Antioxidant activity by headspace gas chromatography of volatile oxidation products of omega-6 and omega-3 polyunsaturated lipids.
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Antioxidants↗
Biomedical subjects
E N Frankel
Publications and source records attributed to E N Frankel.
Reduced oxidative susceptibility of LDL from patients participating in an intensive atherosclerosis treatment program.
The goal of this investigation was to determine whether participation in an atherosclerosis treatment program would reduce the oxidative susceptibility of LDL from patients with coronary artery disease. The treatment program included intensive exercise therapy, stress management, and consumption of a diet containing 10% fat. The size and antioxidant and lipid contents of LDL particles from 25 patients were analyzed at baseline and after 3 mo of therapy. The susceptibility of LDL to copper-mediated oxidation was measured by a conjugated diene assay and headspace gas chromatography (HSGC). Atherosclerosis treatment significantly reduced plasma total cholesterol and apolipoprotein B concentrations and the molar ratio of LDL cholesterol ester to apolipoprotein B (P < 0.01). The LDL content of alpha-tocopherol and beta-carotene was increased (27% and 17%, respectively, P < 0.04) and the molar ratio of LDL cholesterol ester the sum of LDL alpha-tocopherol and LDL beta-carotene decreased from 159 at baseline to 122 at 3 mo (P < 0.01). The lag phase of LDL conjugated diene formation increased 24%, whereas the maximum rate of oxidation slowed 29% (P < 0.01). As assessed by HSGC, copper-catalyzed formation of volatile lipid oxidation products was reduced 15% (P < 0.007); the reduction in volatiles was correlated with an increase in the alpha-tocopherol content of LDL (r=-0.48, P < 0.01). The principal determinants of reduced LDL oxidative susceptibility were the particle contents of alpha-tocopherol and beta-carotene. To our knowledge, this is the first report to document a reduction in LDL oxidation in coronary artery disease patients undergoing atherosclerosis-reversal therapy.
Aged↗
Determination of hydroperoxides and structures by high-performance liquid chromatography with post-column detection with diphenyl-1-pyrenylphosphine.
A high-performance liquid chromatographic method, using post-column detection with diphenyl-1-pyrenylphosphine (DPPP), was developed for the quantitative and qualitative determination of isomeric lipid hydroperoxides (OOH). The OOH eluted from a normal-phase column were passed through a photodiode array detector and then mixed with DPPP solution in a reaction coil heated at 80 degrees C. DPPP oxide formed by the reaction with OOH was determined by monitoring the fluorescence intensity at 380 nm and excitation at 352 nm. The conjugated diene OOH (13-cis,trans- and 9-cis,trans-OOH) and nonconjugated OOH (12-cis-trans-and 10-cis,trans-OOH) from photosensitized oxidation of methyl linoleate were determined in a molar ratio of 31:29:19:21, respectively. However, only the two conjugated hydroperoxides were detected by ultraviolet absorption at 234 nm. Further applications were carried out for the determination of OOH of methyl oleate and methyl linolenate. This method proved to be useful for the determination of the OOH containing both conjugated and nonconjugated diene structures.
Chromatography, High Pressure Liquid↗
Effect of alpha-tocopherol and Trolox on the decomposition of methyl linoleate hydroperoxides.
To clarify the mechanisms of antioxidant action, the effect of alpha-tocopherol and its water-soluble carboxylic acid derivative, Trolox, was studied on the decomposition of methyl linoleate hydroperoxides (MeLoOOH). Decomposition rate and the distribution of autoxidation products formed from MeLoOOH were followed by analyzing the volatile and non-volatile products by static headspace gas chromatography and normal-phase high-performance liquid chromatography, respectively. Both alpha-tocopherol and Trolox markedly inhibited the decomposition of MeLoOOH in a concentration-dependent way. In the absence of antioxidants, MeLoOOH was completely decomposed after incubation for 48 h at 60 degrees C, and in the presence of equal molar concentration of antioxidants only 6-7% of initial MeLoOOH was decomposed even after 280 h of incubation. MeLoOOH produced 1.2% methyl linoleate hydroxy compounds (MeLoOOH) in the presence of alpha-tocopherol and 3.8% in the presence of Trolox. Both antioxidants inhibited the formation of volatile decomposition products and the formation of ketodiene compounds. The hydroxy compounds may be formed by the reaction of alkoxy radical and hydrogen donating antioxidants. Conversion of MeLoOOH into stable MeLoOH demonstrated that the antioxidants alpha-tocopherol and Trolox trap alkoxyl radicals by H-donation.
Antioxidants↗
Older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease.
