β-Oxidation of conjugated linoleic acid isomers and linoleic acid in rats

To assess the oxidative metabolism of conjugated linoleic acid (CLA) isomers, rats were force-fed 1.5–2.6 MBq of [1-¹⁴C]-linoleic acid (9c,12c-18∶2),-rumenic acid (9c,11t-18∶2), or-10trans, 12cis-18∶2 (10t, 12c-18∶2), and ¹⁴CO₂ production was monitored for 24 h. The animals were then necropsied and the radioactivity determined in different tissues. Both CLA isomers were oxidized significantly more than linoleic acid. Moreover, less radioactivity was recovered in most tissues after CLA intake than after linoleic acid intake. The substantial oxidation of CLA isomers must be considered when assessing the putative health benefits of CLA supplements.     

Dietary comparison of conjugated linolenic acid (9 cis, 11 trans, 13 trans) and α-tocopherol effects on blood lipids and lipid peroxidation in alloxan-induced diabetes mellitus in rats

The present study investigated the dietary effect of conjugated linolenic acid (CLnA) on lipid profiles and lipid peroxidations in alloxan-induced diabetes mellitus in rats. Diabetic rats were fed with 20% sunflower oil (diabetic control), sunflower oil supplemented with 0.5% CLnA, sunflower oil supplemented with 0.15% α-tocopherol, and sunflower oil containing 0.25% CLnA+0.15% α-tocopherol. The results demonstrated that 0.5% CLnA, 0.15% α-tocopherol, and 0.25% CLnA+0.15% α-tocopherol each on supplementation significantly lowered total cholesterol and non-HDL-cholesterol in comparison with the diabetic control group. The TAG level was significantly lowered in both the 0.15% α-tocopherol and 0.25% CLnA+0.15% α-tocopherol groups. LDL lipid peroxidation and erythrocyte membrane lipid peroxidation were reduced significantly in each of the experimental groups vs. the control group. The CLnA+α-tocopherol diet induced a greater reduction in membrane lipid and liver lipid peroxidation than the α-tocopherol diet alone. In conclusion, dietary CLnA exerts antioxidant activity as evidenced by reduced lipid peroxidation in chemically induced diabetes mellitus.     

Antioxidant synergy of α-tocopherol and phospholipids

The prevention of oxidation of a refined sardine oil by α-tocopherol at 0.04%, by several phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL)] at 0.5%, as well as by combinations of α-tocopherol with each phospholipid, was investigated. The evolution of the oxidation process during 1 mon at 40±2°C was followed by a series of methods, measuring peroxide value (PV), diene, triene, and polyene index, and absorbance at 430 nm, while α-tocopherol and phospholipid content were being monitoried. Among these indices, PV was found to be the most adequate to follow the process. PC was the most effective individual antioxidant as shown by the PV values obtained at the end of the storage period, which were 54.0, 83.4, 87.9, and 97.7 meq O₂/kg for PC, CL, PE, and α-tocopherol, respectively. The highest synergistic effect was obtained with a mixture of α-tocopherol and PE, and the second and third best by mixtures made with PC and CL, respectively. The corresponding PV values recorded at the end of the period were 27.0, 35.0, and 58.0 meq O₂/kg. The high degree of synergy between PE and tocopherol is probably due to the occurrence of a simultaneous antioxidant mechanism involving Maillard compounds.     

The effect of α-linolenic acid and linoleic acid on the growth and development of formula-fed infants: A systematic review and meta-analysis of randomized controlled trials

This systematic review and meta-analysis aimed to evaluate the effect of modifying 18-carbon PUFA [18-C PUFA: α-linolenic acid (ALA, 18∶3n−3) and linoleic acid (LA, 18∶2n−6)] in the diets of term and preterm infants on DHA (22∶6n−3) status, growth, and developmental outcomes. Only randomized controlled trials (RCT) involving formula-fed term and preterm infants, in which the 18-C PUFA composition of the formula was changed and growth or developmental outcomes were measured, were included. Differences were presented as control (standard formula) and treatment (18-C PUFA-supplemented formula). Primary analyses for term infants were 4 and 12 mon and for preterm infants 37–42 and 57 wk postmenstrual age. Five RCT involving term infants and three RCT involving preterm infants were included in the systematic review. Infants fed ALA-supplemented formula had significantly higher plasma and erythrocyte phospholipid DHA levels than control infants. There was no effect of ALA supplementation on the growth of preterm infants. In term infants, ALA supplementation was associated with increased weight and length at 12 mon, which was at least 4 mon after the end of dietary intervention. Developmental indices of term infants did not differ between groups. There was a transient improvement in the retinal function of preterm infants fed ALA-supplemented diets compared with controls. The findings suggest that ALA-supplemented diets improve the DHA status of infants. Further studies are needed to provide convincing evidence regarding the effects of ALA supplementation of formula on infant growth and development.     

