R L Maas

Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man.

To assess the pharmacologic effects of aspirin on endogenous prostacyclin and thromboxane biosynthesis, 2,3-dinor-6-keto PGF1 alpha (PGI-M) and 2,3-dinor-thromboxane B2 (Tx-M) were measured in urine by mass spectrometry during continuing administration of aspirin. To define the relationship of aspirin intake to endogenous prostacyclin biosynthesis, sequential urines were initially collected in individuals prior to, during, and subsequent to administration of aspirin. Despite inter- and intra-individual variations, PGI-M excretion was significantly reduced by aspirin. However, full mass spectral identification confirmed continuing prostacyclin biosynthesis during aspirin therapy. Recovery of prostacyclin biosynthesis was incomplete 5 d after drug administration was discontinued. To relate aspirin intake to indices of thromboxane biosynthesis and platelet function, volunteers received 20 mg aspirin daily followed by 2,600 mg aspirin daily, each dose for 7 d in sequential weeks. Increasing aspirin dosage inhibited Tx-M excretion from 70 to 98% of pretreatment control values; platelet TxB2 formation from 4.9 to 0.5% and further inhibited platelet function. An extended study was performed to relate aspirin intake to both thromboxane and prostacyclin generation over a wide range of doses. Aspirin, in the range of 20 to 325 mg/d, resulted in a dose-dependent decline in both Tx-M and PGI-M excretion. At doses of 325-2,600 mg/d Tx-M excretion ranged from 5 to 3% of control values while PGI-M remained at 37-23% of control. 3 d after the last dose of aspirin (2,600 mg/d) mean Tx-M excretion had returned to 85% of control, whereas mean PGI-M remained at 40% of predosing values. Although the platelet aggregation response (Tmax) to ADP ex vivo was inhibited during administration of the lower doses of aspirin the aggregation response returned to control values during the final two weeks of aspirin administration (1,300 and 2,600 mg aspirin/d) despite continued inhibition of thromboxane biosynthesis. These results suggest that although chronic administration of aspirin results in inhibition of endogenous thromboxane and prostacyclin biosynthesis over a wide dose range, inhibition of thromboxane biosynthesis is more selective at 20 than at 2,600 mg aspirin/d. However, despite this, inhibition of platelet function is not maximal at the lower aspirin dosage. Doses of aspirin in excess of 80 mg/d resulted in substantial inhibition of endogenous prostacyclin biosynthesis. Thus, it is unlikely that any dose of aspirin can maximally inhibit thromboxane generation without also reducing endogenous prostacyclin biosynthesis. These results also indicate that recovery of endogenous prostacyclin biosynthesis is delayed following aspirin administration and that the usual effects of aspirin on platelet function ex vivo may be obscured during chronic aspirin administration in man.

Intravenous Prostacyclin in Thrombotic Thrombocytopenic Purpura

A therapeutic trial of prostacyclin (PGI), was done in a patient with thrombotic thrombocytopenic purpura resistant to treatment with antiplatelet drugs and plasmapheresis. Despite marked thrombocytopenia and continued treatment with aspirin, sulfinpyrazone, and dipyridamole, the urinary excretion of 2,3-dinor-thromboxane B2, a major thromboxane urinary metabolite, was within the normal range (90.3 to 368 pg/mg creatinine) at 96 pg/mg creatinine. Because of its potent antiaggregatory properties and the possibility of a defect in endogenous PGI2 production in thrombotic thrombocytopenic purpura, synthetic PGI2 (4 to 10 ng/kg-1 . min-1) was infused intravenously, first for 72 hours and then continuously for 18 days. Prostacyclin markedly reduced the excretion of 2,3-dinor-thromboxane B2, and the platelet count rose steadily to reach 100 000/mm3 by the eighth day of the second infusion. The patient remains in clinical remission, on no therapy, 7 months later. A controlled evaluation of PGI2 in thrombotic thrombocytopenic purpura is warranted. Apparent therapeutic failure in previous cases may have resulted from inadequate prolongation of PGI2 infusion.

