suggests thatagalsidase alfa may slow or halt the progression of kidney disease when used before extensive kidney damage occurs. No significant side effects were observed with ERT during the course of the study.

Angiotensin II Binding to Angiotensin I-Converting Enzyme Triggers Calcium Signaling

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system. (Hypertension. 2011;57:965-972.) . Online Data Supplement

Angiotensin-converting enzyme inhibition augments the expression of rat elastase-2, an angiotensin II-forming enzyme

Becari C, Teixeira FR, Oliveira EB, Salgado MC. Angiotensin-converting enzyme inhibition augments the expression of rat elastase- 2, an angiotensin II-forming enzyme. Am J Physiol Heart Circ Physiol 301: H565-H570, 2011. First published May 20, 2011; doi:10.1152/ajpheart.00534.2010.-Mounting evidence suggest that tissue levels of angiotensin (ANG) II are maintained in animals submitted to chronic angiotensin-converting enzyme (ACE) inhibitor treatment. We examined the expression levels of transcripts for elastase-2, a chymostatin-sensitive serine protease identified as the alternative pathway for ANG II generation from ANG I in the rat vascular tissue and the relative role of ACE-dependent and -independent pathways in generating ANG II in the rat isolated carotid artery rings of spontaneously hypertensive rats (SHR) and Wistar normotensive rats (WNR) treated with enalapril for 7 days. Enalapril treatment decreased blood pressure of SHR only and resulted in significantly more elastase-2 mRNA expression in carotid artery of both enalapril-treated WNR and SHR. Captopril induced a comparable rightward shift of concentration-response curves to ANG I in vehicle and enalapril-treated rats, although this effect was of lesser magnitude in SHR group. Chymostatin induced a rightward shift of the dose response to ANG I in vehicle-treated and a decrease in maximal effect of 22% in enalapril-treated WNR group. Maximal response induced by ANG I was remarkably reduced by chymostatin in enalapril-treated SHR carotid artery (by 80%) compared with controls (by 23%). Our data show that chronic ACE inhibition was associated with augmented functional role of non-ACE pathway in generating ANG II and increased elastase-2 gene expression, suggesting that this protease may contribute as an alternative pathway for ANG II generation when ACE is inhibited in the rat vascular tissue.

Effect of angiotensin converting enzyme inhibitor enalapril on body weight and composition in young rats

Obesity is considered a worldwide public health problem showing an increased prevalence in developing countries, with urgent need for new and more efficient drugs and therapies. Enalapril, an angiotensin-I converting enzyme inhibitor (ACEi), is classically used in antihypertensive therapies, however, earlier publications have shown that this drug could also have significant impact on body weight in rats as well as in humans, besides reducing blood pressure. The effect of this drug in the white adipose tissue has been neglected for long time, even considering that most components of the renin-angiotensin and kallikrein-kinin system are expressed in this tissue. Furthermore, the adipose tissue is considered today as one of the most important sites for endocrine/inflammatory regulation of appetite and energy output and AngII has been linked to the metabolism in this tissue. Therefore, we analyzed the influence of chronic enalapril treatment in normotensive rats at earlier ages, evaluating body weight, energy homeostasis, lipid profile and serum levels of the hormones leptin and insulin, in the presence of a standard or a palatable hyperlipidic diet regimen for one month. Our results show that enalapril treatment is able to reduce body fat on both diets, without alteration in serum lipid profile. Furthermore, animals receiving enalapril showed reduction in food intake, leptin level and energy intake. In summary, these findings show for the first time that the ACEi enalapril reduces body fat in young normotensive rats and highlights a novel target to treat obesity and associated diseases. (c) 2007 Elsevier B.V. All rights reserved.

Debaryomyces hansenii UFV-1 Intracellular alpha-Galactosidase Characterization and Comparative Studies with the Extracellular Enzyme

Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher k(cat) than the intracellular enzyme (7.16 vs 3.29 s(-1), respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The K(m) for hydrolysis of pNP alpha Gal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was acompetitively inhibited by galactose (K(i) = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.