Fractional conversion of thromboxane A2 and B2 to urinary 2,3-dinor-thromboxane B2 and 11-dehydrothromboxane B2 in the cynomolgus monkey

Following the intravenous administration of thromboxane (TX) B2, the stable hydration product of TXA2, to human and nonhuman primates the most abundant urinary metabolites are 2,3-dinor-TXB2 and 11-dehydro-TXB2. However, it is not known whether fractional conversion of TXB2 to its enzymatic metabolites is an accurate representation of TXA2 metabolism. Thus, we have compared the metabolic disposition of synthetic TXA2 and TXB2 via the beta-oxidation and 11-OH-dehydrogenase pathways in vivo in the monkey. TXA2 or TXB2 (20 ng/kg) was intravenously administered to four cynomolgus monkeys pretreated with aspirin in order to suppress endogenous TXA2 production. Urinary TXB2, 2,3-dinor-TXB2 and 11-dehydro-TXB2 were measured before, during and up to 24 h after thromboxane administration by means of reversed-phase high-performance liquid chromatography radioimmunoassay. Aspirin treatment suppressed urinary 2,3-dinor-TXB2 and 11-dehydro-TXB2 by approx. 75%. A similar fractional conversion of TXA2 and TXB2 into 2,3-dinor-TXB2 and 11-dehydro-TXB2 was found. These results suggest that TXA2 is hydrolyzed to TXB2 prior to enzymatic degradation and that metabolites of the latter represent reliable indices of TXA2 biosynthesis. Due to the variability in the conversion of thromboxanes into 2,3-dinor-TXB2 and 11-dehydro-TXB2, the measurement of both metabolites seems to represent a more reliable index of acute changes in TXA2 production.     

Antibody-mediated extraction/negative-ion chemical ionization mass spectrometric measurement of thromboxane B2 and 2,3-dinor-thromboxane B2 in human and rat urine

An antibody-mediated extraction method for gas chromatographic-mass spectrometric analysis of thromboxane A2 (TXA2) urinary metabolites is reported. An antibody (Ab) raised against thromboxane B2 (TXB2) (35% cross-reacting with 2,3-dinor-TXB2) was coupled to CNBr-activated Sepharose 4B (Se) and used as stationary phase for simultaneous extraction of both compounds from urine. After addition of deuterium-labeled TXB2 as internal standard, rat or human urine was percolated through a small Ab-Se column. After being washed, the eluate was directly derivatized to the pentafluorobenzyl ester, methyloxime, and trimethylsilyl ether. Quantitation was performed by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry, monitoring the carboxylate anions. This method was applied to evaluate the urinary excretion of TXB2 and 2,3-dinor-TXB2 in humans and rats. We report on the excretion of 2,3-dinor-TXB2 in the rat. This novel approach to the extraction of urinary thromboxanes is more convenient than currently available methods in terms of simplicity, rapidity, and recovery. This method could be extended to any other prostanoid for which an antibody could be obtained.     

Increased renal TXA2 synthesis in diabetes mellitus: simultaneous determination of urinary TXB2 and 2,3-dinor-TXB2

The present study was designed to determine urinary excretion of kallikrein(KAL)-kinin as well as prostaglandin (PG) E2, TXB2 and 2,3-dinor-TXB2, a major urinary metabolite of TXA2 synthesized in platelets, by specific RIAs in patients with diabetes mellitus (DM). KAL or kinin excretion in 26 type II DM did not differ from control values obtained in 18 age-matched healthy subjects (C), although DM with HbA1 greater than 11% excreted less KAL. Urinary PGE2 excretion (7.6 +/- 2.8 ng/mg creatinine, mean +/- SE) was significantly lower in DM compared to C (17.5 +/- 3.9, p less than 0.05), while DM excreted more TXB2 (0.57 +/- 0.09, p less than 0.01) and 2,3-dinor-TXB2 (0.56 +/- 0.12, N.S.) than C (0.19 +/- 0.02 or 0.33 +/- 0.01). DM with or without mild proteinuria demonstrated lower PGE2, but higher TXB2 and 2,3-dinor-TXB2 excretion. A positive correlation of TXB2/2,3-dinor-TXB2 with proteinuria was observed in this group. However, in DM with massive proteinuria over 500 micrograms/mg creatinine, TXB2 and 2,3-dinor-TXB2 excretion decreased to levels almost identical to C. As a whole, a ratio of TXB2 to PGE2 or 2,3-dinor-TXB2 in DM was significantly higher than in C. The results suggest that a relative preponderance of TXB2 to 2,3-dinor-TXB2 may indicate an augmented renal, in addition to platelet, TXA2 synthesis. An excessive vasoconstrictive and proaggregatory TXA2 renal synthesis, concomitant with a decrease in vasodilatory and antiaggregatory PGE2, may have profound effects on renal functions such as protein excretion in DM.     

