Olav M. Bakke

Hydroxylation of aromatic hydrocarbons in the rat

Abstract The hydroxylation of benzene, toluene, ethylbenzene, styrene, cumene, o -xylene, m -xylene, p -xylene, p -cymene, pseudocumene, and mesitylene was studied following their oral administration to rats. The animals were fed a purified diet containing neomycin in order to reduce the levels of normally occurring simple urinary phenols. Phenolic metabolites were quantitatively estimated in hydrolyzed urine samples by gas chromatography. The aromatic hydrocarbons administered at a dose of 100 mg/kg were metabolized to the following monohydric phenols (% of dose): benzene to phenol (3.7), toluene to o -cresol (0.04–0.11) and p -cresol (0.4–1.0), ethylbenzene to p -ethylphenol (0.3), styrene to 4-vinylphenol (0.1), o -xylene to 3,4-dimethylphenol (0.1) and very small amounts of 2,3-dimethylphenol, m -xylene to 2,4-dimethylphenol (0.9), p -xylene to 2,5-dimethylphenol (1.0), pseudocumene to 2,4,5-trimethylphenol (0.05), and mesitylene to 2,4,6-trimethylphenol (0.4). Cumene and p -cymene were not metabolized to phenols. The analytical methods allowed the detection of several alcoholic metabolites. The following compounds were shown to be urinary metabolites: benzyl alcohol from toluene, 1- and 2-phenylethanol from ethylbenzene and styrene, 2-phenyl-1-propyl alcohol and 2-phenyl-2-propyl alcohol from cumene, 2-methylbenzyl alcohol from o -xylene, and p-isopropylbenzyl alcohol from p -cymene. The possible significance of the formation of phenolic metabolites to the toxicity of aromatic hydrocarbons is briefly discussed.

Changes in lipid metabolizing enzymes of hepatic subcellular fractions from rats treated with tiadenol and clofibrate

Abstract the levels of hepatic lipid metabolizing enzymes including palmitoyl-CoA hydrolase, palmitoyl- l -carnitine hydrolase as well as some other enzymes were studied in the 100,000 g × 1 hr sediment, the corresponding supernatant and lipid-rich floating layer from rats fed tiadenol or clofibrate-containing diets (0.3 per cent w w ). Tiadenol administration resulted in a large increase of the total activity of palmitoyl-CoA hydrolase, and of peroxisomal-CoA oxidation, while only a moderate enhancement was obtained after clofibrate feeding. the total activity of palmitoyl- l -carnitine hydrolase was increased more by tiadenol than by clofibrate. the specific activity of the two former enzymes was decreased in the particulate MLP-fraction (100,000 g × 1 hr sediment containing mitochondria, peroxisomes and microsomes) after treatment with tiadenol. The specific activity of palmitoyl-CoA hydrolase was increased more than 10-fold in the cytosolic fraction after administration of tiadenol. Tiadenol increased the specific activity of palmitoyl- l -carnitine hydrolase considerably in the cytosolic fraction, but the activity of this enzyme was little affected by clofibrate treatment. the specific activity of palmitoyl-CoA hydrolase and palmitoyl- l -carnitine hydrolase increased in the lipid-rich floating layer. Since there was also a shift in the distribution of peroxisomal palmitoyl-CoA oxidation and catalase, but not of urate oxidase after treatment with the drugs, it is suggested that the drugs induce peroxisomes with altered membrane characteristics.

Hepatic enzymes, coash and long-chain acyl-coa in subcellular fractions as affected by drugs inducing peroxisomes and smooth endoplasmic reticulum

