Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF(2 alpha)-III, 15-epi-iPF(2 alpha)-III, iPF(2 alpha)-VI, and 8,12-iso-iPF(2 alpha)-VI along with the prostaglandin PGF(2 alpha) and 2,3-dinor-iPF(2 alpha)-III, a metabolite of iPF(2 alpha)-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF(2 alpha)-III obtained by our method were significantly correlated with results by an ELISA, although an approximately 2-fold high bias was observed for the ELISA data. For iPF(2 alpha)-III and its metabolite 2,3-dinor-iPF(2 alpha)-III, smokers had significantly higher concentrations than nonsmokers (513 +/- 275 vs. 294 +/- 104 pg/mg creatinine; 3,030 +/- 1,546 vs. 2,046 +/- 836 pg/mg creatinine, respectively). The concentration of iPF(2 alpha)-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents.     

Effect of dietary sources of n-3 fatty acids on pig performance and technological,nutritional and sensory qualities of pork

In France, animal products (dairy products, meat and eggs) are the main source of n-3 polyunsaturated fatty acids (PUFA) in the human diet; however, many individuals do not consume enough of this nutrient. The objective of this study was to increase n-3 PUFA precursor and derivative contents in tissues and test how they influence technological and sensory qualities of meat without negatively affecting growth performances of pigs. A total of 60 male pigs [(Large White×Landrace)×Pietrain] were assigned according to their initial liveweight (50.7±2.7 kg) to five experimental groups corresponding to five different diets that they received from 14 to 22 weeks of age. Dietary lipid supplements were composed of soybean and palm oil (SP), dehulled and extruded linseed (EL-), docosahexaenoic acid (DHA)-rich microalgae (MAG) or a mixture of linseed and microalgae at 75%/25% (3EL-/MAG) and 50%/50% (EL-/MAG), respectively. Diet did not influence growth performances of pigs or the technological quality of the meat. The n-3 PUFA content in the longissimus dorsi muscle, subcutaneous backfat (SCB) and liver increased with a dietary supply of linseed and microalgae and corresponded to circulating fatty acids (FA). The amount of malondialdehyde, representative of FA lipid peroxidation measured in SCB, increased significantly with the supply of microalgae, meaning that PUFA from the microalgae included in the diet increased the meat’s susceptibility to oxidation. The MAG diet scored highest for ‘abnormal’ flavor, similar to that of fish or organ meat, but the n-3 PUFA-rich diet had no effect on other sensory characteristics. Results of this study indicate benefits of enriching animal feed with n-3 PUFA, but the inclusion of long-chain n-3 PUFA such as DHA must be limited to avoid oxidation susceptibility and development of an off-odor.     

The effect of vitamin E supplementation on reduction of lymphocyte DNA damage induced by T-2 toxin and deoxynivalenol in weaned pigs

The objective of the present study was to establish the effect of deoxynivalenol (DON) and T-2 toxin on lipid peroxidation, lymphocyte DNA fragmentation and immunoglobulin production in weaned pigs, and furthermore, to evaluate the potential of vitamin E (α-tocopheryl acetate) in prevention of toxin mediated changes. Forty-eight weaned castrated male crossbred pigs (mean live weight at the beginning of the experimental period was 11.7 kg) were randomly assigned to five experimental groups: control (without toxin and vitamin E), T-2 (3 mg/kg T-2 toxin), T-2 + E (3 mg/kg T-2 toxin + 100 mg/kg vitamin E), DON (4 mg/kg DON) and DON + E (4 mg/kg DON + 100 mg/kg vitamin E). After 14 days of treatment blood was collected for analysis. Lipid peroxidation was studied by assays of malondialdehyde (MDA), total antioxidant status (TAS) of plasma and erythrocyte glutathione peroxidase (GPx). DNA damage in lymphocytes was measured by comet assay. Serum immunoglobulin levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and the hepatotoxicity was studied by measuring plasma liver enzyme levels (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl-transferase (GGT). Production parameters of both DON groups were significantly impaired in comparison to the control. DON significantly increased the amount of DNA damage in lymphocytes by 28%. Moreover, the levels of TAS were lowered by addition of DON. T-2 toxin significantly impaired daily live weight gain and feed conversion, increased the amount of DNA damage in lymphocytes by 27%, decreased total serum IgG and did not alter plasma TAS. Plasma and 24-h urinary malondialdehyde (MDA) excretion rate and erythrocyte Gpx levels did not differ among the groups. Supplementation with vitamin E did not improve production parameters impaired by DON and T-2 toxin and only partially protected lymphocyte DNA from toxin impact. To our knowledge, these are the first data on genotoxic effects of moderate doses of DON and T-2 toxin on pig lymphocytes. The effect of DON and T-2 toxin on the immune system was reflected as a change in immunoglobulin synthesis, which might be toxin and species specific. According to other results no major induction of oxidative stress could be proven. Enhancement of antioxidant status with vitamin E in the case of DON and T-2 toxin intoxication can be beneficial for remaining the lymphocyte DNA integrity.     

