Intravascular filarial parasites elaborate cyclooxygenase-derived eicosanoids.

The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Since prostanoids formed from arachidonic acid can modulate interactions among platelets, leukocytes, and endothelial cells, we examined whether intravascular nematode parasites can elaborate prostanoids. Microfilariae of Brugia malayi utilize exogenous and endogenous arachidonic acid to generate and release two predominant prostanoids, prostacyclin and prostaglandin E2. Filarial metabolism of host fatty acids to form these vasodilatory, antiaggregatory, and immunomodulatory eicosanoids provides a means by which these helminthic parasites may influence host immune and other cellular responses.

Evidence for prostanoid biosynthesis as a biochemical feature of certain subclasses of non-small cell carcinomas of the lung as determined in established cell lines derived from human lung tumors.

Detectable levels (greater than or equal to 0.2 pmol/10(6) cells) of one or more prostanoid species resultant to calcium ionophore A23187-induced biosynthesis from endogenous arachidonic acid were distributed in 28 cell lines derived from different histological classes of lung tumors as follows: large cell undifferentiated carcinoma (3 of 3 cell lines); adenosquamous carcinoma (1 of 2 cell lines); squamous cell carcinoma (0 of 2 cell lines); adenocarcinoma (9 of 10 cell lines); bronchioloalveolar cell carcinoma (2 of 2 cell lines); and small cell carcinoma (1 of 9 cell lines). Using the mean levels of 9 alpha,11 beta-prostaglandin F2, prostaglandin F2 alpha, prostaglandin D2, prostaglandin E2, thromboxane B2 and 6-keto-prostaglandin F1 alpha as an index of prostaglandin H (PGH) synthase activity, the distribution in cell lines representative of the different histological classes of human lung tumors exhibiting PGH synthase activity exceeding mean values greater than or equal to 2 pmol/10(6) cells was as follows: large cell undifferentiated carcinoma (3 of 3 cell lines), adenosquamous carcinoma (1 of 2 cell lines), adenocarcinoma (8 of 10) cell lines), bronchioloalveolar cell carcinoma (2 of 2 cell lines) and small cell carcinoma (0 of 9 cell lines). Three different prostanoid species accumulated to mean levels greater than or equal to 2 pmol/10(6) cells. Prostaglandin E2 levels exceeded 2 pmol/10(6) cells in 14 of the 16 cell lines in which this prostanoid accumulated to detectable levels. Cumulative levels of prostaglandin F2 alpha exceeded 2 pmol/10(6) cells in 9 of the 15 cell lines in which prostaglandin F2 alpha reached detectable levels. Detectable levels of thromboxane B2 were observed in five cell lines with thromboxane B2 accumulation exceeding 2 pmol/10(6) cells in two of the five cell lines. 9 alpha,11 beta-prostaglandin F2 and 6-keto-prostaglandin F1 alpha accumulated to detectable levels in the culture medium of one cell line, while prostaglandin D2 accumulation to detectable levels was observed in two cell lines. Stimulation of cultured human lung tumor cells exhibiting PGH synthase activity greater than or equal to 2 pmol/10(6) cells in the presence of 10(-5) M exogenous arachidonic acid resulted in a 2- to 4-fold increase in the accumulation of individual prostanoids, while the inclusion of a 10(-5) M exogenous concentration of arachidonic acid failed to stimulate detectable prostanoid production in human lung tumor cells in which PGH synthase activity was not previously expressed.(ABSTRACT TRUNCATED AT 400 WORDS)

Cigarette smoking: profiles of thromboxane- and prostacyclin-derived products in human urine.

Thromboxane (TX) B2, 2,3-dinor-TXB2, 11-dehydro-TXB2, 6-oxoprostaglandin (PG)F1 alpha and 2,3-dinor-6-oxo-PGF1 alpha were measured in 24 h urine samples obtained from 30 apparently healthy chronic cigarette smokers and 37 closely matched non-smoking control subjects. Samples were analysed using a newly developed assay based on immunoaffinity chromatography and capillary column gas chromatography/electron capture negative ion chemical ionisation mass spectrometry. There were significant and comparable increases in the excretion rates of both 2,3-dinor-TXB2 and 11-dehydro-TXB2 in the smoking compared with the non-smoking group (2P less than 0.001). Excretion rates of 2,3-dinor-TXB2 were 418 +/- 35 and 265 +/- 26 pg/mg creatinine in the two groups, respectively. 11-Dehydro-TXB2 excretion rates were 440 +/- 54 and 221 +/- 18 pg/mg creatinine, respectively (mean +/- S.E.). There were significant (2P less than 0.05) positive correlations between average reported cigarette consumption and excretion of both thromboxane metabolites. There were small but significant (2P less than 0.02) increases in the excretion rates of both 6-oxo-PGF1 alpha and 2,3-dinor-6-oxo-PGF1 alpha in the smoking compared with the non-smoking group. There was no significant difference in the rates of excretion of TXB2 in the two groups. The effects of acute cigarette smoke exposure (five cigarettes in 2 h) was also studied in four normally non-smoking healthy volunteers. There was no significant change in the excretion rate of any of the eicosanoids measured during control and smoking periods (at least 2 weeks apart), indicating that increased TXA2 biosynthesis in chronic smokers is unlikely to be a consequence of acute platelet activation.

