Characterization of γ-linolenic acid geometrical isomers in borage oil subjected to heat treatments (deodorization)

Heating of borage oil, either under vacuum as a model or during steam-vacuum deodorization, produces artifacts that are geometrical isomers of γ-linolenic acid (cis-6,cis-9,cis-12 18∶3 acid). In a first approach, we have studied the behavior of these fatty acids in the form of either methyl or isopropyl esters on two capillary columns (CP-Sil 88 and DB-Wax). From this study, it appears that the DB-Wax capillary column is the best suited analytical tool to study in some detail γ-linolenic acid geometrical isomers. In a second approach, the structure of these isomers was formally established by combining several analytical techniques: Argentation thin-layer chromatography, comparison of the equivalent chainlengths with those of isomers present in NO₂-isomerized borage oil on two different capillary columns, partial hydrazine reduction, oxidative ozonolysis, gas chromatography coupled with mass spectrometry and gas chromatography coupled with Fourier transform infrared spectroscopy. The two main isomers that accumulate upon heat treatments are thetrans-6,cis-9,cis-12 andcis-6,cis-9,trans-12 18∶3 acids with minor amounts ofcis-6,trans-9,cis-12 18∶3 acid. One di-trans isomer, supposed to be thetrans-6,cis-9,trans-12 18∶3 acid, is present in low although noticeable amounts in some of the heated oils. The content of these artificial fatty acids increases with increasing temperatures and duration of heating. The degree of isomerization (DI) of γ-linolenic acid is less than 1% when the oil is deodorized at 200°C for 2 h. Heating at 260°C for 5 h increases the DI up to 74%. Isomerization of γ-linolenic acid resembles that of α-linolenic (cis-9,cis-12,cis-15 18∶3) acid in several aspects: The same kinds and numbers of isomers are formed, and similar degrees of isomerization are reached when the octadecatrienoic acids are heated under identical conditions. It seems that the reactivity of a double-bondvis-à-vis cis-trans isomerization is linked to its relative position, central or external, and not to its absolute position (Δ6, 9, 12 or 15).     

Heated fat-based oil substitutes, oleic and linoleic acid-esterified propoxylated glycerol

Four oils [triolein, trilinolein, oleic acid-esterified propoxylated glycerol (EPG-08 oleate), and linoleic acid-esterified propoxylated glycerol (EPG-08 linoleate)], each without added antioxidants, were heated for 12 h/d at approximately 190°C in a small deep-fat fryer until the polymer concentration exceeded 20%, as determined by high-performance size-exclusion chromatography. Increases in the free fatty acid content, total acid value, food oil sensor value, and p-anisidine value during heating indicated that significant thermal oxidation had occurred in each oil. Capillary supercritical fluid chromatography (SFC) was used to determine the substrate concentration of each oil after each heating interval. The average, apparent first-order reaction rate constant (as determined by SFC) for trilinolein was 0.0348±0.0034 h⁻¹, while the rate for EPG-08 linoleate was 0.0253±0.0032 h⁻¹. The average apparent reaction rate constant for triolein was 0.0256±0.0011 h⁻¹, while the rate for EPG-08 oleate was 0.0252±0.0008 h⁻¹. Triolein contained >20% polymer after 60 h of heating, EPG-08 oleate contained >20% polymer after 36 h of heating, and both trilinolein and EPG-08 linoleate contained >20% polymer after 24 h of heating.     

Kinetic model of asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> CGMCC No.2266

The kinetic model of asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester using Saccharomyces cerevisiae CGMCC No.2266 with 10% glucose as co-substrate to realize the regeneration of NADPH was established. The effect factors on reduction, the type and the content of co-substrate and coenzyme, and the changes of the substrate and product content vs. time during the reaction process were investigated. The results indicate that 10% glucose can increase the reaction conversion from 23.0% to 98.4% and NADPH is reducer. The reduction process conforms with sequence mechanisms. The model parameters are as follows: v _m =5.0×10⁻⁴ mol·L⁻¹·h⁻¹, k₁=1.5×10⁻⁶ mol·L⁻¹·h⁻¹, k₂=3.0×10⁻³ mol·L⁻¹·h⁻¹. The kinetic model is in good agreement with the experimental data.     

