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 (TxB2) was coupled with beta-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 immunocomplex 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-beta-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 X 1.927, r = 0.9895, and enzyme immunoassay (y) was y = 0.9749 X -0.94808, r = 0.9887. Thus, the enzyme-immunoassay shows specificity and sensitivity comparable to radioimmunoassay making use of radioactive tracer unnecessary.     

Enzyme immunoassay of thromboxane B2

An immunoassay for thromboxane B2 was developed in which the hapten molecule was labeled with beta-galactosidase. The immunoprecipitate formed after competition between enzyme-labeled and unlabeled thromboxane B2 was subjected to a fluorometric assay of beta-galactosidase. Thromboxane B2 was detectable in the range of 0.1-30 pmol. Both enzyme immunoassay and radioimmunoassay showed essentially the same cross-reactivities with other prostaglandins and their metabolites when the same antibody was used. Known amounts of thromboxane B2 were added to human plasma, and the sample was applied to an octadecyl silica column. The extract was analyzed by enzyme immunoassay to examine the correlation between the added (x) and measured (y) thromboxane B2 (y = 1.09x + 11.07 pmol/ml, r = 0.99). A satisfactory correlation was observed between radioimmunoassay (x) and enzyme immunoassay (y) (y = 0.92x + 4.64 pmol/ml, r = 0.96). The validity of enzyme immunoassay was also confirmed by gas chromatography-mass spectrometry of a dimethylisopropylsilyl ether derivative of thromboxane B2 methyl ester. The method was applicable to the assay of thromboxane B2 produced from endogenous precursor during thrombin-induced aggregation of human platelets.     

In vitro enhanced throboxane B2 release by polymorhonuclear leukocytes and macrophages after treatment with human recombinant interleukin 1

Macrophages (MØ) and polymorphonuclear leukocytes (PMNs) are cell types that interact with bacterial endotoxin and play key roles in mediating the inflammatory reaction. We examined the ability of rat peritoneal macrophages and human PMNs to synthesize thromboxane A₂ (detected as TxB₂) in response to human recombinant interleukin 1 (hrIL1). TxB₂ levels were measured by radioimmunoassay in the cell-free supernatant of cell suspensions after 1 hr incubation at 37°C. TxB₂ release by both PMNs and MØs was increased in a dose-dependent manner by hrIL1.     

TxA2 production by human arteries and veins

Human arterial and venous segments from patients under-going operations when incubated in Tris buffer both alone and with arachidonic acid were able to produce thromboxane B₂ (assessed by radioimmunoassay). Thromoboxane B₂ (TxB₂) production was progressive in time (till 40 min.) and was enhanced by the addition of 1mM norepinephrien. Contamination of tissues by platelet was checked and platelets did not contribute to thromboxane formation. The investigation of the conversions of 1-¹⁴C arachidonic acid by vascular tissue indicated that human vascular tissues produce the metabolites of the cyclooxygenase dependent pathway and that prostacyclin is the main metabolite with a PGI₂/TxA₂ ratio of 4:1. The arterial wall was found to posses an active lipoxygenase dependent pathway. Thromboxane production by intimal cells was neglible and the main source of thromboxane was the media. The production of thromboxane did not change in relation to age, but arterial segments from men produced significantly larger amounts of thromboxane than those from women.     

Comparison of substrate specificities of the human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases

A study of the relative activity of the purified placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases with various prostaglandins and thromboxane B₂(TxB₂) suggests that most, if not all, oxidation in the placenta of the 15-hydroxyl group of prostaglandins of the A, E, and F series as well as PGI₂ (prostacyclin) and 6-keto PGF_1α is catalyzed by the NAD-linked enzyme. Prostaglandin B₁ is an excellent substrate for the NADP-linked enzyme. Despite the conformational similarities between PGB₁ and PGI₂, the latter molecule is a poor substrate for the NADP-linked enzyme. Thromboxane B₂ is not oxidized by the NAD-linked enzyme and is oxidized slowly by the NADP-linked enzyme.     

