Homocysteine: validation and comparison of two methods using samples from patients with pulmonary hypertension / Homocisteína: validação e comparação entre dois métodos utilizando amostras de pacientes com hipertensão pulmonar

Introduction and objective: The determination of homocysteine plasma levels has been reported as a risk marker of interest in severe diseases involving endothelial injury and associated with the development or progression of atherosclerotic lesions and thrombus formation. The aims of this study were to validate method for quantification of plasma homocysteine by high performance liquid chromatography (HPLC) with fluorimetric detection, and to compare the results obtained from patients with pulmonary hypertension by HPLC with those obtained by spectrophotometric enzymatic cycling (S-Ec) method. Materials and methods: The validation parameters, such as linearity, matrix effect, precision, accuracy, detection and quantitation limits, and robustness of the method were evaluated aiming to demonstrate that it is suitable for the intended use. The data obtained in the quantification of homocysteine using the validated method (HPLC) and the spectrophotometric enzymatic cycling (S-Ec) method, were compared. Results: The method was precise, accurate, and robust; it also had good recovery and showed no matrix effect. The linearity covered a range of 5.0-85.0 µmol/l and the limits of detection and quantification were 1.0 µmol/l and 3.4 µmol/l, respectively. The results obtained for homocysteine determination by HPLC and S-Ec methods were comparable. Conclusion: The validated HPLC method showed good performance for quantification of plasma homocysteine levels, while S-Ec method provided results for homocysteine comparable with those obtained by the validated method; therefore, this methodology is a potential alternative of automated method for clinical laboratories. .

Introdução e objetivo: A determinação dos níveis plasmáticos de homocisteína tem sido relatada como um marcador de risco de interesse em doenças graves que cursam com lesões endoteliais, estando associada ao desenvolvimento ou à progressão de lesões ateroscleróticas e formação de trombos. Os objetivos do presente estudo compreenderam validar o método de dosagem de homocisteína plasmática por cromatografia líquida de alta eficiência (CLAE) com detecção fluorimétrica, analisar amostras de pacientes com hipertensão pulmonar e comparar os resultados obtidos por CLAE com aqueles obtidos com a metodologia espectrofotométrica enzimática cíclica (E-Ec). Materiais e métodos: Os parâmetros de validação linearidade, efeito de matriz, precisão, exatidão, limites de detecção e quantificação, além de robustez do método foram avaliados visando demonstrar que este está apropriado para o uso pretendido. Os dados obtidos na quantificação de homocisteína pelo método validado (CLAE) e pela metodologia espectrofotométrica enzimática cíclica (kit da Labtest) foram comparados. Resultados: O método mostrou-se preciso, exato, robusto, com boa recuperação e não apresentou efeito de matriz. A linearidade abrangeu a faixa de 5 a 85 µmol/l, e os limites de detecção e quantificação foram 1 µmol/l e 3,4 µmol/l, respectivamente. Quanto à comparação dos resultados da determinação de homocisteína por CLAE e por E-Ec, eles foram comparáveis. Conclusão: O método validado por CLAE apresentou desempenho adequado para mensuração dos níveis plasmáticos de homocisteína, enquanto o uso da metodologia E-Ec forneceu resultados para homocisteína comparáveis com aqueles obtidos pelo método validado, sendo esta metodologia uma opção de método automatizado para laboratórios clínicos. .

Preeclampsia: are platelet count and indices useful for its prognostic?

INTRODUCTION: Preeclampsia (PE) is characterized by hypertension and proteinuria after the 20th week gestation. The aim of this study was to investigate whether platelet count (PC) and platelet indices (mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit (PCT)) could predict severe form of preeclampsia (sPE). METHODS: Three groups were evaluated; G1-pregnant with sPE (N = 29); G2-normotensive pregnant (N = 28) and Group 3: non-pregnant women (N = 30). Platelet parameters were obtained using the same automatic blood cells count. Statistical analysis was performed by analysis of variance, t-test, and receiver operating characteristic (ROC) curve. P ≤ 0.05 was considered significant. RESULTS: Lower PC and PCT were observed in sPE comparing to normal pregnant (P = 0.031 and 0.035, respectively) and to non-pregnant women (P < 0.001 and 0.004, respectively). PDW was higher in sPE comparing to normotensive pregnant (P = 0.028) and to non-pregnant women (P < 0.001). MPV was higher in sPE comparing to normotensive pregnant and non-pregnant women (P = 0.05 and P < 0.001, respectively). Analysis from the ROC curve and its areas for each variable showed that the parameters have regular diagnostic significance, except for PCT, considered as not good for this purpose. CONCLUSION: PC emerges as a good candidate for sPE diagnosis, since it is a simple and habitually done method, with lower cost and greater accessibility in the clinical laboratory. Further studies evaluating sequential PC and platelet indices throughout pregnancy are necessary to clarify the role of platelet parameters in PE development and severity.

