Lack of interference by sorbitol with aminopyrine breath test

Summary The effect of sorbitol on the aminopyrine breath test was investigated in 13 patients with various liver diseases given a bolus dose of sorbitol 20 mg · kg^–1 i. v. 30 min after ¹⁴C-aminopyrine and 13 patients without the sorbitol bolus who also underwent an aminopyrine breath test.The time course of ¹⁴CO₂ exhalation after the bolus of sorbitol did not differ from that in the control group.     

Biotransformation enzymes in nasal mucosa and liver of Sprague-Dawley rats

The metabolism of hexamethylphosphoramide (HMPA), aminopyrine, ethoxycoumarin, ethoxyresorufin and pentoxyresorufin, by the monooxygenase cytochrome P-450-dependent system, was studied in microsomes from nasal epithelial membranes and liver tissue of Sprague-Dawley rats. Nasal metabolism rates for the different substrates ranged from 9% of liver values for aminopyrine to 83% for ethoxycoumarin. HMPA-demethylase activity followed Michaelis-Menten kinetics in nasal mucosa microsomes but was biphasic in those from liver. SKF 525A, metyrapone, dioxolane and α-naphthoflavone (ANF), inhibitors of various P-450 monooxygenases, were examined with regard to inhibition of nasal and liver ethoxycoumarin deethylase. In addition, activity of epoxide hydrolase, glutathione S-transferase, DT-diaphorase and UDP-glucuronyltransferase (UDP-GT) in nasal tissue homogenates were investigated. These activities were generally lower than those present in the liver. Various attempts to increase the activity of oxidative enzymes in nasal tissue by PB, 3-MC and ethanol failed, 3-MC and PB doubled the microsomal UDP-GT and the epoxide hydrolase activities. The results together with data from the literature suggest that the balance between P-450 isozymes and detoxifying enzymes differs in the nose compared with the liver. The activities of these enzymes in nasal tissue of different strains of rats also varies substantially with implications regarding the metabolic fate and activitation of inhaled xenobiotics.     

The comparison of in vivo plasma radioactivity clearance and 14CO2 breath elimination of model drugs in the rat: A study in regional hepatocyte function

We systematically related the in vivo plasma radioactivity clearance and the ¹⁴CO₂ breath elimination of two drugs ([4-dimethyl-¹⁴C]aminopyrine and [1-methyl-¹⁴C]caffeine), whose major sites of hepatic metabolism correspond to the microsomal mixed-function oxidase system (cytochromes P-450 and P-448, respectively), and two compounds ([1-¹⁴C]ethanol and [¹⁴C]methanol], whose major sites of hepatic metabolism correspond to the cytosol. All studies were carried out in the freely moving rat. Measurements of plasma radioactivity clearance and ¹⁴CO₂ breath elimination were done with normal animals as well as with animals pretreated with agents that either stimulate or inhibit the model drugs' in vivo metabolism. Following pretreatment with phenobarbital, the aminopyrine plasma radioactivity clearance was significantly increased over normal, and this was paralleled by a significant increase of the ¹⁴CO₂ breath elimination as well. The ratio (plasma elimination/breath elimination) was unchanged between the normal and phenobarbital-pretreated animals and approached unity. by contrast, neither the aminopyrine plasma radioactivity clearance nor the ¹⁴CO₂ breath elimination was significantly altered by pretreatment with 3-methylcholanthrene. When the plasma radioactivity clearance and the ¹⁴CO₂ breath elimination were studied after administration of [¹⁴C]caffeine, both the plasma radioactivity clearance and ¹⁴CO₂ breath elimination showed a significant increase in the 3-methylcholanthrene-treated animals as compared to normal. The ratio (plasma elimination/breath elimination) was unchanged, however, between the normal and 3-methylcho-lanthrene-pretreated animals. Neither the caffeine plasma radioactivity clearance nor ¹⁴CO₂ breath elimination was significantly altered by pretreatment with phenobarbital. In animals pretreated with pyrazole, the ethanol plasma radioactivity clearance and ¹⁴CO₂ breath elimination were both significantly decreased. However, neither measurement was significantly altered after pretreatment with 3-amino-1,2,4-triazole. By contrast, the methanol plasma radioactivity clearance and ¹⁴CO₂ breath elimination were significantly decreased in both the pyrazole and 3-aminio-1,2,4-triazole-pretreated animals. These studies illustrate that for the model compounds selected and the dosages utilized, the ¹⁴CO₂ breath elimination rate closely reflected the plasma radioactivity elimination rate.     

