F2-isoprostane excretion rate and diurnal variation in human urine

8-Iso-PGF2alpha is formed in vivo by non-enzymatic free radical catalysed oxidation of arachidonic acid. Urinary measurement of this compound has previously been shown to reflect the oxidative stress of the body in human and animal studies. To investigate the normal excretion rate and a possible diurnal variation of 8-iso-PGF2alpha excretion in humans urinary samples were collected from ten healthy volunteers of both sexes at different times during a day and as a 24-h urine sample. The samples were analyzed by a newly developed radioimmunoassay with a specific antibody against free 8-iso-PGF2alpha. There was no diurnal variation in the urinary levels of 8-iso-PGF2alpha during the day in this study. Neither was there any statistically significant difference between the 8-iso-PGF2alpha levels at any time of the day or in the morning urine samples compared to the 24-h urine samples. In conclusion, all urine samples collected at any time of the day, preferably a morning urine sample (representative urine from 6-8 hours), can thus be used to obtain a reliable and adequate value of the amount of the 8-iso-PGF2alpha excretion in urine in healthy individuals.     

Urinary excretion of immunoreactive prostaglandin F2 alpha in healthy children and adults

The 24-hours urinary excretion of immunoreactive prostaglandin F2 alpha (U-iPGF2 alpha) in normal children on a free diet was not significantly different in 30 boys (aged 3-15 years; geometric mean 589 ng/24 h) compared to 27 girls (aged 4-14 years; mean 473 ng/24 h). In both sexes this excretion rose with age until adolescence where it reached a plateau. In normal adults the men had significantly higher (p less than 0.001) excretions of U-iPGF2 alpha than the women; also body weight and urinary creatinine excretion were higher in men (p less than 0.001). In the children, as well as in the total population, U-iPGF2 alpha correlated best with body weight (r = 0.44 and r = 0.48 respectively; p less than 0.001) and the urinary creatinine excretion (r = 0.53 and 0.57 respectively; p less than 0.001); both body weight and urinary creatinine excretion are reflections of total body development. After the correction for urinary creatinine excretion or for body weight, the sex difference in the adult U-iPGF2 alpha totally disappeared.     

Intra-day variation of in vivo prostaglandin F2alpha formation in healthy subjects

Prostaglandin F(2alpha) (PGF(2alpha)) is a major stable prostaglandin formed in vivo in physiological and pathophysiological situations and has mainly potent vasoconstrictive and pro-inflammatory properties. PGF(2alpha) is now used as an indicator of acute and chronic inflammation in human clinical settings but the extent of daily variation of PGF(2alpha)in vivo in healthy humans is unknown. We quantified levels of the PGF(2alpha) metabolite 15-keto-dihydro-PGF(2alpha) in 10 healthy males and females in spot urine samples during the day (including morning urine sample) and in 24-h urine during the same day. The intra-day coefficient of variation was 20.9%. However, the total mean value of 15-keto-dihydro-PGF(2alpha) in urine collected in the morning did not significantly differ from the mean level of 15-keto-dihydro-PGF(2alpha) in the 24-h urine samples in the 10 subjects. 15-Keto-dihydro-PGF(2alpha) levels in morning urine showed a positive linear correlation with levels of 15-keto-dihydro-PGF(2alpha) in 24-h urine (R=0.72, P<0.05). In conclusion, formation of PGF(2alpha) shows a biological variation within the day in healthy humans which should not be overlooked when planning a clinical study. Single morning urine samples can be used as an alternative to 24-h urine collections for quantification of PGF(2alpha) formation to simplify the sampling regime in larger clinical studies.     

Urinary excretion of prostaglandin E2 and prostaglandin F2alpha in potassium-deficient rats

Potassium-deficiency was induced in rats by dietary deprivation of potassium. The animals became polyuric and urine osmolality decreased more then three-fold compared to controls. Urinary excretion of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) did not increase during 2 weeks of potassium depletion. Partial inhibition of renal prostaglandin synthesis by meclofenamate did not increase the urine osmolality after water deprivation. These results make unlikely the hypothesis that the polyuria of potassium-deficiency, is the result of enhanced renal synthesis of prostaglandins with subsequent antagonism of the hydro-osmotic effect of vasopressin. Male animals consistently excreted less PGE2 than female animals.     

