Elisabeth Granström

The release of prostaglandin F2α as reflected by 15-keto-13,14-dihydroprostaglandin F2α in the peripheral circulation during normal luteolysis in heifers

Abstract Progesterone and the main plasma metabolite of PGF2α, 15-keto-13,14-dihydro-PGF2α, were determined at hourly intervals in the peripheral circulation during luteolysis in two heifers. The prostaglandin release was found to occur during 2–3 days as rapid pulses with a duration of 1–5 hours prior to and during luteolysis, which was indicated by decreasing levels of progesterone.

[44] Radioimmunoassay of the major plasma metabolite of PGF2α, 15-keto-13,14-dihydro-PGF2α

Publisher Summary This chapter discusses radioimmunoassay of the major plasma metabolite of PGF 2α , 15-Keto-13,14-dihydro-PGF 2α . In this particular assay, plasma volumes of as much as 0.5 ml may be assayed without disturbing interferences from other plasma constituents. After the addition of the labeled ligand, the tubes are left for incubation. The next step is the separation of the free and the antibody-bound fractions. Of all methods tried, 3 the polyethylene glycol method gave the most reliable results. The tubes are vigorously vortexed, the formed grayish-white pellet contains the precipitated γ-globulins including the antibody-bound fraction of the prostaglandin metabolite, whereas the free fraction is found in the supernatant. The yellow color of unextracted plasma gives about 10% reduction in counting efficiency in the scintillation counter as compared to the standard vials without plasma. It is important that the counted values for counts per minute are corrected for quenching. This can easily be done if the scintillation counter is equipped with automatic quench correction facilities, such as automatic external standard channels ratio. Before setting up routine radioimmunoassay runs, the optimal titer of the antibody must be established. This should always be done also with the commercial preparations, as experience shows that the recommended procedure from the manufacturer is often far from optimal.

Vasopressin and Prostaglandins in Premenstrual Pain and Primary Dysmenorrhea

Abstract. Both vasopressin and PGF2alpha are effective uterine stimulants in the non‐pregnant human uterus, especially around the onset of menstruation. In order to clarify the relationship of these hormones to menstrual pain, plasma concentrations of vasopressin and two prostaglandin metabolites (lS‐keto‐B.H‐dihydro‐PGF2alpha and 11‐ketotetranor PGF metabolites) were measured in serial blood samples taken premenstrually and during menstruation. Five women with premenstrual pain gave 7‐9 blood samples at intervals of 30 minutes on the day preceding the onset of menstruation. From 5 women with severe primary dysmenorrhea a corresponding series of blood samples were taken during the first day of menstruation. Two groups of 5 women with no symptoms served as controls, either premenstrually or during menstruation. In the women with premenstrual pain the vasopressin concentrations were significantly higher than in the corresponding control group. Even higher and markedly fluctuating vasopressin levels were found in the women with dysmenorrhea who, in general, had more intense pain than the women with premenstrual symptoms. In the group with dysmenorrhea there was also a significant rise in plasma concentration of the PG metabolites. No such increase was seen in the group with premenstrual pain. It is concluded that the pathophysiology of premenstrual pain could imply increased vasopressin secretion. The more severe pain in primary dysmenorrhea seems to be the result of a combined effect of vasopressin and PGF2alpha

[43] Radioimmunologic determination of 15-keto-13,14-dihydro-PGE2: A method for its stable degradation product, 11-deoxy-15-keto-13,14-dihydro-11β,16ξ-cyclo-PGE2

Publisher Summary This chapter describes the chemical instability of 15-keto- 13,14-dihydro-PGE2 under various conditions, the development of a radioimmunoassay (RIA)for the stable bicyclic degradation product, and conditions for the induced quantitative conversion of 15-ketodihydro-PGE2 in biological samples into this product prior to measurements. 15-ketodihydro-PGA2 undergo a different fate cyclization into a bicyclic product, l l-deoxy-15-keto-13,14-dihydro- 1 lβ,16ξ-cyclo-PGE2. This cyclization product contains at least two epimeric sites, at C-8 and C-16; furthermore, the cyclization of the PGA2 derivative might also involve a transient formation of the corresponding PGC2 compound, resulting in additional epimeric sites at C-11 and C-12. The radioimmunoassay described in this chapter is developed using a racemic mixture of these epimers for the preparation of the immunogen. The crucial point RIA is the prior quantitative conversion of all 15-ketodihydro-PGE2 present in the sample into the bicyclic derivative before analysis.

