Structure and quantitative determination of the major urinary metabolite of thromboxane B2 in the guinea pig

2,3-Dinor-thromboxane B₂ was the major urinary metabolite of thromboxane B₂ in the guinea pig. The structure was assessed mainly by mass spectrometric analysis of a number of derivatives of the metabolite and by chemical degradation by oxidative ozonolysis. A method for quantitative determination of 2,3-dinor-thromboxane B₂ in guinea pig urine based on multiple ion analysis and octadeuterated 2,3-dinor-thromboxane B₂ as internal standard was developed. The basal excretion of the metabolite was 65 ± 36 (S.D.) ng/kg × 24 h (n = 19; range 19–140 ng). This level corresponded to an endogenous synthesis of 543 ± 300 ng of TXB₂. No increase in the excretion was seen after anaphylaxis, in contrast to what has earlier been reported for PGF_2α.     

Structure and quantitative determination of the major urinary metabolite of thromboxane B2 in the guinea pig.

2,3-Dinor-thromboxane B2 was the major urinary metabolite of thromboxane B2 in the guinea pig. The structure was assessed mainly by mass spectrometric analysis of a number of derivatives of the metabolite and by chemical degradation by oxidative ozonolysis. A method for quantitative determination of 2,3-dinor-thromboxane B2 in guinea pig urine based on multiple ion analysis and octadeuterated 2,3-dinor-thromboxane B2 as internal standard was developed. The basal excretion of the metabolite was 65 +/- 36 (S.D.) ng/kg x 24 h (n = 19; range 19--140 ng). This level corresponded to an endogenous synthesis of 543 +/- 300 ng of TXB2. No increase in the excretion was seen after anaphylaxis, in contrast to what has earlier been reported for PGF2 alpha.     

Studies on the urinary excretion of thromboxane B2 in Zellweger patients and control subjects: evidence for a major role for peroxisomes in the β-oxidative chain-shortening of thromboxane B2

In this paper we studied the urinary excretion of thromboxane B₂ and its β-oxidation product 2,3-dinor-thromboxane B₂ in urines from control subjects and four Zellweger patients, which lack morphologically distinguishable peroxisomes. In the urine of three classical Zellweger patients we found a ratio of 2,3-dinor-thromboxane B₂/thromboxane B₂ of 0.35, 0.48 and 0.62 respectively, whereas in healthy children and adults values were found of 3.1–10 and 5.5–40 respectively. These data strongly suggest that peroxisomes are a major site for β-oxidation of thromboxane B₂.     

Determination of 6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2α in human urine by gas chromatography—negative ion chemical ionization mass spectrometry

A method for quantification of 6-keto-PGF_1α, 2,3-dinor-6-keto-PGF_1α, TXB₂, 2,3-dinor TXB₂, PGE₂, PGD₂ and PGF_2α in human urine samples, using gas chromatography—negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography—mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF_1α and 55% for tritiated TXB₂. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.     

Urinary excretion of 2,3-dinor-thromboxane B2 in man under normal conditions, following drugs and during some pathological conditions

Quantitation of 2,3-dinor-thromboxane B₂ (2,3-dinor-TxB₂) was performed by gas chromatography - mass spectrometry. Under normal conditions the urinary excretion of 2,3-dinor-TxB₂ was relatively constant in the same individual from day to day but during a 24-hour period a somewhat higher excretion rate was found during the first few hours after awakening. A pronounced reduction of the urinary excretion of 2,3-dinor-TxB₂ was found after oral administration of 500 mg of aspirin or 50 mg of indomethacin, while 500 mg of paracetamol did not affect the urinary excretion. Increased excretion of 2,3-dinor-TxB₂ was found in normal pregnancies and in diseases such as diabetes mellitus and homocysteinuria in comparison to the urnary excretion in normal healthy subjects. We also report one case, where the urinary excretion of 2,3-dinor-TxB₂ was increased for a short period following the first symptoms of a infarction and those data indicate that thromboxane A₂ (TxA₂ may be of pathophysiological importance in human myocardial infarction. The results strongly indicate that measurements of the urinary excretion of 2,3-dinor-TxB₂ should be meaningful as a tool for investigation of the involvement of thromboxane in various pathophysiological processes in man.     

Metabolism of thromboxane B2 in the monkey.