Fractional conversion of thromboxane B2 to urinary 11-dehydrothromboxane B2 in man

Thromboxane (TX) B2, the chemically stable hydration product of pro-aggregatory TXA2, undergoes two major pathways of metabolism in man, resulting in the formation of 2.3-dinor-TXB2 and 11-dehydro-TXB2, respectively. We have measured the excretion of the latter during the infusion of exogenous TXB2 over a 50-fold dose range in order to examine the fractional conversion of TXB2 to urinary 11-dehydro-TXB2 and to re-assess the rate of entry of endogenous TXB2 into the circulation. Four healthy male volunteers received 6-h intravenous infusions of the vehicle alone and TXB2 at 0.1, 1.0 and 5.0 ng.kg-1.min-1 in random order. They were pretreated with aspirin 325 mg/d in order to suppress endogenous TXB2 production. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured before, during and up to 24 h after the infusions and in aspirin-free periods, by means of NICI-GC/MS-validated radioimmunoassays. Aspirin treatment suppressed urinary 11-dehydro-TXB2 by 91%. The fractional elimination of 11-dehydro-TXB2 was independent of the rate of TXB2 infusion and averaged 6.8 +/- 0.7%, as compared to 6.4 +/- 0.9% for 2,3-dinor-TXB2. Interpolation of 11-dehydro-TXB2 values obtained in aspirin-free periods onto the linear relationship between the quantities of infused TXB2 and the amount of metabolite excreted in excess of control values (y = 0.0058x, r = 0.94, P less than 0.001) permitted calculation of the mean rate of entry of endogenous TXB2 into the circulation as 0.12 ng.kg-1.min-1. We conclude that: (a) urinary 11-dehydro-TXB2 is at least as abundant a conversion product of exogenously infused TXB2 as 2,3-dinor-TXB2; (b) its excretion increases linearly as a function of the rate of entry of TXB2 into the circulation up to approx. 40-fold the calculated rate of secretion of endogenous TXB2; (c) the latter is consistent with previous estimates based on monitoring of the beta-oxidation pathway of TXB2 metabolism.     

Role for thromboxane A2 from glomerular thrombi in nephropathy with type 2 diabetic rats

We used rats (the Otsuka Long-Evans Tokushima Fatty strain) as a model of type 2 diabetes to find whether thromboxane (TX) A2 is involved in diabetic nephropathy, and if so, to identify where it is synthesized. We measured urinary excretion of TXB2 and 2,3-dinor-TXB2 in rats up to 60 weeks of age as markers of renal and platelet synthesis of TXA2, respectively. Some diabetic rats were given daily oral doses of OKY-046 (100 mg/kg), a TXA2 synthase inhibitor, starting when they were 10 weeks of age. Healthy Long-Evans Tokushima Otsuka rats served as the controls. Urinary excretion of protein was greater in diabetic rats at 26 weeks than in controls, and the difference increased with age. Urinary excretion of TXB2 by diabetic rats was about 150% that of controls at 14 weeks, and remained at that level. In diabetic rats, urinary excretion of 2,3-dinor-TXB2 increased with age in parallel to increases in proteinuria, but in controls, excretion of these metabolites did not change with age. In diabetic rats, OKY-046 prevented the increase in urinary excretion of both metabolites, and decreased the proteinuria. Histologic examination at 60 weeks showed intraglomerular thrombi in diabetic rats but not in controls. OKY-046 reduced intraglomerular thrombi formation and the score for glomerulosclerosis. When platelet aggregation began, more TXA2 than before was released from the thrombi that formed, and the TXA2 contributed to the progress of nephropathy in this rat model of type 2 diabetes.     

