Analysis of novel hydroperoxides and other metabolites of oleic, linoleic, and linolenic acids by liquid chromatography-mass spectrometry with ion trap MSn

Linoleate is oxygenated by manganese-lipoxygenase (Mn-LO) to 11S-hydroperoxylinoleic acid and 13R-hydroperoxyoctadeca-9Z,11E-dienoic acid, whereas linoleate diol synthase (LDS) converts linoleate sequentially to 8R-hydroperoxylinoleate, through an 8-dioxygenase by insertion of molecular oxygen, and to 7S,8S-dihydroxylinoleate, through a hydroperoxide isomerase by intramolecular oxygen transfer. We have used liquid chromatography-mass spectrometry (LC-MS) with an ion trap mass spectrometer to study the MSn mass spectra of the main metabolites of oleic, linoleic, alpha-linolenic and gamma-linolenic acids, which are formed by Mn-LO and by LDS. The enzymes were purified from the culture broth (Mn-LO) and mycelium (LDS) of the fungus Gaeumannomyces graminis. MS3 analysis of hydroperoxides and MS2 analysis of dihydroxy- and monohydroxy metabolites yielded many fragments with information on the position of oxygenated carbons. Mn-LO oxygenated C-11 and C-13 of 18:2n-6, 18:3n-3, and 18:3n-6 in a ratio of approximately 1:1-3 at high substrate concentrations. 8-Hydroxy-9(10)epoxystearate was identified as a novel metabolite of LDS and oleic acid by LC-MS and by gas chromatography-MS. We conclude that LC-MS with MSn is a convenient tool for detection and identification of hydroperoxy fatty acids and other metabolites of these enzymes.     

Conjugated linoleic acid reduces arachidonic acid content and PGE2 synthesis in murine keratinocytes

Using a T-maze, the influence of transient global cerebral ischemia on working memory in gerbils was investigated. Furthermore, it was examined whether a correlation exists between impairment in choice accuracy in the T-maze and neuron loss in the hippocampus. In two experiments, male Mongolian gerbils were tested in a previously learned delayed alternation T-maze task 1 week after a 4 min occlusion of the common carotid arteries. In both experiments memory was significantly impaired and in the second experiment, where the design allowed a separation between working and reference memory deficits, a selective impairment in working memory was seen. The results suggest that ischemia-induced disruption of delayed alternation in the T-maze in gerbils is a model which is relevant to the clinical manifestations of vascular dementia.     

Effect of dietary linoleic to linolenic acid ratio and vitamin E supplementation on vitamin E status of poults

Two experiments were conducted to determine the effect of dietary linoleic to linolenic acid (LO:LN) ratio and dl-alpha-tocopheryl acetate (TA) supplementation on selected characteristics of the liver and cerebellum and on vitamin E status of turkey poults from hatch through 22 d of age. In Experiment 1, 1-d-old poults were fed diets containing no supplemental TA (0E) or 150 IU TA/kg diet (150E). Poults fed the 150E diet had greater (P < 0.001) concentrations of alpha-tocopherol (TOC) in the liver and plasma than those fed the 0E diet from 7 to 22 d of age. The 150E diet, however, did not completely overcome the decrease in liver and plasma TOC concentrations observed at these ages. The 150E diet had no effect on poult BW, feed efficiency, or on the weight, protein, lipid, or fatty acid concentrations of the liver. Thiobarbituric acid reactive substances assay of liver and hemolysis assay of red blood cells (RBC) showed that the 150E diet decreased the susceptibility of liver and RBC to in vitro peroxidation at 13 and 22 d of age. In Experiment 2, 1-d-old poults were fed the 0E and 150E diets in a complete factorial arrangement with decreasing ratios of LO:LN (10, 5, and 1). Dietary LO:LN ratio had no effect on RBC hemolysis or cerebellum TOC concentration. As the ratio of LO:LN decreased, the arachidonic acid content of liver and cerebellum lipids decreased. Ratios of n-6 to n-3 fatty acids in liver and cerebellum were directly related to dietary LO:LN at 13 and 22 d of age.     

