Biomarkers of oxidative damage in cigarette smokers: Which biomarkers might reflect acute versus chronic oxidative stress?

Cigarette smoking predisposes to the development of multiple diseases involving oxidative damage. We measured a range of oxidative damage biomarkers to understand which differ between smokers and nonsmokers and if the levels of these biomarkers change further during the act of smoking itself. Despite overnight abstinence from smoking, smokers had higher levels of plasma total and esterified F(2)-isoprostanes, hydroxyeicosatetraenoic acid products (HETEs), F(4)-neuroprostanes, 7-ketocholesterol, and 24- and 27-hydroxycholesterol. Levels of urinary F(2)-isoprostanes, HETEs, and 8-hydroxy-2'-deoxyguanosine were also increased compared with age-matched nonsmokers. Several biomarkers (plasma free F(2)-isoprostanes, allantoin, and 7β-hydroxycholesterol and urinary F(2)-isoprostane metabolites) were not elevated. The smokers were then asked to smoke a cigarette; this acute smoking elevated plasma and urinary F(2)-isoprostanes, plasma allantoin, and certain cholesterol oxidation products compared to presmoking levels, but not plasma HETEs or urinary 8-hydroxy-2'-deoxyguanosine. Smokers showed differences in plasma fatty acid composition. Our findings confirm that certain oxidative damage biomarkers are elevated in smokers even after a period of abstinence from smoking, whereas these plus some others are elevated after acute smoking. Thus, different biomarkers do not measure identical aspects of oxidative stress.     

Culture of mesothelial cells from bovine pericardium and characterization of their arachidonate metabolism

Cultures of mesothelial cells from bovine pericardium were established and their arachidonate metabolism was characterized. The identification of the cultured cells was based on morphological observations, and by electrophoretic analysis of cytoskeletal proteins, which demonstrated a pattern previously reported for mesothelial cells. Factor VIII-related antigen was present by indirect immunofluorescence, but the cells had no thrombomodulin activity. The cultured pericardial cells metabolized arachidonic acid to 6-ketoprostaglandin F1 alpha and a small amount of prostaglandin E2. The same metabolites were produced by pieces of intact parietal pericardium but not by pieces from which mesothelium had been removed. The cultured mesothelial cells produced 94.6 +/- 60.4 (mean +/- S.D.) ng/mg (n = 3) cell protein of 6-ketoprostaglandin F1 alpha in response to the calcium ionophore A23187, 117.3 +/- 13.6 ng/mg (n = 3) with exogenous arachidonic acid, 18.3 +/- 11.3 ng/mg (n = 5) with bradykinin, 8.4 +/- 4.3 ng/kg (n = 4) with histamine and 11.2 +/- 9.7 ng/mg (n = 5) with thrombin. All of these values were significantly higher (P less than 0.05) than the control (2.1 +/- 1 ng/mg; n = 5). From these results, we conclude that the mesothelial cells account for the arachidonate metabolism in the pericardium. The production of prostaglandin I2 occurs in response to physiological or pathological, agonists, and is substantial. That is, it is approximately the same as endothelial cells. The release of eicosanoids by mesothelial cells into the pericardial space may have a significant role in cardiac physiology and pathology.     

Clinical use of unbound plasma cortisol as calculated from total cortisol and corticosteroid-binding globulin

