Diagnostic Accuracy of Griess Test for Asymptomatic Bacteriuria in Pregnancy.

Introduction: Asymptomatic bacteriuria (ASB) is a microbiological diagnosis based on the isolation of a specified quantitative count of bacteria in a properly collected specimen of urine from pregnant women without signs and symptoms, which are referable to urinary tract infection. Global prevalence of ASB in pregnancy is 1.9-9.5%. Objectives: 1. To evaluate the accuracy of Griess test as a tool for screening of ASB in pregnancy. 2. To measure the validity (sensitivity and specificity) of Griess test in comparison with urine culture (Gold standard) and its diagnostic ability by ROC curve. Methodology: A Cross sectional study was done during June and July 2012. Predesigned, pretested questionnaire was used for collection of data regarding demographic profile. Midstream urine sample was collected in sterile container, Griess test was done and urine sample sent for culture. Results: In the present study, the prevalence of asymptomatic bacteriuria was 18% among the pregnant women. The sensitivity of Griess test was 92.3% and specificity was 99%. Area under the curve is 0.96. Hence Griess test has good diagnostic value when compared to urine culture for detecting bacteriuria, it is statistically highly significant with p=0.000001. Conclusion: ASB is not uncommon among antenatal mothers in the population studied. Hence routine urine examination and Griess test for detecting ASB should be included in the ANC on routine basis.

Optimization of cell density and LPS concentration for the evaluation of nitric oxide production on BV-2 in a Griess assay

Introduction: Production of nitric oxide (NO) is one of the main responses elicited by a variety of immune cells such as macrophages (e.g. microglia, resident macrophages of brain), during inflammation. Evaluation of NO levels in the inflammatory milieu is considered important to the understanding of the intensity of an immune response; and has been performed using different methods including the Griess assay. To assay NO in culture, an appropriate number of cells are stimulated into an inflammatory phenotype. Common stimuli include lipopolysaccharide (LPS), IFN-γ and TNF-α. However, overt stimulation could cause cell cytotoxicity therefore an ideal concentration of LPS should be used. Objective: To set-up a model of BV-2 cell activation that allows the assay of detectable levels of NO. Optimization of BV-2 microglia cell density and LPS concentrations after stimulation by bacterial lipopolysaccharide (LPS) for the Griess assay is demonstrated in this study. Methods: BV-2 microglia were cultured at different cell densities, and treated with LPS at three concentrations (1, 5, 10 µg/ml). NO production in culture supernatants were then measured at 18, 24, 48 and 72 hours. Moreover, methyl tetrazolium assay (MTT) was also performed to ensure that NO measurement is performed at no-cytotoxic concentrations of LPS. Results and Conclusions: NO production follows a temporal pattern. The density of 25000 cells/ well was the ideal seeding density for NO evaluation in BV-2 cells. BV-2 stimulation by LPS is dose dependent, and NO levels are increased proportional to the LPS concentration up to 1.0µg/ml, whereas the higher LPS concentrations are associated with decreased cell viability may be caused by the high toxic levels of LPS or NO. Although Griess assay has been commonly used by the scientists, however, optimization of its parameters on BV-2 cells will be useful for the experiments which will be performed on this particular cell line. The optimized pattern of Griess assay on BV-2 cells was achieved in this study, hence easier and more practical for the future scientists to perform Griess assay on BV-2 cells.

Evaluación de los métodos de la normatividad mexicana para la determinación de nitritos en alimentos infantiles

Se evaluaron los dos métodos para la determinación de nitritos, que solicita la normatividad mexicana, en alimentos infantiles cárnicos con verduras. Se determinó el contenido de nitritos a los alimentos infantiles, materias primas y productos intermedios del proceso de elaboración; en cada corrida analítica se incluyeron un blanco de reactivos y una muestra testigo; además se determinó la sensibilidad, porcentaje de recuperación y precisión de las metodologías. Los resultados en los alimentos infantiles indicaron una importante diferencia en los contenidos de nitritos obtenidos entre las metodologías, debido a la persistente presencia de turbidez en los extractos. Se propusieron diferentes tratamientos físicos para eliminarla, pero únicamente la redujeron; tal turbidez se atribuyó a los hidratos de carbono; las concentraciones de nitritos informadas presentaron una dispersión grande y estuvieron por debajo del límite de cuantificación de ambos métodos, por lo que no es recomendable la aplicación de estas técnicas para alimentos que se sospechan contienen trazas de nitritos.

Evaluation of the methods for the determination of nitrites in baby foods according Mexican legislation. We evaluated the two methods accepted by the Mexican norm for the determination of nitritesin infant meatbased food with vegetables. We determined the content of nitrites in the infant food, raw materials as well as products from the intermediate stages of production. A reagent blank and a reference sample were included at each analytical run. In addition, we determined the sensitivity, recovery percentage and accuracy of each methodology. Infant food results indicated an important difference in the nitrite content determined under each methodology, due to the persistent presence of turbidity in the extracts. Different treatments were proposed to eliminate the turbidity, but these only managed to reduce it. The turbidity was attributed to carbohydrates which disclosed concentration exhibit a wide dispersion and were below the quantifiable limit under both methodologies; therefore it is not recommended to apply these techniques with food suspected to contain traces of nitrites.

