Analysis of oxidized heme proteins and its application to multiple antioxidant protection.

Oxidation in tissues and homogenates can be determined by the analysis of oxidized heme proteins. Oxidation of heme proteins can be measured by spectral changes and the deconvolution of the spectra of mixtures of heme proteins by a spreadsheet heme spectra analysis program (HSAP), incorporating the spectra of the individual pure heme proteins. HSAP also is used to analyze the spectra of mixtures of heme proteins found in the literature. HSAP is applied in measuring the protective effects in rats of multiple antioxidants suitable for use in humans for protection against diseases.

Fluorescent lipid oxidation products and heme spectra index antioxidant efficacy in kidney tissue of hamsters.

Studies ranging from epidemiological to molecular suggest that dietary antioxidants protect against chronic diseases. The hypothesis was investigated that hamster kidney homogenate are better protected against induced oxidative damage after animals were fed a purified vitamin E deficient diet supplemented with 30 international units (IU) vitamin E/kg (30 IU diet) than fed the minimum required 3 IU vitamin E/kg (3 IU diet). The addition of 200 mg (+)-catechin to the 3 IU diet may offer protection. The effects of dietary (+)-catechin and alpha-tocopherol on tissue oxidizability were investigated by measuring (i) fluorescent products in lipid extracts, (ii) heme protein oxidation and (iii) heme destruction. A rapid initial increase of oxidation markers was measured over the 4 h incubation period for iron + ascorbate induced oxidation and a constant increase for lipoxygenase catalyzed products. Analysis of covariance over time and comparison at specific incubation times showed that iron + ascorbate induced homogenates from hamsters fed the 30 IU diet generated less fluorescent products and oxidized heme proteins than homogenates from the 3 IU or the 3 IU plus (+)-catechin fed animals. Incubation with lipoxygenase produced more lipid fluorescent products and heme protein oxidation in the 3 IU than in the 30 IU vitamin E group. In conclusion, supplementary dietary vitamin E but not supplementary (+)-catechin in a diet containing the minimum requirement of vitamin E for the species enhances oxidative resistance of kidney tissue.

Measuring the oxidation of heme compounds in heart homogenates from rats supplemented with dietary antioxidants.

Protection by antioxidant nutrients against oxidative damage in rat heart homogenates was studied. Following spontaneous oxidation of heart homogenates from rats fed vitamin E, selenium, or beta-carotene, oxidized heme proteins (OHP) and thiobarbituric acid reactive substances (TBARS) were measured. The absorbance spectra of oxidized and reduced heme proteins were analyzed with a heme spectral analysis program (HSAP) developed in this laboratory. HSAP is a multicomponent analysis program that uses successive approximations and computer spread-sheet solver functions to deconvolute a complex absorbance spectrum into individual heme protein spectra. Vitamin E markedly decreased formation of OHP, and vitamin E, selenium, or beta-carotene significantly lowered the production of TBARS during spontaneous oxidation of heart homogenates compared with homogenates from rats fed antioxidant-deficient diets. Pyridine hemochrome analysis showed that the total amounts of heme proteins present in the homogenates decreased during the oxidative incubation period. The formation of OHP correlated significantly with the amount of TBARS produced and could be simulated as a function of the oxidative and protective reactions involved in the oxidation of rat heart homogenates.

Multiple antioxidants protect against heme protein and lipid oxidation in kidney tissue.

The effect of antioxidant nutrients in rat kidney homogenates was studied by measuring the formation of oxidized heme proteins (OHP) and thiobarbituric acid reactive substances (TBARS) during spontaneous oxidation at 37 degrees. OHP were analyzed using a modified spreadsheet protocol; the Heme Protein Spectra Analysis Program (HPSAP). Male Sprague-Dawley (SD) rats were fed a basal diet fortified with vitamin E, selenium, or beta-carotene, or a combination of all three antioxidants. A second group of male SD rats received a basal diet fortified with Trolox, ascorbic acid palmitate, acetylcysteine, beta-carotene, coenzyme Q10, coenzyme Q0, and (+)-catechin. A control group of rats was given a vitamin E- and selenium-deficient basal diet. The amount of TBARS production during a 1 h reaction decreased as the relative antioxidant effectiveness of the dietary treatments increased. Dietary treatments providing nine antioxidants significantly reduced the formation of OHP and methemoglobin during the 1 h reaction compared to the dietary treatment providing only two antioxidant nutrients. These data suggest that increasing the diversity and quantity of antioxidants in the diet provides significantly more protection for heme proteins and lipids in kidney tissue than individual antioxidants or a combination of vitamin E and selenium.

