Formation of a thiamine disulfide complex with fatty acid: Mechanism of formation of the complex

The formation of complexes between thiamine disulfide (TDS) orO-acetyl thiamine disulfide (O-acetyl TDS) and fatty acid or fatty acid methyl ester in methanol has been studied by fluorescence quenching and¹³C NMR relaxation (T₁) measurements. The association constants (K-values) of TDS andO-acetyl TDS with fatty acids (from 11∶0 to 18∶0, and 18∶1, 18∶2, 18∶3 and 20∶4) and fatty acid methyl esters have been determined. These values do not depend on either the number of carbon atoms or the degree of unsaturation of the fatty acid. The K-values of TDS andO-acetyl TDS with fatty acid were 7.8 M⁻¹ and 5.1 M⁻¹, respectively. The K-values of TDS andO-acetyl TDS with fatty acid methyl ester were very small. These results show that the-OH moiety in TDS and the-COOH moiety in the fatty acid are necessary for formation of the complex     

Molecular heterogeneity of platelet-activating factor (PAF) in rat glandular stomach determined by gas chromatography/mass spectrometry. PAF molecular species changes upon water-immersion stress

The molecular heterogeneity of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylacetyl-GPC) in normal rat glandular stomach was studied by gas chromatography/mass spectrometry (GC/MS) and tandem mass spectrometry. The percentage compositions of the molecular species of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC in the antrum were, respectively. 1-alkyl [16∶0 (34%) and 18∶0 (66%)]-2-acetyl-GPC and 1-acyl [16∶0 (60%), 18∶0 (14%) and 18∶1 (26%)]-2-acetyl-GPC. The alkyl chain composition of 1-alkyl-2-acyl-GPC was quite different from that of 1-alkyl-2-acyl-GPC in both the antrum and corpus, demonstrating a high degree of selectivity of alkyl chain utilization in PAF biosynthesis. The amount of 1-acyl-2-acetyl-GPC was much greater than that of 1-alkyl-2-acetyl-GPC. The molecular heterogeneity of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC in the corpus was similar to that in the antrum. Water-immersion stress affected not only the amount of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC, but also their molecular heterogeneity in the antrum and corpus. Whereas the amounts of 1-hexadecyl-2-acetyl-GPC and 1-acyl [16∶0, 18∶0 and 18∶1]-2-acetyl-GPC decreased markedly (to less than one-fifth) in the antrum after such stress for 1 hr, the amount of 1-octadecyl-2-acetyl-GPC increased markedly (up to 4-fold) in the corpus and severe lesions were observed after stress for 7 hr. The changes may be associated with the pathogenicity of gastric ulcers. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Molecular species of 1-O-alk-1′-enyl-2-acyl-, 1-O-alkyl-2-acyl- and 1,2-diacyl glycerophospholipids in Japanese oysterCrassostrea gigas (Thunberg)

Molecular species of 1-O-alk-1′-enyl-2-acyl-, 1-O-alkyl-2-acyl-, and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (EPL) andsn-glycero-3-phosphocholine (CPL) of Japanese oysterCrassostrea gigas were analyzed by selectedion monitoring gas chromatography/mass spectrometry using electron impact ionization. The characteristic fragment ions, [RCH=CH+56]⁺ due to the alkenyl residue in thesn-1 position and [RCO+74]⁺ due to the acyl residue in thesn-2 position of alkenylacylglycerols, [R+130]⁺ due to the alkyl residue in thesn-1 position and [RCO+74]⁺ due to the acyl residue in thesn-2 position of alkylacylglycerols, [RCO+74]⁺ due to the acyl residues in thesn-1 and/orsn-2 positions of diacylglycerols, and [M−57]⁺ being indicative of the corresponding molecular weight, were used for structural assignments. For alkenylacyl EPL and CPL, 19 and 16 molecular species were determined, respectively. Two molecular species, 18∶0alkenyl-22∶6n−3 and 18∶0-alkenyl-22∶2-non-methylene interrupted diene (NMID), amounted to 53.2% and 47.9%, respectively. The alkylacyl EPL and CPL consisted of 16 and 20 molecular species, respectively, and the prominent components were 18∶0alkyl-22∶2NMID, 20∶1alkyl-20∶1n−11 (27.4%) and 20∶1alkyl-20∶2NMID (16.3%) in the former, and 16∶0alkyl-20∶5n−3 (23.0%) and 16∶0alkyl-22∶6n−3 (21.6%) in the latter. For the diacyl EPL and CPL, 14 and 51 molecular species were determined, respectively. The major molecular species were 18∶0–20∶5n−3 (37.4%), 16∶0–20∶5n−3 (14.2%) and 18∶1n−7–22∶2NMID (13.2%) in the former, and 16∶0–20∶5n−3 (33.4%) and 16∶0–22∶6n−3 (22.3%) in the latter. It was found that there were significant differences in the molecular species between the alkylacyl and diacyl EPL and the alkylacyl and diacyl CPL; the number of molecular species was larger in CPL than in EPL, while the number of total carbons and double bonds of the major molecular species were larger in the EPL than in the CPL. Alkenylacyl EPL were similar to alkenylacyl CPL in molecular species composition.     

