Padronização do tempo e temperatura para determinação da atividade da enzima G6PD em amostras de papel filtro

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) is a cytoplasmic enzyme present in all cells of the body, responsible for catalyzing the first step of the pentose phosphate pathway, which generates the cofactor NADPH. This cofactor plays a fundamental role in protecting against oxidative damage, particularly in erythrocytes, which lack alternative pathways for NADPH production. Individuals with reduced enzymatic activity are classified as G6PD-deficient. Currently, various methods are available for diagnosing G6PD deficiency, including qualitative, quantitative, semi-quantitative, and molecular tests, each with its own limitations. To ensure reliable results in enzymatic activity testing, careful control of pre-analytical variables such as time and storage conditions is essential. This study aimed to standardize the time and temperature conditions between sample collection and processing for the quantitative determination of G6PD activity in filter paper samples using a colorimetric enzymatic method. It also aimed to conduct the first population-based screening for G6PD deficiency in patients treated at Primary Health Care Units in Campo Grande, Mato Grosso do Sul, Brazil. For the standardization process, filter paper samples impregnated with whole blood were collected from 17 individuals with G6PD deficiency and 19 with normal enzymatic activity. The biological material was stored under three different conditions: room temperature, 5 °C, and -20 °C, and tested daily over a period of 15 days. For the population screening, 426 dried blood spot samples were collected and tested using a colorimetric assay to determine enzymatic activity. The results showed that room temperature caused significant instability, reducing enzymatic activity by approximately 61%. It was observed that enzyme degradation was more strongly influenced by temperature than by storage time, as accurate identification of G6PD status was no longer possible after 48 hours at room temperature. Samples stored under refrigeration remained stable for up to seven days, while those frozen at -20 °C showed even greater stability, allowing for storage of up to 14 days. Linear regression analysis indicated that 58% of the variation in G6PD enzymatic activity could be explained by storage time and temperature, suggesting that additional variables may also influence sample stability. It was concluded that storage at room temperature is a risk factor for false-positive results, and prompt analysis is recommended for such samples. Samples stored under refrigeration or freezing conditions exhibited greater stability and diagnostic reliability. In the screening performed, a prevalence of 1.1% of G6PD-deficient individuals and 15.2% with intermediate enzymatic activity was identified.