Formation of red pigment by a two-step 2-thiobarbituric acid reaction of alka-2,4-dienals. Potential products of lipid oxidation

Reaction of 2,4-hexadienal, 2,4-nonadienal and 2,4-decadienal with 2-thiobarbituric acid (TBA) in aqueous acetic acid produced a 532-nm absorbing red pigment. While the 1∶1 reaction of the aldehyde and TBA produced little pigment, reaction of the aldehyde with an excess amount of TBA produced significant amounts. Instant heating of the reaction mixture did not produce the pigment. However, initial reaction at 5°C and subsequent heating to 100°C produced the pigment efficiently (two-step reaction). Pigment formation required water and dissolved oxygen. The yield of the pigment from the alka-2,4-dienals was 1/10–1/20 of that from malonaldehyde. In the first step of the reaction at 5°C, the 1∶1 adducts of the aldehydes at the5-position of TBA and several other uniden-tified adducts were formed. In the second step, these adducts were converted at 100°C, in the presence of water and oxygen, into the red pigment. The structure of the red pigment from 2,4-hexadienal was elucidated to be the 1∶2 adduct of malonaldehyde and TBA. 2-Hexenal andt-butylhydroperoxide showed marked synergistic effects on the pigment formation from the alka-2,4-dienals. Red pigment formation due to the alka-2,4-dienals may be enhanced by the presence of other aldehydes and hydro-peroxides.     

Thiobarbituric acid-reactive substances from peroxidized lipids

The thiobarbituric acid (TBA) reaction was performed on linoleic acid 13-monohydroperoxide, autoxidized fatty esters, edible fats and oils, rat liver microsomal lipids, and on human erythrocyte ghost lipids in order to determine which substances from peroxidized lipids are TBA-reactive. The reaction was carried out in 2% acetic acid containing butylated hydroxytoluene using two different reaction modes: a one-step mode which involves heating at 100°C, and a two-step mode which involves first treatment at 5°C and subsequent heating at 100°C. Yields of the red 1∶2 malonaldehyde/TBA adduct, as estimated by absorbance, fluorescence intensity and high-performance liquid chromatography, were much higher than the malonaldehyde content as determined by direct chemical analysis. Yields of red pigment obtained by the two-step mode were slightly higher than those obtained by the one-step mode. Pigment yields were dramatically increased by addition oft-butyl hydroperoxide. Red pigment formation from alkenals and alkadienals was similarly enhanced by the two-step mode or by addition oft-butyl hydroperoxide, whereas pigment formation from malonaldehyde was not. It appears likely that a component of the total red pigment formed from the peroxidized lipids was due to aldehyde species other than malonaldehyde.     

Specific determination of malonaldehyde by N-methyl-2-phenylindole or thiobarbituric acid

Reactivity of a commercially available test kit (LPO-586), based on N-methyl-2-phenylindole, toward aldehydes was characterized and compared with that of thiobarbituric acid (TBA). In hydrochloric acid, LPO-586 produced a violet pigment with malonaldehyde (MA) but not with other tested aldehydes. In methane sulfonic acid, LPO-586 produced the violet pigment with MA and 4-hydroxynonenal (HNE), but not with other tested aldehydes. Pigment formation with MA was not inhibited by other aldehydes, but that with HNE was inhibited by alka-2,4-dienals. TBA produced a red pigment with MA but not with other tested aldehydes in hydrochloric acid or in acetate with ethylenediaminetetraacetic acid (EDTA). Both the LPO-586 test in hydrochloric acid and the TBA test in hydrochloric acid or in acetate with EDTA can be used for specific measurement of MA in oxidized lipid samples.     

