Al L. Tappel

Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes

Liver slices were used to measure lipid peroxidation induced by bromotrichloromethane, tert-butyl hydroperoxide (t-BOOH), or ferrous iron. The responses of liver homogenates and microsomes to oxidative conditions were compared with the response of tissue slices. Lipid peroxidation was evaluated by the production of thiobarbituric acid-reactive substances (TBARS). As was observed in homogenates and microsomes, TBARS production by liver slices depended upon the amount of tissue, the incubation time, inducer, the amount of inducer, and the presence of antioxidant. Control liver slices incubated at 37 degrees C for 2 h produced 19 nmol of TBARS per g of liver. When slices were incubated in the presence of 1 mM BrCCl3, 1 mM t-BOOH, or 50 microM ferrous iron, TBARS production increased 4.6-, 8.2-, or 6.7-fold over the control value, respectively. Comparable induction of TBARS by liver homogenates and microsomes was observed when these preparations were incubated with the same inducers. Addition of 5 microM butylated hydroxytoluene (BHT) prevented the induction of TBARS by 50 microM ferrous iron by liver slices. The results indicate the usefulness of tissue slices to measure lipid peroxidation. The usefulness of tissue slices is emphasized when a number of compounds or tissues are studied and tissue integrity is desired as in toxicological, pharmacological, and nutritional studies where reduced numbers of experimental animals is a relevant issue.

Measurement of fluorescent lipid peroxidation products in biological systems and tissues

Abstract A sensitive fluorometric assay for measurement of lipid peroxidation damage to biological preparations and tissues is described. The method consists of a chloroform-methanol extract of tissue followed by measurement of fluorescence characteristics of products derived from lipid peroxidation. Fluorescence analysis has been used successfully with rats and mice in vivo with stress induced during dietary and aging experiments and in peroxidation of subcellular organelles in vitro. Replicate extractions of fluorescent tissue showed a standard error of 2%. In a test of linearity with concentration, the amount of lipid-soluble fluorescent material in extracted samples was directly proportional to that added before extraction. Specific and general applications to in vivo and in vitro lipid peroxidation experiments are diseussed.

An enzymatic protective mechanism against lipid peroxidation damage to lungs of ozone-exposed rats

The effects of whole animal exposure to ozone and of dietary α-tocopherol on the occurrence in rat lung of lipid peroxidation and alteration of the activity of enzymes important in detoxification of lipid peroxides were studied. Exposure to 0.7 and 0.8 ppm ozone continuously for 5 and 7 days, respectively, significantly elevated the concentration of TBA reactants, primarily malonaldehyde, produced by lipid peroxidation, as well as the activities of glutathione (GSH) peroxidase, GSH reductase and glucose-6-phosphate (G-6-P) dehydrogenase. As a logarithmic function of dietary α-tocopherol (0, 10.5, 45, 150 and 1500 mg/kg), the increase in formation of malonaldehyde and the increase in activities of GSH peroxidase and G-6-P dehydrogenase were partially inhibited. The activity of GSH reductase was not affected by dietary α-tocopherol. The concentration of malonaldehyde and the activity of GSH peroxidase in lung were linearly correlated (p<0.001). This study confirmed the occurrence of lipid peroxidation in the lung during ozone exposure and revealed an enzymatic mechanism against damage. An apparent compensation mechanism is that with increased lipid peroxides there is increased activity of GSH peroxidase, which in turn increases lipid peroxide catabolism. The increased activities of GSH reductase and G-6-P dehydrogenase also function in the protective chain by providing increased levels of GSH and NADPH, respectively.

Sensitive fluorometric method for tissue tocopherol analysis

A sensitive, highly reproducible method for tissue tocopherol analysis that combines saponification in the presence of large amounts of ascorbic acid to remove interfering substances, extraction of the nonsaponifiable lipids with hexane, and fluorometric measurement of the tocopherol is presented. The nonsaponifiable lipid phase contained only one fluorochrome in the 290 nm excitation and 330 nm emission range, and it was identified as tocopherol by thin layer and column chromatography. Column chromatography of the hexane extract of a saponified,14C-tocopherolspiked microsomal fraction showed that no measurable oxidation to tocopherylquinone has occurred. The fluorometric method for tocopherol analysis was applied to homogenates and subcellular fractions from rat liver, kidney, lung, and heart and red blood cells. The heavy mitochondrial and microsomal fractions had the highest subcellular concentrations of tocopherol.

Hydrocarbon gases produced during in vitro peroxidation of polyunsaturated fatty acids and decomposition of preformed hydroperoxides.

Hydrocarbon gases have been used previously as an index of lipid peroxidation in vivo and in vitro. In vitro experiments are reported on the formation of hydrocarbon gases from peroxidizing ω-3 and ω-6 fatty acids. Hydrocarbon gases were not related during a 20-hr peroxidation phase but were released following the decomposition of hydroperoxides by addition of excess ascorbic acid. The major hydrocarbon gas products in iron, copper, or hematin catalyzed peroxidation systems were ethane or ethylene from linolenic acid, and pentane from linoleic acid and arachidonic acid. Calculations of the ratios of hydrocarbon gases formed were based on fatty acid decrease and/or change in diene conjugation and peroxide values. Depending on the fatty acid, catalyst, and calculation basis used, pentane formation was a high as 1.3 mol %, ethanol 4.3 mol %, and ethylene 10.6 mol %.