Effects of margarine and butter consumption on distribution of trans-18∶1 fatty acid isomers and conjugated linoleic acid in major serum lipid classes in lactating women

Trans FA (TFA) have at least one trans double bond and comprise several isomers and types, including many of the CLA (e.g., c9, t11–18∶2 CLA). Some TFA may have adverse effects (e.g., cardiovascular disease), whereas some are though to have beneficial effects (e.g., anticarcinogenicity). The presence of TFA in human tissues and fluids is related to dietary intake, although this relationship is not completely understood—especially in regard to serum lipid fractions. This study was conducted as part of an investigation designed to test the influence of butter (B), “low TFA” margarine (LT), and regular margarine (RM) on milk fat content. Here we tested the secondary hypothesis that consumption of B, LT, and RM by lactating women would result in differential distribution of TFA and CLA in major serum lipid classes. Breastfeeding women (n=11) participated in this randomized Latinsquare study consisting of five periods: intervention I (5 d), washout I (7 d), intervention II (5 d), washout II (7 d), and intervention III (5 d). Extracted serum lipid was separated into cholesterol ester (CE), TAG, and phospholipid (PL) fractions and analyzed for total and isomeric TFA and CLA concentrations. Data indicate that TAG consistently contained the highest concentration of total t-18∶1. No interaction between treatment and fraction was found for any of the t-18∶1 isomers identified. Absolute concentration of each t-18∶1 isomer was greatest during the RM period, regardless of fraction. On a relative basis, concentrations of t10–18∶1 and t12–18∶1 were most responsive to treatment in the CE fraction. The concentration of c9, t11–18∶2 CLA was highest in the TAG fraction and lowest in the PL fraction, regardless of treatment. In summary, these results indicate (i) that there is a differential distribution of some isomeric TFA and CLA among human serum lipid fractions and (ii) that dietary TFA intake influences absolute and relative concentrations of some of the isomers in selected fractions.     

Large-scale purification of γ-linolenic acid by selective esterification using Rhizopus delemar lipase

γ-Linolenic acid (GLA) is a physiologically valuable fatty acid, and is desired as a medicine, but a useful method available for industrial purification has not been established. Thus, large-scale purification was attempted by a combination of enzymatic reactions and distillation. An oil containing 45% GLA (GLA45 oil) produced by selective hydrolysis of borage oil was used as a starting material. GLA45 oil was hydrolyzed at 35°C in a mixture containing 33% water and 250 U/g-reaction mixture of Pseudomonas sp. lipase; 91.5% hydrolysis was attained after 24 h. Film distillation of the dehydrated reaction mixture separated free fatty acids (FFA; acid value 199) with a recovery of 94.5%. The FFA were selectively esterified at 30°C for 16 h with two molar equivalents of lauryl alcohol and 50 U/g of Rhizopus delemar lipase in a mixture containing 20% water. The esterification extent was 52%, and the GLA content in the FFA fraction was raised to 89.5%. FFA and lauryl esters were not separated by film distillation, but the FFA-rich fraction contaminated with 18% lauryl esters was recovered by simple distillation. To further increase the GLA content, the FFA-rich fraction was selectively esterified again under similar conditions. As a result, the GLA content in the FFA fraction was raised to 97.3% at 15.2% esterification. After simple distillation of the reaction mixture, lauryl esters contaminating the FFA-rich fraction were completely eliminated by urea adduct fractionation. When 10 kg of GLA45 oil was used as a starting material, 2.07 kg of FFA with 98.6% GLA was obtained with a recovery of 49.4% of the initial content.     

Preparation and fractionation of conjugated trienes from α-linolenic acid and their growth-inhibitory effects on human tumor cells and fibroblasts

Conjugated α-linolenic acid (ClnA) was prepared from α-linolenic acid (9,12,15–18∶3n−3, LnA) by alkaline treatment; we fractionated CLnA into three peaks by reversed-phase column-HPLC as evidenced by monitoring absorbance at 205, 235, and 268 nm. Peak I was a conjugated dienoic FA derived from LnA, whereas Peaks II and III were conjugated trienoic LnA. Proton NMR analysis showed that Peak III consisted of the all-trans isomer. The methylated Peak III was further divided into five peaks (Peaks IV–VIII) by silver ion column-HPLC. Peak V, a major constituent in the Peak III fraction, was identified as conjugated 10t,12t,14t-LnA by GC-EIMS and ¹H NMR analysis. Peaks III and V, which consisted of conjugated all-trans trienoic LnA, had stronger growth-inhibitory effects on human tumor cell lines than the other collected peaks and strongly induced lipid peroxidation as compared with Peaks I, II, and LnA. We propose that conjugated all-trans trienoic FA have the strongest growth-inhibitory effect among the conjugated trienoic acids and conjugated dienoic acids produced by alkaline treatment of α-LnA, and that this effect is mediated by lipid peroxidation.     

