Dendritic cells as a major source of macrophage-derived chemokine/CCL22 in vitro and in vivo.

Macrophage-derived chemokine (MDC)/CCL22 is a CC chemokine active on dendritic cells (DC), NK cells and Th2 lymphocytes. The present study was aimed at comprehensively investigating MDC production in vitro and in vivo. DC were the most potent producers of MDC among leukocytes tested. Endothelial cells did not produce MDC under a variety of conditions. Signals that induce maturation (lipopolysaccharide, IL-1, TNF, CD40 ligand, recognition of bacteria and yeast) dramatically augmented MDC production, and dexamethasone and vitamin D3 blocked it. Prostaglandin E(2), which blocked the acquisition of IL-12 production and the capacity to promote Th1 generation, did not affect MDC production. Using mass spectrometry-based techniques, DC supernatants were found to contain N-terminally truncated forms of MDC [MDC(3-69), MDC(5-69) and MD(C7-69)] as well as the full-length molecule. In vivo, CD1a(+), CD83(+), MDC(+) DC were found in reactive lymph nodes, and in Langerhans' cell histiocytosis. Skin lesions of atopic dermatitis patients showed that CD1a(+) or CD1b(+) DC, and DC with a CD83(+) phenotype were responsible for MDC production in this Th2-oriented disorder. Thus, DC are the predominant source of MDC in vitro and in vivo under a variety of experimental and clinical conditions. Processing of MDC to MDC(3-69) and shorter forms which do not recognize CCR4 is likely to represent a feedback mechanism of negative regulation.

Identification and measurement of endogenous beta-oxidation metabolites of 8-epi-Prostaglandin F2alpha.

F2-isoprostanes are prostaglandin-like compounds derived from nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. 8-epi-Prostaglandin (PG) F2alpha, a major component of the F2-isoprostane family, can be conveniently measured in urine to assess noninvasively lipid peroxidation in vivo. Measurement of major metabolites of endogenous 8-epi-PGF2alpha, in addition to the parent compound, may be useful to better define its formation in vivo. 2,3-Dinor-5,6-dihydro-8-epi-PGF2alpha is the only identified metabolite of 8-epi-PGF2alpha in man, but its endogenous levels are unknown. In addition to this metabolite, we have identified another major endogenous metabolite, 2,3-dinor-8-epi-PGF2alpha, in human and rat urine. The identity of these compounds, present at the pg/ml level in urine, was proven by a number of complementary approaches, based on: (a) immunoaffinity chromatography for selective extraction; (b) gas chromatography-mass spectrometry for structural analysis; (c) in vitro metabolism in isolated rat hepatocytes; and (d) chemical synthesis of the enantiomer of 2,3-dinor-5, 6-dihydro-8-epi-PGF2alpha as a reference standard. In humans, the urinary excretion rate of both dinor metabolites is comparable with that of 8-epi-PGF2alpha. Both metabolites increase in parallel with the parent compound in cigarette smokers, and they are not reduced during cyclooxygenase inhibition. Another beta-oxidation product, 2, 3,4,5-tetranor-8-epi-PGF2alpha, was identified as a major product of rat hepatocyte metabolism. In conclusion, at least two major beta-oxidation products of 8-epi-PGF2alpha are present in urine, which may be considered as additional analytical targets to evaluate 8-epi-PGF2alpha formation and degradation in vivo.

Reduction of urinary 8-epi-prostaglandin F2 alpha during cyclo-oxygenase inhibition in rats but not in man.