[3H8]Thromboxane B2 was biosynthesized and infused into an unanesthetized monkey. Several urinary metabolites were isolated and their structures elucidated using gas chromatography-mass spectrometry. In addition to the major urinary metabolite, dinor-thromboxane B2, a series of metabolites resulting from dehydrogenetion of the alcohol group at C-11 were identified: 11-dehydro-thromboxane B2, 11-dehydro-15-keto-13,14-dihydro-2,3-dinor-thromboxane B2, and 11-dehydro-15-keto-13,14-dihydro-19-carboxyl-2,3,4,5-tetranor-thromboxane B2. 6-(1,3-dihydroxypropyl)-7-hydroxy-10-oxo-3-pentadecaenoic acid was also identified. Three mono-O-ethylated metabolites were formed from thromboxane B2, which in this study was infused in an ethanolic solution. A small quantity of thromboxane B2 was excreted unchanged into the urine.     

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.     

Enzyme-immunoassay of thromboxane B2 at the picogram level

A highly sensitive and reproducible enzyme-immunoassay for the measurement of thromboxane B₂ was developed. Thromboxane B₂ (T×B₂) was coupled with β-D- galactosidase by mixed anhydride reaction. Thromboxane B₂-antiserum was generated in rabbits and used at a final dilution of 1:480,000. The separation of immuno- complex from the free form of TxB₂ was accomplished by the double antibody method. The second antibody was sheep anti rabbit IgG. The precipitated enzyme activity was measured fluorometrically with 4-methyl-umbelliferyl-gb-D-galactoside as substrate.This method allowed to measure TxB₂ in the range of 0.002 - 5 picomole per tube. The cross-reactivity of the anti-thromboxane B₂-antiserum with 2,3-dinor thromboxane B₂ was about 20%, but it was less than 0.2% for the other prostanoids tested.TxB₂ extracted from human urine was measured by enzyme-immunoassay (y) and radioimmunoassay (x) which has been found closely correlated to values obtained by gas chromatography-mass spectrometry. Regression analysis of the data comparing enzyme-immunoassay and radioimmunoassay gave the equation y = 0.996 x + 0.470, correlation coefficient r = 0.9947. Inter-assay coefficient of variation was 3.1%.The assay was further simplified by coating the second antibody on glass beads. The regression equation between this solid-phase enzyme immunoassay (y) and radioimmunoassay ( (x) was y = 0.9860 × 1.927, r = 0.9895, and enzyme immunoassay (y) was y = 0.9749 × −0.94808, r = 0.9887. Thus, the enzyme-immunoassay shows specificity and sensitivity comparable to radioimmunoassay making use of radioactive tracer unnecessary.     

Synthesis of thromboxane B2 metabolites

This paper reports the synthesis of 11-dehydrothromboxane B₂ methyl ester (II), 15-dehydrothromboxane B₂ methyl ester (III), 15-dehydro-13,14-dihydrothromboxane B₂ (XII) and 2,3-dinorthromboxane B₂ methyl ester (XV). These compounds, as their free acids, have been reported to be thromboxane metabolites.     

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

Human alveolar macrophages, obtained during diagnostic bronchoscoy, were maintained in monolayer culture. Challenge of these cells (>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.D⁴, 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, F_2α and the lysosomal enzyme N-acetyl-β-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 zymosanA, n = 4), and the synthesis of thromboxane B₂ was inhibited by >90% in 1 μM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B₄ synthesis.     

Regulation of urinary thromboxane B2 in man: Influence of urinary flow rate and tubular transport

Thromboxane B₂ (TxB) is excreted in human urine, but the mechanism of renal excretion and the quantitative relationship of urinary TxB to the active parent compound, thromboxane A₂, of renal or extrarenal origin is not established. To determine the effects of vasoactive hormones, uricosuric agents and urinary flow rate on TxB excretion, urinary TxB was measured by radioimmunoassay and mass spectrometry, and renal metabolism of blood TxB was determined by radiochromatography of urine after i.v. [³H]-TxB infusions. Basal TxB was 6.7 ± 1.1 ng/h during an oral water load, and TxB fell with s.q. antidiuretic hormone (to 3.4 ± 0.4 ng/h, P<0.01) and with fluid restriction (to 2.6 ± 0.5 ng/hr, P=0.001) in parallel with urinary volume. Urinary excretion of unmetabolized [³H]-TxB also fell (by 56%) with fluid restriction, implicating altered metabolism rather than synthesis as the mechanism of the urinary flow effect. Angiotensin II infusions slightly reduced both TxB and urine volume, consistent with a flow effect. In contrast, probenecid did not alter urine volume, but increased urinary uric acid (by 244%), TxB (from 5.6 ± 0.9 to 11.1 ± 2.9 ng/h) and urinary excretion of blood [³H]-TxB (by 243%) by similar amounts (all P<0.05), suggesting that TxB is actively reabsorbed in the proximal tubule, similarly to uric acid. Thus, urinary excretion of TxB of renal and extrarenal origin is regulated by proximal and distal tubule factors.     