Thiobarbituric acid reaction of aldehydes and oxidized lipids in glacial acetic acid

Thiobarbituric acid (TBA) reaction of several aldehydes and oxidized lipids in glacial acetic acid was performed. All the samples were freely soluble in the solvent used. Saturated aldehydes produced a stable yellow pigment with an absorption maximum at 455 nm, a red pigment derived from malonaldehyde at 532 nm, and an orange pigment due to dienals at 495 nm. The absorbance maximum was 7–9 per μmol for saturated aldehydes, 27.5 per μmol for malonaldehyde and about 2 per μmol for dienals. Autoxidation of unoxidized lipids increased progressively in glacial acetic acid. When the TBA test was performed under nitrogen, autoxidation of unoxidized lipids was inhibited completely. While saturated aldehydes produced no yellow pigment under nitrogen, oxidized lipids produced a considerable amount of stable yellow pigment. The value for absorbance at 455 nm as a function of autoxidation time paralleled those of peroxide values. The absorbance of most oxidized lipids at 455 nm was higher than at 532 nm. Yellow pigment formation in the TBA test under nitrogen could not be ascribed to free saturated aldehydes but rather to unspecified closely related substances. The stable yellow pigment was found to be an excellent indicator of lipid oxidation.     

Studies on peroxidized lipids. VI. Fluorescent products derived from the reaction of primary amines,malonaldehyde and monofunctional aldehydes

Monofunctional aldehydes such as acetaldehyde,n-propylaldehyde,n-butylaldehyde,n-hexylaldehyde,n-heptylaldehyde and benzaldehyde affected the reaction between primary amines and malonaldehyde. While the reaction of primary amines and malonaldehyde at pH 7 produced fluorescent 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehydesIa-f, the reaction of the primary amines, malonaldehyde and the aldehydes listed above gave fluorescent 4-substituted 1,4-dihydropyridine-3,5-dicarbaldehydesIIa-j. The primary amines used for this reaction included alkylamines, amino acids and alkanolamines. The optimal ratio of the amine, malonaldehyde and the aldehyde was 1:2:1–2, at which compoundsII were produced quantitatively. Peroxidized lipids which may contain malonaldehyde and other aldehydes could react with the primary amines to produce highly fluorescentII. Fluorescence spectra ofII showed excitation maxima at 386–403 nm and emission maxima at 444–465 nm in phosphate similar to those ofI. The spectra of these 1,4-dihydropyridinesI andII were roughly similar to those of lipofuscin pigment, but they exhibited different characteristics in acid and alkaline media from those of lipofuscin pigment. CompoundsII may be useful as model compounds to elucidate the chemical structure of lipofuscin pigment.     

Reaction of thiobarbituric acid with saturated aldehydes

The reaction of thiobarbituric acid (TBA) with saturated aldehydes, i.e., 1-butanal, 1-hexanal and 1-heptanal, produced a 455-nm yellow and a 532-nm red pigment. Formation of the pigments depended on the reaction conditions. The yellow pigment was unstable in the presence of excess amounts of the saturated aldehydes. The red pigment was formed only when the reaction was performed at a TBA/aldehyde ratio of 1∶1 in aqueous acetic acid. Formation of the yellow and red pigments required molecular oxygen. The colorless adducts, intermediates for the yellow and the red pigments, were isolated from the reaction mixtures. Aldol condensation and dehydration of 2 mol of the saturated aldehydes initially gave the α,β-unsaturated aldehydes, which in turn reacted with TBA to form the colorless adducts, pyranopyrimidine derivatives. The adducts were then converted into the yellow and red pigments under aerobic conditions.     

Studies on the TBA test for rancidity grading: II. TBA reactivity of different aldehyde classes

The TBA reactivities of several aldehydes, most of them known as ordinary products of lipid autoxidation, have been investigated systematically. Gas liquid chromatography-purified alkanals, 2-alkenals and 2,4-alkadienals were reacted with TBA in water solution. The formation of pigments with maximum absorbance at 450 and 530 nm was measured at optimum time-temperature conditions-different for readings at 450 and 530 nm- and values for absorbance per mole aldehyde were calculated. These values show that on reaction with TBA all studied aldehydes build a yellow 450 nm pigment, while only 2,4-alkadienals and, to a lesser extent, 2-alkenals produce the red 530 nm pigment. Consequently both pigments are measures of aldehydic products of lipid autoxidation: In the case of predominant unsaturated aldehyde formation, determination of the pigment with maximum absorbance at 530 nm is preferable. However, if alkanals are predominant, the determination of the yellow pigment at 450 nm is more apporpriate, as it grants higher sensitivity. The first paper in this series was published inFette, Seifen, Anstrichmittel 72:635 (1970).     