Flavanol-rich cocoa drink lowers plasma F(2)-isoprostane concentrations in humans

Flavan-3-ols are potent antioxidants in vitro, but convincing evidence for antioxidant action in vivo is lacking. We examined whether an oxidative stress-mediated increase in plasma F(2)-isoprostanes is counteracted by a flavanol-rich cocoa beverage. Twenty volunteers were examined in a comparative randomized double-blind crossover design with respect to ingestion of high-flavanol cocoa drink (HFCD; 187 mg flavan-3-ols/100 ml) vs. low-flavanol cocoa drink (LFCD; 14 mg/100 ml). With 10 individuals, the treatment was combined with strenuous physical exercise. Total (esterified plus nonesterified) F(2)-isoprostanes were analyzed by GC/MS. LFCD caused a slight increase in the mean (+/- SEM) plasma concentrations of F(2)-isoprostanes 2 and 4 h after intake (2.16 +/- 0.19 nM at 4 h vs. 1.76 +/- 0.11 nM at 0 h, n = 10), which may be attributable to postprandial oxidative stress. This increase did not occur with HFCD (1.57 +/- 0.06 nM at 4 h vs. 1.65 +/- 0.10 nM at 0 h, n = 10). The difference in F(2)-isoprostanes 2 and 4 h after intake of HFCD vs. LFCD became statistically significant when the intake was combined with physical exercise (P < 0.01, ANOVA). We conclude that dietary flavanols, using cocoa drink as example, can lower the plasma level of F(2)-isoprostanes, indicators of in vivo lipid peroxidation.     

The response of muscle interstitial F2-isoprostane (8-ISO-PGF2alpha) during dynamic muscle contractions in humans

8-Iso-prostaglandin F2alpha (8-iso-PGF2alpha) is a characteristic F2-isoprostane which is produced in humans via a free radical-catalysed lipid peroxidation mechanism of arachidonic acid, independent of the cycloxygenase pathway. The measurement of the plasma levels of 8-iso-PGF2alpha was shown to be the most reliable biochemical index of oxidant stress status in the human body. However, there is no reference in literature of local muscle interstitial 8-iso-PGF2alpha production during dynamic muscle contractions. The aim of the present study was to evaluate the response of 8-iso-PGF2alpha during intensive exercise with a cycle ergometer. Two microdialysis probes with CMA-60 microdialysis catheters were inserted into the vastus lateralis muscle of the right leg of six healthy male volunteers. After insertion, these microdialysis probes were attached to a perfusion pump that perfused ringer acetate solution at a rate of 0.3 microl/min. The dialysate fluid samples were collected: (a) during a 30 min rest period and (b) during a 30 min period of dynamic exercise with a cycle ergometer at 150 Watts. Our measurements showed that the levels of 8-iso-PGF2alpha in the interstitial fluid (IF) of the vastus lateralis muscle increase significantly during exercise (from 113.5 +/- 30.2 to 329.9 +/- 69.8 pg/ml, P = 0.05). In conclusion, dynamic muscle exercise produces a local increase of the IF levels of 8-iso-PGF2alpha due to local peroxidation injury of the contractive muscle. The microdialysis method is widely applied, easily repeated and it could contribute in evaluating the local lipid muscle peroxidation during intensive exercise.     