1. 8-epi-prostaglandin (PG) F2 alpha, a major F2 isoprostane, is produced in vivo by free radical-dependent peroxidation of lipid-esterified arachidonic acid. Both cyclo-oxygenase isoforms (COX-1 and COX-2) may also form free 8-epi-PGF2 alpha as a minor product. It has been recently seen in human volunteers that the overall basal formation of 8-epi-PGF2 alpha in vivo is mostly COX-independent and urinary 8-epi-PGF2 alpha is therefore an accurate marker of 'basal' oxidative stress in vivo. 2. To test the validity of this marker in the rat, we evaluated in vivo the effect of COX inhibition on the formation of 8-epi-PGF2 alpha vs prostanoids. Two structurally unrelated COX inhibitors (naproxen: 30 mg kg-1 day-1; indomethacin: 4 mg kg-1 day-1) were given i.p. to rats kept in metabolic cages. In vivo formation of 8-epi-PGF2 alpha was assessed by measuring its urinary excretion. Prostanoid biosynthesis was assessed by measuring urinary excretion of major metabolites of thromboxane (TX) and prostacyclin (2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha). All compounds were selectively measured by immunopurification/gas chromatography-mass spectrometry. 3. Naproxen reduced urinary excretion of 2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha but, unexpectedly, also that of 8-epi-PGF2 alpha (82, 49 and 52% inhibition, respectively). Indomethacin had a similar effect (77, 69 and 55% inhibition). Esterified 8-epi-PGF2 alpha in liver and plasma remained unchanged after indomethacin. 4. These findings prompted us to re-assess the contribution of COX activity to the systemic production of 8-epi-PGF2 alpha in man. We gave naproxen (1 g day-1) to healthy subjects (four nonsmokers and four smokers). Urinary 8-epi-PGF2 alpha remained unchanged in the two groups (9.63 +/- 0.99 before vs 10.24 +/- 1.01 after and 20.14 +/- 3.00 vs 19.03 +/- 2.45 ng h-1 1.73 m-2), whereas there was a marked reduction of major urinary metabolites of thromboxane and prostacyclin (about 90% for both 11-dehydro-TXB2 and 2,3-dinor-TXB2; > 50% for 2,3-dinor-6-keto-PGF1 alpha). 5. To investigate whether rat COX-1 produces 8-epi-PGF2 alpha more efficiently than human COX-1, we measured the ex vivo formation of 8-epi-PGF2 alpha and TXB2 simultaneously in whole clotting blood. Serum levels of 8-epi-PGF2 alpha and TXB2 were similar in rats and man. 6. We conclude that a significant amount of COX-dependent 8-epi-PGF2 alpha is present in rat but not in human urine under normal conditions. This implies that urinary 8-epi-PGF2 alpha cannot be used as an index of near-basal oxidant stress in rats. On the other hand, our data further confirm the validity of this marker in man.

Measurement of urinary 8-Epi-prostaglandin F2alpha, a novel index of lipid peroxidation in vivo, by immunoaffinity extraction/gas chromatography-mass spectrometry. Basal levels in smokers and nonsmokers.

8-Epi-prostaglandin F2alpha (8-epi-PGF2alpha) is an F2-isoprostane recently identified as a marker of free radical-catalyzed lipid peroxidation in vivo and potential mediator of oxidative damage. Currently, endogenous 8-epi-PGF2alpha is measured by gas chromatography-mass spectrometry after lengthy sample preparation. We extracted and purified 8-epi-PGF2alpha in one step from biological samples on immunoaffinity columns prepared with an anti-8-epi-PGF2alpha antiserum, raised in our laboratory. Quantitation was done by stable-isotope dilution gas chromatography/negative-ion chemical ionization mass spectrometry, with selected ion recording. Carboxylate anions of the pentafluorobenzyl ester trimethylsilyl ether derivative of 8-epi-PGF2alpha and [2H4]8-epi-PGF2alpha were monitored (m/z 569 and 573). Basal urinary excretion of 8-epi-PGF2alpha can be accurately and rapidly measured by this method. Under normal conditions rats (n = 30) excreted 2.18 +/- 0.68 ng/24 h. In healthy nonsmoking young volunteers, urinary excretion of 8-epi-PGF2alpha, measured three times on alternate days, was fairly constant (CV 2-10%). Nonsmokers excreted significantly less 8-epi-PGF2alpha than age-matched smokers (8.08 +/- 2.3 vs. 18.40 +/- 4.77 ng/h/1.73 m2; n = 6; p < 0.005), as reported by others using different methods.

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.

Rapid, direct enzyme immunoassay of 11-keto-thromboxane B2 in urine, validated by immunoaffinity/gas chromatography-mass spectrometry.

