E. Mussini

Species difference in diazepam metabolism and anticonvulsant effect.

Abstract Diazepam showed an antimetrazol activity which was longer-lasting in mice than in rats. The levels of diazepam in blood and brain were similar in both species but N-desmethyl metabolites ofdiazepam accumulated in mouse brain while they were practically undetectable in rat brain. The three main metabolites of diazepam, namely N-methyloxazepam, N-desmethyldiazepam and oxazepam showed strong anticonvulsant activity in mice.

A specific gas chromatographic method for the determination of microsomal styrene monooxygenase and styrene epoxide hydratase activities.

A gas chromatographic (GC) method for the determination of the metabolite resulting from the activities of microsomal styrene monooxygenase (epoxide synthetase) and epoxide hydratase using styrene or styrene epoxide as substrates has been developed. The determination of the activities of both enzymes is based on the GC determination of phenylethylene glycol after its esterification with n-butylboronic acid. Kinetic parameters for both enzymes are given.

ATPase Activity Defects in Alloxan-Induced Diabetic Sciatic Nerve Recovered by Ganglioside Treatment

ATPase activities were measured in sciatic nerves from rats with alloxan-induced diabetes (ALX-D) of various duration (2 wk, 5 wk, 9 wk, and 6 mo). Our data confirm that sciatic nerve Na+-K+-ATPase abnormalities are present very early in ALX-D rats, similar to results previously described in streptozocin-induced diabetic rats, spontaneously diabetic BB Wistar rats, and ALX-D rabbits. Na+-K+-ATPase activity decreased by 26–47% in ALX-D rats compared with age-matched controls. Ganglioside treatment (10 mg/kg i.p. for 10 or 30 days starting 1 wk after ALX injection) completely impeded the enzyme reduction. The effect observed at the end of either 10 or 30 days of treatment lasted ≥1 mo. Chronic diabetic groups treated for 30 days before killing also presented normal ATPase activity at the end of treatment. Therefore, gangliosides are effective on Na+-K+-ATPase even in animals with a longer duration of diabetes. The maintenance of fairly normal ATPase activity by ganglioside treatment could mirror a more general recovery from early metabolic dysfunction and/or late structural abnormalities in diabetic nerve fibers.

Further studies on species difference in diazepam metabolism

Abstract The anticonvulsant activity of diazepam lasts longer in mice than in rats. The brain levels of diazepam are similar in both species but N-demethyl diazepam and oxazepam accumulate in mouse brain while they are practically undetectable in rat brain. This difference occurs also in blood and adipose tissue. N-methyloxazepam was not detected in rats or mice after an intravenous administration of diazepam. It is suggested that the N-methyl diazepam and oxazepam formed in mice may be responsible for the prolonged anticonvulsant action of diazepam in this species.

The metabolism of diazepam by liver microsomal enzymes of rats and mice

Abstract The metabolic pathways by which diazepam and its metabolites are transformed in vitro by rat and mouse liver microsomes were studied in detail. Using a gas chromatographic technique to measure benzodiazepines, it was demonstrated that the predominant reactions were N-demethylation in rat liver microsomes and C 3 hydroxylation in mouse liver microsomes. When N-demethyldiazepam or N-methyloxazepam was used as a substrate there was a clear formation of oxazepam. No exazepam was found in vitro when diazepam was incubated with liver microsomes. Some preliminary data indicate that the presence of diazepam inhibits the further hydroxylation of N-demethyldiazepam and the N-demethylation of N-methyloxazepam.

Microsomal Styrene Mono-oxygenase and Styrene Epoxide Hydrase Activities in Rats

1. Styrene epoxide formation and styrene epoxide hydration have been studied in liver, lung, kidney, heart, spleen and brain of female and male rats. 2. Styrene epoxide formation is NADPH-dependent although it is enhanced when NADH is added together with NADP. This enzymic activity is inhibited by metyrapone and SKF 525-A but not by the effective inhibitors of epoxide hydrase, 1,2-epoxy-3,3,3-trichloropropene and cyclohexene oxide. 3. Known inducers of liver microsomal mono-oxygenases show a different activity on the two enzymes. Phenobarbital increases both formation and hydration of styrene epoxide; and carbamazepine increase the hydration but not the formation of styrene epoxide; a steroid contraceptive combination (lynestrenol+ mestranol) increases styrene epoxide formation while it inhibits epoxide hydrase; 3-methylcholanthrene does not affect either of the activities.

Respiratory excretion of hydrogen and methane in Italian subjects after ingestion of lactose and milk

Abstract. Lactose malabsorption, by the breath hydrogen test, and lactose intolerance (presence of symptoms) were studied in twenty healthy Italian subjects after intake of 12.5, 25 and 50 g lactose, whole milk and low‐lactose milk. A rise in respiratory concentration of hydrogen (>20 ppm) (malabsorption) was found in fifteen subjects after 50 g lactose, in thirteen after 25 g and in seven after 12.5 g. Symptoms generally occurred in subjects presenting a rise in respiratory hydrogen excretion, but such a rise was often observed without symptoms. Thirteen subjects presented symptoms after 50 g lactose, but only three after 25 g and one after 12.5 g. Whole milk (500 ml) gave a lower incidence of lactose malabsorption than 25 g lactose (7/20 versus 13/20, P < 0.05) and more subjects developed symptoms (7/20 versus 3/20, NS). Low‐lactose milk produced no malabsorbers and one intolerant. Breath methane was detected constantly in seven subjects and in three on some of the days of observation. Respiratory methane excretion generally appeared to be unrelated to lactose ingestion.

Brain levels of metrazol determined with a new gas chromatographic procedure

Abstract A gas chromatographic method for the determination of metrazol in the brain after the administration of this convulsant drug in rats and mice is presented. The peak levels of brain metrazol were reached at the same time in both species, but they were lower and declined faster in mice than in rats, after comparable doses of injected metrazol. The threshold for convulsions was different in the two animal species; in rats it was approximately 45 μg of metrazol per g of brain and in mice approximately 37 μg/g.

Studies on nitrazepam reduction in vitro.

The solvents decreased the reduction rate of nitrazepam by rat liver homogenate; the lowest interference was observed with ethanol. High concentrations of nicotinamide used in media for nitroreductase determination lowered enzyme activity. Reduction of nitrazepam to 7-amino derivative decreased during the preservation of liver centrifugal fractions in 1.15% KCl at 0°C. The highest enzyme activity was found in rat liver, followed by the activity of kidney, heart, lung, skeletal muscle and spleen preparations. The substance was not reduced in brain. The Michaelis constants Km of nitrazepam reduction did not differ substantially, being of the same order of 10−4M in preparations from rat, mouse, rabbit and guinea pig liver. The maximal velocities of the reduction Vmax were 10−6moles per hour per 1 g of liver tissue. Chlorpromazine, diazepam and imipramine influenced the reduction only in high concentrations. EDTA, cysteine and ascorbic acid did not change substantially the enzyme activity. KCN was a strong non-competitive inhibitor (Ki = 10−4 M of the nitrazepam reduction.

The effect of injections of fluorocitrate into the brains of rats.

Abstract Intracerebral injection of fluoroacetate and fluorocitrate were made in rats by manual and stereotaxic technique. Fluorocitrate was found to be at least 100 times more toxic than fluoroacetate under various experimental conditions. Fluorocitrate was more toxic when injected in the third cerebral ventricle, or in the nucleus reuniens thalami than when injected in the lobus frontalis. Gas chromatographic determination of fluoroacetate shows that this compound is rapidly removed as such from the brain. These results and their implications are discussed in relation to current views of the action of these compounds.