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).     

Characteristics of the thiobarbituric acid reactivity of human urine as a possible consequence of lipid peroxidation

A 532 nm red pigment formed in the thiobarbituric acid (TBA) assay of human urine was characterized after separation of the pigment by high-performance liquid chromatography. The yield of the red pigment was some-what higher at pH 2 than at pH 5; its development was not inhibited by ethylenediaminetetraacetic acid. The characteristics of the pigment were similar to those of the pigment derived from standard malonaldehyde. The amount of the pigment formed was roughly equal to the content of malonaldehyde derivatives estimated as 1-(2,4-dinitrophenyl)pyrazole. Pigment formation was significantly enhanced byt-butyl hydroperoxide (t-BuOOH) and ferric ions, which may be due to pigment formed from aldehydes other than malonaldehyde; the presence of these aldehydes was confirmed by the formation of the corresponding 2,4-dinitrophenylhydrazones. The amount of pigment produced from 24-h urine samples of 12 healthy subjects was estimated to be 26–95 nmol/kg, and 65–182 nmol/kg in the presence oft-BuOOH. These values are lower than those for urine of rabbit or rat. The TBA reactivity in the absence and presence oft-BuOOH of human urine was not related to age or sex. The TBA reactivity of human urine collected in the afternoon and in the evening was higher than that of urine collected in the morning.     

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.     

Separation of oxidized and unoxidized molecular species of phosphatidylcholine by high pressure liquid chromatography

Soy phosphatidylcholine (PC) has been separated into its major molecular species by reversephase high pressure liquid chromatography (HPLC). An aqueous methanol gradient was used that allowed detection of the various species by their absorbance at 206 nm. Oxidized species were detected by their absorbance at 234 nm and were resolved from the unoxidized species. This technique has been used to separate and purify unoxidized dilinoleyl phosphatidylcholine (di 18∶2 PC) from other species of soy PC and to monitor the autoxidation of an aqueous suspension of the purified di 18∶2 PC. Two oxidized products were formed from di 18∶2 PC. Further analysis showed that they were PC, but one of the products contained an oxidized and an unoxidized fatty acid; in the other product, both fatty acids were oxidized. Present in part at FASEB 63rd Annual Meeting, Dallas, Texas, April 1979.     

A study of the applicability of a modified thiobarbituric acid test to flavor evaluation of fats and oils

A modified thiobarbituric acid test has been devised for the determination of certain components of oxidized fat. The reaction was carried out in a single phase solvent designed to eliminate the color extraction or fat removal steps of other TBA tests. A moderate temperature of 60C, with no added acid, was used to minimize breakdown of hydroperoxides and TBA. Under certain reaction conditions, temperature, sunlight, ferric ion, and oxygen can influence results. Of the aldehydes examined, the TBA reaction products of dienals showed an absorption peak at 532 m dm only, while the TBA reaction products of the saturated aldehydes showed a peak at 452 mμ only. The spectral behavior of TBA reaction products of monoenals was found to vary with the location of the double bond. Fat samples, aged at either 60C or 37.8C, were examined organoleptically and by the modified TBA test. The 452 mμ peak was of value in assessing the flavor of beef fat, cottonseed oil, and used frying fat, whereas the 532 mμ wavelength was of value in assessing the flavor of soybean oil, used frying fat, and pork fat.     

The 2-thiobarbituric acid reagent for determination of oxidative rancidity in fish oils

A method has been devised for the quantitative determination of malonaldehyde in oxidized fish oils by means of the 2-thiobarbituric acid reagent in alcoholic solution. TBA numbers and peroxide values have been determined at intervals during hydroperoxidation of pilchard oil. The curves follow the same general pattern but the numerical relationship between them depends upon the temperature of oxidation. In alcoholic solution, the reaction between thiobarbituric acid and malonaldehyde is carried out in the dark, because sunlight causes a decrease in optical density of the TBA-malonaldehyde complex at 532 mμ with the appearance of a second maximum at 452 mμ. The nature of the compounds responsible for absorption at these two wave-lengths is discussed.     

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.     

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.     

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.     

