Rolf Blomstrand

Inhibitory effect on ethanol oxidation in man after administration of 4-methylpyrazole

Abstract The effect of 4-methylpyrazole on the metabolism of ethanol has been examined in 5 healthy male volunteers after oral administration of 5 μC ethanol-1- 14 C with a carrier dose of 9 g ethanol and measuring the 14 CO 2 excreted in the expired air. The degree of inhibition reached a maximum 1.5 – 2 hours after oral administration of 4-methylpyrazole and then the inhibitory effect decreased slowly. Thus 1000 mg of methylpyrazole inhibited the elimination of 14 CO 2 in expired with 49.6%, 1.5 hours after oral administration of the compound compared to the control experiment. The inhibitory effect decreased slowly to 28 per cent 6 hours after administration of methylpyrazole. The results of this investigation indicate a dose-response relationship between degree of inhibition and dose of 4-methylpyrazole. The results also suggest an individual response to 4-methylpyrazole. Experimental data with larger doses of alcohol to alcoholics suggest that 4-methylpyrazole acts as a competitive LADH-inhibitor. The data obtained in alcoholic volunteers suggest that 4-methylpyrazole might have an inhibitory effect on the ethanol induced rise in lactate/pyruvate ratio in blood.

Serum cholesterol determination by mass fragmentography

Abstract A highly sensitive and accurate reference method for estimation of total cholesterol in serum is described. A fixed amount of [2,2,3,4- 2 H 4 ] cholesterol is added to a fixed amount of serum (usually corresponding to 10 μl). After saponification and extraction with hexane, the amount of unlabelled cholesterol is determined from the ratio between the recordings at m / e 386 and m / e 389 obtained after analysis with a mass spectrometer equipped with an MID (multiple ion detector) unit. The relative standard deviation of the method is about 1.3%. The method was compared with the Liebermann—Burchard method using sera with a range of cholesterol levels. The method can be used for determination of cholesterol down to a level of 10 pmoles (4 ng).

Studies on the Intestinal Absorption of Radioactive β-Carotene and Vitamin A in Man: Conversion of β-carotene into vitamin A

β-carotene-15, 151-14C, β-carotene-15, 151-3H, vitamin A alcohol-15-14C, and vitamin A-15-3H acetate were fed to eight patients in whom polyethylene cannulae had been inserted in the thoracic duct in the neck. The lymph lipids were extracted and chromatographed on columns and on thin-layer plates of alumina. Only 8.7-16.8 per cent of the fed labelled β-carotene is normally absorbed via the lymphatic pathway and mainly recovered as retinyl esters. Unchanged labelled β-carotene comprised only 1.7 to 27.9 per cent of the radioactivity after ingestion of labelled β-carotene.The recovery of the radioactivity in the lymph lipids after feeding labelled vitamin A alcohol or acetate was in the range of 6.7 to 66.9 per cent. Absorption of radioactivity into the lymph followed the appearance of the chylomicron triglycerides.Labelled retinyl esters, mainly retinyl palmitate, represented 75 to 90 per cent of the absorbed radioactivity after feeding labelled vitamin A, and 60 to 75 per cent after feeding labelled β-car...

Studies on the metabolic interactions between 4-methylpyrazole and methanol using the monkey as an animal model☆☆☆

Abstract Young cynomolgus monkeys, Macaca fascicularis , were chosen as a model to investigate the metabolism of 4-methylpyrazole and its interaction with methanol. Following a single dose of 4-methylpyrazole, the elimination of 4-methylpyrazole from the plasma followed a linear course; at a dose of 50 mg/kg (424 μmol/kg) the constant rate of elimination of 4-methylpyrazole was approximately 13 μmol/kg/h with disappearance of the dose within 35 h. This rate of elimination was slower when the smaller dose of 20 mg 4-methylpyrazole was administered. The primary metabolite of 4-methylpyrazole, 4-hydroxymethylpyrazole, accumulated to levels at most 10% of those of 4-methylpyrazole and did not persist in the plasma after the elimination of 4-methylpyrazole. The concurrent administration of methanol with 4-methylpyrazole inhibited the disappearance of 4-methylpyrazole from the plasma about 25%, which suggested an interaction between the metabolism of the two compounds. As expected from its potent inhibitory effect on alcohol dehydrogenase, 4-methylpyrazole produced a profound and dose-dependent inhibition of the rate of elimination of methanol from the plasma. In addition, 4-methylpyrazole at a plasma level of greater than 10 μ m was capable of preventing the accumulation of formic acid in methanol poisoned monkeys. In such a way 4-methylpyrazole repeatedly injected in various dose combinations was able to reverse methanol toxicity in the monkey. These studies indicated the usefulness of the monkey as a model to study 4-methylpyrazole activity and toxicity in light of its possible use to treat methanol poisoning in man.

