Determination of urinary 8-epi-prostaglandin F(2alpha) using liquid chromatography-tandem mass spectrometry: increased excretion in diabetics

Liquid chromatography-tandem mass spectrometry (LC/MS-MS) was applied to the quantitative analysis of urinary 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) level. 8-Epi-PGF(2alpha) and its internal standard, [(2)H(4)]-8-epi-PGF(2alpha), were extracted from urine by using a solid phase extraction cartridge and loaded to LC/MS-MS in selected reaction monitoring (SRM) mode. The standard curve showed good linearity in the range of 40 pg to 10 ng (r = 0. 997). The accuracy of the added 8-epi-PGF(2alpha) ranged from 96.8 to 104.9% with a mean +/- SD of 99.5+/-2.5%. The average level +/- SD of urinary 8-epi-PGF(2alpha) in 13 healthy volunteers (five women and eight men, 31+/-7.4 years old) was 429.4+/-149.6 pg/mg creatinine. The level of seven patients with noninsulin dependent diabetes mellitus (two women and five men, 40+/-13.6 years old), 630.9+/-275.6 pg/mg creatinine, was statistically higher than that of healthy volunteers (P<0.05). This finding suggested that diabetics are in a highly oxidative condition. This simple and rapid LC/MS-MS method can be used to elucidate the pathophysiological feature of diabetes or for monitoring the curative effect.     

Urinary 8-epi-PGF2alpha and its endogenous beta-oxidation products (2,3-dinor and 2,3-dinor-5,6-dihydro) as biomarkers of total body oxidative stress

Although measurements of plasma F2-isoprostanes are established markers of oxidative stress, their quantification only reflects acute non-enzymatic lipid peroxidation. In this study, a new approach is described for the rapid isolation and measurement of urinary 8-epi-PGF2alpha and its endogenous beta-oxidation metabolites (2,3-dinor-8-epi-PGF2alpha and 2,3-dinor-5,6-dihydro-PGF2alpha) for use as index of total body oxidative stress. Isoprostanes were partitioned with ethyl acetate and subsequently purified by chromatography on an aminopropyl (NH2) and silica (Si) cartridge. Final analysis of F2-isoprostanes as trimethylsilyl-ester/pentafluorobenzyl ester derivatives was carried out by stable isotope dilution mass spectrometry. Overall recovery of F2-isoprostanes was 80+/-4%. Inter- and intra-assay coefficients of variation were 5% and 7%, respectively. In a group of healthy humans, the mean excretion rates expressed as nmol/mmol creatinine for 2,3-dinor-8-epi-PGF2alpha, 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha, and 8-epi-PGF2alpha were 5.43+/-1.93, 2.16+/-0.71, and 0.36+/-0.16, respectively. Correlations were obtained between 8-epi-PGF2alpha and 2,3-dinor-8-epi-PGF2alpha or 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha (r=0.998 and r=0.937, respectively). A strong relationship was also seen between 2,3-dinor-8-epi-PGF2 and 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha (r=0.949). The new technique allows for high sample throughput and avoids the need for HPLC and/or other expensive equipment required for the initial sample preparation. Simultaneous analysis of urinary 8-epi-PGF2alpha and its metabolites should provide unique tool in clinical trials exploring the role of oxidant injury in human disease.     

Salivary isoprostanes indicate increased oxidation injury in periodontitis with additional tobacco abuse

Isoprostanes (IPs) are indicators of in-vivo oxidative stress, and have been successfully used as markers for chronic inflammatory processes. The presence of chronic periodontal disease and cigarette smoking has been individually linked to the development of atherosclerosis, yet data regarding oxidative stress in this context are not available yet. The aim of this study was to evaluate levels of the salivary prostaglandins (PGs) 8-epi-PGF(2alpha), 6-oxo-PGF(1alpha), thromboxane B(2) (TXB(2)) and PGF(2alpha) in association with periodontal disease status with and without additional cigarette smoking. We analyzed saliva samples from 121 adults, (aged 21-73 years, 90 non-smokers, 31 smokers) for levels of 8-epi-PGF(2alpha), 6-oxo-PGF(1alpha), TXB(2) and PGF(2alpha). On the basis of periodontal disease indices the periodontal status of each subject was assessed and outcomes were then correlated with smoking status and laboratory findings. Salivary 8-epi-PGF(2alpha) levels increased with deteriorating plaque index, and were significantly higher (115.5 +/- 23.5 pg/ml) in smoking individuals, when compared to non-smokers (70.2 +/- 20.4 pg/ml, p<0.0001). In addition, smokers showed higher TXB(2) and PGF(2alphas) and lower 6-oxo-PGF(1alpha) levels p<0.0001). Oxidative stress, as reflected by elevated salivary 8-epi-PGF(2alpha) levels, is associated with the extent of periodontal disease and is significantly aggravated by concomitant tobacco abuse. Chronic inflammation and smoking have been individually associated with the development of atherosclerosis. The results of this study indicate that: 1) salivary IPs can reliably assess the degree of oxidative stress, and: 2) smoking and periodontal disease are two modifiable cardiovascular risk factors, able to potentiate each other.     

