Reductive activation of halothane by human haemoglobin results in the modification of the prosthetic haem

The metabolic activation of halothane by human haemoglobin (Hb) under reducing conditions in vitro is reported. Absolute spectra of sodium dithionite-reduced Hb, recorded during its anaerobic incubation in the presence of the substrate, showed decreasing concentrations of reduced Hb (Hb 2+) with time. The loss of Hb 2+ was accompanied, although only to some extent, by a concurrent oxidation to methaemoglobin (Hb 3+), suggesting that electron transfer from Hb to the substrate had occurred. Reductive halothane metabolism was observed under these conditions as indicated by a dose-dependent inorganic fluoride (F −) production, which was, however, lower than that observed with heated Hb or a water soluble haem preparation (methaemalbumin). A rapid, partial loss of Hb was found upon addition of the substrate to the incubation mixture, as indicated by a decrease of the typical peak at 418 nm in the absolute spectra recorded in the presence of carbon monoxide (CO). This effect was associated with a loss of the Hb prosthetic group, haem, as shown by a decrease of the pyridine-haemochromogen reaction. Both effects were time and dose dependent. The inhibition of the Hb inactivation reaction by adding exogenous CO or the spin trapping agent N- t-butyl- α-phenylnitrone (PBN) to the incubation mixture beforehand indicated that (a) a reduced and free haem iron is required by Hb to activate halothane, and (b) the formation of free radical reactive metabolites of halothane is likely to be responsible for Hb inactivation. The mechanism of the reaction may involve the attack of these metabolites on the haem group of Hb, as indicated by the detection, with a reverse-phase ion-pairing HPLC system, of two Hb-derived products showing a typical haem-like absorption spectrum. The present results resemble those obtained recently with carbon tetrachloride (Ferrara etal., Alternatives to Laboratory Animals 21: 57–64, 1993) and suggest a common mechanism of activation of the two polyhalogenated alkanes by Hb.