Chemotactic activity of thromboxane B2, prostaglandins and their metabolites for polymorphonuclear leucocytes

(1) The chemotactic activities of thromboxane B₂ (TxB₂, PGE₂, PGF_2α, the 15-oxo, 15-oxo-13,14-dihydro and 13,14-dihydro metabolites of PGE₂, PGF_2α, and a metabolite of TxB₂ for polymorphonuclear leucocytes (PMN) have been investigated.(2) Thromboxane B₂ increased the directional migrationm of rat peritoneal PMN at a concentration of 2.0 μg/ml and of human peripheral neutrophils at a concentration of 0.5 μg/ml.(3) Neither PGE₂, PGF_2α nor their metabolites showed chemotactic activity for rat peritoneal PMN.(4) PGF_2α and 15-oxo-13,14-dihydro-thromboxane B₂ showed no chemotactic activity for human peripheral PMN.(5) The possible role of thromboxane B₂ in inflammation is discussed.     

Inhibition of platelet thromboxane synthetase by L-1-tosylamido-2-phenylethyl chloromethyl ketone

L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) was found to inhibit several aspects of arachidonic acid (20:4) metabolism in human platelets; the primary effect being inhibition of thromboxane synthetase. Thromboxane B₂ (TxB₂) formation from exogenous 20:4 or PGH₂, or from endogenous 20:4, was inhibited by TPCK at concentrations between 0.1 and 0.5 mM. Formation of malondialdehyde (MDA) and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), products which also arise from PGH₂, was inhibited to a similar extent. Inhibition of formation from 20:4 of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), the product of the lipoxygenase pathway, was observed; although the extent of this inhibition was less than that of TxB₂ formation. A small inhibitory effect of TPCK on the release of 20:4 from platelet phospholipids was also observed. This evidence indicated that while a number of reactions are inhibited by TPCK, the primary effect appears to be inhibition of thromboxane synthetase.     

Prostacyclin and thromboxane synthesis by endometrial cancer and leiomyomas

To study the role of prostacyclin (PGI₂) and thromboxane A₂ (TxA₂) in uterine tumors, pieces of endometrial cancer (n=12) and leiomyomas (n=12)_were incubated in vitro, and the productions of 6-keto-prostaglandin F_1a (6-keto-PGF_1a, a hydration product of PGI₂) and thromboxane B₂ (TxB₂, a hydration product of TxA₂), measured by radioimmunoassay, were compared to those of corresponding healthy tissues. The production of 6-keto-PGF_1a by endometrial cancer (20.8; 1.5–85 ng/mg protein/min, median and interquartile range), by healthy endometrium (25.5; 10.0–55.0), by healthy myometrium (34.9; 25.0–59.9) and by leiomyoma (20.3; 10.2–45.1) was similar. The production of TxB₂ was increased by endometrial cancer (55.5; 10.5–155.2, p < 0.02) in comparison with endometrium (9.8; 4.3–35.1), myometrium (3.8; 2.1–8.0) and leiomyoma (1.9; 1.0–3.8). The 6-keto-PGF_1a/TxB₂ ratio in endometrial cancer (0.9; 0.3–1.5) was smaller (p < 0.02) than that in healthy endometrium (3.3; 1.9–4.8). Thus, TxA₂ may be a factor in endometrial cancer.     

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 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.     

Some interrelationships between glucose levels thromboxane production and prostacyclin production in normal and diabetic mice

We investigated the relationship between glucose levels and platelet thromboxane production or aortic prostacyclin production, using radioimmunoassay (RIA) to measure thromboxane B₂ and 6-keto-PGF_2α. We found a direct relationship (p<.05) between plasma glucose levels and thromboxane A₂ production by arachidonate stimulated platelets in platelet rich plasma of normal mice. However, when mice were deprived of food overnight, the glucose level fell but the TxB₂ production rose significantly. Moreover, mice with streptozotocin diabetes had significantly elevated glucose levels. but normal TxB₂ production, which also rose significantly after fasting. Thus in our laboratory both fasting and diabetes nullify or reverse the direct relationship between glucose levels and TxB₂ production seen in normal fed mice. This makes it diffisult to ascribe the correlation between glucose and TxB₂ levels in normal fed animals to cause and effect. RIA revealed an inverse correlation between glucose levels and 6-keto-PGF_1α production which was highly significant in aortas taken from fasted mice and stimulated for 10 minutes with 0.1mM arachidonate. This inverse correlation was present with either normal or diabetic aortas. Moreover, fasting increased the production of 6-keto-PGF_1α. However there was a significant elevation of 6-keto production by aortas of mice with diabetes of 5–6 weeks duration, compared to aortas of normal mice. Therefore either diabetes in these mice reversed a normal inhibitory effect of glucose on 6-keto production, or else the inverse correlation between glucose levels and 6-keto production does not represent a cause and effect relationship between the two variables.     