A method to calculate unbound cortisol from total cortisol (measured by competitive protein binding) and CBG (measured by radial immunodiffusion) based on the binding equilibrium has been evaluated. The calculated results (y) correlate well with those (x) obtained by centrifugal ultrafiltration at 37 degrees C (y = 1.04 x - 2.11 ng/ml; r = 0.975; n = 150). The concentration of CBG is similar in normal men (37.7 +/- 3.5 (SD) micrograms/ml; n = 12) and women (39.5 +/- 3.7 (SD) micrograms/ml; n = 7) and shows no diurnal variation, but marked diurnal variation is observed for total cortisol (193.7 +/- 35.0 (SD) ng/ml at 08.00 h vs 43.2 +/- 23.3 (SD) ng/ml at 22.00 h; n = 19) and particularly for unbound cortisol (16.5 +/- 5.6 (SD) ng/ml at 08.00 h vs 2.3 +/- 1.8 (SD) ng/ml at 22.00 h; n = 19). The concentration of CBG (89.1 +/- 11.2 (SD) micrograms/ml) and of total cortisol (395.6 +/- 103.3 (SD) ng/ml at 08.00 h; 110.3 +/- 16.6 (SD) ng/ml at 22.00 h) are clearly elevated in estrogen treated women (n = 11) but unbound cortisol levels (17.2 +/- 7.7 (SD) ng/ml at 08.00 h; 2.5 +/- 0.5 (SD) ng/ml at 22.00 h) are similar to the control group. The concentration of CBG is significantly decreased in patients with Cushing's syndrome (33.2 +/- 5.6 micrograms/ml; n = 17) and unbound cortisol is relatively more elevated than total cortisol in these patients. In adrenal insufficiently CBG is normal, but total and unbound cortisol are markedly decreased. There is a significant decrease of CBG in hyperthyroidism (35.7 +/- 5.5 micrograms/ml; n = 22), in cirrhosis (32.0 +/- 8.0 micrograms/ml; n = 14) and in renal disease and a significant increase in patients treated with antiepileptic drugs (47.5 +/- 6.3 micrograms/ml; n = 14), but total and unbound cortisol are normal in all these conditions. We conclude that unbound cortisol can be calculated in a simple and reliable way from total cortisol and CBG and permits a better evaluation of adrenal function, particularly in patients with altered CBG concentrations.     

Antioxidant supplementation prevents exercise-induced lipid peroxidation, but not inflammation, in ultramarathon runners

To determine if 6 weeks of supplementation with vitamins E and C could alleviate exercise-induced lipid peroxidation and inflammation, we studied 22 runners during a 50 km ultramarathon. Subjects were randomly assigned to one of two groups: (1) placebos (PL) or (2) antioxidants (AO: 1000 mg vitamin C and 300 mg RRR-alpha-tocopheryl acetate). Blood samples were obtained prior to supplementation (baseline), after 3 weeks of supplementation, 1 h pre-, mid-, and postrace, 2 h postrace and for 6 days postrace. Plasma levels of alpha-tocopherol (alpha-TOH), ascorbic acid (AA), uric acid (UA), F2-isoprostanes (F2-IsoPs), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and C-reactive protein (CRP) were measured. With supplementation, plasma alpha-TOH and AA increased in the AO but not the PL group. Although F2-IsoP levels were similar between groups at baseline, 28 +/- 2 (PL) and 27 +/- 3 pg/ml (AO), F2-IsoPs increased during the run only in the PL group (41 +/- 3 pg/ml). In PL women, F2-IsoPs were elevated postrace (p <.01), but returned to prerace concentrations by 2 h postrace. In PL men, F2-IsoP concentrations were higher postrace, 2 h postrace, and 1, 2, 3, 4, and 6 days postrace (PL vs. AO group, each p <.03). Markers of inflammation were increased dramatically in response to the run regardless of treatment group. Thus, AO supplementation prevented endurance exercise-induced lipid peroxidation but had no effect on inflammatory markers.     

Changes in plasma angiotensin-converting enzyme activity and noradrenaline responses to long-term nitric oxide inhibition vary depending on their basal values in chickens

