Sulfur-substituted and α-methylated fatty acids as peroxisome proliferator-activated receptor activators

FA with varying chain lengths and an α-methyl group and/or a sulfur in the β-position were tested as peroxisome proliferator-activated receptor (PPAR)α,-δ(β), and-γ ligands by transient transfection in COS-1 cells using chimeric receptor expression plasmids, containing cDNAs encoding the ligand-binding domain of PPARα,-δ, and-γ. For PPARα, an increasing activation was found with increasing chain length of the sulfur-substituted FA up to C₁₄-S acetic acid (tetradecylthioacetic acid=TTA). The derivatives were poor, and nonsignificant, activators of PPARδ. For PPARγ, activation increased with increasing chain length up to C₁₆-S acetic acid. A methyl group was introduced in the α-position of palmitic acid, TTA, EPA, DHA, cis9,trans11CLA, and trans10,cis12 CLA. An increased activation of PPARα was obtained for the α-methyl derivatives compared with the unmethylated FA. This increase also resulted in increased expression of the two PPARα target genes acyl-CoA oxidase and liver FA-binding protein for α-methyl TTA, α-methyl EPA, and α-methyl DHA. Decreased or altered metabolism of these derivatives in the cells cannot be excluded. In conclusion, saturated FA with sulfur in the β-position and increasing carbon chain length from C₉−S acetic acid to C₁₄−S acetic acid have increasing effects as activators of PPARα and-γ in transfection assays. Furthermore, α-methyl FA derivatives of a saturated natural FA (palmitic acid), a sulfur-substituted FA (TTA), and PUFA (EPA, DHA, c9,t11 CLA, and t10,c12 CLA) are stronger PPARα activators than the unmethylated compounds.     

An <Emphasis Type="Italic">in vitro</Emphasis> method for studying the proliferation and differentiation of Atlantic salmon preadipocytes

The aim of the present study was to develop a cell culture system for studying the proliferation and differentiation of preadipocytes isolated from Atlantic salmon adipose tissue. The expression of proliferating cell nuclear antigen (PCNA) was used as a marker for cell proliferation. The cells started to proliferate within 48 h after seeding and continued to proliferate throughout the culture period of 2 wk. Undifferentiated preadipocytes showed a fibroblast-like morphology with a homogeneous cytoplasm devoid of lipid droplets. At confluence, an exogenous lipid mixture was added to the cell cultures. The preadipocytes became larger and rounder during the subsequent days, and the cytoplasm gradually filled with lipid-rich droplets. These droplets were revealed by oil red O staining. Immunocytochemical staining showed that differentiated adipocytes expressed detectable levels of the three regulatory proteins associated with adipocyte differentiation: peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), and leptin. The cells also showed activity of glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8), a biochemical marker of adipocyte differentiation. The morphological and biochemical data presented here show that fish preadipocytes have properties that are similar to those of preadipocytes in mammals. We conclude therefore that salmon adipose tissue contains a sizable population of preadipocytes. Exogenous lipids promote the activation of adipose-related genes and induce the differentiation of fish preadipocytes in vitro.     

Inhibition of oxidation in 10% oil-in-water emulsions by β-carotene with α- and γ-tocopherols

The effects of low concentrations of β-carotene, α-, and γ-tocopherol were evaluated on autoxidation of 10% oil-in-water emulsions of rapeseed oil triacylglycerols. At concentrations of 0.45, 2, and 20 μg/g, β-carotene was a prooxidant, based on the formation of lipid hydroperoxides, hexanal, or 2-heptenal. In this emulsion, 1.5, 3, and 30 μg/g of γ-tocopherol, as well as 1.5 μg/g of α-tocopherol, acted as antioxidants and inhibited both the formation and decomposition of lipid hydroperoxides. Moreover, at a level of 1.5 μg/g, γ-tocopherol was more effective as an antioxidant than α-tocopherol. At levels of 0.5 μg/g, both α- and γ-tocopherol significantly inhibited the formation of hexanal but not the formation of lipid hydroperoxides. Oxidation was effectively retarded by combinations of 2 μg/g β-carotene and 1.5 μg/g γ- or α-tocopherol. The combination of β-carotene and α-tocopherol was significantly better in retarding oxidation than α-tocopherol alone. While γ-tocopherol was an effective antioxidant, a synergistic effect between β-carotene and γ-tocopherol could not be shown. The results indicate that there is a need to protect β-carotene from oxidative destruction by employing antioxidants, such as α- and γ-tocopherol, should β-carotene be used in fat emulsions.     