Aminopyrine disposition: A sensitive index of acetaminophen-induced hepatocellular damage

Rates of ¹⁴CO₂ excretion in breath and of plasma decay of [¹⁴C]aminopyrine were measured after intraperitoneal administration of trace doses of [¹⁴C]aminopyrine in adult male Wistar rats: Both measurements were shown to be sensitive indices of hepatic aminopyrine N-demethylation. Thus, changes in the disposition of aminopyrine administered ip could be used to investigate effects of different doses of acetaminophen on hepatic drug-metabolizing capacity. After the administration of different doses of acetaminophen, liver damage was assessed by measurement of glutathione content, cytochrome P-450 content, and microsomal demethylation capacity as well as by liver histology. Decreased ¹⁴CO₂ excretion, suggesting decreased hepatic aminopyrine N-demethylase activity, occurred even in animals with minimal acetaminophen-induced hepatic injury. Further reduction of ¹⁴CO₂ excretion occurred with increasing doses of acetaminophen, which caused more extensive liver damage. These results suggest that measurement of ¹⁴CO₂ accumulation in breath can serve as a convenient, sensitive, and noninvasive laboratory test to assess in vivo hepatic drug-metabolizing capacity under a variety of conditions, including liver damage secondary to exposure to toxic compounds.     

Metabolism of Progesterone by the Nasal Mucosa in Mice and Rats

Abstract: The metabolism of 4-¹⁴C-progesterone by the nasal mucosa and lung from mice and rats was investigated in vitro. Using thin-layer radiochromatography at least 10 and 7, yet unidentified, metabolites were found at incubation with slices from the nasal mucosa and lung, respectively. The results further indicated that the rate of progesterone metabolism was higher in the nasal mucosa than in the lung. Autoradiography of lung slices incubated with ¹⁴C-progesterone showed that a selective localization of radioactivity occurred in the bronchial mucosa.     

Epithelial binding of 1,1,2,2-tetrachloroethane in the respiratory and upper alimentary tract

The bioactivation and binding of¹⁴C-labelled 1,1,2,2-tetrachloroethane (TCE) in the tissues of C57B1 mice were studied. As shown by autoradiography with heated and organic solvent-extracted tissue sections of i.v. injected mice, a high and selective localization of bound metabolites occurred in the nasal olfactory mucosa, preferentially in the Bowman's glands. High levels of bound metabolites were also present in epithelia of the trachea, bronchi and bronchioli and in the squamous epithelia of the oral cavity, tongue and esophagus. An epithelial binding was observed in tissue slices incubated with¹⁴C-TCE. Incubation of¹⁴C-TCE with homogenates of the olfactory mucosa and liver showed that the olfactory mucosa had a higher ability to activate¹⁴C-TCE into products that become irreversibly bound to protein. Addition of metyrapone, glutathione or sodium dithionite to the incubations decreased the level of irreversible binding, suggesting that the activation of TCE to reactive products is mediated via an oxidative cytochrome P-450 dependent process in the olfactory mucosa.     

The role of glutathione and cytochrome P-450 in the metabolism of methyl chloride