In this study, we investigated the effects of N(omega)-nitro-L-arginine (L-NNA) on arterial blood pressure (BP), plasma noradrenaline (NA) and adrenaline (A) levels and angiotensin-converting enzyme (ACE) activity. L-NNA was applied with tap water (1 mg/ml) from the 3rd to the 8th week of age (group L-NNA1). In Experiment 1, long-term L-NNA application increased BP compared to the control group (group C1) (L-NNA1 = 131.4 +/- 6.3, n = 6; C1 = 82.7 +/- 4.7 mm Hg, n = 7) but decreased plasma noradrenaline and adrenaline levels and ACE activity (NA levels: C1 = 15.5 +/- 0.8, n = 7; L-NNA1 = 8.6 +/- 0.5 ng/ml, n = 7; A levels: C1 = 15.5 +/- 0.8, n = 7; L-NNA1 = 6.0 +/- 0.5 ng/ml, n = 7; ACE activities: C1 = 87.3 +/- 3.1, n = 6; L-NNA1 = 46.2 +/- 1.9 U/l, n = 5). On the other hand, in Experiment 2 (carried out under the same conditions and in age-matched chickens), blood pressure, plasma noradrenaline levels and ACE activity were found to differ in the control group (C2) (BP = 141.4 +/- 15.5 mm Hg, n = 7; NA = 1.1 +/- 0.4 ng/ml, n = 7; ACE = 57.2 +/- 5.3 U/l, n = 7) as compared to C1, while plasma adrenaline levels were similar. In this series, long-term L-NNA application (group L-NNA2) did not change the BP, but surprisingly increased noradrenaline and ACE values (values of L-NNA2: BP = 165.7 +/- 15.6 mm Hg, n = 7; NA = 9.3 +/- 1.3 ng/ml, n = 8; ACE = 149.4 +/- 16 U/l, n = 8) while decreasing plasma adrenaline levels. L-arginine addition to L-NNA treatment completely reversed plasma noradrenaline and ACE activity values. These results indicate the modulatory activity of an L-arginine-NO pathway on adrenaline release as well as on the renin-angiotensin system in chickens.     

Systemic elevations of free radical oxidation products of arachidonic acid are associated with angiographic evidence of coronary artery disease

Oxidant stress is widely believed to participate in cardiovascular disease pathogenesis. However, progress in defining appropriate systemic antioxidant targeted therapies has been hindered by uncertainty in defining clinically relevant systemic oxidant stress measures. In a case control study, 50 subjects with CAD (>50% stenosis in one or more major coronary vessels) and 54 without CAD (<30% stenosis in all major coronary vessels) were tested. Plasma was isolated and stored under conditions designed to prevent artificial lipid peroxidation. Systemic levels of multiple (n=9) specific fatty acid oxidation products including individual hydroxyoctadecadienoic acids (HODEs), hydroxyeicosatetraenoic acids (HETEs), and F(2)-isoprostanes were simultaneously measured by high-performance liquid chromatography (HPLC) with on-line tandem mass spectrometry, along with traditional risk factors and C-reactive protein (CRP) levels. Of the markers monitored, only 9-HETE and F(2)-isoprostanes, both products of free radical-mediated arachidonic acid oxidation, were significantly elevated in patients with angiographically defined CAD (9-HETE, 8.7 +/- 4 vs 6.8 +/- 4 micromol/mol arachidonate, P = 0.011; and F(2)-isoprostanes, 9.4 +/- 5 vs 6.2 +/- 3 micromol/mol arachidonate, P < 0.001). In multivariable analyses with simultaneous adjustment for Framingham risk score and C-reactive protein, 9-HETE (4th quartile OR = 4.8, 95% CI=1.3 to 17.1; P = 0.016) and F(2)-isoprostanes (4th quartile OR=9.7, 95% CI=2.56 to 36.9; P < 0.001) remained strong and independent predictors of CAD risk. Systemic levels of 9-HETE and F(2)-isoprostanes are independently associated with angiographic evidence of CAD and appear superior to other specific oxidation products of arachidonic and linoleic acids as predictors of the presence of angiographically evident coronary artery disease.     

Influence of stage of the estrous cycle on endogenous opioid modulation of luteinizing hormone, prolactin, and cortisol secretion in the gilt