High performance liquid chromatography and glass capillary gas chromatography of geometric and positional isomers of long chain monounsaturated fatty acids.

Positional and geometrical isomers of monounsaturated long chain fatty acids were analyzed by the combination of high performance liquid chromatography (HPLC) and glass capillary gas chromatography (GC). A preparative group separation ofcis andtrans isomers of the monounsaturated fatty acid methyl esters was achieved according to chain length by reversed-phase HPLC, and using a highly sensitive interference refractive index detector. After collection of the different fractions containingcis andtrans forms of the monounsaturated fatty acid methyl esters, the fractions were analyzed for their content of positional isomers using glass capillary GC with Silar-5 CP as stationary phase. The preparative step in the HPLC was also used analytically for the determination of the ratio between thecis andtrans monounsaturated fatty acids. A comparison was made between the results obtained with the HPLC technique and the results of a GLC technique with a packed OV-275 column. There was a good correlation between the 2 techniques with a tendency to highertrans values with the HPLC technique (4%). It was shown with reference substances that 18∶1ω6-cis to ω11-cis and 18∶1ω5-trans to ω12-trans, the most common monounsaturated fatty acid isomers in partially hydrogenated vegetable oils, could be almost quantitatively recovered in the HPLC step. Most of the individual positional isomers of monounsaturated fatty acids of varying chain length could be separated and determined in the glass capillary GC step with the exception of those isomers containing the double bond in a relatively high ω-position. The relative standard deviation of the technique as determined with reference substances was better than 4%. The described technique was applied to the analysis of the isomeric monounsaturated fatty acid content in partially hydrogenated vegetable and marine oils, and about 5 samples a day could be executed.

Studies on phospholipids with particular reference to cardiolipin of rat heart after feeding rapeseed oil

The influence of dietary rapeseed oil on the lipid classes and fatty acid pattern of rat heart homogenate and mitochondria has been investigated after feeding a diet with 9.8% by wt erucic acid for 10 days and 1.4% and 2.6% erucic acid for 28 days. The rats treated with 9.8% erucic acid showed a significant increase in the triglycerides of the heart mitochondria. This tendency was much less pronounced in rats treated with 1.4 and 2.6% erucic acid, respectively. In all experiments, the triglycerides of the heart mitochondria showed a high content of erucic acid. The fatty acids of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin were all influenced by the dietary rapeseed oil, but the erucic acid seemed to have a specific affinity to cardiolipin. Cardiolipin of rat heart mitochondria was isolated and identified with gas chromatography and mass spectrometry. The isolated cardiolipin was found to contain 12% erucic acid after feeding 9.8% erucic acid as rapeseed oil for 10 days. Similar results were obtained after feeding glyceryl trierucate for 5 days to rats. The incorporation of erucic acid into cardiolipin was followed by a corresponding decrease of linoleic acid. This observation is of great interest because the molecular structure of fatty acids in lipid molecules has a profound influence on the packing of these molecules in a bilayer. Since cardiolipin is a component of the inner membrane of mitochondria its high affinity for erucic acid might influence the normal function of the inner membrane of heart mitochondria.

Biological effects and metabolic interactions after chronic and acute administration of 4-methylpyrazole and ethanol to rats☆☆☆

Abstract 4-Methylpyrazole in a dose producing an inhibition of alcohol dehydrogenase of about 60% was given alone or in combination with ethanol (10%) as sole drinking fluid to growing rats in periods up to 38 weeks. No effects were observed on the weight curves. Hematologic analyses showed normal values for blood and bone marrow. Studies of liver function with transaminase, bilirubin and albumin did not reveal any functional changes. Kidney function was normal as judged by creatinine and normal electrolytes. Electronmicroscopy of liver, kidney, and heart did not reveal any changes related to treatment. Combined treatment of ethanol and 4-methylpyrazole caused an increase of the microsomal drug-metabolizing activity. Chronic administration of ethanol and 4-methylpyrazole indicated that there is a mutual interaction in the metabolism of ethanol and 4-methylpyrazole, leading to a higher concentration of both ethanol and 4-methylpyrazole in the blood. Acute experiments, where alcohol dehydrogenase is saturated with ethanol, indicated a much slower elimination of 4-methylpyrazole. Administration of ethanol and 4-methylpyrazole in acute experiments showed a lower concentration of 4-hydroxymethylpyrazole in the blood indicating that ethanol interferes with the 4-methylpyrazole- and/or 4-hydroxymethyl-pyrazole-metabolizing enzymes. The present investigation has shown that the acute and chronic toxicity of 4-methylpyrazole alone or in combination with ethanol is minimal at doses that are effective in blocking ethanol metabolism in the rat. Because of its low toxicity and powerful inhibitory capacity, 4-methylpyrazole should be a potential tool for experimental clinical investigation of alcohol metabolism and its effects. 4-Methylpyrazole is also a potential therapeutic agent in methanol or ethylene glycol poisoning.

