A reversed-phase high-performance liquid chromatography (RP-HPLC) has been developed to analyze the metabolites of arachidonic acid based on the specificities of ultraviolet absorption of these various metabolites and is sensitive to the nanogram level. This procedure makes it possible to extract complex mixtures of eicosanoids efficiently with a single step and to analyze them simultaneously by RP-HPLC from biological samples using octadesylsilyl silica extraction column and PGB2 as an internal standard. The cyclooxygenase, products (prostaglandin (PG)D2, PGE1, PGE2, PGF1alpha, PGF2alpha, 6-keto-PGF1alpha, and thromboxane B2 (TXB2)) and lipid peroxidation product, isoprostanes, of arachidonic acid were monitored by one isocratic HPLC system at 195 nm wavelength. The lipoxygenase products (leukotriene(LT)B4, LTC4, LTD4, and 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, 15-HETE) were measured by another isocratic HPLC system at 280 nm for LTs and 235 nm for HETEs. This method provides a simple and reliable way to extract and assess quantitatively the final arachidonic acid metabolites.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid ; Absorption ; Alprostadil ; Arachidonic Acid* ; Chromatography, High Pressure Liquid* ; Chromatography, Liquid ; Complex Mixtures ; Dinoprost ; Dinoprostone ; Eicosanoids ; Hydroxyeicosatetraenoic Acids ; Isoprostanes ; Leukotriene C4 ; Leukotriene D4 ; Lipid Peroxidation ; Lipoxygenase ; Prostaglandin-Endoperoxide Synthases ; Silicon Dioxide ; Solid Phase Extraction* ; Thromboxane B2

BACKGROUND AND OBJECTIVES: It is known that various inflammatory mediators released from the eosinophils and mast cells play important roles in the pathogenesis of nasal polyp. Among those mediators, the arachidonic acid has particular importance as a precursor of other mediators. By assaying the tissue concentration of the6-keto-PGF(1alpha), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETE) in the nasal polyp, we aimed to investigate the role of arachidonic acid metabolite in the pathogenesis of antrochoanal polyp and nasal polyp associated with chronic paranasal sinusitis. MATERIALS AND METHODS:Three turbinate tissues taken during the septoplasty were served as the control. The experimental group consisted of 3 antrochoanal polyps and 7 inflammatory polyps. The tissue level of the 6-keto-PGF(1alpha), LTC(4), LTD(4), LTE(4), 15-HETE, and 12-HETE were measured using high performance liquid chromatography. RESULTS: The level of 6-keto-PGF(1alpha), LTC4, 15-HETE, 12-HETE were significantly lower in antrochoanal polyp than in the control turbinate. In the inflammatory polyp, the levels of 6-keto-PGF(1alpha) and LTC(4) were lower than the control. However, in the inflammatory polyp, LTD(4) and LTE(4) were detectable, which were not detected in the control turbinate and antrochoanal polyp. CONCLUSION: The results of this study indicate that the decreased arachidonic acid metabolism may underlie the pathogenesis of the antrochoanal polyp. However, in the pathogenesis of inflammatory polyp, the increased production of LTD(4) and LTE(4) may have an important role.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid ; Arachidonic Acid* ; Chromatography, Liquid ; Eosinophils ; Hydroxyeicosatetraenoic Acids ; Leukotriene C4 ; Leukotrienes ; Mast Cells ; Metabolism ; Nasal Polyps* ; Polyps* ; Sinusitis* ; Turbinates

