Chemiluminescent determination of cholesterol hydroperoxides in human erythrocyte membrane

A method for separating, detecting, and quantifying cholesterol hydroperoxide (Ch-OOH) based on extraction, purification by solid-phase extraction cartridge, high-performance liquid chromatography with chemiluminescent detection (HPIC-CI), and liquid chromatography mass spectrometry has been developed for human erythrocyte membrane. We prepared standard compounds of the cholesterol 5α-, 7α-, and 7β-hydroperoxides (Ch 5α-OOH, Ch 7α-OOH, and Ch 7β-OOH). An octyl silica column with methanol/water/acetonitrile 89∶9∶2 (by vol) as eluent was used to determine Ch-OOH. HPLC-CL that incorporated cytochrome c and luminol as the post-column luminescent reagent was used. We also investigated the optimal assay conditions and how to prevent formation of artifact Ch-OOH. Analysis of erythrocyte membranes from seven healthy volunteers identified Ch 7α-OOH and Ch 7β-OOH, but not Ch 5α-OOH, as commonly occurring components. The respective mean concentrations of Ch 7α-OOH and Ch 7β-OOH were 2,5±1.6 and 5A±3.5 pmol/mL blood.     

Characterization of lipid hydroperoxides generated by photodynamic treatment of leukemia cells

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits <0.5 pmol for cholesterol hydroperoxides and <50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells. L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm² (which reduced clonally assessed survival by 30%) showed 12–15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis. Three peaks of relatively low retention time (<12 min) were assigned to the following species by virtue of comigration with authentic standards: 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide (5α-OOH), 3β-hydroxycholest-4-ene-6β-hydroperoxide (6β-OOH), and 3β-hydroxycholest-5-ene-7α/7β-hydroperoxide (7α/7β-OOH). Formation of 5α-OOH and 6β-OOH (singlet oxygen adducts) was confirmed by subjecting [¹⁴C]cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection. Material represented in a major peak at 18–22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18–22 min peak during Ca²⁺/phospholipase A₂ treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts ofPCOOH and ChOOH at a light fluence that clonally inactivated <10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing. This paper is based on a dissertation submitted by G.J. Bachowski in partial fulfillment of the requirements for a Ph.D. degree in Biochemistry at the Medical College of Wisconsin (Milwaukee, WI).     

Effect of dietary restriction on age-related increase of liver susceptibility to peroxidation in rats

Dietary restriction (DR) increases life span and decreases age-related diseases in experimental animals. It has received a great deal of attention in connection with the relationship between aging, nutrition, and oxidative stress because oxidative injury in several organ systems is a prominent feature in aging. We investigated the possibility that DR can protect vulnerable liver lipids against age-related increases of peroxidation. Male Fischer 344 rats fed ad libitum (AL) or dietarily restricted (maintained on 60% of AL food intake) were killed by decapitation at 4 (young) or 12 mon (adult) of age. Phosphatidylcholine hydroperoxide (PCOOH) concentration of liver was determined using a chemiluminescent high-performance liquid chromatographic method. Liver PCOOH increased with age in adult rats, but less of an increase of PCOOH was seen in DR rats, which is consistent with results on production of thiobarbituric acid-reactive substances and oxygen-derived free radicals. No significant differences were found in liver superoxide dismutase and catalase activity between AL and DR groups of young and adult rats. Liver triglyceride and cholesterol contents were lower in DR than AL rats at 12 mon. Fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine indicated that the ratio of (20∶3n−6+20∶4n−6)/18∶2n−6, an index of linoleic acid (18∶2n−6) desaturation, was lower in DR than in AL rats. We concluded that DR suppresses age-related oxidative damage in liver by modulating the amount of lipid as well as fatty acid composition.     