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.     

Circulating and urinary thromboxane B2 metabolites in the rabbit: 11-dehydro-thromboxane B2 as parameter of thromboxane production

The metabolism of thromboxane B2 was studied in the rabbit. The aim of the study was to identify metabolites in blood and urine that might serve as parameters for monitoring thromboxane production in vivo. [5,6,8,9,11,12,14,15-3H8]-Thromboxane B2 was administered by i.v. injection to rabbits, and blood samples and urine were collected with brief intervals. The metabolic profiles were visualized by two-dimensional thin layer chromatography and autoradiography, and the structures of five major metabolites were determined using chromatographic and mass spectrometric methods. In urine the major metabolites were identified as 11-dehydro-TXB2 and 2,3,4,5-tetranor-TXB1, and other prominent products were 11-dehydro-2,3,4,5-tetranor-TXB1, 2,3-dinor-TXB1 and 2,3-dinor-TXB2. In the circulation, TXB2 was found to disappear rapidly. The first major metabolite to appear was 11-dehydro-TXB2, which also remained a prominent product in blood for the remainder of the experiment (90 min). With time, the profile of circulating products became closely similar to that in urine. TXB2 was not converted into 11-dehydro-TXB2 by blood cells or plasma. The dehydrogenase catalyzing its formation was tissue bound and was found to have a widespread occurrence: the highest conversion was found in lung, kidney, stomach and liver. The results of the present study suggest that 11-dehydro-TXB2 may be a suitable parameter for monitoring thromboxane production in vivo in the rabbit in blood as well as urinary samples, and possibly also several tissues. This was also demonstrated in comparative studies using radioimmunoassays for TXB2 and 11-dehydro-TXB2.     

Effects of intravenous oxygen on prostacyclin and thromboxane formation in patients with peripheral occlusive arterial disease.

Oxygen infusion is used in complementary medicine for treatment of peripheral occlusive arterial disease. The mechanism of action is unknown. Thus, we determined the effects of oxygen infusion on prostacyclin, thromboxane and nitric oxide synthesis. Twelve patients with peripheral occlusive arterial disease received oxygen 40 ml/d intravenously for 3 weeks. Study parameters, analyzed by gas chromatography-mass spectrometry on day 1, 3, 10, 16, 21: 2,3-dinor-6-oxo-PGF(1alpha), colour invisible 2,3-dinor-TXB2 and nitrate in one-hour-urine before and after oxygen infusion, reflecting prostacyclin, thromboxane and nitric oxide synthesis. Urinary 8-iso-PGF2alpha, indicating oxidative stress, was assessed in one patient. Urinary 2,3-dinor-6-oxo-PGF1alpha rose from baseline more than 4-fold after oxygen infusion. In contrast, urinary 2,3-dinor-TXB2 excretion remained unchanged. Oxygen infusion had no effect on urinary nitrate excretion. Urinary 8-iso-PGF(2alpha) was not influenced by oxygen infusion with and without diclofenac pretreatment. Our data demonstrate a shift of the prostacyclin/thromboxane ratio toward prostacyclin by oxygen infusion. Thus, a mechanism of action is provided and clinical trials with intravenous oxygen find a rational basis.     