Inhibition by cardiolipins of platelet-activating factor-induced rabbit platelet activation

Evidence is presented that cardiolipin, a naturally occurring phospholipid, inhibits the aggregatory effect of platelet-activating factor (paf) on rabbit plateletsin vitro. Bovine heart cardiolipin was shown to inhibit the aggregation of washed rabbit platelets induced by 1×10⁻¹⁰ M and 2×10⁻¹⁰ M paf with IC₅₀ values (doses for half-maximal inhibition) of 8.4±0.8×10⁻⁷ M and 2.6±0.6×10⁻⁶ M, respectively. Phosphonocardiolipin was also able to inhibit platelet aggregation induced by 1× 10⁻¹⁰ M paf with an IC₅₀ value of 3±1×10⁻⁷M. Both compounds, in concentrations up to 1×10⁻⁵ M, were unable to aggregate washed rabbit platelets and failed to inhibit the aggregation induced by 0.9 and 1.8 μM adenosine diphosphate or 0.2–1.0 μM arrchidonic acid. By contrast, the acetylated derivative of cardiolipin exerted an aggregatory effect on aspirin-treated rabbit platelets in the presence of creatine phosphate/creatine phosphokinase. This aggregation was inhibited by the specific paf antagonists BN 52021 and WEB 2086. Also, platelets treated with acetyl-cardiolipin were insensitive to the aggregatory effect of paf. Phosphatidic acid, phosphatidylglycerol,bis(dipalmitoylglycero)phosphate and their phosphono analogues were totally inactive. Similar data were obtained when platelet-rich plasma was used instead of washed rabbit platelets. Our results support the hypothesis that the effect of cardiolipin is mediated through specific paf receptors that act on the rabbit platelet membrane.     

Cis-trans isomerization of octadecatrienoic acids during heating. Study of pinolenic (cis-5,cis-9,cis-12 18∶3) acid geometrical isomers in heated pine seed oil

To understand the heat-inducedcis-trans isomerization of ethylenic bonds in octadecatrienoic acids, pine seed oil, which contains the unusual nonmethylene-interrupted pinolenic (cis-5,cis-9,cis-12 18∶3) acid as a major component, was heated under vacuum at 240°C for 6 h together with linseed and borage oils. As a results, a small percentage of pinolenic acid undergoescis-trans isomerization. The main isomer that accumulates is thetrans-5,cis-9,trans-12 18∶3 acid. Minor amounts of the three mono-trans isomers are also present. Identification of isomers was realized by combining gas-liquid chromatography on a CP Sil 88 capillary column, argentation thin-layer chromatography and comparing the equivalent chainlengths of artifacts to those of isomers present in NO₂-isomerized pine seed oil. Hydrazine reduction was used to demonstrate that there was no positional shift of double bonds. Heat-induced geometrical isomerization of pinolenic acid differs from that of α- and γ-linolenic acids in at least two aspects. The reaction rate is slower (about one-fourth), and mono-trans isomers are formed in low amounts.     

Reaction of dipyridamole with the hydroxyl radical

Dipyridamole [2,6-bis-diethanolamino-4,8-dipiperidinopyrimido-(5,4-d) pyrimidine], a well known platelet aggregation inhibitor, shows powerful hydroxyl radical scavenging activity by inhibiting OH-dependent salicylate and deoxyribose degradation. Steady-state competition kinetics experiments with deoxyribose were carried out to evaluate the second-order rateconstant for the reaction between hydroxyl radical and dipyridamole. OH· radicals were generated either by a Fenton-type reaction or by X-ray irradiation of water solutions. A second-order rate constant k_{(Dipyridamole+OH·)} of 1.72±0.11×10¹⁰M⁻¹ s⁻¹ and of 1.54±0.15×10¹⁰ M⁻¹ s⁻¹ was measured by Fenton chemistry and by radiation chemistry, respectively. Mannitol was used as an internal standard for hydroxyl radicals in steady-state competition experiments with deoxyribose. A rate constant k_{(Mannitol+OH·)} of 1.58±0.13×10⁹ M⁻¹ s⁻¹ and 1.88±0.14×10⁹ M⁻¹ s⁻¹ was measured in the Fenton model and in the water radiolysis system, respectively. Both these rate constants are in good agreement with the published data obtained by the “deoxyribose assay” and by pulse radiolysis.     