Rat liver microsomes metabolized methyl chloride to formaldehyde at a rate 15-fold less than the rate of benzamphetamine demethylation. The reaction rate was stimulated approximately 2-fold in microsomes from phenobarbital-pretreated rats and was inhibited by addition of SKF-525A, carbon monoxide, metyrapone, and hexobarbital to the microsomal suspension, indicating dependence on cytochrome P-450. The in vivo incorporation of ¹⁴CH₃Cl into liver macromolecules, previously shown to reflect metabolism to CH₃Cl to formate, was not significantly altered by SKF-525A, Aroclor 1254, or 3-methylcholanthrene pretreatment of rats, although pretreatment with phenobarbital produced a 35 and 28% increase in ¹⁴CH₃Cl uptake into liver lipid and acid-insoluble material, respectively. Pretreatment with phenobarbital increased the in vivo metabolism of ¹⁴CH₃Cl to ¹⁴CO₂ (also derived from a formate intermediate) by 19%, but had no effect on urinary metabolites derived from ¹⁴CH₃Cl. SKF-525A inhibited ¹⁴CO₂ production from ¹⁴CH₃Cl by 30% and also had no effect on urinary excretion of ¹⁴C. In contrast, pretreatment with diethylmaleate inhibited ¹⁴CH₃Cl incorporation into liver macromolecules by 70 to 85%, and lowered ¹⁴CO₂ expiration and urinary ¹⁴C excretion by 52 and 60%, respectively. S-Methylcysteine pretreatment produced a similar inhibition of ¹⁴CH₃Cl incorporation and metabolism to ¹⁴CO₂; urinary excretion of ¹⁴C, however, was approximately doubled. ¹⁴CH₃Cl uptake into liver was also stimulated by cysteine pretreatment. These results indicate a strong dependence of CH₃Cl metabolism on tissue nonprotein sulfhydryl content and suggest a possible role for cytochrome P-450 in the in vivo metabolism of CH₃Cl. A scheme for the metabolism of CH₃Cl is postulated which involves initial reaction with glutathione, and sequential metabolism of the conjugate to S-methylcysteine, methanethiol, and formaldehyde.     

l-Methionine ordinarily does not interfere with the aminopyrine breath test: Studies in dogs and rats

The influence of l-methionine on ¹⁴CO₂-exhalation resulting from administration of [¹⁴C]aminopyrine was studied in rats and dogs. l-Methionine given i.v. one hour after i.v. [¹⁴C]aminopyrine modified ¹⁴CO₂ output neither in normal rats nor in normal dogs. Only in methionine depleted rats subjected to enzyme induction by phenobarbital, and to subtoxic doses of [¹⁴C]aminopyrine, l-methionine increased ¹⁴CO₂ production. These results are consistent with the idea that excess or deficiency of l-methionine ordinarily does not interfere with the aminopyrine breath test.     

Assessment of drug metabolism in hepatic disease: Comparison of plasma kinetics of oral cyclobarbital and the intravenous aminopyrine breath test

Summary The exhalation of ¹⁴CO₂ derived from an i.v. tracer dose of [dimethylamine-¹⁴C]aminopyrine has been investigated in normal controls and patients. They subsequently ingested 200 mg cyclobarbital calcium in the evening and the decline in the plasma drug level over the following 2 days was measured by thin-layer chromatography. The peak specific activity of exhaled ¹⁴CO₂ occurred 0.5–2 h after ¹⁴C-aminopyrine injection in the absence of liver disease and in non-cirrhotic liver disorders. It was delayed in certain patients with cirrhosis. Compared to 8 medically healthy subjects, 10 patients with acute viral hepatitis, 8 with cirrhosis and 10 with fatty liver exhibited a significantly increased half-life of ¹⁴CO₂ exhalation. Normal mean values were found in 12 patients with non-cirrhotic alcoholic liver disease and in 14 patients with non-hepatic diseases. The cyclobarbital (CB) half-life was prolonged and the clearance reduced in patients with viral hepatitis, cirrhosis, or alcoholic liver damage as compared to data from 17 control subjects. Due to a larger apparent volume of distribution, patients with fatty liver disease had an increased CB half-life, although its clearance was normal. A close negative correlation was detected between the clearance and the logarithm of the CB level measured 36 h after drug ingestion. The oral CB test evaluated from a single blood sample taken about 36 h after drug administration appears to be a useful indicator of human drug metabolising capacity. Discrimination between patients with and without disordered liver function was similar in the two drug elimination tests.     