Unsaturated lipid peroxidation catalyzed by hematin compounds and its inhibition by vitamin E1

Evidence favoring hematin catalysis over autoxidation as the dominant mechanism of lipid peroxidation in animal tissues is presented. Lipid peroxidation in Erlich ascites tumor cells and isolated electron transport particles has been studied. Random destruction of the cytochromes and a loss of catalytic activity correlate with peroxidation of the electron transport particle. Mixtures of α-, β-, and γ-tocopherols show no synergistic effect. Synergism with ascorbate and citrate greatly enhance the antioxidant activity of α-tocopherol. A tocopherol-ascorbate-glutathione-triphosphopyridine nucleotide couple could act synergistically to inhibit lipid peroxidation in animal tissues. Supported in part by the Bureau of Commercial Fisheries.     

Peroxisome proliferator-activated receptor α is not the exclusive mediator of the effects of dietary cyclic FA in mice

Cyclic FA monomers (CFAM) formed during heating of α-linolenic acid have been reported to interfere in hepatic metabolism in a putatively peroxisome proliferator-activated receptor α (PPARα)-dependent manner. In the present work, CFAM (0.5% of the diet) were administered for 3 wk to wild-type and PPARα-null mice of both genders to elucidate the role of PPARα in mediating the effects of CFAM on the activity of acyl-CoA oxidase (ACO) and ω-laurate hydroxylase (CYP4A), the regulation of which is known to be dependent on the PPARα. Dietary CFAM enhanced CYP4A activity threefold in male and female wild-type mice. This effect was abolished in PPARα-null mice. A twofold induction of ACO activity was found in wild-type female mice fed CFAM; however, no effect was seen in males. In wild-type animals, (ω-1)-laurate hydroxylase (CYP2E1) activity, the expression of which has not been shown to be PPARα dependent, was not affected by the CFAM diet. In contrast, stearoyl-CoA desaturase activity was reduced in wild-type mice. CFAM feeding reduced the activities of ACO, CYP2E1, and stearoyl-CoA desaturase and caused accumulation of lipids in the livers of female PPARα-null mice. These data show that CFAM apparently activate gene expression via the PPARα and have profound effects on lipid homeostasis, exacerbating the disturbances preexisting in mice lacking functional PPARα. Although the data emphasize the importance of PPARα in the metabolism of the CFAM, these results show that PPARα is not the exclusive mediator of the effects of CFAM in lipid metabolism in mice.     