Effect of temperature on linolenic acid loss and 18:3 Δ9-cis, Δ12-cis, Δ15-trans formation in soybean oil

Oil was extracted from soybeans, degummed, alkalirefined and bleached. The oil was heated at 160, 180, 200, 220 and 240°C for up to 156 h. Fatty acid methyl esters were prepared by boron trifluoride-catalyzed transesterification. Gas-liquid chromatography with a cyanopropyl CPSil88 column was used to separate and quantitate fatty acid methyl esters. Fatty acids were identified by comparison of retention times with standards and were calculated as area % and mg/g oil based on 17:0 internal standard. The rates of 18:3ω3 loss and 18:3 Δ9-cis, Δ12-cis, Δ15-trans (18:3c,c,t) formation were determined, and the activation energies were calculated from Arrhenius plots. Freshly prepared soy oil had 10.1% 18:3ω3 and no detectable 18:3c,c,t. Loss of 18:3ω3 followed apparent first-order kinetics. The first-order rate constants ranged from .0018±.00014 min⁻¹ at 160°C to .083±.0033 min⁻¹ at 240°C. The formation of 18:3c,c,t did not follow simple kinetics, and initial rates were estimated. The initial rates (mg per g oil per h) of 18:3c,c,t formation ranged from 0.0031±0.0006 at 160°C to 2.4±.24 at 240°C. The Arrhenius activation energy for 18:3ω3 loss was 82.1±7.2 kJ mol⁻¹. The apparent Arrhenius activation energy for 18:3c,c,t formation was 146.0±13.0 kJ mol⁻¹. The results indicate that small differences in heating temperature can have a profound affect on 18:3c,c,t formation. Selection of appropriate deodorization conditions could limit the amount of 18:3c,c,t produced.     

Long-term behavior of oil-based varnishes and paints I. Spectroscopic analysis of curing drying oils

The curing of drying oils at 60°C has been investigated by Fourier transform infrared spectroscopy and Fourier transform Raman analysis of linseed oil and poppyseed oil. In the first step, hydroperoxides are formed (broad vibration band centered around 3425 cm⁻¹) with concomitant conjugation and cis-trans isomerization of the double bonds (disappearance of cis bands at 3011 and 716 cm⁻¹, appearance of trans conjugated and trans nonconjugated bands at 987 and 970 cm⁻¹). The subsequent decomposition of hydroperoxides in the presence of oxygen leads to the formation of alcohols (nitrite band at 779 cm⁻¹ after nitrogen monoxide treatment), aldehydes (bands at 2810 and 2717 cm⁻¹ in gas phase), ketones (saturated and unsaturated at 1720 and 1698 cm⁻¹, respectively), carboxylic acids (saturated and unsaturated acid fluorides identified at 1843 and 1810 cm⁻¹ after SF₄ treatment), and peresters or γ-lactones (near 1770 cm⁻¹). A rapid decrease in the double-bond concentration is recorded when curing continues, and the formation of epoxides, characterized by a vibration band at 885 cm⁻¹, is observed. Thermolysis experiments have suggested the proposal of a reaction of addition of peroxyl radicals on the conjugated double bonds as a probable mechanism. This mechanism explains both the rapid disappearance of conjugated double bonds and the formation of epoxides as intermediate products observed in the initial step of curing.     

