Asunto(s)
Galactosemias/sangre , Galactosemias/terapia , Uridina Difosfato Galactosa/deficiencia , Niño , Cromatografía Líquida de Alta Presión , Eritrocitos/química , Galactosemias/complicaciones , Humanos , Espectroscopía de Resonancia Magnética , Uridina/uso terapéutico , Uridina Difosfato Galactosa/sangre , Uridina Difosfato Galactosa/aislamiento & purificación , Uridina Difosfato Glucosa/sangre , Uridina Difosfato Glucosa/aislamiento & purificación , Uridina Difosfato Glucosa DeshidrogenasaRESUMEN
To settle the ongoing controversy regarding differential uridine diphosphoglucose (UDPG) and uridine diphosphogalactose (UDPGal) content of erythrocytes, which may be important in evaluating the metabolic abnormality in patients with galactosemia, we derived a combined enzymatic-high-performance liquid chromatography (HPLC) assay. Uridine diphosphoglucuronate (UDPGA), the unique product of UDPG dehydrogenase activity, was separated and quantified by HPLC in extracts of human erythrocytes. The quantity of UDPGA produced in cell filtrates incubated with the enzyme corresponds to the amount of UDPG directly determined by HPLC. The amount of UDPGA produced was independent of the enzyme purity or activity used. On the other hand, the amounts of UDPG estimated by fluorometric measurement of the production of reduced nicotinamide adenine dinucleotide varied with the enzyme purity and activity. The combined enzymatic-HPLC method confirms the direct determinations of UDPG content of normal erythrocytes. The results indicate that, under appropriate conditions, the fluorometric-based assay will give accurate estimates of UDPG, but the direct HPLC method yields consistent and correct UDPG and UDPGal determinations.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Eritrocitos/química , Uridina Difosfato Glucosa/sangre , Humanos , Valores de Referencia , Uridina Difosfato Galactosa/sangre , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Glucosa DeshidrogenasaRESUMEN
Impurities in a reagent dehydrogenase caused overestimates of erythrocytic uridine diphosphate glucose and accounted for clinically important differences in results between those of one group of investigators using enzymatic methods and those of two other groups using enzymatic methods, high-performance liquid chromatography, and nuclear magnetic resonance. These data have relevance for the current debate regarding the pathophysiologic changes in galactosemia.
Asunto(s)
Eritrocitos/química , Galactosemias/sangre , Uridina Difosfato Galactosa/sangre , Uridina Difosfato Glucosa Deshidrogenasa , Uridina Difosfato Glucosa/sangre , Contaminación de Medicamentos , Femenino , Glucosa 1-Deshidrogenasa , Glucosa Deshidrogenasas/metabolismo , Humanos , Indicadores y Reactivos , L-Lactato Deshidrogenasa/metabolismo , Masculino , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Glucosa Deshidrogenasa/metabolismoRESUMEN
The first recognized case of a Rennes-like variant form of galactosemia in a Caucasian individual is described. Galactose-1-phosphate uridyl transferase activity was approximately 10% of the normal in both erythrocytes and cultured skin fibroblasts. Electrophoretic mobility of the variant enzyme in erythrocytes was slower than that of normal individuals and identical to that of the two cases originally reported from Rennes, France. In normal cultured skin fibroblasts, four transferase bands were found. In this tissue, the patient again had a slower moving transferase. It is proposed that in transferase variants an altered subunit results in a specifically altered enzyme mobility analogous for each tissue.