Pär Westlund

Identification of 11-dehydro-TXB2 as a suitable parameter for monitoring thromboxane production in the human

In order to identify suitable parameters for measurement of thromboxane production in vivo, the metabolism of TXB2 was studied in the human. [3H8]-TXB2 was given intravenously to a healthy human volunteer. Blood samples were collected for 50 min after the injection, and urine was collected for 24 hours. The urinary and blood metabolic profiles were visualized by the use of two-dimensional TLC and autoradiography. Identification of metabolites was achieved with GC/MS and in some cases by cochromatography with reference compounds in TLC and GC. In blood, unmetabolized TXB2 was the dominating compound during the first 30 min. Three less polar metabolites appeared, two of which were identified as 11-dehydro-TXB2 and 11,15-didehydro-13,14-dihydro-TXB2, respectively. The third compound was tentatively identified as 15-dehydro-13,14-dihydro-TXB2. Since 11-dehydro-TXB2 was one of the major metabolites in blood as well as urine, it was deemed suitable as target for measurement of thromboxane production in vivo. The advantages of 11-dehydro-TXB2 over its parent compound, TXB2, were demonstrated in experiments where unlabeled TXB2 was injected i.v. to a human volunteer, and the blood and urinary levels of both compounds were then followed by radioimmunoassay. Measured levels of 11-dehydro-TXB2 were found to give a more reliable picture of metabolic events than TXB2, the latter compound to a large extent reflecting technical difficulties during blood sample collection.

Identification of wild type and variants of oestrogen receptors in polymorphonuclear and mononuclear leucocytes

Objective   Leucocytes play an important role in the pathogenesis of autoimmune and cardiovascular diseases. Clinical and epidemiological observations indicate that the sex steroid hormones, particularly oestrogens, may regulate leucocyte functions. The assumption that oestrogens have a direct effect on leucocytes has to be supported by identification of functional oestrogen receptors (ER) in leucocytes. This study aimed at investigating the presence of ER subtypes in different types of leucocytes isolated from peripheral blood of female and male donors.

Actions of Lipoxin A4 and Related Compounds in Smooth Muscle Preparations and on the Microcirculation in Vivo

The present chapter summarizes our findings with lipoxins (LX) in spasmogenic assays and in the intact microvasculature of the hamster cheek pouch. The initial observations1,2 were made in experiments using lipoxins isolated from human leukocytes. With the aid of synthetic compounds3, it has been possible to further explore the pharmacodynamics and structure activity relationships for lipoxins in smooth muscle preparations.

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. [5,6,8,9,11,12,14,15-3H8]-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 may be 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.

Evidence for a novel pathway of leukotriene formation in human platelets.

The metabolism of arachidonic acid via lipoxygenase-catalyzed reactions in washed human platelets was investigated. In addition to the previously discovered lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 8,15-dihydroxyeicosatetraenoic acid and 14,15-dihydroxyeicosatetraenoic acid, several other products were formed. The compounds were all dihydroxylated metabolites of arachidonic acid, containing a conjugated triene structure, and identified as 11,12-dihydroxyeicosatetraenoic acid (two isomers) and 5,12-dihydroxyeicosatetraenoic acid (four isomers). The identification was based on ultraviolet spectroscopy and gas chromatography-mass spectrometry of native and hydrogenated compounds. Stereochemical analysis of the hydroxyl groups of the 5,12-dihydroxyeicosatetraenoic acids and experiments with 18O2 indicated that the compounds were formed by the 12-lipoxygenase pathway, probably via an unstable epoxide.

Measurement of Thromboxane Production in Vivo: Metabolic and Analytical Aspects

In studies aiming at establishing a role for thromboxanes in various physiological or pathological conditions, it is often desirable to measure the endogenous thromboxane production. Since TXA2 is difficult to measure as such, a common approach in such studies is to monitor its stable hydrolysis product, TXB2, instead, and to compare obtained TXB2 levels in, for example, plasma samples in patient and control groups. Numerous such studies have been published to date; however, even the so called “basal” TXB2 levels reported therein vary widely. Not infrequently, “basal” TXB2 levels were found to be around 100–200 pg/ml plasma or even higher, however, others claim to find values below 15, 10 or even 2 pg/ml (e.g., refs. 1–6). It is difficult to explain such differences between studies where identical assay methods have been used (for example a commercially available TXB2 radioimmunoassay); that is, if the obtained data really reflect the endogenous situation.

Albumin prevents metabolism of 12-hydroxyeicosatetraenoic acid by leukocytes in vitro.