Mechanisms of dimer and trimer formation from ultraviolet-irradiated α-tocopherol

α-Tocopherol (α-TH) undergoes ultraviolet (UV)-induced photooxidation on the surface of mouse skin to produce a dihydroxydimer, a spirodimer, and trimers as the major products. To study the photochemistry involved, we UV-irradiated α-TH in a thin film on a glass petri dish. Photooxidation yielded a mixture of dihydroxydimer, spirodimer, and trimers. In the time-course studies, the dihydroxydimer accumulated and then was further oxidized, whereas the spirodimer and trimers accumulated more gradually. Reaction of two tocopheroxyl radicals forms the dihydroxydimer, whereas the spirodimer may be formed either by photooxidation of α-TH to an orthoquinone methide (o-QM) followed by a Diels-Alder reaction or by photooxidation of α-TH to the dihydroxydimer, followed by two-electron oxidation. Irradiation of a mixture of d ₁₀-labeled and unlabeled (d ₀) dihydroxydimer produced a mixture of labeled and unlabeled spirodimers as detected by positive atmospheric pressure chemical ionization-mass spectrometry. The absence of mixed label spirodimers among products indicated that direct oxidation of the dihydroxydimer is a facile route to the spirodimer and is probably the major spirodimer-forming reaction in α-TH photooxidations. Trimer formation from the dihydroxydimer and the spirodimer was observed, however, and requires an o-QM intermediate. Photooxidation of d ₁₀-labeled and unlabeled (d ₀) dihydroxydimers yielded mixed isotopomers of the trimer products, thus demonstrating that the dihydroxydimer and spirodimers underwent conversion to o-QM intermediates. Photochemical conversion of α-TH to UV-absorbing dimer and trimer products may contribute to photoprotection by topically applied α-TH.     

Protection of α-tocopherol in nonpurified and purified fish oil

Menhanden oil was purified by column chromatography to remove minor components. The effect of α-tocopherol (α TOH) (50–500 ppm) on the rate of formation of hydroperoxides in the original menahaden oil and in the purified menhaden triacylglycerol (TAG) fraction was studied at 30°C in the dark. An increase in the initial rate of formation of hydroper-oxides was observed at αTOH concentrations above 100 ppm in both substrates. The original menhaden oil oxidized more rapidly than the purified menhaden, TAG at all antioxidant levels tested, and the presence of minor components in the menhaden oil was found to contribute only to a limited extent to the peroxidizing effect of αTOH. The αTOH did not display any prooxidant activity at either of the concentrations tested when the control oil was the purified menhaden TAG. Addition of ascorbyl palmitate eliminated the initial peroxidizing effect of αTOH, and this emphasizes the participation of the α-toco-pheroxyl radical in the reactions causing an accumulation of hydroperoxides at high concentrations of αTOH. Presented in part at the Annual Meeting of the American Oil Chemists’ Society in San Diego, April 25–28, 2000.     

What is the role of α-linolenic acid for mammals?

This review examines the data pertaining to an important and often underrated EFA, α-linolenic acid (ALA). It examines its sources, metabolism, and biological effects in various population studies, in vitro, animal, and human intervention studies. The main role of ALA was assumed to be as a precursor to the longer-chain n-3 PUFA, EPA and DHA, and particularly for supplying DHA for neural tissue. This paper reveals that the major metabolic route of ALA metabolism is β-oxidation. Furthermore, ALA accumulates in specific sites in the body of mammals (carcass, adipose, and skin), and only a small proportion of the fed ALA is converted to DHA. There is some evidence that ALA may be involved with skin and fur function. There is continuing debate regarding whether ALA has actions of its own in relation to the cardiovascular system and neural function. Cardiovascular disease and cancer are two of the major burdens of disease in the 21st century, and emerging evidence suggests that diets containing ALA are associated with reductions in total deaths and sudden cardiac death. There may be aspects of the action and, more importantly, the metabolism of ALA that need to be elucidated, and these will help us understand the biological effects of this compound better. Additionally, we must not forget that ALA is part of the whole diet and should be seen in this context, not in isolation.     