Effects of dietary α-linolenic acid on the conversion and oxidation of13C-α-linolenic acid

The effects of a diet rich in α-linolenic acid vs. one rich in oleic acid on the oxidation of uniformly labeled¹³C-α-linolenic acid and its conversion into longer-chain polyunsaturates (LCP) were investigatedin vivo in healthy human subjects. Volunteers received a diet rich in oleic acid (n=5) or a diet rich in α-linolenic acid (n=7; 8.3 g/d) for 6 wk before and during the study. After 6 wk, subjects were given 45 mg of¹³C-α-linolenic acid dissolved in olive oil. Blood samples were collected att=0, 5, 11, 24, 96, and 336 h. Breath was sampled and CO₂ production was measured each hour for the first 12 h. The mean (±SEM) maximal absolute amount of¹³C-eicosapentaenoic acid (EPA) in plasma total lipids was 0.04 ±0.01 mg in the α-linolenic acid group, which was significantly lower (P=0.01) than the amount of 0.12±0.03 mg¹³C-EPA in the oleic acid group. Amounts of¹³C-docosapentaenoic acid (DPA) and¹³C-docosahexaenoic acid (DHA) tended to be lower as well. The mean proportion of labeled α-linolenic acid (ALA) recovered as¹³CO₂ in breath after 12 h was 20.4% in the ALA and 15.7% in the oleic acid group, which was not significantly different (P=0.12). The cumulative recovery of¹³C from¹³C-ALA in breath during the first 12 h was negatively correlated with the maximal amounts of plasma¹³C-EPA (r=−0.58,P=0.047) and¹³C-DPA (r=−0.63,P=0.027), but not of¹³C-DHA (r=−0.49,P=0.108). In conclusion, conversion of¹³C-ALA into its LCP may be decreased on diets rich in ALA, while oxidation of¹³C-ALA is negatively correlated with its conversion into LCP. In a few pilot samples, low¹³C enrichments of n−3 LCP were observed in a diet rich in EPA/DHA as compared to oleic acid.     

Effects of conjugated linoleic acid isomers on the hepatic microsomal desaturation activities in vitro

The influence of individual conjugated linoleic acid (CLA) isomers on the Δ6 desaturation of linoleic and α-linolenic acids and on the Δ9 desaturation of stearic acid was investigated in vitro, using rat liver microsomes. The Δ6 desaturation of 18∶2n−6 was decreased from 23 to 38% when the ratio of 9cis,11trans-18∶2 to 18∶2n−6 increased from 0.5 to 2. The compound 10trans,12cis-18∶2 exhibited a similar effect only at the highest concentration. The Δ6 desaturation of α-linolenic acid was slightly affected by the presence of CLA isomers. The sole isomer to induce an inhibitory effect on the Δ9 desaturation of stearic acid was 10trans,12cis-18∶2.     

Kinetics of halogen-exchange fluorination of 2,6-dichlorobenzaldehyde

Under the conditions of phase transfer catalysis and nitrobenzene as the solvent, the halogen-exchange fluorination of 2,6-dichlorobenzaldehyde using KF as fluorinating agent was studied. The kinetics was investigated and the reaction rate constants were obtained under the optimum conditions of n(KF):n(2,6-dichlorobenzaldehyde): n(Ph₄PBr):n(acetone-furan crown ether) = 4:1:0.1:0.05 and temperatures of 433 K, 443 K, 453 K and 463 K. The results illustrated the activation energy of the first and the second step is 4.57 × 10⁴ J·mol⁻¹ and 3.53 × 10⁴ J·mol⁻¹, respectively. The pre-exponential factor is 4.50 × 10⁵ h⁻¹ and 1.08 × 10⁴ h⁻¹, respectively. Thus a reliable kinetics data could be obtained for further research. __________ Translated from Chemical Engineering (China), 2007, 35(8): 33–36 [译自: 化学工程]     

Linoleic acid uptake by isolated enterocytes: Influence of α-linolenic acid on absorption

