H. Kindahl

Post-partum release of prostaglandin F2α and uterine involution in the cow

Peripheral plasma levels of the main blood plasma metabolite of PGF2α (15-keto-13,14-dihydro-PGF2α) and progesterone were investigated during the immediate, post-partum period in 59 normally calving cows. Uterine involution was monitored by weekly rectal palpations. The levels of the prostaglandin metabolite were high at parturition and remained thereafter elevated for periods varying up to 7–23 days. Uterine involution was completed during periods ranging from 16–53 days. According to the clinical findings, the animals were divided into three groups. Group A comprises 46 animals which had an uncomplicated, puerperal period. A significant (p<0.001) correlation between the duration of elevated prostaglandin levels and the time for completed uterine involution (Y=29.6 − 1.3 (X − 13.5)) was found for these animals. Group B animals (n=8) had periods of varying length with uterine discharge during the first 30 days post-partum. When compared to group A animals, the animals in group B had comparatively longer periods of prostaglandin release and also longer periods for completion of uterine involution. Group C animals (n=5) at times had palpable, thin-walled, cystlike structures in the ovaries during the first 30 days post-partum. In this group of animals, the periods of high prostaglandin levels, as well as for the completion of uterine involution, were similar to those for the animals in group A. Progesterone levels remained low during the immediate post-partum period and in no case were elevated levels found until the prostaglandin release had ceased.

[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.

Oral Neospora caninum inoculation of neonatal calves

Four calves born to cows seronegative for Neospora caninum were dosed orally within 6 h after birth with tachyzoites of the bovine N. caninum Nc-SweB1 isolate added to colostrum. Two of the calves were dosed via stomach tube and two by feeding bottle. The latter two calves showed transient fever and passed blood-stained diarrhoea 1-2 weeks after inoculation. From 5 weeks after inoculation they developed a significant antibody response which remained high until the calves were euthanised and necropsied at 15 and 19 weeks after inoculation, respectively. The two calves inoculated by stomach tube showed no clinical signs and they remained seronegative throughout the study. At necropsy of the seropositive calves, no pathological lesions were seen, and parasites were not detected by immunohistochemistry. Neospora caninum was not re-isolated in cell culture from the brains of the seropositive calves; however, N. caninum DNA was detected in brain from both of them by PCR. The data suggest that oral infection of N. caninum via colostrum might be a possible route of vertical transmission in newborn calves, in addition to transplacental infection.

Serum antibody profile and reproductive performance during two consecutive pregnancies of cows naturally infected with Neospora caninum.

The objective of this study was to record how the antibody levels change over time during pregnancy in dairy cows naturally infected with the protozoan parasite Neospora caninum, and relate this to the reproductive performance. Eighteen cows with antibodies to N. caninum were serum sampled monthly during their first pregnancy and 13 of them were also followed for a second pregnancy. In all, five pregnancies ended in abortion and two in stillbirth. Antibodies to N. caninum in serum were demonstrated by immune stimulating complex enzyme-linked immunosorbent assay (iscom ELISA). The N. caninum antibody titres remained well above the 1:100 cut-off limit for the test used during 2 years in all cows. In the non-aborting cows, mean values of antibody titres to N. caninum rose 1.5-2.5 dilution steps to reach a plateau 4-5 months before parturition, and thereafter decreased from 2 months before parturition. These changes were statistically significant (p < 0.001). The same pattern was seen in the aborting cows. The consistent pattern of rise in antibody titres observed during both pregnancies in all cows indicated a reactivation rather than a reinfection of the parasite at mid-gestation.

Blood levels of 15-keto-13, 14-dihydroprostaglandin F2α during the postpartum period in primiparous cows

Plasma levels of the PGF(2alpha) metabolite 15-keto-13, 14-dihydroprostaglandin F(2alpha) were determined postpartum in 51 primiparous Black and White Lowland cows. The highest geometric mean was 1702 pmol/l on day 3 and lowest 190 pmol/l on day 21. It should be noted that variation between animals in the concentration of the metabolite is high. For instance, on days 4, 10, 16 and 22, concentration of metabolite ranged from 775-2500, 209-2450, 45-851 and 30-398 pmol/l, respectively. The duration of the massive postpartum release of PGF(2alpha) could be determined in only 29 cows. Significant correlations were found between the duration of elevated PGF(2alpha) metabolite levels and the time required for completion of uterine involution (r = -0.41, P < 0.05) and between the duration of increased PGF(2alpha) metabolite levels and the interval from parturition to occurrence of the first ovulation followed by a normal luteal phase length (r = -0.37, P < 0.05). The occurrence of the first ovulation followed by a normal luteal phase length in the 29 cows was positively correlated with the time needed for completion of uterine involution (r = 0.54, P < 0.01).

Chemical instability of 15-keto-13,14-dihydro-PGE2: the reason for low assay reliability.