Effect of dietary fats rich in lauric, myristic, palmitic, oleic or linoleic acid on plasma, hepatic and biliary lipids in cholesterol-fed hamsters

Effects of different dietary fats on plasma, hepatic and biliary lipids were determined in male golden Syrian hamsters (Mesocricetus auratus) fed on purified diets for 7 weeks. Diets were made by blending different fats containing characteristic fatty acids: butter (14:0 + 16:0), palm stearin (16:0), coconut oil (12:0 + 14:0), rapeseed oil (18:1), olive oil (18:1) and sunflowerseed oil (18:2). In all diets except the sunflowerseed oil diet dietary 18:2 was held constant at 2% energy. Total fat supplied 12% of energy and cholesterol was added at 4 g/kg diet. Plasma cholesterol and triacyglycerol concentrations were increased by dietary cholesterol. After 7 weeks, plasma cholesterol concentrations were highest with the palm stearin, coconut oil and olive oil diets (8.9, 8.9 and 9.2 mmol/l) and lowest with the rapeseed oil and sunflowerseed oil diets (6.7 and 5.5 mmol/l) while the butter diet was intermediate (8.5 mmol/l). Hepatic cholesterol concentration was highest in hamsters fed on the olive oil diet and lowest with the palm stearin diet (228 v. 144 mumol/g liver). Biliary lipids, lithogenic index and bile acid profile of the gall-bladder bile did not differ significantly among the six diets. Although the gallstone incidence was generally low in this study, three out of 10 hamsters fed on the palm stearin diet developed cholesterol gallstones. In contrast, no cholesterol gallstones were found with the other diets. Rapeseed and sunflowerseed oils caused the lowest plasma cholesterol and triacyglycerol concentrations whereas olive oil failed to demonstrate a cholesterol-lowering effect compared with diets rich in saturated fatty acids. Since 18:2 was kept constant at 2% of energy in all diets, the different responses to rapeseed and olive oils could possibly be attributed to their different contents of 16:0 (5.6% v. 12.8% respectively). Other possible explanations are discussed.     

Effects of arachidonic acid metabolism on hypoxic vasoconstriction in rabbit lungs

Hypoxic pulmonary vasoconstriction is an essential mechanism that matches lung perfusion to ventilation, thus optimising pulmonary gas exchange. Despite its pathophysiological relevance, the mechanism of hypoxic pulmonary vasoconstriction still remains enigmatic. We investigated whether arachidonic acid metabolism is involved in the regulation of hypoxic pulmonary vasoconstriction in isolated, buffer-perfused rabbit lungs. Seven inhibitors were employed to determine the contribution of different vasoactive lipoxy- and cyclooxygenase mediators as well as cytochrome P450 products on the magnitude of hypoxic pulmonary vasoconstriction. Hypoxic pulmonary vasoconstriction was not affected by (i) the cyclooxygenase inhibitor acetylsalicylic acid, (ii) the thromboxane A2 receptor antagonist BM13.505, (iii) the 5'-lipoxygenase inhibitor MK886, and (iv) the lipoxygenase and cyclooxygenase inhibitor BW755c. The hypoxia-elicited pressor response was prominently inhibited by (i) nordihydroguaiaretic acid (50-150 microM), an inhibitor of lipoxygenase and cyclooxygenase and (ii) methoxsalen (100 microM) and 1-aminobenzotriazole (1-10 mM), two inhibitors of cytochrome P450-derived metabolites. However, no specificity for the regulation of hypoxic pulmonary vasoconstriction was found, as corresponding inhibitory potency of these agents was noted when vasoconstriction was achieved by the stable thromboxane analogue U46619 under conditions of normoxia. We conclude that there is no evidence for a specific involvement of different pathways of arachidonic acid metabolism in the mechanism of hypoxic pulmonary vasoconstriction in rabbits.     

Inhibition of arachidonic acid induced mortality in rabbits with several non-steroidal anti-inflammatory agents

The intravenous administration of arachidonic acid to rabbits is an effective in vivo model for evaluating potential anti-thrombotic drugs. Most of the non-steroidal anti-inflammatory agents (NSAIFA) inhibit this arachidonic acid induced mortality (except sodium salicylate and acetaminophen). However, there is a lack of correlation between the relative potencies from various assays (rabbit anti-thrombotic, anti-inflammatory, alalgesic, ulcerogenic and inhibition of prostaglandin synthetase evaluations). These studies imply other actions with NSAIFA than an effect solely on the prostaglandin biosynthetic pathway.     

