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In Vitro Technology

Early studies of drug metabolism indicated the central role of the liver in modifying ingested substances. As the only organ positioned between the intestine and the systemic circulation the liver was the logical site of metabolism.

Techniques to examine the role of the liver in vitro arose with the development of nondestructive homogenization procedures by Potter and Elvehjem1 in 1936 and the utilization of differential centrifugation for the separation of liver organelles initially developed by Claude2 and Schneider3. Gerald Mueller, working in the laboratory of James and Elizabeth Miller applied these techniques to the study of the metabolism of dimethylaminoazobenzene. This rat carcinogen had been shown by the Millers to bind to the liver4 and they sought to explore in vitro the pathways of metabolism that had originally been described by Stevenson and co-workers5. Mueller and Miller showed that DAB was metabolized by rat liver homogenates and required oxygen and a reductant NADPH.6,7

The wide-ranging role that this enzyme system has in drug metabolism was uncovered by Julius Axelrod. At the age of 35 Axelrod initiated a long and illustrious career at NIH where he began his studies on sympathomimetic amines and, in particular, amphetamine. Axelrod was fascinated by the rabbit's ability to rapidly metabolize amphetamine and wanted to find the mechanism of these conversions. When he broached his concern about lack of training in enzymology to co-worker Gordon Tomkins, Tomkins responded that there was no big mystery to being an enzymologist that all you had to have was a razor blade and a liver.8

 

Julius Axelrod (pictured lower right) started his career working in a food testing laboratory in New York. The lab was asked to find the reason that the popular pain remedy, acetanilide, was causing methemoglobinemia in a small percentage of patients. Axelrod consulted with B.B.Brodie at NIH and he and Brodie found that the problem was due to the metabolic conversion of the active ingredient acetanilide to aniline. They also found that acetaminophen was a major metabolite and a safe analgesic alternative. This project launched Axelrod's career as a metabolism scientist.

With that encouragement Axelrod quickly showed that amphetamine was rapidly metabolized in rabbit liver slices. He went on to define the co-factor requirements for the reaction finding that NADPH was required; similar to observations of Bert LaDu in his studies on aminopyrine demethylation9.Axelrod then determined the sub-cellular location of the activity by using methods developed by Hogeboom and Schneider10. This newly defined microsomal oxidizing system was soon shown to be responsible for the metabolism of a wide variety of drugs and other chemicals.

 

Ref: 1V.R.Potter and C.A.Elvehjem, J.Biol. Chem. ,114:495-504 (1936) 2A.Claude, Cold String Harbor Symposium Quant.Biol.,9:263-270 (1941)3 W.C.Schneider,J.Biol.Chem. 165:585-593 (1949) 4E.C.Miller and J.A.Miller, Cancer Res. 7:468-480 (1947)5 E.S.Stevenson, K. Dobriner, and C.P.Rhoads, Cancer Research 2:160-167 (1942)6 G.C.Mueller and J.A.Miller, J.Biol.. Chem. 176:535-544 (1948) 7G.C.Mueller and J.A. Miller, J.Biol. Chem. ,180:1125-1236 (1949)8Axelrod, J. Ann.Rev.Pharmacol.Toxicol. 28:1-23 (1988)9 La Du, B.N., Trousof, N. and Brodie, B.B., Fed. Proc. Fed. Am. Soc.Exp.Biol. 12:339 (1953) 10Hogeboom, G.H., Schneider, W.C. and Striebich, M.J. Cancer Res. 13:617-632 (1953)

Pictures: James and Elizabeth Miller in Miller, J. Drug Met.Rev. 26:1-36 (1994), Julius Axelrod-Ann.Rev.Pharm.Tox. 28:1-23 (1988)

 

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