In the present paper we studied the influence of albumin on the in vitro metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) and arachidonic acid in leukocytes and aspirin-treated platelets. In the presence of physiological concentrations of albumin, the metabolism of both 12-HETE and arachidonic acid was substantially altered, implicating the importance fatty acid binding proteins might have on the profile of products formed both in vitro and in vivo. The results clearly showed that albumin effectively withdraws arachidonic acid and 12-HETE from further metabolism by the leukocytes but does not influence the conversion of arachidonic acid to 12-HETE by the platelets. Thus, some of the hypotheses concerning transcellular metabolism raised from in vitro data within the eicosanoid field might have little relevance for the in vivo situation.

Synthesis, structural identification and biological activity of 11,12-dihydroxyeicosatetraenoic acids formed in human platelets

An enantiospecific route for the synthesis of 11,12-dihydroxyeicosatetraenoic acids was developed and used to synthesize 11,12-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acids. The 11,12-DHETEs were synthesized with the stereochemistry of the hydroxyl group being 11(R),12(S) and 11(S),12(S). The synthetic compounds were used to elucidate the structure of 11,12-DHETEs formed in human platelets by comparison of the chromatographic retention time in HPLC and GC as well as their ion fragmentation pattern in GC-MS. The major 11,12-DHETE formed in human platelets was found to be identical with 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid. Two more compounds were tentatively identified as 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid and 11,12-dihydroxy-5(E),7(E),9(E),14(Z)-eicosatetraenoic acid. Furthermore, the 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid was found to possess biological activity on neutrophil functional responses. However, the major compound, 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid, formed in platelets lacks biological activity in the test systems used. The present data further support that 11,12-dihydroxy-eicosatetraenoic acids are formed in human platelets via a leukotriene like mechanism presumably by the 12-lipoxygenase. Furthermore, the biological effects of one of the compounds showed a unique activity profile compared to other lipoxygenase products.

12-hydroxyeicosatetraenoic acid is a long-lived substance in the rabbit circulation.

12-Hydroxyeicosatetraenoic acid (12-HETE) is one of the major metabolites formed from arachidonic acid in platelets. We have recently shown that the in vitro metabolism of 12-HETE by human leukocytes, with and without stimulation, is effectively inhibited by the addition of physiological concentrations of albumin, probably by sequestration of the compound. In the present paper, we have studied the in vivo metabolism of 12-HETE in the rabbit, using either [1-14C]- or [14C(U)]12-HETE. Distribution of radioactivity was followed in urine, plasma, and bile, as well as in a number of tissues. In most of the tissues examined, the hydrophilic radioactivity constituted more than 50% of the total radioactivity after 20 min. When the lipophilic fraction was analyzed, around 15% of the radioactivity was shown to be unesterified 12-HETE, and only a very minor part could be detected as metabolites. The dominating lipophilic compound in the circulation after i.v. administration of radiolabeled 12-HETE was at all time points (1-60 min.) the parent compound, as analyzed by HPTLC and HPLC. A comparison of the plasma metabolite profiles obtained when [1-14C]- and [14C(U)]12-HETE were used displayed almost identical patterns, thus indicating that beta-oxidized metabolites either were not formed or were rapidly removed from the circulation. The appearance of large amounts of water-soluble radioactivity with time supported the latter conclusion. Several minor metabolites were seen that chromatographed in the dihydroxy acid region as judged by HPLC and TLC. The major one of these compounds represented about 10% of the lipophilic plasma radioactivity after 60 min., while unmetabolized 12-HETE at this stage still represented about 30%. The metabolite had a polarity similar to 12,20-dihydroxyeicosatetraenoic acid; however, when chromatographed together, these two compounds separated, indicating a different structure of the metabolite. Our findings are in agreement with in vitro data concerning the protective effect of albumin on the metabolism of 12-HETE and is the first extensive metabolic study of 12-HETE in vivo covering all metabolic possibilities involving the carbon skeleton.

Eicosanoid Metabolism in Human Platelets is Modified by Albumin

Activation of human platelets leads to release of arachidonic acid from cellular phospholipids, the step which is believed to be the rate limiting for the metabolism of this fatty acid. Once released arachidonic acid can be metabolised by two platelet enzymes. Cyclooxygenase produces endoperoxides with the subsequent formation of thromboxane A2 (TXA2). Another enzyme, 12-lipoxygenase, catalyses conversion of arachidonic acid to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) which is rapidly reduced to 12-hydroxyeico-satetraenoic acid (12-HETE)1. The physiological significance of the latter pathway is poorly understood to date. Previously we have shown that albumin prevents metabolism of 12-HETE by polymorphonuclear leukocytes in vitro and protects it from metabolism by blood cells in vivo 2,3. In the present study we have investigated the influence of albumin on the activity of 12-lipoxygenase in the presence of phospholipase A2 and C inhibitors. In addition, we studied the influence of 15-hydroxyeicosatetraenoic acid (15-HETE), which has been reported to be a selective platelet 12-lipoxygenase inhibitor4, and 5-hydroxyeico-satetraenoic acid (5-HETE), which is a substrate for 12-lipoxygenase, on the endogenous 12-HETE production in the presence of albumin.