Effects of dietary acetate and bicarbonate on experimental atherosclerosis in rabbits

Scanning electron microscopic evaluation of the resected plaques from 11 patients with symptomatic and asymptomatic lesions of the carotid artery bifurcation were carried out. The most common findings were mounds of atheromatous material, many of which appeared to be fractured and capped with fibrin-platelet exudates, projecting into the lumen of the vessels. Both small and large ulcer craters were identified, and attached bodies were present in three-fourths of the specimens studied. These consisted most commonly of platelet clumps and fibrin sheets and were intimately related to the atheroma.     

Effects of guan-fu base a on experimental cardiac arrhythmias and myocardial contractility

Guan-fu base A (GFA) is a terpenoid alkaloid isolated from the tuber of Aconitum coreanum in our institute. GFA (20-30 mg.L-1) reduced the ventricular tachycardia (VT) and ventricular fibrillation (VF) rate induced by K(+)-free and high Ca2+ solution in Langendorf heart of rats. Pretreatment of conscious rats with GFA 2.5-10 mg.kg-1 iv, increased the amount of beiwutine necessary to produce arrhythmias. Ouabain-induced VT in conscious dogs was reverted to sinus rhythm in 1-2 min by iv GFA 9-10 mg.kg-1. GFA 10-20 mg.kg-1 iv was also found to be effective in protecting anesthetized dogs from atrial fibrillation induced by topical application of ACh. GFA 10 mg.kg-1 iv obviously decreased heart rate and prolonged the P-R interval, but slightly affected the myocardial contractility in anesthetized dogs. GFA showed no obvious effect on stroke volume and cardiac output in conscious dogs. In conclusion, GFA showed therapeutic and prophylactic effect on different models of experimental arrhythmias without causing marked effect on myocardial contractility.     

Effects of medium chain fatty acids (MCFA), myristic acid, and oleic acid on serum lipoproteins in healthy subjects

In this study we investigated the effects on lipoproteins of medium chain fatty acids (MCFA) and myristic acid relative to those of oleic acid. Thirty-seven women and 23 men consumed a 3-wk run-in diet enriched in oleic acid followed by a 6-wk test diet rich in MCFA (n = 21), myristic (n = 20), or oleic acid (n = 19). Experimental fats were incorporated into solid foods. Total fat intake was 40 En% fat. The dietary compositions were the same except for 10 En%, which was provided by MCFA, myristic, or oleic acids, respectively. With the myristic acid diet, low density lipoprotein (LDL) cholesterol was 0.37 mmol/L higher compared with the oleic acid diet (P = 0.0064 for difference in changes). The MCFA diet increased LDL cholesterol, though not significantly, with 0.23 mmol/L relative to the oleic acid diet (P = 0.0752). Compared with the oleic acid diet, HDL cholesterol concentrations increased with the myristic acid diet by 0.10 mmol/L (P = 0.0273) but not with the MCFA diet. The MCFA diet slightly elevated triacylglycerol concentrations, but responses did not significantly differ between the diets. The MCFA diet significantly decreased the apoA-I to apoB ratio compared with both other diets (P < 0.02). We conclude that MCFA raise LDL cholesterol concentrations slightly and affect the apoA-I to apoB ratio unfavorably compared with oleic acid. Myristic acid is hypercholesterolemic, although less than predicted earlier, and raises both LDL and HDL cholesterol concentrations compared with oleic acid.     