Separation of major prostaglandins, leukotrienes, and monoHETEs by high performance liquid chromatography.

A procedure using high performance liquid chromatography (HPLC) is described for the separation of major primary cyclooxygenase metabolites (prostacyclin metabolite-6ketoPGF1 alpha, thromboxane B2, and prostaglandins F2 alpha, E2, and D2), leukotrienes (C4, B4, and D4), monohydroxyeicosatetraenoic acids (15-, 11-, 12-, and 5HETEs), and free arachidonic acid. It is therefore possible to quantitate major arachidonic acid metabolites by a single chromatographic procedure. Using this technique we have determined that a major arachidonic acid metabolite of human lung macrophages co-elutes with leukotriene B4.

Optimization of an enzyme immunoassay for 11-dehydro-thromboxane B(2) in urine: comparison with GC-MS.

The urinary excretion of stable metabolites of thromboxane A2, such as 11-dehydro-thromboxane B2, reflects platelet activity in vivo. Efficient sample purification is required before analysis of thromboxane metabolites, due to the presence of large amounts of interfering material in urine. Analysis by gas chromatography-mass spectrometry after extensive sample work-up procedures provides the most reliable data, but detection by enzyme immunoassay may be reliable if sample cleanup is adequate. We describe an improved immunoassay procedure for 11-dehydro-thromboxane B2, which is based on a simple one-step solid phase extraction, by using Bond-Elut Certify II columns, followed by enzyme immunoassay by using commercially available reagents. 11-Dehydro-thromboxane B2 exists in two forms, with different chemical and immunological characteristics, which are in pH-dependent equilibrium. We kept 11-dehydrothromboxane B2 in its open ring form throughout the assay, by incubating and handling samples at pH 8.6. The extraction step achieved a recovery of 83% (95% confidence interval 74-92%), the sensitivity of the enzyme immunoassay was doubled, and the reproducibility of the assay improved under these conditions. Intra- and interassay coefficients of variation were 3 and 13.8%, respectively. A single 500-mg dose of aspirin reduced the excretion of 11-dehydro-thromboxane B2 by 77+/-14%, suggesting good specificity. Comparison with gas chromatography-mass spectrometry in 28 urine samples showed excellent agreement between the two methods (r2 = 0.94; p<0.0001), and a regression line with a slope close to 1.0. The presently modified enzyme immunoassay for 11-dehydro-thromboxane B2 is suitable for clinical studies evaluating platelet function in vivo and has the advantage of being simpler and less expensive to use than gas chromatography-mass spectrometry.

An improved method for separation of thromboxane B2 by reversed phase liquid chromatography.

Column efficiency for thromboxane B2 (TXB2) is 10 times lower than for prostaglandins when chromatographed on octadecyl-silica columns. We described the use of a new non-silica reversed phase support which brings the column efficiency for TXB2 in the range of the prostaglandins.

Extraction of thromboxane B2 from urine using an immobilized antibody column for subsequent analysis by gas chromatography-mass spectrometry.

This paper describes an antibody affinity (immunoaffinity) column which, in one step, extracts and sufficiently purifies urinary thromboxane B2 (TXB2) for quantitative analysis by high resolution gas chromatography-negative ion chemical ionization-selected ion monitoring-mass spectrometry (HRGC-NICI-SIM-MS). Polyclonal TXB2 antibody from rabbit was partially purified using immobilized Staphylococcus aureus Protein A. The purified IgG fraction was then immobilized using an N-hydroxysuccimidyl silica gel. The resulting matrix bound 570 ng TXB2 per ml of gel. TXB2 was quantitatively eluted with acetonitrile-water (19:1). Columns constructed from the gel could be used repeatedly since binding capacity was reconstituted using 0.01 M phosphate buffer (pH 7.4) with no apparent loss of activity. Using these columns, urinary TXB2 was sufficiently purified in one step such that in subsequent analysis by HRGC-NICI-SIM-MS interference free chromatograms were observed.