We have developed a direct enzyme immunoassay (EIA) for quantifying immunoreactive 11-keto-thromboxane B2 (iKTXB) in unprocessed human urine. Cross-reactivity with other thromboxane metabolites and prostanoids was negligible. Analytical recovery of 11-keto-TXB2 in urine specimens was 97.4% to 99.8%. Total imprecision for two clinical specimens was 8.5% and 12.2%. Intake of acetylsalicylic acid decreased the measured concentration of iKTXB. Cardiopulmonary bypass, a procedure known to activate platelets, increased the mean excretion rate of iKTXB 10-fold. Simultaneous gas chromatography-mass spectrometry analysis of 11-keto-TXB2 and 11-keto-2,3-dinor TXB2 in urine specimens (n = 17) from healthy subjects indicated that urinary iKTXB concentrations measured by EIA represented a sum of the two 11-keto metabolites. We conclude that the direct EIA is sufficiently sensitive, rapid, simple, and specific to allow screening for alterations in thromboxane biosynthesis in patients.

Urinary excretion and origin of 11-dehydro-2,3-dinor-thromboxane B2 in man.

The in vivo biosynthesis of thromboxane B2 (TXB2) in man is currently evaluated by measuring urinary excretion of its major urinary metabolites, 11-dehydro-TXB2 and 2,3-dinor-TXB2. 11-Dehydro-2,3-dinor-TXB2, another prominent metabolite of exogenous TXB2 in man, has never been measured in human urine. We measured urinary 11-dehydro-2,3-dinor-TXB2 in parallel with 11-dehydro-TXB2 and 2,3-dinor-TXB2 by immunoaffinity extraction/gas chromatography-mass spectrometry in healthy non-smokers (n = 12) and age-matched smokers (n = 11). In non-smokers, urinary excretion of 11-dehydro-2,3-dinor-TXB2, 11-dehydro-TXB2 and 2,3-dinor-TXB2 was 29.7 +/- 11.1, 53.6 +/- 15.0 and 13.5 +/- 2.8 ng/h (mean +/- SD), respectively. In smokers, only urinary excretion of 2,3-dinor-TXB2 was significantly different (19.7 +/- 6.7 ng/h, p < 0.01). Selective inhibition of platelet thromboxane biosynthesis by chronic low-dose aspirin (30 mg/day for 8 days, 4 subjects) comparably reduced platelet-derived metabolites and 11-dehydro-2,3-dinor-TXB2, suggesting that the latter also derives from platelets in healthy subjects.

Urinary excretion of thromboxane and prostacyclin metabolites during chronic low-dose aspirin: evidence for an extrarenal origin of urinary thromboxane B2 and 6-keto-prostaglandin F1 alpha in healthy subjects.

In vivo biosynthesis of thromboxane and prostacyclin is currently evaluated by measuring urinary excretion of selected metabolites. Urinary thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) (non-enzymatic hydrolysis products of thromboxane and prostacyclin) are thought to derive from renal biosynthesis of the parent compounds, while enzymatic metabolites such as 2,3-dinor-TXB2 and 2,3-dinor-6-keto-PGF1 alpha appear to be mainly derived from systemic (platelet) thromboxane and (vascular) prostacyclin, respectively. Using immunoaffinity extraction and high-resolution gas chromatography-negative ion chemical ionization mass spectrometry (HRGC-NICIMS), we measured the paired excretion of non-enzymatic and enzymatic metabolites of thromboxane and prostacyclin in healthy subjects before, during and after an eight-day schedule of oral low-dose aspirin (30 mg/day), a treatment known to inhibit platelet and perhaps vascular but not renal cyclooxygenase. Low-dose aspirin cumulatively reduced urinary excretion of TXB2 and 2,3-dinor-TXB2 (about 80% inhibition on day 8 of aspirin treatment, P less than 0.01), as well as 6-keto-PGF1 alpha and 2,3-dinor-6-keto-PGF1 alpha (about 45% inhibition on day 8 of aspirin treatment, P less than 0.01). Excretion of all metabolites recovered slowly after aspirin withdrawal. Urinary PGE2, taken as an index of renal cyclooxygenase activity, was not inhibited by aspirin. A highly significant correlation was found between paired excretion values of non-enzymatic vs. enzymatic metabolites of thromboxane and prostacyclin in all individuals studied (TXB2 vs. 2,3-dinor-TXB2 (r = 0.91 +/- 0.03); 6-keto-PGF1 alpha vs. 2,3-dinor-6-keto-PGF1 alpha (r = 0.92 +/- 0.06], irrespective of aspirin treatment. TXB2/2,3-dinor-TXB2 and 6-keto-PGF1 alpha/2,3-dinor-6-keto-PGF1 alpha mean ratios remained unchanged throughout the experiment. These data do not support the view that urinary TXB2 and 6-keto-PGF1 alpha derive mainly from renal biosynthesis in healthy subjects, but rather suggest that they may represent a fraction of systemic (platelet) thromboxane and (vascular) prostacyclin escaping metabolism. These data also suggest that chronic low-dose aspirin may partly inhibit vascular prostacyclin in addition to platelet thromboxane biosynthesis.