Multicomponent analysis of heme protein spectra in biological materials.

Absorption spectra of heme proteins in a tissue homogenate contain information about the oxidation status of the biological sample. Deconvolution of absorption spectra can be used to quantitate the amount of individual heme proteins present in the mixture. The heme spectral analysis program (HSAP), a computer spreadsheet program, was used to quantitatively calculate values for heme proteins measured spectrally (390 to 450 and 500 to 640 nm) in tissue homogenates undergoing oxidation. The amount of oxidized heme proteins obtained by HSAP can be compared to other measurements of tissue oxidation. Precise quantitation of the amount of heme proteins present in a homogenate sample provided accurate assessment of the oxidized heme proteins calculated by HSAP. This quantitation was achieved through modification of existing pyridine hemochrome methods. Input into HSAP of the total heme protein content via the pyridine hemochrome value generated reproducible values for oxidized heme proteins. The program has broad potential as a multicomponent analysis tool. Modification of HSAP led to the development of a difference spectra analysis program (DSAP) which was used to quantitate the type and amount of heme proteins observed in mitochondrial difference spectra. In the present application, HSAP and DSAP provide methods for interpreting complex spectral information of multicomponent biological samples that undergo oxidation.

Vitamin E protects chick tissues against ex vivo oxidation of heme protein.

Protection by dietary vitamin E against ex vivo oxidation of heme proteins in liver and heart tissues of chicks was studied. A previously developed assay consisting of deconvoluting spectra obtained from tissue homogenates to measure oxidation of heme proteins proved successful. Compared to liver and heart from chicks fed the basal diet, the tissues from chicks receiving vitamin E exhibited less oxidation.

Protection of vitamin E, selenium, trolox C, ascorbic acid palmitate, acetylcysteine, coenzyme Q0, coenzyme Q10, beta-carotene, canthaxanthin, and (+)-catechin against oxidative damage to rat blood and tissues in vivo.

Male Sprague-Dawley rats were fed either a vitamin E and selenium deficient diet, a diet supplemented with vitamin E and selenium, or a diet supplemented with vitamin E, selenium, trolox C, ascorbic acid palmitate, acetylcysteine, Beta-carotene, canthaxanthin, coenzyme Q0, coenzyme Q10, and (+)-catechin. Rats were injected with CBrCl3 (0.05 mmol/100 g body weight) intraperitoneally. Oxidative damage to tissues was measured by formation of oxidized heme proteins (OHP) in blood, liver, kidney, heart, lung, and spleen. Diets supplemented with antioxidants showed protection against oxidative damage caused by CBrCl3. The protection was dependent on the diversity and quantity of antioxidants in the diet. In general, diets supplemented with both fat soluble and water soluble antioxidants provided better protection than diets supplemented only with vitamin E and selenium or with vitamin E, selenium, and fat soluble antioxidants.

Vitamin E, selenium, trolox C, ascorbic acid palmitate, acetylcysteine, coenzyme Q, beta-carotene, canthaxanthin, and (+)-catechin protect against oxidative damage to kidney, heart, lung and spleen.

Male Sprague-Dawley rats were fed diets that varied qualitatively and quantitatively in antioxidants. Kidney, heart, lung, and spleen homogenates were incubated at 37 degrees C with and without hydroperoxide or Fe+2. Protection of antioxidants against oxidative damage to tissue was determined by measurement of oxidized heme proteins. Tissues from rats supplemented with dietary vitamin E and selenium showed protection compared to tissues from rats on the basal diet. Tissues from rats with diets containing larger quantities of antioxidants and both fat soluble antioxidants: vitamin E, beta-carotene, coenzyme Q10, ascorbic acid 6-palmitate and water soluble antioxidants: selenium, trolox C, acetylcysteine, coenzyme Q0, (+)-catechin, showed the highest protection.

Hemichrome formation, lipid peroxidation, enzyme inactivation and protein degradation as indexes of oxidative damage in homogenates of chicken kidney and liver.

The change in relative hemichrome formation (RHF) was investigated as a potential marker of oxidative damage in kidney and liver homogenates prepared from chicks fed diets deficient or adequate in vitamin E. RHF gave an earlier indication of oxidative damage in tissue homogenates than the formation of thiobarbituric acid reactive substances (TBARS) or decrease in glutathione peroxidase activity (GPXA). RHF correlated significantly with both TBARS and GPXA. The correlations were 0.64 (P < 0.0001) and -0.57 (P = 0.0002) in kidney homogenates and 0.53 (P = 0.0006) and -0.71 (P < 0.0001) in liver homogenates. The correlation between RHF and the sum of TBARS and GPXA was also highly significant in both kidney and liver homogenates.

Protection by vitamin E selenium, trolox C, ascorbic acid palmitate, acetylcysteine, coenzyme Q, beta-carotene, canthaxanthin, and (+)-catechin against oxidative damage to liver slices measured by oxidized heme proteins.

Male SD rats were fed a vitamin E- and selenium-deficient diet, a diet supplemented with vitamin E and selenium, and diets supplemented with vitamin E, selenium, trolox C, ascorbic acid palmitate, acetylcysteine, beta-carotene, canthaxanthin, coenzyme Q0, coenzyme Q10, and (+)-catechin. Liver slices were incubated at 37 degrees C with and without CBrCl3, t-butyl-hydroperoxide, Fe+2, or Cu+2. The effect of antioxidant nutrients on the oxidative damage to rat liver was studied by measurement of the production of oxidized heme proteins (OHP) during the oxidative reactions. Diet supplemented with vitamin E and selenium showed a strong protection against heme protein oxidation compared to the antioxidant-deficient diet. Furthermore, increasing the diversity and quantity of antioxidants in the diets provided significantly more protection.

Binding of plasma selenoprotein P to cell membranes.

Competitive binding assays were performed to determine the amount of binding of 75Se-labeled plasma selenoprotein P (PSP) to membranes from different rat tissues at physiologic pH. 75Se PSP for use as a ligand in the binding assays was labeled in vivo by injecting rats with 75Se selenious acid. PSP was obtained from plasma by salt precipitation and affinity and ion-exchange chromatography. Membranes for receptor assays were prepared from liver, kidney, testes, and brain of rats fed diets with either 0.01, 0.1, or 2 ppm selenium. At pH 7.4 PSP was bound differentially to tissues in the following order: brain > kidney > testes > liver. Specific binding of PSP to tissue membranes from rats fed the different levels of selenium increased with increasing amounts of dietary selenium. Saturation assays indicated apparent saturation of membrane receptors by the 75Se-labeled PSP. Another significant new finding was a 134-kDa complex of PSP and membrane receptor, identified by gel-filtration chromatography of cross-linked samples from binding assays. This provides evidence for a membrane receptor for PSP in rat tissues.

Ferrous-iron-induced oxidation in chicken liver slices as measured by hemichrome formation and thiobarbituric acid-reactive substances: effects of dietary vitamin E and beta-carotene.

Hemichrome formation in chicken liver slices was determined by employing a Heme Protein Spectra Analysis Program (HPSAP) on the visible spectrum of the liver tissue. Relative hemichrome formation (RHF) in liver tissue exposed to ferrous iron for 1 h at 37 degrees C could be predicted according to the general catalytic equation RHF = k.[Fe2+]/(Ap + [Fe2+]), with k = 132 +/- 30, where the factor Ap represents the additive antioxidative potential in the liver tissue. RHF in Fe2+ exposed liver slices incubated at 37 degrees C for 1 h correlated significantly with formation of thiobarbituric acid-reactive substances (TBARS) (r = .77, P < .0001). RHF was found to decrease significantly with increasing vitamin E concentration in liver tissue exposed to ferrous iron (1 h, 37 degrees C). However, the influence of beta-carotene on RHF in ferrous-iron exposed liver slices (1 h, 37 degrees C) was less evident, as the concentration of Fe2+ was found to be decisive for whether beta-carotene acted as an antioxidant or a prooxidant under the conditions in question. Results in the liver slice model system regarding the effect of vitamin E and beta-carotene on iron overload were supported in a subsequent in vivo iron injection experiment with chicks. These observations indicate that RHF is a sensitive marker for ferrous-iron-induced oxidative damage in the present tissue slice system.

Protection by vitamin E, selenium, and beta-carotene against oxidative damage in rat liver slices and homogenate.