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.     

Effects of different medium-chain fatty acids on intestinal absorption of structured triacylglycerols

To study the effect of the chain length of medium-chain fatty acids on the intestinal absorption of long-chain fatty acids, we examined the lymphatic transport of fat following administration of five purified structured triacylglycerols (STAG) containing different medium-chain fatty acids in the sn-1, 3 positions and long-chain fatty acids in the sn-2 position in a rat model. Significant amounts of medium-chain fatty acids were found in lymph samples after intragastric administration of 1,3-dioctanoyl-2-linoleyl-sn-glycerol (8∶0/18∶2/8∶0), 1,3-didecanoyl-2-linoleyl-sn-glycerol, and 1,3-didodecanoyl-2-linoleyl-sn-glycerol. The accumulated lymphatic transport of medium-chain fatty acids increased with increasing carbon chain length. The recoveries of caprylic acid (8∶0), capric acid (10∶0), and lauric acid (12∶0) were 7.3±0.9, 26.3±2.4, and 81.7±6.9%, respectively. No significant differences were observed for the maximal intestinal absorption of linoleic acid (18∶2n−6) when the chain length of medium-chain fatty acids at the primary positions was varied, and the absorption of 18∶2 and oleic acid (18∶1) from 8∶0/18∶2/8∶0 and 1,3-dioctanoyl-2-oleyl-sn-glycerol was similar. We conclude that the chain length of the medium-chain fatty acids in the primary positions of STAG does not affect the maximal intestinal absorption of long-chain fatty acids in the sn-2 position in the applied rat model, whereas the distribution of fatty acids between the lymphatics and the portal vein reflects the chain length of the fatty acids. Presented in part at the 3rd ISSFAL Conference, Lyon, France, June 1–5, 1998.     

Ether lipid content and fatty acid distribution in rabbit polymorphonuclear neutrophil phospholipids

This study was undertaken to determine if rabbit neutrophils contain sufficient ether-linked precursor for the synthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activatin factor) by a deacylation-reacylation pathway. The phospholipids from rabbit peritoneal polymorphonuclear neutrophils were purified and quantitated, and the choline-containing and ethanolamine-containing phosphoglycerides were analyzed for ether lipid content. Choline-containing phosphoglycerides (37%), ethanolamine-containing phosphoglycerides (30%), and sphingomyelin (28%) were the predominant phospholipid classes, with smaller amounts of phosphatidylserine (5%) and phosphatidylinositol (<1%). The choline-linked fraction contained high amounts of 1-O-alkyl-2-acyl-(46%) and 1,2-diacyl-sn-glycero-3-phosphocholine (54%), with a trace of the 1-O-alk-1′-enyl-2-acyl species. The ethanolamine-linked fraction contained high amounts of 1-O-alk-1′-enyl-2-acyl-(63%) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (34%), and a low quantity of the 1-O-alkyl-2-acyl species (3%). The predominant 1-O-alkyl ether chains found in thesn-1 position of the choline-linked fraction were 16∶0 (35%), 18∶0 (14%), 18∶1 (26%), 20∶0 (16%), and 22∶0 (9%). The major 1-O-alk-1′-enyl ether chains found in thesn-1 position of the ethanolamine-linked fraction were 14∶0 (13%), 16∶0 (44%), 18∶0 (27%), 18∶1 (12%) and 18∶2 (3%). The major acyl groups in thesn-1 position of 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine were 16∶0, 18∶0 and 18∶1. The most abundant acyl group in thesn-2 position of all classes of choline- and ethanolamine-linked phosphoglycerides was 18⩺2. Although this work does not define the biosynthetic pathway for platelet activating factor, it does show that there is ample precursor present to support its synthesis by a deacylation-reacylation pathway.     

