通过离子对反相高效液相色谱法荧光测定腺嘌呤核苷酸和腺苷。

Fluorometric determination of adenine nucleotides and adenosine by ion-paired, reverse-phase, high-performance liquid chromatography.

作者信息

Ramos-Salazar A, Baines A D

出版信息

Anal Biochem. 1985 Feb 15;145(1):9-13. doi: 10.1016/0003-2697(85)90319-7.

Abstract

A sensitive and specific assay for measurement of adenine nucleotides and adenosine by paired-ion high-performance liquid chromatography is described. The 1,N6-ethenoderivatives of ATP (epsilon-ATP), ADP (epsilon-ADP), AMP (epsilon-AMP), and adenosine (epsilon-Ado), formed by reaction with chloroacetaldehyde at 37 degrees C, were separated under isocratic conditions in 20 min. These compounds are strongly fluorescent at an emission wavelength of 280 nm, rendering a lowest detection limit of 2-5 pmol per injection. The detector responded linearly over the measured ranges (5-100 pmol for epsilon-Ado and 5-4000 pmol for nucleotides). Specificity was confirmed enzymatically. alpha, beta-Methyleneadenosine 5'-diphosphate could be used as an internal standard for measurement of the nucleotides. Significant amounts of NADH appeared as a separate peak in hypoxic tissue. Recoveries from snap-frozen kidney were 88, 92, 76, and 63% for AMP, ADP, ATP, and adenosine, with SD for recovery of 1.0, 10.5, 8.3, and 5.6%, respectively. This method was successfully used to measure adenine nucleotides and adenosine in oxygenated and hypoxic perfused rat kidneys.

摘要

描述了一种通过离子对高效液相色谱法测定腺嘌呤核苷酸和腺苷的灵敏且特异的分析方法。在37℃下与氯乙醛反应形成的ATP（ε-ATP）、ADP（ε-ADP）、AMP（ε-AMP）和腺苷（ε-Ado）的1,N6-乙烯基衍生物，在等度条件下20分钟内分离。这些化合物在280nm发射波长处具有强荧光，每次进样的最低检测限为2-5pmol。检测器在测量范围内呈线性响应（ε-Ado为5-100pmol，核苷酸为5-4000pmol）。通过酶法确认了特异性。α,β-亚甲基腺苷5'-二磷酸可作为核苷酸测量的内标。大量的NADH在缺氧组织中表现为一个单独的峰。速冻肾脏中AMP、ADP、ATP和腺苷的回收率分别为88%、92%、76%和63%，回收率的标准差分别为1.0%、10.5%、8.3%和5.6%。该方法成功用于测定氧合和缺氧灌注大鼠肾脏中的腺嘌呤核苷酸和腺苷。