Radioimmunoassay of 13,14-dihydro-15-keto prostaglandin F in bovine peripheral plasma

A radioimmunoassay has been developed for 13,14-dihydro-15-keto-prostaglandin F in bovine peripheral plasma. Acidified plasma samples were extracted with diethyl ether and the dried extracts assayed for 13,14-dihydro-15-keto-prostaglandin F using antiserum raised against a 13,14-dihydro-15-keto-prostaglandin F_2α-albumin complex. The tracer used for the assay was prepared enzymatically from tritiated prostaglandin F_1α. Polyethylene glycol was employed to separate free and bound 13,14-dihydro-15-keto-prostaglandin F. The inter-assay coefficient of variation based on 9 determinations of control plasma was 13.8%. The detection limit of the assay was 25 pg 13,14-dihydro-15-keto-prostaglandin F/ml plasma.In 3 cows around estrus there was a complex series of peaks of 13,14-dihydro-15-keto-prostaglandin F concentrations coincident with luteolysis and declining progesterone concentrations. Changes in peripheral plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F in the pregnant cow near term showed a close correlation with prostaglandin F levels in utero-ovarian venous plasma. The concentration of 13,14-dihydro-15-keto-prostaglandin F in 12 men was 114±20 pg/ml plasma. It is concluded that the measurement of peripheral 13,14-dihydro-15-keto-prostaglandin F concentrations may offer a simple and convenient method for monitoring uterine prostaglandin F production in the cow.     

Radioimmunoassay of 13, 14-dihydro-15-keto prostaglandin F in bovine peripheral plasma.

A radioimmunoassay has been developed for 13, 14-dihydro-15-keto-prostaglandin F in bovine peripheral plasma. Acidified plasma samples were extracted with diethyl ether and the dried extract assayed for 13, 14-dihydro-15-keto-prostaglandin F using antiserum raised against a 13, 14-dihydro-15-keto-prostaglandin F2alpha-albumin complex. The tracer used for the assay was prepared enzymatically from tritiated prostaglandin F1alpha. Polyethylene glycol was employed to separate free and bound 13, 14-dihydro-15-keto-prostaglandin F. The inter-assay coefficient of variation based on 9 determinations of control plasma was 13.8%. The detection limit of the assay was 25 pg 13, 14-dihydro-15-keto-prostaglandin F/ml plasma. In 3 cows around estrus there was a complex sseries of peaks of 13, 14-dihydro-15-keto-prostaglandin F concentrations coincident with luteolysis and declining progesterone concentrations. Changes in peripheral plasma concentrations of 13, 14-dihydro-15-keto-prostaglandin F in the pregnant cow near term showed a close correlation with prostaglandin F levels in utero-ovarian venous plasma. The concentration of 13, 14-dihydro-15-keto-prostaglandin F in 12 men was 114 +/- 20 pg/ml plasma. It is concluded that the measurement of peripheral 13, 14-dihydro-15-keto-prostaglandin F concentrations may offer a simple and convenient method for monitoring uterine prostaglandin F production in the cow.     

Maternal and fetal prostaglandin concentrations during late gestation in dairy cattle

A study was conducted to measure the blood plasma concentrations of prostaglandin F_2α (PGF_2α), 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM), prostaglandin E₂ (PGE₂) and 13,14-dihydro-15-keto-prostaglandin E₂ (PGEM) in the jugular vein, umbilical vein and artery and uterine vein of 18 Holstein Friesian cows during late gestation. A caesarean section was performed on all cows before term in order to obtain blood samples from the different sources. Plasma PG concentrations in the uterine or fetal circulation were significantly higher than in jugular vein plasma. Correlations between peripheral PG metabolite concentrations and primary PG concentrations in the various sources of the uterus or fetus were not significant (r = .17 − .47) and demonstrated that prostaglandin values based upon peripheral blood alone are of limited value.     

Plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F during pregnancy in sheep

A specific and sensitive radioimmunoassay is described for 13,14-dihydro-15-keto-prostaglandin F in ovine plasma. Using this assay it has been shown that, in sheep, jugular venous 13,14-dihydro-15-keto-prostaglandin F concentrations increase at parturition and correlate well with concentrations of prostaglandin F in the utero-ovarian vein. It is suggested that uterine prostaglandin F production under these conditions may be assessed by measuring peripheral venous 13,14-dihydro-15-keto-prostaglandin F, thereby avoiding the need for chronic utero-ovarian venous catheters.     