Variation in fatty acid content and seed weight in some lauric acid richCuphea species

Significant genetic variation for lauric acid (12∶0) and capric acid (10∶0) composition and seed weight was measured within lauric acid-rich, self-pollinating germplasm accessions ofCuphea wrightii, C. tolucana, andC. lutea. Means and ranges of individual plant progenies for 12∶0 content ofC. wrightii accessions was 60.5±.63% (49.8±65.8%), 10∶0 content was 23.7±.54% (18.6±33.0%), and 1000-seed weight was 1.50±.03 g (1.20–2.47 g). Progenies of single plant selections carried to the S₂ generation exhibited reduced variability within selections, but significant variation among selections for 12∶0, 10∶0 and 1000-seed weight. Variation among single plant selections ofC. tolucana was less than that ofC. wrightii and attributed to a restricted germplasm base. Means and ranges for 12∶0 content were 61.6±.47% (59.2–69.9%), 10∶0 was 22.3±.62% (11.7–25.3%), and 1000-seed weight was 1.40±.05 g (0.90–1.69 g).Cuphea lutea has a significantly different 12∶0−10∶0 profile than the other lauric acid-rich species. Means and ranges for 12∶0 were 36.8±.14% (33.7–40.8%), 10∶0 was 21.8±.08% (16.4–23.9%), 1000-seed weight was 2.26±.02 g (1.82–272 g). The 1000-seed weight was highly positively correlated with 8∶0, 10∶0, 18∶1 and 18∶2 contents and highly negatively correlated with 12∶0, 14∶0 and 16∶0 in bothC. wrightii andC. tolucana. No such relationship was found forC. lutea. A highly significant negative correlation was also measured for 12∶0 and 10∶0 contents inC. wrightii andC. tolucana.     

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 chain compositon of ether and ester glycerophospholipids in the Japanese oysterCrassostrea gigas (Thunberg)

The fatty chain compositions of 1-O-alk-1′-enyl-2-acyl, 1-0-alkyl-2-acyl, and 1,2-diacyl glycerophospholipids of the Japanese oysterCrassostrea gigas (Thunberg) were investigated. Major fatty chains in thesn-1 position of 1-alk-1′-enyl-2-acyl ethanolamine phospholipids (EPL) were 18∶0 (64.7%) and 20∶1 (11.1%). Majorsn-1 chains of alkenylacyl choline phospholipids (CPL) were 18∶0 (63.3%) and 16∶0 (22.2%). In the case of 1-alkyl-2-acyl EPL, the predominant fatty chains in thesn-1 position were 18∶0 (51.5%), 16∶0 (16.0%) and 20∶1 (12.5%); in the case of 1-alkyl-2-acyl CPL, the majorsn-1 chains were 16∶0 (44.0%) and 14∶0 (23.4%). Saturated fatty chains were predominant in both EPL and CPL. Prominent fatty acids in thesn-2 position of the alkenylacyl EPL were 22∶6n−3 (29.0%), 20∶5n−3 (19.0%) and 22∶2 NMID (non-methylene interrupted dienes, 16.6%) contributing to about 65% of the total fatty acids, while alkenylacyl CPL was rich in the saturated acids 16∶0 (32.0%) and 18∶0 (9.2%). In the alkylacyl EPL, 16∶0, 18∶1n−9, 18∶0 and 16∶1n−7 were prominentsn-2 fatty acids and accounted for 30.6%, 10.0%, 9.8%, and 8.3%, respectively. Polyunsaturated fatty acids were detected, but were present at extremely low percentages. Majorsn-2 fatty acids in alkylacyl CPL were 16∶0 (25.4%), 22∶6n−3 (16.0%) and 20∶5n−3 (8.4%). The major fatty acids of diacyl EPL were 20∶5n−3 (22.3%), 16∶0 (17.9%), and 18∶0 (16.1%), and those of diacyl CPL were 16∶0 (30.4%), 20∶5n−3 (17.6%) and 18∶1n−7 (7.4%).     