Changes in phosphatidylcholine molecular species in the shrimp <Emphasis Type="Italic">Macrobrachium borellii</Emphasis> in response to a water-soluble fraction of petroleum

The effect of the water-soluble fraction (WSF) of crude oil on lipid contents, lipid classes, FA, and PC molecular species was studied in high-phospholipid (hepatopancreas) and low-phospholipid (egg) tissues of a freshwater crustacean. After a 21-d exposure to a sublethal concentration of WSF, a significant decrease in shrimp total lipids was observed, although no alterations could be detected in the hepatopancreas or egg lipid contents. TAG/phospholipid ratios increased in the hepatopancreas and decreased in the eggs, suggesting alterations either in the mobilization of TAG to phospholipid pools or in the energy balance. The FA composition of phosphoglycerides in the hepatopancreas and eggs was dominated by PUFA, whereas the n−3/n−6 ratio was not affected by WSF exposure, although there was a significant increase in hepatopancreas 18∶1n−9. Analysis of the PC molecular species by HPLC-ELSD showed the presence of 15 species, with 16∶0/18∶1, 18∶1/18∶2, 16∶0/20∶5, and 16∶1/20∶5 being the major species in the hepatopancreas. The PC molecular species in the eggs showed a different pattern, dominated by 16∶0/18∶1 and 18∶1/18∶2. Of the PC molecular species, 10 contained 22∶6n−3, 20∶5n−3, and 20∶4n−6. Small amounts of di-PUFA species were also found. Exposure to WSF altered the PC molecular species in both tissues. The four major hepatopancreas molecular species and most of the ones containing PUFA decreased. This was compensated for by an increase in 16∶1/18∶1 (152%) and 18∶1/18∶1 (50%). The two major egg PC molecular species decreased, whereas the PUFA-containing ones increased. The contrasting responses of both tissues of WSF contamination suggests the presence of different homeostatic mechanisms.     

Improved HPLC assay for lipid peroxides in human plasma using the internal standard of hydroperoxide

We have developed a sensitive reversed-phase chemiluminescence HPLC approach for simultaneous quantitative and qualitative analyses of hydroperoxides of cholesteryl ester and TC in human plasma. Standard hydroperoxides of cholesteryl ester and TG and a novel internal standard (1-tetradecanyl 3-octadecenoyloxy-5β-cholan-24-oate monohydroperoxide) (I.S.) were chemically synthesized and the standard curves confirmed to be linear throughout the calibration range (1–1000 pmol). Within-day and between-day CV were less than 7%, and the recoveries were within the range of 84–93%. With sample size minimized to 0.1 mL of plasma for each run, plasma cholesteryl ester hydroperoxide levels were 189±87 nM (mean±SD) in healthy young (22–25 yr old; n=15, male/female=6∶9) and 210 ±69 nM in healthy elderly (39–60 yr old; n=6, male/female= 3∶3). TG hydroperoxide was not detected in healthy subjects. In patients with advanced liver failure (36–67 yr old; n=4, male/female=2∶2), hydroperoxide levels of plasma cholesteryl ester and TG were 11,903±9,553 nM and 3,318±1,590 nM, respectively, indicating an involvement of lipid oxidation. Sensitive and specific monitoring of plasma lipid peroxides using the present chemiluminescence HPLC approach with the synthesized I.S. may help our understanding of chemical and pathophysiological aspects of lipid peroxidation.     

Rapid transmethylation of microgram amounts of phosphatidylcholine on potassium methoxide/celite columns1,2,3

A rapid and convenient method for the determination of acyl groups in phosphatidylcholine (PC) has been developed. Transmethylation reactions were carried out on potassium methoxideimpregnated Celite microcolumns that were readily prepared from Pasteur pipettes. The methods were tested on microgram amounts of synthetic L-α-phosphatidylcholines (di-14∶0; di-18∶2; α-16∶0-β-14∶0) and on egg yolk lecithin, in methylene chloride or hexane solution. Transmethylation of these lipids occurred rapidly at room temperature. Gas liquid chromatographic (GLC) analysis of the product methyl esters demonstrated that the reaction was neither selective for one acyl position of PC over the other, nor sensitive to the amount of unsaturation within the acyl group. The results of the acyl group analysis of natural egg yolk lecithin compared favorably with the results from an established procedure. Presented at AOCS 68th Annual Meeting, New York, May 1977. Reference to brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned. Chloroform and carbon disulfide have been classified by OSHA as carcinogens and/or toxic materials; they should be handled with care under proper ventilation.     