Oxidative stress in patients with multiple sclerosis

It is well known that brain and nervous system cells are prone to oxidative damage because of their relatively low content of antioxidants, especially enzymatic ones, and of the high levels of both membrane polyunsaturated fatty acids (PUFA) and iron easily released from injured cells. We have investigated the oxidative stress in the blood (plasma, erythrocytes and lymphocytes) of 28 patients affected with multiple sclerosis (MS) and of 30 healthy age matched controls, by performing a multiparameter analysis of non-enzymatic and enzymatic antioxidants--Vitamin E (Vit. E), ubiquinone (UBI), reduced and oxidized glutathione (GSH, GS-SG), superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and fatty acid patterns of phospholipids (PL-FA). PL-FA and Vit. E were assayed by GC-MS; UBI and GSH/GS-SG by HPLC; SOD, GPX and CAT by spectrophotometry. In comparison to controls, patients with MS showed significantly reduced levels of plasma UBI (0.21 +/- 0.10 vs. 0.78 +/- 0.08 mg/ml, p < 0.001), plasma Vit. E (7.4 +/- 2.1 vs. 11.4 +/- 1.8 mg/ml, p < 0.01), lymphocyte UBI (8.1 +/- 4.0 vs. 30.3 +/- 7.2 ng/ml blood, p < 0.001) and erythrocyte GPX (22.6 +/- 5.7 vs. 36.3 +/- 6.4 U/g Hb, p < 0.001). This blood antioxidant deficiency was associated with plasma levels of PL-PUFA--especially C20:3 n-6 and C20:4 n-6--significantly higher than controls. In conclusion, the blood of patients with MS shows the signs of a significant oxidative stress. The possibility of counteracting it by antioxidant administration plus an appropriate diet, might represent a promising way of inhibiting the progression of the disease. Antioxidant supplements should include not only GSH repleting agents, but also Vit. E, ubiquinol, and selenium.     

Control of matrix effects in the analysis of urinary F2-isoprostanes using novel multidimensional solid-phase extraction and LC-MS/MS

F(2)-isoprostanes (F(2)-iPs), established markers of oxidative stress, exist as four sets of regioisomers. Simultaneous and specific determination of F(2)-iPs can be achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed novel methods for urine sample preparation and HPLC to control matrix-related ion suppression effects in the LC-MS/MS analysis of F(2)-iPs. A selective solid-phase extraction (SPE) wash protocol was developed with an Oasis HLB (hydrophilic-lipophilic balance) SPE cartridge using an elution profile of [(3)H]8-iso-prostaglandin (PG)F(2alpha) (iPF(2alpha)-III) when the methanol concentration was increased under acidic, neutral, and base wash conditions. A multidimensional (MD)-SPE method that incorporated size exclusion chromatography [corrected] reverse-phase chromatography, and normal-phase chromatography was developed using an Oasis HLB SPE cartridge and an HLB microElution SPE plate. Average extraction recoveries of the deuterated internal standards of iPF(2alpha)-III and iPF(2alpha)-VI were 62 +/- 8% and 60 +/- 10%. A buffer-free HPLC method for the separation of F(2)-iP isomers was developed on base-deactivated C8 columns. Average matrix effects for iPF(2alpha)-III and iPF(2alpha)-VI were 95 +/- 6% and 103 +/- 5%. The clean extraction of urine F(2)-iPs using MD-SPE and the separation of F(2)-iP isomers using a novel HPLC method did not cause apparent ion suppression in the analysis of iPF(2alpha)-III and iPF(2alpha)-VI using LC-MS/MS. These findings should be useful for establishing a routine LC-MS/MS method for the analysis of F(2)-iPs.     