Tandem mass spectrometric determination of 11-dehydrothromboxane B2, an index metabolite of thromboxane B2 in plasma and urine

11-Dehydrothromboxane B2 is one of the major enzymatic metabolites of thromboxane B2 (TXB2), a biologically inactive product of thromboxane A2. The short half-life of thromboxane A2 and ex vivo production of thromboxane B2 by platelet activation make these prostanoid metabolites inappropriate as indices of systemic thromboxane biosynthesis, whereas 11-dehydro-TXB2 has been shown to reflect the release of thromboxane A2 in the human blood circulation. Analysis of 11-dehydro-TXB2 in plasma and urine was performed by gas chromatography-mass spectrometry-mass spectrometry using the chemically synthesized tetradeuterated compound as an internal standard. The high selectivity of triple-stage quadrupole mass spectrometry (tandem mass spectrometry) considerably facilitates sample purification as compared to single quadrupole mass spectrometric determination. Plasma concentrations in five healthy male volunteers were in the range 0.8-2.5 pg/ml. Urinary excretion of 11-dehydro-TXB2 was higher than that of 2,3-dinor-TXB2: 1.2 +/- 0.36 micrograms/24 h vs 0.53 +/- 0.33 micrograms/24 h (n = 5). Thus 11-dehydro-TXB2 appears at present to be the best index metabolite of systemic TXA2 activity in plasma as well as in urine.     

A chemiluminescent immunoassay for urinary thromboxane B2.

Because of the vasoactive properties of thromboxane A2 and other related prostaglandins, much research has been conducted on drugs which alter their levels. Urinary levels of thromboxane B2 and 2,3-dinor thromboxane B2 (major urinary metabolite of thromboxane B2) are used as an indication of thromboxane production in-vivo. In order to accurately measure urinary TXB2 levels of subjects on investigative drugs which lower TXA2 and subsequently TXB2, a simple and sensitive analytical tool becomes necessary. We have thus developed a non-radioisotopic (chemiluminescent) assay for urinary TXB2. Sensitivity has been demonstrated to 5 pg/ml. The method correlates well with gas chromatography/mass spectrometry (the accepted reference method) even without column chromatographic purification prior to the conduct of the chemiluminescent assay (r = 0.96). In addition, we have demonstrated feasibility for a chemiluminescent assay to measure urinary 2,3-dinor TXB2.     

Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10-15%) of the anti-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 diluted 1/200,000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6-12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).     

Role of platelet and vascular eicosanoids in the pathophysiology of ischemic heart disease

The role of platelet and vascular arachidonate metabolism in ischemic heart disease can be derived from direct measurements and/or inhibitor trials. Direct measurements have yielded somewhat conflicting results, largely related to analytical problems and ex vivo platelet activation during blood sampling. On the other hand, inhibitor trials have clearly established the following: 1) thromboxane (TX) A2-dependent platelet activation plays an important role in the dynamic process of coronary thrombosis in unstable angina, 2) TXA2 does not appear to mediate coronary vasospasm, as seen in variant angina, 3) endogenous prostacyclin (PGI2) is not released in response to myocardial ischemia and is unlikely to regulate coronary blood flow, and 4) exogenous PGI2 is of limited therapeutic benefit. The demonstration that low-dose aspirin (0.5-1.0 mg/(kg X day] is a selective inhibitor of TXA2-dependent platelet function provides a conceptual and practical framework for the rational design of future trials. Moreover, the identification of major enzymatic metabolites of TXB2 in plasma (11-dehydro-TXB2) and urine (2,3-dinor-TXB2) and development of appropriate analytical techniques offer the opportunity for better defining the pathophysiological role of TXA2 in humans.     

Influence of morphology and malignancy of three new human melanoma cell lines on the cyclooxygenase pathway