Correlation of suppressed linoleic acid metabolism with the hypocholesterolemic action of eritadenine in rats

The dose-dependent effects of dietary eritadenine on the metabolism of linoleic acid and on the plasma cholesterol concentration were investigated to clarify the mechanism of the hypocholesterolemic action of eritadenine in rats. Rats were fed control or eritadenine-supplemented (2 to 20 mg/kg) diets for 14 d. Eritadenine supplementation significantly decreased both the plasma cholesterol concentration and the 20∶4n−6/18∶2n−6 ratio of liver microsomal and plasma phosphatidylcholine (PC) in a dose-dependent manner. Eritadenine was also found to decrease the activity of Δ6 desaturase in liver microsomes; these was significant correlation between the Δ6-desaturase activity and the 20∶4n−6/18∶2n−6 ratio in the PC of liver microsomes (r=0.989, P<0.001) or plasma (r=0.986, P<0.001). Certain plasma PC molecular species, as represented by 16:0-18:2, were increased by eritadenine in a dose-dependent manner, and certain plasma PC molecular species, as represented by 18:0-20:4, were conversely decreased by eritadenine. There was a significant correlation between the plasma total cholesterol concentration and the proportion of the sum of plasma PC molecular species which contain 18:1 or 18:2 in the sn-2 position. These results support the idea that the suppression of linoleic acid metabolism by eritadenine might be associated with the hypocholesterolemic action of eritadenine.     

Selective deposition oftrans-8- andcis-9-octadecenoates in egg and tissue lipids of the laying hen

The deposition oftrans-8-octadecenoate-8(9)-³H (8t-18∶1-³H) was compared tocis-9-octadecenoate-10-¹⁴C (9c-18∶1-¹⁴C) in the major egg yolk neutral lipids and phospholipids and in organ lipids from the laying hen.trans-8-Octadecenoate was preferentially incorporated into only the phosphatidylethanolamines (PE), whereas discrimination against 8t-18∶1-³H occurred in the phosphatidylcholines (PC), triglycerides (TG) and cholesteryl esters (CE). The 1-acyl position of both PE and PC contained three times more 8t-18∶1-³H than 9c-18∶1-¹⁴C. Almost total exclusion of the 8t-18∶1-³H from the 2-acyl position of these phospholipids was found. Preferential incorporation of 9c-18∶1-¹⁴C occurred at the combined 1- and 3-acyl positions and at the 2-acyl position of yolk TG. Tissue lipid analyses indicated that there was preferential deposition of 9c-18∶1-¹⁴C into all organs. Individual liver lipid classes displayed the same relative order of discrimination against 8t-18∶1-³H as did egg yolk lipids (CE>TG>PC>PE).     

Fatty acid composition of the adipose tissue and yolk lipids of a bird with a marine-based diet, the emperor penguin (Aptenodytes forsteri)

