Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC.
Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC.
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DOI：
10.1007/s11302-012-9321-8
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发表时间：
2012-12
影响因子：
3.5
通讯作者：
Wildman, Scott S. P.
中科院分区：
文献类型：
--
作者：
Contreras-Sanz, Alberto;Scott-Ward, Toby S.;Gill, Hardyal S.;Jacoby, Jennifer C.;Birch, Rebecca E.;Malone-Lee, James;Taylor, Kevin M. G.;Peppiatt-Wildman, Claire M.;Wildman, Scott S. P.
核苷酸和核苷不仅参与细胞代谢，而且还通过P1和P2受体在细胞外起作用，通过旁分泌和自分泌信号传导途径引起各种生理和病理生理反应。我们首次采用离子对反相高效液相色谱紫外（UV）耦合法对12种不同的核苷酸和核苷进行了快速、同时的定量（三磷酸腺苷、二磷酸腺苷、单磷酸腺苷、腺苷、三磷酸尿苷、二磷酸尿苷、单磷酸尿苷、尿苷、三磷酸鸟苷、二磷酸鸟苷、单磷酸鸟苷、鸟苷）：（1）从小鼠肾细胞系（M1皮质集合管）释放和（2）在人生物样品（即，尿）。为便于尿液样本分析，纳入了固相萃取步骤（总回收率≥ 98%）。所有样品均在进样（100 μl）至Synergi Polar-RP 80 μ m（250 × 4.6 mm）反相色谱柱（粒度为10 μm，用保护柱保护）后进行分析。使用移动的流动相（磷酸盐缓冲液加离子配对剂四丁基硫酸氢铵; pH 6）在2- 30%乙腈（v/v）中以1 ml min−1流速运行梯度洗脱曲线35 min（包括平衡时间）。通过254 nm处的UV吸光度检测洗脱的化合物，并使用已知浓度的核苷酸和核苷混合物的标准曲线进行定量。在验证（专属性、线性、检测限和定量限、系统精密度、准确度和中间精密度参数）后，该方案成功且可重复地用于定量瞬时浸浴M1细胞的等渗和低渗细胞缓冲液以及正常受试者和膀胱过度活动症患者的尿液样本中皮摩尔至纳摩尔浓度的核苷和核苷酸。本文的在线版本（doi：10.1007/s11302-012-9321-8）包含补充材料，可供授权用户使用。
Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ≥ 98 %). All samples were analyzed following injection (100 μl) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 μm, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2–30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min−1 flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients. The online version of this article (doi:10.1007/s11302-012-9321-8) contains supplementary material, which is available to authorized users.
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影响因子：
4.1
作者：
Liebich, HM;DiStefano, C;Schmid, HR
通讯作者：
Schmid, HR
影响因子：
5.5
作者：
Ferguson, DR;Kennedy, I;Burton, TJ
通讯作者：
Burton, TJ
影响因子：
23.4
作者：
Kumar, Vivek;Chapple, Christopher R.;Chess-Williams, Russell
通讯作者：
Chess-Williams, Russell
DOI：
10.1016/s0378-4347(01)00265-1
发表时间：
2001-08-25
期刊：
JOURNAL OF CHROMATOGRAPHY B
影响因子：
--
作者：
Katayama, M;Matsuda, Y;Sato, H
通讯作者：
Sato, H
影响因子：
19.6
作者：
Jankowski, V.;Karadogan, S.;Jankowski, J.
通讯作者：
Jankowski, J.