Distribution of α-and γ-tocopherols in Atlantic salmon (Salmo salar) tissues

Groups of Atlantic salmon parr (mean initial weight 9.5 g) were fed three diets, the first containing no tocopherol supplement, the others supplemented with either all-rac-α-tocopherol (A-T) or RRR-γ-tocopherol (G-T). Tocopherol concentrations in the liver, serum, testes, kidney, brain, gill, muscle, and perivisceral fat were measured after 36 wk. Despite a higher dietary intake of G-T, compared to A-T, deposition of γ-tocopherol (γT) was less efficient than of α-tocopherol (αT) in most tissues except in the perivisceral fat, an adipose tissue. In fish fed the G-T diet, the γT/αT ratio was highest in the perivisceral fat and lowest in the liver, indicating that the liver is the most discriminatory organ for retaining αT as compared to γT, and the perivisceral fat is more suitable for the storage of γT. A negative correlation (P<0.01) was observed between the γT/αT ratio and the corresponding tissue phospholipid content, suggesting that γT is less efficiently deposited compared to αT in the phospholipid-rich membranes which are presumed to be the functional site for lipid antioxidants in vivo. During restricted intake of αT, the liver and muscle exhibited the greatest reduction of this tocopherol among the tissues analyzed. The presence of minimal αT in the muscle from fish fed the tocopherol-unsupplemented diet led to greater susceptibility to lipid peroxidation after frozen storage than was the case for muscle containing higher concentrations of either αT or γT. However, both αT and γT were effective stabilizers of salmon muscle lipids during frozen storage. Presented in part at the Annual Meeting of the American Oil Chemists' Society, San Antonio, Texas, May 1995.     

Solvent crystallization of α, β and γ polymorphs of oleic acid

The features of the solvent crystallization of α, β and γ polymorphs of ultra-pure oleic acid were examined in acetonitrile and decane. The solubilities of the three polymorphs were measured precisely, yielding a lower value for β than for the other two forms at any temperature. Thesolution- mediated transformation either from α to β or from γ to β was observed directly in the nearly saturated solution. It was found that this transformation occurred predominantly via the nucleation of the stable β form at the expense of the less stable ones. Finally the habit of the single crystal of each polymorph was determined for the first time. The results obtained were compared to the similar experiments of stearic acid polymorphs.     

Polymorphic transformation of 1,3-distearoyl-sn-2-linoleoyl-glycerol

Polymorphic transformation behavior of sub-α₁, sub-α₂, α, and γ in 1,3-distearoyl-sn-2-linoleoyl-glycerol (SLS) has been studied with X-ray diffraction, differential scanning caloremetry, and Fourier-transform infrared spectroscopy. Synchrotron radiation X-ray beam was employed to observe rapid transformation processes from the sub-α and α forms to the γ form. The chain length structures were double in sub-α₁, sub-α₂, and α, whereas γ was of triple chain-length structure. The subcell packing was pseudohexagonal for the two sub-α forms, hexagonal for the α form, and parallel type for the γ form. In comparison with 1,3-distearoyl-sn-2-oleoyl-glycerol (SOS), the occurrence behavior of sub-α, α, and γ of SLS was the same as that of SOS. However, the absence of β′ and β was unique for SLS. The chain-chain interactions between the linoleoyl moieties may stabilize the γ form, prohibiting the transformation into β′ and β forms. The presence of two cis double bonds may cause this stabilization, revealing the disordered chain conformation of the unsaturated chains.     

Epidermal Lipoxygenase Products of the Hepoxilin Pathway Selectively Activate the Nuclear Receptor PPARα

Arachidonic acid can be transformed into a specific epoxyalcohol product via the sequential action of two epidermal lipoxygenases, 12R-LOX and eLOX3. Functional impairment of either lipoxygenase gene (ALOX12B or ALOXE3) results in ichthyosis, suggesting a role for the common epoxyalcohol product or its metabolites in the differentiation of normal human skin. Here we tested the ability of products derived from the epidermal LOX pathway to activate the peroxisome proliferator-activated receptors PPARα, γ, and δ, which have been implicated in epidermal differentiation. Using a dual luciferase reporter assay in PC3 cells, the 12R-LOX/eLOX3-derived epoxyalcohol, 8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid, activated PPARα with similar in potency to the known natural ligand, 8S-hydroxyeicosatetraenoic acid (8S-HETE) (both at 10 μM concentration). In contrast, the PPARγ and PPARδ receptor isoforms were not activated by the epoxyalcohol. Activation of PPARα was also observed using the trihydroxy hydrolysis products (trioxilins) of the unstable epoxyalcohol. Of the four trioxilins isolated and characterized, the highest activation was observed with the isomer that is also formed by enzymatic hydrolysis of the epoxyalcohol. Formation of a ligand for the nuclear receptor PPARα may be one possibility by which 12R-LOX and eLOX3 contribute to epidermal differentiation.     