The emperor penguin (Aptenodytes forsteri) is an Antarctic seabird feeding mainly on fish and therefore has a high dietary intake of n-3 polyunsaturated fatty acids. The yolk is accumulated in the developing oocyte while the females are fasting, and a large proportion of the fatty acid components of the yolk lipids are derived by mobilization from the female's adipose tissue. The fatty acid composition of the total lipid of the yolk was characterized by high levels of n-3 polyunsaturated fatty acids. However, it differed in several respects from that of the maternal adipose tissue. For example, the proportions of 14∶0, 16∶1n−7, 20∶1n−9, 22∶1n−9, 20∶5n−3, and 22∶6n−3 were significantly greater in adipose tissue than in yolk. Thus adipose tissue lipids contained 7.6±0.3% and 8.0±0.3% (wt% of total fatty acids; mean ±SE; n=5) of 20∶5n−3 and 22∶6n−3, respectively, whereas the yolk total lipid contained 1.6±0.1 and 5.5±0.3% of these respective fatty acids. The proportions of 16∶0, 18∶0, 18∶1n−9, 18∶2n−6, and 20∶4n−6 were significantly lower in the adipose tissue than in the yolk lipids. The proportions of triacylglycerol, phospholipid, free cholesterol, and cholesteryl ester in the yolk lipid were, respectively, 67.0±0.2, 25.4±0.3, 5.3±0.2, and 1.8±0.2% (wt% of total yolk lipid). The proportions of 20∶4n−6, 20∶5n−3, 22∶5n−3, and 22∶6n−3 were, respectively, 5.7±0.3, 2.8±0.2, 1.4±0.1, and 11.7±0.5% in phospholipid and 0.4±0.0, 1.2±0.1, 0.8±0.1 and 3.6±0.3% in triacylglycerol. About 95% of the total vitamin E in the yolks was in the form of α-tocopherol with γ-tocopherol forming the remainder. Two species of carotenoids, one identified as lutein, were present.     

Fatty acid positional distribution in egg yolk triglycerides from various avain species

The fatty acid composition and distribution in egg yolk triglycerides and phosphatides from the turkey, duck, prairie chicken, bobwhite quail, Japanese quail, and inbred-hybrid and midget mutant hens were determined after all species had been fed diets of similar fat and fatty acid content for 90 days. Total volk lipids were composed of ca. two-thirds neutral lipids and one-third polar lipids. The predominant fatty acids were palmitic and stearic. There were statistically significant differences in the my ristic, palmitic, palmitoleic, linoleic, and linolenic acids in the yolk triglycerides and in the proportion of 16∶1, 18∶0, 18∶2, arachidonic, docosanoic, docosahexaenoic, and tetracosanoic acids in the phosphatides among the various species. Linoleic acid predominantly was linked at the 2-position in the yolk triglycerides followed by the 20∶4 acid. The 18∶1 acid also was found preferentially at the 2-position. There was a low level of 18∶2 in the yolk triglycerides and phosphatides from the duck and an especially high level of 20∶4 acid in the phosphatides. The triglycerides in the species studied have essentially the same distribution of fatty acids in the 2-position. In all the species, the affinity for the fatty acids at the 2-position is in the following order: 18∶2=20∶4>18∶1 =18∶3>18∶0=16∶1>14∶0>16∶0 Differences observed among the various genera did not appear to follow taxonomic boundaries. The duck has an efficient system for converting 18∶2 into 20∶4 by elongation and desaturation. The prairie chicken apparently has a high requirement for 18∶2 but an inadequate system for its conversion into 20∶4.     

Omega-3 fatty acid and cholesterol content of newly hatched chicks from α-linolenic acid enriched eggs

Egg yolk was enriched with α-linolenic acid (18∶3n−3) by feeding laying hens diets containing flax, canola or soybean seeds. Fertilized eggs were incubated and the fatty acid composition of whole body, liver, plasma, brain and the cholesterol content of plasma and liver tissue of the hatched chicks were studied. Eggs enriched with 18∶2n−6 fatty acids by feeding hens diets containing sunflower seeds were used as the controls. Feeding flax enriched (P<0.05) egg yolk and the developing progeny with 18∶3n−3, 20∶5n−3, 22∶5n−3 and 22∶6n−3. Feeding sunflower seeds resulted in an increase (P<0.05) of 18∶2n−6, 20∶4n−6, 22∶4n−6 and 22∶5n−6. The predominant polyunsaturated fatty acid of the brain was docosahexaenoic acid (22∶6n−3) which was higher (P<0.05) in the flax and canola fed group. The cholesterol content of the liver tissue was lower (P<0.05) in chicks hatched from hens fed flax seeds. This study indicates that 18∶3n−3 and 18∶2n−6 in the maternal diet are potent modulators of long-chain polyunsaturated n−3 or n−6 fatty acid and of cholesterol content in the developing progeny.     