Binding of 3H-Metronidazole in Olfactory,Respiratory and Alimentary Epithelia in Rats

Abstract: Whole-body autoradiography of ³H-metronidazole in rats showed retention of bound metabolites in the epithelia lining the olfactory part of the nose, the tongue, the gingiva, the palate, the pharynx, the oesophagus and the forestomach. In vitro microautoradiography in O₂- and N₂-atmosphere with some of these tissues indicated reductive formation of bound metabolites in specific cells of the epithelia. Studies with subcellular fractions of the nasal olfactory mucosa showed formation of DNA- and protein-bound metronidazole metabolites. A lower bioactivating capacity was found in experiments with the liver. The bioactivation was dependent on N₂-atmosphere, and presence of the P450-inhibitor metyrapone or GSH in the incubation media depressed the protein-binding of metronidazole both in the nasal olfactory mucosa and the liver. These data indicate that the bioactivation is partly P450-dependent and GSH may play an important role in scavaging the bioactivated drug. The epithelial cells with a capacity to bioactivate metronidazole may be potential targets for negative effects of the drug. Whole-body autoradiography also showed a strong binding of radioactivity in the contents of caecum and colon. This can be considered to be due to reductive bioactivation of metronidazole by the intestinal microorganisms and reflects the principal site of action of the drug.     

Effect of famotidine on oxidative drug metabolism

Summary Famotidine, a new H₂-receptor antagonist was tested for drug interactions using ¹⁴C-aminopyrine and antipyrine. Elimination of these model drugs was studied before and during 8 days of famotidine dosing in 8 healthy volunteers. Famotidine 40 mg b.d. did not inhibit aminopyrine ¹⁴CO₂ half-life or antipyrine clearance although an unexpected mild enzyme inducing effect could not be excluded. It is unlikely that famotidine will inhibit hepatic drug metabolism during routine clinical use as the daily dose is expected to be 40 mg/day but interactions should be looked for if more prolonged or larger doses are used.     

Tissue specificity of N-nitrosomorpholine metabolism in Sprague-Dawley rats

The distribution and metabolism of N-nitroso[14C]morpholine ([14C]NMOR) in Sprague-Dawley rats were studied by autoradiographic and in vitro techniques. Low-temperature whole-body autoradiography indicated that the non-metabolized compound is able to pass freely through the cellular membranes and distribute evenly in most tissues of the body. Experiments in vitro showed that, in addition to the liver, the nasal mucosa had a high capacity to degrade the [14C]NMOR and a localization of metabolites was also observed in this tissue in vivo. Microautoradiography of the nasal olfactory mucosa showed the highest labelling over the subepithelial glands in the lamina propria mucosae. The nasal mucosa is, apart from the liver, the most prevalent site of NMOR carcinogenesis in the rat and the results indicate that a local bioactivation occurs in this tissue. Further experiments showed that NMOR metabolism in the nasal mucosa and the liver was inhibited by metyrapone and by a carbon monoxide atmosphere, indicating that the metabolism is cytochrome P-450 dependent. In addition to the nasal mucosa and the liver, a localization of metabolites was demonstrated in the oesophageal mucosa in vivo, while in vitro this tissue showed a capacity to degrade the [14C]NMOR. A low incidence of tumours of the oesophagus has been reported in rats treated with NMOR. It is concluded that NMOR is metabolized in the nasal and oesophageal mucosa as well as in the liver, and that local bioactivation at these sites may give rise to tumours.     

Disposition and pharmacokinetics of inhaled dimethylamine in the Fischer 344 rat

The disposition and pharmacokinetics of [14C]dimethylamine [( 14C] DMA) following 6-hr inhalation of either 10 or 175 ppm were determined in male Fischer 344 rats. Seventy-two hours after termination of exposure, the disposition of recovered radioactivity was similar for each airborne concentration, with more than 90% in the urine and feces, 7 to 8% in selected tissues and the carcass, and 1.5% exhaled as 14CO2. Over 98% of the radioactivity in the urine was unmetabolized DMA. Analysis of tissue radioactivity immediately after exposure to [14C]DMA showed that the respiratory nasal mucosa contained the highest concentration of 14C, followed by the olfactory nasal mucosa; concentrations of 14C in liver, lung, kidney, brain, and testes were approximately 2 orders of magnitude less than in the nasal mucosal tissues. Radioactivity in plasma of rats exposed by inhalation to 175 ppm of [14C]DMA decayed in a biphasic manner. The terminal half-life for plasma radioactivity was similar to the half-lives of some plasma proteins, suggesting incorporation of 14C into proteins subsequent to metabolism of [14C]DMA. The results indicate that, while most of the inhaled DMA is excreted unchanged, a small amount of oxidative metabolism of DMA occurs.     

