Oxylipin patterns in human colon adenomas.

OBJECTIVE: Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2) is an important lipid mediator in colorectal carcinoma (CRC) pathogenesis. Other lipid mediators derived from lipoxygenases (LOX) have also been implicated in neoplastic processes in the colon. In this study we aimed to characterize lipid mediators, so called oxylipins, in human colon adenomatous polyps. DESIGN: We quantified oxylipins in healthy colon tissue and colorectal adenoma tissue procured during routine colonoscopy examinations. Lipid metabolite profiles were analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: Adenoma tissue showed a distinct prostaglandin profile as compared to normal colon mucosa. Interestingly, PGE2 was not higher in adenoma tissue as compared to normal mucosa. In contrast, we found significantly lower levels of prostaglandin D2, prostaglandin J2, and prostaglandin D1 in adenoma tissue. Furthermore, levels of 5-LOX and 12-LOX pathway products were clearly increased in adenoma biopsy samples. We also investigated the effect of aspirin treatment on prostaglandin profiles in adenoma tissue in a subset of patients and found a trend towards decreased prostaglandin levels in response to aspirin. CONCLUSION: The human data presented here show specific changes of oxylipin profiles in colon adenoma tissue with decreased prostaglandin D2 levels as well as increased 5- and 12-LOX metabolites.

Combined Targeted Proteomics and Oxylipin Metabolomics for Monitoring of the COX-2 Pathway.

The important role of inducible cyclooxygenase-2 (COX-2) in several diseases necessitates analytical tools enabling thorough understanding of its modulation. Analysis of a comprehensive oxylipin pattern provides detailed information about changes in enzyme activities. In order to simultaneously monitor gene expression levels, a targeted proteomics method for human COX-2 is developed. With limits of detection and quantification down to 0.25 and 0.5 fmol (on column) the method enables sensitive quantitative analysis via LC-MS/MS within a linear range up to 2.5 pmol. Three housekeeping proteins are included in the method for data normalization. A tiered approach for method development comprised of in silico and experimental steps is described for choosing unique peptides and selective and sensitive SRM transitions while avoiding isobaric interferences. This method combined with a well-established targeted oxylipin metabolomics method allows to investigate the role of COX-2 in the human colon carcinoma cell lines HCT-116, HT-29, and HCA-7. Moreover, the developed methodology is used to demonstrate the time-dependent prostanoid formation and COX-2 enzyme synthesis in lipopolysaccharide-stimulated human primary macrophages. The described approach is a helpful tool which will be further used as standard operation procedure, ultimately aiming at comprehensive targeted proteomics/oxylipin metabolomics strategies to examine the entire arachidonic acid cascade.

Effects of chronic low-dose aspirin treatment on tumor prevention in three mouse models of intestinal tumorigenesis.

Although early detection and treatment of colorectal cancer (CRC) have improved, it remains a significant health-care problem with high morbidity and mortality. Data indicate that long-term intake of low-dose aspirin reduces the risk of CRC; however, the mechanisms underlying this chemopreventive effect are still unclear. Different mouse models for inflammation-associated, sporadic, and hereditary CRC were applied to assess the efficacy and mechanism of low-dose aspirin on tumor prevention. An initial dosing study performed in healthy mice indicates that aspirin at a dose of 25 mg/kg/d has a similar pharmacodynamic effect as low-dose aspirin treatment in human subjects (100 mg/d). Chronic low-dose aspirin treatment suppresses colitis-associated and to a lesser extent spontaneous tumorigenesis in mice. Aspirin's antitumor effect is most pronounced in a preventive approach when aspirin administration starts before the tumor-initiating genotoxic event and continues for the duration of the experiment. These effects are not associated with alterations in cell proliferation, apoptosis, or activation of signaling pathways involved in CRC. Aspirin-induced reduction in tumor burden is accompanied by inhibition of thromboxane B2 formation, indicating reduced platelet activation. Aspirin treatment also results in decreased colonic prostaglandin E2 formation and tumor angiogenesis. With respect to colitis-triggered tumorigenesis, aspirin administration is associated with a reduction in inflammatory activity in the colon, as indicated by decreased levels of pro-inflammatory mediators, and tumor-associated iNOS-positive macrophages. Our results suggest that low-dose aspirin represents an effective antitumor agent in the context of colon tumorigenesis primarily due to its well-established cyclooxygenase inhibition effects.