Fatty acid metabolism and cell proliferation. VII. Antioxidant effects of tocopherols and their quinones

The antioxidant capacities of α- and γ-tocopherols (α-E and γ-E) and their quinones (α-EQ and γ-EQ) were determined in non-biological and biological systems. The non-biological system consisted of arachidonic acid [20∶4 (n−6)], the oxidant cumene hydroperoxide, and a Fe³⁺ catalyst to facilitate malondialdehyde (MDA) formation from lipid peroxides. α-E and γ-E had similar antioxidant capacities in this system. α-EQ also functioned as an antioxidant, while γ-EQ exhibited a crossover effect by functioning as an antioxidant at low concentrations and a prooxidant at high concentrations. Biological lipid peroxidation in smooth muscle cells challenged with 20∶4 (n−6) was measured both by MDA formation in confluent cultures and by cell growth in proliferating cultures. α-E, γ-E and α-EQ had similar antioxidant capacities, but γ-EQ was highly cytotoxic for cells in both confluent and proliferating cultures. Cellular retention of antioxidants was estimated indirectly from MDA formation when cells were loaded with an antioxidant (preincubation) and then incubated for varying periods of time in fresh media containing 20∶4 (n−6). Cellular retention also was measured directly with tritiated α-E and tritiated αEQ. These studies showed that cellular retention decreased in the sequence γ-E>α-E>α-EQ. Thus, cellular retention does not explain the enhanced antioxidant capacity of α-E compared to γ-E that has been reported for animal systems. The antioxidant capacity of αE evidently is enhanced by its metabolism to a quinone which, unlike the quinone from γ-E, functions as a biological antioxidant.     

Antioxidant activities of tocopherols on Fe2+-ascorbate-induced lipid peroxidation in lecithin liposomes

The antioxidant activities of 4 tocopherols, tocol, and a water-soluble model analog of α-tocopherol were compared. Egg lecithin liposomes were used and oxidation was catalyzed by Fe²⁺-ascorbate. The activities decreased in the order α->β->γ->δ-tocopherol>tocol, in agreement with their potencies in vivo. The water-soluble analog was the least effective. Activity depended on the molar ratio of antioxidant to unsaturated lipid, with one molecule each of the α-, β-, γ-, δ-tocopherol and tocol capable of protecting, respectively, 220, 120, 100, 30 and 20 molecules of polyunsaturated fatty acid. The mechanism of possible antioxidant effect of the compounds used is discussed.     

Polymorphism of POP and SOS. I. occurrence and polymorphic transformation

The polymorphic modifications of POP and SOS were identified with X-ray diffraction (XRD), DSC and Raman spectroscopy by using pure samples (99.9%). In POP, six polymorphs, α,γ, pseudo-β′₂, pseudo-β′₁, β′₂ and β′₁, were obtained, whereas five polymorphs, α, γ, pseudo-β′, β₂ and β₁, were isolated in SOS. Thermodynamic stability increased from α to β₁ straightforwardly both in POP and SOS, because the polymorphic transformation went monotropically in the order described above. Additionally, the 99.2% sample of POP crystallized another form, δ, but the 99.9% sample did not, implying subtle influences of the impurity. The four forms, α, γ, β₂ and β₁, of POP, revealed XRD and DSC patterns identical to the four forms of SOS designated by the same symbols. The chain length structure was double inα and triple in the other three forms in both POP and SOS. Peculiarity of POP was revealed partly in the chain length structure of pseudo-β′₂ and pseudo-β′₁ which were double, whereas pseudo-β′ of SOS was triple. This apparently showed contrast to the facts that the three forms revealed rather similar XRD short spacing patterns. Another peculiarity of POP was revealed in enthalpy value of the melt crystallization of α: ΔH_c (α) = 68.1 kJ/mol which was much larger than that of SOS (47.7 kJ/mol), and also than AOA and BOB. These peculiarities mean that the double chain length structures of POP are more stabilized than the others. Raman bands of CH₂ scissoring mode of SOS indicated parallel packing in γ, β₂ and β₁, and orthorhombic perpendicular packing in pseudo-β′. The polymorphic transformation mechanisms were discussed based on the proposed polymorphic structure models. Presented at the AOCS annual meetings in New Orleans, Louisiana in May 1987 and Phoenix, Arizona in May 1988.     

