Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB₂ by HPLC on silica columns is reported.     

Rapid determination of amino acids in biological samples using a monolithic silica column

A high-performance liquid chromatography method in which fluorescence detection is used for the simultaneous determination of 21 amino acids is proposed. Amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and then separated on a monolithic silica column (MonoClad C18-HS, 150 mm × 3 mm i.d.). A mixture of 25 mM citrate buffer containing 25 mM sodium perchlorate (pH 5.5) and acetonitrile was used as the mobile phase. We found that the most significant factor in the separation was temperature, and a linear temperature gradient from 30 to 49°C was used to control the column temperature. The limits of detection and quantification for all amino acids ranged from 3.2 to 57.2 fmol and 10.8 to 191 fmol, respectively. The calibration curves for the NBD-amino acid had good linearity within the range of 40 fmol to 40 pmol when 6-aminocaproic acid was used as an internal standard. Using only conventional instruments, the 21 amino acids could be analyzed within 10 min. This method was found to be suitable for the quantification of the contents of amino acids in mouse plasma and adrenal gland samples.     

Analysis of leukotrienes, prostaglandins, and other oxygenated metabolites of arachidonic acid by high-performance liquid chromatography

High-performance liquid chromatography procedures were developed which separate leukotrienes (LTs), hydroxy-fatty acids (HETEs), prostaglandins (PGs), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), the stable metabolite of thromboxane A2 (TXB2), 12-hydroxyheptadecatrienoic acid (HHT), and arachidonic acid (AA). Two methods employing reverse-phase columns are described. One method uses a radial compression system, the other a conventional steel column. Both systems employ methanol and buffered water as solvents. The radial compression system requires 60 min for separation of the AA metabolites, while the conventional system requires 100 min. Both methods provide good separation and recovery of 6-keto-PGF1 alpha, TXB2, PGE2, PGF2 alpha, PGD2, LTC4, LTB4, LTD4, LTE4, HHT, 15-, 12-, and 5-HETE; and AA. The 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-diHETE), a stereoisomer of LTB4, coelutes with LTB4. To determine the applicability of the methods to biologic systems, AA metabolism was studied in two models, guinea pig lung microsomes and rat alveolar macrophages. Both HPLC systems demonstrated good recovery and resolution of eicosanoids from the two biological systems. A simple evaporation technique for HPLC sample preparation, which avoids the use of chromatographic and other time-consuming methodology, is also described.     

Rapid extraction of phycobiliproteins from cultured cyanobacteria samples

Cyanobacteria are a valuable and ubiquitous component of marine picophytoplankton that contribute significantly to total carbon biomass and primary productivity of the oceans. They contain water soluble, natively highly fluorescent proteins, phycobiliproteins, that can be considered ideal marker pigments for understanding the distribution and trophic dynamics of picoplankton populations. However, there is no standard protocol for extracting and quantitating these proteins from cyanobacterial cells. Ideally, the cells would be disrupted quickly and efficiently with complete extraction and recovery of the released proteins. For that purpose, we describe a method for extracting phycobiliproteins from a Synechococcus CCMP 833 cyanobacteria culture that utilizes 3% 3-[(3-cholamidopropyl)dimethyammonio]propanesulfonic acid (Chaps) 0.3% asolectin combined with nitrogen cavitation. Extraction efficiencies of greater than 85% were achieved by this method, which requires less than 3h. The analysis of the extracted samples was carried out by capillary electrophoresis with laser-induced fluorescence detection.     

Rapid isolation of DNA from complex biological samples using a novel capture reagent--methidium-spermine-sepharose.