1-Acyl-2-acetyl-sn-glycero-3-phosphocholine from stimulated human polymorphonuclear leukocytes

1-Acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl GPC) was found in the fraction of platelet-activating factor obtained from stimulated human polymorphonuclear leukocytes (PMN). The amount of 1-acyl-2-acetyl GPC obtained from 1×10⁷ PMN stimulated with ionophore A23187 at 37 C for 15 min ranged from 8 to 56 pmol (32±10 pmol, mean±standard error; n=4). The main species was 16∶0 palmitoyl (17±5 pmol), followed by 18∶0 stearoyl (8±3 pmol) and 18∶1 oleoyl (7±3 pmol). Although the physiological significance is unknown, 1-acyl-2-acetyl GPC was always detected when 1-alkyl-2-acetyl GPC was detected.     

Distributions of hydroperoxide positional isomers generated by oxidation of 1-palmitoyl-2-arachidonoyl-<Emphasis Type="Italic">sn</Emphasis>-glycero-3-phosphocholine in liposomes and in methanol solution

The differences in distribution of geometric isomers of unsaturated PC hydroperoxides generated by free radical oxidation were compared, as corresponding hydroxy analogs, in heterogeneous liposomes and in a homogeneous methanol solution by using HPLC with UV detection due to the presence of conjugated dienes. Identification of fractionated peak components was carried out by GC-MS. When the oxidation of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, PC(16∶0/18∶2), was initiated in liposomes by a hydrophilic azo radical initiator, and in a methanol solution by a hydrophobic azo radical initiator, there was no significant difference in the relative percentages of 1-palmitoyl-2-(9-hydroxy-trans-10,trans-12-octadecadienoyl)-sn-glycero-3-phosphocholine (9-t,t-OH PC) and 1-palmitoyl-2-(13-hydroxy-trans-9,trans-11-octadecadienoyl)-sn-glycero-3-phosphocholine (13-t,t-OH PC) between the PC oxidized in liposomes and in the methanol solution. For the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, PC(16∶0/20∶4), the relative percentage of 1-palmitoyl-2-(5-hydroxy-trans-6,cis-8,11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (5-OH PC) was significantly higher (P<0.01) than that of 1-palmitoyl-2-(15-hydroxy-cis-5,8,11,trans-13-eicosatetraenoyl)-sn-glycero-3-phosphocholine (15-OH PC) in liposomes. For the homogeneous methanol solution of PC(16∶0/20∶4), the relative percentage of 5-OH PC was close to that of 15-OH PC. For the PC(16∶0/20∶4) oxidized in bulk with added pentamethylchromanol, the individual amount of 15-OH PC, 1-palmitoyl-2-(11-hydroxy-cis-5,8trans-12,cis-14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (11-OH PC), 1-palmitoyl-2-(12-hydroxy-cis-5,8,trans-10,cis-14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (12-OH PC), 1-palmitoyl-2-(8-hydroxy-cis-5,trans-9,cis-11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (8-OH PC), 1-palmitoyl-2-(9-hydroxy-cis-5,trans-7,cis-11,14-eicosatetraenoyl)-sn-glycero-3-phosphocholine (9-OH PC), and 5-OH PC were close to each other compared to the corresponding values in liposomes and in methanol solution. The results obtained by gel permeation chromatography of the PC liposomes containing hydrophilic 2,2′-azobis-2-amidinopropane) dihydrochloride (AAPH) suggest that the AAPH added to the liposomes of PC(16∶0/20∶4) was partitioned into the water phase and out of the hydrophobic region of the fatty acyl moieties of the PC. These results confirm that the distance that exists in the bis-allylic carbons of the unsaturated fatty acyl moieties of PC from the interface between the hydrophilic region of PC and the water phases played an important role in influencing hydrogen abstraction to form a symmetrical distribution of hydroperoxide isomers in both the heterogeneous liposomes and the homogeneous methanol solution.     