Effect of phosphatidylcholine structure on the adenylate cyclase activity of a murine fibroblast cell line

To determine which structural characteristics of membrane phospholipids influence adenylate cyclase activity, we measured basal and sodium fluoride- or forskolin-stimulated activity in a murine fibroblast cell line,i.e., Balb/c3T3 cells grown in media supplemented with fetal calf serum (FCS), lipid-depleted FCS (LD-FCS) or LD-FCS complexed with different phosphatidylcholine (PC) molecular species. Cells grown in the presence of LD-FCS showed a substantial decrease in their basal and NaF-stimulated adenylate cyclase activities; however, their forskolin-stimulated activity was not altered, suggesting that the enzyme's catalytic site is not affected by changes in membrane lipids. Media supplemented with different LD-FCS/PC complexes were shown to prevent the LD-FCS-mediated reduction of basal and NaF-stimulated adenylate cyclase activity to different extents. Addition ofcis-9-16∶1/cis-9-16∶1,cis-9-18∶1/cis-9-18∶1 orcis-9-18∶1/cis-9,12-18∶2sn-glycerophosphocholine (GPC) completely restored adenylate cyclase activity, whilecis-11-18∶1/cis-11-18∶1 GPC was not effective and only a partial recovery was observed with 16∶0/16∶0, 16∶0/cis-9-18∶1 andtrans-9-18∶1 GPC. Considering the structural features of these seven PC molecular species, the findings suggest that an optimal lipid environment is conferred to the enzyme by the presence of thecis double bonds, each located in Δ9 position of the PC acyl chains. The limited effect ofcis-9-16∶1/cis-9-18∶1 GPC andcis-9-18∶1/cis-9-16∶1 GPC suggests that an equal length of the terminal hydrocarbon chains extending bevond the Δ9 double bonds is also important. Moreover, complete restoration of adenylate cyclase activity in cells exposed to 16∶0/cis-9,12-18∶2 GPC suggests that twocis-9,12 double bonds located on the same chain are as effective as twocis-9 double bonds each located on two different chains of PC. As the four double bonds of 16∶0/cis-5,8,11,14-20∶4 GPC had no effect, a mere increase in the number of double bonds seems insufficient to build an optimal lipid microenvironment for the enzyme.     

Effect of clofibric acid on the molecular species composition of diacyl glycerophosphocholine of rat liver microsomes

The effect of administeringp-chlorophenoxyisobutyric (clofibric) acid to rats on the molecular species composition of diacyl-glycerophosphocholine (GPC) of rat liver microsomes was studied. Microsomal choline glycerophospholipids were converted to 1,2-diradyl-3-acetylglycerol and were separated into molecular species by reverse-phase high performance liquid chromatography. Diacyl-GPC consisted of 17 different molecular species. The predominant species were arachidonoyl derivatives, such as 18∶0–20∶4 (22.2% of the total) and 16∶0–20∶4 (22.0%). Administration of clofibric acid to rats caused a marked increase in 16∶0–18∶1 species of diacyl-GPC from 8% to 30%, making these the predominant species of diacyl-GPC in clofibric acid-fed rats. Also, a significant decrease (50% of controls) in 18∶0–18∶2 and 18∶0–20∶4 species was observed, whereas the decrease in molecular species containing 16∶0 at the 1-position such as 16∶0–18∶2 and 16∶0–20∶4 was small (approximately 85% of control). The results show that clofibric acid caused marked changes in the molecular species composition of diacyl-GPC. The participation of 1-acyl-GPC acyltransferase and stearoyl-CoA desaturase in the regulation of the molecular species composition of diacyl-GPC is discussed.     

Composition of mouse peritoneal macrophage phospholipid molecular species

The individual molecular species composition of diacyl, alkylacyl and alkenylacyl glycerophospholipids was determined in mouse peritoneal macrophages. A marked deterogeneity in the relative composition (mol%) of macrophage ether and ester phospholipid individual species was noted. High concentrations of 16∶0–20∶4 were found in ether phospholipids such as alkenylacyl glycerophosphoethanolamine (GPE; 27.5 mol%) and alkylacyl glycerophosphocholine (GPC; 16.6%) as compared to mol % levels of 16∶0–20∶4 in diacyl GPE (5.7%) and diacyl GPC (8.1%), respectively. Interestingly, alkenylacyl GPE was highly enriched in 1-ether (16∶0) relative to alkylacyl GPC. The predominant diacyl molecular species in glycerophosphoinositol (GPI) and glycerophosphoserine (GPS) were 18∶0–20∶4 (59.1%) and 16∶0–18∶1 (41.1%), respectively. It is noteworthy that the level of 18∶0–20∶4 was several times higher in diacyl GPI (59.1%) than in diacyl GPS (11.1%), diacyl GPE (25.7%), and diacyl GPC (3.7%). The most abundant molecular species in diacyl GPC and diacyl GPE were 16∶0–18∶1 (29.9%) and 18∶0–20∶4 (25.7%), respectively. The abundance of 20∶4 in ether phospholipids, specifically 16∶0–20∶4 and 18∶0–20∶4, in alkylacyl GPC is significant in view of the role these antecedents play in the biosynthesis of platelet-activating factor (PAF) and 20∶4-derived eicosanoids in stimulated macrophages. The unique molecular species composition of the peritoneal macrophage distinguishes this cell type from others.     