F2-isoprostane induced prostaglandin formation in the rabbit

F(2)-isoprostanes, non-enzymatic free radical mediated products of arachidonic acid, have shown to form during various oxidant stress status and have potent biological effects. This study investigates to what extent 8-iso-PGF(2alpha) (a major F(2)-isoprostane), a bioactive product of lipid peroxidation can modify endogenous prostaglandin F(2alpha) (PGF(2alpha)) formation since prostaglandins are inflammatory as well as potent vasoregulatory substances that modulate diverse important physiological functions, and also form during acute and chronic inflammation. An immediate appearance and disappearance of 8-iso-PGF(2alpha) was seen in both plasma and urine within a short interval after i.v. administration of 43 microg/kg of 8-iso-PGF(2alpha) to the rabbits. A successive but differential formation of PGF(2alpha) resulted in a rapid and pulsatile increase of plasma 15-keto-dihydro-PGF(2alpha), a major metabolite of primary PGF(2alpha). Later, this compound was excreted efficiently as intact compound into the urine during the 3 h of experiment. A 8-fold increase of PGF(2alpha) metabolite in plasma at 10 min and 12-fold increase in the urine at 30-60 after the i.v. administration of 8-iso-PGF(2alpha) was observed which continued throughout the 3 h of experiment. This observation suggests that pharmacologically administered or endogenously produced 8-iso-PGF(2alpha) during oxidant stress induces prostaglandin formation presumably through the classical cyclooxygenase-catalysed arachidonic acid oxidation which might be inflammatory itself to the cells and exerts further vasoconstrictive effects.     

Coupling between cyclooxygenases and prostaglandin F(2alpha) synthase. Detection of an inducible,glutathione-activated,membrane-bound prostaglandin F(2alpha)-synthetic activity

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF(2alpha) only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF(2alpha) from endogenous AA, even though significant increase in PGF(2alpha) production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF(2alpha)-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.     

Canine prostaglandin F2alpha receptor (FP) and prostaglandin F2alpha synthase (PGFS): molecular cloning and expression in the corpus luteum

In the dog luteolysis is not affected by hysterectomy. This observation led to the hypothesis that paracrine/autocrine rather than endocrine mechanisms of PGF2alpha are responsible for luteal regression in the dioestric bitch. The present experiments tested for the capacity of canine CL to produce and respond to PGF2alpha by qualitatively and quantitatively determining the expressions of PGFS, the enzyme converting PGH2 into PGF2alpha, and the PGF2alpha-receptor (FP) in CL of non-pregnant dogs during dioestrus. Canine PGFS and FP were isolated and cloned; both genes show a high homology (82-94%) when compared to those of other species. Relatively weak FP mRNA expression was detected on day 5 of dioestrus. It had increased by day 25 and remained constant thereafter. In situ hybridization (ISH) localized FP solely to the cytoplasm of the luteal cells, suggesting that these cells are the only luteal targets of PGF2alpha in this species. Only negative results were obtained for the expression of PGFS in canine CL by routine qualitative RT-PCR. When Real Time (TaqMan) PCR was applied, repetitively more negative than positive results were obtained at all timepoints. Any positive measurements observed at any point were neither repeatable nor related to the stage of dioestrus. This led us to conclude that expression of PGFS is either absent or present at very low level only. These data suggest that luteal regression in non-pregnant bitches is not modulated by PGF2alpha. However, the FP seems to be constitutionally expressed, explaining the receptivity of canine CL to exogenous PGF2alpha.     

Increased urinary F2-isoprostane excretion in alcoholic liver disease

Free radical-induced lipid peroxidation (LP) is thought to be important in alcoholic liver disease (ALD), however, direct demonstration of increased LP in patients with ALD has been difficult. Quantification of F2-isoprostanes (F2-isoP), prostanoids produced by peroxidation of arachidonic acid, in plasma and urine are sensitive and specific indices of LP in vivo. To determine if LP is increased in ALD, 24-h urinary excretion of F2-isoPs were measured in 10 patients hospitalized because of ALD. The mean urinary excretion of the F2-isoP in the ALD patients' urine was 9.6+/-3.5 ng/mg creatinine, which was significantly elevated compared to controls' urinary excretion, which was 1.7+/-0.2 ng/mg creatinine (p<.01). The urinary excretion of F2-isoP decreased to 3.6+/-1.1 ng/mg creatinine as the patients improved clinically with abstinence over the 1-month period. These data suggest that lipid peroxidation, as assessed by this noninvasive method, is increased in patients with acute ALD and decreases with time as the patients improve clinically with abstinence.     