Dose dependence of the14C-aminopyrine breath test

Summary Although the aminopyrine breath test has received much attention, the question has not yet been settled whether pharmacological or tracer doses of the drug should be used. Nine volunteers were given¹⁴C-aminopyrine 9 mg/kg or a tracer amount, in a randomized sequence and according to a crossover design. The specific activity of¹⁴CO₂ in breath was significantly different only during the first hour. Up to the 8th hour the disappearance of¹⁴CO₂ from breath was smaller after the pharmacological (28.5±SD 5.4%/h) than after the tracer dose (36.2±10.6%/h; p<0.05). The overall disappearance of¹⁴C-atoms from the subjects was significantly slower after the higher dose. In view of the smaller radiation exposure and the decreased risk of agranulocytosis, the use of a tracer dose appears preferable.Supported by the Swiss National Science Foundation     

Alteration of the inhibitory effect of metyrapone by reduction to metyrapol during the metabolism of methacetin in vivo in mice

Summary Metyrapone is known as an inhibitor of the oxidative drug metabolism in vitro. We have used the exhalation analysis as a tool to study the influence of this inhibitor on the demethylation of¹⁴C-methacetin in vivo. In parallel we investigated the reductive metabolism of metyrapone in mice by measuring the concentrations of metyrapone and its reduced metabolite metyrapol with a HPLC-method.50 mg of metyrapone/kg b. wt. resulted in a 90% inhibition of¹⁴CO₂ exhalation when given 2 min before the exhalation analysis was started. The prolongation of the intervals between i.p. metyrapone and substrate administration leads to a diminution of the in vivo inhibition. We found that the hepatic metyrapone concentration falls rapidly and passes the detection limit at 120 min. Transiently metyrapol reaches a maximal concentration 15 min after the application of metyrapone. The rapid reduction of metyrapone was confirmed in vitro with fresh mouse liver homogenates.The administration of metyrapol itself in vivo causes a decrease in¹⁴CO₂ exhalation, too. The¹⁴CO₂ exhalation curves after metyrapol correspond to the curves after metyrapone, when sufficient time was allowed for its reduction to metyrapol. It can be concluded that not only metyrapone itself but also its reductive metabolite metyrapol is an effective, however weaker inhibitor.     

Influence of pyridine and some pyridine derivatives on spectral properties of reduced microsomes and on microsomal drug metabolizing activity

Addition of pyridine, 3-(3-pyridyl)-propanol, reduced metyrapone and some other pyridine derivatives to dithionite reduced rat liver musomes results in the formation of a six banded spectrum with absorption maxima at 425, 446, 527, 539, 555 and 568 nm. With metyrapone as the ligand, a 425 nm band can only be observed during development of the spectrum. The ratio of the two Soret bands depends on the pH. The 446 nm band is maximal at pH 7·8 in phenobarbital stimulated musomes and decreases on both lowering and increasing the pH. For metyrapone the apparent spectral dissociation constant K_s for the absorbance change at 446 nm was 16 μM in musomes from untreated animals and was decreased to 2·4 μM after phenobarbital pretreatment. In 3-methylcholantrene stimulated musomes the K_s value for metyrapone was increased about 25 fold and amounted to 390 μM. The K_s value for reduced metyrapone in unstimulated musomes was similar to that for metyrapone. Less pronounced pretreatment effects were, however, observed with this ligand leading to decreased K_s values in both PB and MC stimulated musomes. Pyridine and 3-(3-pyridyl)-propanol were only bound to reduced cytochrome P-450 in millimolar concentrations. All tested ligands were able to inhibit musomal drug metabolism. Metyrapone was the most potent inhibitor of drug demethylation exerting 50 per cent inhibition of p-nitroanisole demethylation at 4 μM and of aminopyrine demethylation at 86 μM. Aniline hydroxylation was inhibited by 50 per cent by millimolar concentrations of all inhibitors at aniline concentrations of 4 mM.     

