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The metabolism of ethane and pentane in man is demonstrated to occur from the uptake of an enriched atmosphere of these gases in a rebreathe spirometer circuit. Dithiocarb, an inhibitor of alkane metabolism, reduced uptake and increased the respiratory excretion of these gases. This effect was least marked for the slowly metabolised ethane. Therefore the endogenous production of ethane as measured by respiratory excretion is less affected. However pentane is rapidly metabolised and this limits the use of simple respiratory excretion of pentane as a measure of in vivo lipid peroxidation.

Sprague-Dawley male rats were treated with either 100 mg/kg Nifurtimox or Benznidazole p.o. and pentane evolution was measured at different periods of time. No significant increase in pentane evolution was observed in animals treated with Benznidazole during periods of time up to 10 h. In animals treated with Nifurtimox, a significant increase in pentane evolution was observed at 10 h but not at 3 or 6 h. The pentane evolution effect of Nifurtimox was compared to that of carbon tetrachloride. The latter was very intense up to 1 h and ceased thereafter. The possible participation of lipid peroxidation in the unwanted toxic side effects of Nifurtimox and Benznidazole is discussed.

The effect of a single dose of ethanol on lipid peroxidation in three groups of rats fed different amounts of vitamin E was determined by the measurement of pentane in the breath. All rats had increased pentane production above basal levels by 15 min following oral administration of 6 g ethanol/kg body wt. The increase in total pentane production during a 13-hr test period after intragastric administration of ethanol was greater in the rats fed the vitamin E-deficient diet than in the rats fed vitamin E-supplemented diets (alpha = 2P = 0.02). The results support the hypothesis that acute ethanol toxicity involves lipid peroxidation and further demonstrate the usefulness in toxicological studies of monitoring pentane as an index of lipid peroxidation in vivo.

Primary rat hepatocyte suspension cultures (approximately 2 X 10(6) cells) exposed to solubilized 2,3,4-trimethylpentane at concentrations ranging from 7.9 to 31.5 mM under two different culture conditions resulted in a linear dose response, as determined by lactate dehydrogenase leakage and viability data. A significant increase in the 2,3,4-trimethylpentane effective concentration 50 for primary hepatocytes occurred when exposures were implemented in medium containing 0.05% albumin. The effective concentration 50 for hepatocytes exposed to 2,3,4-trimethylpentane in medium lacking and containing albumin were 17.1 and 20.7 mM, respectively. Metabolite analysis by gas chromatography-mass spectrometry of supernatant (lacking or containing albumin) and cell extracts from hepatocyte cultures exposed to 2,3,4-trimethylpentane for 4 h indicated the presence of three metabolites: 2,3,4-trimethyl-1-pentanol, 2,3,4-trimethyl-2-pentanol, 2,3,4-trimethyl-2-pentanol, and 2,3,4-trimethyl-1-pentanoic acid. Electron microscopic examination of 2,3,4-trimethylpentane-exposed primary hepatocytes indicated ultrastructural changes which included abnormal condensed chromatin association with the nuclear membrane, swollen mitochondria, increased amounts of cytoplasmic lipid, significant loss of microvilli from the cell surface, increased vacuolation, and increased numbers of peroxisomes. Although these changes were observed under both culture conditions, they were more severe in cultures lacking albumin. This study indicates that primary hepatocyte suspension cultures provide a useful system for rapidly identifying liver metabolites of selected test compounds of interest.

Measurements of pentane and ethane as indices of in vivo lipid peroxidation were made on samples of breath from vitamin C-sufficient and vitamin C-deficient guinea pigs injected with 23 microliters carbon tetrachloride (CCl4)/100 g body wt. Vitamin C-deficient animals produced significantly more pentane and ethane after CCl4 treatment than did vitamin C-sufficient guinea pigs. Pretreatment of vitamin C-deficient animals with 75 mg ascorbic acid/100 g body wt significantly lowered both pentane and ethane evolution. Protection against in vivo lipid peroxidation similar to that provided by ascorbic acid was also found when vitamin C-deficient guinea pigs were pretreated with isoascorbic acid, reduced glutathione, alpha-tocopherol or beta-carotene. When animals were pretreated with the radical scavenger mannitol, a protective effect was also observed as measured by pentane evolution.

