Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Abstract

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. Electronic supplementary material The online version of this article (doi:10.1007/s11302-012-9321-8) contains supplementary material, which is available to authorized users.

A Novel Method for Measuring the ATP-Related Compounds in Human Erythrocytes

“…The r values indicated a good linearity according to the ICH guidelines (Épshtein 2004), and the ranges of CVs were within the precision in the literature for water based solutions because the previous data showed CVs up to 9.1% (Tekkanat and Fox 1988;Maessen et al 1988;Taniai et al 2006;Contreras-Sanz et al 2012). The present limits sug- The r values were obtained using the average of three replicated curves.…”

“…gest that our method had an appropriate sensitivity for measuring ATP-related compounds in human whole blood samples, including improved sensitivities for IMP, inosine, hypoxanthine and uric acid with respect to prior measurements (Anderson and Murphy 1976;Harmsen et al 1981;Crescentini and Stocchi 1984;Stocchi et al 1985Stocchi et al , 1987Tekkanat and Fox 1988;Maessen et al 1988;Smolenski et al 1990;Caruso et al 2004;Taniai et al 2006;Coolen et al 2008;Yeung et al 2008;Contreras-Sanz et al 2012). Moreover, our recoveries were satisfactory because they were within the literature values ranging 58.6 to 108.2% in the blood matrix (Harmsen et al 1981;Crescentini and Stocchi 1984;Stocchi et al 1985Stocchi et al , 1987Tekkanat and Fox 1988;Caruso et al 2004;Coolen et al 2008).…”

“…However, the ADP peak in blood samples might be eluted with peptides because purity factors were < 900, their spectra had an additional peak and the peptide bond typically absorbs from 180 to 240 nm in the UV region (Kelly and Price 2000). This impure ADP peak was not reported in other methods because this is the first time that the ATP-related compounds were evaluated using the 3D spectra and purity factors (Scholar et al 1973;Anderson and Murphy 1976;Schweinsberg and Loo 1980;Harmsen et al 1981;Ericson et al 1983;Crescentini and Stocchi 1984;de Korte et al 1985;Stocchi et al 1985Stocchi et al , 1987Werner et al 1987;Tekkanat and Fox 1988;Maessen et al 1988;Smolenski et al 1990;Formato et al 1990;Nishikawa et al 1991;Guieu et al 1994;Caruso et al 2004;Taniai et al 2006;Coolen et al 2008;Yeung et al 2008;Contreras-Sanz et al 2012).…”

“…The complete interpretation of the energetic homeostasis of RBCs under stressful conditions might be performed with a liquid chromatographic (LC) method using ultraviolet (UV) detection that measures all the compounds involved in the degradation pathway for adenine nucleotides alongside the uric acid (Anderson and Murphy 1976;Schweinsberg and Loo 1980;Harmsen et al 1981;Crescentini and Stocchi 1984;Stocchi et al 1985Stocchi et al , 1987Bontemps et al 1986;Werner et al 1987;Maessen et al 1988;Tekkanat and Fox 1988;Smolenski et al 1990;Nishikawa et al 1991;Guieu et al 1994;Smoleńska et al 1999;Caruso et al 2004;Taniai et al 2006;Coolen et al 2008;Yeung et al 2008;Contreras-Sanz et al 2012). The commercial kits (ab65313, abcam; K255-200, BioVision; TB288, Promega; FL-AA, Sigma-Aldrich) and LC methods using fluorometric detection or mass spectrometry (Levitt et al 1984;Ramos-Salazar and Baines 1985;Fujimori et al 1990; Kawamoto et al 1998;Katayama et al 2001;Tuytten et al 2002;Xing et al 2004;Klawitter et al 2007;Wang et al 2009;Birkler et al 2010;Pabst et al 2010;Jiang et al 2012) can only detect some of these compounds simultaneously.…”

Analysis of Intra- and Extracellular Levels of Purine Bases, Nucleosides, and Nucleotides in HepG2 Cells by High-performance Liquid Chromatography

“…To date, several methods have been developed for the analysis of purines, including high-performance liquid chromatography (HPLC), [10][11][12][13][14][15] and LC-MS-based methods. 16 These biochemicals have now been measured in a variety of systems, including cultured cells, [17][18][19] isolated tissues, 15 food substances, [12][13][14][20][21][22][23][24][25] fungi, 26 and biological samples.…”