Degradation of monocarbonyls from autoxidizing lipids

In an attempt to account for carbonyls found in oxidized lipid systems, but not theoretically predicted from the decomposition of lipid hydroperoxides, a member from each of the monocarbonyl classes commonly observed in oxidizing lipids was oxidized at 45C in a Warburg apparatus and the carbonyl products studied. The carbonyl compounds used weren-nonanal,n-non-2-enal,n-hepta-2,4-dienal andn-oct-1-en-3-one. Nonanal was relatively stable to oxidation and was oxidized to nonanoic acid. Oct-1-en-3-one did not absorb oxygen during a 52-hr period; however, the unsaturated aldehydes oxidized at faster rates than methyl linoleate or linolenate. Non-2-enal upon absorption of 0.5 mole of oxygen was oxidized almost quantitatively to non-2-enoic acid. Hepta-2,4-dienal was polymerized at 0.5 mole of oxygen uptake. In addition both of the unsaturated aldehydes produced shorter chain mono- and dicarbonyls as oxidative degradation products. The identification of these compounds helps to explain the presence of carbonyls in oxidizing lipids and model systems that are not accountable through the decomposition of theoretically predictable isomeric hydroperoxide esters. The relatively large yield of malonaldehyde from the oxidized dienal suggests that these carbonyls may serve as a major source of malonaldehyde in oxidizing diene esters. Significant quantities of malonaldehyde are not observed in methyl linoleate until late stages of oxidation, and the dienals formed through degradation of primary hydroperoxides may in turn oxidize to give malonaldehyde. Technical Paper No. 1804, Oregon Agricultural Experiment Station. Submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy.     

Characteristics of the thiobarbituric acid reactivity of oxidized fats and oils

The thiobarbituric acid (TBA) reactivity of oxidized methyl linoleate, soybean oil, sesame oil, lard, chicken oil and sardine oil was characterized by using four different methods with 0.01% butylated hydroxytoluene (BHT). Optimal pH for the reactivity of most of the oxidized samples was 3–4, and that of some samples was above 5. Introduction of 2 mMt-butyl hydroperoxide (t-Bu00H) or 0.2 mM ferric ion in the reaction markedly enhanced the reactivity. Introduction of 0.2 mM ethylenediamine tetraacetic acid suppressed the reactivity. The characteristics of the TBA-reactivity of the samples were similar to those of alkadienals or alkenals. The most preferable method for the estimation of the TBA-reactive substances of the oxidized fats and oils was that using solvents at pH 3.5 with introduction of BHT, andt-Bu00H or ferric ion.     

Lipophilic aldehydes and related carbonyl compounds in rat and human urine

Rat and human urine samples were analyzed for lipophilic aldehydes and other carbonyl products of lipid peroxidation. The following compounds were identified as their 2,4-dinitrophenyl hydrazones by cochromatography with pure standards using three solvent systems: butanal, butan-2-one, pentan-2-one, hex-2-enal, hexanal, hepta-2,4-dienal, hept-2-enal, octanal, non-2-enal, deca-2,4-dienal, 4-hydroxyhex-2-enal, and 4-hydroxynon-2-enal. In general, fasted rats excreted less of these compounds than fed rats, indicating they were partially of dietary origin or that the endogenous compounds were excreted in a form not susceptible to hydrazone formation. The compounds excreted in human urine were similar to those excreted in rat urine but were present in lower concentrations. Identification of the conjugated forms fo the lipophilic aldehydes and related carbonyl compounds excreted in urine may be a source of information about their reactions in vivo.     