The spontaneous degradation of 15-keto-13,14-dihydro-PGE2 was studied under various conditions. In aqueous media, dehydration rapidly occurred, particularly at high or very low pH. The acid catalyzed dehydration product was identified as 15-keto-13,14-dihydro-PGA2. This compound was also formed at alkaline pH, however, at higher pH a bicyclic compound, 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE2, was formed in addition (cf. Fig. 1). The amounts of the latter compound increased with time and with increasing pH. In samples containing albumin, 15-keto-dihydro-PGE2 was degraded even more rapidly than in buffer of the same pH. In addition to the formation of the dehydration products, a considerable part of the metabolite was bound to albumin to yield water soluble adducts. A similar binding occurred to a low molecular weight fraction of plasma. The compound that was bound was 15-keto-dihydro-PGA2, whereas both 15-keto-dihydro-PGE2 and the bicyclic product were relatively inert in this respect. Due to its chemical stability, the bicyclic degradation product, 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE2, is suggested as a suitable target for measurements instead of the labile parent compound, 15-keto-dihydro-PGE2, or the reactive dehydration product, 15-keto-dihydro-PGA2.

Characterization of a Swedish bovine isolate of Neospora caninum

Abstract  The brain of a stillborn calf, seropositive to Neospora caninum and born to a seropositive cow, was homogenized and cultured on Vero cells, where growth of Neospora-like tachyzoites was detected after 8 weeks. The ultrastructural features of the new isolate (Nc-SweB1) corresponded to those of previously published Neospora isolates. In indirect immunofluorescence tests, antigens on Nc-SweB1 tachyzoites were recognized by antibodies raised to a canine N. caninum isolate (Nc-1) but not by antibodies to Toxoplasma gondii, Sarcocystis cruzi, S. tenella, Eimeria alabamensis, Babesia divergens, or B. motasi. Immunoblot analyses revealed no major antigenic difference between Nc-SweB1 and Nc-1, whereas several differences were seen between Nc-SweB1 and protozoa related to N. caninum. The sequences of 16S-like rRNA and the internal transcribed spacer 1 of Nc-SweB1 revealed complete homology with corresponding sequences of two canine N. caninum isolates. Thus, no dissimilarity between Nc-SweB1 and the canine isolates was found, confirming that Nc-SweB1 is N. caninum and suggesting that Neospora-like organisms isolated from cattle are indeed N. caninum.

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

Stimulation of vasopressin release in women with primary dysmenorrhoea and after oral contraceptive treatment--effect on uterine contractility.

Objective To study aspects of the aetiology of primary dysmenorrhoea and mechanisms underlying the therapeutic effect in this condition of an oral contraceptive.

Species differences in circulating prostaglandin metabolites. Relevance for the assay of prostaglandin release.

The profiles of circulating metabolites of prostaglandin F2 alpha were investigated in a number of species, viz. rat, rabbit, guinea pig, cattle and sheep. The aim of the study was to identify in each animal major plasma metabolites that outlast the initially formed, short-lived 15-keto-13, 14-dihydroprostaglandin F2 alpha and might thus serve as better parameters for monitoring prostaglandin production in vivo. Tritium-labeled prostaglandin F2 alpha was injected intravenously and frequent blood samples were collected. The metabolic profiles at different stages were visualized using two-dimensional thin-layer chromatography and autoradiography. Identification of circulating products was achieved by comparison with reference compounds using several chromatographic methods, and by gas chromatography-mass spectrometry in cases where larger amounts of the prostaglandin had been administered. In the rabbit a similar study was also done with tritium-labeled prostaglandin E2. Certain species differences were seen in the removal of labeled compounds from the circulation, the elimination being most efficient in the guinea pig. Further differences were seen in the profiles of circulating prostaglandin metabolites. The first appearing major prostaglandin F2 alpha metabolite was always 15-ketodihydroprostaglandin F2 alpha. However, this compound was later replaced in the circulation by a number of more degraded products, the profiles of which were relatively typical for each species. Thus, in cattle, rat and guinea pig, the earliest-formed metabolites, 15-ketodihydroprostaglandin F2 alpha and its tetranor counterpart, 5 alpha, 7 alpha-dihydroxy-11-ketotetranorprostanoic acid, remained comparatively prominent plasma products, whereas highly polar dicarboxylic acids rapidly dominated the metabolite spectrum in the ovine and lapine circulation. These differences were further supported by separate kinetic experiments, using unlabeled prostaglandin F2 alpha and radioimmunological determination of formed products. These latter experiments also demonstrated further pronounced species differences in the rat of elimination of the different prostaglandin metabolites. A considerable interconversion between prostaglandin E and F compounds was also demonstrated in some species. In conclusion, the traditional prostaglandin parameters in plasma, the 15-ketodihydrometabolites, were found not to be the best parameters in all species. It is suggested that species differences in prostaglandin metabolism are taken ito consideration when the optimal analytical protocol is sought for future biological studies. Some alternatives are suggested in the present paper.