HPTLC ANALYSIS OF SPHINGOMYLEIN, CERAMIDE AND SPHINGOSINE IN ISCHEMICREPERFUSED RAT-HEART/

Since the sphingomylein-ceramide-sphingosine pathway, especially ceram ide, has been shown to induce programmed cell death (apoptosis), and s ince apoptosis may be involved with ischemic/reperfused injury in the heart, it became desirable to quantitate the three components in ische mic/reperfused rat heart. One group of rat hearts (n = 6) was isolated and perfused with Krebs-Henseleit buffer using the Langendorff non-re circulating mode. The hearts were perfused for 10 min, made ischemic f or 30 min and reperfused for 120 min. Hearts were collected and stored at -70 degrees C before ischemia, after ischemia and after 30, 60 and 120 min of reperfusion. The hearts were homogenized, and lipids were extracted using the Folch method. The lipids were then chromatographed on Whatman silica gel 60 Angstrom high-performance thin-layered chrom atography (HPTLC) plates. The plates were developed with iodine. photo graphed using Photoshop software and quantitated using NIH Imaging sof tware. The results show a 50% decrease of sphingomylein during reperfu sion with a corresponding increase in ceramide with sphingosine showin g a smaller decrease as compared with the ceramide increase. (C) 1998 Elsevier Science B.V. All rights reserved.