A new radioimmunoassay for urinary thromboxane B2 with prior purification by high performance liquid chromatography.

In order to assess the role of the renally formed thromboxane A2 in humans, a new radioimmunoassay was developed for the determination of the urinary excretion of thromboxane B2, the stable metabolite of thromboxane A2. Urines were extracted with ethylacetate and thromboxane B2 was separated from the other primary prostaglandins by a reversed phase high performance liquid chromatography using water, acetonitrile and acetic acid (71:29:0.1) as mobile phase with an overall recovery of 44.1% +/- 1.3 (n = 42). The radioimmunoassay, performed on the fraction containing thromboxane B2 used a sensitive (less than 1pg) and highly specific antibody (final dilution: 1/460 800) that we have raised in the rabbit. The coefficient of variation was found lower than 5% in the usable part of the standard curve and the inter-assay reproducibility was at 7.3%. The 24-hour urinary excretion of thromboxane B2 measured in 26 healthy adults established at 109 +/- 12.9 ng/24h in men and at 99.5 +/- 9.2 ng/24 h in women. Therefore, it differed from that of the other prostaglandins by lower values devoid of sex-related differences.

Thromboxane B2 (TXB2) formation in clotting whole blood of healthy and diabetic humans in vitro.

We investigated the ability of platelets from two groups of diabetics type I and two groups of healthy volunteers matched of age to generate thromboxane B2 (TXB2) during spontaneous clotting of whole blood. The serum concentration of TXB2, reflecting the ability of the platelets to generate TXA2 during clotting, was measured by gas liquid chromatography. Platelets from old diabetics with more than 40 years duration of diabetes mellitus formed significantly less TXB2 than those from old healthy controls. Platelets from juvenile diabetics (9 years duration of disease) formed nearly the same amount of TXB2 as those from young healthy volunteers. The importance of these results is discussed.

Identification of thromboxane B2 in guinea-pig uterine homogenates.

On the basis of gas chromatographic and mass spectrometric evidence, thromboxane B2 has been identified in incubates of homogenised guinea-pig uterus.

Synthesis of leukotriene B4, and prostanoids by human alveolar macrophages: analysis by gas chromatography/mass spectrometry.

Human alveolar macrophages, obtained during diagnostic bronchoscopy, were maintained in monolayer culture. Challenge of these cells (greater than 95% purity) with 1.2 mg/ml zymosan A particles (opsonized with human serum) was followed by a rapid release of leukotriene B4 into the medium, 7.28 +/- 5.99 ng/mg cell protein at 2 h (mean +/- S.D.4, n = 4). Leukotriene B4 was identified and measured by a novel technique employing capillary column gas chromatography coupled to negative ion chemical ionization mass spectrometry. The release of thromboxane B2, prostaglandins D2, E2, F2 alpha and the lysosomal enzyme N-acetyl-beta-D-glucosaminidase was also measured. Thromboxane B2 was the most abundant metabolite of arachidonic acid released into the culture medium (65.2 +/- 14.8 ng/mg cell protein 2 h after the addition of zymosan A, n = 4), and the synthesis of thromboxane B2 was inhibited by greater than 90% in 1 microM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B4 synthesis.

Identification of 11-dehydro-2,3-dinorthromboxane B3 in human urine based on the mass spectral properties of 11-dehydrothromboxane B3 and related compounds.

11-Dehydro-2,3-dinorthromboxane B3 (an enzymatic metabolite of TXB3) was identified. Urine from a healthy male adult who had received daily administration of 1.8 g (0.6 g three times) of eicosapentaenoic acid (EPA) ethyl ester was collected. A urine sample was extracted with an affinity column of anti-11-dehydro-TXB2 monoclonal antibody. Conversion of 11-dehydro-2,3-dinor-TXB3 in the urine extract into the methyl ester (ME)-11-n-propylamide (PA)-9, 12, 15-dimethylisopropylsilyl (DMIPS) ether derivative was followed by gas chromatography with selected-ion monitoring (GC/SIM) (resolution: 8000) and GC/mass spectrometry (MS). High-resolution SIM result revealed that a major component corresponded to the [M - C3H7]+ (m/z 668.4198) ion, which may be specific for the structural integrity by analogy with the corresponding 11-dehydro-TXB3 derivative. Conventional GC/MS analysis confirmed that the peak of the interest was the title compound. The mass spectrum of the 11-dehydro-2,3-dinor-TXB3 ME-PA-DMIPS ether derivative was dominated by the ions of [M - CH3]+ (m/z 696) and [M - C3H7]+ (m/z 668, base peak) and ions containing a protected 11-carboxylic acid moiety together with the unidentified additional ions which were considered to derive from urinary endogenous substances. However, the quality was sufficient for use of mass spectral identification of 11-dehydro-2,3-dinor-TXB3, the beta-oxidation product of 11-dehydro-TXB3. Characteristic fragment ions of [M - C5H9-(dimethylisopropylsilanol)n]+ (m/z 524 and 406) yielding information about the position of the incorporated double bond were prominent, the same as the corresponding ions found in the mass spectrum of the 11-dehydro-TXB3 derivative. Consequently, the fragmentation products were closely related to those of the 11-dehydro-TXB3 derivative, except for an obvious shift produced by the lack of C-2/C-3 hydrocarbon units. The results demonstrate the endogenous formation of TXA3 in humans after administration of EPA ethyl ester and excretion of 11-dehydro-2,3-dinor-TXB3 into urine as one of the enzymatic metabolites of TXB3, together with the previously reported 11-dehydro-TXB3.