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.     

Autoxidation of methyl linoleate initiated by the ozonide of allylbenzene

Allylbenzene ozonide (ABO), a model for polyunsaturated fatty acid (PUFA) ozonides, initiates the autoxidation of methyl linoleate (18∶2 ME) at 37°C under 760 torr of oxygen. This process is inhibited by d-α-tocopherol (α-T) and 2,6-di-ert-butyl-4-methylphenol (BHT). The autoxidation was followed by the appearance of conjugated diene (CD), as well as by oxygen-uptake. The rates of autoxidation are proportional to the square root of ABO concentration, implying that the usual free radical autoxidation rate law is obeyed. Activation parameters for the thermal decomposition of ABO were determined under N₂ in the presence of radical scavengers and found to be E_a=28.2 ±0.3 kcal mol⁻¹ and log A=13.6±0.2; k_d (37°C) is calculated to be (5.1±0.3)×10⁻⁷ sec⁻¹. Autoxidation data are also reported for ozonides of 18∶2 ME and methyl oleate (18∶1 ME).     

Selective increase in pinolenic acid (all-<Emphasis Type="Italic">cis</Emphasis>-5,9,12–18∶3) in Korean pine nut oil by crystallization and its effect on LDL-receptor activity

The aims of this study were to obtain concentrated pinolenic acid (5,9,12–18∶3) from dietary Korean pine (Pinus koraiensis) nut oil by urea complexation and to investigate its cholesteroi-lowering effect on the LDL-receptor activity of human hepatoma HepG2 cells. Pine nut oil was hydrolyzed to provide a low-pinolenic acid-containing FA extract (LPAFAE), followed by crystallization with different ratios of urea in ethanol (EtOH) or methanol (MeOH) as a solvent to produce a high-pinolenic acid-containing FA extract (HPAFAE). The profiles of HPAFAE obtained by urea complexation showed different FA compositions compared with LPAFAE. The long-chain saturated FA palmitic acid (16∶0) and stearic acid (18∶0) were decreased with urea/FA ratios (UFR) of 1∶1 (UFR1), 2∶1 (UFR2), and 3∶1 (UFR3). Linoleic acid (9,12–18∶2) was increased 1.3 times with UFR2 in EtOH, and linolenic acid (9,12,15–18∶3) was increased 1.5 times with UFR3 in MeOH after crystallization. The crystallization with UFR3 in EtOH provided the highest concentration of pinolenic acid, which was elevated by 3.2-fold from 14.1 to 45.1%, whereas that of linoleic acid (9,12–18∶2) was not changed, and that of oleic acid (9–18∶1) was decreased 7.2-fold. Treatment of HepG2 cells with HPAFE resulted in significantly higher internalization of 3,3′-dioctadecylindocarbocyanine-LDL (47.0±0.15) as compared with treatment with LPAFAE (25.6±0.36) (P<0.05). Thus, we demonstrate a method for the concentration of pinolenic acid and suggest that this concentrate may have LDL-lowering properties by enhancing hepatic LDL uptake.     

Phosphatidylcholine hydroperoxide levels in human plasma are lower than previously reported

The quantification of PC hydroperoxide (PCOOH) in human plasma was studied by HPLC with chemiluminescence detection (HPLC-CL). We identified for the first time the monohydroperoxide of 1-palmitoyl-2-linoleoyl-PC hydroperoxide (PC 16∶0/18∶2-OOH) in plasma by LC-MS and HPLC-CL. The standard compound, PC 16∶0/18∶2-OOH (synthetic PCOOH), as well as PCOOH from egg yolk, was used. Comparison of the PCOOH concentration in each participant's plasma as determined by use of a Finepak SIL NH₂ column with 2-propanol/methanol/water as the mobile phase (system A, the conventional method) gave a higher concentration than did an LC-18-DB column with methanol containing 0.01% triethylamine (system B). The mean PCOOH concentration for the 43 healthy volunteers was 55.1±30.4 pmol/mL (mean±SD) for system A and 16.3±9.9 pmol/mL for system B. Moreover, the main peak of the plasma extract appeared at a different time from that of synthetic PCOOH or egg yolk PCOOH in system A, whereas in system B plasma sample retention time practically corresponded to that of standard PCOOH. These findings confirm that the PCOOH plasma concentration is not so high as previously reported.     

