The role of cytochrome P-450 in in vitro methemoglobin formation by primaquine was investigated through a series of experiments with golden-Syrian hamsters, treated before sacrificing with phenobarbital or 20-methylcholanthrene to increase the cytochrome P-450 content or with cobaltous chloride to cause its decrease. To determine the extent of induction in the liver microsomal cytochrome P-450-dependent mixed-function oxidases, cytochrome P-450 content and formaldehyde from the 0-demethylation of p-nitroanisole were measured. A 90% increase occurred in cytochrome P-450 content with phenobarbital pretreatment and an increase of 102% was observed in p-nitroanisole 0-demethylation. Formaldehyde from primaquine increased 64%. The methylcholanthrene treatment produced a 55% increase in P-448. In vitro methemoglobin-forming ability was examined by incubating washed human red blood cells with primaquine, hamster liver microsomes and a NADPH-generating cofactor mixture. In the control flasks, the cofactor mixture was omitted. After the incubations, the red blood cells were separated from the other components of the incubation flask by differential centrifugation and the methemoglobin and oxyhemoglobin contents were measured. The ability to produce methemoglobin by primaquine was greatly enhanced by microsomes with cofactors and only traces of methemoglobin were produced in their absence. Hamster liver microsomes, in the presence of the cofactor mixture, were 2.5 times more active in producing methemoglobin than mouse liver microsomes in comparable experiments. This process is induced to a limited extent (15%) with phenobarbital, while methylcholanthrene failed to produce induction. Hamsters pretreated with cobaltous chloride demonstrated decreases in both P-450 levels and the ability to produce methemoglobin. With the combination of the phenobarbital and cobaltous chloride pretreatments, a decrease from phenobarbital-induction levels was noted. The methemoglobin-forming ability of primaquine correlated well with increasing cytochrome P-450 in the non-phenobarbital-treated hamsters. The possible primaquine metabolite, 5,6-dihydroxyprimaquine, was an active methemoglobin-former in the system without cofactors but its activity was tripled in the presence of cofactors. The metabolism of dihydroxyprimaquine was also induced with phenobarbital particularly at a low microsomal protein concentration. A negative relationship was observed between methemoglobin formation from dihydroxyprimaquine with non-phenobarbital-induced microsomes. In conclusion, cytochrome P-450 does appear to be involved in the metabolism of primaquine by hamster liver microsomes to methemoglobin-forming compounds, but its involvement appears to be in only a part of the overall process.
Ian M. Fraser
R. Bruce Wilcox
Master of Science (MS)
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This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Hicks, Tommy L., "Role of Cytochrome P-450 in Methemoglobin Formation by Primaquine with Hamster Liver Microsomes" (1977). Loma Linda University Electronic Theses, Dissertations & Projects. 1387.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives