Water-soluble inhibitor(s) of tumor respiration formed from ultraviolet-induced oxidation of linoleic and linolenic acids

Inhibition of Ehrlich ascites carcinoma respiration by aqueous extracts of oxidized linoleic or linolenic acid (aqueous emulsions UV-irradiated, 90 min) was associated entirely with relatively involatile compounds which were both thiobarbituric acid (TBA)-reactive and peroxidase-reactive. Inhibitory compounds were heat stable and migrated in thin-layer chromatography with aldehydes, "hydroperoxides," and TBA-reactive compounds. Peroxidase-catalyzed reduction of the "hydroperoxide" diminished the inhibition. At 4.7 x 10(-5) M "hydroperoxide" concentration, the residues from both linoleic and linolenic acid inhibited tumor oxygen consumption to a similar degree. However, at this concentration of "hydroperoxide" only the dried extract from linolenic acid was able to produce inhibition (100%) of aerobic glucose utilization by tumor cells. No glycolytic inhibition by the dried residue of oxidized linoleic acid was observed. At least 12 compounds (approximate chain length, 7C-13C) containing alpha,Beta-unsaturated carbonyl groups were isolated by gas-liquid chromatography (GLC) of dried extracts of oxidized linolenic acid. No single fraction inhibited tumor respiration, but the recombined mixture of all compounds caused complete respiratory inhibition of ascites tumor cells. Less material was required to inhibit oxygen consumption before than after GLC presumably because the more highly inhibitory components of the extract (along with "hydroperoxides" and TBA-reactive compounds) were lost during GLC. Extracts from oxidized linolenic acid were found to produce in all tumor cells cytoplasmic evaginations which were readily detected by phase microscopy.     

Mechanism of horseradish peroxidase-catalyzed oxidation of malonaldehyde

The mechanism of malonaldehyde oxidation by horseradish peroxidase in the presence of manganese(II) and acetate was investigated. Our results show that an apparent oxygenase behavior demonstrated by peroxidase in this system can be explained in terms of normal peroxidase activity. A free radical is generated from the reaction of malonaldehyde with compounds I and II of peroxidase; this radical is scavenged by dissolved molecular oxygen to give the appearance of peroxidase acting as an oxygenase. Oxygen consumption, absorbance spectra, and kinetic results show that the reaction is initiated by autoxidation of malonaldehyde to give a free radical. The radical reacts with oxygen and through the action of manganese(II), a peroxide is generated. This peroxide drives the peroxidase cycle to generate more free radicals which propagate the oxygen consumption reaction.     

The Induction of Lipid Peroxidation in Rat Liver by Oral Intake of 9-Oxononanoic Acid Contained in Autoxidized Linoleic Acid

A component in the autoxidation products of linoleic acid (LA) which induced the endogenous lipid peroxidation was searched for. Secondary autoxidation products (SP) of LA induced the production of thiobarbituric acid reactive substances (TBARS) in liver as well as the LA hydroperoxides stimulated. SP were separated chromatographically into some fractions which were administered orally to rats. One fraction composed mainly of 9-oxononanoic acid (9-ONA) markedly stimulated the TBARS production in liver. Authentic 9-ONA also increased the lipid peroxide level as determined by a new test, as well as the activities of glutathione peroxidase and reductase in liver. Thus, hepatic lipid peroxidation was induced by orally given 9-ONA which was present in the autoxidation products of LA.     

Heme degradation during autoxidation of oxyhemoglobin

Two fluorescent heme degradation compounds are detected during autoxidation of oxyhemoglobin. These fluorescent compounds are similar to fluorescent compounds formed when hydrogen peroxide reacts with hemoglobin [E. Nagababu and J. M. Rifkind, Biochem. Biophys. Res. Commun. 247, 592-596 (1998)]. Low levels of heme degradation in the presence of superoxide and catalase are attributed to a reaction involving the superoxide produced during autoxidation. The inhibition of most of the degradation by catalase suggests that the hydrogen peroxide generated during autoxidation of oxyhemoglobin produces heme degradation by the same mechanism as the direct addition of hydrogen peroxide to hemoglobin. The formation of the fluorescent degradation products was inhibited by the peroxidase substrate, ABTS, which reduces ferrylhemoglobin to methemoglobin, indicating that ferrylhemoglobin is produced during the autoxidation of hemoglobin. It is the transient formation of this highly reactive Fe(IV) hemoglobin, which is responsible for most of the heme degradation during autoxidation.     

Protective Effects of a Culture Supernatant of Lactobacillus acidophilus and Antioxidants on Ileal Ulcer Formation in Rats Treated with a Nonsteroidal Antiinflammatory Drug

Ileal ulcers and thiobarbituric acid (TBA)-reactive substances in the ileal mucosa were induced in rats treated with a nonsteroidal antiinflammatory drug, 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonylphenyl)thiophene (BFMeT), at a dose of 1,000 mg/kg administered with tap water as drinking water. However, the formation of ileal ulcers and TBA-reactive substances in the ileal mucosa was repressed by giving the animals a culture supernatant of Lactobacillus acidophilus as drinking water. We measured the antioxidative activity of the culture supernatant and found that the supernatant inhibited the formation of t-butyl hydroperoxide-induced TBA-reactive substances in erythrocyte membrane ghosts. Therefore, the effects of various known antioxidative compounds on the ileal ulcer formation induced by BFMeT were investigated. While α-tocopherol, t-butyl-1,4-hydroxyanisole and allopurinol did not repress ulcer formation after BFMeT treatment, ascorbic acid, dimethyl sulfoxide, glutathione and β-carotene significantly inhibited formation. Among these compounds, ascorbic acid was the most effective. Accumulation of TBA-reactive substances in the ileal mucosa after BFMeT treatment also decreased significantly in rats treated with ascorbic acid. In addition, the percentage of Gram-negative rods in the ileal contents of rats treated with BFMeT and tap water was dramatically increased, but it was not increased in rats treated with BFMeT and these antioxidants. A positive correlation between the percentage of Gram-negative rods and the number of ileal ulcers was also observed. These results suggest that lipid peroxidation mediated by oxygen radicals plays an important role in the induction of ileal ulcers by BFMeT in rats, and that lipopolysaccharide-activated neutrophils probably produce highly reactive hypochlorous acid and hydrogen peroxide, which are inactivated by ascorbic acid and glutathione, respectively.     

Effect of UV-light on formation of fluorescent products of lipid peroxidation

The rate of fluorescent product formation during the peroxidation of polyunsaturated linolenic acid or egg phosphatidylethanolamine and also during the oxidation of linolenic acid together with a phenylalanine and synthetic phosphatidylethanolamine 1,5--3 times more intensive after previous UV-irradiation of the unsaturated fatty acid. Schiff bases are fluorescent products in amine containing systems which are produced in the reaction of the malonaldehyde with amines. It is possible that fluorochromes produced during the only unsaturated acid oxidation are the result of the radical recombination. Accumulation of the oxidated products determined by TBA-reactive substances does not inevitably correlate with the fluorescent intensity in explored systems.     

Purification, partial characterization, and possible role of catalase in the bacterium Vitreoscilla

Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin that is relatively autoxidizable. It also contains a catalase whose primary function may be to remove hydrogen peroxide produced by this autoxidation. This enzyme was purified and partially characterized. It is a protein of 272,000 Da with a probable A2B2 subunit structure, in which the estimated molecular size of A is 68,000 Da and that of B, 64,000 Da, and an average of 1.6 molecules of protoheme IX per tetramer. The turnover number for its catalase activity was 27,000 s-1 and the Km for hydrogen peroxide was 16 mM. The peroxidase activity measured using o-dianisidine was 0.6% that of the catalase activity. Cyanide, which inhibited both catalase and peroxidase activities, bound the heme in a noncooperative manner. Azide inhibited the catalase activity but stimulated the peroxidase activity. An apparent compound II was formed by the reaction of the enzyme with ethyl hydrogen peroxide. The enzyme was reducible by dithionite, and the ferrous enzyme reacted with CO. The cellular content of Vitreoscilla hemoglobin varies during the growth cycle and in cells grown under different conditions, but the ratio of hemoglobin to catalase activity remained relatively constant, indicating possible coordinated biosynthesis and supporting the putative role of Vitreoscilla catalase as a scavenger of peroxide generated by Vitreoscilla hemoglobin.     

Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylenediaminetetraacetic acid.

The thiobarbituric acid (TBA) reactivity of rat liver and brain homogenates was characterized to elucidate what kinds of aldehyde species contributed to the reactivity. Characteristic pH dependence of the reactivity with a maximum at around pH 3 and marked enhancement of the reactivity by t-butyl hydroperoxide (t-BuOOH) and ferric ion were similar to those of alkadienals. The amounts of aldehyde species, including alkadienals determined as 2,4-dinitrophenylhydrazones, were high enough to account for the enhanced reactivity. The reactivity was inhibited by ethylenediaminetetraacetic acid (EDTA) but not completely, suggesting the presence of malonaldehyde whose reactivity was not affected by EDTA. The amounts of malonaldehyde determined as 1-(2,4-dinitrophenyl)pyrazole could account for a part of the reactivity in the presence of EDTA. Hence, the TBA reactivity of liver and brain homogenates at around pH 3 in the presence of t-BuOOH and ferric ion may be accounted for by alkadienals and malonaldehyde and that in the presence of EDTA by malonaldehyde.     

Enzymatic transformations of morphinane alkaloids

Horseradish peroxidase transforms morphinane alkaloids into N-oxides and morphine to pseudomorphine in the presence of hydrogen peroxide. The crude poppy enzyme fraction shows the same activities. The rates of reactions were influenced by phenolic compounds and their relation controlled by the concentration of hydrogen peroxide and the presence of ascorbic acid.     

The autoxidation of glyceraldehyde and other simple monosaccharides under physiological conditions catalysed by buffer ions

Glyceraldehyde and other simple monosaccharides autoxidise under physiological conditions generating 1-hydroxyalkyl (carbon-centred) free radicals and intermediates of dioxygen reduction: superoxide, hydrogen peroxide and hydroxyl radicals. The major glyceraldehyde-derived product is the alpha-ketoaldehyde, hydroxypyruvaldehyde. Close similarities between the temperature dependence of the kinetics of glyceraldehyde autoxidation and glyceraldehyde enolisation to an ene-diol indicates that enolisation is the rate-determining step in the autoxidative process. Inspection of a wide range of carbonyl compounds showed that the monosaccharide moiety -CH(OH)-C- is conserved in carbonyl compounds reactive towards autoxidation, indicating that the ability to form an ene-diol is a prerequisite to monosaccharide autoxidation. The ene-diol intermediate autoxidises rapidly to the products: hydrogen peroxide, water and alpha-ketoaldehydes: beta-hydroxypyruvaldehyde is produced from glyceraldehyde and dihydroxyacetone, glyoxal from glycolaldehyde autoxidation. Ene-diol autoxidation is catalysed by hydrogen peroxide and trace metal ion contaminants; removal of either of these factors sufficiently retards ene-diol autoxidation such that ene-diol autoxidation rather than enolisation becomes the rate determining step in the overall autoxidative process. Under enolisation control, the rate of monosaccharide autoxidation is influenced by pH and the buffer system used for pH control.     

Reaction conditions affecting the relationship between thiobarbituric acid reactivity and lipid peroxides in human plasma.

The thiobarbituric acid (TBA) reactivity of human plasma was studied to evaluate its adequacy in quantifying lipid peroxidation as an index of systemic oxidative stress. Two spectrophotometric TBA tests based on the use of either phosphoric acid (pH 2.0, method A) or trichloroacetic plus hydrochloric acid (pH 0.9, method B) were employed with and without sodium sulfate (SS) to inhibit sialic acid (SA) reactivity with TBA. To correct for background absorption, the absorbance values at 572 nm were subtracted from those at 532 nm, which represent the absorption maximum of the TBA:MDA adduct. Method B gave values of TBA-reactive substances (TBARS) 2-fold higher than those detected with method A. SS lowered TBARS by about 50% with both methods, indicating a significant involvement of SA in plasma TBA reactivity. Standard SA, at a physiologically relevant concentration of 1.5 mM, reacted with TBA, creating interference problems, which were substantially eliminated by SS plus correction for background absorbance. When method B was carried out in the lipid and protein fraction of plasma, SS inhibited by 65% TBARS formation only in the latter. Protein TBARS may be largely ascribed to SA-containing glycoproteins and, to a minor extent, protein-bound MDA. Indeed, EDTA did not affect protein TBARS assessed in the presence of SS. TBA reactivity of whole plasma and of its lipid fraction was instead inhibited by EDTA, suggesting that lipoperoxides (and possibly monofunctional lipoperoxidation aldehydes) are involved as MDA precursors in the TBA test. Pretreatment of plasma with KI, a specific reductant of hydroperoxides, decreased TBARS by about 27%. Moreover, aspirin administration to humans to inhibit prostaglandin endoperoxide generation reduced plasma TBARS by 40%. In conclusion, reaction conditions affect the relationship between TBA reactivity and lipid peroxidation in human plasma. After correction for the interfering effects of SA in the TBA test, 40% of plasma TBARS appears related to in vivo generated prostaglandin endoperoxides and only about 60% to lipoperoxidation products. Thus, the TBA test is not totally specific to oxidant-driven lipid peroxidation in human plasma.     

The characterisation of thiobarbituric acid reactivity in human plasma and urine

The specificity of the thiobarbituric acid reaction (TBA) has been investigated using techniques of high-performance thin-layer chromatography and spectrofluorimetry. It was found that malondialdehyde (MDA) derived from different lipid and nonlipid origins formed the same MDA-TBA complex. This complex could be separated from other TBA-reactive compounds by both chromatography and spectrofluorimetry. Normal human plasma and urine both formed an MDA-TBA complex along with other TBA-reactive compounds. In plasma this was associated mainly with phosphatidylcholine and appeared to be peroxidic in reaction. Urine, however, contained polar MDA-forming compounds probably resulting from the oxidation of 2-deoxyaldoses during the acid-heating stage of the TBA test.     

Ex situ volatile survey of ground almond and pistachio hulls for emission of spiroketals: Analysis of hull fatty acid composition,water content,and water activity

The spiroketal conophthorin has recently been implicated as an important semiochemical of the navel orangeworm moth (Amyelois transitella), a major insect pest to California tree nuts. Additionally, new evidence demonstrates that fungal spores in the presence of linoleic acid produce conophthorin. Numerous investigations have analyzed the volatile emissions of almonds and pistachios under varying conditions, yet there are few reports of conophthorin as a volatile component. Previous studies by our laboratories have suggested almond hulls may be a source of conophthorin production. Accordingly, the volatile emissions of ex situ almond and pistachio ground hulls were surveyed at several developmental stages. Each ground sample was analyzed at various intervals to determine if conophthorin was produced. The almond and pistachio samples were presumed to have a natural fungal bouquet present. Additionally, the fatty acid composition, water content, and water activity of the hulls were analyzed for each sample. Conophthorin and the structurally similar compound chalcogran were detected from almond hulls and shells, but not from the pistachio samples. The almond and pistachio hulls were investigated for four fatty acid components – palmitic, oleic, linoleic, and linolenic. The fatty acid composition of almond hulls varied greatly throughout the growing season, whereas the composition of pistachio hulls remained relatively constant. Both water content and activity were constant in early stages of almond growth then dropped in the later stages of hull split. Spiroketal emission along with other associated volatiles is discussed. This is the first report of the fatty acid composition, water content, and water activity of developing almond and pistachio hulls.     

Metabolism of unsaturated fatty acids in tomato fruit: linoleic and linolenic acid as precursors of hexanal

Incubation of linoleic or linolenic acid with tissue slicesas well as cell-free extracts of tomato fruits produced hexanal.Biogenesis of hexanal from these fatty acids was further substantiatedby the use of uniformally labelled ¹⁴C substrates. Based onthe fact that hydrogen peroxide inhibited oxygen uptake andalso production of carbonyls, it is apparent that lipoxidaseis involved in these reactions. The activity of the crude solubleextract was increased by dialysis and ammonium sulphate fractionation. In general, ripe fruits contained greater enzymatic activitiesbut smaller amounts of linoleic and linolenic acid than greenfruits. The enzymatic activity was enhanced by metal ions andcompounds containing free -SH groups. (Received December 27, 1971; )     

Manganese inhibits benzoyl peroxide/copper-dependent lipid peroxidation in the microsomal fraction of rabbit dental pulp

1. Of all the cations tested only manganese ion inhibited the benzoyl peroxide/Cu2+-dependent formation of thiobarbituric acid (TBA)-reactive substance. 2. Ki of Mn2+ for the formation of TBA-reactive substance was 5.0 microM. 3. The inhibitory manner of manganese was non competitive against copper.     

On the mechanism of phenylhydrazine-induced hemolytic anemia

The thiobarbituric acid (TBA)-reactants have been measured in the circulating red blood cells (RBC) and RBC trapped in the spleens of normal and phenylhydrazine-treated rats. There was significant TBA-reactivity in the circulating RBC of phenylhydrazine-treated rats which was increased 3-fold in RBC obtained from the spleen. Since lipid peroxidation accompanies formation of TBA-reactive malonyldialdehyde, it is suggested that phenylhydrazine induces anemia as a consequence of peroxidation of RBC membrane lipids and this effect may be a result of the autoxidation of the drug and the interaction of oxygen radicals with membrane lipids.     

