Estrogen receptor αlpha gene (ESR1) PvuII and XbaI polymorphisms are associated to metabolic and proinflammatory factors in polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that involves multiple factors. Although the etiology of PCOS is unknown, there is an involvement of sex steroid hormones in the pathophysiology of this syndrome. Therefore, polymorphisms in genes involved in the action of estrogen may contribute to a woman's susceptibility to PCOS. AIM: This study aimed to evaluate the association between the polymorphisms PvuII and XbaI in the estrogen receptor alpha (ESR1) gene and the occurrence of PCOS. The study also aimed to assess the influence of these polymorphisms on the metabolic and inflammatory profiles of women with PCOS. MATERIAL AND METHODS: This case-control study included 99 women with PCOS, diagnosed according to the Rotterdam criteria, and 104 age-matched healthy women. The polymorphisms were evaluated using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: No association between the ESR1 gene polymorphisms and the presence of PCOS was observed. However, we found associations between the PvuII polymorphism and C-reactive protein levels, testosterone levels, family history of diabetes, and waist circumference. The XbaI polymorphism was associated with fasting glucose and a family history of hypertension. CONCLUSION: These polymorphisms are not associated with PCOS development, but they are involved in the phenotype of complications of the syndrome. Therefore, prior knowledge of these genomic variants might contribute to taking preventive measures that could delay the metabolic and reproductive complications commonly seen in women with PCOS.

Correlation between plasminogen activator inhibitor-1 (PAI-1) promoter 4G/5G polymorphism and metabolic/proinflammatory factors in polycystic ovary syndrome.

Polycystic Ovary Syndrome (PCOS) is the most common cause of subfertility associated to metabolic disorders. The aim of this study was to correlate metabolic and proinflammatory factors in women with PCOS. The frequency of Plasminogen Activator Inhibitor-1 (PAI-1) promoter 4 G/5 G polymorphism was also compared to healthy controls. We evaluated 79 PCOS and 79 healthy women. PAI-1 levels are positively correlated with proinflammatory factors in PCOS group. 4 G allele in PAI-1 gene was more frequent in PCOS and the 4G/4 G genotype was associated with increased PAI-1 levels. A correlation between insulin resistance and proinflammatory and overweight was also observed. C-reactive protein, serum levels of vascular cell adhesion molecule-1 (sVCAM-1), Lipid Accumulation Product (LAP) and vitamin D are good tools to evaluated factors associated to cardiovascular risk in women with PCOS.

Apolipoprotein A5-1131T>C polymorphism, but not APOE genotypes, increases susceptibility for dyslipidemia in children and adolescents.

Apolipoprotein A5 (APOA5) and apolipoprotein E (APOE) play important roles in the metabolism of cholesterol and triglycerides. The aim of this study was to determine the allelic and genotypic distributions of the APOA5-1131T>C (rs 662799) and the APOE HhaI polymorphisms and to identify the association of both individual and combined APOA5-APOE genetic variants and the risk for dyslipidemia in children and adolescents. We genotyped 53 dyslipidemic and 77 normolipidemic individuals. The total cholesterol, triglycerides and HDL cholesterol were determined enzymatically. For APOA5 polymorphism, the presence of the allele C confers an individual risk for dyslipidemia (OR = 2.38, 95% CI = 1.15-4.89; P = 0.018). No significant differences were observed for lipid parameters among the APOA5 groups, except for a higher value of HDLc (P = 0.024) in C-carriers. The allelic and genotypic frequencies of APOE polymorphism were similar between groups and did not increase the susceptibility for dyslipidemia. None of the combined APOA5-APOE polymorphisms increased risk for dyslipidemia. We demonstrated an association between APOA5-1131T>C polymorphism and dyslipidemia in children and adolescents. This finding may be useful to guide new studies with genetic markers down a path toward a better characterization of the genetic risk factors for dyslipidemia and atherosclerotic diseases.

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline.