In a previous study we showed that intestinal uptake of α-linolenic acid (18∶3n−3) was carrier-mediated and we suggested that a plasma membrane fatty acid protein was involved in the transport of long-chain fatty acids. To further test this hypothesis, the mechanism of linoleic acid (18∶2n−6) uptake by isolated intestinal cells was examined using a rapid filtration method and 20 mM sodium taurocholate as solubilizing agent. Under these experimental conditions transport of [1-¹⁴C]linoleic acid monomers in the concentration range of 2 to 2220 nM was saturable with a V_m of 5.1±0.6 nmol/mg protein/min and a K_m of 183±7 nM. Experiments carried out in the presence of metabolic inhibitors, such as 2,4-dinitrophenol and antimycin A, suggested that an active, carriermediated mechanism was involved in the intestinal uptake of this essential fatty acid. The addition of excess unlabeled linoleic acid to the incubation medium led to a 89% decrease in the uptake of [1-¹⁴C]linoleic acid, whiled-glucose did not compete for transport into the cell. Other long-chain polyunsaturated fatty acids added to the incubation mixture inhibited linoleic acid uptake by more than 80%. The presence of α-linolenic acid (18∶3n−3) in the incubation medium caused the competitive inhibition (K_i=353 nM) of linoleic acid uptake. The data are compatible with the hypothesis that intestinal uptake of both linoleic, and α-linolenic acid is mediated by a membrane carrier common to long-chain fatty acids.     

FA composition of plasma,red blood cell,and liver phospholipids of newborn piglets fed trans isomers of alpha-linolenic acid

The effects of dietary cis and trans α-linolenic acid (18∶3n−3) on the FA composition of plasma, red blood cell, and liver phospholipids were studied in newborn piglets. Animals were fed for 14 d with one of three diets: a control diet (group A) containing cis 18∶3n−3 at a level of 2.0% of total FA, a diet (group B) in which a part of the 18∶3n−3 acid was isomerized (1.3% of cis 18∶3n−3 and 0.7% of trans 18∶3n−3), or a diet (group C) with 2.0% cis 18∶3n−3 and 0.7% trans 18∶3n−3. Feeding animals with diets containing trans 18∶3n−3 resulted in the presence of trans isomers of 18∶3n−3, trans isomers of EPA, and trans isomers of DHA in phospholipids; however, the level of total trans n−3 PUFA in tissues was less than 0.3% of total tissue FA. In group B, the reduction of dietary amounts of cis 18∶3n−3 was associated with a decrease in individual and total cis n−3 PUFA. In contrast, in group C there was no decrease in tissue n−3 PUFA despite the increased dietary level of trans 18∶3n−3. These results suggest that the isomerization of a part of dietary n−3 PUFA, leading to the reduction of their levels in the diet, could induce a decrease in n−3 PUFA in phospholipids. The physiological effects of trans PUFA are not known and should be considered in future studies.     

Chia seeds as a source of natural lipid antioxidants

Chia (Salvia sp) seeds were investigated as a source of natural lipid antioxidants. Methanolic and aqueous extracts of defatted chia seeds possessed potent antioxidant activity. Analysis of 2 batches of chia-seed oils demonstrated marked difference in the fatty acid composition of the oils. In both batches, the oils had high concentrations of polyunsaturated fatty acids. The major antioxidant activity in the nonhydrolyzed extract was caused by flavonol glycosides, chlorogenic acid (7.1 × 10⁻⁴ mol/kg of seed) and caffeic acid (6.6 × 10⁻³ m/kg). Major antioxidants of the hydrolyzed extracts were flavonol aglycones/kaempferol (1.1 × 10⁻³ m/kg), quercetin (2.0 × 10⁻⁴ m/kg) and myricetin (3.1 × 10⁻³ m/kg); and caffeic acid (1.35 × 10⁻² m/kg). Two methods were used to measure antioxidant activities. Both were based on measuring bleaching ofβ-carotene in the coupled oxidation ofβ-carotene and linoleic acid in the presence of added antioxidants.     

