FLUOROMETRIC-DETERMINATION OF ADENINE-NUCLEOTIDES AND ADENOSINE BY ION-PAIRED, REVERSE-PHASE, HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
FLUOROMETRIC-DETERMINATION OF ADENINE-NUCLEOTIDES AND ADENOSINE BY ION-PAIRED, REVERSE-PHASE, HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
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DOI：
10.1016/0003-2697(85)90319-7
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发表时间：
1985-01-01
影响因子：
2.9
通讯作者：
BAINES, AD
中科院分区：
文献类型：
--
作者：
RAMOSSALAZAR, A;BAINES, AD
建立了一种灵敏、特异的成对离子高效液相色谱法测定腺嘌呤核苷酸和腺苷的方法。与氯乙醛在37度反应生成的1，N6-三磷酸腺苷(？-ATP)、二磷酸腺苷(？-ADP)、腺苷(？-AMP)和腺苷(？-ADO)的1，N6-亚甲基衍生物。C，在等度条件下20min内分离。这些化合物在280 nm的发射波长下是强荧光的，最低检测下限为2-5pmol/进样。检测器在测量范围内线性响应(β-ADO为5-100pmo1，核苷酸为5-4000pmo1)。酶学分析证实了其特异性。α、β-亚甲基腺苷5‘’-二磷酸可作为测定核苷酸的内标。大量的NADH在低氧组织中表现为一个单独的峰值。冰冻肾中AMP、ADP、ATP和腺苷的回收率分别为88%、92%、76%和63%，SD分别为1.0%、10.5%、8.3%和5.6%。该方法成功地用于氧合和低氧灌流大鼠肾脏中腺嘌呤核苷酸和腺苷的测定。
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.degree. 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/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.