Metabolism of prostaglandin F2α in swine kidney

Abstract [9β-3H] Prostaglandin F2α was incubated with the high-speed supernatant from a homogenate of swine kidney. Two less polar metabolites were found which were identified as 9α,11α,15-trihydroxyprost-5-enoic acid and 9α,11α-dihydroxy-15-ketoprost-5-enoic acid. The elucidation of these structures was based on gas-chromatographic and mass-spectrometric analyses of the trimethylsilyl ether derivatives and acetates of the methyl esters, and by borohydride reduction of 9α,11α-dihydroxy-15-ketoprost-5-enoic acid.

[60] Two-dimensional thin-layer chromatography of prostaglandins and related compounds

Publisher Summary This chapter describes two-dimensional thin layer chromatography assay systems, suitable for several different types of analytical problems in the field of eicosanoid biochemistry. Detection of radioactive spots is achieved by autoradiography, employing either the traditional X-ray type of film or a more recently developed, highly sensitive film. This latter product, LKB Ultrofilm 3H, renders the detection of even small amounts of tritium labeled products possible, and reduces the necessary exposure time also for 14C-labeled substances. For analytical purposes the two-dimensional assay offers further possibilities. Identification of compounds can be facilitated, not only by the increased resolution as discussed above, but also by the so-called spot shift technique. The sample components are then derivatized in situ after the first run minor differences in properties between two compounds may thus be enhanced, or they may be separated according to a different principle in the second system.

Metabolism of prostaglandin F1α in the guinea pig

Abstract [9β- 3 H]Prostaglandin F 1α was injected subcutaneously to male guinea pigs. Six metabolites were isolated from urine; three C 18 compounds: 7α,9α-dihydroxy-13-ketodinorprostanoic acid, 7α,9α,13-trihydroxy-dinorprost-11-enoic acid (dinorprostaglandin F 1α ) and 7α,9α,13-trihydroxy-dinorprostanoic acid (dihydro-dinorprostaglandin F 1α ); and three C 16 compounds: 5α,7α-dihydroxy-11-ketotetranorprostanoic acid, 5α,7α,11-trihydroxy-tetranorprost-9-enoic acid (tetranorprostaglandin F 1α ) and 5α,7α 11-trihydroxy-tetranorprostanoic acid (dihydro-tetranorprostaglandin F 1α ). Identification was based on gas chromatography-mass spectrometry of several derivatives, as well as comparison with reference compounds.

[45] Radioimmunoassay of 5α,7α-dihydroxy-11-ketotetranorprostane-1,16-dioic acid, a major prostaglandin F metabolite in blood and urine

Publisher Summary This chapter describes a radioimmunoassay for a major prostaglandin (PG) F product, 5α,7α-dihydroxy-11-ketotetranorprostane-1,16-dioic acid, and its application to measurements in urine as well as in plasma. The assay is sufficiently nonspecific to allow measurements also of structurally related tetranor metabolites, for example, major PGF metabolites in other species than the human. In preparation of the metabolite, the metabolite from biological material is isolated , generally from urine from patients receiving PGF 2α for termination of pregnancy. In order to obtain a comparatively specific antibody it is desirable to perform the coupling exclusively at one of the two carboxyls. The labeled ligand is prepared accordingly; the infusion is done with [ 3 H]PGF 2α of the highest possible specific activity after indomethacin pretreatment. For performance of the radioimmunoassay, the high precision LKB diluter is normally used both for preparation and for the later analysis of these dilutions. Assay of the 11-ketotetranor metabolites may be carried out in unextracted plasma. To improve separation of the free and antibodybound fraction after the incubation, we normally add bovine γ-globulin, 0.2 ml of a 0.5% solution , to all sample tubes containing less than 0.3 ml of unextracted plasma.

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.

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.