Elevated plasma levels of human urotensin-II immunoreactivity in congestive heart failure

Human urotensin-II (hU-II) is the most potent endogenous cardiostimulant identified to date. We therefore determined whether hU-II has a possible pathological role by investigating its levels in patients with congestive heart failure (CHF). Blood samples were obtained from the aortic root, femoral artery, femoral vein, and pulmonary artery from CHF patients undergoing cardiac catheterization and the aortic root from patients undergoing investigative angiography for chest pain who were not in heart failure. Immunoreactive hU-II (hU-II-ir) levels were determined with radioimmunoassay. hU-II-ir was elevated in the aortic root of CHF patients (230.9 +/- 68.7 pg/ml, n = 21; P < 0.001) vs. patients with nonfailing hearts (22.7 +/- 6.1 pg/ml, n = 18). This increase was attributed to cardiopulmonary production of hU-II-ir because levels were lower in the pulmonary artery (38.2 +/- 6.1 pg/ml, n = 21; P < 0.001) than in the aortic root. hU-II-ir was elevated in the aortic root of CHF patients with nonischemic cardiomyopathy (142.1 +/- 51.5 pg/ml, n = 10; P < 0.05) vs. patients with nonfailing hearts without coronary artery disease (27.3 +/- 12.4 pg/ml, n = 7) and CHF patients with ischemic cardiomyopathy (311.6 +/- 120.4 pg/ml, n = 11; P < 0.001) vs. patients with nonfailing hearts and coronary artery disease (19.8 +/- 6.6 pg/ml, n = 11). hU-II-ir was significantly higher in the aortic root than in the pulmonary artery and femoral vein, with a nonsignificant trend for higher levels in the aortic root than in the femoral artery. The findings indicated that hU-II-ir is elevated in the aortic root of CHF patients and that hU-II-ir is cleared at least in part from the microcirculation.     

Evaluation of the postprandial effects of a fast-food meal on human plasma F(2)-isoprostane levels

Measurement of the F(2)-isoprostane, 8-epi-PGF(2alpha) is increasingly used as a sensitive and reliable marker of lipid peroxidation in vivo. Because the majority of 8-epi-PGF(2alpha) in plasma is associated with lipoproteins, it is possible that 8-epi-PGF(2alpha) derived from polyunsaturated fatty acid-rich food may become incorporated within these lipoproteins during synthesis and could contribute to the levels detected in plasma. In this study, we evaluated the postprandial effect of a single fast-food meal (McDonald's Big Mac meal, McDonald's Corp., London, England) on plasma total 8-epi-PGF(2alpha) in nine healthy subjects. Blood was collected before and 2 h postprandially. 8-Epi-PGF(2alpha) was measured by immunoaffinity extraction and gas chromatography-mass spectrometry. Fasting plasma 8-epi-PGF(2alpha) (875 +/- 25 pM) increased postprandially (956 +/- 23 pM, p <.05), although no significant change was observed in the normalized concentrations (2. 78 +/- 0.1 vs. 2.95 +/- 0.3 nmol/mmol arachidonic acid). Plasma lipid hydroperoxides, fatty acids, vitamin E, total antioxidant status, cholesterol, and triglycerides were not altered. Plasma glucose increased postmeal (4.4 +/- 0.1 vs. 4.9 +/- 0.1 mM, p <.05). These results indicate that the overall contribution of this lipid-rich meal to plasma 8-epi-PGF(2alpha) and other lipid peroxidation markers was small.     

