High-performance liquid chromatographic technique for detection of a fluorescent analogue of ADP-ribose in isolated blood vessel preparations
High-performance liquid chromatographic technique for detection of a fluorescent analogue of ADP-ribose in isolated blood vessel preparations
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DOI：
10.1006/abio.2002.5667
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发表时间：
2002-06-15
影响因子：
2.9
通讯作者：
Mutafova-Yambolieva, VN
中科院分区：
文献类型：
--
作者：
Bobalova, J;Bobal, P;Mutafova-Yambolieva, VN
生物培养基中内源核苷酸的分析因快速降解和难以检测的低最终浓度而受到阻碍。描述了一种反相高效液相色谱（HPLQ 技术），可有效检测血管组织灌流液中低飞摩尔浓度范围内稳定的腺苷 5'-二磷酸核糖 (ADPR) 荧光衍生物 1,N-6-etheno-ADPR (epsilon-ADPR)。epsilon-ADPR 是由 ADPR 与氯乙醛在 80°C 和 pH 条件下反应形成的采用 0.1 M KH2PO4 (pH 6.0)、增量甲醇（18 分钟内 0-35%）和 25 cm x 4.5 mm (5 mum) 硅胶 ODS-AM 柱进行梯度洗脱，激发波长为 230 nm，发射波长为 410 nm，检测灵敏度约为 10。 fmol. HPLC 检测方法的线性范围为 0.0125 至 1 pmol epsilon-ADPR。该方法在标准核苷酸的保留时间和峰面积的日内和日间重现性方面得到验证，证实了 ADPR 与氯乙醛反应后存在乙烯环。 （8 Hz，0.3 ms，15 V，1-2 分钟）犬和豚鼠分离的肠系膜动脉段 (C) 2002 Elsevier Science（美国）。
Analysis of endogenous nucleotides in biologic media is hampered by rapid degradation and low final concentrations that are difficult to detect. A reversed-phase high-performance liquid chromatographic (HPLQ technique is described that efficiently detects a stable fluorescence derivative of adenosine 5'-diphosphoribose (ADPR), 1,N-6-etheno-ADPR (epsilon-ADPR), at low femtomolar concentration range in vascular tissue superfusates. epsilon-ADPR was formed by the reaction of ADPR with chloroacetaldehyde at 80degreesC and pH 4.0. Gradient elution with 0.1 M KH2PO4 (pH 6.0), increasing methanol (0-35% over 18 min), and a 25-cm by 4.5-mm (5 mum) silica ODS-AM column were employed. epsilon-ADPR was detected by fluorescence at an excitation wavelength of 230 nm and an emission wavelength of 410 nm. The detection sensitivity for epsilon-ADPR was approximately 10 fmol. Linearity of the HPLC detection method was demonstrated in the range from 0.0125 to 1 pmol epsilon-ADPR. The method was validated in terms of within-day and between-day reproducibility of retention times and peak areas of standard nucleotide. Matrix-assisted laser desorption/ionization mass spectrometry measurements confirmed the presence of an etheno ring after reaction of ADPR with chloroacetaldehyde. The method was applied to quantitate the overflow of ADPR upon electrical field stimulation (8 Hz, 0.3 ms, 15 V, 1-2 min) of both canine and guinea-pig isolated mesenteric artery segments. (C) 2002 Elsevier Science (USA).