Impact of food polyphenols on oxylipin biosynthesis in human neutrophils.

The intake of food polyphenols is associated with beneficial impacts on health. Besides anti-oxidative effects, anti-inflammatory properties have been suggested as molecular modes of action, which may result from modulations of the arachidonic acid (AA) cascade. Here, we investigated the effects of a library of food polyphenols on 5-lipoxygenase (5-LOX) activity in a cell-free assay, and in human neutrophils. Resveratrol, its dimer (ε-viniferin), and its imine analogue (IRA) potently blocked the 5-LOX-mediated LT formation in neutrophils with IC50 values in low µM-range. Among the tested flavonoids only the isoflavone genistein showed potent 5-LOX inhibition in neutrophils (IC50 = 0.4 ±â€¯0.1 µM), however was ineffective on isolated 5-LOX. We exclude an interference with the 5-LOX-activating protein (FLAP) in HEK_5-LOX/±FLAP cells and suggest global effects on intact immune cells. Using LC-MS based targeted oxylipin metabolomics, we analyzed the effects of 5-LOX-inhibiting polyphenols on all branches of the AA cascade in Ca2+-ionophore-challenged neutrophils. While ε-viniferin causes a clear substrate shunt towards the remaining AA cascade enzymes (15-LOX, cyclooxygenase - COX-1/2, cytochrome P450), resveratrol inhibited the COX-1/2 pathway and showed a weak attenuation of 12/15-LOX activity. IRA had no impact on 15-LOX activity, but elevated the formation of COX-derived prostaglandins, having no inhibitory effects on COX-1/2. Overall, we show that food polyphenols have the ability to block 5-LOX activity and the oxylipin pattern is modulated with a remarkable compound/structural specificity. Taken the importance of polyphenols for a healthy diet and their concentration in food supplements into account, this finding justifies further investigation.

Aspirin alone and combined with a statin suppresses eicosanoid formation in human colon tissue.

Eicosanoids, including prostaglandins (PGs) and thromboxanes, are broadly bioactive lipid mediators and increase colon tumorigenesis possibly through chronic inflammatory mechanisms. Epidemiological and experimental data suggest that acetylsalicylic acid (ASA) helps prevent colorectal cancer (CRC), possibly through cyclooxygenase (COX)-mediated suppression of eicosanoid, particularly PGE2, formation. Recent studies suggest that statins prevent CRC and improve survival after diagnosis. We identified patients on ASA and/or statin treatment undergoing routine colonoscopy and measured eicosanoid levels in colonic mucosa with targeted metabolomics technology (LC-MS/MS). ASA-treated individuals (n = 27) had significantly lower tissue eicosanoid levels of most COX-derived metabolites than untreated individuals (n = 31). In contrast, COX-derived lipid metabolites tended to be higher in patients with statin treatment (n = 7) as compared with those not receiving statins (n = 24). This effect was not discernible in subjects treated with ASA and statins (n = 11): Individuals treated with both drugs showed a pronounced suppression of COX-derived eicosanoids in colon tissue, even compared with subjects treated with ASA alone. Our data from a routine clinical setting support the hypothesis that ASA and statins could inhibit CRC development via lipid mediator modification. Further studies should directly investigate the effect of dual ASA and statin treatment on colon tumorigenesis in humans.

Effect of Omega-3 Fatty Acid Supplementation on Oxylipins in a Routine Clinical Setting.