1. The activities of acyl-CoA hydrolase, catalase, urate oxidase and peroxisomal palmitoyl-CoA oxidation as well as the protein content and the level of CoASH and long-chain acyl-CoA were measured in subcellular fractions of liver from rats fed diets containing phenobarbital (0.1% w/w) or clofibrate (0.3% w/w). 2. Whereas phenobarbital administration resulted in increased microsomal protein, the clofibrate-induced increase was almost entirely attributed to the mitochondrial fraction with minor contribution from the light mitochondrial fraction. 3. The specific activity of palmitoyl-CoA hydrolase in the microsomal fraction was only slightly affected while the mitochondrial enzyme was increased to a marked extent (3-4-fold) by clofibrate. 4. Phenobarbital administration mainly enhanced the microsomal palmitoyl-CoA hydrolase. 5. The increased long-chain acyl-CoA and CoASH level observed after clofibrate treatment was mainly associated with the mitochondrial, light mitochondrial and cytosolic fractions, while the slight increase in the levels of these compounds found after phenobarbital feeding was largely of microsomal origin. 6. The findings suggest that there is an intraperoxisomal CoASH and long-chain acyl-CoA pool. 7. The specific activity of palmitoyl-CoA hydrolase, catalase and peroxisomal palmitoyl-CoA oxidation was increased in the lipid-rich floating layer of the cytosol-fraction. 8. The changes distribution of the peroxisomal marker enzymes and microsomal palmitoyl-CoA hydrolase after treatment with hypolipidemic drugs may be related to the origin of peroxisomes.

Enzymatic changes in rat liver associated with low and high doses of a peroxisome proliferator.

The activities of a number of lipid-metabolizing and subcellular marker enzymes were measured in total homogenates and subcellular fractions prepared from the livers of male rats fed diets containing 0.05, 0.1, 0.3, and 0.5% of the hypolipidemic drug tiadenol, resulting in mean drug intake of 45, 90, 330, and 530 mg/day/kg body wt, respectively. In the total homogenates, a massive induction of palmitoyl-CoA hydrolase and peroxisomal palmitoyl-CoA oxidation accompanied by increased free CoASH and long-chain acyl-CoA content was observed at the highest dose levels whereas little change occurred up to 90 mg/day/kg/body wt. The palmitoyl-CoA synthetase activity increased slightly up to 90 mg/day/kg body wt, but higher doses resulted in decreased enzyme activity. Catalase activity increased with the dose to be elevated by a factor of approximately 1.6 at 330 mg/day/kg, whereas the activities of urate oxidase decreased. The specific activities of palmitoyl-CoA hydrolase and peroxisomal palmitoyl-CoA oxidation increased in all fractions, but most markedly in the cytosol. The changes in the activities and the distribution of subcellular marker enzymes and the increase of the peroxisome-associated polypeptide (PPA-80) are in keeping with a peroxisome proliferating effect resulting in formation of premature organelles with altered properties. Since high doses of many hypolipidemic drugs produce hepatic tumors and peroxisomal proliferation in rodents and since no increase in peroxisomes is found in human liver after therapeutic use of lower doses, the dose-response relationship is of interest for the evaluation of the toxicology of this class of agents.

Disposition and oxidative metabolism of phenylbutazone in man

SummaryThe absorption and elimination of orally administered14C-phenylbutazone and the role of oxidation in its metabolism have been studied. The main routes of excretion of14C-phenylbutazone and its metabolites were investigated in 3 patients with rheumatoid arthritis, and in 1 patient with a T-tube in the common bile duct. Up to 9 days after an oral dose of14C-phenylbutazone 600 mg (30 µCi) 63% of the radioactivity was found in the urine and 14% had appeared in the faeces. The cumulative excretion of radioactivity in bile amounted to 9.5% of the dose in 4 days. Only 1% of the radioactivity in the urine and bile was due to unchanged phenylbutazone. The role of oxidative metabolism of phenylbutazone in healthy human subjects was studied by gas chromatography. In 3 subjects given a single dose of phenylbutazone 600 mg, only 8.3% of the dose was excreted in urine as oxidized metabolites after 5 days. However, in 5 patients who had taken phenylbutazone for more than 5 weeks, these metabolites accounted for 23.4% of the dose. These results suggest that oxidative metabolism becomes more important after continued administration of the drug. After a single dose of phenylbutazone, the side-chain oxidized metabolite (II) was the major free derivative excreted in urine, but the ring oxidized metabolite, oxyphenbutazone (I), was much more important than the former in plasma. However, after prolonged treatment there was little difference between the concentration of the two metabolites in plasma. This finding suggests that side-chain oxidation is increased relative to ring oxidation on prolonged treatment with phenylbutazone. A third derivative containing hydroxyl groups both in the phenyl ring and in the side-chain (metabolite III) was found in urine in experiments with phenylbutazone, but in only one out of 3 volunteers given repeated doses of oxyphenbutazone.