Levels of 13,14-dihydro-15-keto-PGE2 in some biological fluids as measured by radioimmunoassay

A rabbit antiserum directed toward the prostaglandin E₂ metabolite 13,14-dihydro-15-keto-prostaglandin E₂ (KH₂PGE₂) was produced by immunization with a human albumin-KH₂PGE₂ conjugate. The antiserum recognized the 15-keto-group (it cross reacts with 13,14-dihydro-prostaglandin E₂ 0.2%); the saturated 13,14-bond (it cross reacts with 15-keto-prostaglandin E₂ 7%); the 9-keto group (it cross reacts with 13,14-dihydro-15-keto-prostaglandin F_2α 5%); and the 11-hydroxy group (it cross reacts with 13,14-dihydro-15-keto-PGA₂ 0.4%).By subjecting the antiserum to preparative isoelectric electrofocusing, populations of antibodies that varied in their cross reaction with 13,14-dihydro-15-keto-prostaglandin F_2α (KH₂PGF_2α) from 20% to 1% were obtained. The levels of KH₂PGE₂ in plasma of rat and mouse as measured by radioimmunoassay of the unfractionated plasma were 0.39 ± 0.07 ng/ml and 0.41 ± 0.13 ng/ml, respectively. Recovery of exogenously added KH₂PGE₂ from human plasma was 100%. Radioimmunoassay with two antisera; an antiserum directed toward KH₂PGF_2α that cross reacts with KH₂PGE₂ 1% and the antiserum to KH₂PGE₂, demonstrated that KH₂PGE₂, not KH₂PGF_2α, was being measured with the anti-KH₂PGE₂. The levels of KH₂PGE₂ in rat plasma did not vary with sex. In rats, the levels of KH₂PGE₂ markedly increased after exercise stress.In mice carrying a spindle-cell sarcoma (SAI) and a fibrosarcoma (SaD2), the levels of KH₂PGE₂ in the plasma increased with time after transplantation. The increase was not observed in the plasma of mice carrying a transplantable anaplastic carcinoma (15091AK), a lymphatic leukemia (AW5147), two mammary adenocarcinomas (CADI, CAD2), a myeloid leukemia (C1498), and a hepatoma (BW7756).     

Jugular levels of 13,14-dihydro-15-keto-prostaglandin F and progesterone around luteolysis and early pregnancy in the ewe

Six non-pregnant ewes at day 12 of the estrous cycle each had a day-12 embryo transferred into the uterine horn ipsilateral to the corpus luteum, and 4 non-pregnant ewes at day 13 each had a day-13 embryo similarly transferred. Four control ewes, 2 at day 12 and 2 at day 13 received sheep serum into the uterine horn ipsilateral to the corpus luteum. Jugular blood samples were taken at 2-hourly intervals for 3 days post-surgery, then twice-daily for a further 4 days, and the plasma radioimmunoassayed for progesterone and 13,14-dihydro-15-keto-prostaglandin F. All control ewes exhibited estrus within the expected time range and pulsatile peaks of 13,14-dihydro-15-keto-prostaglandin F occurred coincident with declining progesterone levels. With one exception, the recipient ewes had prolonged cycles and those ewes found pregnant at necropsy, 30 days after transfer, showed no progesterone decline and no pulsatile peaks of prostaglandin during days 12 to 16 after estrus. These observations suggest that the presence of the embryo at a critical stage after mating suppresses the release of uterine prostaglandin F_2α.     

Peripheral plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2α and progesterone around luteolysis and during early pregnancy in the goat

Peripheral plasma concentrations of 13,14-dihdyro-15-keto-prostaglandin F_2α (PGFM) and progesterone were determined during both luteolysis in the oestrous cycle and early pregnancy in four goats. Luteal regression, characterised by decreasing progesterone concentrations, began on day 12 or 13. PGFM concentrations showed a pulsatile pattern around this time, with peak concentrations increasingly markedly as progesterone levels fell and oestrus approached. During early pregnancy progesterone concentrations did not fall after day 12 and no marked elevation of PGFM above basal values of 50–150 pg/ml was detected.     

Fetal-maternal secretion of prostaglandins in the cow

A study was conducted to measure the blood plasma concentrations of prostaglandin F₂α (PGF₂α), 13,14-dihydro-15-keto-prostaglandin F (PGFM), 6-keto-prostaglandin F₁α (6-keto), prostaglandin E₂ (PGE₂), and thromboxane B₂ (TBX₂) in the ovarian vein, uterine artery, uterine vein, umbilical artery and umbilical vein in 24 cows from days 80 to 260 of pregnancy. Blood was collected during surgery and all prostaglandins were measured using specific radioimmunoassay procedures. Results indicate that PGF₂α blood levels are higher in the umbilical vessels and uterine vein than in the ovarian vein and uterine artery. PGFM and PGE₂ showed a trend towards higher values in the umbilical than in the maternal vessels, but the levels of 6-keto and TBX₂ were not different among the vessels studied. No differences across time couls be observed in any of the prostaglandins measured, partly due to the great variability in blood levels among animals during the same stage of pregnancy.     