BACKGROUND AND OBJECTIVES: Both the granulation tissue and cholesterol granuloma can erode the surrounding bone and ossicles. However, the etiology of bone resorption in the granulation tissue and cholesterol granuloma has not been evident. The aim of this study was to assay the concentrations of arachidonic acid metabolites (AAMs) in cholesterol granuloma and the mastoid granulation tissue in order to better understand the possible role of AAMs in the pathophysiology of cholesterol granuloma in comparison with the granulation tissue. MATERIALS AND METHOD: Cholesterol granuloma tissues were obtained from eight patients who had underwent tympanomastoidectomy. Granulation tissues, which served for comparison, were taken from 12 patients who had underwent tympanomastoidectomy. Tissue concentrations of prostagladin (PG)E2, 6-keto-PGF1alpha, leukotriene (LT)C4, LTD4, LTE4, 15-hydro xyeicosatetraenoic acid (HETE), 12-HETE, 5-HETE, and thromboxane (TXB)2 were calculated using high performance liquid chromatography and compared between cholesterol granuloma and granulation tissue. RESULT: The level of 12-HETE was higher in cholesterol granuloma than in the granulation tissue. Among the PGs, the tissue concentration of PGE2 was particularly high in cholesterol granuloma than in the granulation tissue. LTD4 was the only LT detectable in cholesterol granuloma. In comparison to the AAMs in cholesterol granuloma, the lipoxygenase pathway products such as 12-HETE, 15-HETE, and 5-HETE were present in lower concentrations in the granulation tissue. LT was undetectable in the granulation tissues. CONCLUSION: Our results suggest that cholesterol granuloma and the granulation tissue is different not only in terms of histology, but also in terms of biochemical properties such as arachidonic acid metabolism.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid ; Arachidonic Acid* ; Arachidonic Acids ; Bone Resorption ; Cholesterol* ; Chromatography, Liquid ; Cytochrome P-450 CYP1A1 ; Dinoprostone ; Granulation Tissue* ; Granuloma* ; Humans ; Leukotriene D4 ; Leukotriene E4 ; Lipoxygenase ; Mastoid ; Metabolism ; Otitis Media* ; Otitis*

Arachidonic acids (AA) widely exist in multiple organs and can be metabolized into small lipid molecules with strong biological functions through several pathways. Among them, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE), which are produced by cytochrome P450 enzymes, have attracted a lot of attentions, especially in vascular homeostasis. The regulation of vascular function is the foundation of vascular homeostasis, which is mainly achieved by manipulating the vascular structure and biological function. In the past 30 years, the roles of EETs and 20-HETE in the regulation of vascular function have been widely explored. In this review, we discussed the effects of EETs and 20-HETE on angiogenesis and vascular inflammation, respectively. Generally, EETs can dilate blood vessels and inhibit vascular inflammation, while 20-HETE can induce vasoconstriction and vascular inflammation. Interestingly, both EETs and 20-HETE can promote angiogenesis. In addition, the roles of EETs and 20-HETE in several vascular diseases, such as hypertension and cardiac ischemia, were discussed. Finally, the therapeutic perspectives of EETs and 20-HETE for vascular diseases were also summarized.
Arachidonic Acid ; Arachidonic Acids ; Cytochrome P-450 Enzyme System ; Humans ; Hydroxyeicosatetraenoic Acids ; Hypertension ; Vasoconstriction

This study aims to explore the effects of arachidonic acid lipoxygenase metabolism in vascular calcification. We used 5/6 nephrectomy and high-phosphorus feeding to establish a model of vascular calcification in mice. Six weeks after nephrectomy surgery, vascular calcium content was measured, and Alizarin Red S and Von Kossa staining were applied to detect calcium deposition in aortic arch. Control aortas and calcified aortas were collected for mass spectrometry detection of arachidonic acid metabolites, and active molecules in lipoxygenase pathway were analyzed. Real-time quantitative PCR was used to detect changes in the expression of lipoxygenase in calcified aortas. Lipoxygenase inhibitor was used to clarify the effect of lipoxygenase metabolic pathways on vascular calcification. The results showed that 6 weeks after nephrectomy surgery, the aortic calcium content of the surgery group was significantly higher than that of the sham group (P < 0.05). Alizarin Red S staining and Von Kossa staining showed obvious calcium deposition in aortic arch from surgery group, indicating formation of vascular calcification. Nine arachidonic acid lipoxygenase metabolites were quantitated using liquid chromatography/mass spectrometry (LC-MS) analysis. The content of multiple metabolites (12-HETE, 11-HETE, 15-HETE, etc.) was significantly increased in calcified aortas, and the most abundant and up-regulated metabolite was 12-HETE. Furthermore, we examined the mRNA levels of metabolic enzymes that produce 12-HETE in calcified blood vessels and found the expression of arachidonate lipoxygenase-15 (Alox15) was increased. Blocking Alox15/12-HETE by Alox15 specific inhibitor PD146176 significantly decreased the plasma 12-HETE content, promoted calcium deposition in aortic arch and increased vascular calcium content. These results suggest that the metabolism of arachidonic acid lipoxygenase is activated in calcified aorta, and the Alox15/12-HETE signaling pathway may play a protective role in vascular calcification.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid ; Animals ; Arachidonate 12-Lipoxygenase ; Arachidonate 15-Lipoxygenase/metabolism* ; Arachidonic Acid ; Hydroxyeicosatetraenoic Acids ; Lipoxygenase/metabolism* ; Mice ; Signal Transduction ; Vascular Calcification

OBJECTIVETo observe the changing contents of leukotriene B4 ( LTB4 ), leukotriene C4 ( LTC4 ), and leukotriene D4 (LTD4 ) of lung tissue in asthmatic rats, and explore the effect of Shuanglong Capsule (SLC) on it.