α-亚麻酸对高脂模型大鼠组织脂肪酸代谢的影响

研究不同ALA含量油脂对高脂模型大鼠组织脂肪酸代谢的影响.60只雄性Wistar大鼠分为正常组、高脂组、花生油组、13％、27％和55％ ALA含量油脂组,除正常组和高脂组外,其余各组在饲喂高脂饲料的同时采用灌胃方式连续给予2 mL/kg.bw剂量的受试油.试验6周后分别测定大鼠各组织脂肪酸组成.结果表明,高脂饮食能够降低大鼠各组织n-3脂肪酸含量,但摄入不同ALA油脂可显著增加组织n-3脂肪酸含量,并具有一定的剂量效应关系;但ALA及其代谢产物EPA、DPA和DHA的累积具有组织特异性,其中肾和心组织中ALA累积高于血浆、脑及肝组织,肝和脑组织中EPA和DPA含量增加较显著,而肾和心组织中EPA含量不变,各组织DHA含量增加不显著.不同ALA油脂组C18:3(n-6)和C20:3 (n-6)差异不显著,但与花生油组相比,其血浆、脑和肾组织C20:4含量显著降低.因此,富含ALA含量的油脂能够增加组织中ALA及其代谢产物在组织中的含量,提高其在脑组织中的分布比例,这可能是ALA具有心血管保护作用和促进脑生长发育的作用机制之一.     

Fatty acid transfer from sow to piglet differs for different polyunsaturated fatty acids (PUFA)

Polyunsaturated fatty acids (PUFA) are essential for the development of the nervous system in animals. It is known that pigs are good models for human in many aspects. The aim of the study was to investigate how fat content and FA composition in sows' diet influence FA composition in brain of newborn and in liver and brain of one-day-old piglets, respectively. High fat (6 %) feeds were designed with regard to saturated or polyunsaturated fat content and n-6/n-3 ratio by adding either oats rich in linoleic acid (LA) or linseed oil rich in alpha-linolenic acid (ALA). The ratio n-6/n-3 PUFA was 11 in all three diets (the low fat (3 %), high fat saturated and high fat oats diet), while the ratio in the linseed oil diet was 2. Increased proportion of ALA in the diet increased ALA and eicosapentaenoic acid (EPA) in piglets' neutral and polar liver lipids and the long chain PUFA, EPA, docosapentaenoic and docosahexaenoic acid in piglet brain. The results suggest that transport of n-3 PUFA from sow to piglet was higher via milk than via bloodstream in the uterus and that increased content of ALA in sows' feed led to an increased accumulation of n-3 FA in piglets' liver and brain.     

Urinary F₂-isoprostanes, obesity, and weight gain in the IRAS cohort

Obesity has been associated with increased F(2)-isoprostane (F(2)-IsoP) levels cross-sectionally. However, the prospective association may be inverse, based on our earlier finding that elevated urinary F(2)-IsoP levels predict lower risk of diabetes. This earlier finding led us to hypothesize that urinary F(2)-IsoPs reflect the intensity of oxidative metabolism and as such predict lower risk of both diabetes and weight gain. We examined cross-sectional relationships with obesity and prospective relationships with weight gain using the data from 299 participants of the Insulin Resistance Atherosclerosis Study (IRAS), all of whom were free of diabetes at baseline. Four urinary F(2)-IsoPs were assayed in stored baseline urine samples using liquid chromatography with tandem mass spectrometry: iPF(2α)-III, 2,3-dinor-iPF(2α)-III, iPF(2α)-VI, and 8,12-iso-iPF(2α)-VI (F(2)-IsoP 1-4, respectively). Baseline F(2)-IsoPs were positively associated with baseline measures of obesity; the strongest associations were found with two F(2)-IsoPs: odds ratios (95% confidence intervals) for overall and abdominal obesity were 1.74 (1.26-2.40) and 1.63 (1.18-2.24) for F(2)-IsoP2 and 1.47 (1.12-1.94) and 1.64 (1.22-2.20) for F(2)-IsoP4. F(2)-IsoP2 showed the strongest and significant inverse association with weight gain during the 5-year follow-up period: increase in F(2)-IsoP2 equal to 1 s.d. was associated with 0.90 kg lower weight gain (P = 0.02) and the odds ratios for relative (≥5%) and absolute (≥5 kg) weight gain were 0.67 (0.47-0.96) and 0.57 (0.37-0.87), respectively. The other three F(2)-IsoPs were consistently inversely associated with weight gain, although not significantly, suggesting that different F(2)-IsoPs vary in their ability to detect the association with weight gain.     