Male Sprague-Dawley (SD) rats were fed a vitamin E and selenium deficient diet and diets supplemented with vitamin E, selenium, beta-carotene, and a combination of the three. Tissue slices and homogenate of liver were incubated at 37 degrees C with and without the presence of prooxidants. The effect of vitamin E, selenium, beta-carotene, and the combination of the three antioxidants on the oxidative damage to rat liver tissue was studied by measuring the production of oxidized heme proteins in both tissue slices and homogenate during spontaneous and prooxidant-induced oxidation. The diet with the combination of all three antioxidants showed a strong protective effect against oxidative damage to heme proteins in contrast to the antioxidant-deficient diet. In general, diets with vitamin E, selenium, and beta-carotene were less effective than the combination of all three antioxidants. The protective effect of antioxidants on the heme protein oxidation was correlated with their inhibitory effect on lipid peroxidation measured as the production of thiobarbituric acid-reactive substance (TBARS). The protection of antioxidants on heme proteins was also dependent on the type of oxidation inducer. Possible mechanisms of antioxidants against oxidation in liver tissues are discussed.

Oxidation of heme proteins as a measure of oxidative damage to liver tissue slices.

Oxidative damage to heme proteins in rat liver tissue slices was studied. Tissue slices were incubated in Krebs-Ringer phosphate (KRP) buffer at 37 degrees C with and without the presence of prooxidants. The absorbance spectra (500-640 nm) of heme proteins of tissue slices obtained from both spontaneous and prooxidant-induced oxidation were analyzed with a heme protein spectra analysis program (HPSAP) developed in this laboratory. The dominant heme proteins in a fresh nonperfused tissue slice were hemoglobin and reduced cytochromes of mitochondria. In an oxidized tissue slice, the major oxidized product was hemichrome. Bromotrichloromethane, t-butyl hydroperoxide, and ferrous ion accelerated the oxidative reactions, and the amount of oxidized products was dependent on the incubation time as well as the type and concentration of prooxidants.

Protection of heme proteins by vitamin E, selenium, and beta-carotene against oxidative damage in rat heart, kidney, lung and spleen.

Effects of the combination of vitamin E, selenium, and beta-carotene on oxidative damage to rat heart, kidney, lung, and spleen were studied by measurement of the production of oxidized heme proteins (OHP) during spontaneous and prooxidant-induced oxidation. Male SD rats were fed with a vitamin E and selenium deficient diet or a diet supplemented with vitamin E, selenium, and beta-carotene. Homogenates of heart, kidney, lung, and spleen were incubated at 37 degrees C with and without the presence of bromotrichloromethane (CBrCl3). The diet supplemented with antioxidants showed a strong protective effect against oxidative damage to heme proteins during the early stages of both spontaneous and CBrCl3-induced oxidation in contrast to the antioxidant deficient diet. Synergism of multiple antioxygenic nutrients against oxidative damage to various animal tissues is discussed.

Potentiation of oxidative damage to proteins by ultraviolet-A and protection by antioxidants.

We have studied the damage of alcohol dehydrogenase (ADH) and glyceraldehyde 3-phosphate dehydrogenase (GAPD) induced by Fe++/EDTA + H2O2 in combination with UV-A (main output at 365 nm). Enzyme inactivation, formation of hydroxyl radicals (measured in the absence of enzymes), increase in protein carbonyls, oxidation of sulfhydryl (SH) groups, loss of native protein fluorescence, and enhanced protease degradation were used to determine protein damage. Hydroxyl radical production was greatly enhanced by the combination of UV-A with Fe++/EDTA + H2O2. The combined treatment increased protein carbonyls but decreased native protein fluorescence and SH groups. The combined treatment caused turbidity in GAPD but not in ADH, whereas trypsin susceptibility was increased more in ADH than in GAPD. These measurements of protein oxidation correlated well with enzyme activities. Glyceraldehyde 3-phosphate dehydrogenase and dithiothreitol were most protective against such damage, while hydroxyl radical and singlet oxygen scavengers were partially effective. Superoxide dismutase had no effect. Thus, UV-A potentiation of protein damage induced by FE++/EDTA + H2O2 appeared to involve hydroxyl radicals and perhaps singlet oxygen but not superoxide radicals. The damage to proteins induced by combination of UV-A with physiological oxidants, iron ions and H2O2 may be relevant to UV-A-induced skin and tissue damage.

Glutathione and antioxidants protect microsomes against lipid peroxidation and enzyme inactivation.