Influência dos anticoagulantes e da temperatura de armazenamento sobre os níveis sanguíneos de nitrito / Effects of anticoagulants and storage temperature on blood nitrite levels

A mensuração dos metabólitos do óxido nítrico pode ser útil para a melhor compreensão de diferentes processos fisiopatológicos. Este estudo avaliou a influência de diferentes anticoagulantes (ácido etilenodiaminotetracético [EDTA], citrato e heparina) e do armazenamento a -20ºC por quatro meses sobre os níveis de nitrito, sendo estes mensurados em amostras de soro e plasma pelo método de Griess. Os tipos de amostra utilizados (soro ou plasma) e de anticoagulante usado na coleta não influenciaram significativamente os níveis de nitrito, independentemente de as dosagens serem realizadas em amostras frescas ou naquelas mantidas a -20ºC por quatro meses.

The measurement of nitric oxide metabolites may be useful for better understanding of different physiopathological processes. Thus, the aim of this study was to evaluate the influence of different anticoagulants (EDTA, citrate and heparin) and four-month storage at -20ºC on nitrite levels. The serum and plasma samples were analyzed by using the Griess method. The type of sample (serum or plasma) or anticoagulant used in the collection did not influence on nitrite levels significantly, regardless the fact they were fresh or four-month samples stored at -20ºC.

Avaliação da produção de óxido nítrico em ratos, submetidos aos exercícios aeróbio e anaeróbio / Evaluation of the production of nitric oxide in mice, submitted to aerobic and anaerobic exercises

Óxido nítrico (NO) exerce influências muito importantes em vários processos fisiológicos. Neste trabalho avaliamos a produção de NO sanguíneo em ratos Wistar, submetidos ao nado aeróbio e anaeróbio agudos. A formação do óxido nítrico foi verificada através da dosagem dos produtos de oxidação estáveis do metabolismo do óxido nítrico (nitratos). Para isso utilizamos o método colorimétrico de Griess. Verificamos a existência de uma diferença significativa (p = 0,000261) na produção de óxido nítrico entre a realização do nado aeróbio e o anaeróbio, na qual o aeróbio mostrou-se mais eficiente na promoção de níveis mais elevados. O exercício aeróbio agudo com duração de no mínimo 10 minutos mostrou-se mais eficaz no quesito produção de NO em relação ao exercício de 5 minutos. A positiva relação observada entre o exercício aeróbio e a formação de NO pode ajudar a explicar os efeitos benéficos do exercício na saúde cardiovascular. Sabemos que a prática de exercício aeróbio e sua duração aumentam a biodisponibilidade de NO, o qual é considerado importante regulador fisiológico da pressão arterial.

Nitric Oxide (NO) exerts important influences in several physiological processes. In this work we evaluated the production of sanguine NO in Wistar rats, submitted to the acute aerobic and anaerobic exercises. The formation of nitric oxide was verified through the dosage of the end products of oxidation of the metabolism of nitric oxide (nitrates). For this we used the colorimetric Griess method. We verified the existence of a significant difference (p = 0.000261) in the production of NO among the accomplishment of the aerobic swimming and the anaerobic, where the aerobic was shown more efficient in the promotion of higher levels. The acute aerobic exercise with duration of at least 10 minutes was shown more effective in the requirement production of NO in relation to the 5 minutes exercise. The positive relationship observed between the aerobic exercise and the formation of NO can help to explain the beneficial effects of the exercise in the cardiovascular health. We know that the practice of aerobic exercise and your duration increases the biodisponibility of NO, which is an important physiologic regulator of the blood pressure.

Effect of Mitomycin C on the Proliferation and Nitric Oxide Production in the Cultured Trabecular Meshwork Cells

PURPOSE: To investigate the effect of mitomycin C on the proliferation of cultured trabecular cells and its relation to nitric oxide (NO) production. METHODS: The effect of NO donor, SIN-1, on the proliferation of primarily cultured porcine trabecular meshwork cells was studied with MTT assay. After treatment with mitomycin C at various concentrations for 1 hour, the proliferation was assessed by MTT assay and the production of nitrite was assessed by Griess reaction after 24 hours and 3 days respectively. RESULTS: SIN-1 significantly inhibited proliferation of cultured trabecular meshwork cells (p0.05). CONCLUSIONS: Both NO donor and mitomycin C have an inhibitory effect on the proliferation of trabecular meshwork cells. However, inhibitory effect of mitomycin C was not related to the production of NO.

Role of Nitric Oxide on the Proliferation of Human Tenon Capsule Fibroblasts

PURPOSE: To investigate the role of nitric oxide(NO) on the survival and proliferation of human Tenon capsule fibroblasts(HTCF) in tissue culture. METHODS: Following exposure to NO donor such as sodium nitroprusside(SNP) or to NO synthase inhibitor such as N omega-Nitro-L-arginine methyl ester(L-NAME) at various concentrations for 24 hr in the media with or without serum, the cellular proliferation and nitrite production were assessed by rapid colorimetric assay (MTT assay) and Griess assay, respectively. RESULTS: L-NAME decreased the cellular proliferation of HTCF in a dose-dependent manner. In contrast, SNP increased the proliferation in a dose-dependent manner. The differences of nitrite production were more remarkable in the serum-deprived condition with the L-NAME or SNP administration. CONCLUSIONS: NO donor has a proproliferative, and NOS inhibitor has a antiproliferative activities on the proliferation of HTCF. This suggests that NO may be act as an important modulator in wound healing.