Measurement of plasma F2-isoprostanes as an index of lipid peroxidation does not appear to be confounded by diet

F2-isoprostanes (F2-IPs) are formed by the free radical-catalysed oxidation of arachidonic acid. The measurement of F2-IPs, especially 8-epi-PGF2alpha, is recognised as a reliable marker of lipid peroxidation and is currently used as a sensitive index of oxidative stress in vivo. The majority of 8-epi-PGF2alpha present in the circulation occurs in association with lipoproteins which are synthesised in the liver. Since lipoproteins are derived from dietary fatty acids and triglycerides, it is possible that 8-epi-PGF2alpha generated in polyunsaturated fatty acid-rich food (during initial processing/packaging or during meal preparation) may become incorporated within these lipoproteins during synthesis. In view of the growing use of 8-epi-PGF2alpha as a marker of lipid peroxidation in vivo in nutritional or clinical studies, it is therefore important to investigate the possibility that the circulating levels measured could be confounded by the presence of 8-epi-PGF2alpha in food. In this study we evaluated the levels of 8-epi-PGF2alpha present in several popular fast-foods, using a combination of solid phase extraction and gas chromatography-mass spectrometry. Fast-foods were selected to represent meals prepared from vegetable-, chicken-, fish- and meat-derived ingredients. Total (free + esterified) 8-epi-PGF2alpha levels ranged from 0.09 to 0.73 pmol/g (122-644 pmol/mmol arachidonic acid), with the highest levels present in beef-derived meals. Further investigation of hamburgers and cheeseburgers revealed 8-epi-PGF2alpha levels of 1.83 +/- 0.24 and 0.84 +/- 0.03nmol/mmol arachidonic acid, respectively. Lower concentrations of vitamin E were found in the hamburgers. The postprandial contribution to plasma 8-epi-PGF2alpha levels following ingestion of 100 g portions of these fast-foods would therefore be expected to be no greater than the low picomole range, and would be unlikely to influence the normal endogenous levels of 8-epi-PGF2alpha and those produced during oxidative stress.     

Production of 6-oxo-prostaglandin F1 alpha and prostaglandin E2 by isolated glomeruli from normal and diabetic rats.

Production of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) and prostaglandin E2 (PGE2) was measured by radioimmunoassay in supernatants of isolated glomeruli from rats with streptozocin-induced diabetes and non-diabetic rats. Production of 6-oxo-PGF1 alpha by discs of aortas from these rats was measured at the same time. As shown before, aortic discs from diabetic rats produced significantly less 6-oxo-PGF1 alpha than aortic discs from non-diabetic rats (diabetic 1.99 +/- SEM 0.27 ng v non-diabetic 2.92 +/- 0.46 ng/mg net weight aorta; p less than 0.05). In contrast production of 6-oxo-PGF1 alpha by isolated glomeruli was not reduced in the diabetic rats (diabetic 77 +/- 7 pg v non-diabetic 70 +/- 8 pg/micrograms glomerular DNA). Similarly production of PGE2 was not diminished in the diabetic glomeruli (diabetic 1.20 +/- 0.15 ng v non-diabetic 0.91 +/- 0.12 ng/microgram glomerular DNA). It is concluded that regional differences in production of prostacyclin and 6-oxo-PGF1 alpha occur in experimental diabetes. Diminished prostacyclin production may contribute to the increased susceptibility of diabetic patients to atherosclerosis but is less likely to have a role in the pathogenesis of microangiopathy.     

Effect of cyclooxygenase blockade on gas exchange and hemodynamics in Pseudomonas pneumonia

Acute bilateral Pseudomonas aeruginosa pneumonia was induced in 10 anesthetized dogs, after which five dogs received intravenous indomethacin (2 mg/kg) (indomethacin group), whereas five others were infused with saline (2 ml/kg) (control group). Plasma levels of 6-ketoprostaglandin F1 alpha(6-keto-PGF1 alpha) and thromboxane B2 (TxB2), stable metabolites of prostacyclin (PGI2) and thromboxane A2 (TxA2), respectively, were measured by radioimmunoassay. Although TxB2 levels were not different before and after inoculation in either group, 6-keto-PGF1 alpha levels increased from their base-line value in each animal as pneumonia developed (indomethacin group: less than 100 to 330 +/- 90 pg/ml; control group: less than 100 to 630 +/- 300 pg/ml). Both prostaglandins fell to less than 100 pg/ml in each dog after indomethacin infusion, whereas they remained elevated in the control group after infusion of normal saline. Perfusion of consolidated lung regions (Qp/QT), measured with radioactive microspheres and expressed as a percent of total pulmonary blood flow, was dramatically reduced after indomethacin (35 +/- 3 to 16 +/- 1%) with consequent improvement in pulmonary shunt (Qs/QT: 30 +/- 8 to 18 +/- 6%) and arterial O2 tension (PaO2: 123 +/- 25 to 274 +/- 77 Torr). These parameters remained unchanged or deteriorated further in the control group after infusion of saline. Three additional dogs with Pseudomonas pneumonia were studied in which the indomethacin-induced reduction in Qp/QT was substantially but not completely reversed by intravenous infusion of PGI2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological modulation of prostacyclin and thromboxane production of rat and cat venous tissue slices.

