Absence of trans-N-methylation of morphine in the rat
Author: S. J. MULE' , G. J. MANNERING
Pages: 27 to 28
Creation Date: 1965/01/01
G. J. MANNERING 2
From the Department of Pharmacology and Toxicology University of Wisconsin Medical School Madison, Wisconsin/USA
The finding of considerable amounts of C 14O 2 in the breath of rats [ 1] and man following the administration of N-methyl labeled morphine suggested that morphine was converted to normorphine, a view that gained support when it was shown that both slices [ 1] and a microsomal enzyme preparation [ 3] [ 4] from rat livers mediated the N-demethylation of morphine. The rat has also been shown to excrete normorphine in the urine after receiving morphine. [ 5] [ 6] However, it has not been possible to demonstrate the presence of normorphine in the urine of man given morphine, although highly sensitive techniques were employed. [ 7] Trans-N-methylation would provide one explanation for the apparent discrepancy between the pulmonary excretion of labeled CO 2 and the failure to reveal the end product of N-demethylation in the urine. Trans-N-methylation would be demonstrated if the specific activity of the morphine isolated from the urine were lower than that of the N-methyl labeled morphine administered. Rapoport [ 7] conducted such a study in humans and concluded that any difference between the specific activities of injected morphine and morphine isolated from the urine was within experimental error. This being the case in humans, it seemed pertinent to determine whether or not trans-N-methylation occurs in the rat, a species known to N-demethylate morphine in vitro and to excrete normorphine in the urine. [ 6] Two experimental approaches were employed: (1) the specific activity of injected N-methyl-C 14 labeled morphine was compared with that isolated from the urine of rats, and [ 2] non-labeled morphine was injected with l-methioninemethyl-C 14 and the morphine isolated from the urine was checked for any radioactivity it would have acquired from the labeled methionine, if trans-N-methulation had occurred in the expected manner.
In the first experiment ten male rats of the Holtzman strain weighing about 200 gm each were injected intraperitoneally with N-C 14-methyl labeled morphine sulfate (20 mg/kg, calculated as free base). The urine was collected for 24 hours and pooled. Crystalline morphine was isolated from the pooled urine after acid hydrolysis by employing solvent extraction, paper chromatography and sublimation as described previously. [ 8] The crystalline morphine was dissolved in a known volume of methanol and aliquots of this solution were processed for measurement of radioactivity using a liquid scintillation spectrometer and a gas flow counter. A third aliquot was used to measure the concentration of morphine in the solution. This was accomplished spectrophotometrically [ 9] using the difference between the maximum (285 mμ) and minimum (260 mμ) absorption at acid pH as well as the difference in maximum (297 mμ) and minimum (278 mμ) absorption under alkaline conditions. The specific activity of the morphine isolated from the urine as determined by the liquid scintillation techniques was 38.6 cpm/mg, while that of the injected morphine was 38.7 cpm/mg. There was also no difference between the specific activities of injected and isolated morphine as measured by the gas flow technique.
In the second experiment six male rats were injected intraperitoneally with 20 mg/kg (free base) of non-labeled morphine sulfate and 1.2 mg/kg of 1-methionine-methyl-C 14 (specific activity: 4.9 mc/mM) and their urines were collected for 24 hours. Similar quantities of morphine and labeled methionine were added to the urines from control animals which received no injections. Crystalline morphine was isolated from pooled experimental and control urines, and its specific activity was measured as described previously. The data on the specific activity determinations appear in table 1. The first crystalline morphine from the experimental urine suggested that some trans-N-methylation had occurred, but with subsequent second and third sublimations, essentially all radioactivity, was lost. The morphine isolated from control urine showed no radioactivity following the second and third sublimation.
Comparison of the specific activity of morphine isolated from control and experimental rat urine
Specific activity of morphine (cpm/mg) sublimations
A total of 20 μc of methionine-C 14 and 6 mg of non-labeled morphine was added to beakers and the urine collected from non-injected rats for 24 hours.
These experiments demonstrate that trans-N-methylation does not play a significant role in the overall metabolism of morphine in the rat. This is not to argue against theories which implicate N-demethylation in the chain of biochemical events leading to the analgesia produced by narcotic drugs [ 10] , for the overall metabolism of morphine may not reveal the quantitatively small but pharmacologically important metabolic alterations occurring at vital sites in the central nervous system.
Footnote 1. Postdoctoral research fellow of the National Institute of Neurological Diseases and Blindness. Present address: NIMH, Addiction Research Center, Lexington, Ky.
Footnote 2. Present address: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota.
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