The effects of partially hydrogenated marine oils on the mitochondrial function and membrane phospholipid fatty acids in rat heart

The influence of dietary partially hydrogenated marine oils containing docosenoic acid on rat heart mitochondrial membrane phospholipid fatty acid composition was studied with particular reference to cardiolipin and oxidative phosphorylation. Five groups of male weanling rats were fed diets containing 20% (w/w) peanut oil (PO), partially hydrogenated peanut oil (HPO), partially hydrogenated Norwegian capelin oil (HCO), partially hydrogenated herring oil (HHO), and rapeseed oil (RSO) for 10 weeks. All the cardiac phospholipids investigated were influenced by the experimental diets. An increased amount of arachidonic acid observed in phosphatidylethanolamine (PE) after feeding partially hydrogenated oils suggests a changed regulation of the arachidonic acid metabolism in comparison with PO treatment. 22∶1 originating from the dietary oils was incorporated only to a small extent into phosphatidylcholine (PC) and PE. A selective incorporation of 18∶1 isomers into the 1- and 2-positions of PC and PE with respect to geometry and position of the double bond was observed. Large amounts of 18∶1trans were incorporated into the 1-position of PC and PE, irrespective of the amount of 18∶2 supplemented to the diets, replacing a considerable proportion of stearic acid in this position. After feeding HHO and RSO, the content of 22∶1 in mitochondrial cardiolipin of rat heart was found to be 3% (mainly cetoleic acid) and 10% (mainly erucic acid), respectively, indicating a high affinity forcis isomers of 22∶1, but also a considerable resistance against incorporation oftrans isomers was observed. The ability of rat cardiac mitochondria to oxidize palmitoylcarnitine and to synthesize ATP was depressed after feeding HHO and RSO. Dietarycis isomers of 22∶1 seem to have a specific ability to interfere with cardiac ATP synthesis and also to alter the fatty acid composition of cardiolipin of rat heart.

Plasma cortisol determination by mass fragmentography

Abstract A reference method for the estimation of plasma cortisol with high accuracy and specificity is described. A fixed amount of [4- 14 C] cortisol (usually 0.2 μg) is added to a fixed amount of plasma (usually 2 ml) and the mixture is extracted with dichloromethane. The extract is treated with methoxylamine hydrochloride and trimethylsilylimidazol to yield the dimethoxime-tri-trimethylsilyl derivative of cortisol. The amount of unlabeled cortisol is determined from the ratio between the recordings at m / e 605 and m / e 607 obtained after analysis with a combined gas Chromatograph—mass spectrometer equipped with an MID unit (multiple ion detector). The two ions used correspond to the M-31 peak in the mass spectrum of unlabeled and (4- 14 C) labeled cortisol, respectively. The relative standard deviation of the method is about 3.5%. The method was compared with a fluorometric method and a competitive protein binding method used in routine analysis. The results indicate that the competitive protein binding technique is to be preferred in routine work.

The combustion of triolein-1-14C and its inhibition by alcohol in man

Abstract In the present investigation monitoring of expired 14CO2 in man has been used in order to determine the immediate effect of alcohol on the formation of 14CO2 from 14C-labelled oleic acid administrated as triolein. From the output of 14CO2 a picture of the rate of oxidation of the labelled substrate could be obtained. Labelled triolein was given perorally or intravenously to male volunteers, and the excretion of 14CO2 in expired air was registered. The same experiment was repeated in the next day, but, in addition, alcohol was given perorally or intravenously. There was then a significant depression of the 14CO2 excretion. In the same volunteers 4-methylpyrazole, administered intravenously, prevented to a variable extent the inhibitory effect of alcohol on the fatty acid oxidation. The results suggest that ethanol inhibits β-oxidation of the fatty acids in the liver in man.