Indirect evidence has suggested that lipid peroxidation is associated with iron overload in vivo. As a measure of lipid peroxidation, pentane expired in the breath of rats loaded with an accumulated dose of either 100 mg or 186-200 mg of iron injected intraperitoneally as iron dextran was measured over a 7 to 8 week period, and the effect on pentane production of feeding antioxidant-supplemented diets was determined. By the seventh week of feeding the diets, rats fed 0.3% L-ascorbic acid produced 17% less (P = 0.03) pentane than did rats fed the basal antioxidant-deficient diet, whereas rats fed 0.004% dl-alpha-tocopherol acetate produced 92% less (P less than 0.001). After being fed the basal diet for 7 weeks, iron-loaded rats produced 76 +/- 9 pmol pentane/100 g body wt/min. When synthetic antioxidants were added to the diet at a concentration of 0.25%, the order of effectiveness in decreasing pentane production after 1 week was: N,N'-diphenyl-p-phenylenediamine greater than ethoxyquin greater than butylated hydroxyanisole greater than butylated hydroxytoluene greater than propyl gallate approximately equal to no antioxidant. After removal of either ethoxyquin or N,N'-diphenyl-p-phenylenediamine from the diets for 1 week, pentane production increased to a high level. The total amount of lipid soluble fluorophores in individual spleens of rats fed N,N'-diphenyl-p-phenylenediamine, ethoxyquin, dl-alpha- tocopherol acetate, ascorbic acid and no antioxidant were correlated significantly with the corresponding total integrated amount of pentane produced by the individual rats over the 7 to 8 week period. This study has provided some of the most direct evidence to date that lipid peroxidation is associated with iron overload in vivo.

Oxidant damage to red cell membranes could play a part in the pathogenesis of acute and chronic haemolysis in glucose-6-phosphate dehydrogenase deficiency. Therefore, we studied the substrate for red cell membrane lipid peroxidation, i.e. the content of various polyunsaturated fatty acids in ghosts, and the susceptibility of red cells to lipid peroxidation in normal subjects and in subjects deficient in glucose-6-phosphate dehydrogenase. The fatty acid composition of red cell membranes and plasma was analysed by capillary column gas chromatography. The sensitivity of red cells to lipid peroxidation was evaluated after hydrogen-peroxide-induced oxidant stress. The degree of lipid peroxidation was monitored by measuring the release of pentane and ethane formed during the breakdown of n-6 and n-3 fatty acids. The red cell sensitivity to lipid peroxidation was found to be higher in subjects with glucose-6-phosphate dehydrogenase deficiency than in normal subjects. In the former, saturated fatty acids, in particular palmitic and stearic acid, were found to be decreased, whereas the proportion of arachidonic acid showed a clear increase. Fatty acid analysis of plasma did not reveal significant abnormalities in enzyme-deficient patients, which could explain the alteration of membrane fatty acids. Our results suggest that the increased content of substrate for lipid peroxidation, particularly arachidonic acid, in red cell membranes of subjects deficient in glucose-6-phosphate dehydrogenase, should be considered in an evaluation of an enhanced sensitivity to red cell lipid peroxidation.

Weanling rats were fed one of 3 diets containing 0, 11 or 200 international units (IU) dl-alpha-tocopherol acetate/kg diet for 4 weeks. Following this period, the drinking water was replaced with an 18% solution of ethanol (v/v). An isocaloric D-glucose solution was substituted for the drinking water of a control group of rats fed the vitamin-E-deficient diet for 4 weeks. The 4 treatment groups were maintained on the diet and drinking regimen for 20 weeks. Basal levels of expired pentane were determined at weeks 0, 1, 3, 5, 7 and 9. Chronic ethanol consumption did not influence basal pentane production during the 9-week treatment. Basal levels of expired pentane were affected by dietary vitamin E. Rats supplemented with vitamin E had basal pentane levels less than one-half of the level of rats fed a vitamin-E-deficient diet (p less than 0.001). After 14 weeks of treatment, the 2 groups of rats fed a vitamin-E-deficient diet were administrated p.o. an acute dose of 6 g of ethanol/kg body wt. Pentane expired above basal levels during the following 4-hr period correlated with the amount of hepatic triglycerides determined at the conclusion of the experiment. The etiology of ethanol toxicity is a complex and multifactorial system made up of many biological variables that influence lipid peroxidation. The appropriate choices of experimental designs and methods are important in examining the role of lipid peroxidation.