Fourteen gilts that had displayed one or more estrous cycles of 18-22 days (onset of estrus = Day 0) and four ovariectomized (OVX) gilts were treated with naloxone (NAL), an opiate antagonist, at 1 mg/kg body weight in saline i.v. Intact gilts were treated during either the luteal phase (L, Day 10-11; n = 7), early follicular phase (EF, Day 15-17; n = 3), or late follicular phase (LF, Day 18-19; n = 4) of the estrous cycle. Blood was collected at 15-min intervals for 2 h before and 4 h after NAL treatment. Serum luteinizing hormone (LH) concentrations for L gilts averaged 0.65 +/- 0.04 ng/ml during the pretreatment period and increased to an average of 1.3 +/- 0.1 ng/ml (p less than 0.05) during the first 60 min after NAL treatment. Serum prolactin (PRL) concentrations for L gilts averaged 4.8 +/- 0.2 ng/ml during the pretreatment period and increased to an average of 6.3 +/- 0.3 ng/ml (p less than 0.05) during the first 60 min after NAL treatment. Serum PRL concentrations averaged 8.6 +/- 0.7 ng/ml and 7.6 +/- 0.6 ng/ml in EF and LF gilts, respectively, prior to NAL treatment, and decreased (p less than 0.05) to an average of 4.1 +/- 0.2 ng/ml and 5.6 +/- 0.4 ng/ml in EF and LF gilts, respectively, during the fourth h after NAL. Naloxone treatment failed to alter serum LH concentrations in EF, LF, or OVX gilts and PRL concentrations in OVX gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Changes in the concentrations of plasma steroid hormone and plasma 13, 14-dihydro-15-oxo-prostaglandin F2 alpha in late pregnancy rabbits treated with an inhibitor of 3 beta-hydroxysteroid dehydrogenase

Changes in the concentrations of progesterone, 17 beta-estradiol and 13, 14-dihydro-15-oxo-prostaglandin F2 alpha (PGFM) were evaluated in the peripheral plasma of rabbits during late pregnancy by treating trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, in an attempt to obtain further insight into the involvement of progesterone and prostaglandin (PG) in the initiation of parturition. The concentrations of progesterone were 18.8 +/- 2.2 ng/ml (mean +/- SE, n = 6) before administration of the inhibitor, significantly (p less than 0.05) fell to 7.6 +/- 1.0 ng/ml (n = 6) at 30 min, and remained low until 10 h after the drug administration. The concentrations of progesterone were still low (5.4 +/- 0.5 ng/ml, n = 6) at 20-24 h after administration of the inhibitor, and were also low (4.9 +/- 2.2 ng/ml, n = 6) at delivery. Premature deliveries occurred at 28.8 +/- 2.0 h after injection of trilostane (on days 29 of gestation). Increased concentrations of PGFM were observed at delivery. However, administration of trilostane induced no discernible changes in the concentration of estradiol. These findings suggest that delivery is induced by progesterone withdrawal and that synthesis prostaglandin F2 alpha is remarkably increased at delivery in the rabbit.     

Oxidative damage in Parkinson disease: Measurement using accurate biomarkers

Oxidative damage has been implicated in the pathogenesis of Parkinson disease (PD) but the literature data are confusing. Using products of lipid and DNA oxidation measured by accurate methods, we assessed the extent of oxidative damage in PD patients. The levels of plasma F₂-isoprostanes (F₂-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), cholesterol oxidation products, neuroprostanes (F₄-NPs), phospholipase A₂ (PLA₂) and platelet activating factor–acetylhydrolase (PAF-AH) activities, urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), and serum high-sensitivity C-reactive protein were compared in 61 PD patients and 61 age-matched controls. The levels of plasma F₂-IsoPs, HETEs, 7β-and 27-hydroxycholesterol, 7-ketocholesterol, F₄-NPs, and urinary 8-OHdG were elevated, whereas the levels of plasma PLA₂ and PAF-AH activities were lower, in PD patients compared to controls (p <  0.05). The levels of plasma F₂-IsoPs, HETEs, and urinary 8-OHdG were higher in the early stages of PD (p trend <  0.05). There was a significant negative correlation between the cumulative intake of levodopa and urinary 8-OHdG (r =  ?0.305, p =  0.023) and plasma total HETEs (r =  ?0.285, p =  0.043). Oxidative damage markers are systemically elevated in PD, which may give clues about the relation of oxidative damage to the onset and progression of PD.     

Study of plasma aldosterone in normal pregnancy, in pre-eclamptic women and in cord plasma of newborns.