The measurement of malondialdehyde in peroxidised ox-brain phospholipid liposomes

The preparation of ox-brain phospholipid liposomes and their peroxidation using different catalysts have been described in detail. The degree of peroxidation is related to the formation of thiobarbituric acid (TBA)-reacting compounds and expressed as malondialdehyde (MDA). As confirmatory data, fluorescent MDA-phospholipid complexes were measured in parallel. Close agreement between the polar TBA-reactive compounds and the nonpolar fluorescent compounds confirmed the usefulness of the simple TBA test as a measure of peroxidative activity in pure lipid liposomes. Relative differences in the catalytic activity of ascorbic acid and cupric ions when assayed by the two methods are discussed.     

日光温室黄瓜发育过程中主要芳香物质和脂肪酸含量的变化

以'新泰密刺'为试验材料,比较分析了日光温室条件下,不同月份黄瓜商品成熟果实及其发育过程中的主要芳香物质及亚麻酸、亚油酸含量差异.结果显示:(1)黄瓜果实中挥发性物质主要有2E,6Z-壬二烯醛、2E-壬烯醛、2E-己烯醛、2,6-壬二烯醇、正己醛、戊醛、2-戊烯醛和3,6-壬二烯醇.其中2E,6Z-壬二烯醛相对含量以5月份最高,1月份其次,11月份最低;具有黄瓜香气的2,6-壬二烯醇和3,6-壬二烯醇,以及与黄瓜风味品质密切相关的2E,6Z-壬二烯醛/2E-壬烯醛比值以1月份最高,5月份其次,11月份最低.(2)1月份黄瓜果实的亚麻酸和亚油酸含量显著大于5月份,说明低温弱光有利于不饱和脂肪酸的形成和积累.(3)随着果实发育天数的增加,黄瓜2E,6Z-壬二烯醛、2,6-壬二烯醇和3,6-壬二烯醇相对含量,2E,6Z-壬二烯醛/2E-壬烯醛比值均较大幅度升高,商品成熟期达到高峰,若延迟采收,上述指标多趋于下降;2E-壬烯醛、2E-己烯醛、正己醛、戊醛等的相对含量多随果实发育而降低,商品成熟期2E-壬烯醛、2E-己烯醛和正己醛降到最低,之后快速升高.研究表明,果实发育过程中亚麻酸和亚油酸含量及二者的比例与2E,6Z-壬二烯醛和2E-壬烯醛相对含量及其比值的变化规律基本相似,说明黄瓜风味品质与其亚麻酸和亚油酸含量有一定相关性.     

Anthracycline-Induced Oxygen Consumption and Oxidative Damage in Rat Liver Microsomes are not Necessarily Coupled: A study with 8 structurally related anthracyclines

The stimulative effect of 8 anthracyclines (the parent compounds daunorubicin and doxorubicin and 6 structurally closely related anthracyclines) on the production of thiobarbituric acid (TBA)-reactive material was investigated in liver microsomes. Except for daunorubicinone and doxorubicinone, all derivatives stimulated NADPH-dependent production of TBA-reactive material. Doxorubicinone had no effect, daunorubicinone inhibited TBA-reactivity at concentrations up to 50 μM. However, the latter two compounds stimulated oxygen consumption in the presence of EDTA to a degree comparable to that induced by the parent compounds. Since the oxygen uptake under these circumstances represents redox cycling of the drugs, apparently redox cycling and production of TBA-reactive material were not coupled for these compounds.

Spectral measurements showed no decisive role for interaction with free iron (Fe³⁺) ions in the non-coupling of redox cycling and production of TBA reactive material. Evidence for a role of bound iron ions was not obtained.

It is discussed that for the aglycones oxygen consumption and production of TBA reactive material might be non-coupled through their different interaction with microsomal RNA.     

Heligmosomoides polygyrus: peroxidase activity

Peroxidase activity in Heligmosomoides polygyrus was located primarily in the mitochondrion. The enzyme was active with a range of organic and inorganic electron donors and, in addition to hydrogen peroxide, it could utilize cumene peroxide, but the highest activity was obtained with linoleic acid peroxide. The effects of electron chain substrates and inhibitors on H. polygyrus mitochondrial peroxidase activity was consistent with the enzyme being linked functionally to cytochrome c, although in vivo, this may not be the only electron donor. The interaction of the peroxidase with electron transport is discussed.