Uptake and metabolic rate of liposome-entrapped [U-14C]glutamate in rats

The metabolic fate of [U-¹⁴C]glutamate administered intravenously to rats in aqueous solutions or entrapped in vescicles of phospholipids extracted from rat brain has been studied. When [U-¹⁴C]glutamate was administered entrapped in liposomes, higher serum radioactivity levels and higher radioactivity uptake by liver and brain were observed. Rats treated with [U-¹⁴C]glutamate entrapped in liposomes exhibited an increased glutamate oxidation shown by increased expired [¹⁴C]CO₂ and a modified elimination of the radioactivity in urine.     

Extrahepatic sites of metabolism of N-nitrosopyrrolidine in mice and rats

1. By the use of whole-body autoradiography, the localization of N-nitroso[¹⁴C]pyrrolidine and its metabolites was demonstrated in the tissues of C57B1-mice and Sprague—Dawley rats.

2. On the basis of the autoradiorgraphic data, tissues were selected and tested for their ability to metabolize N-nitroso[¹⁴C]pyrrolidine to ¹⁴CO₂ and tissue-bound metabolites.

3. The results indicated that only a few tissues had a marked ability to degrade N-nitrosopyrrolidine. These were the liver and the nasal mucosa in both species, the tracheo-bronchial mucosa in mice, and to a lower extent the kidneys in both species.     

Kinetic alterations in rat liver microsomal cholecalciferol 25-hydroxylase associated with phenobarbital administration

The kinetics of cholecalciferol 25-hydroxylase in vitamin D-depleted rat liver microsomes, before and after phenobarbital induction, were studied. Three days of pretreatment with phenobarbital altered significantly both the apparent K_m and the V_max of the hydroxylase. Untreated vitamin D-repleted rats had lower cytochrome P-450 content and aminopyrine demethylase activity than the vitamin D-depleted animals. Phenobarbital administration reversed this nutritional effect on aminopyrine demethylase but not on cytochrome P-450 content. Furthermore, vitamin D deficiency potentiated the phenobarbital inductive effect upon microsomal protein. No inhibition of aminopyrine demethylase could be elicited in the presence of cholecalciferol or 25-hydroxycholecalciferol either prior to or after phenobarbital treatment, suggesting that these two oxidases are different entities.     

Effects of Aroclor 1254 on disposition and hepatotoxicity of ethylene in the rat

The purpose of this study was to investigate the disposition of ethylene (C₂H₄) in the F344 rat and to determine the effects of pretreatment of animals with Aroclor 1254 on the disposition and hepatotoxicity of ethylene. Control or Aroclor 1254-pretreated (500 mg/kg, 5 days before exposure) male rats were exposed to ¹⁴C₂H₄ (10,000 ppm, 0.04–0.11 μCi/μmol) for 5 hr. Urine, feces, expired CO₂, and expired ¹⁴C₂H₄ were collected for 36 hr following the exposure. Aroclor pretreatment did not affect the amount of C₂H₄ expired (17–18 μmol/rat) but did cause a 4-fold increase in expired ¹⁴CO₂ and a doubled urinary excretion of radioactivity. In control animals the blood, gut, kidney, liver, and lung all had detectable ¹⁴C concentrations (20, 83, 14, 77, and 6 nmol-eq of C₂H₄, respectively) 36 hr after exposure. Aroclor pretreatment increased the concentrations in these tissues by factors of 1.5-, 8-, 6-, 17-, and 6-fold, respectively. In contrast to control rats. Aroclor-pretreated animals had detectable concentrations of radioactivity in brain, fat, heart, and muscle. Rats exposed to 10,000 ppm C₂H₄ exhibited no light microscopic evidence of hepatotoxicity. However, Aroclor-pretreated animals had centrolobular hepatic necrosis as assessed microscopically 36 hr after exposure to C₂H₄. These results demonstrate that the metabolism of C₂H₄ is stimulated by Aroclor pretreatment, and that the hepatic necrosis seen in Aroclor-pretreated rats is the result of a greater amount of metabolism or the production of a metabolite(s) not seen in control animals.