Wehrli S L, Palmieri M J, Berry G T, Kirkman H N, Segal S
Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia 19104.
Anal Biochem. 1992 Apr;202(1):105-10. doi: 10.1016/0003-2697(92)90214-r.
The levels of uridine diphosphogalactose (UDPGal) and uridine diphosphoglucose (UDPGlu) in trichloroacetic acid extracts of human red blood cells (RBC) were measured by 31P NMR spectroscopy. Individual determinations were compared to results obtained by enzymatic and high-pressure liquid chromatographic (HPLC) methods. The characteristic doublet of the P beta resonance signals of both UDPGal and UDPGlu were detected in proton-decoupled spectra of extracts. Quantitative analyses were obtained by employing a standard, methylene diphosphonate, in an external capillary tube during data acquisition for periods of 14 to 24 h using an "inverse-gated" pulse sequence. The ratio of the integrated area of each of the uridine sugar nucleotide doublets to the area of the external reference peak was linear with concentrations between 0.03 and 0.50 mM. There was no difference between the mean value obtained by 31P NMR of 6.6 +/- 1.4 mumol UDPGlu/100 g Hgb or 2.1 +/- 0.6 mumol UDPGal/100 gHgb and the corresponding levels determined enzymatically or by HPLC in identical RBC extracts. When analyzed as paired data, only UDPGlu by NMR was found to be lower than the value obtained by HPLC. As a quantitative analytical tool, NMR spectrometry validated both the enzymatic and HPLC methods used for measurement of uridine sugar nucleotides in our laboratories.
采用³¹P核磁共振波谱法测定了人红细胞（RBC）三氯乙酸提取物中尿苷二磷酸半乳糖（UDPGal）和尿苷二磷酸葡萄糖（UDPGlu）的水平。将个体测定结果与酶法和高压液相色谱（HPLC）法获得的结果进行了比较。在提取物的质子去耦谱中检测到了UDPGal和UDPGlu的Pβ共振信号的特征双峰。在数据采集期间，使用“反门控”脉冲序列，通过在外部毛细管中加入标准品亚甲基二膦酸盐，进行了14至24小时的定量分析。每个尿苷糖核苷酸双峰的积分面积与外部参考峰面积的比值在0.03至0.50 mM的浓度范围内呈线性关系。通过³¹P NMR获得的平均值，即6.6±1.4 μmol UDPGlu/100 g血红蛋白或2.1±0.6 μmol UDPGal/100 g血红蛋白，与在相同红细胞提取物中通过酶法或HPLC测定的相应水平之间没有差异。当作为配对数据进行分析时，仅发现通过NMR测定的UDPGlu低于通过HPLC获得的值。作为一种定量分析工具，核磁共振光谱法验证了我们实验室用于测量尿苷糖核苷酸的酶法和HPLC法。