We have synthesized and analyzed the functional properties of a novel DNA capture reagent containing a methidium moiety attached to a sepharose bead by a spermine linker. DNA present in a biological fluid or other complex sample binds to the reagent. The DNA-capture reagent complex is then separated from the sample by centrifugation and the DNA is released from the reagent by brief incubation in 0.1 to 0.5 N NaOH or KOH. Capture of DNA from complex samples is independent of the salt concentration of the sample, and occurs in the presence of high concentrations of EDTA, proteinase K and detergents. Many samples can be processed simultaneously. The following specific applications, in which denatured DNA is quantitated or characterized, are demonstrated: 1). Isolation of hepatitis B virus DNA from serum and quantitation by dot-blot hybridization, 2). Isolation and quantitation of DNA from urine, 3). Isolation of human genomic DNA from one microliter of blood or 100 HeLa cells followed by amplification of a specific gene sequence using the Polymerase Chain Reaction, 4). Isolation of single stranded phage M13 sequencing templates from bacterial cultures. These investigations suggest that a capture reagent containing an intercalating moiety bound to a solid support may be useful for many applications in molecular biology and molecular diagnostics.     

Metabolism of oxygenated derivatives of arachidonic acid by Caco-2 cells.

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated separately with 3H8-labeled preparations of three different lipid mediators of inflammation: 5-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B4. Both [3H8]5-hydroxyeicosatetraenoic and [3H8]12-hydroxyeicosatetraenoic acids were taken up and metabolized by Caco-2 cells, but [3H]leukotriene B4 remained unmetabolized in the incubation medium. [3H]5-hydroxyeicosatetraenoic acid was esterified into cellular phospholipids (15%) and triglycerides (4%) but did not undergo beta-oxidation. When [3H]12-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, 14% underwent two cycles of beta-oxidation to form [3H]8-hydroxyhexadecatrienoic acid, and 3% underwent three cycles of beta-oxidation to form [3H]6-hydroxytetradecadienoic acid, both of which were released into the media. [3H]12-Hydroxyeicosatetraenoic acid was also esterified into cellular phospholipids (13%), but none was esterified into cellular triglycerides.     

花生四烯酸代谢物对呼吸道感受器的作用

迷走神经与神经．免疫交互作用密切相关。在呼吸道中,迷走神经传入纤维末梢上的伤害性感受器能被多种细胞因子、炎性介质和免疫调质激活。在炎症过程中,细胞膜磷脂在磷脂酶A2作用下释放花生四烯酸（arachidonic acid,AA）,后者再经不同的酶促反应产生多种脂类代谢物。与其它的炎性介质一样,这些代谢物在炎症反应中可以激活呼吸道感受器并发挥重要作用。有些AA代谢物直接与感觉神经末梢上的受体结合,产生神经冲动并传向中枢,引起反射性效应;有些作用于感觉末梢周围的组织,改变肺的机械特性,从而刺激感觉传入神经;有些提高感觉神经末梢对机械或化学刺激的敏感性,从而增强其反应;还有些可以产生其它介质或调质而触发反射效应,或者引起炎症细胞的聚集而产生局部效应。总之,AA代谢产物有多种来源,并通过多种途径刺激呼吸道中的伤害性感受器。本综述对此进行了探讨,希望有助于阐明呼吸道炎症反应的机理。     

Effects of selenium-deficient diets on the production of prostaglandins and other oxygenated metabolites of arachidonic acid and linoleic acid by rat and rabbit aortae

Selenium is an essential component of glutathione peroxidase, which reduces free and esterified hydroperoxides of polyunsaturated fatty acids. Adequate glutathione peroxidase activity could be important for the maintenance of prostacyclin synthesis by blood vessels, since hydroperoxides can inhibit the formation of this substance. We have investigated the effects of dietary selenium deficiency on glutathione peroxidase activity and the synthesis of 6-oxoprostaglandin F1 alpha and monohydroxy and trihydroxy metabolites of polyunsaturated fatty acids by aorta. The latter products can be formed either by the actions of cyclooxygenase or lipoxygenase or by lipid peroxidation. Aortic glutathione peroxidase activity was reduced by over 80% by feeding rats a selenium-deficient diet for 4 weeks, and to undetectable levels after 6 weeks. There were no appreciable differences in the levels of free and esterified oxygenated metabolites of linoleic acid or arachidonic acid between the control and treated groups after 4 weeks. However, after 6 weeks, there were modest, but statistically significant reductions in the formation of 6-oxoprostaglandin F1 alpha and monohydroxy products formed by cyclooxygenase. On the other hand, the amounts of esterified 18:2 metabolites appeared to be higher in aortae from animals on the selenium-deficient diet, although only the increase in esterified 9-hydroxy-10,12-octadecadienoic acid was statistically significant. These results suggest that selenium deficiency can affect the formation of prostacyclin and other oxygenated metabolites of polyunsaturated fatty acids by aorta, possibly by increasing lipid peroxidation. However, the differences between control and selenium-deficient rats after 6 weeks were not very dramatic, in spite of the fact that glutathione peroxidase activity was undetectable. It would therefore appear that additional mechanisms are also involved in controlling the levels of lipid hydroperoxides in aorta.     

