The effect of 19-hydroxy prostaglandins on the human myometrium in vitro

Human semen contains high amounts of different prostaglandins of which the quantitatively dominating compounds are 19-hydroxy PGE and 19-hydroxy PGF. The effect of PGE and PGF on the contractility of the human uterus is well known whereas the corresponding 19-hydroxy derivatives have not previously been examined in detail. In the present investigation 19-hydroxy PGE1 was shown to dose-dependently decrease the frequency and the amplitude of the contractions in human myometrial tissue in vitro. The effect of seminal 19-hydroxy prostaglandins may thus be of importance for sperm transport through the female genital tract.     

11, 15, 19-trihydroxy-9-ketoprost-13-enoic acid and 11, 15, 19-trihydroxy-9-ketoprosta-5, 13-dienoic acid in human seminal fluid.

Two novel prostaglandins (PG) have been found in human seminal fluid which had been frozen immediately after collection. They were characterized by combined gas-liquid chromatography-mass spectrometry of various derivatives as 19-hydroxy prostaglandin E1 (11, 15, 19-trihydroxy-9-ketoprost-13-enoic acid) and 19-hydroxy prostaglandin E2 (11, 15, 19-trihydroxy-9-ketoprosta-5, 13-dienoic acid). They were present in three to five times the quantity of prostaglandins E1 and E2. Incubation of seminal fluid for 3 hr at 25 degrees C reduced levels of 190H-PGEs2.5-fold and PGE22-fold, while increasing levels of PGAs and PGBs 2-fold. No 190H PGA or 190H PGB was detected in extracts of unincubated fluid. The PGAs, PGBs and their 19-hydroxy analogs are probably artifacts arising metabolically or as a result of classical isolation techniques.     

The measurement of E and 19-hydroxy E prostaglandins in human seminal plasma.

A method is described which measures the four main prostaglandins of human semen (PGE1, E2, 19-hydroxy PGE1, and 19-hydroxy PGE2). For routine measurements E1 and E2 are measured together as are 19-OH E1 and 19-OH E2. These are measured by forming oximes in aqueous solution extraction, methylation and trimethyl silylation followed by gas chromatography. The method has sufficient sensitivity to measure the levels found in the majority of semen samples. The normal range in men with proven fertility was 90 to 260 mug/ml of 19-hydroxy Es and 30-200 mug/ml of Es.     

Rapid and slow hydroxylators of seminal E prostaglandins

Human seminal fluid contains prostaglandin (PG) E1, PGE2, 19-hydroxy-PGE1 and 19-hydroxy-PGE2 in large and variable amounts. 19-Hydroxy-PGE1 and 19-hydroxy-PGE2 are formed from PGE1 and PGE2 by prostaglandin 19-hydroxylase, a cytochrome P-450 enzyme, in seminal vesicles. The hypothesis that genetic polymorphism of this enzyme might contribute to the variable concentrations of PGE1, PGE2, 19-hydroxy-PGE1 and 19-hydroxy-PGE2 was examined by analysis of seminal fluid of 40 normal men. E prostaglandins were measured with 17-phenyl-PGE2 as an internal standard by high-performance liquid chromatography on beta-cyclodextrin silica. Using the ratios of substrate/product, i.e., R1 = PGE1/19-hydroxy-PGE1 and R2 = PGE2/19-hydroxy-PGE2, as indicators of prostaglandin 19-hydroxylase capacity, a bimodal distribution of R values was found: nine men (23%) were slow hydroxylators (R1 greater than 0.45 and R2 greater than 0.45), while the remaining men were rapid hydroxylators (both R1 and R2 less than 0.45). Semen of slow hydroxylators and semen of the five most rapid hydroxylators (both R1 and R2 less than 0.10) differed in absolute amounts of PGE1 and PGE2 but not in 19-hydroxy-PGE1 and 19-hydroxy-PGE2. 20-Hydroxy-PGE1 and 20-hydroxy-PGE2 are formed from PGE1 and PGE2 by cytochrome P-450 in the vesicular glands and the ampullae of deferent ducts of the ram. Seminal fluid of five rams was analyzed for PGE1, PGE2, 20-hydroxy-PGE1 and 20-hydroxy-PGE2, and a large variation in substrate/product ratios was found. Polymorphism of cytochrome P-450 might contribute to variations in seminal prostaglandins in man and in sheep.     