Solubility of linoleic acid in aqueous solutions and its reaction with water

In a study of stable emulsions of linoleic acid in 0.1M-KH₂PO₄/Na₂HPO₄ buffer solutions prepared by sonic vibrations, the influence of linoleic acid on pH was manifested in buffer solutions of pH 8.00 and decreased gradually till it became negligible in pH 4.50. This change in pH values was due to differences in solubility of linoleic acid in the buffer solutions. Ultraviolet spectra of soluble linoleic acid in buffer solutions indicated the presence of conjugated dienes, which increased with the increasing of the pH of the system. Unbuffered aqueous emulsions of linoleic acid had a pH value which ranged between 4.69 and 5.10. Saturated aqueous solutions, obtained by high-speed centrifugation, had concentrations of 15.80 to 16.00 mg. linoleic acid per 100 ml. of D.I. water. From the solubility data and conductivity values of linoleic acid the apparent classic and thermodynamic ionization constants were calculated to be 6.974±0.023×10⁻⁶ and 6.905±0.017×10⁻⁶ at 0.7°C. and 1.730±0.009×10⁻⁵ and 1.689±0.007×10⁻⁵ at 25°C., respectively. The result of the chemical interaction of linoleic acid and water is a saturated hydroxy fatty acid. This acid gave a positive test for glycol groups with periodic acid oxidation test and appeared to be a tetrahydroxy compound with the exact structure unknown. Presented at the 51st Annual Meeting, American Oil Chemists' Society at Dallas, Tex., April 4–6, 1960. American Meat Institute Foundation Journal Paper No.204.     

Analysis of alpha-linolenic acid geometrical isomers in deodorized oils by capillary gas-liquid chromatography on cyanoalkyl polysiloxane stationary phases: A note of caution

Analysis of alpha-linolenic acid geometrical isomers in deodorized or heated oils by capillary gas-liquid chromatography (GLC) on polar cyanoalkyl polysiloxane stationary phases requires some care to avoid interferences with other fatty acids. Depending on the temperature of the column, thecis-11 20∶1 acid may elute before, with or after thecis-9,cis-12,cis-15 18∶3 acid during GLC. In some instances [temperature higher than 180°C with a CP Sil 88 column (Chrompack, Middelburg, The Netherlands)], the 20∶1 acid coelutes with thetrans-9,cis-12,cis-15 18∶3 acid, leading to abnormally high levels of this last isomer. Consequently, the degree of isomerization of alpha-linolenic acid will be over-estimated under such conditions. It is recommended that the behavior ofcis-11 20∶1 acid relative to temperature be checked carefully prior to the determination of alpha-linolenic acid geometrical isomers by GLC. Temperatures lower than 160°C seem appropriate to separate all of these components from each other and fromcis-11 20∶1 acid in a 50 m×0.25 mm i.d. CP Sil 88 capillary column.     

Structural changes of sinapic acid during alkali-induced air oxidation and the development of colored substances

Structural changes of sinapic acid were induced by air oxidation in aqueous solutions at pH 7–10 and followed by spectral and high-performance liquid chromatographic (HPLC) analysis. Color properties of the sinapic acid solutions were determined by taking the transmittance spectra, calculating the Commission Internationale de l’Eclairage (CIE) 1931 tristimulus values, and converting to Hunter L a b values. Reaction rate constants for sinapic acid were determined by a kinetic study based on the quantitative results from HPLC analysis. These reactions were first order with respect to sinapic acid and fit the appropriate equation with a coefficient of R ² >0.97. Sinapic acid was converted to thomasidioic acid with reaction rate constants (k) of 8.54×10⁻⁶, 2.51×10⁻⁵, and 4.87×10⁻⁵ s⁻¹ in phosphate-boric acid buffers of pH 7, 8.5, and 10, respectively. Similar reactions in ammonium bicarbonate buffers were more than 10 times faster. With time, thomasidioic acid further converted to 2,6-dimethoxy-p-benzoquinone and 6-hydroxy-5,7-dimethoxy-2-naphthoic acid. Air oxidation of sinapic acid aqueous solutions caused darkening of the color for the system, with the 2,6-dimethoxy-p-benzoquinone as a major color contributor.     

Kinetic study of the reaction between tocopheroxyl radical and unsaturated fatty acid esters in benzene