Purification and properties of rat lung soluble glutathione peroxidase.

Gluthathione peroxidase (gluthatione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) has been purified approximately 2700-fold from rat lung soluble fraction. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate/urea polyacrylamide gel electrphoresis. Selenium-75 tracer cochromatographed with the enzyme activity, indicating that rat lung soluble gluthathione peroxidase is a selenium enzyme. The enzyme had an approximate molecular weight of 80000 and contained four identical subunits. The optimal activity of the enzyme was at between pH 8.8 and 9.1. The enzyme had general specificity toward hydroperoxides, and high specificity for reduced glutathione. The kinetic behavior or the purified lung soluble glutathione peroxidase followed a ping-pong-like mechanism; the enzyme first reduced the lipid hydroperoxide substrate to the corresponding hydroxy fatty acid, then was regenerated to the native form by reduced glutathione.

The mechanism of the oxidation of unsaturated fatty acids catalyzed by hematin compounds

Abstract The mechanism of linoleate oxidation in the presence of the catalysts hemin, cytochrome c, hemoglobin, and catalase was the subject of this investigation. Catalytic activity decreased in the order: cytochrome c > hemin > hemoglobin > catalase. Kinetically, the oxidation of colloidal linoleate followed the relationship: d o 2 dt = K ( hematin catalyst ) 1 2 The linoleate peroxides resulting from hemin-catalyzed linoleate oxidation were characterized by production of carbonyl compounds. The hemin-catalyzed oxidation of methyl oleate, methyl linoleate, and methyl linolenate was zero order. Induction periods decreased and reaction rates increased as the reactivity of the fatty acid esters increased. The postulated mechanism of linoleate-oxidation initiation involves a direct reaction of linoleate peroxide with hematin catalysts. Propagation may take place in a manner similar to autocatalytic linoleate oxidation. The termination reaction probably involves interaction of linoleate peroxide radicals and hematin compounds.

Fluorescent products of lipid peroxidation of mitochondria and microsomes

Liver microsomes and mitochondria and heart sarcosomes from rats fed diets with varying α-tocopherol concentrations and lipid contents were peroxidized over a 6 hr time period. Lipid peroxidation was measured by absorption of oxygen, production of thiobarbituric acid (TBA) reactants and by development of fluorescence. The spectral characteristics of the fluorescent compounds were the same for all peroxidizing systems; the excitation maximum was 360 nm and the emission maximum was 430 nm. As time of peroxidation increased, uptake of oxygen and production of fluorescent compounds increased. These two parameters as well as production of TBA reactants were dependent upon dietary antioxidant and all three had an inverse relationship with the amount of dietary α-tocopherol. The relationship between absorption of oxygen and development of fluorescent compounds was also dependent upon dietary polyunsaturated fats (PUFA). Subcellular particles from animals fed higher levels of PUFA produced more fluorescent products per mole of oxygen absorbed than did those from animals on a diet with lower PUFA content. TBA reacting products increased with time during the initial phase of peroxidation: in the microsomal systems their production stabilized or decreased by 4–6 hr of peroxidation. Using the synthetic 1-amino-3-iminopropene derivative of glycine as standard for quantitation of fluorescence, the molar ratios of oxygen absorbed per fluorescent compound produced were calculated. This ratio for subcellular particles isolated from rats fed diets with PUFA ratios similar to those in the average American human diet was 393∶1. The fluorescent compounds had the same spectral characteristics as the lipofuscin pigment that accumulates in animal tissues as a function of age, oxidative stress or antioxidant deficiency. The fluorescent molecular damage represented by that accumulated in human heart age pigment by 50 years of age was calculated to have been caused by approximately 0.6 μmole of free radicals per gram of heart tissue.

DAMAGE TO PROTEINS, ENZYMES, AND AMINO ACIDS BY PEROXIDIZING LIPIDS

Abstract Transient free-radicals are produced in peroxidizing lipid-protein reaction systems. The pattern of damage to proteins, induced by these radicals, is similar to that observed in the case of radiation damage; proteins and enzymes lose solubility and constituent amino acids are destroyed. Lipid peroxidation damage appears to be about one-tenth as effective as radiation damage. Amino acid destruction was measured in lipid-peroxidation damaged γ-globulin, catalase, serum albumin, hemoglobin, and and ovalbumin. Among the most labile amino acids are methionine, histidine, cystine, and lysine. Major products of lipid peroxidation-cysteine interaction are hydrogen sulfide and cystine.

A new sensitive assay for the measurement of hydroperoxides

The enzyme glutathione (GSH) peroxidase can be used to measure hydroperoxides quantitatively, easily, and specifically. A timed reaction of GSH peroxidase, coupled with the oxidation of NADPH by GSH reductase, allows a direct spectrophotometric measurement of hydroperoxide. Addition of catalase prior to the addition of GSH peroxidase permits the distinction between hydrogen peroxide and organic hydroperoxides. The solvents that can be used with the assay include methanol, ethanol, water, and aqueous solutions of detergents such as Brij 35, Triton X-100, and cetyl trimethyl ammonium bromide. The utility of the method is demonstrated by the measurement of hydrogen peroxide and organic hydroperoxides formed upon ozonolysis of an unsaturated fatty acid.