A radioimmunoassay without chromatography was used for the determination of plasma aldosterone in pregnancy. The mean values (+/- S.D.) of aldosterone concentration increased consistently from 23.2 +/- 5.3 ng/100 ml (n = 14) during the first trimester to 37.2 +/- 10.6 ng/100 ml (n = 17) during the second trimester and 64.0 +/- 18.8 ng/100 ml (n = 29) during the third trimester of pregnancy. The highest values were found at delivery (71.9 +/- 14.2 ng/100 ml; n = 21) and in the cord plasma of newborns (83.4 +/- 14.9 ng/100 ml; n = 21). Significantly lower plasma aldosterone values were found in the plasma of pre-eclamptic women during the third trimester of pregnancy (41.9 +/- 21.3 ng/100 ml; n = 11).     

Prostacyclin metabolism in adults and neonates. Urinary profiles of 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha studied by gas chromatography-mass spectrometry

Metabolism of endogenous prostacyclin was studied in adults and neonates by measuring urinary levels of 6-ketoprostaglandin F1 alpha (spontaneous hydrolysis product) and 2,3-dinor-6-ketoprostaglandin F1 alpha (enzymatically formed by beta-oxidation). Quantification of prostanoids was achieved by capillary gas chromatography-mass spectrometry using the stable isotope dilution technique. Purification of the urinary lipid extract included silicic acid column chromatography and reverse- and straight-phase high-pressure liquid chromatographies. Accuracy of the method was proven by recovery experiments for both metabolites. Partial mass spectra of endogenous 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha were obtained from urine samples. In neonates (third day of life, n - 5 pooled urines) levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.28 +/- 0.18 ng/ml) were much lower than those of 6-ketoprostaglandin F1 alpha (2.13 +/- 1.10 ng/ml), indicating low beta-oxidation activity at high prostacyclin formation. In adults (n = 7), levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.27 +/- 0.21 ng/ml) and levels of 6-ketoprostaglandin F1 alpha (0.20 +/- 0.11 ng/ml) were about the same, indicating relatively high beta-oxidation at low prostacyclin formation. Values are expressed as mean +/- S.D.     

Rapid preparation of human urine and plasma samples for analysis of F2-isoprostanes by gas chromatography-mass spectrometry

Reliable MS-based methods have been developed for the measurement of free and esterified F2-isoprostanes. However, prior to sample analysis several steps of purification, including solid-phase extraction followed by TLC or HPLC, are usually required, making it tedious to analyze large sample numbers, e.g., for population studies. We report a quick sample purification method using anion exchange solid phase extraction (SPE), which is highly selective for acidic compounds. Urine and hydrolyzed plasma of healthy individuals were acidified before SPE extraction, washed with 4 different solvent mixtures and finally eluted with ethyl acetate. The eluted samples were first derivatized with pentafluorobenzyl bromide followed by a second derivatization with bis-(trimethylsilyl)trifluoroacetamide. F2-isoprostanes were analyzed by GC-MS-NCI. The method was highly sensitive; the limit of detection at 5:1 signal-to-noise ratio was 0.037 ng/ml and 0.007 ng/mg creatinine for plasma and urine, respectively. Anion exchange SPE extraction for F2-isoprostane showed recovery of 55-65% and high linearity for concentration 0-1.0 ng/ml for urine (CV=4.08%, r2=0.990) and 0-0.5 ng/ml for plasma (CV=4.07%, r2=0.998). Fasting for 6h significantly increased plasma F2-isoprostanes levels, which has implications for the design of intervention studies using this biomarker.     

Prostaglandin F in reproductive tissues collected during the luteal phase of the estrous cycle and at parturition in Virginia opossums (Didelphis virginiana )