Competitive deposition oftrans-12- andcis-9-octadecenoates into egg yolk lipids

The deposition oftrans-12-octadecenoate-12(13)-³H (12t-18∶1-³H) was compared tocis-9-octadecenoate-10-¹⁴C (9c-18∶1-¹⁴C) in the major egg yolk neutral lipids and phospholipids.trans-12-Octadecenoate was preferentially incorporated into cholesteryl esters (CE), phosphatidylcholines (PC), and phosphatidylethanolamines (PE) but was discriminated against in triglycerides (TG). Isotopic ratios indicate that 5.9 and 5.6 times more 12t-18∶1-³H than 9c-18∶1-¹⁴C was esterified at the 1-acyl position of PE and PC, respectively. The combined 1- and 3-acyl positions of TG and the 2-acyl position of TG, PE and PC were each preferentially esterified with 9c-18∶1-¹⁴C.     

Influence of dietary arachidonic acid on metabolism in vivo of 8 cis, 11 cis, 14-eicosatrienoic acid in humans

This study investigated the influence of dietary arachidonic acid (20∶4n-6) on Δ5 desaturation and incorporation of deuterium-labeled 8cis, 11cis, 14-eicosatrienoic acid (20∶3n-6) into human plasma lipids. Adult male subjects (n=4) were fed diets containing either 1.7 g/d (H120∶4 diet) or 0.21 g/d (LO20∶4 diet) of arachidonic acid for 50 d and then dosed with a mixture containing ethyl esters of 20∶3n-6[d4] and 18∶1n-9[d2]. A series of blood samples was sequentially drawn over a 72-h period, and methyl esters of plasma total lipid, triacylglycerol, phospholipids, and cholesteryl ester were analyzed by gas chromatography-mass spectrometry. Based on the concentration of 20∶3n-6[d4] in total plasma lipid, the estimated conversion of 20∶3n-6[d4] to 20∶4n-6[d4] was 17.7.±0.79% (HI20∶4 diet) and 2.13±1.44% (LO20∶4 diet). The concentrations of 20∶4n-6[d4] in total plasma lipids from subjects fed the HI20∶4 and LO20∶4 diets were 2.10±0.6 and 0.29±0.2 μmole/mL plasma/mmole of 20∶3n-6[d4] fed/kg of body weight. These data indicate that conversion of 20∶3n-6[d4] to 20∶4n-6[d4] was stimulated 7-8-fold by the HI20∶4 diet. Phospholipid acyltransferase was 2.5-fold more selective for 20∶3n-6[d4] than 18∶1n-9[d2], and lecithin:cholesterol acyltransferase was 2-fold more selective for 18∶1n-9[d2] than 20∶3n-6[d4]. These differences in selectivity were not significantly influenced by diet. Absorption of ethyl 20∶3n-6[d4] was about 33% less than ethyl 18∶1n-9[d2]. The sum of the n-6 retroconversion products from 20∶3n-6[d4] in total plasma lipids was about 2% of the total deuterated fatty acids. Neither absorption nor retroconversion appears to be influenced by diet.     

Proportion of geometrical hydroperoxide isomers generated by radical oxidation of methyl linoleate in homogeneous solution and in aqueous emulsion