Preparation, separation, and confirmation of the eight geometrical cis/trans conjugated linoleic acid isomers 8,10-through 11,13–18∶2

Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or I₂ catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8,10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18∶2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag⁺-HPLC) and gas chromatography (GC). Ag⁺-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12–18∶2. However, one of the 8,10 isomers (8cis, 10trans-18∶2) coeluted with the 9trans,11cis18∶2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag⁺-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13–18∶2 geometric isomers (trans,cis before cis,trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by GC. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18∶1 18∶2, and 18∶3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.     

Effects of α-tocopherol and ascorbyl palmitate on the isomerization and decomposition of methyl linoleate hydroperoxides

In order to study antioxidant action on lipid hydroperoxide decomposition, the effects of α-tocopherol (TOH) and ascorbyl palmitate on the decomposition rate and reaction sequences of 9- and 13-cis,trans methyl linoleate hydroperoxide (cis,trans ML-OOH) decomposition in hexadecane were studied at 40°C. Decomposition of cis,trans ML-OOH as well as the formation and isomeric configuration of methyl linoleate hydroxy and ketodiene compounds were followed by high-performance liquid chromatographic analysis. TOH effectively inhibited the decomposition of ML-OOH. The decomposition rate was two times slower at 0.2 mM and more than 10 times slower at 2 and 20 mM of TOH. Ascorbyl palmitale (0.2, 2, and 20 mM) slightly accelerated the decomposition of ML-OOH. Both compounds had an effect on the reaction sequences of ML-OOH decomposition. At high levels TOH inhibited the isomerization of cis,trans ML-OOH to trans,trans ML-OOH through peroxyl radicals and increased the formation of hydroxy compounds. Further, the majority of the hydroxy and ketodiene compounds formed had a cis,trans configuration, indicating that cis,trans ML-OOH decomposed through alkoxyl radicals without isomerization. These results suggest that when inhibiting the decomposition of hydroperoxides, TOH can act as a hydrogen atom donor to both peroxyl and alkoxyl radicals. In the presence of ascorbyl palmitate, cis,trans ML-OOH decomposed rapidly but without isomerization. In contrast to TOH, the majority of hydroxy compounds were cis,trans, but the ketodiene compounds were trans,trans isomers. This indicates that ascorbyl palmitate reduced cis,trans ML-OOH to the corresponding hydroxy compounds. However, the simultaneous formation of trans,trans ketodiene compounds suggests that ML-OOH decomposition, similar to the control sample, also occurred in these samples. Thus, under these experimental conditions, the reduction of ML-OOH to more stable hydroxy compounds did not occur to an extent significant enough to inhibit the radical chain reactions of ML-OOH decomposition.     

Effect of temperature and addition of α-tocopherol on the oxidation of trilinolein model systems

The effects of temperature and addition of α-tocopherol were evaluated in trilinolein model systems through quantification of oxidized TAG monomers, dimers, and polymers following oxidation at different temperatures. Samples of trilinolein without and with 250 and 500 mg/kg α-tocopherol added were stored at 25, 60, and 100°C. Quantification of oxidized monomers, dimers, and polymers by a combination of adsorption and exclusion chromatography provided a useful measurement for studying the evolution of oxidation. Results showed that the amounts of primary oxidation compounds (trilinolein oxidized monomers) that accumulated during the induction period decreased as the temperature increased, indicating that the slope of the initial linear stage of oxidation depended on temperature. The end of the induction period was marked by a sharp increase in the levels of total oxidation compounds, the initiation of polymerization, and the loss of α-tocopherol. Addition of α-tocopherol did not prevent, but rather delayed, formation of trilinolein oxidized monomers and the initiation of polymerization.     

Enzymatic esterification of (−)-menthol with lauric acid in isooctane by sorbitan monostearate-coated lipase from Candida rugosa

Esterification of (−)-menthol and (±)-menthol with lauric acid in isooctane was successfully catalyzed by a commercial nonioic surfactant (sorbitan monosterate)-coated lipase from Candida rugosa (Lipase AY “Amano” 30) at the molar ratio of 1∶1 and at 35°C using 1.5 g enzyme/g (−)-menthol and 0.1-g molecular sieves. After 1 h, molar conversion of (−)-menthol reached 81%. Equilibrium was reached after ca. 4 h, giving a (−)-menthol molar conversion of 94%. Under the same conditions, native lipase catalyzed the esterification of (−)-menthol and lauric acid to yield a molar conversion of 93% after 72 h. Coating the lipase with sorbitan monosterate increased the esterification rates of both (−)-menthol and (±)-menthol with lauric acid. After 6 h, the molar conversions of (−)-menthol and (±)-menthol were 94, and 62%, respectively.     