The influence of inclusions of vitamin E and corn oil on semen traits of Japanese quail (Coturnix coturnix japonica)

Reported was an investigation of the effect of vitamin E (Vit.E) and corn oil on semen traits of male Japanese quail (Coturnix coturnix japonica). From 8 to 20 wk of age, birds were raised on corn-based diets supplemented with corn oil (0 and 3%) and Vit.E (National Research Council (NRC) recommended 25mg/kg/day/dry matter and 150 mg/kg/day/dry matter) in a 2×2 factorial manner. The diet was supplemented with corn oil and Vit.E (E2C2) which provided additional n-6 polyunsaturated fatty acids in the form of 20:4n-6 and 22:4n-6 in spermatozoa phospholipid. The left testes weights were increased (P<0.01) in groups that received Vit.E in the diet (3.95 and 4.12 g, respectively) (P=0.03) and combined testes weight was the greatest in E2C2 group (7.57g) (P=0.02). Semen volume increased throughout the experiment in the E2C2 group. E2C1 and E2C2 birds had the greatest (90.05% and 92.1%, respectively) live sperm percent by comparison with other groups. The susceptibility of semen to lipid peroxidation in vitro was increased in quail fed E1C1 and E1C2, but was reduced when 150 mg Vit.E kg/day/dry matter feed was provided in the diet. The amount of Vit.E in the seminal plasma of E1C1 and E1C2 groups was (P<0.01) less than that in the other two groups (E2C1 and E2C2). From this study, it may be concluded that increasing diet n-6/n-3 ratio can be beneficial for semen traits, however, this application increased sperm peroxidation sensitivity but it can be controlled by inclusion of antioxidant such as Vit.E (150 mg/kg/day/dry matter) to diet.     

Effects of whole linseed supplementation and treatment duration on fatty acid profile and endogenous bioactive compounds of beef muscle

Diet supplementation with oilseeds is known to improve the fatty acid profile of meat, but few studies have been carried out to determine the time required for the incorporation of a significant quantity of n-3 polyunsaturated fatty acids (PUFA) into meat from steers. Therefore, the present study aimed to assess the effects of linseed supplementation and feeding duration on the fatty acid profile, cholesterol and bioactive compounds of bovine meat. In total, 54 Friesian steers were randomly allocated during the finishing period into six experimental treatments following a 2×3 factorial design. The six treatments consisted of two diets, the control diet (CO) with no supplemental fat and the linseed diet (LS) containing 10% whole linseed, fed 40, 75 or 120 days before slaughter. At the end of each finishing period, steers from the CO and LS groups were slaughtered. After 8 days of ageing chemical analysis, the fatty acid profile, cholesterol content and bioactive compounds were determined from the longissimus thoracis muscle. Including linseed in the diet increased the content of monounsaturated fatty acids, CLA and n-3 PUFA, and reduced the proportion of saturated fatty acids and n-6 PUFA. The percentage of myristic fatty acid increased with the duration of feeding, regardless of diet and a decrease in PUFA and n-6 PUFA was observed in the CO and LS diets, respectively. Furthermore, meat from steers fed linseed showed an increased percentage of n-3 PUFA, linolenic acid, and EPA from 40 to 75 days of feeding, whereas vaccenic acid, CLA 9c,11t, and total CLA increased from 40 and 75 days but declined at 120 days. Beef from the linseed group had a higher content of bioactive substances such as creatine, carnosine and anserine than beef from the control group. The duration of feeding significantly affected the creatine concentrations, with an increase in the LS group from 40 to 75 days of feeding. Feeding linseed did not modify the cholesterol content, on average and the lowest cholesterol content was found in meat after 75 days of linseed administration. This study demonstrates that a short-term diet manipulation is sufficient to improve the nutritional properties of meat, including n-3 PUFA and bioactive compounds.     

