Joseph R. Bianchine

The production of the arachidonate metabolite HETE in vascular tissue.

Blood platelets contain two enzyme systems which oxygenate arachidonic acid (AA). First, a cyclooxygenase1 which metabolises AA into the biologically active prostaglandin endoperoxides (PGG2 and PGH2) which are precursors for the platelet prostaglandins PGE2, PGF2α and thromboxane A2 (TxA2). And second, a lipoxygenase that metabolises AA into 12-L-hydroperoxy-5, 8, 10, 14-eicosatetraenoic acid (HPETE)1,2. HPETE is a labile intermediate which is nonenzymatically reduced in aqueous environments to a more stable metabolite, 12-L-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (HETE). HPETE is produced in significant quantities by aggregated platelets, but its exact biological role is not known. Turner et al.3 has suggested that HETE may be a mediator of neutrophil chemotaxis. Vane and coworkers demonstrated that 15-hydroperoxy-5, 8, 10, 14-eicosatetraenoic acid4,5 as well as HPETE were potent inhibitors of prostacyclin (PGI2), the major arachidonate metabolite in vascular tissue. These data suggest that HPETE, if present in vascular tissue, might act as endogenous regulator of PGI2 production. Using radiochemical techniques and mass sspectrometry we have now identified a lipoxygenase enzyme in vascular tissue (rabbit aorta) in which biosynthesis of HETE also occurred. The detection of HETE in this suggests a potential role of the lipoxygenated product as an endogenous regulator of PGI2 biosynthesis.

Formation of antibodies to prostaglandins in the yolk of chicken eggs

Abstract The critical step in the development of a radioimmunoassay procedure is the production of a specific, sensitive antibody. Such antibodies are usually obtained from the serum of immunized animals. A major disadvantage of this technique is that a limited amount of antibody can be obtained at any one time from an animal which can be practically maintained in a small vivarium. In addition, the acquisition of the antibody requires the use of an invasive technique which may be difficult for both the experimental animal and the investigator. We report here on a technique which takes advantage of the fact that the antibodies produced by immunized hens is secreted into the yolk of their eggs. Using this procedure, we have produced antibodies to several prostaglandins and used these antibodies to develop radioimmunoassay procedures. Our results indicate that antibody directed against a hapten can be obtained in a relatively inexpensive animal model system without the use of an invasive technique.

The Relationship Between the Stimulation of Dopamine Synthesis and Release Produced by Amphetamine and High Potassium in Striatal Slices

The effects of amphetamine (amph) and high K+ on the synthesis and release of dopamine (DA) were compared in striatal slices. Both agents stimulated DA synthesis as well as release. For both agents, Ca2+ was required for the initiation of synthesis stimulation as well as for the maintenance of this stimulation. The addition of EGTA to medium containing slices that were already stimulated by 1.0μm‐amph or 55 mm‐K+ markedly reduced the stimulation of DA synthesis. Although it has been reported that high K+ activates soluble tyrosine hydroxylase (TH), neither high K1 nor amph appeared to increase the affinity of the synthetic cofactor, 6‐MPH4, or decrease the affinity of the catechol, DA, for TH. This finding was supported by the observation that the inhibitory effect of L‐DOPA on DA synthesis in slices, in which synthesis was stimulated by either agent, was not decreased. Although both 1.0μM‐amph and 55 mm‐K® stimulated the release of [3H]DA from striatal slices, the release produced by K+ was Ca2+‐dependent, whereas release produced by amph did not occur at any concentration tested. Studies on pH requirements for both synthesis and release also confirmed a similarity between amph and K1 in stimulating synthesis but not in stimulating release. These results suggest that depolarizing agents, such as high K1, couple synthesis and release of DA by a Ca2+‐dependent mechanism. In contrast, the simultaneous stimulation of synthesis and release by amph is not regulated by Ca2+.

Improved method for the determination of aspirin and its metabolites in biological fluids by high-performance liquid chromatography: applications to human and animal studies

An improved method has been developed for the determination of acetylsalicylic acid, salicylic acid, gentisic acid, and salicyluric acid in plasma and urine of rabbits and man. Samples are extracted with dichloromethane containing mephenytoin as an internal standard, the solvent is evaporated under reduced pressure, the residue reconstituted and analyzed by high-performance liquid chromatography. Extraction efficiencies, linearity and assay precision were determined. This method has been applied to human bioavailability studies and the data are presented.