Temporal relationships between peripheral plasma concentrations of oxytocin, progesterone and 13, 14-dihydro-15-keto-prostaglandin F2α during the oestrous cycle and early pregnancy in the ewe

Peripheral plasma concentrations of oxytocin, 13,14-dihydro-15-keto-prostaglandin F_2α(PGFM), progesterone and LH were determined at 3 hourly intervals during the oesterous cycle (n = 3) and in early pregnancy (n = 4) in sheep. The progesterone and LH concentrations showed that the cycling ewes were samples during the periods of luteal regression (decreasing progesterone concentrations), the preovulatory gonadotrophin surge and the beginning of the next luteal phase (increasing progesterone concentrations). The pregnant ewes had basal LH concentrations and luteal phase concentrations of progesterone (>lng/ml afte day 5 following mating) throughout the whole of the sampling period. Oxytocin concentrations in the non-pregnant ewes decreased around the time of luteal regression to reach low concentrations (mean concentrations of approximately 18pg/ml) during the preovulatory period and then increased after the preovulatory surge. PGFM concentrations exhibited a pulsatile pattern with increasing concentrations as progesterone levels fell. In the pregnant ewes oxytocin concentrations gradually fell until approximately 16 days post-mating (approximately 7–8pg/ml). The magnitude of the pulses in PGFM concentrations were also lower than in the cycling ewes. These results demonstrate that the increased concentrations of PGFM which are found during the period of luteal regression are not caused by increased peripheral concentrations of oxytocin.     

Uterine activity and prostaglandin production following intraamniotic hyperosmolar urea

The relationship between endogenous prostaglandin (PG) production and uterine activity was studied in hyperosmolar urea induced abortion patients. Polygraphic recordings of intraamniotic pressure were obtained at periodic intervals following intraamniotic injection of 80 gm urea. At 0, 0.25, 1, 4 and 8 hours amniotic fluid and blood samples were obtained for PGE, PGF and 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) analysis by radioimmunoassay. Blood was also sampled at time of absorption. In eight patients studies, uterine tone was elevated by 0.25 hour although no rhythmic contractions were observed by 1 hour. At 4 hours, amniotic fluid PGF concentration increased significantly (P < .01) over the pre-injection value and continued to increase at 8 hours. Amniotic fluid PGE, PGFM and all plasma PG's showed no change during the 8 hour period following urea administration. At time of abortion the plasma PGFM concentration was significantly greater than at the time of injection (238 ± 54.4 vs. 86.7 ± 7.3 pg/ml). There was no significant differences between pre-injection and absorption plasma PGF or PGE concentrations. In the present study, there is no evidence that increased prostaglandin production precedes urea induced contractions. The possible role of PG's in uterine contractions is discussed.     

Plasma levels of 13,14-dihydro-15-keto prostaglandin F2α, estrogens, and progesterone during stretch-induced labor at term

The uterus of six healthy multiparous women at term was mechanically stretched by a rubber catheter and balloon. Apparent labor was inaugurated in all cases within 5 hours and increased progressively with time. Advanced cervical softening and dilatation were also evident after the stretch treatment. Significant increases in the levels of 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) were observed with the progress of treatment (P < 0.01). Plasma estrogens and progesterone levels did not change significantly during the treatment (P > 0.05). Stretching and/or resulting uterine contractions appear to induce the secretion of prostaglandin F_2α (PGF) from the organ, which in turn seems to be involved in both cervical softening, and the onset and progress of labor, under stable conditions of plasma estrogens and progesterone.     

Prevention of the luteolytic action of oxytocin in the goat by inhibition of prostaglandin synthesis

Oxytocin-induced luteolysis in goats was associated with significant increases in peripheral plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM). Oral administration of the prostaglandin (PG) synthetase inhibitor meclofenamic acid (lg/day) prevented both the luteolytic action of oxytocin and the increase in PGFM concentrations. These results confirm that the luteolytic effect of oxytocin is mediated via the production and release of PGF(2alpha).     

