Reiss P D, Zuurendonk P F, Veech R L
Anal Biochem. 1984 Jul;140(1):162-71. doi: 10.1016/0003-2697(84)90148-9.
A high-performance liquid-chromatographic (HPLC) method for the rapid separation of purine and pyrimidine nucleotides, NAD+, NADP+, FAD, FMN, UDP-Glc, UDP-glucuronate, and ADP-ribose found in neutralized perchloric acid extracts of rat liver is described. Separation was achieved within 26 min on a radially compressed column of Partisil 10-SAX. The column was eluted with a gradient of sodium phosphate and sodium chloride. The sodium phosphate was purified by passage through tandem columns of anion- and cation-exchange resins to remove uv-absorbing impurities. The sensitivity of this procedure is such that an amount of ATP contained in 10 micrograms of liver can be measured. The recoveries of all nucleotides were between 87 and 107%. In extracts of rat liver interfering substances were found to elute with GDP, and UDP eluted with NADP. Consequently, the tissue contents of UDP and GDP were determined in a second run by measuring the increase in UTP and GTP, respectively, following sample pretreatment with pyruvate kinase (PK). The tissue level of NADP+ was calculated as the difference between the total UDP and NADP+ peak and the increase in UTP following PK treatment. In those nucleotides amenable to enzymatic analysis, namely NAD+, AMP, UDP-Glc, UTP, and ATP, the tissue contents measured enzymatically were not significantly different from those determined by HPLC. However, ADP as measured with PK was found to be 15% higher compared to the HPLC determination.
本文描述了一种高效液相色谱（HPLC）方法，用于快速分离大鼠肝脏中和高氯酸提取物中发现的嘌呤和嘧啶核苷酸、NAD⁺、NADP⁺、FAD、FMN、UDP-葡萄糖、UDP-葡萄糖醛酸和ADP-核糖。在Partisil 10-SAX径向压缩柱上26分钟内实现了分离。用磷酸钠和氯化钠梯度洗脱该柱。磷酸钠通过串联的阴离子和阳离子交换树脂柱以去除紫外线吸收杂质进行纯化。该方法的灵敏度使得能够测量10微克肝脏中所含的ATP量。所有核苷酸的回收率在87%至107%之间。在大鼠肝脏提取物中，发现干扰物质与GDP一起洗脱，而UDP与NADP一起洗脱。因此，在丙酮酸激酶（PK）对样品进行预处理后，通过分别测量UTP和GTP的增加，在第二轮实验中测定UDP和GDP的组织含量。NADP⁺的组织水平通过总UDP和NADP⁺峰与PK处理后UTP增加量之间的差值计算得出。在适用于酶分析的那些核苷酸中，即NAD⁺、AMP、UDP-葡萄糖、UTP和ATP，酶法测定的组织含量与HPLC测定的结果无显著差异。然而，发现用PK测定的ADP比HPLC测定的结果高15%。
