High performance liquid chromatography method for the detection of released purinergic and biogenic amine signaling molecules from in vitro ileum tissue.
Adenosine triphosphate (ATP) and serotonin (5-HT) are known to play key roles in the function and activity of the gastrointestinal tract; however, no methods have been established for the monitoring of these signaling molecules within one assay. We have developed a simple chromatographic methodology using UV/visible detection for the analysis of purinergic and biogenic amine signaling molecules. The chromatographic separation was achieved in an isocratic mode, where the mobile phase consisted of 5% methanol and 95% ammonium phosphate buffer with 10 mM tetrabutylammonium bisulfate. Column temperature of 45 degrees C provided the means to separate all analytes within 14.7 min. Good resolution and tailing factors were observed for all components within the separation. The LOD for ATP and 5-HT was 30 and 317 nM, respectively, with a linear range from 10-0.02 microM. In vitro measurements were carried out by using aliquots from the buffer the tissue was stored in after 30 min to measure released molecules. In vitro assay of ileum tissue in the presence and absence of endogenous ATP was carried out. Results showed that ATP can elevate 5-HT release. This method can be used to study alterations in these key signaling molecules with gastrointestinal disease.
Lwin
Ayemon
A
Department of Bioengineering, Imperial College London, London, UK.
Patel
Bhavik Anil
BA
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001679:Biogenic Amines / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007082:Ileum / Q000473:pathology*; D066298:In Vitro Techniques; D008297:Male; D011983:Receptors, Purinergic / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012701:Serotonin / Q000737:chemistry; D015398:Signal Transduction; D013696:Temperature; D014466:Ultraviolet Rays
D001679:Biogenic Amines; D011983:Receptors, Purinergic; D012701:Serotonin; D000255:Adenosine Triphosphate