Maximal .OH production is seen upon reoxygenation of viable anoxic cultured cardiomyocytes but not of compromised cells.

Hydroxyl radicals (.OH) in isolated cultured cardiomyocytes upon reoxygenation (reoxy) were measured after graded anoxia (A) using high performance liquid chromatography (HPLC). Isolated myocytes were subjected to A for 15, 30, 60, 90 and 120 minutes and reoxy for 120 seconds. Supernatant was collected after reoxy, extracted with ether and injected into HPLC for measuring hydroxylation products of salicylic acid (2,5-DHBA) as an indicator of .OH formation. 2,5-DHBA was detected maximally after 15 minutes of A and 120 seconds of reoxygenation (34.2 +/- 3 pmol/mg protein), at which time 80% of cells had maintained their rod shape and 99% of cells excluded both trypan blue (TB) and horseradish peroxidase (HP). There was significantly less (P < 0.05) 2,5-DHBA in the group subjected to 15 minutes of A only without reoxy (7.95 +/- 1.2 pmol/mg protein). 2,5-DHBA decreased to 13.1 +/- 2 pmol/mg protein at 120 minutes of A/120 seconds reoxy. With increasing anoxic time, the number of rod-shaped cells decreased from 80% at 15 minutes to 30% at 120 minutes, while the number of TB/HP positive cells increased from 0% at 15 minutes to 100% at 120 minutes. The cell membrane blebs were nonexistent at 15 minutes, but at 120 minutes A intense bleb formation was observed. These data suggest that .OH is produced upon reoxygenation of anoxic cultured cardiomyocytes and their production is maximum when majority of myocytes are viable.