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.     

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.     

Biosynthesis of thromboxanes in human platelets. I. Characterization and assay of thromboxane systhetase

An enzyme system which catalyzes the rapid conversion of prostaglandin endoperoxide to thromboxane B₂ was found in the microsomal fraction of human platelet homogenate. The products of the reaction were identified by gas chromatography-mass spectrometry as thromboxane B₂ and the C-17 hydroxy fatty acid HHT. A simple radiometric TLC method was developed for the determination of the enzyme activity. Various parameters affecting the enzyme activity have been defined. Thromboxane synthetase was strongly inhibited by its substrate analogs. The activity was completely abolished when low amounts (5 × 10^?5$\text{M}$) of the 9,11 (epoxymethano) prostanoic acid was included in the assay mixture. The enzyme reaction was not affected by nonsteroidal antiinflammatory agents.     

Effect of acute ethanol intake on thromboxane and prostacyclin in human

To investigate the effects of acute ethanol administration on the production of proaggregatory thromboxane A₂ (TxA₂) and anti-aggregatory prostacyclin (PGI₂), ethanol (1.5 g/kilogram body weight) was given to eight healthy nonsmoking men, and the stable metabolites thromboxane B₂ (TxB₂) and 6-keto-prostaglandin F_1α (6-keto-PGF_1α), respectively, measured by radioimmunoassay from serial blood samples before drinking and during the ensuing 18 hours. Each subject was studied as his own control on another occasion when only an equivalent volume of water was given. Serum TxB₂ level decreased (p < 0.01) from 206 + 31 ng/ml (mean) ± S.E. to 167² ± 24 and 161 ± 23 ng/ml (two and four hours after beginning of the drinking, respectively) concomitantly with the attainment of maximal blood ethanol concentrations (about 120 mg/100 ml), whereas no changes occurred in plasma 6-keto-PGF_1α concentrations. Our results may provide an explanation for known effects of ethanol on platelet aggregation. They also raise speculation whether TxA₂-inhibition and the antiatherogenic effect of alcohol intake are somehow related.     

OKY-1581: A selective inhibitor of thromboxane synthesis and

OKY-1581 is an effective inhibitor of thromboxane synthesis and . The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either or caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

A comparative study of thromboxane and prostacyclin release from ex vivo cultured bovine vascular endothelium

Thromboxane B₂, 6-keto-Prostaglandin F_1α, and Prostaglandin E₂ release have been quantitated from cultured adult by bovine endothelial cell monolayers and from $\text{ex Vivo}$ vascular segments employing specific radioimmunoassay and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF_1α released was diminished compared to amounts released from the vessel segments while thromboxane B₂ and prostaglandin E₂ release were similar in the two endothelial model systems. However, the amount of thromboxane B₂ assayed was small and the quantity of thromboxane A₂ it represents is probably of little $\text{in Vivo}$ significance to prostacyclin.     

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.     

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D₂ (PGD₂) was developed in which PGD₂ was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labelled and free PGD₂, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)- propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3 – 100 pg for PGD₂. The IC₅₀ value for PGD₂ in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enyzme immunoassay was applied to the measurement of PGD₂ content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD₂. PGD₂ contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD₂. The level of PGD₂ in rat brain was extremely low but determined to be 0.11 ± 0.03 ng/g tissue (mean ± S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.     

Identification of prostaglandin D2 as a major prostaglandin in homogenates of rat brain

Prostaglandin (PG) E₂, D₂, F_2α and thromboxane B₂ (TxB₂) were determined in homogenates of rat brain by gas-chromatography - mass spectrometry. The level of PGD₂ was 735 ± 19 ng/g, of PGF_2α 150 ± 13 ng/g, of TxB₂ 112 ng/g and of PGE₂ 86 ± 8 ng/g. The same relative proportions of cyclo-oxygenase products were found in incubates of unstimulated sliced rat brain. ¹⁴C-PGH₂ was converted in high yield into PGD₂ by enzyme(s)_present in the soluble fraction of the homogenate. These results indicate that PGD₂ is the major cyclooxygenase product in the central nervous system of the rat.