Thromboxane B2 urinary metabolites in patients undergoing cardiopulmonary bypass.

The urinary excretion of selected markers for renal injury and thromboxane metabolites was studied in 16 patients undergoing cardiopulmonary bypass (CPB). Excretion of both tubular and glomerular markers sharply increased on day 1 after CPB and remained elevated throughout the observation period (five days). Immunoreactive thromboxane B2 (i-TXB2, mainly reflecting 2,3-dinor-TXB2) and immunoreactive 11-keto-thromboxane B2 (i-11-keto-TXB2) were measured by direct enzyme immunoassays. TXB2, 2,3-dinor-TXB2 and 11-keto-TXB2 were also measured in selected samples by GC-MS. Urinary excretion rates of both i-TXB2 and i-11-keto-TXB2 markedly increased on day 1 after surgery and decreased thereafter. Following CPB, excretion rates of 2,3-dinor-TXB2 and TXB2 displayed parallel changes, suggesting that in these patients most urinary TXB2 derives from blood platelets rather than the kidney. Taken together, our observations do not support the hypothesis that acute renal injury observed after CPB is caused by exaggerated thromboxane biosynthesis in the kidney.

A study on the biological behavior of human brain tumors. Part I. Arachidonic acid metabolism and DNA content.

The study of proliferative characteristics and biochemical aspects seem to be of great importance in order to define brain neoplastic behavior. The purpose of this study is to verify the existence of any possible correlation between Arachidonic Acid (AA) metabolism and proliferative characteristics in 30 meningiomas and 30 neuroepithelial tumors. The most represented metabolite in neuroepithelial tumors is TxB2, while 6-Keto-PGF1 alpha is the lowest represented product. Unimodal DNA distribution was observed in 66% of neuroepithelial tumors and in 87% of meningiomas. Aneuploidy was more frequent in glioblastomas and anaplastic meningiomas as previously reported; AA overall synthesis capacity and profile were similar between unimodal and bimodal cases of neuroepithelial tumors. Total AA metabolite, as well as TxB2 and PGD2, synthesis capacity are significantly higher in cases with S-phase cell percentage greater than or equal to 3% than in cases with S-phase % less than 3%. Total production of AA metabolites via the cyclooxygenase pathway is significantly higher in meningiomas with bimodal DNA distribution than in cases with unimodal DNA content; when considering S-phase cell percentage, similarly to what observed in neuroepithelial tumors, meningiomas with S% greater than 3% shows a significantly higher overall synthesis capacity for AA. AA metabolism capacity well correlates with proliferative patterns in neuroepithelial tumors: the relationship depends preferentially on TxB2 and PGD2 synthesis capacity. In cases of meningiomas, the amount of AA metabolites seem to be related to DNA content and proliferative activity when anaplastic features are histologically demonstrated.

A study on the biological behavior of human brain tumors. Part II: Steroid receptors and arachidonic acid metabolism.

The significance of steroid receptors (SR) in human brain tumors is presently a field of intense investigation in order to clarify some aspects of the biological behavior of these neoplasms. We studied the relationship between the presence of steroid receptors and the production of metabolites of the arachidonic acid cascade which have been reported to have a role in the biological behavior of some human tumors. We found that some metabolites of arachidonic acid are produced in different amounts in brain tumors which either did or did not express some steroid receptors. In particular the PGE2 were higher in estrogen receptors (ER) positive meningiomas than in ER negative ones and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, is significantly higher in androgen receptors (AR) negative meningiomas than in AR positive ones. In neuroepithelial tumors the glucocorticoid receptors (GR) positive cases synthesized more TxB2 and less PGE2 than the GR negative ones. Our data seem to suggest that some correlations exist between the presence of some steroid receptors and arachidonic acid metabolite production.

Metabolism of thromboxane B2 in the isolated perfused rat kidney: mass spectrometric identification of urinary products.