A study of three vasodilating agents as selective inhibitors of thromboxane A2 biosynthesis

Abstract Three clinically efficacious vasodilatory drugs were found to be selective inhibitors of thromboxane A 2 biosynthesis. Hydralazine, dipyridamole, and diazoxide inhibited platelet aggregation at 1 × 10 −4 M, 1.75 × 10 −4 M, and 2 × 10 −3 M respectively. Their relative inhibitory potencies on thromboxane B 2 production in human platelet microsomes were examined and found to be similar to that observed for their inhibition on human platelet aggregation. At 10 −3 M, hydralazine, dipyridamole, and diazoxide inhibited thromboxane B 2 formation by 65 percent, 27 percent and 18 percent respectively. These compounds were examined in the sheep vesicular gland system, and they were shown not to be inhibitors of the cyclooxygenase enzyme. Thus, the inhibition of thromboxane A 2 biosynthesis by these three drugs in human platelet microsomes appeared to be specific at the thromboxane synthetase level.

Identification of long chain dicarboxylic acids in the serum of two patients with Reye's syndrome

Sera from two patients with Reyes Syndrome were analysed by computerized capillary gas chromatography--mass spectrometry profiling techniques. The most striking abnormalities were the accumulation of long chain dicarboxylic acids. Four saturated dicarboxylic acids (dodecanedioic, tetradecanedioic, hexadecanedioic, and octadecanedioic), and six unsaturated long chain dicarboxylic acids (dodecenedioic, tetradecenedioic, tetradecadienedioic, hexadecenedioic, octadecadienedioic, and octadecenedioic) were identified. The C16 and C13 dicarboxylic acids have never been reported for Reyes Syndrome or any other dicarboxylic acidemias. The data might reflect marked increase of extramitochondrial omega-oxidation of long chain fatty acids or impaired metabolism of omega-dicarboxylic acids formed in Reyes patients.

Prostaglandin inhibition of apomorphine-induced circling in mice

The effect of prostaglandins (PGs) on apomorphine (apo)-induced circling was examined in unilaterally lesioned mice. Intraventricularly injected PGD2, PGE2, and PGF2 alpha at a dose of 1.0 nmole/g all inhibited apo-induced circling. When injected directly into the striatum, these same PGs also inhibited circling in a dose range of 0.01-0.1 nmole/g, while the PGE2 metabolite, 13,14-dihydro-15-keto-PGE2, was inactive at 0.1 nmole/g. For both routes of administration, PGF2 alpha appeared to be the most potent of the PGs tested. PGs administered alone by either route to unilaterally lesioned mice did not produce circling. Pretreatment with the PG synthetase inhibitor, indomethacin, caused the apo treated mice to circle at significantly higher rates than control animals. These results are the first report suggesting that within dopamine (DA)-mediated pathways PGs act at sites postsynaptic to the dopaminergic synapse.

Prostaglandin inhibition of amphetamine-induced circling in mice

The effect of prostaglandins (PG) on amphetamine(AMPH)-induced circling was examined in mice unilaterally lesioned with 6-hydroxy-dopamine. At doses of 0.03–1.0 nmol/g, intraventricularly injected PGD2, PGE2, and PGF2α all inhibited AMP-induced circling, while thromboxane-B2 (TxB2) was inactive at 1.0 nmol/g. The inhibition of circling was not due to alterations in body temperature as measured by rectal temperature changes. When injected intrastriatally, the same major PG inhibited AMP-induced circling at the lower doses of 0.01–0.1 nmol/g, while the PGE2 metabolite 13,14-dihydro-15-keto-PGE2 was inactive at 0.1 nmol/g. PG administered alone did not procude circling. For both routes of administration, the order of potency was PGE2>PGD2>PGF2α. These results suggest that PG can alter motor function governed by central dopaminergic pathways.

The Excretion of Caffeine in the Semen of Men: Pharmacokinetics and Comparison of the Concentrations in Blood and Semen

Abstract: The concentration of caffeine in the blood and semen of men was measured after an oral dose of 200 or 400 mg caffeine. Caffeine was rapidly absorbed (mean tmax = 0.76 ± 0.12 hour), with an average maximum concentration in the blood of 7.4 μg/ml for the 400‐mg dose and 3.4 μg/ml for the 200‐mg dose. The mean clearance of caffeine was 161 and 156 ml/min, while the mean volume of distribution was 50 and 47 liters for the 400‐ and 200‐mg doses, respectively. Distribution of caffeine into the semen was rapid, with a concentration of caffeine in the semen almost identical to that observed concurrently in the blood (blood/semen concentration ratio = 0.97). The mean half‐life of caffeine in the blood and semen was 3.7 and 3.6 hours, respectively, indicating that the decline of caffeine in the blood is very similar to that in semen. Thus, caffeine partitions rapidly into and out of the prostatic and seminal vesicular secretions, which contribute to the formation of the ejaculate.

Role of ferric iron in platelet lipoxygenase activity

Abstract Four iron chelating agents, EDTA, EGTA, ferron and orthophenan-throlene, were found to inhibit human platelet lipoxygenase activity in a dose-dependent manner. The inhibition produced by these chelators could be selectively reversed by the addition of ferric ion but not ferrous ion. The ID 50 for lipoxygenase activity directly correlated with the avidity of these compounds for ferric ion. Thus, human platelet lipoxygenase requires ferric ion for activity.