The study investigated the relationship between lipid peroxidation and enzyme inactivation in rat hepatic microsomes and whether prior inactivation of aldehyde dehydrogenase (ALDH) exacerbated inactivation of other enzymes. In microsomes incubated with 2.5 microM iron as ferric sulfate and 50 microM ascorbate, ALDH, glucose-6-phosphatase (G6Pase) and cytochrome P450 (Cyt-P450) levels decreased rapidly and concurrently with increased levels of thiobarbituric acid-reactive substances. Microsomal glutathione S-transferase and nicotinamide adenine dinucleotide phosphate-cytochrome c reductase were little affected during 1 hr of incubation. Addition of reduced glutathione partially protected and N,N'-diphenyl-p-phenylenediamine and butylated hydroxytoluene completely protected microsomes against inactivation of ALDH, G6Pase and Cyt-P450, as well as lipid peroxidation induced by iron and ascorbate. ALDH was more susceptible than G6Pase to inactivation by iron and ascorbate, and was thus an excellent marker for oxidative stress. Inhibition of ALDH by cyanamide injection of rats exacerbated the inactivation of G6Pase in microsomes incubated with 0.1 mM, but not 25 microM 4-hydroxynonenal (4-HN). 4-HN did not stimulate lipid peroxidation. Thus, 4-HN may play a minor role in microsomal enzyme inactivation. In contrast, lipid peroxyl radicals play an important role in microsomal enzyme inactivation, as evidenced by the prevention of both lipid peroxidation and enzyme inactivation by chain-breaking antioxidants.

Protein damage and lipid peroxidation: effects of diethyl maleate, bromotrichloromethane and vitamin E on ammonia, urea and enzymes involved in ammonia metabolism.

Changes in ammonia and urea were investigated as potential marker products of free radical damage to protein and subsequent metabolism of those damaged proteins in vivo. Both serum and liver lipid peroxidation products as measured by thiobarbituric-acid-reactive substances (TBARS) were increased by feeding rats a vitamin-E-deficient diet. The acute injection of diethyl maleate and bromotrichloromethane (DEM/BrCCl3) increased TBARS in liver of rats fed a vitamin-E-deficient diet. The concentrations of ammonia and urea in the serum and liver did not correlate with lipid peroxidation. The activities of liver glutaminase and arginase were decreased by DEM/BrCCl3 treatment in rats fed vitamin-E-deficient diet. Glutamate-ammonia ligase activity was decreased by vitamin-E-deficient diet but not by DEM/BrCCl3 treatment. Ornithine carbamoyltransferase, arginosuccinate synthase, argininosuccinate lyase and glutamate dehydrogenase (NAD(P)+) were not affected by dietary vitamin E or by DEM/BrCCl3. The data suggest that the concentrations of ammonia and urea, major by-products of nitrogen metabolism, are unchanged by the oxidant damage and lipid peroxidation, and that their control in vivo is a dynamic equilibrium of various metabolic pathways.

Headspace gas chromatography of volatile lipid peroxidation products from human red blood cell membranes.

An improved headspace capillary gas chromatographic (GC) method was developed to measure the oxidative susceptibility of human red blood cell (RBC) membranes. This method analyzed volatile peroxidation products of both n-6 (hexanal and pentane) and n-3 (propanal) polyunsaturated fatty acids. Oxidative susceptibility tests were standardized by incubating in a sealed 10-mL headspace bottle 0.25 or 1 mL of human RBC membrane in 40 mM phosphate buffer for 1 hr at 37 degrees C with a mixture of Fe++, ascorbic acid and H2O2. Sodium dodecyl sulfate increased significantly the amount of hexanal measured by headspace GC. By this standard headspace method, in one series of red blood cell membranes (RBCM) samples a four-fold variation in oxidative susceptibility was observed in RBCM from blood freshly drawn from six healthy subjects. In another series of RBCM samples a sixteen-fold variation in oxidative susceptibility was noted in frozen RBCM from blood freshly drawn from five healthy subjects. Correlation between hexanal formation and polyunsaturated fatty acids (PUFA) depletion provided good evidence that under these standard conditions hexanal is exclusively derived from the oxidation of arachidonic acid. Hydroperoxides of arachidonic acid are more readily formed and decomposed than those of linoleic acid in the presence of Fe++, ascorbic acid and H2O2 to produce hexanal as the main product that can be readily analyzed by headspace GC. This method may provide a useful tool to study susceptibility toward lipid peroxidative damage in human RBC membranes.