Further observations on the 2-thiobarbituric acid method for the measurement of oxidative rancidity

The validity of the 2-thiobarbituric acid (TBA) procedure for the measurement of oxidative rancidity by the determination of malonaldehyde (MA) as the red pigment with an absorption max at 532 mμ has been questioned [J. Am. Oil Chem. Soc.39, 34 (1962)]. Side reactions were reported to occur yielding degradation of TBA to products which absorb at the same wave length as the TBA-MA complex and at 450 mμ. Results reported in the present paper stress the importance of reagent purification. Little or no decomposition of TBA to produce interfering colors was found after heating with acids, oxidizing agents or hydroperoxides. Technical Paper No. 1669, of the Oregon Agricultural Experiment Station. Research supported in part by the National Institutes of Health RG 7006.     

Trans,n-3, and n-6 fatty acids in canadian human milk

The presence oftrans fatty acids in human milk may be a concern because of their possible adverse nutritional and physiological effects on the recipient infant. The mother's diet is the source of human milktrans fatty acids, and since these fatty acids are prevalent in many common foods of the Canadian diet, thetrans fatty acid content and the fatty acid composition of Canadian human milk were measured by gas-liquid chromatography coupled with silver nitrate-thin layer chromatography. In samples obtained from 198 lactating mothers across Canada, the average percentage of totaltrans (sum oft18∶1,t18∶2, andt18∶3) was 7.2% of breast milk fatty acids with a range of 0.1–17.2%. Analysis oft18∶1 isomer distribution indicated that partially hydrogenated vegetable oils are the major source of thesetrans fatty acids in human milk, whereas contribution from dairy products appeared to be relatively minor. Linoleci and α-linolenic acid levels were inversely related to the totaltrans fatty acids, indicating that the elevation oftrans fatty acids in Canadian human milk is at the expense of n-3 and n-6 essential fatty acids. Levels of arachidonic and docosahexaenoic acids did not correlate with their parent fatty acids, indicating that it might be difficult to elevate the levels of n-6 and n-3 C_20–22 polyunsaturated fatty acids in breast milk by increasing levels of linoleic and α-linolenic acids in the mother's diet.     

Manganese(II)-Induced Oxidation of Limonene by Dioxygen

Manganese(II) complexes {[Mn^II(bpy)₂ ²⁺]_MeCN} in acetonitrile activate dioxygen for the oxidation of limonene to produce mainly carvone, carveol, limonene oxide, and perillaldehyde. The reaction efficiencies after 24 h reaction time are approximately 5-times higher than those obtained for analogous iron(II) complexes. However, the 5 h long induction period is observed for the common conditions of the reaction. The simultaneous presence of the catalyst, dioxygen and the substrate is necessary for the active species to be formed. When t-BuOOH is present in the reaction mixture, the induction period does not appear. In contrast, the replacement of t-BuOOH by HOOH completely inhibits the reaction. We have proposed a putative mechanism in which a manganese(IV)–hydroperoxo adduct with incorporated substrate is formed during the induction period and it becomes an active catalyst for limonene oxidation.     

Amine-malonaldehyde condensation products and their relative color contribution in the thiobarbituric acid test

Different malonaldehyde-amine condensation products were tested for their relative color contribution to the thiobarbituric acid (TBA) test, an indicator for oxidative rancidity of polyunsaturated lipids. The open chain mono- and disubstituted malonaldehyde (M) addition products (R-N=CH-CH=CHOH and R-N=CH-CH=CH-NH-R) gave complete (100%) recovery ofM on a mole basis. When theM residue was incorporated into cyclic products which formed between the ureido- or guanidino- substituents of α-amino acids such as citrulline or arginine andM, recovery ofM by the TBA color test was 30% and 6%, respectively. Products, from imine-amine type interactions, containing theM residue as in pyrazoline or pyrazole ring systems, released from 4% to noM.     