When we incubated rat liver microsomes with ferrous ions and an NADPH-regenerating system, ethane and n-pentane formation increased correspondingly with decreasing concentrations of oxygen in the atmosphere above the incubation, whereas malondialdehyde increased with increasing oxygen concentrations up to a plateau. At very low oxygen concentrations - 100% helium as atmosphere, but presumably traces of oxygen were present in the microsomes - ethane and n-pentane formation were maximal and dependent on the concentrations of ferrous ions, in the case of ethane, a peak being reached at about 20 microM Fe2+, whereas n-pentane continuously increased with increasing concentrations of Fe2+. It is suggested that the inverse relationship of ethane or n-pentane and malondialdehyde is due to two different reaction sequences of microsomal lipid peroxidation with different oxygen sensitivities.

Pentane is one decomposition product of omega 6-unsaturated lipid hydroperoxides. The measurement of respiratory pentane is one of the most sensitive in vivo tests of lipid peroxidation. This measurement was applied to test the ability of a low level, short-term exposure of rats of nitrogen dioxide to induce lipid peroxidation. When exposed to 4.48 ppm nitrogen dioxide for 60 min, no increase in pentane production was detected. For 10 weeks prior to exposure, the rats were fed Torula yeast-based diets with 10% stripped lard or 10% stripped corn oil. The ratios of basal pentane production by rats fed the following antioxidants were for corn oil- and lard-fed rats, respectively: 40 I.U. vitamin E/kg and 0 selenium: 0 vitamin E and 0.1 ppm selenium: 0 vitamin E and 0 selenium, 1:2.2:5.8 and 1:1.9:3.5. Pentane production was significantly (P<0.05) greater by corn oil-fed rats than by lard-fed rats only when both vitamin E and selenium were absent from the diet.

Incubation of rat hepatocytes with 14 mM dimethyl sulfoxide (DMSO) produced an increase in the formation of ethane, measured by capillary column gas chromatography, to 18.0 pmoles/hr/10(7) cells from 11.2 pmoles/hr/1-(7) cells from 5.6 pmoles/hr/10(7) cells in control hepatocytes. This was about one-third the stimulation of ethane and n-pentane formation produced by incubation of hepatocytes with 13 mM carbon tetrachloride. DMSO-stimulated ethane and n-pentane formation was inhibited up to 63% by 0.1 microM alpha-tocopherol and up to 89% by N2. Formation of dimethylsulfide from DMSO by hepatocytes was the same in air and N2. DMSO increased methane production by hepatocytes to 31.3 pmoles/hr/10(7) cells from 6.9 pmoles/hr/10(7) cells in control hepatocytes. Although DMSO apparently stimulated lipid peroxidation by hepatocytes, as measured by ethane and n-pentane formation, there was no increase in the formation of thiobarbituric acid reactive material. DMSO was not toxic to hepatocytes, measured by release of cytosolic lactate dehydrogenase, over a 2-hr incubation. Possible mechanisms for the increase in alkane formation by DMSO are discussed.

Pentane measurement in exhaled gases was proposed as a safe method to evaluate the importance of lipoperoxidation in vivo, in man and in animals. However we have observed that pentane, which arises from lipoperoxide decomposition, is significantly consumed by cytochrome P-450-rich liver microsomes. This pentane consumption is completely inhibited after heating the microsomes at 100 degrees C, and is considerably reduced by metyrapone, a cytochrome P-450 inhibitor. Hepatic metabolism of pentane, particularly after cytochrome P-450 induction, constitutes a risk of error, when pentane exhalation is taken as an index of lipoperoxidation in vivo.