Increases in plasma cholesterol are associated with progressive increases in the risk of atherosclerotic cardiovascular disease. In humans plasma cholesterol is contained primarily in apolipoprotein B-based low density lipoprotein (LDL). Cells stop making the high-affinity receptor responsible for LDL removal as they become cholesterol replete; this slows removal of LDL from plasma and elevates plasma LDL. As a result of this delayed uptake, hypercholesterolemic individuals not only have more LDL but have significantly older LDL. Oxidative modification of LDL enhances their atherogenicity. This study sought to determine whether increased time spent in circulation, or aging, by lipoprotein particles altered their susceptibility to oxidative modification. Controlled synchronous production of distinctive apolipoprotein B lipoproteins (yolk-specific very low density lipoproteins; VLDLy) with a single estrogen injection into young turkeys was used to model LDL aging in vivo. VLDLy remained in circulation for at least 10 days. Susceptibility to oxidation in vitro was highly dependent on lipoprotein age in vivo. Oxidation, measured as hexanal release from n-6 fatty acids in VLDLy, increased from 13.3 +/- 5.5 nmol of 2-day-old VLDLy per ml, to 108 +/- 17 nmol of 7-day-old VLDLy per ml. Oxidative instability was not due to tocopherol depletion or conversion to a more unsaturated fatty acid composition. These findings establish mathematically describable linkages between the variables of LDL concentration and LDL oxidation. The proposed mathematical models suggest a unified investigative approach to determine the mechanisms for acceleration of atherosclerotic cardiovascular disease risk as plasma cholesterol rises.
Aging↗
Effect of n-3 fatty acid-rich fish oil supplementation on the oxidation of low density lipoproteins.
This study was aimed at determining the effect of fish oil supplementation on copper-catalyzed oxidation of low density lipoproteins (LDL) from nine hypertriglyceridemic human subjects. A rapid headspace gas chromatographic method was used to measure the volatile oxidation products from LDL. Propanal and hexanal were the major volatile products formed in the oxidation of n-3 and n-6 polyunsaturated fatty acids (PUFA), respectively. Fish oil supplementation resulted in a significant increase in propanal formation from 3.7 to 13.4 nmol/mL LDL (P < 0.01); it also resulted in small decreases in pentanal formation from 14.7 to 11.4 nmol/mL LDL and in hexanal formation from 138 to 108 nmol/mL LDL (P < 0.05). The changes in peroxidation products paralleled the changes in LDL composition, which showed a significant increase in n-3 PUFA from 3.2 to 14.6% (P < 0.01) and a decrease in n-6 PUFA from 43.7 to 35.0% (P < 0.05). Propanal formation was highly and significantly correlated with n-3 PUFA content (r = 0.950, P < 0.001). Since total volatiles remained unchanged, this indicated that the two groups of LDL samples did not differ in overall oxidative susceptibility. Although fish oil intake did not alter the oxidative susceptibility of LDL, the chemically modified LDL particles generated a distinct pattern of volatile oxidation products that reflected changes in their fatty acid composition.
Chromatography, Gas↗
Effects of a carotene-deficient diet on measures of oxidative susceptibility and superoxide dismutase activity in adult women.
The effect of consuming a low carotene diet (approximately 60 micrograms carotene/day) on oxidative susceptibility and superoxide dismutase (SOD) activity in women living in a metabolic research unit was evaluated. The diet had sufficient vitamins A, E, and C. The women ate the diet supplemented with 1500 micrograms/day beta-carotene for 4 days (baseline), then the unsupplemented diet for 68 days (depletion), followed by the diet supplemented with > 15,000 micrograms/day carotene for 28 days (repletion). Production of hexanal, pentanal, and pentane by copper-oxidized plasma low density lipoproteins from carotene-depleted women was greater than their production of these compounds when repleted with carotene. Erythrocyte SOD activity was depressed in carotene-depleted women; it recovered with repletion. Thiobarbituric acid reactive substances in plasma of carotene-depleted women were elevated and diminished with repletion. Dietary carotene seems to be needed, not only as a precursor of vitamin A, but also to inhibit oxidative damage and decrease oxidation susceptibility.
Adult↗
Inhibition of human LDL oxidation by resveratrol.
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Coronary Disease↗
Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine.
The "French paradox" (apparent compatibility of a high fat diet with a low incidence of coronary atherosclerosis) has been attributed to the regular drinking of red wine. However, the alcohol content of wine may not be the sole explanation for this protection. Red wine also contains phenolic compounds, and the antioxidant properties of these may have an important role. In in-vitro studies with phenolic substances in red wine and normal human low-density lipoprotein (LDL) we found that red wine inhibits the copper-catalysed oxidation of LDL. Wine diluted 1000-fold containing 10 mumol/L total phenolics inhibited LDL oxidation significantly more than alpha-tocopherol. Our findings show that the non-alcoholic components of red wine have potent antioxidant properties toward oxidation of human LDL.