Relative autoxidative and photolytic stabilities of tocols and tocotrienols

The relative stabilities of selected individual tocols and tocotrienols and of equimolar mixtures of either α- plus γ- or α- plus δ- tocopherols were determined in methyl myristate and methyl linoleate during autoxidation and photolysis. Solutions containing 0.05% of the appropriate tocopherol(s) or tocotrienols were subjected to UV light (254 nm) or to a flow of 4.3 ml/min of oxygen, both at 70 C. Tocopherols (T) and tocotrienols (T−3) were determined by gas chromatography without preliminary separation or purification. Under photolytic conditions, stabilities in increasing order in methyl myristate were γ-T−3<α-T−3<δ-T<α-T <γ-T<5,7-T<β-T and in methyl linoleate were α-T<α-T−3≤γ-T−3≤β-T≤5,7-T <γ-T<δ-T. A solvent effect on the initial rate of photolysis was observed for 5-methyl substituted tocols but not for the tocols with an unsubstituted 5-position or for the tocotrienols. Under autoxidative conditions, stabilities in increasing order in methyl myristate were α-T=α-T−3 <β-T−3<γ-T−3<δ-T−3<γ-T<δ-T=β-T and in methyl linoleate were α-T<α-T−3 <γ-T−3<β-T<γ-T<δ-T. Tocopherols were much more stable during autoxidation in methyl myristate than they were in methyl linoleate. In mixtures, there was no significant protection of α-tocopherol by either γ- or δ-tocopherol under any of the conditions used. However, α-tocopherol was highly effective in protecting γ- and δ-tocopherols in methyl myristate during both photolysis and autoxidation and in methyl linoleate during photolysis. During autoxidation in methyl linoleate, α-tocopherol protection of γ- and δ- tocopherols after 24 hr was slight tough measurable.     

Tocopherol Content and Profile of Soybean: Genotypic Variability and Correlation Studies

Plant seed oils, including soybean seed oil, represent the major source of naturally derived tocopherols, the antioxidant molecules that act as free radical quenchers preventing lipid peroxidation in biological systems and vegetable oil products. All four isomers of tocopherols, i.e. α, β, γ, δ tocopherols that exist in nature are found in soybean seeds. The biological activity and the contribution of these isomers in improving the oxidative stability of vegetable oil are in reverse order. Because of the nutritive value and the importance for oil stability, enhancement of tocopherol content, through breeding programs, in soybean seeds has become a new and an important objective. Genotypic variability, which is the basis of every breeding program, is scarcely reported for tocopherol content and profile in soybean seeds. In the present investigation, the tocopherol content and profile in seed samples of 66 genotypes of Indian soybean were determined. The ratios observed between the lowest and the highest values for α, β, γ, δ, total tocopherol content were 1:13.6, 1:10.4, 1:7.5, 1:9.1, 1:7.9, respectively. The mean contents for α, β, γ, δ and total tocopherols were 269, 40, 855, 241 and 1,405 μg/g of oil, respectively. Total tocopherol content was the highest in ‘Co Soya2’ followed by ‘Ankur’. Concentration of α-tocopherol was the highest (27%) in ‘Ankur’ followed by ‘MACS124’ (26%) whereas gamma tocopherol concentration was the highest (69%) in ‘VLS1’ and ‘PK327’ followed by ‘MACS13’ (67%). In view of the fact that levels of unsaturated fatty acids, apart from tocopherols, also determine the oxidative stability of vegetable oils, the relationship of four isomers of tocopherols with each other as well as with different unsaturated fatty acids and oil content was also investigated in the present study. All the four isomers of tocopherols exhibited highly significant correlations with each other (p < 0.001) whereas γ-tocopherol and total tocopherol content showed a significant relationship with linoleic acid (p < 0.05).     