Radioimmunoassay of main urinary metabolite of prostaglandin F2alpha in normal subjects

Radioimmunoassay of 5alpha, 7alpha-dihydroxy-11-keto-tetranor-prosta-1,16-dioic acid, main urinary metabolite of prostaglandin F2alpha (PGF2alpha-MUM), was performed in normal subjects. Twenty-four hours secretion of PGF2alpha-MUM were 29.04 +/- 9.73 microgram in males and 18.22 +/- 5.88 microgram in females on an average. And an oral administration of aspirin resulted in the remarkable decrease of PGF2alpha-MUM in both sexes.     

Fetal viability does not affect the onset of stretch-induced labor and the increase in amniotic fluid prostaglandin F2 alpha and plasma prostaglandin F2 alpha metabolite levels.

The role of the fetus in the onset and progress of stretch-induced labor and in the change in amniotic fluid prostaglandin F2 alpha and plasma prostaglandin F2 alpha metabolite levels was evaluated in six normal pregnant women (group 1) and six women whose fetuses had been dead for more than one week (group 2). The uterus was distended by a balloon inflated with physiologic saline. Regular uterine contractions occurred, and increased in all patients. Within 21 hours, all patients delivered a normal baby in group 1 and a macerated fetus in group 2. There was no significant difference in induction-delivery interval between the two groups. Both groups showed a significant and similar range of increases in the levels of amniotic fluid prostaglandin F2 alpha and plasma prostaglandin F2 alpha metabolite during treatment (P less than 0.001). Thus, the fetus has no functional role in the onset and progress of stretch-induced labor or in the rise of amniotic fluid prostaglandin F2 alpha and plasma prostaglandin F2 alpha metabolite levels.     

Spontaneous uterine infections are associated with elevated prostaglandin F(2)alpha metabolite concentrations in postpartum dairy cows

Postpartum Holstein (n=21) and Jersey (n=4) cows were used to determine if uterine infections are associated with elevated plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F(2)alpha (PGFM). Based upon clinical examinations and bacterial content of intrauterine fluid samples, cows detected with uterine infections between 21 and 28 d post partum were used (infected; n=14). These cows were matched with herdmates that were free of infection (control; n=11). Beginning on the day the cows were assigned to the experiment (Day 1), blood samples were collected on alternate days for the next 14 to 15 d. Plasma samples were stored at -20 degrees C until assayed. From Day 1 until the end of the experiment, uterine fluid samples were collected transcervically twice weekly for aerobic bacterial culture. Endometrial biopsies were collected between Days 6 and 8 and Days 13 and 15. Control cows did not show signs of uterine infection throughout the trial, and bacterial cultures indicated that there were no significant bacterial populations in the uteri of the control cows. The uteri of infected cows harbored numerous microbes. Actinomyces pyogenes was most prominent. Various species of Streptococcus and Pasteurella were also prevalent in the infected cows. Escherichia coli was present in the uterus of both infected and control cows. Biopsies showed that infected cows had more (P<0.05) neutrophils, plasma cells and lymphocytes in the endometrium than did the control cows. As determined by plasma progesterone concentrations, 83% of the control and 50% of the infected cows had functional luteal tissue during the 2-wk sampling period. Plasma PGFM profiles were linear (P<0.03) and did not differ between treatment groups (P>0.01). However, average plasma PGFM concentrations were greater (P<0.0001) in infected than in control cows. These data indicate that plasma PGFM concentrations are greater in postpartum cows with spontaneous uterine infections then in herdmates free of infection.     