Adiposidade e perfil metabólico em crianças de escolas da zona urbana de Ouro Preto-MG / Adiposity and metabolic profile of schoolchildren in the urban areas of Ouro Preto, Minas Gerais

A obesidade, doença multifatorial definida como excesso de gordura corporal, apresenta concomitância entre fatores de risco genéticos e ambientais. O diagnóstico precoce e as intervenções no período crítico do desenvolvimento da obesidade - infância e adolescência - têm sido recomendados, buscando-se evitar desfechos desfavoráveis na idade adulta. Este estudo transversal teve como objetivo caracterizar perfil lipídico, glicemia, adiponectina, leptina e grelina de escolares entre seis e nove anos, portadores de sobrepeso e obesidade, do município de Ouro Preto-MG. Os dados foram analisados a partir do teste de normalidade Shapiro Wilk; e nas comparações entre os grupos foi aplicado o teste paramétrico (Teste t) ou não paramétrico (Teste Mann Whitney), adotando-se intervalo de confiança de 95% e nível de significância para valores ≤ 0,05. A idade média da população escolar foi de 7,8 ± 1,1 anos, com prevalência de 8,9% de sobrepeso e 3% de obesidade. Foram identificados hipercolesterolemia em 5,5%, HDL alterado em 98,7%, taxa limítrofe de LDL em 32,4% e glicemia alterada em 46,6% das crianças. Na análise estratificada quanto ao gênero, foram observados valores maisaltos para leptina em meninas (p=0,032) e grelina nos meninos (p=0,033), não havendo diferença para as demais variáveis. Os resultados demonstram ser o excesso de peso entre escolares problema de saúde relevante no município, ressaltando-se a importância de implementação de programa de intervenção precoce por parte dos gestores. Elucidar os precursores da obesidade na infância pode levar a intervenções capazes de atenuar ou impedir suas consequências na juventude e fase adulta.

Obesity, defined as an excess in body fat, is a multifactorial disease involving both genetic and environmental risk factors. Early diagnosis and interventions during critical periods of development of obesity - childhood and adolescence - have been recommended, aiming at preventing unfavorable outcomes at a later age. This cross-sectional study sought tocharacterize the lipid profile, glucose, adiponectin, leptin and ghrelin in schoolchildren between six and nine years of age with overweight and obesity in the city of Ouro Preto (MG). The data was analyzed with the Shapiro Wilk normality test, groups comparisons were made with either a parametric (T test) or a nonparametric (Mann Whitney) test, adopting confidence intervals of 95% and a significance level of ≤ 0.05. The average age of the school population was 7.8 ± 1.1 years, with a prevalence of 8.9% of overweight and 3% of obesity. Hypercholesterolemia was found in 5.5%, HDL was abnormal in 98.7%, LDL levels were borderline in 32.4% and altered glucose levels were present in 46.6% of the children. In stratified analysis by gender, higher values of leptin in girls (p = 0.032) and ghrelin in boys (p = 0.033) were found, with no difference for the other variables. The results show that overweight/obesity among schoolchildren should be considered a significant health problem in this population, highlighting the importanceof implementing early intervention programs. Uncovering the precursors of childhood obesity could lead to interventions so as to prevent or mitigate its consequences in youth and adulthood.

Preeclampsia and ABO blood groups: a systematic review and meta-analysis.