Identification of lipoxygenase products from arachidonic acid metabolism in stimulated murine eosinophils

The presence of arachidonic acid lipoxygenase pathways in murine eosinophils was demonstrated by the isolation and identification of several lipoxygenase products from incubations of these cells. The most abundant arachidonate metabolite from murine eosinophils stimulated with ionophore A23187 and exogenous arachidonic acid was 12-S-hydroxyeicosatetraenoic acid (12-S-HETE), and the next most abundant was 15-HETE. Two families of leukotrienes were also recovered from these incubations. One family comprised the hydrolysis products of leukotriene A4, and the other included products derived from the 14,15-oxido analog of leukotriene A4 (14,15-leukotriene A4). Two double oxygenation products of arachidonate were also identified. These compounds were a 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and a 5,12-dihydroxyeicosatetraenoic acid (5,12-diHETE). Eosinophil stimulation promoter is a murine lymphokine which enhances the migration of eosinophils. When murine eosinophils were incubated with eosinophil stimulation promoter in concentrations sufficient to produce a migration response, a 2-3-fold increase in the production of 12-HETE was observed compared to unstimulated cells. Coupled with the recent demonstration that arachidonic acid lipoxygenase inhibitors suppress the migration response to eosinophil stimulation promoter and that 12-HETE induces a migration response, this observation provides further evidence in support of the hypothesis that eosinophil stimulation promoter stimulation of eosinophils results in the generation of lipoxygenase products which modulate the migratory activity of the cells.

[70] Quantitative assay of urinary 2,3-dinor thromboxane B2 by GCMS

Publisher Summary Quantification of endogenous thromboxane A 2 (TxA 2 ) production in man may provide a reliable index of platelet activation in vivo and have considerable relevance to understanding the role of platelets in these conditions. The quantification of urinary dinor-TxB 2 discussed in this chapter is based on stable isotope dilution and gas chromatography–mass spectrometry (GC-MS) analysis with selected ion monitoring. This method offers the usual advantages of such metabolite assays, including avoidance of artifactual elevations in parent compound levels upon sampling, measurement of a compound that is concentrated in the blood or urine, and high specificity. On the other hand, the method requires (a) preparation of a suitable low blank internal standard; and (b) extensive sample purification to ensure accurate GC-MS analysis, particularly when relatively low endogenous levels of dinor-TxB 2 are being quantified in patients taking inhibitors of the cyclooxygenase enzyme.

A secondary isotope effect in the lipoxygenase reaction

Fatty acids containing a prochiral tritium label have often been used in the study of enzymatic reactions which involve an obligatory step of hydrogen abstraction. In the lipoxygenase reaction, the primary isotope effect associated with this approach is detected as an isotopic enrichment of the substrate. Herein we characterize a previously unrecognized secondary isotope effect which changes the specific activity of both the substrate and product. The 12-lipoxygenase of human platelets removes the 10-LS hydrogen of arachidonic acid in the formation of 12-hydroperoxyeicosatetraenoic acid. We studied the specific activity changes associated with conversion of the enantiomerically labeled [10-DR-3H]arachidonic acid to 12-[10-3H]hydroxyeicosatetraenoic acid in aspirin-treated platelets. [3-14C]Arachidonic acid served as internal standard. The most pronounced change in 3H/14C ratio in the early stages of reaction was a 15-20% deficiency of tritium in the product. Later, the remaining arachidonate showed a marked increase in 3H/14C ratio. The changes in specific activity closely matched those predicted for a secondary isotope effect. Comparison of these data with the theoretical equations for a secondary isotope effect indicated the 10-DR-3H substrate reacted at about 84% of the rate of unlabeled molecules. Interestingly, this secondary isotope effect is similar in magnitude to the secondary isotope effect in autoxidation reactions, a finding compatible with a basic similarity in reaction mechanisms in enzymatic and non-enzymatic oxygenation of lipids.