Urinary excretion of 2,3-dinor-thromboxane B1, a major metabolite of thromboxane B2 in the rat.

Urinary 2,3-dinor-thromboxane B2 (2,3-dinor-TXB2), an enzymatic degradation product of TXB2, is currently measured for evaluating in vivo thromboxane biosynthesis in rats. We simultaneously measured 2,3-dinor-TXB2 and 2,3-dinor-TXB1, another product of TXB2 metabolism, in the urine of rats by immunoaffinity extraction/gas chromatography negative ion chemical ionization mass spectrometry (GC-NICIMS). In rats under basal conditions, urinary excretion of 2,3-dinor-TXB1 was much higher than that of 2,3-dinor-TXB2 (19.22 +/- 4.86 and 1.64 +/- 0.29 ng/24 h, respectively). The relative abundance of the two metabolites in each animal was fairly constant (91.9 +/- 1.6 and 8.1 +/- 1.6% of their sum, respectively). Urinary excretion of both 2,3-dinor-TXB1 and 2,3-dinor-TXB2 increased in rats undergoing in vivo hepatic ischemia-reperfusion. Other thromboxane metabolites, including 11-dehydro-TXB2 and 11-dehydro-2,3-dinor-TXB2, were measured by GC-NICIMS in selected urines. The resulting profile was: 2,3,4,5-tetranor-TXB1 > 2,3-dinor-TXB1 >> 11-dehydro-TXB2 > 2,3-dinor-TXB2 = TXB2. This study shows that urinary 2,3-dinor-TXB1 is a suitable parameter of TXB2 biosynthesis in vivo in rats. The possible cross-reactivity of 2,3-dinor-TXB1 in immunoassays of urinary 2,3-dinor-TXB2 or even TXB2 in rats should be considered in future studies.

Separation and quantitative determination of radiolabeled prostaglandins, thromboxanes, 6-keto-prostaglandin F1 alpha and other arachidonic acid metabolites produced in biological material.

Evaluation of several thin-layer chromatographic procedures for the separation of various labeled arachidonic acid metabolites (including 6-keto-prostaglandin F1 alpha) produced in the biological system is described. Manual scanning and autoradiography of the plates developed by two-dimensional thin-layer chromatography was also done for locating the radioactivities due to arachidonic acid metabolites other than thromboxane B2 and the classical prostaglandins (PGF2 alpha, PGE2, and PGD2).

Increased thromboxane B2 excretion in diabetes mellitus.

Thromboxane (TX) A2 is a potent vasoconstrictor as well as a proaggregator of platelets. Augmented TXB2 platelet synthesis and attenuated vascular prostacyclin formation have been demonstrated in diabetes mellitus. We undertook to establish a simple method of extracting urinary TXB2 (UTXB2) and to elucidate the pathophysiologic role of renal TXA2 in diabetes mellitus. One-step extraction of UTXB2 with an octadecylsilyl-silica column was sufficient as pretreatment for TXB2 radioimmunoassay because recovery of UTXB2 was good, the eluate was parallel with the dose-response curve, and the value coincided with that obtained by the conventional method. When platelet TXA2 synthesis was completely suppressed by administration of 100 mg aspirin, urinary TXB2 excretion (UTXB2V) declined to 41% of the initial levels, suggesting that renal TXA2 formation contributes significantly to UTXB2V. UTXB2V was 94.5 +/- 14.0 ng/day or 108.8 +/- 17.3 ng/gm creatinine in controls. Approximately half of the patients with diabetes demonstrated a UTXB2 level higher than the mean + 2 SD level of controls. Although UTXB2V did not show a significant correlation with protein excretion, UTXB2V in patients with diabetes with proteinuria greater than 100 mg/day was augmented (224.4 +/- 30.5 ng/day) compared with that in patients with diabetes without proteinuria greater than 100 mg/day. Furthermore, UTXB2V correlated negatively with the p-aminohippuric acid clearance rate, but not with the creatinine clearance rate. The results suggest that renal TXA2 synthesis may be augmented in diabetic nephropathy and may play a pathophysiologic role in renal hemodynamics as well as in protein excretion.