Preeclampsia (PE) is a multifactorial pregnancy-specific syndrome which represents one of the leading causes of maternal mortality worldwide. Inherited thrombophilia have been investigated as risk factor for the development of PE and it is currently known that ABO blood group may impact haemostatic balance, having the non-O blood groups (A, B or AB) subjects increased risk for thrombus formation, as compared to those of group O. We performed a systematic review of the literature for published studies investigating whether ABO blood groups could influence PE developing. A sensitive search of four databases identified 45 unique titles. The retrieved papers were assessed independently by authors and a rigorous process of selection and data extract was conduct. Methodological quality of the included studies was also evaluated. Two studies met eligibility criteria. As a main finding of our systematic review, an association between the AB blood group and the occurrence of PE was detected based on two original studies. Considering the role of ABO blood groups on the hemostatic process and thrombus formation, special attention should be given to pregnant patients carrying the AB blood group in order to prevent the syndrome and improve prognosis.

Association between methylation in mismatch repair genes, V600E BRAF mutation and microsatellite instability in colorectal cancer patients.

Colorectal cancer (CRC) corresponds to the third most prevalent type of cancer. Its origins can either be sporadic or inherited, being Lynch syndrome the most common form of hereditary CRC. The activation of BRAF oncogene, inactivation of mismatch repair genes by methylation of CpG islands, and microsatellite instability (MSI) have been reported to be involved in CRC development. The goal of the study was to characterize CRC tumors using clinical and molecular criteria through association and cluster analysis. Amsterdam II and Bethesda guidelines and molecular variables were analyzed in 77 patients from Brazil. The replication error (RER) status, based in microsatellite instability, showed association with metachronous tumor, MLH1 gene methylation and inverse association with left-sided and synchronous tumors. The PMS2 gene was considered the best predictor for differentiating levels of methylation and the mononucleotide were considered the best markers to evaluate RER status. The cluster 1 was characterized of individuals over 60 years of age, female, right-sided tumor, high microsatellite instability, and metachronous or synchronous tumors. The individuals in cluster 2 were younger than 45 years of age, male and showed left sided or rectum tumors, and microsatellite stability. Even though it was not observed a significant association, a higher number of individuals with family history of cancer and tumors without promoter methylation were found in cluster 2. The V600E mutation did not show association with clinical or molecular characteristics. Evaluation of MSI and methylation of MLH1 and PMS2 genes should be considered in order to assist with clinical diagnosis.

Urinary 11-dehydro thromboxane B2 levels in type 2 diabetic patients before and during aspirin intake.

BACKGROUND: Diabetic patients commonly present an increased risk for cardiovascular events, for which aspirin is the most frequently used medication for primary prevention. Urinary 11-dehydro thromboxane (11-dhTXB2) concentrations assess the effect of aspirin on platelets and identify patients who are at risk of cardiovascular events. The present study investigated whether or not type 2 diabetic patients who took a daily dose of 100mg of aspirin had a significant reduction in urinary 11-dhTXB2 concentrations and whether these results were associated with clinical and laboratory variables. METHODS: Eighty-one type 2 diabetic patients were enrolled in the study. Laboratory tests included the determination of lipidic profile, glycated hemoglobin, platelets count, molecular analysis for both GPIIbIIIa and COX-1 polymorphisms, and urinary 11-dhTXB2. RESULTS: Patients' median value for urinary 11-dhTXB2 before aspirin intake was 179 pg/mg of creatinine. After 15days taking aspirin, the patients presented median of 51 pg/mg of creatinine, thus revealing a significant difference between medians (p=0.00). A reduction of 95% in urinary 11-dhTXB2 concentrations could only be identified in 4 patients (5%). A BMI of ≥ 26 presented a significant association with a reduction of urinary 11-dhTXB2 concentrations (p=0.010), as shown by the multiple logistic regression model. Other clinical and laboratory variables showed no association. CONCLUSIONS: Regardless of the mechanisms related to aspirin non-responsiveness, most patients enrolled in the present study also presented a reduced or minimal response to low-dose aspirin therapy, thereby indicating a clear variability related to aspirin effectiveness. Moreover, BMI appears to be independently associated to the reduction of urinary 11-dhTXB2 concentrations in type 2 diabetic patients taking aspirin.