The proportion of geometrical hydroperoxide isomers generated by aerobic oxidation of methyl linoleate (18∶2 Me) in either aqueous emulsion consisting of Tris-HCl buffer (pH 7.4) or in a homogeneous dichloromethane solution was determined to understand the mechanism of lipid oxidation in different reaction systems. Four geometrical isomers were generated after oxidation of 18∶2 Me in dichloromethane: methyl 13-hydroperoxy-cis-9,trans-11-octadecadienoate, methyl 13-hydroperoxy-trans-9,trans-11-octadecadienoate, methyl 9-hydroperoxy-trans-10,cis-12-octadecadienoate, and methyl 9-hydroperoxy-trans-10,trans-12-octadecadienoate in the ratios of 1∶4∶1∶4, respectively. The ratios between each isomer did not change until the peroxide value (PV) increased to 58 meq/kg. Oxidation of 18∶2 Me in aqueous emulsion yielded the same geometrical isomers of hydroperoxide. However, the ratios were different: 3∶2∶3∶2 until the PV increased to 110 meq/kg. Predominant (60%) formation of trans,trans hydroperoxide isomers was obtained in the oxidation of a mixture of 18∶2 Me and methyl laurate (12∶0 Me). These results are interpreted to reflect the importance of the concentration of hydrogen atom-donating equivalents to the kinetic preference for different products. The high effective concentration of hydrogen donors in the oxidation of 18∶2 Me in emulsions favored the formation of the less stable cis,trans isomers. The lower concentration of hydrogen donor in the dichloromethane solution effectively slowed hydrogen donation and led to the strong preference for the more stable trans,trans isomers. This interpretation was further tested by preparing emulsions of 18∶2 Me and 12∶0 Me to dilute concentration of hydrogen-donating species using the nonhydrogen-donating 12∶0 Me. Consistent with the proposed hypothesis, the proportion of trans,trans isomers increased as a result of 12∶0 Me addition.     

Hepatic phospholipid molecular species in the guinea pig adaptations to pregnancy

Incorporation of polyunsaturated fatty acids (PUFA), particularly 22∶6n−3, into fetal brain at specific gestational ages is critical for development of normal brain function. We have studied adaptations to maternal liver phospholipid molecular species compositions that may be related to the supply of PUFA to fetal brain. The increment of 22∶6n−3 in brain phosphatidylethanolamine (PE) was maximal at day 25 to day 35 of gestation, consistent with early prenatal development of guinea pig brain. At the same gestational ages, there was a transient increase in maternal liver concentration of 16∶0/22∶6 phosphatidylcholine (PC), which preceded the progressive increase in total PC concentration toward term (day 68). This effect was specific for thesn-1 16∶0 species, as, there was no significant increase in 18∶0/22∶6 PC concentration. These results are consistent with a specific role for 16∶0/22∶6 PC in the directed supply of 22∶6n−3 from maternal liver to the fetus. Concentrations of all PE species in maternal liver decreased at day 25 and day 35 of gestation. The gradual accumulation of 22∶6n−3 in fetal liver throughout gestation did not correlate with the pattern of acquisition of 22∶6n−3 into fetal brain PE. Maternal plasma PC and cholesterol concentrations decreased dramatically by day 25 of gestation, and remained low until term. This hypolipidemia of pregnancy in the guinea pig may be due to increased lipase-mediated turnover of plasma lipoproteins and contrasts strongly with the well-characterized hyperlipidemia in human and rat gestation.     

The effect of dietary supplementation with eicosapentaenoic acid on the phospholipid and fatty acid composition of erythrocytes of marmoset

Adult male marmoset monkeys were fed eicosapentaenoic acid (20∶5n−3) as the ethyl ester in diets containing either 32% (reference diet, no added cholesterol) or 7% (atherogenic diet with 0.2% added cholesterol) linoleic acid (18∶2n−6) for 30 wk. No changes were seen in the level of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) but minor changes were observed in both the sphingomyelin (SPM) and phosphatidylinositol plus phosphatidylserine (PI+PS) fractions of erythrocyte lipids. The extent of total n−3 fatty acid incorporation into membrane lipids was higher in atherogenic diets (polyunsaturated/monounsaturated/saturated (P/M/S) ratio 0.2∶0.6∶1.0) than reference diets (P/M/S ratio 1∶1∶1) and this was true for both PE (33.4±1.0%vs 24.3±1.1%) and PC (9.3±0.5%vs 4.9±0.3%). Although suitable controls for cholesterol effects were not included in the study, earlier results obtained with marmosets lead us to believe such effects were probably small. Regardless of basic diet (atherogenic, reference), 20∶5n−3 was preferentially incorporated into PE (10.8±0.2%, 6.0±0.02%) while smaller amounts were incorporated into PC (6.9±0.4%, 3.2±0.2%). The major n−3 polyunsaturated fatty acid found in PE in response to dietary 20∶5n−3 was the elongation metabolite 22∶5n−3 in both the atherogenic (17.7±0.7%) and reference (14.3±1.0%) dietary groups; 22∶6n−3 levels were less affected by diet (4.7±0.3% and 3.9±0.2%, respectively). The results can be interpreted to indicate an inverse relationship between the amount of dietary 18∶2n−6 and incorporation of 20∶5n−3 into erythrocyte membrane phospholipids regardless of whether the major dietary n−3 fatty acid was α-linolenate (18∶3n−3) or 20∶5n−3. This interpretation is supported by theoretical calculations.     