Omega-6 polyunsaturated fatty acid (n-6 PUFA) is the predominant polyunsaturated fatty acid (PUFA), especially in Western diet. A high omega-6/omega-3 ratio in Western diets is implicated in the development of cardiovascular diseases and inflammatory processes. Studies in animal models and in humans have demonstrated beneficial effects of omega-3 PUFA (n-3 PUFA) in a variety of diseases, including cardiac arrhythmias and inflammatory diseases, as well as breast and colon cancer. The molecular mechanisms underlying the effects of n-3 PUFA are still not well understood. Possible mechanisms include competition between n-3 and n-6 PUFAs at the cyclooxygenase (COX) and lipoxygenase (LOX) and cytochrome P450 levels, and subsequent formation of oxylipins with specific anti-inflammatory or anti-arrhythmic effects. In this study, we report the impact of routine long-term treatment with prescription-grade n-3 PUFA (either 840 mg or 1680 mg per day) on blood cell membrane fatty acid composition, as well as plasma oxylipin patterns, in a patient population with severe hyperlipidemia and cardiovascular disease who are on standard lipid-lowering and cardioprotective medications. Lipidomics analyses were performed by LC/ESI-MS/MS. Supplementation led to a dose-dependent increase in n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the blood cell fraction. We also observed a dose-dependent increase in EPA- and DHA-derived epoxy metabolites, whereas the effect of n-3 PUFA supplementation on LOX-dependent EPA- and DHA-derived hydroxy metabolites was less pronounced, with a tendency towards lower metabolites in subjects with higher n-3 PUFA levels. These data thus generally confirm effects of n-3 PUFA supplementation observed previously in healthy individuals. Additionally, they indicate a suppressive effect of high n-3 PUFA supplementation on the formation of LOX metabolites in the context of concomitant aspirin medication.

Influence of weight reduction on blood levels of C-reactive protein, tumor necrosis factor-α, interleukin-6, and oxylipins in obese subjects.

INTRODUCTION: Obesity is associated with inflammation and weight reduction has been shown to influence the inflammatory process. Besides classic inflammatory markers, oxidized polyunsaturated fatty acid (PUFA) metabolites (oxylipins) are potent mediators of inflammation. Little is known about endogenous levels of oxylipins, e.g. hydroxy, epoxy and dihydroxy FA in obese subjects with persistent low-grade inflammation. We aimed to evaluate levels of inflammatory markers and blood oxylipins in obese subjects before and after weight reduction. SUBJECTS AND METHODS: In the present study, 42 obese (BMI 32.7 ± 0.22 kg/m(2)) men and women were classified in groups according to high-sensitivity C-reactive protein (hsCRP) levels (no inflammation<1mg/L; low-grade inflammation ≥ 3 mg/L). Subjects underwent an intervention for eight weeks, which consisted of two phases: (1) week 1 and 2: total replacement of three meals by a formula diet and (2) six week partial formula diet (replacement of 1-2 meals). Blood samples were taken prior and post intervention for analysis of plasma protein levels of hsCRP, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Plasma Levels of free (unesterified) hydroxy, epoxy, and dihydroxy FAs as well as several prostanoids were analyzed in plasma by means of LC-MS-based targeted metabolomics. RESULTS: At baseline subjects with low-grade inflammation (hsCRP 8.95 ± 1.39 mg/L) showed significant higher levels of IL-6 (22.7 ± 1.15 ng/L) and TNF-α (17.4 ± 0.75 ng/L) compared to subjects with no inflammation (hsCRP: 0.69 ± 0.05 mg/L; IL-6: 15.9 ± 1.18 ng/L; TNF-α: 14.6 ± 0.80 ng/L). In both group's body weight was significantly reduced (p<0.001) after intervention (no inflammation group: -7.19 ± 0.86 kg, -7.3 ± 0.89%, p<0.001; low-grade inflammation group: -6.78 ± 0.87 kg, -6.7 ± 0.81%, p<0.001). Moreover, we observed significant decreases in levels of hsCRP (4.66 ± 0.64 mg/L; p=0.006), IL-6 (6.81 ± 1.15 ng/L; p<0.001) and TNF-α (6.09 ± 0.47 ng/L; p<0.001) in subjects with low-grade inflammation. Of 60 quantified oxylipins, 11 linoleic acid (LA)-, 1 dihomo-γ-linolenic acid (DGLA)-, 7 alpha linolenic acid (ALA)-, 15 arachidonic acid (AA)-, 8 eicosapentaenoic acid (EPA)- and 18 docosahexaenoic acid (DHA)-metabolites could be detected in plasma. For most oxylipins no differences were found between the low and high hsCRP groups before and after weight reduction. Interestingly, in subjects with low- grade inflammation several AA-derived oxylipins (5-, 8-, 12-hydroxyeicosatetraenoic acids (HETE)) were significantly higher compared to subjects with no inflammation before weight reduction and significantly reduced after weight reduction. CONCLUSION: Even moderate weight loss in obese subjects correlates to a significant improvement in the inflammatory state, by reducing hsCRP, IL-6, TNF-α and few oxylipins. The biological consequences of these changes remain to be further investigated.