Effect of endotoxin administration in pregnant camels

Intravenous administration of Escherichia coli endotoxin at a dose of 0.05 μg/kg bodyweight to pregnant camels resulted in abortion. The injection of endotoxin caused significant increases in the plasma concentration of 13,14-dihydro-15-prostaglandin F_2α, the metabolite of prostaglandin F_2α (PG F_2α) and cortisol and a significant decrease in the concentration of progesterone. It is suggested that endotoxin caused abortion in camels was a consequence of endotoxin induced PG F_2α secretion resulting in luteal regression and decreased progesterone concentration.     

Simple direct radioimmunoassay of the F prostaglandins

A simplified and accurate method of determining the F prostaglandins in 0.1 ml of serum without previous extraction is described. The procedure involves addition of anti-prostaglandin F_2α to serum followed by tritiated prostaglandin, equilibration for 4 hours, removal of unbound prostaglandin with dextran-coated charcoal and subsequent liquid scintillation counting of the supernatant. The mean ± S.D. concentration of prostaglandin F_2α in the serum of 15 healthy men was 90 ± 33 pg/ml and in 20 women 108 ± 43 pg/ml.     

The radioimmunological estimation of 13, 14-dihydro-15-ketoprostaglandin E2 in plasma and cervical mucus

Highly specific antibodies to 13,14-dihydro-15-ketoprostaglandin E2 (PGEM) were raised in rabbits. The animals were immunized with PGEM-bovine serum albumin (BSA)-conjugates. The metabolites were extracted with dichloromethane followed by column chromatography. The final antisera dilution was 1:15000 and the cross-reactivity towards prostaglandin A2, F2 alpha, I2, 13,14-dihydro-15-ketoprostaglandin F2 alpha was less than 0.1%. The limit of detection was 7.8 +/- 4.7 pg/ml plasma at the standard range of 3.9 to 500 pg/ml. The intra- and inter-assay variations were 5 and 12%, respectively. PGEM was measured throughout the menstrual cycle in female volunteers. In normal ovulatory women (n = 6) plasma concentrations of PGEM varied between 0.94 to 2.19 ng/ml. A significant increase of plasma PGEM was detected in the preovulatory phase of the cycle (P less than 0.01) over basal levels. In three of these volunteers cervical mucus was analyzed on PGEM and PGFM concentrations showing a fluctuation from 2 pg to 109 pg for PGEM and 0.05 pg to 2.4 pg for PGFM per ml of cervical mucus. The lowest concentrations have been found at the time of ovulation. The application of the radioimmunological method to the measurement of PGEM in addition to the measurement of prostaglandin E2 may be useful for estimating the turnover rates of this fatty acid.     

Determination of prostaglandin F in blood plasma using chemically modified bacteriophage technique

The levels of prostaglandin F found in human and rabbit plasma were determined by the chemically modified bacteriophage assay.Prostaglandin F₂α was coupled covalently to bacteriophage T4 using carbodiimide as cross linking agent and the conjugated phage could be inactivated by anti-prostaglandin F₂α antibodies. Prostaglandins specifically inhibited the modified phage inactivation caused by antiserum and as little as 200 picograms of prostaglandin F₂α could be detected by this system. Anti-prostaglandin F₂α antibodies had a high specificity toward prostaglandin F₂α and exhibited a very low degree of cross reaction to the other prostaglandin derivatives. The concentration of the extracted prostaglandin F₂α from the plasma containing exogenous prostaglandin F₂α could be determined with a high recovery.In normal human males and in male rabbits, 0.300.82 and 0.421.22 nanograms of prostaglandin F per ml of plasma were found, respectively. These levels of prostaglandin F in plasma agree with those determined by the radioimmunoassay.     

Radioimmunoassay of 13, 14-dihydro-15-keto-prostaglandin F2a

A simple method is described for the determination of 13, 14-dihydro-15-keto-prostaglandin F_2a, (PGFM), using a highly specific antiserum raised in New Zealand rabbits. It involves extraction of human peripheral venous plasma with diethyl ether after addition of tritiated PGFM and HCl. Radioimmunoassay is performed on appropriate aliquots; after 2 hours or overnight equilibration the bound and free metabolite are separated using dextran-coated charcoal. The mean values ± S.D. obtained are as follows: healthy males 32 ± 16 pg/ml, females during follicular phase 48 ± 18 pg/ml, luteal phase 37 ± 8 pg/ml, first trimester of pregnancy 66 ± 33 pg/ml, second trimester 67 ± 42 pg/ml and third trimester 72 ± 26 pg/ml.     