The metabolism of thromboxane B2 (TXB2), the stable breakdown product of thromboxane A2, has been studied in isolated perfused kidney preparations using a recirculating system. In a first experiment, TXB2 was infused at a rate of 20 micrograms/kg per min. In a second experiment, a 1:1 mixture of TXB2 and octadeuterated TXB2 (0.4 microgram/kg per min each) was infused. Urinary samples collected during the infusion of TXB2 or vehicle were extracted on C18 cartridges and derivatized to methyl or pentafluorobenzyl ester, methyloxime, trimethylsilyl ether. Samples were analyzed by high-resolution gas chromatography-mass spectrometry in the electron impact and negative ion chemical ionization modes. Products of beta-oxidation, reduction of the delta 5,6 double bond and dehydrogenation at C-11 (2,3-dinor-TXB2, 2,3-dinor-TXB1, 2,3,4,5-tetranor-TXB1 and 11-dehydro-TXB2) were identified in addition to unmetabolized TXB2. 2,3,4,5-tetranor-TXB1 and 2,3-dinor-TXB1 were the most abundant metabolites.

Quantitative profiling of 6-ketoprostaglandin F1 alpha, 2,3-dinor-6-ketoprostaglandin F1 alpha, thromboxane B2 and 2,3-dinor-thromboxane B2 in human and rat urine by immunoaffinity extraction with gas chromatography-mass spectrometry.

A rapid and simple method based on immunoaffinity extraction, stable isotope dilution and gas chromatography-mass spectrometry has been developed for profiling urinary metabolites of prostacyclin and thromboxane. 6-Ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), 2,3-dinor-6-ketoprostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha), thromboxane B2 (TXB2) and 2,3-dinor-thromboxane B2 (2,3-dinor-TXB2) were quantitatively extracted from human or rat urine spiked with deuterated internal standards using mixed-bed columns containing immobilized anti-6-keto-PGF1 alpha and anti-TXB2 antibodies (cross-reacting with 2,3-dinor-6-keto-PGF1 alpha and 2,3-dinor-TXB2, respectively). The extract was directly derivatized to form pentafluorobenzyl ester, methyloxime, trimethylsilyl ether derivatives. Quantitation was performed by stable isotope dilution assay and high-resolution gas chromatography-negative ion chemical ionization mass spectrometry, by monitoring the carboxylate anions (M-181) of the derivatized metabolites. The method was applied to evaluate the urinary excretion of 6-keto PGF1 alpha, 2,3-dinor-6-keto-PGF1 alpha, TXB2 and 2,3-dinor-TXB2 in humans and rats. Results were in accordance with previously reported data obtained by other methods. Novel data on the urinary excretion of 2,3-dinor-6-keto-PGF1 alpha in rats under basal conditions are presented. This sensitive and selective method represents a significant advance in terms of rapidity and simplicity over other immunoaffinity-gas chromatography-mass spectrometry methods for measuring single prostanoids, such as 6-keto-PGF1 alpha or TXB2, since it allows profiling of a group of metabolites whose balance is important in several physiopathological conditions.

Effect of low-dose aspirin on fetal and maternal generation of thromboxane by platelets in women at risk for pregnancy-induced hypertension.

There is evidence that aspirin in low doses favorably influences the course of pregnancy-induced hypertension, but the mechanism, although assumed to involve suppression of the production of thromboxane by platelets, has not been established. We performed a randomized study of the effect of the long-term daily administration of 60 mg of aspirin (n = 17) or placebo (n = 16) on platelet thromboxane A2 and vascular prostacyclin in women at risk for pregnancy-induced hypertension. Low doses of aspirin were associated with a longer pregnancy and increased weight of newborns. Serum levels of thromboxane B2, a stable product of thromboxane A2, were almost completely (greater than 90 percent) inhibited by low doses of aspirin. The urinary excretion of immunoreactive thromboxane B2 was significantly reduced without changes in the level of 6-keto-prostaglandin F1 alpha, a product of prostacyclin. Mass spectrometric analysis showed that aspirin reduced the excretion of the 2,3-dinor-thromboxane B2 metabolite--mainly of platelet origin--by 81 percent and of thromboxane B2, probably chiefly of renal origin, by 59 percent. The urinary excretion of 6-keto-prostaglandin F1 alpha and of its metabolite 2,3-dinor-6-keto-prostaglandin F1 alpha was not affected. Low doses of aspirin only partially (63 percent) reduced neonatal serum thromboxane B2. No hemorrhagic complications were observed in the newborns. Thus, in women at risk for pregnancy-induced hypertension, low doses of aspirin selectively suppressed maternal platelet thromboxane B2 while sparing vascular prostacyclin, but only partially suppressed neonatal platelet thromboxane B2, allowing hemostatic competence in the fetus and newborn.