Impact of dextran sulphate sodium-induced colitis on the intestinal transport of the colon carcinogen PhIP.

Colorectal cancer is one of the most frequent cancers in Western countries. Chronic intestinal diseases such as Crohn's disease and ulcerative colitis, in which the intestinal barrier is massively disturbed, significantly raise the risk of developing a colorectal tumour. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a genotoxic heterocyclic aromatic amine that is formed after strongly heating fish and meat. In this study, the hypothesis that PhIP uptake in the gut is increased during chronic colitis was tested. Chronic colitis was induced by oral administration of dextran sulphate sodium (DSS) to Fischer 344 rats. The transport of PhIP in eight different rat intestinal segments was examined in Ussing chambers. The tissues were incubated with 10 µM PhIP for 90 min, and the concentration of PhIP was determined in the mucosal and serosal compartments of the Ussing chambers as well as in the clamped tissues by LC-MS. Although chronic colitis was clearly induced in the rats, no differences in the intestinal transport of PhIP were observed between control and DSS-treated animals. The hypothesis that in the course of chronic colitis more PhIP is taken up by the intestinal epithelium, thereby increasing the risk of developing colorectal cancer, could not be confirmed in the present report.

Determining the fatty acid composition in plasma and tissues as fatty acid methyl esters using gas chromatography ­ a comparison of different derivatization and extraction procedures.

Analysis of the fatty acid (FA) composition in biological samples is commonly carried out using gas liquid chromatography (GC) after transesterification to volatile FA methyl esters (FAME). We compared the efficacy of six frequently used protocols for derivatization of different lipid classes as well as for plasma and tissue samples. Transesterification with trimethylsulfonium hydroxide (TMSH) led to insufficient derivatization efficacies for polyunsaturated FAs (PUFA, <50%). Derivatization in presence of potassium hydroxide (KOH) failed at derivatizing free FAs (FFAs). Boron trifluoride (BF3) 7% in hexane/MeOH (1:1) was insufficient for the transesterification of cholesterol ester (CE) as well as triacylglycerols (TGs). In contrast, methanolic hydrochloric acid (HCl) as well as a combination of BF3 with methanolic sodium hydroxide (NaOH+BF3) were suitable for the derivatization of FFAs, polar lipids, TGs, and CEs (derivatization rate >80% for all tested lipids). Regarding plasma samples, all methods led to an overall similar relative FA pattern. However, significant differences were observed, for example, for the relative amount of EPA+DHA (n3-index). Absolute FA plasma concentrations differed considerably among the methods, with low yields for KOH and BF3. We also demonstrate that lipid extraction with tert-butyl methyl ether/methanol (MTBE/MeOH) is as efficient as the classical method according to Bligh and Dyer, making it possible to replace (environmentally) toxic chloroform.We conclude that HCl-catalyzed derivatization in combination with MeOH/MTBE extraction is the most appropriate among the methods tested for the analysis of FA concentrations and FA pattern in small biological samples. A detailed protocol for the analysis of plasma and tissues is included in this article.