Maternal dietary n-3 fatty acids alter cardiac ventricle fatty acid composition, prostaglandin and thromboxane production in growing chicks

The effects of feeding n-6 and n-3 fatty acids to broiler hens on cardiac ventricle fatty acid composition, and prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) production of hatched chicks were investigated. Fertile eggs obtained from hens fed diets supplemented with 3.5% sunflower oil (Low n-3), 1.75% sunflower+1.75% fish oil (Medium n-3), or 3.5% fish oil (High n-3) were incubated. The hatched chicks were fed a diet containing 18:3 n-3, but devoid of longer chain n-6 and n-3 fatty acids for 42 days. Arachidonic acid content was lower in the cardiac ventricle of High n-3 and Medium n-3 compared to Low n-3 birds for up to 2 weeks (P<0.002). Long chain n-3 fatty acids were higher in the cardiac ventricle of chicks from hens fed High and Medium n-3 diets when compared to chicks from hens fed the Low n-3 diet. Differences in long chain n-3 fatty acids persisted up to four weeks of age (P<0.001). Peripheral blood mononuclear cells (PBMNC) of 7-day-old High n-3 broilers produced significantly lower PGE2 and TXA2 than PBMNC from Low n-3 and Medium n-3 birds. These results indicate that maternal dietary n-3 fatty acids increases cardiac ventricle n-3 fatty acids while reducing arachidonic acid and ex vivo PGE2 and TXA2 production during growth in broiler chickens.     

Occurrence of prostaglandin E3 in human urine as a result of marine oil ingestion: gas chromatographic-mass spectrometric evidence

This pilot study is an effort to elucidate the metabolic fate of dietary eicosapentaenoate in vivo and its influence on arachidonate cyclooxygenation at the renal level. The ultimate objective is to shed light on the biochemical mechanisms responsible for the physiologic effects of marine oil on humans. We found prostaglandin E3 (PGE3) in urine of a female volunteer who ingested 10-50 g/day of MaxEPA fish oil concentrate for 4 years. PGE3, a cyclooxygenase metabolite of eicosapentaenoate, could not be detected in 24-h urine pools from the same subject 16 weeks after fish oil supplementation ended. The appearance of PGE3 was concurrent with a reduction of urinary PGE2. Identification of the trienoic prostaglandin was based on comparison of chromatographic behavior of three distinct derivatives of endogenous PGE3 with that of authentic material, and on selected ion monitoring mass spectrometry.     

Gammalinolenic acid-enriched diet alters cutaneous eicosanoids

There are reports that vegetable oils containing gammalinolenic acid (GLA) may exert beneficial effects on inflammatory skin disorders. To determine whether or not dietary GLA exerts any modulatory role on cutaneous eicosanoids, guinea pigs were fed either a control diet containing safflower oil (less than 0.5% GLA) or borage oil, a GLA-rich diet containing 25% GLA. After an 8-week feeding period, epidermal samples from both animal groups were analyzed for fatty acid composition and tissue eicosanoids. Analysis of epidermal neutral lipids and phospholipids in borage oil-fed animals showed a marked increase in GLA and its elongase product, dihomogammalinolenic acid (DGLA). Similarly, analysis of epidermal eicosanoids in the borage oil-fed animals revealed significant increases in the amounts of the 15-hydroxy fatty acid (15-OH-20:3n-6) and prostaglandin PGE1, both metabolites of DGLA. Since these metabolites have anti-inflammatory potential, our results suggest that increased dietary GLA could result in the generation of local anti-inflammatory metabolites thus providing a non-toxic approach to suppression of cutaneous inflammatory skin disorders.     