METHODSSD rats were randomly divided into the nomal group, asthmatic model group, Dexamethasone group and the high, middle and low dose SLC groups. All rats except those in the normal group were sensitized by ovalbumin and challenged with the antigen, and the contents of LTB4, LTC4 and LTD4 in lung tissue of all the groups were measured by reverse phase-high performance liquid chromatography (RP-HPLC) and compared.

RESULTSThe levels of LTB4, LTC4, and LTD4 of asthmatic rats were significantly higher than those of rats in the normal group. Dexamethasone and SLC at the dose of 8. 27 g/kg or 4. 13 g/kg could significantly inhibit the production of leukotrienes of lung tissue in asthmatic rats (P <0.05).

CONCLUSIONSLC can significantly inhibit the formation of inflammatory medium LTs of lung tissue in asthmatic rats, it may be one of the key mechanisms of SLC in anti-asthma and anti-inflammatory action.

Animals ; Anti-Asthmatic Agents ; pharmacology ; Asthma ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Leukotriene B4 ; metabolism ; Leukotriene C4 ; metabolism ; Leukotriene D4 ; metabolism ; Leukotrienes ; metabolism ; Lung ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tablets

Conjugated linoleic acid (CLA) is the mixture of positional and geometric isomers of linoleic acid (LA), which is found abundantly in dairy products and meats. This study was performed to investigate the anticarcinogenic effect of CLA in HepG2 hepatoma cells. HepG2 cell were treated with LA and CLA at the various concentrations of 10, 20, 40, 80 uM each at different incubation times. After each incubation times, cell proliferation, fatty acids incorporation into cell, peroxidation and postaglandin E2 (PGE2) and thromboxane A2 (TXA2) for the eicosanoid metabolism were measured. LA treated HepG2 cells were increased cell growth 6 - 70% of control whereas CLA increased cell death the half of those in LA group (p < 0.001). LA and CLA were incorporated very well into the cellular membranes four times higher than in control according to concentration and longer incubation times. Moreover, LA synthesized significantly arachidonic acids corresponding with LA concentration compared to CLA supplementation. The supplementation with LA increased intracellular lipid peroxides concentration corresponding with LA concentration and five times higher than those in CLA significantly at any incubation times (p < 0.001). PGE2 and TXA2 levels were three to twenty times lower in condition of CLA treatments than LA, respectively. Overall, the dietary CLA might change the HepG2 cell growth by the changes of cell composition, production of lipid peroxide. Since CLA have not changed the levels of arachidonic acid of cell membrane, which was sources of eicosanoids, eicosanoid synthesis was not increased in CLA compared to LA. Our results was suggest CLA has a possibility to protect the progress of atherosclerosis because CLA does not produce lipid production and endothelial contraction factors in liver.
Anticarcinogenic Agents ; Arachidonic Acid ; Arachidonic Acids ; Atherosclerosis ; Carcinoma, Hepatocellular* ; Cell Death ; Cell Membrane ; Cell Proliferation ; Dairy Products ; Dinoprostone ; Eicosanoids ; Fatty Acids ; Hep G2 Cells* ; Humans* ; Linoleic Acid ; Lipid Peroxidation ; Lipid Peroxides ; Liver ; Meat ; Membranes ; Metabolism ; Thromboxane A2