A kinetic study of the reaction between a tocopheroxyl radical and unsaturated fatty acid esters has been undertaken. The rates of allylic hydrogen abstraction from various unsaturated fatty acid esters (ethyl oleate2, ethyl linoleate3, ethyl linolenate4, and ethyl arachidonate5) by the tocopheroxyl radical (5,7-diisopropyltocopheroxyl6) in benzene have been determined spectrophotometrically. The second-order rate constants, k₃, obtained are 1.04×10⁻⁵ M⁻¹s⁻¹ for2, 1.82×10⁻² M⁻¹s⁻¹ for3, 3.84×10⁻² M⁻¹s⁻¹ for4, and 4.83×10⁻² M⁻¹s⁻¹ for5 at 25.0°C. Thus, the rate constants, k_abstr/H, given on an available hydrogen basis are k₃/4=2.60×10⁻⁶ M⁻¹s⁻¹ for2, k₃/2=9.10×10⁻³ M⁻¹s⁻¹ for3, k₃/4=9.60×10⁻³ M⁻¹s⁻¹ for4, and k₃/6=8.05×10⁻³ M⁻¹s⁻¹ for5. The k_abstr/H values obtained for the polyunsaturated fatty acid esters3,4, and5 containing H-atoms activated by two π-electron systems are similar to each other, and are about three orders of magnitude higher than that for the ethyl oleate2 containing H-atoms activated by a single π-system. From these results, it is suggested that the prooxidant effect of α-tocopherol in edible oils and fats may be induced by the above hydrogen abstraction reaction.     

Characterization of phospholipase D activity in bovine photoreceptor membranes

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2³H]glycerol-3-phosphocholine (PC), or [¹⁴C]ethanol. Using [³H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [³H]phosphatidic acid and phosphatidylethanol [³H]PtdEt; whereas with [¹⁴C]ethanol or [³H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [¹⁴C]PtdEt or [³H]phosphatidylglycerol, respectively). The use of varying concentrations of [³H]PC and 400 mM of ethanol gave an apparent K _m value for PC of 0.51 mM and a V _max value of 111 nmol × h⁻¹ × (mg protein)⁻¹. The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent K _m of 202 mM and a V _max value for ethanol of 125 nmol × h⁻¹ × (mg protein)⁻¹. A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca²⁺ or Mg²⁺ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

Cultured fish cells metabolize octadecapentaenoic acid (all-cis δ3,6,9,12,15–18∶5) to octadecatetraenoic acid (all-cis δ6,9,12,15–18∶4) via its 2-trans intermediate (trans δ2, all-cis δ6,9,12,15–18∶5)

Octadecapentaenoic acid (all-cis δ3,6,9,12,15–18∶5; 18∶5n−3) is an unusual fatty acid found in marine dinophytes, haptophytes, and prasinophytes. It is not present at higher trophic levels in the marine food web, but its metabolism by animals ingesting algae is unknown. Here we studied the metabolism of 18∶5n−3 in cell lines derived from turbot (Scophthalmus maximus), gilthead sea bream (Sparus aurata), and Atlantic salmon (Salmo salar). Cells were incubated in the presence of approximately 1 μM [U-¹⁴C] 18∶5n−3 methyl ester or [U-¹⁴C]18∶4n−3 (octadecatetraenoic acid; all-cis δ6,9,12,15–18∶4) methyl ester, both derived from the alga Isochrysis galbana grown in H¹⁴CO₃ ⁻, and also with 25 μM unlabeled 18∶5n−3 or 18∶4n−3. Cells were also incubated with 25 μM trans δ2, all-cis δ6,9,12,15–18∶5 (2-trans 18∶5n−3) produced by alkaline isomerization of 18∶5n−3 chemically synthesized from docosahexaenoic acid (all-cis δ4,7,10,13,16,19–22∶6). Radioisotope and mass analyses of total fatty acids extracted from cells incubated with 18∶5n−3 were consistent with this fatty acid being rapidly metabolized to 18∶4n−3 which was then elongated and further desaturated to eicosatetraenoic acid (all-cis δ8,11,14,17,19–20∶4) and eicosapentaenoic acid (all-cis δ5,8,11,14,17–20∶5). Similar mass increases of 18∶4n−3 and its elongation and further desaturation products occurred in cells incubated with 18∶5n−3 or 2-trans 18∶5n−3. We conclude that 18∶5n−3 is readily converted biochemically to 18∶4n−3 via a 2-trans 18∶5n−3 intermediate generated by a Δ³, Δ²-enoyl-CoA-iso-merase acting on 18∶5n−3. Thus, 2-trans 18∶5n−3 is implicated as a common intermediate in the β-oxidation of both 18∶5n−3 and 18∶4n−3.