Prostaglandin F(2alpha) (PGF(2alpha)) plays a role in the regression of the corpus luteum (CL) in a number of placental mammals. However, the mechanism of luteal regression has not been extensively studied in marsupials. The objectives of this study were to characterize changes in concentrations of PGF(2alpha) within utero-ovarian (UO) tissue/venous plasma during the luteal phase of the estrous cycle in Virginia opossums, to correlate these changes with those of plasma progesterone (P(4)), and to characterize the peripheral pattern of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM) in parturient opossums. Ovaries, uteri, UO venous plasma and peripheral plasma were collected on Days 5, 9 and 12 after induced ovulation (n = 3 to 4 opossums/group). In addition, concentrations of PGFM were measured in peripheral plasma collected from two opossums during late gestation (Days 7,9,11 and 12) and at parturition (Day 13). Concentrations of P(4), PGFM and PGF(2alpha) in tissue homogenates and plasma samples were estimated by radioimmunoassay. In nonpregnant opossums, peripheral P(4) levels were highest on Day 5 (38.8 +/- 11.1 ng/ml, x +/- SEM) declined on Day 9 (22.6 +/- 7.4 ng/ml), and were at basal levels by Day 12 (2.4 +/- 0.7 ng/ml). Endometrial concentrations of PGF(2alpha) increased (P = 0.056) from Day 5 (15.7 +/- 4.1 ng/g) to Day 9 (92.1 +/- 61.0 ng/g) and were maintained to Day 12 (97.2 +/- 25.7 ng/g). Prostaglandin F(2alpha) concentrations in UO plasma increased (P < 0.01) from Day 5 (143.1 +/- 32.7 pg/ml) to Day 12 (333.0 +/- 32.4 pg/ml). Prostaglandin F(2alpha) concentrations in ovarian tissue followed a similar pattern and were correlated with UO concentrations (r = 0.708, P < 0.05). In pregnant opossums, the highest levels of peripheral PGFM were recorded in the peripartum period, when luteal regression would also be expected to occur. The negative temporal relationship between peripheral concentrations of P(4) and concentrations of PGF(2alpha) in UO tissue/venous plasma observed in this preliminary study is consistent with the notion that PGF(2alpha) from the ovary and/or uterus may play a role in CL regression in the opossum.     

Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.     

Effect of estradiol-17beta on PGF and total protein content in bovine uterine flushings and peripheral plasma concentration of 13, 14-dihydro-15-keto-PGF(2alpha)

Two experiments were conducted to examine the effect of estradiol-17beta (E(2)-17beta) on content of immunoreactive prostagladin F(2)alpha (PGF, ng) and total protein (TUP, mg) in uterine flushings, as well as concentrations of 13, 14-dihydro-15-keto-PGF(2)alpha (PGFM) in plasma (Pg/ml). In experiment 1, Holstein heifers were utilized in a single reversal trial in which either E(2)-17beta (3 mg in 2 ml saline/ethanol 50:50; n=5) or vehicle alone (n=6) were given intravenously on day 14 or 15 of the estrous cycle (Period 1) following an induced estrus (day of estrus = day 0). Treatment (Trt) groups were reversed in Period 2 (Day 14 or 15 of the second estrous cycle). Jugular venous plasma was obtained before treatment (Oh), and at 5, 6, and 9h posttreatment (PT). Uterine flushings were collected nonsurgically in vivo , per cervix, via Foley catheter at 6h PT (20 ml of .9% saline per uterine horn). E(2)-17beta did not significantly alter (E(2)-17beta vs vehicle; x(-) +/- S.E.M.) PGF (1674 +/- .11 +/- 338.39 vs 1889.91 +/- 400.24 ng; P> .10) or TUP (33.25 +/- 2.57 vs 39.16 +/- 3.04 mg; P > .10). However, E(2)-17beta increased (P < .05) plasma PGFM (E(2)-17beta vs vehicle) after treatment (0h, 113.2 vs 163.8; 5h, 312.5 vs 203.9; 6h, 324.5 vs 198.0; 9h, 323.2 vs 246.8, pg/ml). In experiment 2, crossbred beef cattle received comparable treatments of either E(2)-17beta (n=5) or vehicle (n=5) on day 14 or 15 postestrus. Jugular venous plasma was obtained at 0h PT, and at 6h PT. Uterine flushings (1.9% saline, 20 ml per uterine horn) and peripheral plasma were collected at slaughter. Estradiol-17beta increased PGF (30.07 +/- 5.94 vs 8.46 +/- 2.01 ng; P> <.05) in uterine flushings as well as PGFM in plasma (E(2)-17beta : 55.82 +/- 19.13 pg/ml, at 0h and 89.31 +/- 14.02 pg/ml, at 6h, vs saline: 103.46 +/- 50.73 pg/ml, at 0h and 17.78 +/- 14.22, at 6h). Estradiol-17beta stimulated uterine production and release of PGF and protein as measured in flushings (experiment 2) as well as plasma PGFM responses (experiments 1 and 2). Uterine and/or cervical stimulation of experiment 1 may have masked uterine response to E(2)-17beta.     