Plasma prostaglandin levels and circulating fuel levels in rats with diabetic ketoacidosis: Effects of cyclooxygenase inhibitors and of alpha and beta adrenergic blockade

We studied the effects of two structurally unrelated inhibitors of the fatty acid cyclooxygenase and of alpha and beta adrenergic blockade on the elevated plasma levels of 13,14-dihydro-15-keto-prostaglandin (PG)E₂, 6-keto-PGF_1α and thromboxane(TX)B₂, the stable derivatives of PGE₂, PGI₂ (prostacyclin) and TXA₂, respectively, in rats with streptozotocin-induced diabetic ketoacidosis (DKA). Meclofenamic acid and indomethacin each produced a significant decrease in the elevated plasma levels of 13,14-dihydro-15-keto-PGE₂, 6-keto-PGF_1α and TXB₂. Phentolamine significantly reduced the plasma level of TXB₂ but had no effect on the elevated circulating levels of glucose, free fatty acids, total ketones, 13,14,-dihydro-15-keto-PGE₂ or 6-keto-PGF_1α. Propranolol significantly reduced the elevated circulating levels of glucose, free fatty acids and total ketones but had no effect on the levels of the three prostaglandin derivatives. The ability of meclofenamic acid and indomethacin to reduce the plasma levels of 13,14-dihydro-15-keto-PGE₂, 6-keto-PGF_1α and TXB₂ confirms that the plasma levels of these three derivatives are elevated in rats with DKA. Since abnormalities in the production of PGI₂ and perhaps other cyclooxygenase derivatives may contribute to the pathogenesis of certain important hemodynamic and gastrointestinal features of DKA, cyclooxygenase inhibitors may play a role in the management of selected patients with this disorder. Alpha adrenergic activity is essential for the maintenance of the elevated plasma TXB₂ level in rats with DKA. The fall in the plasma TXB₂ level during alpha adrenergic blockade appears to reflect inhibition of platelet aggregation and platelet TXA₂ production, but other sources of the elevated plasma TXB₂ level in DKA are not excluded. Beta adrenergic activity contributes to the maintenance of elevated circulating levels of glucose, free fatty acids and total ketones in experimental DKA but not to the elevated plasma levels of the prostaglandin derivatives.     

On the metabolism of prostaglandins by human gastric fundus mucosa.

1.Specific radioimmunoassays for the prostaglandins E2, F2alpha and A2 and the metabolites 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2alpha and 13,14-dihydro-15-keto-prostaglandin F2alpha were used to study the metabolism of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric fundus mucosa. 2.Three prostaglandin-metabolizing enzymes were found in the 100 000 X g supernatant of human gastric fundus mucosa, 15-hydroxy-prostaglandin-dehydrogenase, delta13-reductase and delta9-reductase. The specific activity was highest for 15-hydroxy-prostaglandin-dehydrogenase and lowest for delta9-reductase. 3.Formation of prostaglandin A2 (or B2) was not observed under the same conditions. 4.None of the three enzyme activities detected in the 100 000 X g supernatant was found in the 10 000 X g and 100 000 X g pellets of human gastric fundus mucosa. 5.The results indicate that high speed supernatant derived from human gastric mucosa can rapidly metabolize prostaglandin E2 and prostaglandin F2alpha to the 15-keto and 13,14-dihydro-15-keto-derivatives. Furthermore, prostaglandin E2 can be converted to prostaglandin F2alpha, the biological activity of which, on gastric functions, differs from that of prostaglandin E2.     

Secretory diarrhea in villous adenoma of rectum: Effect of treatment with somatostatin and indomethacin

The effects of treatment with the synthetic longacting somatostatin analogue SMS-201-995 were studied in a patient with a fluid and electrolyte secreting villous adenoma of the rectum. The effects of SMS-201-995 on rectal fluid volume and electrolyte loss, and local and general prostanoid production were compared with those of treatment with indomethacin.During treatment with the somatostatin analogue iso-osmolar rectal fluid production increased about 25%; the quantity of prostaglandin E₂ in the rectal fluid rose almost 20-fold. Prostaglandin F_2α, 6-keto-prostaglandin F_1α and 13,14-dihydro-15-keto-prostaglandin F_2α output showed similar, though less impressive increments during somatostatin treatment. The somatostatin analogue did not affect urinary prostanoid excretion except for levels of 2,3-dinor-thromboxane B₂, which doubled. With indomethacin treatment diurnal rectal fluid production dropped by about 50% and all prostanoids measured in urine and rectal fluid decreased below control values.It appears that the somatostatin analogue SMS-201-995 has a marked stimulatory effect on the in vivo prostanoid production by the villous adenoma. Perhaps this stimulation is not confined to the tumor only, but also affects thromboxane synthesis.