Effect of aminoglutethimide on antipyrine, theophylline, and digitoxin disposition in breast cancer

The influence of aminoglutethimide (AG) on antipyrine, theophylline, and digitoxin kinetics was examined. Antipyrine was given as a single test dose before and after 3 mo of AG treatment, whereas theophylline and digitoxin kinetics were investigated at steady state in patients receiving these drugs therapeutically before and after AG therapy. During AG treatment, mean clearance rates for antipyrine, theophylline, and digitoxin increased by 81%, 32%, and 109%. Together with earlier reports of effects of AG on warfarin and dexamethasone disposition and on its own metabolism, these findings indicate that AG is a potent inducer of drug metabolizing microsomal monooxygenases of the liver. Since many drugs known to be metabolized by this enzyme system are frequently used for concomitant conditions in patients with breast cancer, interactions with AG are to be expected.

Analysis of simple phenols of interest in metabolism: II. Conjugate hydrolysis and extraction methods

Abstract Methods for conjugate hydrolysis and ether extraction of simple phenols in urine have been investigated. The losses of monohydric phenols could be compensated for by adding an internal standard and p - or m -methoxyphenol is suggested for this use. However, recoveries of some dihydroxybenzenes were considerably reduced and these losses were shown to occur during the removal of phenolic acids. Both acid and enzymic hydrolysis are recommended, as they do not yield the same quantities of all the phenols. Formation of simple phenols from phenolic acids was shown to occur during treatment with acid but was not significant with the quantities of naturally occurring phenolic acids encountered in the present experiments.

Transplacental passage of diazepam during labor: Influence of uterine contractions

The rate of transplacental passage of diazepam (DZ) has been studied in 33 cases of cephalic presentation where operative forceps delivery was indicated by intrauterine hypoxia or by prolonged second stage of labor. The drug (30 mg) was injected intravenously immediately before delivery either during uterine contractions (Group I) or in the relaxation period (Group II) according to a randomized protocol. As judged by the concentration in the newborn and the child/mother concentration ratio at 2 hr after delivery, and the concentration on the second day, the fetal exposure to the drug was probably less when the injection was timed to coincide with uterine contractions. In the group of patients given the drug in the relaxation period, the injection‐delivery (I‐D) interval was up to 305 sec, and the 2‐hr child/mother concentration ratio was close to unity in some cases. It therefore appears that the transplacental passage of DZ is rapid when the high initial concentrations in the maternal circulation coincide with favorable conditions for transfer in the relaxation period. Although sleep was induced by the injection of DZ in all of the mothers, the amounts of drug transferred during the short I‐D intervals in the present study did not exert deleterious effects on the newborn infants.

Analysis of simple phenols of interest in metabolism: I. Gas chromatographic separation and quantitative determination

Abstract Various gas chromatographic columns have been prepared and tested for the analysis of simple phenols and derivatives. A standard method has been developed using 1% w/w Carbowax 20 M on Chromosorb W (AW-DMCS) and temperature programming (130° → 185°C at 3°C/min). Low stationary phase loading and low operating temperatures made possible a sensitive single run analysis of mono- and dihydroxy-benzenes and derivatives. This was achieved at temperatures at which all compounds, except 4-vinylcatechol, eluted as symmetrical peaks. Additional methods using 15% w/w silicone rubber UC-W98 and 2% w/w tricresyl phosphate at 120° are recommended for special purposes.

Time-course of Transplacental Passage of Diazepam

SummaryNeonatal drug concentrations and neonate/mother concentration ratios are reported in 73 cases of elective Caesarean section and forceps deliveries where diazepam was used as an intravenous sleep-inducing agent. The various parameters were plotted against the injection-delivery interval and the correlation was tested using a non-parametric ranking method.The concentration of diazepam in mixed arteriovenous umbilical cord blood was negatively correlated with the injection-delivery interval in the range of 55 to 810 seconds. Statistically significant positive correlations (p < 0.001) were found between the injection-delivery interval and the neonatal concentrations at 2 and 24 hours. The corresponding neonate/ther concentration ratios varied considerably, and were not so strongly correlated to the duration of antenatal drug transfer.The results suggest that with a slowly eliminated agent like diazepam, the drug concentration in capillary blood obtained from the newborn a few hours after delivery gives a reasonably good indication of the fetal drug exposure. The transplacental passage of diazepam is rapid, with distribution equilibrium between mother and fetus being approached within 5 to 10 minutes after intravenous injection of the drug.