Effects of three trans isomers of eicosapentaenoic acid on rat platelet aggregation and arachidonic acid metabolism

PURPOSE: To compare in a randomized, prospective manner infectious complication rates associated with presacral drainage versus no drainage in the presence of penetrating rectal injury. METHODS: During a 45-month period, 48 patients with penetrating rectal injuries were entered into a randomized, prospective study at an urban Level I trauma center. The patients were randomized to a presacral drainage group or a nondrainage group. Randomization was performed after detection of the rectal injury. Forty-four injuries were identified by proctoscopy (92%), with the rest detected intraoperatively or by physical examination. All patients with rectal injuries were included regardless of age, associated injuries, time from injury to operation, blood loss, severity of rectal injury, other abdominal organs injured, or hemodynamic stability. Rectal injuries were defined as those injuries to the large bowel distal to the peritoneal reflection. All rectal injuries underwent fecal diversion, and all drainage was accomplished using closed Jackson-Pratt drainage. RESULTS: Forty-eight patients were studied, of whom 25 were randomized to no drainage and 23 were randomized to presacral drainage. The average age for the nondrainage group was 21.9 years, and the average age for the presacral drainage group 26.0 years. The average Penetrating Abdominal Trauma Index score was 34.3 for the nondrainage group and 32.4 for the presacral drainage group. There were two (8%) septic complications (one perirectal and one perivesical abscess) associated with the rectal injuries in the presacral drainage group. The abscesses in the drainage group resolved after computed tomography-guided drainage. There was one (4%) septic complication (rectocutaneous fistula) in the nondrainage group, which was associated with a retained missile fragment. The fistula resolved after bedside percutaneous removal of the missile fragment. CONCLUSION: We conclude that presacral drainage for penetrating rectal injuries has no effect on infectious complications associated with the rectal injuries.     

Effects of K+-depolarization, arachidonic acid, ethanol, and aging on the high-affinity choline transport in rat hippocampus

The Na+-dependent high-affinity choline uptake (HACU) transport and the [3H]hemicholinium-3 ([3H]HC-3) specific binding were measured on hippocampal synaptosomes of young (3-6 months) and old (22 months) Wistar rats. In vitro effects of 100-300 microM arachidonic acid (AA) and of 5% ethanol were tested under basal as well as stimulated (55 mM KCl) conditions. The influence of AA (an irreversible decrease of HACU and a reversible increase of [3H]HC-3 binding) was more marked under stimulated rather than basal conditions in brain tissue of young rats. The increased K+-depolarization effect on HACU and the decreased influence of AA on [3H]HC-3 binding were estimated in brain tissue of old compared to young rats. Results suggest the involvement of different pools of the high-affinity choline carrier and marked changes due to aging in the regulation of the HACU transport.     

Influence of dietary linoleic acid on experimental human breast cancer cell metastasis in athymic nude mice

The effects of linoleic acid (LA), an omega-6 fatty acid precursor for prostaglandin biosynthesis, on the later stages of human breast cancer cell metastasis were studied by the intravenous injection of tumor cells into nude mice (). MDA-MB-435 cells were grown as solid tumors in donor mice fed a 12% or 2% LA-containing diet. These cells were harvested, and injected via a tail vein into recipient mice also fed a 12% LA (Group 1) or 2% LA (Group 2) diet. Other groups were fed 12% LA (Group 3) or 2% LA (Group 4), but injected with the cells grown in vitro in a low-LA culture medium. At necropsy 8 weeks later, the incidence of metastatic lung nodules was higher in Group 1 high LA donor/high LA recipient mice (p<0.001), and, to a lesser degree, Group 2 low LA donor/low LA recipient mice (p<0.05) compared with Groups 3 or 4. The extent of metastasis was significantly higher in Group 1 compared with any of the other groups, including metastasis to the ovaries, which occurred in 27% of the Group 1 mice. These findings show that LA, most likely by increased synthesis of cyclooxygenase products, stimulates metastasis, at least in part, by direct effects on the tumor cells, rather than on potential metastatic sites in the host.     

Choline acetyltransferase in ocular tissues of rabbits, cats, cattle, and man

The variation of choline acetyltransferase activity in ocular tissues of four mammalian species, rabbits, cats, cattle, and man, was determined. Enzyme activity of irides and ciliary bodies, i.e., parasympathetically innervated structures, tended to be similar in all four species. Two exceptions were bovine irides and human ciliary bodies; these two tissues had higher enzyme activities. Choline acetyltransferase activity was present in the corneal epithelium of rabbit, bovine, and human eyes, but little or none could be detected in that of cats. Feline retina and pigment epithelium-choroid also contained far less choline acetyltransferase activity than the same tissues in the other three species.     