To reveal a potential modulating effect of vasoactive pharmacological agents on the prostanoid production of the venous wall, prostacyclin and thromboxane release from venous tissue slices was studied. Aortic and caval vein samples from 20 rats as well as from 21 cats were studied. Prostacyclin and thromboxane productions were determined by radioimmunoassay as 6-keto-PGF1 alpha and TxB2 released into the incubation medium. Venous tissue produced significantly less prostacyclin per unit weight than arterial tissue in rats (30.7 +/- 4.6 vs. 52.1 +/- 8.2 pg/mg/min), while in cats an opposite situation was found (16.6 +/- 3.2 vs. 7.06 +/- 1.9 pg/mg/min). Thromboxane production of venous tissue was consequently higher than corresponding values for aortic tissue (3.72 +/- 0.46 vs. 1.54 +/- 0.14 in rats and 3.4 +/- 0.6 vs. 1.33 +/- 0.19 in cats, all values in pg/mg/min). Norepinephrine and dopamine significantly increased both the prostacyclin and the thromboxane release from venous tissue, while isoproterenol had no effect. Vasopressin significantly increased thromboxane release and decreased the ratio of prostacyclin vs. thromboxane production (from 10.4 +/- 1.6 to 7.5 +/- 1.6, in acetylsalicylic acid pretreated cats). Angiotensin and thrombin had no significant effects. Bradykinin (0.5 microgram/ml) significantly augmented prostacyclin release from venous tissue (14.4 +/- 2.6 from 10.9 +/- 2.4 pg/mg/min) and decreased thromboxane release (0.65 +/- 0.18 from 1.35 +/- 0.22 pg/mg/min). Methionine-enkephalin (5 micrograms/ml) significantly reduced the thromboxane release from venous tissue slices. The presented material demonstrates that several vasoactive agents modulate the vasoactive prostanoid release of the venous wall. In some cases, the prostacyclin and the thromboxane productions are influenced separately, which in turn will have its impact on smooth muscle activity and thrombocyte aggregation.     

Selective right, but not left, coronary endothelial dysfunction precedes development of pulmonary hypertension and right heart hypertrophy in rats

We investigated a causal role for coronary endothelial dysfunction in development of monocrotaline (MCT)-induced pulmonary hypertension and right heart hypertrophy in rats. Significant increases in pulmonary pressure and right ventricular weight did not occur until 3 wk after 60 mg/kg MCT injection (34 +/- 4 vs. 19 +/- 2 mmHg and 37 +/- 2 vs. 25 +/- 1% septum + left ventricular weight in controls, respectively). Isolated right coronary arteries (RCA) showed significant decreases in acetylcholine-induced NO dilation in both 1-wk (33 +/- 3% with 0.3 microM; n = 5) and 3-wk (18 +/- 3%; n = 11) MCT rats compared with control rats (71 +/- 8%, n = 10). Septal coronary arteries (SCA) showed a smaller decrease in acetylcholine dilation (55 +/- 8% and 33 +/- 7%, respectively, vs. 73 +/- 8% in controls). No significant change was found in the left coronary arteries (LCA; 88 +/- 6% and 81 +/- 6%, respectively, vs. 87 +/- 3% in controls). Nitro-L-arginine methyl ester-induced vasoconstriction, an estimate of spontaneous endothelial NO-mediated dilation, was not significantly altered in MCT-treated SCA or LCA but was increased in RCA after 1 wk of MCT (-41 +/- 6%) and decreased after 3 wk (-18 +/- 3% vs. -27 +/- 3% in controls). A marked enhancement to 30 nM U-46619-induced constriction was also noted in RCA of 3-wk (-28 +/- 6% vs. -9 +/- 2% in controls) but not 1-wk (-12 +/- 7%) MCT rats. Sodium nitroprusside-induced vasodilation was not different between control and MCT rats. Together, our findings show that a selective impairment of right, but not left, coronary endothelial function is associated with and precedes development of MCT-induced pulmonary hypertension and right heart hypertrophy in rats.     

