David J. Stewart

Cocaine metabolism: Cocaine and norcocaine hydrolysis by liver and serum esterases

The hydrolysis of cocaine and its N‐demethylated product, norcocaine, by esterases was examined in liver and serum. Both liver and serum enzymatically formed ecgonine methyl ester from cocaine. The liver enzyme had a much lower affinity for cocaine than that of serum, indicating that a different form of esterase was present in liver. The liver enzyme had a similar affinity for both norcocaine and cocaine. Likewise, the serum enzyme showed similar affinities for both substrates. The Vmax estimates, however, were consistently higher for norcocaine than cocaine in both liver and serum. Benzoyl ecgonine, a major metabolite of cocaine formed by hydrolysis, was not produced enzymatically in either serum or liver; the rate of spontaneous formation at physiological pH suggests that this metabolite may arise nonenzymatically in the body.

Metabolism of cocaine in man

Following ingestion of [N‐14CH3]cocaine (10 mg, 2.3 µCi) by 2 healthy subjects, breath, saliva, serum, and urine samples were collected serially. Labeled CO2 production was monitored as a measure of N‐demethylation of cocaine. The cumulative excretion of 14CO2 in 5 hr was 2.4% and 6.2% of the administered dose with half‐lives of 2.3 and 1.4 hr, respectively. The greater N‐demethylation was found in a subject with lower plasma cholinesterase activity. Radioactivity excreted in 0 to 28 hr urine reached 65% to 75% of the dose. Ecgonine methyl ester, a product of cocaine hydrolysis by plasma cholinesterase, was identified as a major metabolite in the urine of both subjects and accounted for 32% to 49% of the urinary metabolites.

Hydrolysis of cocaine in human plasma by cholinesterase

Abstract Hydrolysis of cocaine to ecgonine methyl ester in human plasma is mediated by cholinesterase. Cocaine hydrolysis by plasma is blocked by DFP and eserine and partially inhibited by fluoride. Highly purified cholinesterase from human plasma when diluted to the same benzoylcholine hydrolyzing activity as human plasma, shows the same rate of cocaine hydrolysis as human plasma. There was no detectable enzymatic conversion of cocaine to benzoyl ecgonine in plasma.

Induction of the catalytic protein of (Na++K+)-ATPase in the salt gland of the duck

The (Na+ plus K+)-ATPase activities in salt gland homogenates increased 3- to 4-fold after saline treatment of ducks for 3 weeks. The ATPase was purified to a specific activity of 460 and 1015 mumol Pi/mg protein per h, respectively, in control and saline-treated ducks. The catalytic protein was identified on polyacrylamide electrophoresis gels by phosphorylating the enzyme with (32P)ATP. The molecular weight of the protein was estimated to be 98 000. The amount of catalytic unit increased commensurately with the enzyme activity after saline treatment. It is therefore concluded that the increased enzyme activity is due to a de novo enzyme synthesis and is not an activation effect. Phospholipid concentration in the salt gland tissue increased 1.7-fold after the saline treatment. Significant increases occurred in the percentage of the total phospholipids as phosphatidylserine and sphingomyelin. In the partially purified (Na+ plus K+)-ATPase preparation, the percentage composition of phosphatidylserine and phosphatidylethanolamine increased after saline treatment.

N-demethylation of aminopyrine in vivo and in the isolated hepatocyte of the rat

Abstract The N -demethylation of [dimethylamine- 14 C]aminopyrine was monitored in vivo and in isolated hepatocytes of the rat by collection of 14 CO 2 , The isolated hepatocyte is capable of aminopyrine N -demethylation rates similar to those occurring in vivo . The kinetics of N -demethylation in the isolated cells indicate the presence of two separate reactions with K m values of 32 and 710 μM. The kinetics of N -demethylation in vivo , however, indicates the presence of one reaction with an intermediate K m value of 260 μM. It is thought that the two reactions observed in the hepatocytes also occur in vivo but that it is not possible to differentiate between them.

The induction of (Na++K+)-ATPase in the salt gland of the duck

Abstract Hypertonic saline was administered to ducks for 24 h. Protein synthesis was measured in salt-gland slices by a double-isotope technique. Salt-gland slices from saline-treated animals incorporated more radioactivity than those from control animals. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of proteins in the heavy microsomal fraction of the salt glands revealed that the greatest radioactive amino acid incorporation occurred in the catalytic subunit of ( Na + + K + )- ATPase . The results support the tenet that saline treatment increases ( Na + + K + )- ATPase synthesis.

A Mechanism for Cholinergic Stimulation of Sodium Pump in Rat Submandibular Gland

Publisher Summary This chapter presents a mechanism for cholinergic stimulation of sodium pump in rat submandibular gland. Na/K pump activation by carbachol in rat submandibular gland slices does not depend on an influx of sodium into the cells. An evidence presents that pump activation may depend on a cyclic GMP-mediated mechanism. Sodium pump activation by carbachol is dependent on extracellular calcium.

Concentration-dependent ethanol metabolism in perfused liver

Abstract Ethanol at initial concentrations of 9.7, 32.5 and 52 mM was added to a recirculating liver perfusion system in the absence of added substrate. A concentration-dependent increase in ethanol metabolism was observed in perfused liver. The β-hydroxybutyrate/acetoacetate ratio (B/A) increased on addition of a low ethanol concentration (9.7mM). The ratio, however, declined at the higher concentration (52 mM). The decline has been reported previously by others in in vivo studies in the rat. Addition of 10 mM L -alanine to the perfusate increased ethanol metabolism, oxygen consumption and urea formation. The concentration-dependent increase in ethanol metabolism and the decline in B/A ratio did not occur in the presence of alanine. Ethanol metabolism in the presence and absence of alanine was completely blocked by 4-methylpyrazole. The changes in B/A ratio were likewise blocked. It is postulated that the concentration-dependent increase in ethanol metabolism is dependent on the alcohol dehydrogenase pathway. The concentration-dependent increase in ethanol metabolism observed under in vitro conditions was previously reported not to occur in vivo . Since the additon of alanine to the perfusate blocked the concentration-dependent ethanol metabolism, it would appear that the primary difference between in vivo and in vitro observations is the absence of adequate energy substrates under in vitro conditions. Possible mechanisms for the elevated ethanol metabolism under in vitro conditions are discussed.