Humans↗
Chemical changes during the early phase of in vitro oxidative damage to human LDL.
Much of the oxidative damage to human LDL in vivo may lead to only minimal changes in the chemical properties of the LDL. Therefore, chemical changes were evaluated during the initial 3 hours of oxidative attack on human LDL with 5 microM Cu. HPLC analyses were calibrated with a conjugated-diene internal standard. Cholesterol-linoleate-hydroperoxide (Chol-18:2-OOH) accumulated much more rapidly than alpha-tocopherol was lost. Although large amounts of cholesterol arachidonate were destroyed, diene-containing oxidation products of this lipid were not identified by HPLC analysis. Phosphatidyl-choline-hydroperoxides accumulated much more slowly than chol-18:2-OOH. beta-carotene was oxidized relatively slowly, but lycopene was destroyed almost as fast as alpha-tocopherol. The preferential accumulation of chol-18:2-OOH is consistent with a model in which alpha-tocopherol is localized to the surface of the LDL particle, providing minimal protection to hydrophobic components in the core of the LDL.
Adult↗
Headspace gas chromatography to determine human low density lipoprotein oxidation.
We previously described a rapid headspace gas chromatographic method for the determination of hexanal, an important decomposition product of n-6 polyunsaturated fatty acid (PUFA) peroxidation in rat liver samples and human red blood cell membranes. This method was applied to the measurement of Cu2+ catalyzed-oxidation of freshly prepared human low density lipoproteins (LDL) from 10 healthy adult volunteers. A twofold variation in oxidative susceptibility was found by this assay for hexanal and other volatiles. Hexanal values correlated significantly (P < 0.05) with total polyunsaturated fatty acid (PUFA), 18:2 and n-6 PUFA contents of LDL; but poorly with 20:4 and with vitamin E. Therefore, in addition to alpha-tocopherol, other endogenous antioxidants and factors may contribute to LDL's resistance to oxidation. This simple, rapid and sensitive method for oxidative susceptibility provides a useful component in the analysis of the prooxidant/antioxidant status of biological samples. The method is used routinely in our laboratories to determine specific peroxidation products of n-6 and n-3 PUFA.
Adult↗
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.
Aldehydes↗
Preferential hydrolysis of monohydroperoxides of linoleoyl and linolenoyl triacylglycerol by pancreatic lipase.
Four triacylglycerols (TGs) containing palmitoyl and linoleoyl or linolenoyl groups in known positions were synthesized and pancreatic lipase hydrolysis of their monohydroperoxides was investigated. TG monohydroperoxides did not deactivate the lipase and were hydrolyzed at almost the same degrees as their original TGs. In the hydrolysis of unoxidized TGs, pancreatic lipase showed almost the same reactivity on palmitoyl, linoleoyl and linolenoyl groups at the 1(3)-positions. However, this enzyme had fatty acid specificity for TG monohydroperoxides and the molar concentration of hydroperoxy linoleic or linolenic acid liberated from 1(3)-positions of TG monohydroperoxides were 1.6-2.4-times higher than that of the unoxidized fatty acid from the corresponding 3(1)-positions. The susceptibility of hydroperoxy acyl components of TG monohydroperoxides to pancreatic lipase hydrolysis is explained by its molecular structure and hydrophilic property.
Animals↗
Effect of dietary menhaden oil and vitamin E on in vivo lipid peroxidation induced by iron.
Weanling rats were fed diets containing 10% menhaden oil (MO) or 10% corn oil-lard (1:1, COL) with low (less than or equal to 5 IU/kg) or supplementary (35 IU/kg) vitamin E for six weeks. The rats were killed 30 min after injection with 24 mg iron/kg as ferrous chloride because thiobarbituric acid-reactive substances (TBARS) in liver homogenates were highest at 30 min after injection of iron into rats fed a standard diet. Tissue homogenates were used either without incubation (zero-time) or after incubation at 37 degrees C for 1 hr. In addition to TBARS and conjugated dienes, headspace hexanal and total volatiles (TOV) determined by capillary gas chromatography were useful indices of lipid peroxidation since they were decreased by vitamin E supplementation and were increased with increasing iron dose. Regardless of the dietary lipid used, vitamin E supplementation decreased headspace hexanal, TOV, TBARS and conjugated dienes in both zero-time and incubated homogenates of liver and kidney. Dietary MO increased TBARS in both zero-time and incubated homogenates of tissue from rats injected with iron. In contrast, dietary MO decreased hexanal and TOV in incubated tissue homogenates. The study demonstrated the usefulness and limitations of using hexanal and TOV as indices of lipid peroxidation.
Animals↗
Determination of methyl linoleate hydroperoxides by 13C nuclear magnetic resonance spectroscopy.