Argentation thin layer chromatography of arachidonic acid metabolites isolated from human platelets

No thin layer chromatographic system in the literature offers adequate resolution of arachidonic acid and its major metabolites from platelets. We have found that argentation thin layer chromatography gives baseline separation of these compounds. Use of argentation chromatography results in decreased counting efficiency with time when quantitation is done by liquid scintillation methods. Addition of sodium chloride eliminates this complication and gives recoveries comparable to or better than those obtained using nonargented silica gel alone.     

ATP-mediated release of arachidonic acid metabolites from venular endothelium causes arteriolar dilation

This study was designed to test the hypothesis that venular administration of ATP resulted in endothelium-dependent dilation of adjacent arterioles through a mechanism involving cyclooxygenase products. Forty-three male golden hamsters were anesthetized with pentobarbital sodium (60 mg/kg ip), and the cremaster muscle was prepared for in vivo microscopy. ATP (100 microM) injected into venules dilated adjacent arterioles from a mean diameter of 51 +/- 4 to 76 +/- 6 microm (P < 0.05, n = 6). To remove the source of endothelial-derived relaxing factors, the venules were then perfused with air bubbles to disrupt the endothelium. Resting arteriolar diameter was not altered after disruption of the venular endothelium (51 +/- 5 microm), and the responses to venular ATP infusions were significantly attenuated (59 +/- 4 microm, P < 0.05). To determine whether the relaxing factor was a cyclooxygenase product, ATP infusion studies were repeated in the absence and presence of indomethacin (28 microM). Under control conditions, ATP (100 microM) infusion into the venule caused an increase in mean arteriolar diameter from 55 +/- 4 to 78 +/- 3 microm (P < 0.05, n = 6). In the presence of indomethacin, mean resting arteriolar tone was not significantly altered (49 +/- 4 microm), and the response to ATP was significantly attenuated (54 +/- 4 microm, P < 0.05, n = 6). These studies show that increases in venular ATP concentrations stimulate the release of cyclooxygenase products, possibly from the venular endothelium, to vasodilate the adjacent arteriole.     

Leukocyte-derived metabolites of arachidonic acid in ischemia-induced myocardial injury

Within minutes of occlusion of a major coronary artery the polymorphonuclear leukocytes (PMNs) are activated whereby they adhere to the vascular endothelium and migrate through the endothelial layer. Interactions with the endothelium can promote increased vascular resistance, diminished collateral flow, capillary blockade, and predisposition to vasospasm, as well as enhanced vascular permeability. On subsequent reperfusion entrapped leukocytes contribute to the no-reflow phenomenon, while more leukocytes gain access to the previously ischemic region. The leukocytes infiltrate the myocardium where they exacerbate the process of tissue injury and the development of arrhythmias. The release of leukocyte-derived mediators including arachidonic acid (AA) metabolites and oxygen-derived free radicals probably underlies these activities of the leukocytes. PMNs contain active lipoxygenase enzymes capable of metabolizing AA to products that are not normally found in the myocardium, and can dominate the metabolic profile of that tissue, leading to changes in myocardial integrity and function. Inhibitors of the lipoxygenase enzymes suppress the accumulation of leukocytes into the ischemic myocardium and reduce infarct size. However, because the drugs prevent cell invasion it cannot be inferred that a lipoxygenase metabolite per se is deleterious to the ischemic heart, inasmuch as any leukocyte-dependent mechanism of injury will be attenuated whether it is mediated by eicosanoids or by any other leukocyte-derived product. Additional studies with specific inhibitors/antagonists are required to determine the biochemical mechanisms underlying the different aspects of leukocyte-mediated myocardial injury.     

