Reactions of nitrosochloramphenicol in blood

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It has been suggested that nitrosochloramphenicol (NOCAP), a possible metabolite of chloramphenicol (CAP), may be involved in CAP-induced aplastic anemia. We found that NOCAP was rapidly eliminated from human blood in vitro (more than 90% in less than 15 sec). Analysis of the different reactions showed that 5% of NOCAP was covalently bound to plasma proteins, mainly to albumin, the remainder being metabolized in red cells. The most important reaction in red cells was the very rapid adduct formation with GSH ( k = 5,500 M −1s −1), yielding presumably a semimercaptal which either isomerized to a sulfinamide (GSONHCAP, k = 0.05 s −1) or was thiolytically cleaved by another GSH molecule with formation of the hydroxylamine (NHOHCAP) and GSSG ( k = 7.1 M −1s −1). Another important elimination reaction was the covalent binding of NOCAP to the SH groups of hemoglobin ( k = 5 M −1s −1), also yielding a sulfinamide. Besides these reactions with thiols, NOCAP was enzymatically reduced to NHOHCAP in the presence of NADPH ( K m NADPH = 10 −5 M; K m NOCAP = 10 −4 M; V max = 2 μmole/min per ml). This reaction was only effective at NOCAP concentrations below 10 −4M, probably because of limited NADPH-regeneration. Further reduction of NHOHCAP to NH 2CAP was a slow process which did not exceed 0.5 nmole/min per ml. NH 2CAP was mainly formed from GSONHCAP, a reaction which depended on NADPH and the presence of hemolysate, indicating an enzymatic reaction. In contrast to smaller nitrosoarenes, NOCAP was a poor ligand for ferrohemoglobin (probably due to steric hindrance by its bulky molecule) and was therefore much more exposed to biotransformation. NOCAP and NHOHCAP formed ferrihemoglobin at a rate 5000 times slower than did phenylhydroxylamine. In contrast to NOCAP, NHOHCAP penetrated slowly the red cell membrane ( t 1 2 about 5 min), and its disposition in blood was quite ineffective. From these data, it seems likely that most of the NOCAP formed by microorganisms in the intestine or produced in the liver, will be degraded in blood before it can reach the bone marrow.