Dietary fatty acid composition rather than vitamin E supplementation influence ex vivo cytokine and eicosanoid response of porcine alveolar macrophages

This study examined the influence of different dietary fat sources (animal fat, sunflower oil, and fish oil) and supplementation of vitamin E (85, 150 and 300 mg all-rac-alpha-tocopheryl acetate/kg diet) on the ex vivo synthesis of eicosanoids and cytokines by porcine alveolar macrophages. Supplementation of vitamin E provoked an increase in the concentration of alpha-tocopherol of the macrophages irrespective of fat sources. Fish oil increased the macrophage n-3 content with 100% and 40%, and reduced the n-6 with 60% and 53% in comparison with sunflower oil and animal fat, respectively. Fish oil decreased the production of TNF-alpha, IL-8, LTB4, and PGE2 (but not IL-6) relative to the other dietary fat sources, and no difference was observed between sunflower oil and animal fat. Positive correlations were found between the n-6 fatty acid content and the production of PGE2, and the PGE2 production was positively correlated with TNF-alpha and IL-8. Negative correlations were found between the n-3 PUFA content and the concentration of PGE2, TNF-alpha and IL-8. In conclusion, dietary fish oil supplemented at a level of 5%, but not supplemental vitamin E, influenced the inflammatory responses of alveolar macrophages isolated from weaned pigs relatively to animal fat and sunflower oil.     

Isolation and biosynthesis of 20-hydroxyprostaglandins E1 and E2 in ram seminal fluid

Ram semen was found to contain 20-hydroxyprostaglandin E1 and 20-hydroxyprostaglandin E2. The relative amounts of the two compounds were almost equal, although ram semen contained at least 10 times more prostaglandin E1 than prostaglandin E2. The accessory genital glands of the ram were analyzed for their capacity to metabolize [14C]arachidonic acid to prostaglandins. Biosynthesis of prostaglandins was only found in microsomes of the mucosa of the ampulla of vas deferens and in microsomes of the vesicular glands. Ram vesicular glands and the ampulla of vas deferens were also found to contain the two 20-hydroxylated E prostaglandins. Microsomes of ram vesicular glands and NADPH metabolized exogenous prostaglandin E2 to 20-hydroxyprostaglandin E2 albeit in low yields. Prostaglandin E2 appeared to be a better substrate than prostaglandin E1. Microsomes of human seminal vesicles and NADPH metabolized exogenous prostaglandin E2 to 19-hydroxyprostaglandin E2. The results show that 19- and 20-hydroxylation of prostaglandins occurs in human and ram seminal vesicles, respectively, and possibly also in the ampulla of vas deferens of the ram. The ram and human enzymes specifically hydroxylated the terminal and the penultimate carbon of prostaglandin E2, respectively.     

The effect of dietary n-3 long-chain polyunsaturated fatty acids on femur mineral density and biomarkers of bone metabolism in healthy, diabetic and dietary-restricted growing rats

INTRODUCTION: Dietary fish oil promotes bone formation in healthy states, but its effect during insulin deficiency or nutrient restriction is unclear. METHODS: Eighty weanling male rats were randomized to receive an injection of streptozotocin to induce insulin deficiency (diabetes) or saline (control) and a diet containing soy oil or corn + fish oil for 35 days. Half of the saline-injected rats were randomized to 20% dietary restriction. Measurements were growth, biomarkers of bone metabolism and femur bone mass. RESULTS: Density of femur was elevated in the corn + fish group and reduced in the diabetes group. Plasma osteocalcin and bone prostaglandin E2 (PGE2) were reduced by the corn + fish diet. N-telopeptide, IGF-1, bone PGE2 and urinary Ca were highest and calcitriol lowest in the diabetes group. CONCLUSIONS: These data suggest that the benefit of a diet high in n-3 long-chain polyunsaturated fatty acid is most advantageous to long bone density in healthy states.     