Splenic denervation worsens lipopolysaccharide-induced hypotension, hemoconcentration, and hypovolemia

During lipopolysaccharide (LPS)-induced endotoxemia, increased intrasplenic fluid efflux contributes to a reduction in plasma volume. We hypothesized that splenic sympathetic nerve activity (SSNA), which increases during endotoxemia, limits intrasplenic fluid efflux. We reasoned that splenic denervation would exaggerate LPS-induced intrasplenic fluid efflux and worsen the hypotension, hemoconcentration, and hypovolemia. A nonlethal dose of LPS (150 microg x kg(-1) x h(-1) for 18 h) was infused into conscious male rats bearing transit time flow probes on the splenic artery and vein. Fluid efflux was estimated from the difference in splenic arterial inflow and venous outflow (A-V). LPS significantly increased the (A-V) flow differential (fluid efflux) in intact rats (saline -0.01 +/- 0.02 ml/min, n = 8 vs. LPS +0.21 +/- 0.06 ml/min, n = 8); this was exaggerated in splenic denervated rats (saline -0.03 +/- 0.01 ml/min, n = 7 vs. LPS +0.41 +/- 0.08 ml/min, n = 8). Splenic denervation also exacerbated the LPS-induced hypotension, hemoconcentration, and hypovolemia (peak fall in mean arterial pressure: denervated 19 +/- 3 mmHg, n = 10 vs. intact 12 +/- 1 mmHg, n = 8; peak rise in hematocrit: denervated 6.7 +/- 0.3%, n = 8 vs. intact 5.0 +/- 0.3%, n = 8; decrease in plasma volume at 90-min post-LPS infusion: denervated 1.08 +/- 0.15 ml/100 g body wt, n = 7 vs. intact 0.54 +/- 0.08 ml/100 g body wt, n = 8). The exaggerated LPS-induced hypovolemia associated with splenic denervation was mirrored in the rise in plasma renin activity (90 min post-LPS: denervated 11.5 +/- 0.8 ng x ml(-1) x h(-1), n = 9 vs. intact 6.6 +/- 0.7 ng x ml(-1) x h(-1), n = 8). These results are consistent with our proposal that SSNA normally limits LPS-induced intrasplenic fluid efflux.     

Leukotrienes increase levels of prostanoids in cerebrospinal fluid in piglets

We investigated effects of exogenous leukotrienes (C4, D4, or E4) on levels of prostanoids in cerebrospinal fluid in newborn pigs (1-5 days). A "closed" cranial window was placed over the parietal cortex. Pial arterial diameter was measured with a microscope and electronic micrometer system. Levels in cerebrospinal fluid (CSF) of 6-keto-Prostaglandin F1 alpha (6-keto-PGF1 alpha), Thromboxane B2 (TXB2), and Prostaglandin E2 (PGE2) were measured by radioimmunoassay. Topical application of leukotrienes C4, D4, or E4 (5,000 ng/ml) similarly constricted pial arteries by 15 +/- 2% (n = 14) (mean +/- SEM). In addition, leukotrienes increased levels of 6-keto-PGF1 alpha from 806 +/- 136 to 1,612 +/- 304 pg/ml (n = 13), TXB2 from 161 +/- 31 to 392 +/- 81 pg/ml (n = 10), and PGE2 from 2,271 +/- 342 to 4,636 +/- 740 pg/ml (n = 13). Each type of leukotriene had similar effects on prostanoid synthesis. In other experiments (n = 5), we found that 2.0 ng/ml PGE2 in CSF dilated pial arteries by 24 +/- 8% and that 1.0 ng/ml PGI2 dilated pial arteries by 15 +/- 6%. These results indicate that leukotrienes are able to increase levels of prostanoids in cerebral cortex.     