α-Tocopherol prooxidant effect in aqueous media: Increased autoxidation rate of linoleic acid

The prooxidant effect of a-tocopherol was investigated during linoleic acid autoxidation in an aqueous media, at pH 6.9. α-Tocopherol (1.25 x 10⁻⁴ M) was added to linoleic acid (2.5 x 10⁻³ M) and the linoleic acid autoxidation rate was evaluated using 2 methods: spectrophotometric measurement at 234 nm exhibited an important increase of conjugated dienes after α-tocopherol addition, especially during the first 4 days, and gas Chromatographie measurement of unoxidized linoleic acid showed an important degradation of linoleic acid in the presence of a-tocopherol. During the pro-oxidant reaction, α-tocopherol was rapidly oxidized ; it was detected as traces by thin layer chromatography after 4 days of experimentation. Two oxidation products of α-tocopherol have been identified: α-tocopherylquinone and a dimer of α-tocopherol.     

Inhibition of malonaldehyde formation from lipids by an isoflavonoid isolated from young green barley leaves

The inhibitory effect of 2″-O-glycosylisovitexin (2″-O-GIV), isolated from young green barley leaves, on malonaldehyde (MA) formation from lipids was determined by gas chromatography. Two μmol of 2″-O-GIV inhibited MA formation from 0.2 mmol of squalene by almost 100% upon ultraviolet (UV) irradiation. Only 1 μmol of 2″-O-GIV inhibited 90% of MA formation from 0.2 mmol of ethyl linoleate upon UV irradiation. The effective quantities of 2″-O-GIV were much lower than those of either butylated hydroxytoluene or α-tocopherol. When ethyl linoleate, ethyl linolenate and ethyl arachidonate were oxidized by Fenton’s reagent in the presence of 2″-O-GIV or α-tocopherol, both compounds showed similar activity toward MA formation. Both compounds gave maximum inhibition at doses of 0.1–0.3 μmol for ethyl linoleate, 0.1–0.5 μmol for ethyl linolenate and 0.1–0.5 μmol for ethyl arachidonate.     

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.     

Detection of aldehydes with 4-amino-5-hydrazino-1,2,4-triazole-3-thiol as spray reagent

A spray reagent of 4-amino-5-hydrazino-1,2,4-triazole-3-thiol, 2% in aqueous 1N NaOH, was used for the detection of aldehydes on chromatograms of lipids. Purple spots are produced with aldehydes, and 1 μg of palmitaldehyde can be detected easily on thin layer chromatograms. The reagent is specific for aldehydes and does not react with ketones. The aldehyde moiety from plasmalogens is detected only after acidic hydrolysis. Purple or red stains arise also with ozonides. Simple precautions against autoxidation of the lipids should be taken to prevent formation of aldehydes which could lead to misinterpretations.     

Gas chromatographic analysis of reactive carbonyl compounds formed from lipids upon UV-irradiation

Peroxidation of lipids produces carbonyl compounds; some of these, e.g., malonaldehyde and 4-hydroxynonenal, are genotoxic because of their reactivity with biological nucleophiles. Analysis of the reactive carbonyl compounds is often difficult. The methylhydrazine method developed for malonaldehyde analysis was applied to simultaneously measure the products formed from linoleic acid, linolenic acid, arachidonic acid, and squalene upon ultraviolet-irradiation (UV-irradiation). The photoreaction products, saturated monocarbonyl, α,β-unsaturated carbonyls, and β-dicarbonyls, were derivatized with methylhydrazine to give hydrazones, pyrazolines, and pyrazoles, respectively. The derivatives were analyzed by gas chromatography and gas chromatography-mass spectrometry. Lipid peroxidation products identified included formaldehyde, acetaldehyde, acrolein, malonaldehyde, n-hexanal, and 4-hydroxy-2-nonenal. Malonaldehyde levels formed upon 4 hr of irradiation were 0.06 μg/mg from squalene, 2.4 μg/mg from linolenic acid, and 5.7 μg/mg from arachidonic acid. Significant levels of acrolein (2.5 μg/mg) and 4-hydroxy-2-nonenal (0.17 μg/mg) were also produced from arachidonic acid upon 4 hr irradiation.     

Higher Correlation with Sensory Evaluation of Oxidative Rancidity by Modified TBA Test

When carrying out the TBA reaction test with oxidized lipids in the conventional way, a red colour with maximum absorption at 530 nm is measured as degree of rancidity. This colour corresponds mainly to alkadienals formed as products of oxidation. Before the formation of this red colour, a transient yellow colour with maximum absorption at 450 nm is formed, which corresponds to both alkanals, alkenals and alkadienals, consequently having a “broader” and also higher sensitivity. By modifying the conditions of the reaction (heating at a lower temperature, e. g. 2 hours at 50°C) the yellow colour becomes stable enough to be measured. The paper shows that the greater sensitivity of measuring the yellow colour absorption corresponds to higher correlation with sensory evaluation, particularly in the case of low unsaturated samples.     