BACKGROUND: To investigate the effect of manganese on lipid peroxidation and compositional changes of fatty acids in hippocampus of rat brain. METHODS: Seven rats in experimental group were given with MnCl2 intraperitoneally for 4 weeks (4 mg/kg once daily, 5 days per week). Twenty four hours after the last injection, rats were decapitated and, hippocampus were separated from the rat brain. RESULT: In Mn-treated group, manganese concentrations increased significantly in the hippocampus by 222% compared with control group (P<0.01). MDA concentrations increased significantly by 149% compared with control group (P<0.05). Among fatty acids, total n-6 polyunsaturated fatty acids (PUFAs) increased significantly by 237% compared with control group (P<0.05). Linoleic acid (LA) and arachidonic acids (AA) increased by 213%, 238% (P<0.05, P<0.01, respectively). Among n-3 PUFAs except linolenic acids, eicosapentanoic acid(EPA) and docosahexanoic acids (DHA) decreased significantly by 70%, 50% respectively compared with control group (both P<0.01). CONCLUSION: Our results suggest that manganese may cause compositional changes of fatty acids in hippocampus of rat brain. Characteristics of fatty acids compositional changes by manganese were the decrease of EPAs and DHAs (n-3 PUFAs), and increase of AA and LA (n-6 PUFAs). These changes with the increase of MDA, suggest that manganese neurotoxicity is caused by lipid peroxidation.
alpha-Linolenic Acid ; Animals ; Arachidonic Acid ; Arachidonic Acids ; Brain* ; Fatty Acids* ; Fatty Acids, Omega-3 ; Fatty Acids, Unsaturated ; Hippocampus* ; Linoleic Acid ; Linolenic Acids ; Lipid Peroxidation* ; Malondialdehyde ; Manganese* ; Rats*

BACKGROUND: Theophylline has been used in the treatment of asthma for decades as a broncho- dilator, but recent studies suggested that it has anti-inflammatory and immunomodulatory properties. OBJECTIVE: The aim of this study was to determine the effect of theophylline therapy on urinary excretion of leukotriene B4 (LTB4) and C4 (LTC4), which have been known to play a role in the airway inflammation in asthmatic patients. METHODS: Fifty-one patients, aged 13 months to 6 years, who were hospitalized with asthma were randomized in to 3 groups according to treatment regimen. Twenty-one patients were administered theophylline with budesonide and salbutamol inhalation (Group 1). Fifteen patients were treated with budesonide and salbutamol inhalation (Group 2) and the other fifteen patients with salbutamol inhalation only (Group 3). Urine samples for the determination of LTB4 and LTC4 were collected on admission and on the fifth hospital day in each patient. The concentrations of urinary leukotrienes were measured by ELISA (Neogen, U.S.A.) and corrected by urinary creatinine levels. Ten controls were also studied. RESULTS: The initial urinary LTC4 levels in asthmatic children were significantly higher than in controls. Urinary LTC4 was significantly decreased after treatment compared with the initial level in group 1 (p<0.05), but not in groups 2 and 3. Urinary LTB4 did not show significant difference between patients and controls on admission and showed no significant change after treatment compared with the initial levels in all three groups. CONCLUSION: These results suggest that the suppression of LTC4 synthesis is one of the anti-inflammatory mechanisms of theophylline and support the use of theophylline as a the rapeutic agent in asthmatic patients.
Albuterol ; Asthma* ; Budesonide ; Child* ; Creatinine ; Enzyme-Linked Immunosorbent Assay ; Humans ; Inflammation ; Inhalation ; Leukotriene B4* ; Leukotriene C4 ; Leukotrienes ; Theophylline*

The present study was designed to assess the roles of PLA2 activation and arachidonic acid (AA) metabolites in hypoxia-induced renal cell injury. Hypoxia increased LDH release in a dose-dependent manner in rabbit renal cortical slices, and this increase was significant after 20-min hypoxia. The hypoxia-induced LDH release was prevented by amino acids, glycine and alanine, and extracellular acidosis (pH 6.0). Buffering intracellular Ca2+ by a chelator, but not omission of Ca2+ in the medium produced a significant reduction in hypoxia-induced LDH release. The effect of hypoxia was blocked by PLA2 inhibitors, mepacrine, butacaine, and dibucaine. A similar effect was observed by a 85-kD cPLA2 inhibitor AACOCF3. AA increased hypoxia-induced LDH release, and albumin, a fatty acid absorbent, prevented the LDH release, suggesting that free fatty acids are involved in hypoxia-induced cell injury. These results suggest that PLA2 activation and its metabolic products play important roles in pathogenesis of hypoxia-induced cell injury in rabbit renal cortical slices.
Acidosis ; Alanine ; Amino Acids ; Anoxia ; Arachidonic Acid ; Dibucaine ; Fatty Acids, Nonesterified ; Glycine ; Phospholipases A2* ; Phospholipases* ; Quinacrine