RESUMO
Hexokinases play a critical role in the cellular uptake and utilization of glucose. As such, they are of fundamental importance to all cells. By catalyzing glucose to produce glucose-6-phosphate, hexokinases control the first irreversible step of glucose metabolism and initiate all major pathways of glucose consumption. Our objective was to develop and validate highly sensitive and selective high-performance liquid chromatography with photodiode array detector (HPLC-PDA) assays allowing the determination of adenosine diphosphate, which was used for the determination of hexokinase activity. Samples were analyzed by HPLC-PDA using a C18 analytical column (250 × 4.6 mm) for chromatographic separation. Optimal detection was achieved based on isocratic elution with a mobile phase consisting of a mixture of sodium phosphate monobasic buffer and methanol. This method met all of the requirements of specificity, sensitivity, linearity, precision, accuracy and stability generally accepted in bioanalytical chemistry and was successfully applied to a study of hexokinase activity in an alloxan-induced diabetic rat model. Determination of hexokinase activity will permit characterization of cellular metabolic state in many diseases, such as cancer and diabetes.
Assuntos
Difosfato de Adenosina/sangue , Biomarcadores/sangue , Cromatografia Líquida de Alta Pressão/métodos , Diabetes Mellitus Experimental/sangue , Hexoquinase/metabolismo , Animais , Hexoquinase/sangue , Hexoquinase/efeitos dos fármacos , Modelos Lineares , Masculino , Metformina/farmacologia , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
14CO2 production from [1-14C] glucose, the rate of glycolysis measured by the value of lactate production and the activities of various enzymes were determined in buffalo erythrocytes. Buffalo red cell glycolytic metabolites were estimated and used for the calculation of the mass action ratios of reactions catalyzed by the glycolytic enzymes of Bubalus bubalis. A comparison of the values of the mass action ratios with the equilibrium constants of the various glycolytic reactions indicate that hexokinase, phosphofructokinase, phosphoglycerate kinase and pyruvate kinase reactions are displaced from equilibrium, suggesting a regulatory role for each of these enzymes in buffalo erythrocyte glycolysis.
Assuntos
Glicemia/metabolismo , Búfalos/sangue , Eritrócitos/metabolismo , Animais , Dióxido de Carbono/sangue , Glicólise , Hexoquinase/sangue , Cinética , Fosfofrutoquinase-1/sangue , Fosfoglicerato Quinase/sangue , Piruvato Quinase/sangueRESUMO
Cord blood samples from ten term infants were fractionated into populations of young and old erythrocytes and compared with cells prepared in a similar fashion from eight normal adults. The old cell fraction from the newborn infants had very low phosphofructokinase activity and did not display the usual decline of activity of the enzymes phosphoglycerate kinase and enolase. In addition, the old cells from the newborn infants demonstrated impaired glucose consumption, which, upon analysis of the pattern of glycolytic intermediates, appeared to be a result of the phosphofructokinase deficiency. These findings suggest that cells produced earlier in gestation possess the developmental characteristics of fetal blood to a more significant degree and that these biochemical alterations may produce functional impairment.