A lipoic acid-gamma linolenic acid conjugate is effective against multiple indices of experimental diabetic neuropathy

Cell suspensions of Campylobacter jejuni, Escherichia coli, Pseudomonas fluorescens, and Salmonella enteritidis exposed to sublethal concentrations (0.5 to 5 mM) of trisodium phosphate (TSP) for 10 min showed greatly increased susceptibility to lysozyme (10 micrograms ml-1) and/or nisin (1 microM). Under optimal conditions at 37 degrees C, reductions in viable count after 30 min were up to six log cycles. At 4 degrees C, C. jejuni showed greater resistance than at 37 degrees C, and maximal cell kills (95%) were reduced by more than two log cycles. Cells dried on the surface of chicken skin were more resistant than suspended cells to TSP-lysozyme and TSP-nisin treatments; nevertheless, at 37 degrees C, kills varied from approximately 95% for S. enteritidis cells with nisin (30 microM) or lysozyme (100 micrograms ml-1) to > 99.9% for C. jejuni and E. coli cells with nisin. Under the experimental conditions used, nisin also reduced viable counts of skin-attached Staphylococcus aureus by > 99.9%. The results suggest that the high TSP concentrations (approximately 10% wt/vol, 0.25 M) needed for successful decontamination of gram-negative bacteria, on the surface of poultry and other foodstuffs, may be substantially reduced by following TSP treatment with exposure to low lysozyme or nisin concentrations.     

Therapeutic effects of omoconazole nitrate on experimental tinea pedis, an intractable dermatophytosis, in guinea-pigs

OBJECTIVES: To determine the effectiveness and safety of high-dose yohimbine for the treatment of mixed-type impotence. METHODS: Twenty-nine patients who had mixed-type impotence diagnosed on the basis of a sexual history, results of a physical examination, laboratory analysis, assessment of the sensory pain threshold of the dorsal nerve of the penis, and dynamic color Doppler sonography of the cavernosal arteries were entered into a randomized, double-blind, placebo-controlled, crossover comparison of a placebo with high-dose yohimbine hydrochloride (36 mg per day orally). The treatment consisted of two 25-day courses; after a 14-day washout period, the patients who initially received the placebo for 25 days were switched to yohimbine hydrochloride for 25 days. Erectile function, ejaculation, interest in sex, physical examination findings, blood pressure, pulse rate, weight, and audiovisual sexual stimulation test were investigated before treatment and at the end of each drug period. The Mann-Whitney test was used for the statistical analysis. RESULTS: Twenty-seven patients (93%) completed the whole treatment schedule. Positive clinical results (complete and partial responses) were obtained in 12 cases (44%) at the end of the yohimbine phase and in 13 (48%) after the placebo period. No statistical difference was indicated. Drug-related adverse effects occurred in 2 patients in the yohimbine group (7%). CONCLUSIONS: Yohimbine is no better than placebo as a first-line treatment for mixed-type impotence.     

Influence of oleic acid on serum lipoprotein-X in vitro

Lipoprotein-X is no longer detectable in serum by either the agar gel-electrophoresis/polyanion precipitation technique or immunoelectrophoresis with specific antiserum after in vitro addition of oleic acid. Evidence is presented which indicates that this ostensible loss is due not to its destruction, but rather to altered electrophoretic mobility. The findings suggest an explanation for the well known post-heparin "disappearance" of lipoprotein-X, and indicate that caution may be needed in the interpretation of such lipoprotein-X testing of sera from subjects with increased concentrations of lysolipids in their blood.     

Effects of Ce^3＋ on improvement of spectral characteristics and function of chloroplasts damaged by linolenic acid in spinach

Linolenic acid has great effects on the structure and function of chloroplast. We studied the effects of Ce3+ on the improvement of chloroplast spectral characteristics and oxygen evolution damaged by linolenic acid in spinach. Results showed that Ce3+ could decrease the light absorption increased by linolenic acid and promote the distribution of excitation energy to PS Ⅱ and alleviate the decrease of PS Ⅱ fluo-rescence yield caused by linolenic acid. The linolenic acid treatments in various concentrations reduced the oxygen-evolving rate of chloro-plasts, but the rate was accelerated since adding Ce3+.