The tissue disposition and urinary excretion of cadmium, zinc, copper and iron, following repeated parenteral administration of cadmium to rats.

The effect of repeated parenteral administration of cadmium (0.75, 1.5 and 3.0 mg/kg) on tissue disposition and urinary excretion of cadmium, zinc, copper and iron has been studied in the male rat. Cadmium, zinc and copper accumulated in liver and kidney, but the concentration of iron did not alter significantly. The kidney weight relative to body weight showed a dose-related increase in weight of 25--65%. Excretion of cadmium in the urine increased directly with dosage and the increase was most significant when kidney damage had probably occurred. Administration of cadmium also resulted in dose-related increases in the urinary excretion of zinc, copper and iron. The cadmium concentration of blood increased with dosage of cadmium, and the plasma concentrations of zinc and copper were also raised but plasma iron concentration was diminished.     

Oxidative stress induced by chronic administration of sodium dichromate [Cr(VI)] to rats

Chromium occurs in the workplace primarily in the valence forms Cr(III) and Cr(VI). Recent studies have demonstrated that sodium dichromate [Cr(VI)] induces greater oxidative stress as compared with Cr(III), as indicated by the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes, and enhanced excretion of urinary lipid metabolites and hepatic DNA-single strand breaks (SSB) following acute oral administration of Cr(III) and Cr(VI). We have therefore examined the chronic effects of sodium dichromate dihydrate [Cr(VI); 10 mg (33.56 μmol)/kg/day] on hepatic mitochondrial and microsomal lipid peroxidation, enhanced excretion of urinary lipid metabolites including malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT), acetone (ACON) and propionaldehyde (PROP), and hepatic DNA damage over a period of 90 days. The maximal increases in hepatic lipid peroxidation and DNA damage were observed at approximately 45 days of treatment. Maximum increases in the urinary excretion of MDA, FA, ACT, ACON and PROP were 3.2-, 2.6-, 4.1-, 3.3- and 2.1-fold, respectively, while a 5.2-fold increase in DNA-SSB was observed. The results clearly indicate that chronic sodium dichromate administration induces oxidative stress resulting in tissue damaging effects which may contribute to the toxicity and carcinogenicity of hexavalent chromium.     

Chemopreventive effect of bilberry (Vaccinium myrtillus) against cisplatin-induced oxidative stress and DNA damage as shown by the comet assay in peripheral blood of rats

The aim of this study was to evaluate the chemopreventive effect of bilberry on cisplatin induced oxidative stress and DNA damage in rat blood. Twenty-one female Wistar-Albino rats were divided into three groups: group I — untreated; group II — treated with cisplatin (single dose 7.5 mg/kg b.w.); and group III — treated with cisplatin (single dose 7.5 mg/kg b.w.) and bilberry (200 mg/kg b.w. for 10 days). Antioxidant enzyme systems including superoxide dismutase, catalase, glutathione peroxidase and the level of malondialdehyde (MDA) that might occur on erythrocytes have been determined and single cell gel electrophoresis (comet) was utilized to evaluate the DNA damage in lymphocytes. Treatment with cisplatin has increased the levels of MDA and decreased antioxidant enzymes in erythrocytes. Comet assay showed significantly higher values at dose of 7.5 mg/kg cisplatin as the result of oxidative DNA damage when compared to the control group. Cisplatin treatment with bilberry resulted in a highly significant (P < 0.05) decreased in the lymphocytes DNA when compared to the cisplatin group. Bilberry has been effective on antioxidant enzyme systems and MDA level and significantly decreased the comets. Our results indicate that bilberry is capable of preventing genotoxic and cytotoxic damage caused by cisplatin in peripheral blood cells in rats.     