Three newly established human melanoma cell lines (WU-BI, PN-JC, MJ-ZJ) of different morphology and different stage of malignancy were incubated with ionophore A23187 (2.5 to 40 microM) or arachidonic acid (AA, 6.25 to 100 microM). PGF2 alpha, 6-keto-PGF1 alpha, PGE2, TXB2 and 2,3-dinor-TXB2 from isolated cells and supernatants were measured by negative ion chemical ionization gas chromatography/mass spectrometry (GC/MS). PGE2 decreased in the fibroblastoid MJ-ZJ cells from 36.7 ng/mg cell protein about 70% (A23187) and about 20% (AA), respectively. However, in the cell supernatant PGE2 increased up to 295.4 +/- 66.5 ng/mg cell protein. Production of PGF2 alpha and PGE2 increased up to 5.7 +/- 1.2 ng/mg cell protein for polydendritic WU-BI cells and spindle shaped PN-JC cells. Up to 9.3 +/- 4.3 ng PGF2 alpha and 13.4 +/- 4.7 ng PGE2 was measured for WU-BI and PN-JC in the cell supernatants. All three melanoma cell lines completely lacked formation of 6-keto-PGF1 alpha, TXB2, and 2,3-dinor-TXB2.     

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.     

Reversed-phase ion-pair chromatography of straight- and branched-chain dicarboxylic acids in urine as their 2-nitrophenylhydrazides

The derivatization of urinary dicarboxylic acids with 2-nitrophenylhydrazine hydrochloride produced corresponding monohydrazides, which were separated from monocarboxylic acid hydrazides by two step extraction with ethyl acetate at different pH values. Monohydrazides of 11 straight- and branched-chain dicarboxylic acids were eluted isocratically on reversed-phase ion-pair chromatography within 24 min by the combination of pH, the polarity of mobile phase, and the size of counter ion. The analytical results showed good recovery and reproducibility using 3,3-dimethyglutaric acid as an internal standard. The present method provides a notable HPLC method with precolumn derivatization for the analysis of urinary dicarboxylic acids.     

Effects of alveolar hypoxia on the pulmonary circulation and lung mechanics after cromolyn sodium and U-60,257 in lambs.

Because alveolar hypoxia (HYP) triggers pulmonary mast cell degranulation with elaboration of vasoactive mediators such as leukotrienes, we investigated the effects of aerosolized cromolyn sodium (CS), a mast cell stabilizing agent, and U-60,257(U) (a leukotriene blocker) on the circulation, lung mechanics and thromboxane (TXB2) levels in 11 lambs during acute exposure to HYP. Studies were performed in awake, chronically instrumented animals, once after placebo (saline) and again after CS (100 mg; n = 5) or U (90 mg; n = 6). Pulmonary arterial pressure increased 42% during HYP after saline, and 32% and 19% after CS and U, respectively. Pulmonary vascular resistance did not change during HYP after CS or U. Systemic arterial pressure was unchanged after saline and CS but decreased after U; systemic vascular resistance dropped after both CS and U. No changes were seen in tidal volume, lung compliance or airway resistance during HYP after saline or either drug, but minute ventilation increased during HYP in all studies. TXB2 increased during HYP after saline in both studies and was not altered by CS. In contrast, after U, TXB2 decreased. Thus, U more effectively blunted the pulmonary vascular response to HYP than CS and resulted in mild systemic hypotension. The drop in TXB2 after U suggests leukotriene-induced thromboxane synthesis contributes to regulation of pulmonary, and possibly, systemic vasoactivity.     

Identification and Characterization of Cysteinyl-Leukotriene Formation in Tissue Slices from Human Intracranial Tumors: Evidence for Their Biosynthesis Under In Vivo Conditions

In previous studies, it had been shown that human gray and white matter tissue slices have the capacity to synthesize large amounts of cysteinyl-leukotrienes (cysteinyl-LT) in vitro. This study was initiated to investigate cysteinyl-LT formation by tissue slices from intracranial tumors in comparison with cyclooxygenase products such as prostaglandin (PG) F2 alpha and thromboxane (TX) B2. Tissue slices from meningiomas and astrocytomas were found to release large amounts of cysteinyl-LT spontaneously and even higher amounts after ionophore A 23187 stimulation, which could not be accounted for by blood possibly remaining in the tissue slices. Cysteinyl-LT were identified by their immunoreactive characteristics, their biological activity in the guinea pig ileum bioassay, and their retention time on reversed-phase HPLC. With increasing malignancy, astrocytomas were shown to have an increasing biosynthetic capacity for cysteinyl-LT and TXB2 in vitro. In comparison with meningioma patients, malignant astrocytoma patients had an enhanced urinary excretion of LTE4, the major urinary metabolite of cysteinyl-LT in humans, which dropped in level within 7 days after operation by 79%. A correlation exists between the in vivo cysteinyl-LT production in patients with malignant astrocytomas and that observed under basal conditions in the tissue slices in vitro. The results provide evidence that in malignant astrocytoma patients, the tumor tissue produces large amounts of cysteinyl-LT, which may be detected in the patients' urine.     

