High-performance liquid chromatography (HPLC) determination of inosine, a potential biomarker for initial cardiac ischaemia, using isolated mouse hearts

Abstract

Each year in the USA approximately 7–8 million patients with non-traumatic chest pain come to hospital emergency rooms. It is estimated that approximately 2–5% of these patients are experiencing cardiac ischaemia, but due to the shortcomings of the available testing methods they are incorrectly diagnosed and discharged without appropriate therapy having been provided. Preliminary data with a globally ischaemic mouse heart model has demonstrated that endogenous inosine might be a potential biomarker of initial cardiac ischaemia before cardiac tissue necrosis. A high-performance liquid chromatographic diode array detection (HPLC-DAD) method was utilized for the detection and quantification of inosine in Krebs–Henseleit (Krebs) buffer solution perfusing from surgically removed and isolated mouse hearts undergoing global cardiac ischaemia. A C₁₈ column at a flow rate of 0.6 ml min⁻¹ with an aqueous mobile phase of trifluoroacetic acid (0.05% trifluoroacetic acid in deionized water, pH 2.2, v/v) and methanol gradient was used for component separation. The assay detection limit for inosine in Krebs buffer solution was 500 ng ml⁻¹ using a 100-µl neat injection. The HPLC results were used to determine total cardiac effluxed inosine into the Krebs effluent for each mouse during oxidative stress and compared with the per cent cardiac ventricular functional recovery rate to determine if a relationship exists amongst this cardiovascular parameter during periods of cardiac oxidative stress.

Keywords: :

• Inosine
• biomarker
• mouse cardiac ischaemia
• cardiac oxidative stress
• high-performance liquid chromatography (HPLC)

Acknowledgements

The authors would like to thank Dr Bill Gardner, Philip Morris USA, for his technical expertise using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and Philip Morris USA for the use of high-performance liquid chromatographic diode array detection (HPLC-DAD) equipment for the analysis of Krebs samples. The authors also thank Mr Brian Berger for his technical assistance on initial work using isolated mouse hearts undergoing oxidative stress. Dr Lei Xi is supported by a grant from American Heart Association, National Center (No. 0530157N).