Effect of different contents of extruded linseed in the sow diet on piglet fatty acid composition and hepatic desaturase expression during the post-natal period

n-3 polyunsaturated fatty acids (n-3 PUFA) contribute to the normal growth and development of numerous organs in the piglet. The fatty acid composition of piglet tissues is linked to the fatty acid composition of sow milk and, consequently, to the composition of sow diet during the gestation and lactation period. In this study, we investigated the impact of different contents of extruded linseed in the sow diet on the fatty acid composition and desaturase gene expression of piglets. Sows received a diet containing either sunflower oil (low 18:3n-3 with 18:3n-3 representing 3% of total fatty acids) or a mixture of extruded linseed and sunflower oil (medium 18:3n-3 with 9% of 18:3n-3) or extruded linseed (high 18:3n-3 with 27% of 18:3n-3) during gestation and lactation. Fatty acid composition was evaluated on sow milk and on different piglet tissues at days 0, 7, 14, 21 and 28. The postnatal evolution of delta5 (D5D) and delta6 (D6D) desaturase mRNA expression was also measured in the liver of low 18:3n-3 and high 18:3n-3 piglets. The milk of high 18:3n-3 sows had higher proportions of n-3PUFA than that of low 18:3n-3 and medium 18:3n-3 sows. Piglets suckling the high 18:3n-3 sows had greater proportions of 18:3n-3, 20:5n-3, 22:5n-3 and 22:6n-3 in the liver, and of 22:5n-3 and 22:6n-3 in the brain than low 18:3n-3 and medium 18:3n-3 piglets. D5D and D6D mRNA expressions in piglet liver were not affected by the maternal diet at any age. In conclusion, extruded linseed in the sow diet modifies the n-3PUFA status of piglets during the postnatal period. However, a minimal content of 18:3n-3 in the sow diet is necessary to increase the n-3PUFA level in piglet liver and brain. Moreover, modifications in the n-3PUFA fatty acid composition of piglet tissue seem linked to the availability of 18:3n-3 in maternal milk and not to desaturase enzyme expression.     

Recovery of malondialdehyde in urine as a 2,4-dinitrophenylhydrazine derivative after exposure to chloroform or hydroquinone

Malondialdehyde (MDA) excretion in urine as an index for toxicological effects of chloroform and hydroquinone was evaluated. In a first series of experiments three groups of rats were used: non-pretreated rats (group I), starved rats (group II) and starved plus phenobarbital pretreated rats (group III). Chloroform (0.15 or 0.30 ml/kg, p.o.) was given as a single dose. The MDA excretion was related to the pretreatment, and in group III to liver damage. In a second series of experiments control rats were administered hydroquinone (100 or 200 mg/kg, p.o.), which induced a dose-related MDA excretion. These data indicate that the MDA assay was a selective and accurate marker for toxicological effects induced by the tested compounds.     

Modulation of lipid peroxidation and antioxidant enzymes in murine salivary gland by dietary fatty acid ethyl esters

The present study was undertaken to investigate the effect of n-9, n-6, and n-3 dietary fatty acid ethyl esters on basal (uninduced) and Fe2+/ascorbate (induced) lipid peroxidation (LPO) in salivary gland (SG) of mice. Feeding n-3 ethyl ester polyunsaturated fatty acids (PUFA) increased the uninduced and induced LPO in SG homogenates. In contrast, feeding olive oil ethyl esters (n-9) significantly lowered the induced and uninduced LPO in SG tissue. Salivary gland susceptibility to LPO increased in the order of: olive oil < corn oil < safflower oil < n-3 ethyl esters. Olive oil esters in the diet increased primarily the 18:1 levels in SG tissue. Whereas feeding n-3 PUFA notably increased the superoxide dismutase (SOD) and catalase activities in SG homogenates, no significant changes were seen between n-9 and n-6 PUFA-fed mice. Lower levels of Vitamin E (Vit E) in the tissues of n-3 PUFA-fed mice indicate that the higher the dietary lipid unsaturation, the higher the requirement for Vit E in the diet. Our results indicate that, similar to other organs, salivary gland susceptibility to uninduced or induced oxidation depends on the source of dietary PUFA. In conclusion, feeding olive oil increases the resistance of SGs to induced and uninduced LPO.     

