RESUMO
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.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Eritrócitos/química , Uridina Difosfato Glucose/sangue , Humanos , Valores de Referência , Uridina Difosfato Galactose/sangue , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato Glucose DesidrogenaseRESUMO
Two animal models for cystinuria have been examined: the Basenji dog with Fanconi syndrome and cystine stone-forming dogs of various breeds. Brush-border membranes were isolated from these animals and uptake of D-glucose and L-cystine was characterized. Experiments with isolated brush-border vesicles from Basenji dogs with cystinuria as a component of the Fanconi syndrome showed diminished sodium-dependent D-glucose uptake but no decrease in L-cystine uptake even though the cystine defect in vivo was as high as 94% (ie, 6% reabsorption). In contrast, brush-border vesicles isolated from the kidney of a cystine stone-forming dog (Welsh Corgi) with a cystine defect of only 16% (ie, 84% reabsorption) had decreased uptake of cystine compared to values found for Beagle and Basenji vesicles. Thus, cystinuria found in Basenji dogs with the Fanconi syndrome differs from that in classic stone-forming cystinuric dogs. The alteration responsible for the cystinuria of Basenji dogs with Fanconi syndrome does not appear to have a membrane locus and may reflect altered energetics for transport, which are not detected in isolated vesicles. The cystine defect in cystinuric stone-forming dogs does appear to be reflected in the isolated membrane.