Eicosanoid generation and responsiveness of human lymphatics in hyperlipoproteinemia

In this work, the oxidation injury in hyperlipoproteinemia (HLP) was determined by measuring the isoprostane 8-epi-prostaglandin (PG) F2alpha in human lymphatics, lymph fluid, plasma, serum and urine. Lymphatics from 6 patients with HLP generated less PGI2 and contained more 8-epi-PGF2alpha as compared to 6 normolipemics without risk factors. Likewise, plasma (29.3 vs 19.7 pg/ml), lymph fluid (137.3 vs 65.3 pg/ml), serum (286.7 vs 204.1 pg/ml) and urinary (360.8 vs 241.0 pg/mg creatinine) values of 8-epi-PGF2alpha in HLP (as compared to normolipemics) were significantly elevated. Lymphatics from HLP showed an enhanced contractile response, less 14C-arachidonic acid conversion to PGI2 and less PGI2-formation upon various stimuli compared to normolipemics of comparable age. These findings indicate that HLP-induced oxidation injury, resulting in an altered (iso-)eicosanoid production and function, may also significantly affect (patho-) physiology of lymphathics.     

Increased urinary excretion of 8-iso-prostaglandin F2alpha in patients with HFE-related hemochromatosis: a case-control study

The hypothesis according to which iron overload could be harmful has been extensively and controversially discussed in the literature. One underlying pathological mechanism may be elevated oxidative stress. Thus, we studied the correlation between hemochromatosis and an established marker of oxidative stress, 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha, iPF2alpha-III, 15-F2t-IsoP). We enrolled 21 patients with hemochromatosis, positive for the homozygous C282Y mutation in the HFE gene, and 21 healthy controls frequency-matched by age and gender in a case-control study design. The objective was to show that iron overload in HFE-related hemochromatosis is associated with increased oxidative stress assessed through 8-iso-PGF(2alpha) urinary excretion, and that oxidative stress is impacted by iron-removal treatment (phlebotomy). Study parameters were transferrin saturation, 8-iso-PGF(2alpha) urine excretion, transferrin, ferritin, serum iron, and vitamins A and E for all participants. Iron concentration in the liver and non-transferrin-bound iron were measured in patients only. We found a significant difference in 8-iso-PGF2alpha in patients (245 [interquartile range 157-348] pg/mg creatinine) compared with controls (128 [106-191] pg/mg creatinine, P = 0.002). Vitamin A was significantly reduced in cases (0.34 [0.25-1.83] microg/ml compared to 3.00 [2.11-3.39] microg/ml, P < 0.001), while vitamin E did not show a significant difference in cases (14.7 [11.5-18.1] microg/ml) compared with controls (14.9 [13.1-19.2] microg/ml, P = 0.52). After phlebotomy treatment and normalization of the iron parameters in the hemochromatosis group, serum vitamin A levels were significantly increased (1.36 [1.08-1.97] microg/ml, P = 0.035 vs. baseline, P < 0.001 vs. controls) and 8-iso-PGF2alpha urinary excretion was lowered to control levels (146 [117-198] pg/mg creatinine, P = 0.38 vs. controls). In our study, HFE-related hemochromatosis was associated with increased oxidative stress and hypovitaminemia A in C282Y homozygotes. The increased oxidative stress was reversible by normalization of the iron load by phlebotomy. Thus, phlebotomy is an effective and adequate means for reducing oxidative stress in these patients.     

Epidermal growth factor in serum, urine, submandibular glands and kidneys of diabetic mice