Metabolism of prostaglandins, prostaglandin analogs and thromboxane B2 by lung and liver microsomes from pregnant rabbits

Liver microsomes from pregnant rabbits converted prostaglandins F2 alpha, E1, and E2 to their 20-hydroxy metabolites along with smaller amounts of the corresponding 19-hydroxy compounds. Prostaglandins E1 and E2 were also reduced to prostaglandins F1 alpha and F2 alpha, respectively, and prostaglandin E1 was isomerized to 8-isoprostaglandin E1. The above products were also identified after incubation of prostaglandins with liver microsomes from non-pregnant rabbits. In this case, the yield of 20-hydroxy metabolites was much lower. Thromboxane B2 and a number of prostaglandin F2 alpha analogs were also hydroxylated by lung and liver microsomes from pregnant rabbits. The relative rates of hydroxylation by lung microsomes were: prostaglandin E2 approximately prostaglandin F2 alpha approximately 16,16-dimethylprostaglandin F2 alpha approximately 13,14-didehydroprostaglandin F2 alpha greater than thromboxane B2 greater than 15-methylprostaglandin F2 alpha approximately 17-phenyl-18,19,-20-trinorprostaglandin F2 alpha approximately ent-13,14-didehydro-15-epiprostaglandin F2 alpha. Similar results were obtained with liver microsomes except that thromboxane B2 was a relatively poorer substrate for hydroxylation.     

In vitro effect of eicosapentaenoic and docosahexaenoic acids on prostaglandin E2 synthesis in a human lung carcinoma

The capacity to synthesize both prostaglandins E1 (PGE1) and E2 (PGE2) has been determined in human lung mucoepidermoid carcinoma homogenates when [14C]-fatty acid precursors were added to the incubation medium. Only 10% of the total radioactivity recovered in PGs was found in PGF1 alpha and PGF2 alpha. The experiments were principally focused to inhibit the PGE2 synthesis either with pure eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids or with mixtures of both n-3 fatty acids obtained from fish oil. The results demonstrated that significant inhibitions were found when using 25 microM or a higher concentration of pure EPA or DHA in the incubation medium; however, 5 microM of mixtures of different EPA/DHA ratio caused the same inhibition. The results suggest that EPA and DHA, when added together, may enforce their inhibitory effect on PGE2 synthesis.     

The biosynthesis of the 3-series prostaglandins in rat uterus after alpha-linolenic acid feeding: Mass spectroscopy of prostaglandins E and F produced by rat uteri in tissue culture

The abnormal uterine activity associated with dietary n-3 fatty acids may result from competitive inhibition of PG2 production. Uterine synthesis of 2- and 3-series prostaglandins F(PGF) and E(PGE) was studied using mass spectrophotometry in rats fed diets containing predominantly n-3 fatty acid, n-6 fatty acid, or control pelleted diet. Mass spectra of PGF (Me, TMS and Me, TBDMS derivatives) synthesised by uteri of n-3 fed rats were characterised by 8 ions containing the n-3 double bond, and m.i.d. of the 651/653 ions of PGF-Me, TBDMS indicated PGF3 alpha synthesis (44 +/- 8% and 13 +/- 2% of PGF release by uteri incubated + or -5 micrograms/ul calcium ionophore A23187 respectively). In uteri from the control diet group incubated with ionophore, PGF3 alpha ions were detected and PGF 3 alpha represented 9.5 +/- 1.0% of PGF alpha release. Similarly, analysis of PGE from uteri of n-3 fed rats indicated that PGE3 (16 +/- 6% of PGE) was released in the presence of ionophore A23187. Synthesis of 3-series PG by rat uteri was detected after only 3 weeks of n-3 diet. The capacity to synthesise 3-series PG increased at intracellular calcium concentrations which mimicked cell calcium during decidual autolysis at parturition. These experiments suggest that uterine synthesis of 3-series PG is regulated by the specifity of enzymes incorporating fatty acids, rather than by the cyclooxygenase enzyme.     

Isolation and identification of prostaglandins of the E and F series in testes and semen of lowland-black-white breed of bulls

The prostaglandins of E and F series were obtained from testes and semen of sexually mature bulls of lowland-black-white breed. From 1 g of fresh testes tissue we obtained 7.01 X 10(-9) M prostaglandin of the F series (PGF) and 20.65 X 10(-9) M prostaglandin of the E series (PGE): from 11 of semen 3.28 X 10(-6) M of PGF and 10.58 X 10(-6) M of PGE were obtained as well. The prostaglandins thus obtained displayed biological activity in experiments on the isolated small intestine of the rabbit.     