Gas chromatography—High resolution selected-ion mass spectrometric identification of trace 21∶0 and 20∶2 fatty acids eluting with conjugated linoleic acid isomers

High-resolution selected-ion recording (SIR) of the exact molecular ion mass was used to confirm unambiguously the presence of conjugated linoleic acid (CLA) derivatives in biological matrices and standard mixtures and to differentiate non-CLA derivatives from CLA derivatives in the CLA region of the gas chromatogram. The success of this method was based on the selectivity of the SIR technique and its sensitivity, which was comparable to that of flame-ionization detection. A minor fatty acid methyl ester (FAME) was identified as methyl heneicosanoate (21∶0), and six isomers of 20∶2 FAME were found to elute in the CLA region. Isomerization of a standard CLA mixture resulted in a non-CLA flame-ionization response eluting in the CLA region of the gas chromatogram. It is therefore recommended that the identification of minor CLA isomers in natural products of biological matrices should include their direct confirmation by mass spectrometry     

Formation of 8-oxo-2′-deoxyguanosine in the DNA of human diploid fibroblasts by treatment with linoleic acid hydroperoxide and ferric ion

Lipid peroxides are suggested to be related to the occurrence of a variety of diseases including cancer and atherosclerosis. We examined whether lipid peroxides cause oxidative damage to DNA in intact cells. Linoleic acid hydroperoxide (LOOH) and ferric chloride were used at concentrations at which separate treatment had no effect on the formation of 8-oxo-2′-deoxyguanosine (8-oxodG) in DNA or the survival rate of cultured human diploid fibroblasts, TIG-7. The amount of 8-oxodG in the cellular DNA increased significantly when TIG-7 cells were treated concurrently with LOOH and ferric chloride. In a LOOH concentration-dependent manner 8-oxodG was formed. However, no significant induction of the activities of superoxide dismutases, catalase, or glutathione peroxidase was observed under these conditions. The formation of 8-oxodG by lipid hydroperoxides seems to be due to the generation of reactive species other than superoxide radicals and hydrogen peroxide. These results indicate that some species formed during the reaction of lipid hydroperoxides with ferric ion can cause oxidative damage to DNA.     

Separation of α-ketoglutaric acid by salting-out extraction coupled with solar-driven distillation

Alpha-ketoglutaric acid (α-KG), as an essential intermediate in biosynthesis and drug synthesis, has a broad application prospect. However, the lower product concentration and impurities in the α-KG production make the downstream separation more complex and costly. In this study, α-KG was separated from biotransformation broth by salting-out extraction (SOE) combined with solar-driven distillation. First, the aqueous two-phase system (ATPS) consisting of acetone/(NH₄)₂SO₄ was selected by comparison. The effects of acetone/(NH₄)₂SO₄ concentration, temperature, α-KG concentration, and pH on the distribution behavior of α-KG in ATPS were investigated. Under the optimized conditions, higher extraction efficiency and purity of α-KG were obtained in the actual biotransformation broth. In addition, solar evaporation was used to achieve preconcentration of the organic phase and salt recovery, significantly reducing energy consumption. The method is environmentally friendly and easy to operate, providing a new idea for separating low concentration products in biosynthesis.     

Oxidation of α- and β-pinene catalyzed by MnIII (Salen) Cl·H2O

The cationic complex Mn^III (Salen) is a very effective catalyst for the oxidation of both- and-pinene. The higher selectivity towards epoxide formation supports the rebound oxygen mechanism. A turnover of 40 was obtained for both compounds after 16 h of reaction with a molar ratio 10.011 (feedstock: catalyst: iodosobenzene) and conversions between 50 and 60% were observed. A very high selectivity (55%) was determined for epoxide formation from -pinene. The good selectivity observed for myrtanal isomers (6.5 and 23.2%) from-pinene is related to the prior formation of the 1,2-epoxides.     

Preparation of equiaxed α-Al2O3 by adding oxalic acid

The preparation of α-Al₂O₃ powders with equiaxed architecture for the fabrication of advanced ceramics is of great importance but still challenging. A new and facile approach for the fabrication of equiaxed α-Al₂O₃ adopting alumina hydroxide and oxalic acid as the raw materials was reported in this paper for the first time. The current work demonstrated that the adding 0.16 mol/L oxalic acid solution made α-Al₂O₃ heterogeneously nucleate at a temperature as low as 800 °C, and the amount of nucleation was high enough to remove the vermicular microstructures during the morphology evolution of α-Al₂O₃, resulting in the formation of equiaxed α-Al₂O₃ particles with an average size of 205.72 nm at 1300 °C.