Red blood cells from 31 healthy donors were examined for the cholesterol content, the fatty acid composition, and the susceptibility to lipid peroxidation induced by either hydrogen peroxide or phenylhydrazine. Lipid peroxidation was monitored by the release of pentane and ethane. In addition, plasma fatty acids were measured in order to find out, whether plasma and red cell fatty acids were correlated. In experiments with hydrogen peroxide, a significant positive correlation was found between the proportion of arachidonic acid (C 20:4n - 6; r = 0.57, p less than 0.01) and docosahexaenoic acid (C 22:6n - 3; r = +0.71, p less than 0.01), and the release of pentane and ethane, respectively. A significant negative correlation was found between the membrane cholesterol content and the pentane release (r -0.44, p less than 0.05). In experiments performed with phenylhydrazine, red cell membrane lipid composition did not influence the susceptibility of red cells to lipid peroxidation. A close correlation was found between plasma and red cell fatty acids (palmitic acid, r = +0.46, p less than 0.01; linoleic acid, r = +0.41, p less than 0.05; arachidonic acid, r = +0.59, p less than 0.01; docosahexaenoic acid, r = +0.67, p less than 0.01). The results demonstrated that the degree of peroxide-induced oxidation of erythrocyte lipids depends on the content of polyunsaturated fatty acids in the membrane, which on the other hand, is determined by plasma fatty acids. It is suggested that dietary variations may influence the susceptibility of red cells to lipid peroxidation.

The rate of lipid peroxidation, as determined by expired air pentane and ethane measurements, was assessed in suckled or fasted newborn rats exposed to air or hyperoxia (FIO2 greater than 0.95). Pentane and ethane production in suckled newborn rats did not significantly change with exposure to hyperoxia after birth. Pentane production averaged over a 3-day exposure period was 2.8 pmol/100 g/min in hyperoxia versus 2.5 pmol/100 g/min in air. However, significant increases in ethane production occurred in fasted newborn rats over the first 18 h of life (less than 2.5 pmol/100 g/min increasing to greater than 6 pmol/100 g/min), which were not observed in suckled animals. Although hyperoxia did not cause an increase in pentane or ethane production above air-exposed controls, nutritional deprivation in newborn rats appeared to accelerate lipid peroxidation events and resulted in high mortality in newborn rats exposed to hyperoxia.

A method for determining the uptake of inhaled vapors in rats is described. The method uses gas chromatographic data to enable calculations of vapor concentration differences upstream and downstream from a rat exposed to the vapor by the nose-only route. Respiratory data are recorded simultaneously. The data can be used to determine the rate of uptake, uptake as a fraction of vapor inhaled and the effects of vapor inhalation on respiration.

Some of the vitamins and many of the metals that are being considered at this symposium interact in lipid peroxidation. Some of these interactions have been studied in vivo. Measurement of in vivo lipid peroxidation in the rat is accomplished by gas chromatographic analysis of pentane, a minor peroxidation product that is exhaled in the breath. In the rat, lipid peroxidation is proportional to dietary polyunsaturated lipids when the animal is deficient in antioxygenic agents. The chain-breaking antioxidant vitamin E is the main protector against in vivo lipid peroxidation. Dietary selenium, through its involvement in the biosynthesis of glutathione peroxidase, functions in a secondary antioxygenic role as a hydroperoxide reducer. In rats fed a vitamin E-deficient diet, injection of some hydroperoxides, iron, or vitamin C leads to initiation of in vivo lipid peroxidation, apparently by decomposing hydroperoxides to free radicals. Carbon tetrachloride, a toxic halogenated hydrocarbon, is metabolized by liver microsomes and initiates in vivo lipid peroxidation in the liver. These examples show that practical information on interactions involving in vivo lipid peroxidation can be obtained by studies that use the pentane method.

The effect of in vivo lipid peroxidation on the excretion of immunoreactive prostaglandin E2 (PGE2) in the urine of rats was studied. Weanling, male Sprague-Dawley rats were fed a vitamin E-deficient diet containing 10% tocopherol-stripped corn oil (CO) or 5% cod liver oil (CLO) with or without 40 mg dl-alpha-tocopheryl acetate/kg. To induce a high, sustained level of lipid peroxidation, some rats were injected intraperitoneally with 100 mg of iron as iron dextran after 10 days of feeding. Iron overload stimulated in vivo lipid peroxidation in rats, as measured by the increase in expired ethane and pentane. Dietary vitamin E reversed this effect. Rats fed the CLO diet excreted 9.5-fold more urinary thiobarbituric acid-reactive substances (TBARS) than did rats fed the CO diet. Iron overload increased the excretion of TBARS in the urine of rats fed the CO diet, but not in urine of rats fed the CLO diet. Dietary vitamin E decreased TBARS in the urine of rats fed either the CO or the CLO diet. Iron overload decreased by 40% the urinary excretion of PGE2 by rats fed the CO diet, and dietary vitamin E did not reverse this effect. Iron overload had no statistically significant effect on urinary excretion of PGE2 by rats fed the CLO diet. A high level of lipid peroxidation occurred in iron-treated rats, as evidenced by an increase in alkane production and in TBARS in urine in this study, and by an increase in alkane production by slices of kidney from iron-treated rats in a previous study [V. C. Gavino, C. J. Dillard, and A. L. Tappel (1984) Arch. Biochem. Biophys. 233, 741-747]. Since PGE2 excretion in urine was not correlated with these effects, lipid peroxidation appears not to be a major factor in renal PGE2 flux.