Cardiovascular effects of prostaglandin F(2 alpha) and prostaglandin E(2) in Atlantic cod (Gadus morhua)

Little is known of the cardiovascular functions of prostaglandins in non-mammalian vertebrates. There are indications that prostaglandins may have a function in haemostasis by constricting blood vessels in filament arteries in the fish gill after injury. Our aim was to examine the cardiovascular effect of the prostaglandins F(2 alpha) (PGF(2 alpha)) and E(2) (PGE(2)) with emphasis on branchial circulation. Intra-arterial injections of PGF(2 alpha) (10, 40, 160, 400 nmol kg(-1)) in cod caused a dose-dependent increase in ventral aortic blood pressure, a reduction in cardiac output, and an increase in gill vascular resistance. A contraction of filament arteries was observed with in vivo microscopy only seconds after injection. PGF(2 alpha) may therefore possibly be involved in a haemostatic vasoconstriction. In contrast, the most significant effects of PGE(2) appeared to be on the heart. PGE(2) also reduced dorsal aortic blood pressure.     

Quantitative studies on prostaglandin synthesis in man. II. Determination of the major urinary metabolite of prostaglandins F1 alpha and F2 alpha

A method was developed for quantitative determination of 5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F_1α and F_2α in man. The method was based on the use of the O-methyloxime derivative of [5β-³H; 10,10,12,12-²H₄]5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid as internal standard and determination of ratios between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Excretion values found for healthy human subjects were: males, $\text{10.8–59.0 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 24.0 ± 17.2 (SD) μg) and females, $\text{7.6–13.6 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 10.5 ± 2.1 (SD) μg).     

Farrowing induction with a combination of prostaglandin F(2alpha) and a peripherally acting alpha(2)--adrenergic agonist AGN 190851 and a combination of prostaglandin F(2alpha) and oxytocin

We studied the effect of prostaglandin (PG) F(2alpha)-AGN 190851 on farrowing induction and compared it with that of PGF(2alpha)-oxytocin. Eighty crossbred, multiparous sows were randomly assigned to the following 4 treatment groups of 20 sows each: 1) control, saline-saline; 2) PGF(2alpha) (10 mg/sow)-oxytocin (30 IU/sow); 3) PGF(2alpha) (10 mg/sow)-AGN 190851 (0.06 mg/kg); and 4) PGF(2alpha) (10 mg/sow)-AGN 190851 (0.1 mg/kg). Either PGF(2alpha) or saline was administered intramuscularly on Day 111 of gestation at 11:30 h; AGN 190851, oxytocin or saline was administered intramuscularly 20 h after the first injection. The PGF(2alpha)-AGN 190851 (0.1 mg/kg) treated sows had the shortest mean farrowing interval (2.1 +/- 1.6 h, mean +/- SD) compared with the remaining treatment groups (control: 67.1 +/- 26.2 h; PGF(2alpha)-oxytocin: 5.6 +/- 6.7 h; PGF(2alpha)-AGN 190851 [0.06 mg/kg]: 3.0 +/- 2.8 h). Duration of farrowing, litter size, litter weight and interval from weaning to first estrus in sows were not significantly changed by these treatments. The PGF(2alpha)-oxytocin group had a significantly higher stillbirth rate than the control group, whereas the PGF(2alpha)-AGN 190851 (0.1 mg/kg) group had the lowest number of pigs born dead and stillbirth rate among the 4 treatment groups. These results suggested that the PGF(2alpha)-AGN 190851 combination can be used as an alternative method to PGF(2alpha)-oxytocin for synchronizing farrowing.     

Role of prostaglandin F2alpha in ovulation.

Using radioimmunoassay procedures, the levels of plasma, uterine and ovarian prostaglandin (PG) F2alpha, and those of plasma estradiol and progesterone were measured in intact, hysterectomized or ovariectomized immature female rats pretreated with PMS and subsequent HCG. Occurrence of ovulation was confirmed at 8 hours after the HCG administration not only in the intact rats but also in the hysterectomzied rats. The levels of plasma estradiol and progesterone, and of uterine and ovarian PGF2alpha rose with the PMS injection alone, but they did not reach the peaks before the HCG administration. Both plasma estradiol and uterine PGF2alpha showed a peak at 2 hours after the HCG injection. These peaks were antecedent 2 or 6 hours before the peaks of ovarian and plasma PGF2alpha, respectively. However, such increase of uterine PGF2alpha does not seem to be indispensable for ovulation, because ovulation could occur in the hysterectomized rats. The levels of ovarian PGF2alpha showed a high plateau from 4 to 8 hours after the HCG injection, and then rapidly decreased after ovulation. The levels of plasma PGF2alpha peaked not only in the intact rats but also in the hysterectomized rats at 8 hours after the HCG treatment. But in the ovariectomized rats, this plasma PGF2alpha peak at 8 hours disappeared and there was no statistical change of plasma PGF2alpha throughout the PMS-HCG treatment. Plasma progesterone gradually increased and reached the maximum at 10 hours after the HCG injection. These results conclude that the main source of increased plasma PGF2alpha during the ovulatory process induced with the PMS-HCG treatment is the ovary, and it is strongly suggested that a rapid increase of PGF2alpha in the ovary may play some important role(s) in the ovulatory process.     