Crystallization and transformation mechanisms of α, β- and γ-polymorphs of ultra-pure oleic acid

Crystallization and transformation mechanisms of ultrapure (99.999%) oleic acid were examined by Differential Scanning Calorimetry (DSC) and X-ray diffraction. X-ray diffraction spectra revealed three different polymorphs newly named α, β and γ, which differ from each other most significantly in the short spacing spectra. α and β were found to be equivalent to the previous data which Lutton named low and high melting polymorphs, whereas γ was newly identified in the present study. DSC studies have clarified the thermodynamic stability of the three polymorphs in a range of temperature from -20 to 16.2 C. β is always most stable, whereas α and γ are metastable, undergoing a reversible first-order solid-state transformation at -2.2 C (on heating). DSC also showed that the crystallization behaviors are strongly dependent on the polymorphs; α crystallizes at a much higher rate than β; despite the fact that they have close melting points (α, 13.3 C; β, 16.2 C). It was demonstrated for the first time that the above peculiar polymorphic behaviors of oleic acid are quite different from those of stearic acid, a saturated fatty acid with the same carbon chain length. Presented at the AOCS meeting in Philadelphia, May 1985.     

Dietary Marine-Derived Tocopherol has a Higher Biological Availability in Mice Relative to Alpha-Tocopherol

The biologic availability of two kinds of tocomonoenols, marine-derived tocopherol (MDT) and α-tocomonoenol, was investigated in ICR mice. Vitamin E-deficient ICR mice were fed MDT and α-tocomonoenol together with α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, and storage in liver, spleen, lung, and brain was quantified using reverse-phase high-performance liquid chromatography. The vitamin E relative biologic availability (VE-RBA) in liver was 100 for α-tocopherol, 26 ± 3 for β-tocopherol, 4 ± 2 for γ-tocopherol, not detected for δ-tocopherol, 49 ± 6 for MDT, and 30 ± 7 for α-tocomonoenol. The VE-RBA in brain was 100 for α-tocopherol, 5 ± 2 for β-tocopherol, not detected for γ-tocopherol and δ-tocopherol, 8 ± 1 for MDT, and 4 ± 1 for α-tocomonoenol. Tocopherols and tocomonoenols did not accumulate in the spleen or lung. MDT and α-tocomonoenol had high VE-RBA values. The VE-RBA value for MDT was much higher than that for β-tocopherol.     

Binary phase behavior of 1,3-distearoyl-2-oleoyl-sn-glycerol (SOS) and 1,3-distearoyl-2-linoleoyl-sn-glycerol (SLS)

The binary phase behavior of SOS (1,3-distearoyl-2-oleoyl-sn-glycerol) and SLS (1,3-distearoyl-2-linoleoyl-sn-glycerol) was examined by using DSC and conventional and synchrotron radiation X-ray diffraction. The solid-solution phases were observed in the metastable α and γ forms in all concentration ranges. Results indicated that the miscible γ form did not transform to the β′ form when the mixtures were subjected to simple cooling from a high-temperature liquid to a low-temperature solid phase. However, and α-melt-mediated transformation into β′ and β₂ resulted in the formation of immiscible phases in concentration ranges of SLS below 30%. By contrast, at SLS concentration ranges above 30%, the α-melt-mediated transformation caused crystallization of only the γ form, and β′ and β₂ crystals did not appear. These results show that the specific interactions between SOS and SLS are operative in the phase behavior of the mixture states of SOS and SLS.