Measurements were made of pentane, a produce of lipid peroxidation, and chloroform (CHCl3), a product of the reproductive metabolism of carbon tetrachloride (CCl4), expired by rats administered CCl4 plus either phenobarbital (PB), 2-diethylaminoethyl-2,2-diphenyl valerate hydrochloride (SKF-525A), or diethyl maleate (DEM). In rats fed a standard laboratory diet, treatment with PB prior to injection of CCl4 increased expiration of both CHCl3 and pentane. SKF-525A pretreatment decreased the metabolism of CCl4 and CCl4-induced lipid peroxidation. DEM, a depleter of intracellular glutathione, increased lipid peroxidation. In rats fed 10% corn oil diets that contained either 0 or 40 IU of dl-alpha-tocopherol acetate, oxidant stresses caused by glutathione depletion and CCl4-intoxication were found to be additive.

Expired pentane, a product of omega 6-unsaturated fatty acid hydroperoxide decomposition, and foot volume were measured following injection of Freund's adjuvant into the hind feet of vitamin E-deficient rats injected 1 wk previously with either 0 or 100 mg of dl-alpha-tocopherol acetate/100 g body weight. Vitamin E inhibited pentane production after 21 and 28 days. This is the first demonstration in vivo of increased lipid peroxidation and its inhibition by vitamin E during adjuvant-induced chronic inflammation. By 28 days, foot volume was significantly less in vitamin E-injected rats (162%) than in vitamin E-deficient rats (207%).

Effects of two halogenated acetamides (chloroacetamide-iodoacetamide) and of three chlorinated volatile solvents on the ethane-pentane test, as an index of in vivo lipid peroxidation have been studied. Chloroacetamide stimulates alcanes production. Iodoacetamide acts only on pentane exhalation. The three chlorinated solvents seem to induce moderate lipoperoxidation, and the apparent increase of hydrocarbons might also result from decreased metabolism.

Ethane and pentane are evolved during peroxidation of tissue lipids in vivo and are believed to be derived from omega3 and omega6 fatty acids respectively. We present evidence supporting a scission mechanism as the means of forming these hydrocarbons during hydroperoxide decomposition. A fatty acid omega6 hydroperoxide (methyl 13-hydroperoxy-6,9,11-octatrienoate), when incubated with a ferrous ion, yielded pentane as the major hydrocarbon (98%). Reaction with ferrous ion results in an alkoxy capable of undergoing scission to an aldehydic fatty acid and a hydrocarbon.

Pentane was measured in a mixture of guinea pig liver microsomes and carbontetrachloride (CCl4) in the presence of nicotineamide adenine dinucleotide phosphate (NADPH) and under anaerobic conditions by gas chromatography. Pentane of 0.2 pmol/mg protein/min was formed in the presence of NADPH without CCl4. Pentane formation increased to 2.0 pmol/mg protein/min in the presence of CCl4 and NADPH. This reaction required an anaerobic atmosphere, 2.1 mmol/litter of NADPH and 13.4 mmol/litter of CCl4 under optimal conditions. The formation was reduced in the presence of oxygen, glutathione, vitamin E and metyrapone. These results clearly show that pentane is formed by lipid peroxidation initiated by the free radical cleavage products of CCl4, which are anaerobically produced by NADPH-dependent microsomal enzymes. We conclude that pentane is a good index of in vitro lipid peroxidation via microsomal NADPH-P-450 enzyme systems.