Renal metabolism and urinary excretion of thromboxane B2 in the rat.

We wanted to evaluate whether the kidney tissue can metabolize thromboxane (Tx) B2 and, specifically, whether the 2,3-dinor metabolite might be formed in the kidney and excreted in the urine. For this purpose, we used an isolated perfused kidney preparation exposed to vehicle or TxB2 at different infusion rates. Approximately 96% of the total TxB2 infused was recovered in the venous effluent, whereas approximately 1% was found in urine. Isolated perfused kidneys exposed to [3H]TxB2 eliminated in the urine 1.2% of the [3H]TxB2 infused, measured by thin-layer chromatographic analysis, and actively metabolized [3H]TxB2 to 2,3-dinor-TxB2, 11-dehydro-TxB2, and possibly 2,3,4,5-tetranor-TxB1. No metabolites of TxB2 were recovered in the venous effluent. As a marker of renal TxB2 metabolic activity, urinary 2,3-dinor-TxB2 was quantified by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry. The 2,3-dinor-TxB2 was detected both before and during TxB2 infusion in urinary samples but not in the venous effluent. The ratio of 2,3-dinor-TxB2-TxB2 increased during the infusion reaching a peak value immediately after stopping the TxB2 infusion. These results indicate that, in the rat, the kidney tissue metabolizes TxB2 to 2,3-dinor-TxB2, and both TxB2 and 2,3-dinor-TxB2 are excreted in the urine.

Prostaglandin and thromboxane synthesis by human intracranial tumors.

Prostaglandin (PG) and thromboxane (TX) production by homogenates of human intracranial tumors (33 gliomas, 32 meningiomas, six brain metastases) and "normal" brain (n = 26) from tumor-bearing patients was studied. PGF2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha (the hydrolysis product of PGI2) and TXB2 (the hydrolysis product of TXA2) were determined by high-resolution gas chromatography-mass spectrometry after ex vivo metabolism of endogenous arachidonic acid. Prostanoid profiles (relative abundance of each metabolite) were different for gliomas and meningiomas, but similar for gliomas and their nontumoral counterpart, i.e., "normal" brain. Mean overall prostanoid production was significantly higher in gliomas (539 +/- 95) and meningiomas (523 +/- 69) than in "normal" brain (198 +/- 23). Prostanoid synthesis significantly increased with anaplastic grade (glioblastomas greater than anaplastic astrocytomas greater than slow-growing astrocytomas greater than "normal" brain), while profiles did not substantially change (TXB2 was the most and 6-keto-PGF1 alpha the least abundant product). Meningioma profiles showed no marked prevalence of any particular metabolite and no major differences between histological subgroups. All brain metastases from different carcinomas (n = 5) showed a prevalence of TXB2 and PGE2 and very low PGD2 synthesis.

Antiinflammatory action of salicylates: aspirin is not a prodrug for salicylate against rat carrageenin pleurisy.

A current hypothesis postulates that the antiinflammatory effect of aspirin (acetylsalicylic acid) is mediated by its metabolite salicylic acid through inhibition of PGE2 synthesis. We tested this hypothesis in rats with carrageenin-induced pleurisy. Aspirin or salicylate, given orally, reduced exudation and cell migration into the pleural cavity, aspirin being more potent than salicylate. The antiinflammatory effect of aspirin cannot be explained only in terms of salicylate formation. Doses of aspirin and salicylate that inhibit inflammation by 50% result in salicylate levels in the exudate of 70 +/- 12 and 323 +/- 17 micrograms/ml, respectively. At a significant antiinflammatory dose (100 mg/kg), salicylate did not reduce the prostaglandin and thromboxane content of the exudate. This indicates that inhibition of cyclooxygenase is not a likely mechanism for the antiinflammatory effect of salicylate. Salicylate only reduced the amount of 6-keto-PGF1 alpha in the exudate at higher doses (200 mg/kg), while aspirin at an equally antiinflammatory dose (50 mg/kg) reduced the content of 6-keto-PGF1 alpha, TXB2, PGD2 but not of PGE2 in the exudate. It therefore seems unlikely that an inhibition of PGE2 synthesis is the common mechanism by which aspirin and salicylate exert their antiinflammatory effects. These results do not supported the hypothesis that aspirin is a prodrug for salicylate but rather indicate that both compounds may exert their antiinflammatory effects partly by different mechanisms.