Primary odorants of laundry soiled with sweat/sebum: Influence of lipase on the odor profile

Odorants still attached to laundry soiled with human axillary sweat and sebum after a mild washing procedure [European full-scale, short-cycle wash (20 min wash, 15 min rinse) at 30°C, using color detergent, at 3.5 g/L] were extracted and analyzed by aroma extract dilution analysis. Esters (ethyl-2-methylpropanoate and ethylbutanoate), ketones (1-hexen-3-one and 1-octen-3-one) and, in particular, aldehydes [(Z)-4-heptenal, octanal, (E)-2-octenal, methional, (Z)-2-nonenal, (E,Z)-2,6-nonadienal, (E,Z)-2,4-nonadienal, (E,E)-2,4-decadienal, and 4-methoxybenzaldehyde] were identified as primary odorants. Organic acids, which are dominant characteristic odorants in human axillary sweat, were, on the other hand, effectively removed during the washing process. The influence of lipase activity on the odor profile was investigated by analyzing selected sets of textile swatches, sampled from the right/left axillary of male runners, washed in the presence or absence of lipase. The swatches were examined by a sensory ranking analysis prior to the analytical odor analysis. Swatches selected for the subsequent odor analysis possessed greater odor intensity when washed in the presence of lipase than the corresponding swatches washed in the absence of lipase. The aroma extract dilution analysis revealed that aldehydes were present in slightly greater concentrations in swatches washed in the presence of lipase. The aldehydes are believed to be formed through oxidative degradation of triglycerides present in human sebum, which may be facilitated by lipase. Based on sensory panel results and dilution analysis of odorants, the impact of lipase on the odor impression was, however, minor and thus believed to be inadequate as explanation for malodor generation in laundry as experienced by the consumer.     

Incorporation into lipid classes of products from microsomal desaturation of isomerictrans-octadecenoic acids

The microsomal desaturation of positional isomers oftrans-octadecenoic acids is effected by the Δ9-desaturase and, with concomitant geometric isomerization,cis,trans- andcis,cis-octadecadienoic acids of unusual structure are formed. Incorporation of the substrates and their products into lipids varied from 50.5% for incubations with 14–18∶1 to 81.0% for 6–18∶1. A detailed study of the composition of each of the major lipid classes, i.e., phospholipids, triacylglycerol and cholesteryl esters, as well as the composition of the free fatty acid fraction, revealed a complex picture. Generally, thec,c-18∶2 products were enriched in the phospholipid fraction, whereas thec,t-18∶2 appeared preferentially in cholesteryl esters. The 18∶1 substrates themselves did not show marked preferences for any of the lipid classes. Phospholipase A₂ action on phosphatidylcholine and phosphatidylethanolamine demonstrated enrichment of thec,c- and thec,t-18∶2 products in the 2-position, whereas the 18∶1 substrates were preferentially inserted into the 1-positions. Thec,c- andc,t-18∶2 formed by desaturation oft11–18∶1 varied from this pattern, probably due to their conjugated double bond structures. Linoleic acid,c9,c12–18∶2, formed during desaturation oft12–18∶1, surprisingly showed enrichment in the 1-position of phosphatidylcholine. Incubation experiments witht5- andt6-isomers using liver microsomes from rats fed a corn-oil-supplemented diet showed conversion and incorporation rates similar to the rates obtained with microsomes from EFA-deficient rats. The fatty acid composition of lipid classes and the distributions of products and substrate between the 1- and 2-positions of phosphatidylcholine also agreed with results obtained using microsomes from EFA-deficient rats.     

Formation of triacylglycerol core aldehydes during rapid oxidation of corn and sunflower oils with tert-butyl hydroperoxide/Fe2+

