HPTLC analysis of sphingomylein, ceramide and sphingosine in ischemic/reperfused rat heart.

Since the sphingomylein-ceramide-sphingosine pathway, especially ceramide, has been shown to induce programmed cell death (apoptosis), and since apoptosis may be involved with ischemic/reperfused injury in the heart, it became desirable to quantitate the three components in ischemic/reperfused rat heart. One group of rat hearts (n = 6) was isolated and perfused with Krebs-Henseleit buffer using the Langendorff non-recirculating mode. The hearts were perfused for 10 min, made ischemic for 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 A high-performance thin-layered chromatography (HPTLC) plates. The plates were developed with iodine, photographed using Photoshop software and quantitated using NIH Imaging software. The results show a 50% decrease of sphingomylein during reperfusion with a corresponding increase in ceramide with sphingosine showing a smaller decrease as compared with the ceramide increase.