Urinary excretion of 2,3-dinor-thromboxane B2 in man under normal conditions, following drugs and during some pathological conditions

Quantitation of 2,3-dinor-thromboxane B2 (2,3-dinor-TxB2) was performed by gas chromatography-mass spectrometry. Under normal conditions the urinary excretion of 2,3-dinor-TxB2 was relatively constant in the same individual from day to day but during a 24-hour period a somewhat higher excretion rate was found during the first few hours after awakening. A pronounced reduction of the urinary excretion of 2,3-dinor-TxB2 was found after oral administration of 500 mg of aspirin or 50 mg of indomethacin, while 500 mg of paracetamol did not affect the urinary excretion. Increased excretion of 2,3-dinor-TxB2 was found in normal pregnancies and in diseases such as diabetes mellitus and homocysteinuria in comparison to the urinary excretion in normal healthy subjects. We also report one case, where the urinary excretion of 2,3-dinor-TxB2 was increased for a short period following the first symptoms of a myocardial infarction and those data indicate that thromboxane A2 (TxA2) may be of pathophysiological importance in human myocardial infarction. The results strongly indicate that measurements of the urinary excretion of 2,3-dinor-TxB2 should be meaningful as a tool for investigation of the involvement of thromboxane in various pathophysiological processes in vivo in man.     

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.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

Thromboxane B2 transformed from arachidonic acid in carrageenin-induced granuloma

A main product (PI) transformed from arachidonic acid in carrageenin-induced granuloma in rats was structurally analysed. PI was converted to a more polar substance by the reduction with sodium borohydride. On the basis of the data on gas chromatography-mass spectrometry, its structure was identified with the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9,12-heptadecadienoic acid (Thromboxane B2)     

An increase in the ratio of thromboxane A2 to prostacyclin in association with increased blood pressure in patients on cyclosporine A

The aim of this study was to determine the effect of two years of treatment with cyclosporine A on blood pressure and the rates of secretion into the circulation of the vasoconstrictor thromboxane A2 and the vasodilator prostacyclin. Seven patient suffering from multiple sclerosis took part. Their blood pressures and urinary concentrations of 2,3-dinor-thromboxane A2 (a major urinary metabolite of thromboxane A2) and of 2,3-dinor-6-keto-prostaglandin F1 alpha (the major urinary metabolite of prostacyclin) were determined at the end of two years of treatment with cyclosporine A, and once again three months after cessation of this treatment. No other drugs were given during or after cyclosporine A. Mean arterial blood pressure was 113 +/- 5 mmHg (mean +/- SEM) during the cyclosporine A treatment, but fell to 94 +/- 4 mmHg after the three-month's wash-out period. Urinary excretion of the thromboxane metabolite decreased slightly from 674 +/- 150 pg.mg-1 creatinine during cyclosporine A therapy to 503 +/- 90 pg.mg-1-creatinine after the end of therapy. At the same time the prostacyclin metabolite increased significantly from 82 +/- 17 pg.mg-1 creatinine to 113 +/- 23 pg.mg-1 creatinine (P less than 0.05). The ratio of 2,3-dinor-thromboxane B2 to 2,3-dinor-6-keto-prostaglandin F1 alpha (taken as a measure of vasoconstrictor prostanoid activity) fell significantly from 8.4 +/- 0.8 4.7 +/- 0.6 (P less than 0.005). The shift in prostanoid production observed during cyclosporine A treatment could be one causal factor for the hypertensive and thromboembolic events associated with the use of this drug.     

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.     