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.     

The esterified plasma fatty acid profile is altered in early HIV-1 infection

Previous studies have shown that alterations in micronutrient utilization occur in patients with Acquired Immune Deficiency Syndrome. In this study, total plasma fatty acid composition was measured in 36 homosexual men infected with the Human Immunodeficiency Virus 1 (HIV-1) and in 17 HIV-1 seronegative homosexual men in order to evaluate differences associated with early HIV-1 infection. Immunologic assessment included CD4 cell number count and lymphocyte blastogenesis in response to the mitogens phytohemagglutinin (PHA) and pokeweed (PWM). The mean total amount of ω6 polyunsaturated fatty acids (18∶2 and 20∶4) was significantly lower in the HIV-1 seropositive subjects (38±8.1% SD) as compared to HIV-1 seronegative subjects (43±4.2%;P=0.0027). This was also reflected in a higher level of total saturated fatty acids (16∶0 and 18∶0) in HIV-1 seropositive subjects (30±2.2%vs. 26±2.8%;P=0.0001). The ratio of linoleic to arachidonic acid (18∶2 to 20∶4) was higher in the HIV-1 seronegative group (6.76±4.88) compared to the HIV-1 seronegative group (4.86±1.37;P=0.0213). The response to PHA in seropositive subjects correlated inversely with total plasma ω6 fatty acids (r=−0.36;P=0.027), and directly with the 18∶2 to 20∶4 ratio (r=0.33;P=0.046). CD4 cell counts and the response to PWM did not correlate with plasma fatty acid levels in HIV-1 seropositive subjects. We conclude that early HIV-1 infection is associated with lower plasma ω6 polyunsaturated fatty acids, notably arachidonic acid, than are controls, and that the changes in the plasma fatty acid profile correlate with some indices of immune function.     

Synthesis of 1,2 dilinoleoyl and 1,2 dipalmitoylsn-3-glycerophosphorylcholine

The synthesis of 1,2 dilinoleoylsn-3-glycerophosphorylcholine (1,2-18∶2-sn-3-GPC) and 1,2 dipalmitoylsn-3-glycerophosphorylcholine (1,2-16∶0-sn-3-SPC) is described. Synthesis was accomplished by acylating free glycerophosphorylcholine (GPC) with the anhydride and potassium salt of the desired acid. Purification of 1,2-16∶0-sn-3-GPC was accomplished by crystallization, while purification of 1,2-18∶2-sn-3-GPC required the use of alumina column chromatography and then crystallization from acetone at −7C. Scientific contribution N. 491, Agricultural Experiment Station, University of Connecticut, Storrs.     

β-Oxidation of 18∶3n−3 in atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) hepatocytes treated with different fatty acids

To study whether Atlantic salmon β-oxidation was affected by dietary FA composition, an in vitro study with primary hepatocytes was undertaken. Isolated hepatocyte cultures were stimulated with either 16∶0, 18∶1n−9, 18∶2n−6, 18∶3n−3, 20∶5n−3, or 22∶6n−3 in triplicate for 24 h. In addition, a control was included where no FA stimulation was performed, also in triplicate. After stimulation, radiolabeled [1-¹⁴C]18∶3n−3 was added and the cells were incubated for 2 h at 20°C. The cells were then harvested, and radioactivity was determined in the acid-soluble part of the cells and medium, i.e., the end products of the β-oxidation pathway. Specific β-oxidation activity was significantly higher in hepatocytes stimulated with 18∶3n−3. Further, when taking into account the amount of radiolabeled [1-¹⁴C]18∶3n−3 taken up by the cells—the relative amount of β-oxidized [1-¹⁴C]18∶3n−3 of the total FA taken up by the hepatocytes—no significant differences were found. Thus, the regulation of β-oxidation activity in the primary Atlantic salmon hepatocytes seems to be at the level of FA uptake and transport into the cell. This in vitro study shows that the catabolism processes in salmon hepatocytes are affected by the FA available and probably already regulated at the level of FA uptake.