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Carbon Isotopes↗
Effect of alpha-tocopherol on the volatile thermal decomposition products of methyl linoleate hydroperoxides.
alpha-Tocopherol and 1,4-cyclohexadiene were tested for their effect on the thermal decomposition of methyl linoleate hydroperoxide isomers. The volatiles generated by thermolysis in the injector port of a gas chromatograph at 180 degrees C were analyzed by capillary gas chromatography. In the presence of either alpha-tocopherol or 1,4-cyclohexadiene, which are effective donors of hydrogen by radical abstraction, volatile formation decreased in all tests, and significant shifts were observed in the relative distribution of products in certain hydroperoxide samples. When an isomeric mixture of methyl linoleate hydroperoxides (cis, trans, and trans,trans 9- and 13-hydroperoxides) was decomposed by heat, the presence of alpha-tocopherol and 1,4-cyclohexadiene caused the relative amounts of pentane and methyl octanoate to decrease and hexanal and methyl 19-oxononanoate to increase. A similar effect of alpha-tocopherol was observed on the distribution of volatiles formed from a mixture of the trans,trans 9- and 13-hydroperoxides. This effect of alpha-tocopherol was, however, insignificant with pure cis,trans 13-hydroperoxide of methyl linoleate. The decrease in total volatiles with the hydrogen donor compounds, alpha-tocopherol and 1,4-cyclohexadiene, indicates a suppression of homolytic beta-scission of the hydroperoxides, resulting in a change in relative distribution of volatiles. The increase in hexanal and methyl 9-oxononanoate at the expense of pentane and methyl octanoate in the presence of hydrogen donor compounds supports the presence of a heat-catalyzed heterolytic cleavage (also known as Hock cleavage), which seems to mainly affect the trans,trans isomers of linoleate hydroperoxides.
Antioxidants↗
Rapid headspace gas chromatography of hexanal as a measure of lipid peroxidation in biological samples.
A rapid, sensitive and convenient capillary gas chromatographic-headspace method was developed to determine hexanal as an important volatile decomposition product of hydroperoxides formed from n-6 polyunsaturated fatty acids in rat liver samples. Total volatiles were also determined as a measure of overall lipid peroxidation. Samples of headspace taken from sealed serum bottles incubated at 37 degrees C were injected into a gas chromatograph. It was possible to make 15 determinations per hour. This method is convenient because no special sample manipulations are necessary. The addition of 0.5 mM ascorbic acid prior to gas chromatographic analysis significantly increased hexanal production. The applicability of the method was demonstrated in studies of the effect of iron in the presence or absence of hydroperoxides of methyl linoleate and methyl linolenate and tert-butyl hydroperoxide on rat liver homogenates, slices and microsomes. A rapid silica cartridge chromatographic procedure was used to purify hydroperoxides from autoxidized methyl linoleate and methyl linolenate, and hydroperoxy epidioxides (cyclic peroxides) from autoxidized methyl linolenate in 20-40 mg quantities. The hydroperoxides and hydroperoxy epidioxides of methyl linolenate were effective inducers of n-6 polyunsaturated fatty acid peroxidation in liver homogenates. Hexanal and thiobarbituric acid-reacting substances were significantly correlated in liver homogenates and microsomes but not in slices. This specific method for hexanal, a known product of peroxidation of n-6 polyunsaturated fatty acids, can be used as a good measure of lipid peroxidation.
Aldehydes↗
Effect of dietary lipids and vitamin E on in vitro lipid peroxidation in rat liver and kidney homogenates.
Rats were fed for 5 wk 10% (wt/wt) menhaden oil (MO) or a 10% corn oil-lard (COL) mixture (1:1) in diets with a low vitamin E content (less than or equal to 5 mg/kg) or supplemented with d-alpha-tocopheryl succinate to a total of 30 or 150 mg per kg. Thiobarbituric acid-reactive substances (TBARS), conjugated dienes (CD), hexanal and total volatiles (TOV) were measured in tissue homogenates incubated at 37 degrees C for 1 h in the absence (uninduced) and presence of 15 microM ferrous sulfate (induced). The fatty acid composition of liver and kidney reflected that of dietary lipids. For uninduced peroxidation, there was in general a significant inverse correlation of TBARS, CD and TOV with the log of dietary vitamin E content for liver and kidney from rats fed either lipid. For induced peroxidation, the inverse correlation was significant for liver, but not for kidney, from rats fed either lipid. The correlation was generally higher for liver and kidney from rats fed COL than for tissues from rats fed MO. Vitamin E was thus a more effective antioxidant for liver than for kidney regardless of the dietary lipid, and for liver and kidney from rats fed COL than from rats fed MO. Dietary MO enhanced tissue susceptibility to both peroxidation systems. A simulation model developed to mimic the experiments showed good correlations between experimental data and simulated values.
Analysis of Variance↗