Intraluteal infusions of prostaglandins of the E, D, I, and A series prevent PGF2 alpha-induced, but not spontaneous, luteal regression in rhesus monkeys

A luteotropic role for prostaglandins (PGs) during the luteal phase of the menstrual cycle of rhesus monkeys was suggested by the observation that intraluteal infusion of a PG synthesis inhibitor caused premature luteolysis. This study was designed to identify PGs that promote luteal function in primates. First, the effects of various PGs on progesterone (P) production by macaque luteal cells were examined in vitro. Collagenase-dispersed luteal cells from midluteal phase of the menstrual cycle (Day 6-7 after the estimated surge of LH, n = 3) were incubated with 0-5,000 ng/ml PGE2, PGD, 6 beta PGI1 (a stable analogue of PGI2), PGA2, or PGF2 alpha alone or with hCG (100 ng/ml). PGE2, PGD2, and 6 beta PGI1 alone stimulated (p less than 0.05) P production to a similar extent (2- to 3-fold over basal) as hCG alone, whereas PGA2 and PGF2 alpha alone had no effect on P production. Stimulation (p less than 0.05) of P synthesis by PGE2, PGD2, and 6 beta PGI1 in combination with hCG was similar to that of hCG alone. Whereas PGA2 inhibited gonadotropin-induced P production (p less than 0.05), that in the presence of PGF2 alpha plus hCG tended (p = 0.05) to remain elevated. Second, the effects of various PGs on P production during chronic infusion into the CL were studied in vivo. Saline with or without 0.1% BSA (n = 12), PGE2 (300 ng/h; n = 4), PGD2 (300 ng/h; n = 4), 6 beta PGI1 (500 ng/h; n = 3), PGA2 (300 ng/h; n = 4), or PGF2 alpha (10 ng/h; n = 8) was infused via osmotic minipump beginning at midluteal phase (Days 5-8 after the estimated LH surge) until menses. In addition, the same dose of PGE, PGD, PGI, or PGA was infused in combination with PGF2 alpha (n = 3-4/group) for 7 days. P levels over 5 days preceding treatment were not different among groups. In 5 of 8 monkeys receiving PGF2 alpha alone, P declined to less than 0.5 ng/ml within 72 h after initiation of infusion and was lower (p less than 0.05) than controls. The length of the luteal phase in PGF2 alpha-infused monkeys was shortened (12.3 +/- 0.9 days; mean +/- SEM, n = 8; p less than 0.05) compared to controls (15.8 +/- 0.5). Intraluteal infusion of PGE, PGD, PGI, or PGA alone did not affect patterns of circulating P or luteal phase length.(ABSTRACT TRUNCATED AT 400 WORDS)     

Radioimmunoassay of 5-androstene-3 beta,17 beta-diol in plasma and in breast cyst fluid

A simple and reliable radioimmunoassay for the determination of 5-androstene-3 beta, 17 beta-diol in peripheral plasma and in breast cyst fluid, after a chromatography on Celite microcolumn has been described and evaluated. The antiserum used was raised in rabbits injected with dehydroepiandrosterone-15 alpha-(O-carboxymethyl)-bovine serum albumin. In men below 40 years of age the levels ranged from 0.85 to 2.80 ng/ml (mean +/- SEM: 1.52 +/- 0.11; n = 24) and from 0.50 to 2.20 ng/ml (mean +/- SEM: 0.93 +/- 0.09; n = 20) in men aged between 41 and 62 years. The mean level was significantly different (P less than 0.001) between the 2 groups. A significant correlation (r = -0.56; P less than 0.01) was demonstrated between age and all male levels. In females the mean plasma level was in the follicular phase: 0.81 +/- 0.07 ng/ml (range: 0.40-1.50; n = 17; age: 19-41 years) and in the luteal phase: 0.83 +/- 0.05 ng/ml (range: 0.40-1.30; n = 29; age: 18-43 years). No cyclical change and no correlation with age could be evidenced. A significant difference (P less than 0.001) was shown between females and the young male group. In breast cyst fluid the levels ranged from 0.05 to 13.70 ng/ml (mean +/- SEM: 2.36 +/- 0.86; n = 20) whereas the sulfate concentrations ranged from 75 to 7500 ng/ml (mean +/- SEM: 1891 +/- 565; n = 15), thus demonstrating very wide inter-individual variations.