PHOSPHOCREATINE CONCENTRATION ASSESSMENT DURING EARLY REJECTION IN CONCORDANT HAMSTER XENO HEARTS USING HPLC OR IN VITRO 31P MRS YIELDS SIMILAR RESULTS

Abstract

A295 Aims: Changes in the concentrations of high energy phosphates (HEP) post transplantation in concordant xeno heart transplants correlate to functional outcome. The concentrations of HEP can be determined with high accuracy using either in vitro31Phosphorus Magnetic Resonance Spectroscopy (31P MRS) or high pressure liquid chromatography (HPLC). In order to compare the result and accuracy of the two methods we subjected concordant hamster xeno heart transplants to a standardized cold ischemia (CI) and reperfusion (RP) insult known to result in rejection associated biochemical alterations and quantified the concentrations of the adenosine triphosphate (ATP) buffer, phosphocreatine (PCr). Methods: Xenogeneic cervical heart transplantations were performed between Golden Syrian hamsters (70-80 g) and inbred Lewis (RT11) (220 g). All hearts were subjected to 30 minutes of cold ischemia (+ 4°C) and 6 hours of RP before explantation. The excised grafts were snap frozen in liquid nitrogen, freeze dried until stable weight and then subjected to a routine extraction process. The resulting supernatants were used to determine the concentrations of phosphocreatine (PCr) in μmol/g dry heart tissue using high resolution in vitro31P MRS (11.75 T) (group A; n = 8) or HPLC (group B; n = 6). Results: After 30 minutes CI all hearts resumed normal activity and were beating when explanted after 6 hours. The estimated PCr concentration was depending on the method either 9.25 ± 1.53 (MRS) or 12.4 ± 1.52 (HPLC) μmol/g heart tissue respectively (p=ns) (mean ± SEM). Conclusions: Quantification of the HEP PCr, an important buffer for the adenosine triphosphate (ATP) concentrations in cells, using high resolution in vitro31P MRS or HPLC in concordant hamster xeno hearts post transplantation subjected to identical CI/RP protocols yields similar results. Thus, both in vitro methods appears to be equally precise when assessing the concentrations of HEP in rejecting xeno hearts posttransplantation.