Changes in lipid molecular species and sterols of microsomal membranes during aging of potato (Solanum tuberosum L.) seed-tubers

Changes in sterols and the molecular species composition of polar lipids from microsomal membranes were characterized as a prerequisite to determining how lipid chemistry affects membrane susceptibility to peroxidation during aging of potato tubers. Polar lipid content of the microsomal fraction fell 17% (protein basis) as tubers aged from 2 to 38 mon at 4°C. In younger seed-tubers, PC concentration (protein basis) was the highest, followed by digalactosyldiacylglycerol (DGDG), PE, monogalactosyldiacylglycerol (MGDG), and PI. PC and PE increased 14 and 27%, respectively, whereas glycolipids fell 64 and PI 43% with advancing age. These changes resulted in PC and PE dominating the microsomal membrane lipids of 38-mon-old tubers. Nonpositional analysis of lipid molecular species across lipid pools showed an increase in 16∶0/18∶3, 18∶3/18∶3, and 18∶2/18∶3 (PC and PE only), and a decline in 18∶2/18∶2 and 16∶0/18∶2 (except for MGDG) with advancing tuber age. The increase in 18∶3-bearing species effected a linear increase in double-bond index (DBI) of PC and PE during aging. The DBI of DGDG did not change with age; however, it fell 65% for MGDG, resulting in an overall decrease in average microsomal DBI. In addition, Δ5-avenasterol and stigmasterol concentrations increased 1.6- and 3.3-fold, respectively, effecting a significant increase in the sterol/phospholipid ratio with advancing tuber age. The increase in sterol/phospholipid ratio and the possibility that the increased unsaturation of microsomal membranes reflects a compensatory response to maintain optimal membrane function in light of the age-induced loss of galactolipid and PI are discussed.     

Liver phosphatidylcholine hydroperoxidation provoked by ethionine-containing choline-deficient diet in mice

It is shown that peroxidation of phosphatidylcholine (PC) is enhanced in liver of mice fed a hepatocarcinogenic choline-deficient diet containing 0.1% w/w ethionine. Mice were divided into 4 groups and fed for 3 weeks one of the following diets: choline-supplemented; choline-supplemented containing ethionine; choline-deficient; and choline-deficient containing ethionine. Phosphatidylcholine hydroperoxide (PCOOH) of liver lipids was measured by high performance liquid chromatography using a chemiluminescence detector. Mice fed a choline-deficient diet containing ethionine showed 6-fold higher PCOOH levels than the choline-supplemented control mice: the PCOOH/PC molar ratios of liver lipids were 32.3×10⁻⁵ and 5.6×10⁻⁵, respectively. In addition to this remarkable degree of lipid peroxidation in liver of mice fed the choline-deficient diet containing ethionine, we also observed a significant liver fatty infiltration, a decrease in plasma and liver α-tocopherol, and an increase in liver injury-indicative enzyme activities. Also, marker enzymes for hepatocarcinogenesis, glucose-6-phosphatase and γ-glutamyl transpeptidase were affected. These data suggest that enhanced hydroperoxidation of phosphatidylcholine may participate in hepatocarcinogenesis provoked by choline deficiency in the presence of ethionine.