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.     

Identification of the major urinary metabolite of thromboxane B2 in the monkey

Thromboxane B₂ (TxB₂) was biosynthesized from prostaglandin endoperoxides (PGG₂, PGH₂) using guinea pig lung microsomes and infused into an unanesthetized monkey. Urine was collected and TxB₂ metabolites were isolated by reversed phase partition chromatography and high performance liquid chromatography. A major metabolite (TxB₂-M) was found to be excreted in greater than two-fold abundance relative to other metabolites. Its structure was determined by gas chromatography-mass spectrometry to be dinorthromboxane B₂. $\text{In vitro}$ incubation of TxB₂ with rat liver mitochondria yielded a C₁₈ derivative with a mass spectrum identical to that of TxB₂-M, substantiating that the major urinary metabolite of TxB₂ in the monkey is a product of a single step of β-oxidation.     

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, 7, 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 maybe 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.     

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.     

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.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

Metabolism of thromboxane B2 in the cynomolgus monkey

[5,6,8,9,11,12,14,15-³H₈]-Thromboxane B₂ was injected into the saphenous vein of female cynomolgus monkeys, and blood samples were withdrawn from the contralateral saphenous vein. The compound was eliminated from the circulation with a half-life of about 10 min after an initial rapid disappearnace. Some more polar products appeared with time, and also small amounts of material less polar than thromboxane B₂; however, the dominating compound in all blood samples was unconverted thromboxane B₂.About 45% of the given dose of tritium was excreted into urine in 48 hrs. Several metabolites of thromboxane B₂ were found. The major urinary metabolites was identified as dinorthromboxane B₂ (about 32% of urinary radioactivity). Unconverted thromboxane B₂ was also found in considerable amounts (13% of urinary radioactivity).It is concluded that 1) dehydrogenation at C-12 is not a major pathway in the degradation of this compound, in contrast to metabolism at the corresponding C-15 alcohol group of prostaglandins; 2) after having gained access to the circulation, thromboxane B₂ is the main circulating compound; however, assay of thromboxane B₂ in plasma will be complicated or precluded by large artifactual production of the compound by platelets during sample collection.     

Metabolism of leukotriene B4 by guinea pig eosinophils

The metabolism of leukotriene B₄ (5(S),12(R)-dihydroxy-6-cis-8,10-trans-14-cis-eicosatetraenoic acid) by isolated guinea pig eosinophils was investigated. Incubation of guinea pig eosinophils with [³H]-leukotriene B₄ resulted in the rapid conversion of leukotriene B₄ to several more polar metabolites. Two of these metabolites were identified by ultraviolet spectroscopy and gas chromatography-mass spectrometry as the omega oxidation products 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid (20-hydroxy-leukotriene B₄) and 5(S),12(R),19-trihydroxy-6,8,10,14-eicosatetraenoic acid (19-hydroxy-leukotriene B₄). Two novel metabolites, 5(S),12(R),18,19-tetrahydroxy-6,8,10,14 eicosatetraenoic acid (18,19-dihydroxy-leukotriene B₄) and 5(S),12(R)-dihydroxy-1,18-dicarboxylic-6,8,10,14,16-octadecapentaenoic acid (Δ16,17–18-carboxy-19,20-dinor-leukotriene B₄) were tentatively identified. The identification of these compounds indicates that guinea pig eosinophils are capable of metabolizing leukotriene B₄ by both omega and beta oxidation. This catabolic activity may play a role in modulating inflammatory reactions by removing the chemoattractant leukotriene B₄ from inflammatory sites.     

Endogenous formation of the prostaglandin endoperoxine metabolite,thromboxane B2, by brain tissue

Thromboxane B₂ was formed from endogenous precursors during short incubations of guinea pig and rat cerebral cortex. The amount formed by guinea pig brain tissue was 5–6 times the formation of prostaglandin F_2α and E₂. Noradrenalin stimulated and indomethacin and mercaptoethanol inhibited thromboxane B₂ formation. The mass spectrum of the brain compound was identical to thromboxane B₂ formed from arachidonic acid by guinea pig lung and human platelets.     

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.     

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.     

Measurement of urinary 2,3-dinor-thromboxane B2 and thromboxane B2 using bonded-phase phenylboronic acid columns and capillary gas chromatography-negative-ion chemical ionization mass spectrometry

The use of bonded-phase phenylboronic acid columns to selectively extract 2,3-dinor-thromboxane B2 and thromboxane B2 from urine is reported. The compounds were first derivatized as the methoxime and then applied to the phenylboronic acid columns. Subsequent purification by thin-layer chromatography and derivatization to the pentafluorobenzyl ester, trimethylsilyl ether followed by capillary gas chromatography-negative-ion chemical ionization mass spectrometry, monitoring specific ions, allows quantitation in the low-picogram/milliliter range. In healthy male volunteers, the median excretions of 2,3-dinor-thromboxane B2 and thromboxane B2 were 10.3 ng/h (range, 4.5-24 ng/h) and 2.8 ng/h (range, 0.5-7.3 ng/h), respectively. The method offers a noninvasive, specific approach to the study of thromboxane synthesis and platelet function in man. It is much less labor intensive than currently available methods employing electron-impact chromatography-mass spectrometry.     