Biosynthesis of thromboxane B2: Assay, isolation, and properties of the enzyme system in human platelets

The microsomal fraction of human platelets catalyzed the conversion of arachidonic acid to an unstable platelet-aggregating factor and a hydrolyzed product on thin-layer chromatography (TLC). This product was isolated on TLC, purified by silica gel column chromatography and identified by combined gas chromatography-mass spectrometry as the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9, 12L-dihydroxy-5, 10-heptadecatrienoic acid (thromboxane B₂). The enzymatic activity was dependent upon methemoglobin and tryptophan as cofactors. Reduced glutathione had no effect either alone or in combination with other cofactors. Methemoglobin could be replaced by hematin or hemin; and tryptophan by 3-indolacetic acid or catecholamines. The apparent requirement for methemoglobin is due to the reductive activity of ferriprotoporphyrin IX. The reaction, however, catalyzed by the ferriprotoporphyrin IX in the thromboxane synthesizing system is different from that described for the decomposition of lipid peroxides. Certain transition metals and hydrogen donors, such as hydroquinone and ascorbate, which have been shown to stimulate the catalytic activity of ferriprotoporphyrin IX in the decomposition of 15-hydroperoxy-prostaglandin E₁ are inhibitors of thromboxane B₂ formation. This enzyme preparation also transformed eicosa-8, 11, 14-trienoic acid to an unknown product on TLC. The enzyme system was rapidly inactivated upon incubation in the reaction mixture.     

Analytical methods for thromboxane B2 measurement and validation of radioimmunoassay by gas liquid chromatography-mass spectrometry

A sensitive and specific radioimmunoassay using a ¹²⁵I tracer of high specific radioactivity was developed for thromboxane B₂ and was applied to the determination of the biosynthesis of this compound in some systems (i.e. human washed platelets, human platelet rich plasma (PRP) and rat spleen homogenates). The assays were also evaluated by mass spectrometry; levels measured by these two analytical methods were very similar. the results obtained for washed human platelets with a thin layer radiochromatographic method were in good agreement with the two preceeding methods.     

Effect of antiestrogen regimen on prostacyclin and thromboxane A2 in postmenopausal patients with breast cancer: Evidence of significance of hypertension, smoking or previous use of estrogen therapy

To explore the mechanism(s) by which antiestrogens may protect against the development of cardiovascular disorders, we measured the production of vasodilatory, antiaggregatory prostacyclin (PGI₂ and that of vasoconstrictive, proaggregatory thromboxane A₂ (TxA₂) before and after 6 months' use of antiestrogens in postmenopausal patients after operation for stage II breast cancer (n = 38). Urine samples were assayed by high performance liquid chromatography and radioimmunoassays for 2,3-dinor-6-ketoprostaglandin F1α (=metabolite of PGI₂, dinor-6-keto) and for 2,3-dinor-thromboxane B₂ (=metabolite of TxA₂, dinor-TxB₂). In addition, in 35 of these 38 patients we assayed the capacity of platelets to produce thromboxane A2 during standardized blood clotting. The 4 patients using low-dose aspirin had low thromboxane production, and were excluded from further analysis of the data. An antiestrogen regimen consisting either of tamoxifen (n = 15) or of toremifene (n = 19) caused no changes in production of PGI₂ or TxA₂, or in their ratio, and in this regard, these antiestrogens behaved similarly. Hypertensive patients (n = 7) using different antihypertensive agents were characterized by reduced urinary out-put of dinor-6-keto (18.5 ± 6.1 vs 35.5 ± 18.5 ng/mmol, mean ± SD, p < 0.05) and reduced platelet capacity to produce TxA₂ (62.6 ± 67.8 vs 134.6 ± 75.6 ng/mL, p < 0.05). The patients (n = 15) who had used estrogen replacement therapy (ERT) up until diagnosis of breast cancer showed reduced dinor-TxB₂ excretion (15.5 ± 12.7 vs 29.9 ± 20.9 ng/mmol, p < 0.05) before initiation of antiestrogens, and elevated dinor-6-keto output during the antiestrogen regimen (32.4 ± 21.2 vs 22.7 ± 8.7 ng/mmol, p = 0.07). Smokers (n = 6) had elevated dinor-TxB2 output before and during antiestrogen use. Thus we conclude that the cardiovascular protection provided by an antiestrogen regimen is unlikely to be mediated through vaso- and platelet active PGI₂ and TxA₂.