Immunoaffinity purification of 11-dehydro-thromboxane B2 from human urine and plasma for quantitative analysis by radioimmunoassay.

11-Dehydro-thromboxane B2 is now considered to be a reliable parameter of thromboxane A2 formation in vivo. An immunoaffinity purification method was developed for radioimmunoassay of this compound contained in human urine and plasma. Monoclonal anti-11-dehydro-thromboxane B2 antibody was prepared and coupled to BrCN-activated Sepharose 4B. Human urine or plasma was applied to a disposable column of the immobilized antibody. After the column was washed with water, 11-dehydro-thromboxane B2 was eluted with methanol/water (95/5) with a recovery of more than 90%. The purified extract was subjected to a radioimmunoassay utilizing 11-[3H]dehydro-thromboxane B2 methyl ester and the monoclonal anti-11-dehydro-thromboxane B2 antibody. The detection range of the assay was 10-600 fmol (IC50 = 90 fmol). The cross-reactivities of the antibody with thromboxane B2, 2,3-dinor-thromboxane B2, and other arachidonate metabolites were less than 0.05%. These compounds were efficiently separated from 11-dehydro-thromboxane B2 by the immunoaffinity purification. This procedure also allowed the separation of 11-dehydro-thromboxane B2 from unidentified urinary and plasma substances which interfered with the radioimmunoassay. Validity of the results obtained by the radioimmunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.

Quantitative determination of 6-keto-PGF1 alpha released by rat aorta: comparison of mass fragmentography and high resolution gas chromatography with electron capture detection.

Mass fragmentography (MF) and high resolution gas chromatography with electron capture detection (HRGC-ECD) were used for measuring 6-keto-PGF1 alpha, the stable hydrolysis product of prostacyclin (PGI2) released by fresh rings of rat aorta, incubated in the absence of the precursors arachidonic acid or prostaglandin endoperoxide (PGH2). The incubation medium was acidified, extracted, chromatographed on silicic acid column and derivatized. Comparable results were obtained analyzing each sample by MF and HRGC-ECD. Both methods proved to be suitable in terms of sensitivity and specificity for the measurement of 6-keto-PGF1 alpha produce by individual rat aortae.

Separation of prostaglandins, thromboxane, hydroxy fatty acids and arachidonic acid by high pressure liquid chromatography.

Separation of the major metabolites of arachidonic acid (AA) produced by the cyclo-oxygenase and the lipoxygenases was achieved by using reverse phase high-pressure liquid chromatography. Prostaglandins (PGs), thromboxane B2 (TXB2), and AA were separated on a C-18 radial compression column. An initial isocratic elution resolved the PGs and TXB2 which was followed by a linear gradient in order to elute AA. Variations of the gradient elution shape were required to permit the separation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid, 5-12 and 15-hydroxy-5,8,11,14-eicosatetraenoic acid. The recovery of the labeled AA and its metabolites was investigated. Use of these separation methods and radiolabeled substrates should permit investigators to obtain reproducibly in one chromatographic run adequate separation and quantitation of both PGs and hydroxy fatty acid systems.

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.

Production of prostacyclin, 6-keto-PGF1 alpha and thromboxane B2 by incubated samples of umbilical vessels: a comparison of methods for expressing the results on dry weight, wet weight, protein or DNA bases.

The production of PGI2 (determined by bioassay), and of 6-keto-PGF1 alpha and TXB2 (determined by radioimmunoassay) by samples of human umbilical vessels have been measured. The results have been calculated on four bases: dry weight, wet weight, protein and DNA. There was a higher production of PGI2 and 6-keto-PGF1 alpha by umbilical veins than by umbilical arteries; no significant difference in TXB2 production was observed between umbilical veins and arteries. The ratio of 6-keto-PGF1 alpha: TXB2 production was about 100 for the samples of veins and about 40 for the samples of arteries. The best methods of expressing the results were on the bases of protein and DNA, the latter basis being marginally the best. The least satisfactory method for expressing the results was that based on dry weight. The physiological and practical implications of the results are discussed.