Electron capture gas chromatographic detection of thromboxane B2

An electron capture gas chromatographic method is described for the detection of thromboxane B₂. Thromboxane B₂ is esterified with diazomethane, followed by treatment with pentafluorobenzylhydroxylamine hydrochloride and silylation with BSA. In pyridine, the free aldehyde form of the acetal ring is favored allowing rapid formation of a novel thromboxane B₂ pentafluorobenzyloxime. The method has been applied to detect thromboxane B₂ formation during aggregation of washed platelets. It must be emphasized that by ordinary analytical standards, the derivatization reproducibility from 50–375 nanograms is poor (±11% – ±42%); however, the improved selectivity of the method and its ability to detect nanogram levels of thromboxane B₂ make it a useful complement to commonly employed bioassay techniques.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B₂ (TXB₂) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 μg/g tissue) to both PGE₂ (10.7 μg/g tissue) and TXB₂ (6.2 μ/g tissue). Smaller amounts of PGF_2α (0.9 μ/g) and 6-oxoPGF_1α were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE₂, but formed very little TXB₂, PGF_2α or 6-oxoPGF_1α. Homogenates from rabbit lungs converted arachidonic acid (100 μg/g) mainly to PGE₂, both before and after birth. The amount of PGE₂ formed increased during gestation to a maximum of about 6 μg/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 μg/g) which was observed 8 days after birth, followed by an increase to about 4 μg/g in older rabbits.     

A method for measuring the unstable thromboxane A2: Radioimmunoassay of the derived mono-o-methyl-thromboxane B2

A radioimmunoassay was developed for a mono-O-methyl derivative of thromboxane B₂. The antibodies showed high specificity for this compound and cross reacted only 1.2% with thromboxane B₂ and less than 0.1% with prostaglandins and prostaglandin metabolites. The method had a sensitivity of 7 picog. The radioimmunoassay was employed in studies where thromboxane A₂ was generated in human platelets and immediately converted into mono-O-methyl thromboxane B₂ by treatment of the sample with a large volume of methanol. In some of the experiments, thromboxane B₂ was simultaneously measured by a separate radioimmunoassay. Using these two assays it was demonstrated that thromboxane A₂ could be detected only during the earlier stages of the platelet aggregation, whereas thromboxane B₂ rapidly reached a constant level. In a separate experiment, the half-life of thromboxane A₂ in buffer was found to be 32.5±2.5 (S.D.) sec at 37°C; the compound was more stable at lower temperatures. The for thromboxane A₂ was also considerably longer in plasma.     

Measurement of thromboxane B2 in human plasma or serum by radioimmunoassay

A radioimmunoassay for thromboxane B₂ (TxB₂), a stable metabolite of thromboxane A₂, is described. The method consists of extraction of TxB₂ into ethyl acetate from acidified plasma or serum samples and saturation analysis using specific antibodies produced in rabbits against TxB₂-BSA conjugate. The 50 % displacement level of the standard curve was 19.1 ± 2.9 pg/tube (mean ± S.D., n = 19). The method blank was 3.4 ± 3.1 pg/ml (n = 15) and the assay sensitivity thus 9.6 pg/ml (mean blank + 2 S.D.). When 100 to 200 pg of TxB₂ were added to plasma, 96.2–103.6 % were recovered. The intra-assay coefficient of variation varied from 6.7 to 9.7 %, and the inter-assay coefficient of variation was 18.6 % (n = 10). The TxB₂ concentration in the plasma of 14 healthy subjects varied from 29.3 to 120.8 pg/ml with a mean ± S.D. of 70.1 ± 26.1 pg/ml, when the blood was collected into tubes containing acetylsalicylic acid (ASA), whereas significantly higher (p < 0.001) TxB₂ concentrations of 68.3 – 285.3 pg/ml with a mean ± S.D. of 151.8 ± 66.6 pg/ml were obtained from the same subjects in the plasma of blood which was collected into tubes containing no ASA. When blood samples from 10 subjects were allowed to clot at 0, +24 or +37°C for 60 min., the TxB₂ concentrations in the sera were 2053 ± 870 pg/ml, 4001 ± 1370 pg/ml and 178557 ± 54000 pg/ml, respectively. The TxB₂ levels in sera which were separated from blood samples incubated at +37°C, correlated significantly (p < 0.001) with the TxB₂ productions in platelet-rich-plasma (PRP) after an induced aggregation. Our results indicate 1) when TxB₂ is measured in plasma, the use of prostaglandin synthesis inhibitor in the collection tubes is necessary and 2) the measurement of TxB₂ in serum of blood which has been kept at +37°C for a strictly standardized period of time could replace the use of PRP in TxB₂ studies.     

Identification of the major urinary metabolite of thromboxane B2 in the monkey.

Thromboxane B2 (TxB2) was biosynthesized from prostaglandin endoperoxides (PGG2, PGH2) using guinea pig lung microsomes and infused into an unanesthetized monkey. Urine was collected and TxB2 metabolites were isolated by reversed phase partition chromatography and high performance liquid chromatography. A major metabolite (TxB2-M) was found to be excreted in greater than two-fold abundance relative to other metabolites. Its structure was determined by gas chromatography-mass spectrometry to be dinor-thromboxane B2. In vitro incubation of TxB2 with rat liver mitochondria yielded a C18 derivative with a mass spectrum identical to that of TxB2-M, substantiating that the major urinary metabolite of TxB2 in the monkey is a product of a single step of beta-oxidation.     

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.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.