Phospholipids and oxophospholipids in atherosclerotic plaques at different stages of plaque development

We identified and quantified the hydroperoxides, hydroxides, epoxides, isoprostanes, and core aldehydes of the major phospholipids as the main components of the oxophospholipids (a total of 5–25 pmol/μmol phosphatidylcholine) in a comparative study of human atheroma from selected stages of lesion development. The developmental stages examined included fatty streak, fibrous plaque, necrotic core, and calcified tissue. The lipid analyses were performed by normal-phase HPLC with on-line electrospray MS using conventional total lipid extracts. There was great variability in the proportions of the various oxidation products and a lack of a general trend. Specifically, the early oxidation products (hydroperoxides and epoxides) of the glycerophosphocholines were found at the advanced stages of the plaques in nearly the same relative abundance as the more advanced oxidation products (core aldehydes and acids). The anticipated linear accumulation of the more stable oxidation products with progressive development of the atherosclerotic plaque was not apparent. It is therefore suggested that lipid infiltration and/or local peroxidation is a continuous process characterized by the formation and destruction of both early and advanced products of lipid oxidation at all times. The process of lipid deposition appears to have been subject to both enzymatic and chemical modification of the normal tissue lipids. Clearly, the appearance of new and disproportionate old lipid species excludes randomness in any accumulation of oxidized LDL lipids in atheroma.     

Stability of low linolenic acid canola oil to frying temperatures

The effect of heating on the oxidation of low (1.6%) linolenic acid canola oil (C18∶3) at frying temperature (185 ±5°C) under nitrogen and air was examined and then compared to a laboratory deodorized (9.0%, C18∶3) and a commercially deodorized (8.5%, C18∶3) canola oil sample. A significantly lower development of oxidation was evident for the low C18:3 canola oil, based on the measurement of peroxide value (PV), thiobarbituric acid (TBA), free fatty acids (FFA), dienals and carbonyls. The greater stability of the low C18:3 canola oil was also reflected by a corresponding improvement in heated room odor intensity scores. Heating under nitrogen (rather than air) not only improved the odors but limited the oxidation in all oils. While the low C18:3 canola oil heated under nitrogen was acceptable in 94% of odor judgments the same oil heated in air was acceptable in only 44%. This suggests that even low levels of C18:3 may contribute to the development of the heated room odor phenomenon.     

Specific susceptibility of docosahexaenoic acid and eicosapentaenoic acid to peroxidation in aqueous solution

The peroxidation of different polyunsaturated fatty acids (PUFA) after photoirradiation in aqueous solution was evaluated by measuring fatty acid loss and malonaldehyde production in medium. The oxidation rates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two highly unsaturated fatty acids of the n−3 series, were surprisingly lower (14 and 22%, respectively) than the oxidation rates of linoleic, α-linolenic, γ-linolenic, dihomo γ-linolenic, and arachidonic acids (62–90%). The quantities of malonaldehyde (MA) produced were assayed simultaneously by gas chromatography (GC) and high performance liquid chromatography (HPLC). MA production was found to be related to both the degree of unsaturation and the metabolic series of the fatty acid. The maximum value was observed with arachidonic acid (MA production from 2 mM arachidonic acid in aqueous solution was estimated at 44.9±6.0 μM by GC and 46.8 ±4.0 μM by HPLC). Eicosapentaenoic acid and docosahexaenoic acid produced lower MA quantities compared to arachidonic acid (MA production from 2 mM EPA and 2 mM DHA was estimated at 17.9±1.5 μM and 37.9±0.7 μM, respectively, by GC, and 26.3±4.9 μM and 37.3±4.2 μM, respectively, by HPLC). The MA yield, defined as the amount of MA (nmols) produced per 100 nanomoles of oxidized fatty acid, was used to express the susceptibility of individual PUFA to peroxidation. The MA yield correlated well with the degree of unsaturation, but was independent of carbon chain length and metabolic series. The study suggests that adequate assessment of lipid peroxidation cannot be achieved by measuring MA formation alone, but it also requires knowledge of the fatty acid composition of the system studied.