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.     

Modulation of renal hemodynamics by renal eicosanoids during vasopressor infusions in man

Pressor doses of norepinephrine (NE) (n = 8) and angiotensin II (A II) (n = 5) were infused in normal volunteers to determine whether the systemic administration of vasopressor hormones influence renal eicosanoid production and whether, in turn, the eicosanoids produced could modulate renal hemodynamics and electrolyte excretion. At the doses administered, both pressor substances induced the expected rise in blood pressure, a significant decrease (P less than 0.05) in renal blood flow and a proportionally smaller fall in glomerular filtration rate, resulting in a consistent augmentation in filtration fraction. Fractional sodium excretion was concomitantly reduced. NE infusion produced only slight modifications in urinary prostaglandin (PG)E2, 2,3-dinor-6-keto-PGF1 alpha and thromboxane (TX)B2, while urinary 6-keto-PGF1 alpha and PGF2 alpha were increased by 38% and 176% respectively. The increase in urinary 6-keto-PGF1 alpha (the non-enzymatic degradation product of PGI2, predominantly of cortical origin) was proportional to the level of circulating NE (r = 0.78, P less than 0.05) and to the renal vascular resistance (r = 0.85, P less than 0.01), suggesting an immediate compensatory role for PGI2 in response to the NE-induced pressor stimulus. The renal production of PGE2 and PGF2 alpha (predominantly medullary) was inversely correlated with the filtration fraction: the greater the increase in PGE2 and PGF2 alpha the lower the elevation in filtration fraction or the decline in renal blood flow upon NE administration. All infusion variably stimulated the renal eicosanoid production: PGE2, 41%; PGF2 alpha, 102%; 6-keto-PGF1 alpha, 38%; 2,3-dinor-6-keto-PGF1 alpha, 38%; and TXB2, 25%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiologic role for enhanced renal thromboxane production in murine lupus nephritis.

To investigate the physiologic significance of enhanced renal thromboxane production in murine lupus nephritis, we measured renal hemodynamics and eicosanoid production in MRL-lpr/lpr mice from 8 to 20 weeks of age. Over this age range, MRL-lpr/lpr mice develop an autoimmune disease with nephritis similar to human systemic lupus erythematosus (SLE). In these studies, glomerular filtration rate (GFR) and PAH clearance (CPAH) decreased progressively with age in MRL-lpr/lpr mice, but not in controls. This impairment of renal hemodynamics was associated with increased renal thromboxane production, as well as increased excretion of both thromboxane B2 (TxB2) and 2,3-dinor TxB2 in urine. There was an inverse correlation between renal thromboxane production in MRL-lpr/lpr mice and both GFR and CPAH. Furthermore, there were positive correlations between thromboxane production by the kidney and both the severity of renal histopathology and serum anti-DNA antibody levels measured in individual animals. Enhanced urinary excretion of TxB2 and the development of renal dysfunction also coincided temporally with the appearance of increased levels of interleukin 1 beta (IL-1 beta) mRNA in renal cortex. Acute administration of the specific thromboxane receptor antagonist GR32191 to MRL-lpr/lpr mice restored GFR to normal in early stages of the autoimmune disease. However, in animals with more advanced nephritis, the effect of acute thromboxane receptor blockade on renal hemodynamics was less marked. We conclude that thromboxane A2 is an important mediator of reversible renal hemodynamic impairment in murine lupus, especially in the early phase of disease.