Specific analysis in plasma and urine of 2,3-dinor-5, 6-dihydro-isoprostane F(2alpha)-III, a metabolite of isoprostane F(2alpha)-III and an oxidation product of gamma-linolenic acid

F(2)-isoprostanes (iPs) are free radical-catalyzed isomers of prostaglandin F(2alpha). Circulating and urinary iPs have been used as indices of lipid peroxidation in vivo. Utilizing an (18)O-labeled homologous internal standard, we developed a gas chromatography/mass spectrometry assay for the 2,3-dinor-5,6-dihydro (dinor-dihydro) metabolite of iPF(2alpha)-III. Although urinary excretion of iPF(2alpha)-III reflects systemic lipid peroxidation, the metabolite is more abundant (median of 877 (range of 351-1831) versus 174 (range of 56-321) pg/mg of creatinine; p < 0.01) than the parent iP in urine and can be measured in plasma. Metabolite analysis may be preferable in plasma due to the abundance of arachidonic acid as a source of ex vivo lipid peroxidation. Also, iPF(2alpha)-III may be formed in blood samples in a cyclooxygenase-dependent manner by platelets ex vivo. By contrast, the metabolite is not formed by aggregated platelets (0.71 +/- 0.08 versus 0.65 +/- 0.09 pg/ml). Although the metabolite/parent ratio is altered in cirrhosis, urinary dinor-dihydro-iPF(2alpha)-III is elevated and increases further during reperfusion following orthoptic liver transplantation. In addition to its formation as an iPF(2) metabolite, analysis of gamma-linolenic acid autooxidation products and the compound present in freeze-thawed plasma suggests that gamma-linolenic acid may also be an important source of dinor-dihydro-iPF(2alpha)-III.     

Effect of docosahexaenoic acid and alpha-tocopherol enrichment in chicken sperm on semen quality, sperm lipid composition and susceptibility to peroxidation

The aim of the present experiment was to study the effect of fish oil and Vitamin E rich diets on semen production, sperm functions and composition in broiler breeders. The following parameters were measured: semen volume and concentration, sperm motility and viability, sperm susceptibility to induced peroxidation, sperm lipid and alpha-tocopherol contents. Dietary n-3 PUFA were successfully transferred into spermatozoan phospholipid by fish oil feeding according to the following main features: (a) the C22:6n-3 and C22:5n - 3 contents were increased, but C22:4n-6 remained the peculiar and major polyunsaturate; (b) the content and proportion of total PUFA did not change; (c) the proportional increase of n-3 PUFA was compensated by the decrease of n-6 PUFA, an increase in the proportion of n-9 fatty acids was also found. The sperm content of alpha-tocopherol was doubled increasing the dietary availability of the vitamin to 300 mg/kg of feed. The specific n-3 PUFA and Vitamin E enrichment of chicken sperm affected cell functions. Significant interactions between the two treatments were also found for some parameters. The best sperm quality condition in control sperm (rich mainly in n-6 PUFA) was found supplying 200mg Vitamin E/kg of feed to the male breeders, and in contrast in n-3 rich sperm supplying 300 mg Vitamin E/kg.     

Reduction of isoprostanes and regression of advanced atherosclerosis by apolipoprotein E

Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivo oxidant stress, we quantitated the isoprostane iPF(2 alpha)-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF(2 alpha)-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects.     

Role of the Lower and Upper Intestine in the Production and Absorption of Gut Microbiota-Derived PUFA Metabolites

In vitro studies have suggested that isolated gut bacteria are able to metabolize PUFA into CLA (conjugated linoleic acids) and CLnA (conjugated linolenic acids). However, the bioavailability of fatty acid metabolites produced in vivo by the gut microbes remains to be studied. Therefore, we measured intestinal concentration and plasma accumulation of bacterial metabolites produced from dietary PUFA in mice, first injected with a lipoprotein lipase inhibitor, then force-fed with either sunflower oil (200 µl) rich in n-6 PUFA or linseed oil (200 µl) rich in n-3 PUFA. The greatest production of bacterial metabolites was observed in the caecum and colon, and at a much lesser extent in the jejunum and ileum. In the caecal content, CLA proportions were higher in sunflower oil force-fed mice whereas CLnA proportions were higher in linseed oil force-fed mice. The accumulation of the main metabolites (CLA cis-9,trans-11-18:2 and CLnA cis-9,trans-11,cis-15-18:3) in the caecal tissue was not associated with their increase in the plasma, therefore suggesting that, if endogenously produced CLA and CLnA have any biological role in host metabolism regulation, their effect would be confined at the intestinal level, where the microbiota is abundant.