The lipid ester core aldehydes formed during a rapid oxidation (7.8 M tert-butyl hydroperoxide, 90 min at 37°C) of the triacylglycerols of purified corn and sunflower oils were isolated as dinitrophenylhydrazones by preparative thin-layer chromatography and identified by reversed-phase high-performance liquid chromatography with on-line electrospray ionization mass spectrometry and by reference to standards. A total of 113 species of triacylglycerol core aldehydes were specifically identified, accounting for 32–53% of the 2,4-dinitrophenylhydrazine (DNPH)-reactive material of high molecular weight representing 25–33% of the total oxidation products. The major core aldehyde species (50–60% of total triacylglycerol core aldehydes) were the mono(9-oxo)nonanoyl- and mono(12-oxo)-9,10-epoxy dodecenoyl- or (12-oxo)-9-hydroxy-10,11-dodecenoyl-diacylglycerols. A significant proportion of the total (9-oxo)nonanoyl and epoxidized (12-oxo)-9,10-dodecenoyl core aldehydes was found in complex combinations with hydroperoxy or hydroxy fatty acyl groups (6–10% of total triacylglycerol core aldehydes). Identified were also di(9-oxo)nonanoylmonoacylglycerols (0.5% of total) and tri(9-oxo)nonanoylglycerols (trace). The identification of the oxoacylglycerols was consistent with the products anticipated from tert-butyl hydroperoxide oxidation of the major species of corn and sunflower oil triacylglycerols (mainly linoleoyl esters). However, the anticipated (13-oxo)-9,11-tridecadienoyl aldehyde-containing acylglycerols were absent because of further oxidation of the dienoic core aldehyde. A significant proportion of the unsaturated triacylglycerol core aldehydes contained tert-butyl groups linked to the unsaturated fatty chains via peroxide bridges (2–9%). The study demonstrates that rapid peroxidation with tert-butyl hydroperoxide consitutes an effective method for enriching natural oils and fats in triacylglycerol core aldehydes for biochemical and metabolic testing.     

Formation of formaldehyde and malonaldehyde by photooxidation of squalene

Formaldehyde and malonaldehyde were identified upon exposure of squalene to ultraviolet (UV) irradiation at 300 nm. Formaldehyde was derivatized by reaction with cysteamine to form thiazolidine; malonaldehyde was derivatized by reaction withN-methylhdyrazine to produceN-methylpyrazole. The derivatives were subsequently analyzed with a gas chromatography equipped with a fused silica capillary column and a nitrogen/phosphorus detector. The levels of formaldehyde and malonaldehyde produced increased with irradiation time. The amount of formaldehyde produced reached a maximum of 3.40 nmol/mg squalene after 7 hr irradiation; the maximum amount of malonaldehyde generated, 0.92 nmol/mg, was found after 5 hr of irradiation. Prior to this study, formaldehyde had not been reported as a photoproduct of squalene. Acetaldehyde and acetone were also detected in the irradiated squalen,, which may be formedvia a 6-methyl-5-hepten-2-one intermediate. 6-Methyl-5-hepten-2-one can also undergo breakdown to form malonaldehyde.     

Alumina supported MoO3: an efficient and recyclable catalyst for selective oxidation of tertiary nitrogen compounds to N-oxides using anhydrous TBHP as oxidant under mild reaction conditions

Alumina-supported MoO₃ was found to be an efficient heterogeneous and recyclable catalyst for the oxidation of tertiary nitrogen compounds to N-oxides in excellent yields using anhydrous t-BuOOH as oxidant under mild reaction conditions.     

Urinary response toin vivo lipid peroxidation induced by vitamin E deficiency

Experiments were carried out to measure the urinary excretion of free and conjugated malonaldehyde (MDA) and other thiobarbituric acid reactive substances (TBARS) in vitamin E deficient and vitamin E supplemented rats. From both dietary groups, six TBA positive fractions were isolated, in addition to that containing free MDA, by high-performance liquid chromatography (HPLC) on a TSK-GEL G-1000PW column. Three of the fractions isolated were found to be significantly increased in vitamin E deficiency. After acid hydrolysis, only one of the above compounds produced free MDA which indicated the presence of derivatized MDA. Only this fraction exhibited fluorescence at excitation 370 nm and emission 450 nm. The five other fractions formed 2,4-dinitrophenylhydrazones (2,4-DNPH), indicating the presence of carbonyl groups, but the derivatized MDA fraction did not. No significant differences were found in free MDA levels between the vitamin E deficient and the vitamin E supplemented groups.