Modulation of phorbol ester-elicited events in mouse epidermis by dietary n-3 and n-6 fatty acids

Because arachidonic acid-derived eicosanoids are potent modulators of hyperproliferation and inflammation during skin tumor promotion with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) (17, 18), it was hypothesized that dietary modification of epidermal fatty acids might modulate TPA-induced biochemical events in mouse skin. Semipurified diets containing 10% total fat composed of corn oil (CO) or a combination of CO and menhaden oil (MO) or coconut oil (CT) were fed to SENCAR mice for 4 weeks. Fatty acid composition of epidermal phospholipids generally reflected fatty acid composition of dietary oils fed to the mice. Since fatty acid-derived eicosanoids are thought to be essential in tumorigenesis, we compared the effects of dietary fats on prostaglandin E (PGE) production in epidermis treated with a single dose of TPA. TPA-induced PGE production in mouse epidermis from mice fed the MO diet was significantly reduced compared to PGE production in epidermal homogenates from mice fed the CO or CT diets. Type of dietary fats did not appear to modulate TPA-induced vascular permeability, however hyperplasia was slightly elevated in skins of mice fed MO. The subcellular distribution of protein kinase C, the plasma membrane receptor for TPA predominantly located in the cytosol (80%), was altered in epidermis from mice fed the MO diet compared to preparations from mice fed CO or CT diets which exhibited normal protein kinase C distribution. Our results suggest that n-3 rich dietary lipids modulate TPA-elicited events in mouse skin to a greater extent than diets containing higher proportions of saturated or n-6 fatty acids.     

Modification of prostaglandin metabolism in vivo by long-chain omega-3 polyunsaturates

In a pilot study conducted with a healthy male volunteer we determined that short-term dietary supplementation with fish oil markedly suppresses the systemic production of prostaglandin E (P = 0.04). This biochemical effect is observable after an incubation period of several days. The potential consequences of a reduced PGE synthetic rate on renal function, immune system and vascular dynamics, must be considered in the overall evaluation of the safety of fish oil supplements.     

Separation and quantification of PGE3 following derivatization with panacyl bromide by high pressure liquid chromatography with fluorometric detection.

Separation of prostaglandin E3 (PGE3) from prostaglandin E2 (PGE2) and prostaglandin E1 (PGE1) was achieved following derivatization with p-(9-anthroyloxy)phenacyl bromide (panacyl bromide). The eicosanoid esters were analysed by reverse phase high pressure liquid chromatography with fluorometric detection (excitation 360nm and emission 470nm). Human, rat and mouse adherent cells were incubated overnight and the culture medium extracted, derivatized and analysed for PG production. PGE2 was detected from biological samples of each species tested. PGE2 synthesis was reduced when cells were incubated overnight with 5 microM eicosapentaenoic acid. PGE3 was not detectable under these experimental conditions. Studies were also undertaken using adherent cells from mice, rats and human subjects given dietary fish oil supplements rich in EPA. PGE3 production by these cells was not detected although the dietary regimens yielded substantial incorporation of EPA into cell membranes and leukocyte LTB5 production was demonstrable.     

Effect of dietary oils containing different amounts of precursor and derivative fatty acids on prostaglandin E2 synthesis in liver, kidney and lung of rats.

The availability of the fatty acids which are precursors of prostaglandins is affected by dietary intake. We have studied, in particular, the effects of dietary intake of lipids with different amounts of precursor and derivative fatty acids on the synthesis of prostaglandin E2 (PGE2) in rat liver, kidney and lung. Fifteen-month-old rats were fed for 3 months diets containing different amounts of oleic, linoleic, alpha linolenic, gamma linolenic and stearidonic acids. The fatty acid compositions of total phospholipids and prostaglandin E2 levels of liver, kidney and lung were investigated. In the organs studied, the intake of lipids at different amount of precursor/derivative fatty acids caused variations in the fatty acid composition of phospholipids. PGE2 showed different values which did not seem directly affected by tissue availability of arachidonate but by the effect of dietary lipids on the metabolic pool of polyunsaturated fatty acids (PUFAs).     

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.