Effect of prostaglandin F2 alpha (PGF2 alpha) on sources of progesterone and pregnancy in intact, ovariectomized and hysterectomized 90-100 day pregnant ewes.

A single dose of 8 or 16 mg of PGF2 alpha per 58 kg body weight was injected intramuscular into intact, ovariectomized or hysterectomized 90-100 day pregnant sheep in three separate experiments. Both doses of PGF2 alpha decreased the weights of the corpora lutea (P less than or equal to 0.05) and the concentration of progesterone in ovarian venous plasma at 72 hr (P less than or equal to 0.05) compared to the 0 hr sample within treatment groups and to control ewes at 72 hr in intact and hysterectomized pregnant ewes. In hysterectomized pregnant ewes, progesterone in jugular plasma declined (P less than or equal to 0.05) from 0 to 72 hr but never fell below 4 mg/ml and this decrease in progesterone after 8 or 16 mg PGF2 alpha was greater than in control hysterectomized ewes (P less than or equal to 0.05). There was a significant decrease in progesterone over time in jugular or uterine venous plasma in the presence of absence of the ovaries in 90-100 day pregnant ewes (P less than or equal to 0.05) but the profiles of progesterone were not different between vehicle and PGF2 alpha-treated ewes (P greater than or equal to 0.05). Uterine venous progesterone never declined below 30 ng/ml in the presence or absence of the ovaries and there was a significant quadratic increase (P less than or equal to 0.05) in uterine venous progesterone toward the end of the 72 hr sampling period indicating an increase in steroidogenic activity of the placenta. PGF2 alpha did not affect the number of abortions in intact or ovariectomized pregnant ewes (P greater than 0.05). Thus, the corpus luteum of sheep at 90-100 days of pregnancy is functional and responsive to PGF2 alpha, placentomes are functional but do not appear to be responsive to the doses of PGF2 alpha tested and PGF2 alpha was not an abortifacient over the 72 hr treatment period.     

Luteolytic effects of prostaglandin F2alpha on day 8 to 19 corpora lutea in the bitch

Luteolysis was induced in 5 experimental Beagle (8 cycles) and 7 client-owned bitches treated with 150 to 200 microg/kg, sc of prostaglandin F2alpha administered twice daily for 4 d, starting on Days 8 to 19 after the onset of cytological diestrus. Five experimental Beagle bitches had been mated during the estrus preceding treatment, and copulation had been confirmed in 2/7 client-owned bitches presented for termination of unwanted pregnancy. Serum progesterone concentration (mean +/- SD) declined from 26.1 +/- 66 ng/ml before treatment to 0.3 +/- 0.4 ng/ml on the fourth day of treatment One of the 7 client-owned bitches maintained her pregnancy even though serum progesterone concentrations were less than 0.5 ng/ml on the third and fourth day of treatment. Mean (+/- SEM) inter-estrous intervals before and following prostaglandin-induced luteolysis were 207.3 +/- 12.4 (n = 11 cycles in 6 bitches) and 95.5 +/- 20.0 d (n = 6 cycles in the same 6 bitches; P < 0.0001), respectively These results suggest that effective prostaglandin-induced luteolysis can be achieved with administration of 180 microg/kg during the third week of diestrus in pregnant and nonpregnant bitches.