Gibson J B, Reynolds R A, Rogers S, Palmieri M J, Segal S
Division of Biochemical Development and Molecular Diseases, Children's Hospital of Philadelphia, PA 19104.
J Pediatr. 1993 Dec;123(6):906-14. doi: 10.1016/s0022-3476(05)80386-5.
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.
为了解决关于红细胞中尿苷二磷酸葡萄糖（UDPG）和尿苷二磷酸半乳糖（UDPGal）含量差异的持续争议，这在评估半乳糖血症患者的代谢异常中可能很重要，我们开发了一种联合酶促高效液相色谱（HPLC）测定法。尿苷二磷酸葡萄糖醛酸（UDPGA）是UDPG脱氢酶活性的唯一产物，通过HPLC在人红细胞提取物中进行分离和定量。在与该酶孵育的细胞滤液中产生的UDPGA量与通过HPLC直接测定的UDPG量相对应。产生的UDPGA量与所用酶的纯度或活性无关。另一方面，通过荧光法测量还原型烟酰胺腺嘌呤二核苷酸的产生来估计的UDPG量随酶的纯度和活性而变化。联合酶促-HPLC方法证实了正常红细胞中UDPG含量的直接测定。结果表明，在适当条件下，基于荧光法的测定将给出UDPG的准确估计值，但直接HPLC方法可产生一致且正确的UDPG和UDPGal测定结果。
