Hans Kindahl

Radioimmunoassay for Thromboxane B2

Abstract A radioimmunoassay for a prostaglandin endoperoxide metabolite, thromboxane B2, was developed. The antibodies were very specific for this compound, and the method had a sensitivity of 10 pg. Platelets from a human subject were aggregated by addition of collagen and the synthesis of prostaglandin endoperoxides with time was monitored by parallel assay of prostaglandin E2, F2α and thromboxane B2.

Biosynthesis of prostaglandin F2α from arachidonic acid and prostaglandin endoperoxides in the uterus

Formation of prostaglandin F2Alpha in the cow and guinea pig uterus microsomes was studied using 14C-labeled arachidonic acid and prostaglandin H2. The total conversion of arachidonic acid was of a low order and underwent fluctuations during the estrous cycle of the guinea pig, being highest towards the end of the cycle. Injections of beta-estradiol-3-benzoate also resulted in higher activity of the uterine prostaglandin synthetase. The uterine prostaglandin synthesizing system appeared to differ in several respects from that present in seminal vesicles, with regard to the proportions of the products formed and the effects of various agents, e.g. reduced glutathione. An inhibiting factor which supressed the fatty acid cyclo-oxygenase was found to be present in uterine preparations. Prostaglandin endoperoxide (prostaglandin H2) was very efficiently reduced to prostaglandin F2alpha by cow and guinea-pig uterus microsomes. Prostaglandin G2 also gave rise to prostaglandin F2alpha. Prostaglandin E2, on the other hand, was not reduced. Both the inhibiting factor and the endoperoxide reducing activity are likely to be parts of a highly specialized mechanism that modulates prostaglandin F2alpha formation in the uterus.

Release of prostaglandin F2α during the bovine peripartal period

Abstract Progesterone, estrone and 15-keto-13,14-dihydro-PGF 2α levels were determined in the peripheral blood circulation during the peripartal period in 12 cows. Plasma concentrations of progesterone showed a gradual and continuous decrease during the last 60 days before parturition. This gradual decrease was followed by an abrupt decline in the progesterone concentration occurring 24–48 hours before delivery. The plasma levels of estrone started to increase about 30 days prior to parturition with high concentrations attained during the last days of pregnancy. After delivery the estrone content decreased to baseline levels. Increased levels of the PGF 2α metabolite were recorded 24–48 hours before parturition. These increased PGF 2α metabolite levels occurred before or in conjunction with prepartum luteolysis. Prostaglandin metabolite levels remained high during parturition and returned to baseline 10–20 days after delivery.

Albumin stabilizes thromboxane A2.

Recent work on the conversion of arachidonic acid and the prostaglandin endoperoxides in platelets into thromboxane Bz (TXBJ led to the discovery of an unstable, biologically very active intermediate, thromboxane AZ (TXAJ [ 1,2] (fig.1). This compound was shown to constitute the major component of the previously recognized, elusive rabbit aortacontracting substance [3] . The conversion into thromboxanes represents the major pathway in the metabolism of prostaglandin endoperoxides in platelets [4]. TXAz is very potent in inducing irreversible platelet aggregation and has a half-life of around 30 s in aqueous medium at 37°C [2] . Considerably longer half-lives of the compound were found at lower temperatures [5,6] . The half-life was also increased

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 degrees C; the compound was more stable at lower temperatures. The t1/2 for thromboxane A2 was also considerably longer in plasma.

Radioimmunoassay of prostaglandins E2 and F2α in cell culture media utilizing antisera against prostaglandins F2β and F2α

Early determinations of prostaglandins were based on chromatographic separations followed by bioassays [l-3] . More recently, methods such as fluorimetry of NADH generated by prostaglandin dehydrogenase [4], gas-liquid chromatography with electron capture detector [5,6], and quantitative mass spectrometry using deuterium labeled prostaglandins as internal standards [7] have been developed. Several radioimmunoassays for prostaglandins have also been described [ 8161. Although there are reports on radioimmunoassays for prostaglandin El (PGE2)*, several authors have reported difficulties in producing antisera against Etype prostaglandins [8,11,12,14] probably due to dehydration of PGE’s under the conditions used to attach the prostaglandin to a protein. We therefore developed a method to measure PGEZ and PGF,, in cell culture media which is based on NaBHa reduction of the samples followed by radioimmunoassay of PGF,

The effect of cervical dilatation by laminaria on the plasma levels of 15-keto-13,14-dihydro-PGF2α

Laminaria tents were inserted to induce cervical dilatation prior to suction abortion in 42 primigravidae. The plasma level of 15-keto-13,14-dihydro-PGF2 alpha, the principal metabolite of prostaglandin F 2 alpha, increased during the dilatation period. The gentle dilatation by laminaria tents probably induces an endogenous synthesis of prostaglandins causing a softening of the cervix.

Induction of Labor by Oral PGE2 Administration—Evaluation of Different Dose Schedules

The efficacy and safety of different oral doses of PGE2 in tablet form were evaluated in 30 women admitted to the hospital for labor induction at or near term. The aim was to select a recommendable dose schedule based on recording of uterine contractility, clinical outcome and measurement of the resulting plasma levels of the two prostaglandin metabolites 15‐keto‐13.14‐dihydro‐PGE, and 15‐keto‐13,14‐dihydro‐PGF, by gas‐chromatography‐mass spectrometry and by radio‐immunoassay.

Effects of adenosine 3':5'-monophosphate and platelet aggregation on thromboxane biosynthesis in human platelets.

The prostaglandin (PC) endoperoxides, PGGs and PGHz [l], and thromboxane (TX)A, [2] cause rapid aggregation and induce the release reaction in human platelets. Aggregating agents, such as collagen and thrombin, stimulate release of arachidonic acid from platelet phospholipids. This leads to synthesis of endoperoxides, thromboxanes, prostaglandins and hydroxy acids [3-51. Elevation of adenosine 3’: 5’-monophosphate (cyclic AMP) levels in platelets prevents aggregation [6-8] and the simultaneous production of TXBz [9-121. Two mechanisms for the inhibition of thromboxane formation have been proposed: (1) Inhibition of prostaglandin endoperoxide synthase (EC 1.14.99.1) [9]; (2) Inhibition of arachidonic acid release from platelet phospholipids [lo121. In the latter reports no inhibition of prostaglandin endoperoxide synthase was observed [lo-121. We have therefore reinvestigated the effects of cyclic AMP on arachidonic acid release and on the conversion of arachidonic acid to TXBs. The results suggest that elevation of cyclic AMP levels inhibits both of these reactions in platelets and that the inhibitory effect of cyclic AMP on TXBz formation from arachidonic acid is not direct but secondary to inhibition of platelet aggregation.

Dilatation of the Cervix by Oral PGE2 Before First-Trimester Termination of Pregnancy

Abstract. Twentyfour patients—all primigravidae with an especially narrow, rigid cervix were given PGE2 tablets or placebo tablets in order to prime the cervix before first trimester termination of pregnancy. 1.5 mg PGE2 or placebo tablets were administered hourly for 12 hours on the day before vacuum aspiration. The diameter of the cervix was measured by means of a Hegar dilator prior to administering the tablets and also before the vacuum aspiration the following day. The plasma level of 15‐keto‐13,14‐dihydro‐PGF2α was determined before the treatment and again before the vacuum aspiration. All the 12 patients treated with PGE2 tablets had an average change in cervix diameter of 2 mm, whereas in the placebo group the average change was 0.58 mm.