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 microM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37 degrees C was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 microM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 microM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 +/- 0.8 microM and k cat = 48.4 +/- 1.0 min(-1). The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 +/- 10, 1,098 +/- 91, 38.6 +/- 5.2 and 37,340 +/- 5,400 microM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 +/- 92.8 and 310,500 +/- 38,600 microM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 `M) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 `M), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 `M) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 + or - 0.8 `M and k cat = 48.4 + or - 1.0 min-1. The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 + or - 10, 1,098 + or - 91, 38.6 + or - 5.2 and 37,340 + or - 5,400 `M, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 + or - 92.8 and 310,500 + or - 38,600 `M, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds

Kinetic characterization of rat tissue kallikrein using N alpha-substituted arginine 4-nitroanilides and N alpha-benzoyl-L-arginine ethyl ester as substrates.

Hydrolysis of seven N alpha-substituted L-arginine 4-nitroanilides: benzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 microM for p-nitroanilides and 50 to 190 microM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 microM for p-nitroanilides and 200 to 1800 microM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 +/- 2 microM; Vmax = 10.42 +/- 0.28 microM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 +/- 2 microM; Vmax = 3.21 +/- 0.11 microM/min), while Tos-Arg-Nan (Km = 29 +/- 2 microM; Vmax = 0.10 +/- 0.002 microM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 +/- 15 microM; Vmax = 121.3 +/- 7.6 microM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.

Kinetic characterization of rat tissue kallikrein using NÓ-substituted arginine 4-nitroanilides and NÓ-benzoyl-L-arginine ethyl ester as substrates

Hydrolysis of seven N(alpha-substituted L-arginine 4-nitroanilides: henzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N(alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 muM for p-nitroanilides and 50 to 190 muM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 muM for p-nitroanilides and 200 to 1800 muM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 ñ 2 muM; Vmax 10.42 ñ 0.28 muM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 ñ 2 muM; Vmax = 3.21 ñ 0.11 muM/min), while Tos-Arg-Nan (Km = 29 ñ 2 muM; Vmax, = 0. 10 ñ 0.002 muM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 ñ 15 muM; Vmax = 121.3 ñ 7.6 muM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.

Soro-controle de origem bovina e seu emprego no controle de qualidade em bioquímica clínica / Control serum of bovine origin and its usage in quality control in clinical biochemistry

Os autores apresentam estudo de um soro-controle preparadoa partir de material de origem bovina e seu desempenho no controle de qualidade em bioquimica clinica. Esse soro foi preparado na proporção de 6 partes de soro e 4 partes de glicerol adicionando-se 0,1g de azida sódica para cada 100ml. A análise estatística dos resultados obtidos ao longo de oito meses mostrou uma boa estabilidade dos constituintes bioquimicos quando comparados com os valores previamente estabelecidos. As vantagens do uso de soro-controle de origem bovina no laboratório de análises clínicas compreendem a facilidade de obtenção de grandes volumes e proporciona ao analista, segurança em sua manipulação, evitando maiores riscos de contaminação por agentes infecciosos especificamente quanto à hepatite B e AIDS.

Nova formulaçao para o preparo do tampao alcalino utilizado na dosagem de creatinina no sangue e urina metodo de Heinegard-Tiderstrom modificado / New formulation for the preparation of the alkaline buffer used in the determination of blood's and urine's creatinine - modified Heinegard-Tiderstrom's method

Neste trabalho e apresentada uma nova formulaçao para opreparo do tampao alcalino empregado no Metodo de Heinegard-Tiderstrom modificado por LOPES et alii, 1984, para a dosagem de creatinina no sangue e urina. A modificaçao foi proposta com a finalidade de se obter uma metodologia que facilitasse o preparo do tampao alcalino, garantindo a nao precipitaçao em baixas temperaturas, alem da reduçao do custo. A nova formulaçao do tampao alcalino foi avaliada em comparaçao com o reagente de Heinegard-Tiderstrom mod.(8), atraves da dosagem de 100 (cem) amostras de soro com variadas concentraçoes de creatinina. A analise dos resultados demonstrou uma excelente correlaçao linear, sugerindo que este reagente preenche todos os requisitos necessarios ao seu emprego na dosagem de creatinina, apresentando ainda as vantagens acima citadas