Prostaglandins in human brain tumors.

It has been recently observed that arachidonic acid (AA) metabolites may modulate many of the mechanisms involved in tumor growth and metastasis. In order to clarify the role played in human brain tumors, authors have determined AA metabolic profiles in 63 surgical specimen of human intracranial tumors (mostly neuroepithelial tumors and meningiomas). The five metabolites via the cyclooxygenase pathway (PGD2, PGE2, TxB2, PGF2a, 6-Keto-PGF1a) were measured by high resolution gas chromatography-mass spectrometry after "ex vivo" metabolism of endogenous AA by tumor homogenates. The overall synthesis capacity of AA metabolites widely varied among different oncotypes, and, except in two cases of dermoid cysts, was higher than in normal brain tissue. AA metabolism seems more active in neuroepithelial tumors with the highest grade of anaplasia; some changes in the percentage of each metabolite is evident when anaplastic features changed. Thromboxane B2 was the most represented and 6-Keto-PGF1a the less abundant metabolite. Meningiomas and neuroepithelial tumors showed different relative proportion of AA metabolites which have in some cases reported to positively or negatively affect tumor growth. In histological subgroups of meningiomas AA metabolites synthesis capacity did not show any statistical difference. In the six cases of brain metastasis there is a wide range of overall synthesis capacity, with predominant synthesis of thromboxane B2 and prostaglandin E2, while the percentage of prostaglandin D2, reported as antimetastatic, is very low.

Increased urinary excretion of thromboxane B2 and 2,3-dinor-TxB2 in cyclosporin A nephrotoxicity.

Cyclosporin A (CsA) administration to rats is associated with a selective increase in urinary excretion of immunoreactive thromboxane B2 (i-TxB2), the stable breakdown product of TxA2. The exaggerated synthesis of TxA2 may play a role in the reduction of glomerular filtration rate (GFR) observed both in animals and humans undergoing CsA treatment. The present study was designed to get further insight into the origin of the abnormal i-TxB2 urinary excretion. Rats given orally CsA (50 mg/kg/day) for 30 days had a significant increase in the urinary excretion of both 2,3-dinor-TxB2 and TxB2 measured by technique of capillary column gas chromatography-negative ion chemical ionization mass spectrometry (HRGC-NICIMS). Urinary TxB2 is more likely to reflect the renal synthesis of the parent compound, whereas 2,3-dinor-TxB2 is considered to reflect the amount of TxB2 formed in the circulation. Experiments in isolated perfused kidney (IPK) taken from animals given CsA for 30 days showed a lower percentage increase in urinary TxB2 over vehicle treated animals. Moreover in IPK the ratio 2,3-dinor-TxB2/TxB2 was lower than in vivo. The amount of i-TxB2 detectable in serum of animals given CsA was not different from that of control animals. In contrast, isolated glomeruli taken from rats given CsA had an increase in their TxA2 synthesis measured as i-TxB2 in the supernatants. Ultrastructural studies on kidney specimens from animals given CsA showed a focal glomerular endothelial damage together with a marked infiltration of blood borne cells of monocyte-macrophage type in the glomerular tuft. In contrast, kidney specimens taken from IPK preparations were devoid of inflammatory cells. In vitro CsA did not interfere with platelet arachidonic acid (AA) metabolism as shown by a normal i-TxB2 generation in vitro by rat platelet-rich plasma (PRP) exposed to CsA and then challenged with AA or ADP. Similarly isolated glomeruli and isolated proximal tubules from normal rats when challenged with CsA in vitro converted AA into TxA2 normally. It is suggested that the cause of the increased urinary excretion of 2,3-dinor-TxB2 is the consequence of intrarenal platelet and macrophage activation, probably triggered by the endothelial damage. The parallel increase in the urinary excretion of unmetabolized TxB2 is likely to reflect a concomitant activation of resident renal cell AA metabolism induced by CsA.