Release of arachidonic acid metabolites from isolated human alveolar type II cells.

Human alveolar type II cells are thought to play a role in the pathogenesis of lung injury. Patterns of mediator release of arachidonic acid metabolism by type II cells were therefore studied after challenge with calcium ionophore A23187, opsonized zymosan and hydrogen peroxide. A time- and concentration dependent release of cyclooxygenase products was observed, with release of PGE2 greater than 6-keto-PGF1 alpha greater than TxB2. Addition of glutathione or bicarbonate further increased the production of PGE2. N-ethylmaleimide, a sulfhydryl (SH) reactant, induced a dose-dependent increase in the release of TxB2 and 6-keto-PGF1 alpha, but not of PGE2. This relates most likely to the SH-dependency and glutathione requirement of the PGE2 isomerase and SH-independence of thromboxane and prostacyclin isomerase.     

Lipidomics of polyunsaturated-fatty-acid-derived oxygenated metabolites

Nutritionally important PUFAs (polyunsaturated fatty acids) mediate some of their bioactivities through formation of oxygenated metabolites. These bioactive lipids are formed by COX (cyclo-oxygenase), LOX (lipoxygenase) and cytochrome-P450-catalysed reactions, as well as non-enzymatic lipid peroxidation. These reactions produce numerous species, some of which can be formed through more than one pathway. MS-based lipidomics offers the selectivity and sensitivity required for qualitative and quantitative analysis of multiple lipid species, in a variety of biological systems, and can facilitate the study of these mediators.     

Enzymatic hydrolysis and extraction of arachidonic acid rich lipids from Mortierella alpina

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile.     

Plant DNA extraction using silica

Described here is a method that uses silicon dioxide (silica) to extract whole genomic plant DNA of high molecular weight. The protocol is presented in a microcentrifuge format, and yields were approximately 2–4 μg per 200 mg of plant leaf tissue. The method involves fewer steps than many previous extraction protocols and, as shown here for 4 taxonomically distant angiosperms, produces DNA suitable for digestion with restriction endonucleases. The use of commercial kits is not required; the silica costs are comparatively inexpensive (<$0.03 per tube); and CTAB, rather than the more expensive guanidine thiocyanate salt, is used.     

Quantitation of promethazine and metabolites in urine samples using on-line solid-phase extraction and column-switching

A chromatographic method for the quantitation of promethazine (PMZ) and its three metabolites in urine employing on-line solid-phase extraction and column-switching has been developed. The column-switching system described here uses an extraction column for the purification of PMZ and its metabolites from a urine matrix. The extraneous matrix interference was removed by flushing the extraction column with a gradient elution. The analytes of interest were then eluted onto an analytical column for further chromatographic separation using a mobile phase of greater solvent strength. This method is specific and sensitive with a range of 3.75–1400 ng/ml for PMZ and 2.5–1400 ng/ml for the metabolites promethazine sulfoxide, monodesmethyl promethazine sulfoxide and monodesmethyl promethazine. The lower limits of quantitation (LLOQ) were 3.75 ng/ml with less than 6.2% C.V. for PMZ and 2.50 ng/ml with less than 11.5% C.V. for metabolites based on a signal-to-noise ratio of 10:1 or greater. The accuracy and precision were within ±11.8% in bias and not greater than 5.5% C.V. in intra- and inter-assay precision for PMZ and metabolites. Method robustness was investigated using a Plackett–Burman experimental design. The applicability of the analytical method for pharmacokinetic studies in humans is illustrated.