Levels of epidermal growth factor (EGF) in serum were significantly decreased in streptozotocin (STZ)-diabetic mice (446 +/- 168 pg/ml after 1 week and 423 +/- 52 after 4 weeks vs 766 +/- 162 pg/ml in controls, P.002 and less than .001. respectively) and in genetically diabetic ob/ob mice (455 +/- 285 vs 962 +/- 453 pg/ml in nondiabetic ob/+ controls, P.043). The urinary excretion of EGF was significantly increased in STZ mice (104 +/- 53 vs 51 +/- 23 ng/h, P.013) but unchanged in ob/ob mice (33 +/- 9 vs 45 +/- 16 ng/h, P.134). However, when expressed per mg creatinine it was decreased in both cases: in STZ mice to 680 +/- 250 ng/mg at 1 week and 684 +/- 211 at 4 weeks vs 1250 +/- 303 ng/mg in controls (P less than .01); and in the ob/ob mice to 552 +/- 117 vs 1237 +/- 300 ng/mg in ob/+ controls (P less than .01). EGF content of the submandibular glands of STZ mice remained unchanged at 1 week (13.1 +/- 2.9 vs 11.0 +/- 1.8 micrograms/mg protein, P.170) but dropped by 4 weeks (4.7 +/- 1.2 micrograms/mg, P less than .001); in the ob/ob mice it was less than 20% that of controls (2.1 +/- 0.8 vs 12.2 +/- 3.6 micrograms/mg protein). In kidneys, the EGF content was not altered in either ob/ob (524 +/- 50 vs 571 +/- 33 pg/mg protein) or STZ mice (652 +/- 183 vs 665 +/- 80 pg/mg). The preproEGF mRNA level in STZ-treated mice was reduced after 4 weeks in submandibular glands but not in kidneys. The results show that diabetes affects EGF production, utilization and/or excretion in mice and that kidneys are spared from suppression of EGF synthesis that is pronounced in the submandibular glands.     

Leukotrienes C4, D4, and E4 in fetal lamb tracheal fluid

Previously, we demonstrated that either putative leukotriene receptor antagonists or a synthesis inhibitor markedly decreased pulmonary vascular resistance in the near-term fetal lamb and concluded that leukotrienes may play a role in maintaining the high pulmonary vascular resistance in the fetus. To further investigate the role of leukotrienes, we measured concentrations of leukotriene (LT) C4, LTD4, and LTE4 in 17 tracheal fluid samples from 8 of 9 near-term (129-139 days, term = 145 days), chronically-catheterized, fetal lambs during normoxia to evaluate their possible role in regulating resting tone and in seven of the nine before and during hypoxia to evaluate their possible role in hypoxic vasoconstriction. The tracheal fluid samples collected by gravity over 1-3 min, on ice, were immediately treated with cold ethanol, centrifuged, and the supernatant covered with N2 and stored in a -70 degrees C freezer for a maximum of 3 weeks. Purification and separation of leukotrienes was done by reverse-phase high performance liquid chromatography using a gradient elution method, and fractions corresponding to LTC4, LTD4, and LTE4 standards were quantified immediately by radioimmunoassay. During normoxia (descending aortic PaO2 2.9 +/- 0.3 kPa [21.5 +/- 2.5 mmHg]; mean +/- SD), all 3 leukotrienes were detected in 16 of the 17 samples: LTC4 29 +/- 28 pg/ml (range 0-119 pg/ml); LTD4 66 +/- 51 pg/ml (range 9-177 pg/ml); and LTE4 43 +/- 50 pg/ml (range 0-204 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of C-type natriuretic peptide and of its receptor NPR-B in normal and failing heart

C-type natriuretic peptide (CNP) was recently found in the myocardium, but possible insights into differences between atrium and ventricle production are so far lacking. Our aim was to evaluate, in an experimental model of pacing-induced heart failure (HF), plasma and tissue levels of CNP and mRNA expression of the peptide and of its specific receptor, NPR-B. Cardiac tissue was collected from male adult minipigs without (control, n=5) and with pacing-induced HF (n=5). Blood samples were collected at baseline and after pacing (10 min, 1, 2, 3 weeks). CNP in plasma and in cardiac extracts was determined by a radioimmunoassay, while the expression of mRNA by real time PCR. Compared to control, plasma CNP was increased after 1 week of pacing stress (36.9+/-10.4 pg/ml vs.16.7+/-1.1, p=0.013, mean+/-S.E.M.). As to myocardial extract, at baseline, CNP was found in all cardiac chambers and its content was 10-fold higher in atria than in ventricles (RA: 13.7+/-1.9 pg/mg protein; LA: 8.7+/-3.8; RV: 1.07+/-0.33; LV: 0.93+/-0.17). At 3 weeks of pacing, myocardial levels of CNP in left ventricle were higher than in controls (15.8+/-9.9 pg/mg protein vs. 0.9+/-0.17, p=0.01). CNP gene expression was observed in controls and at 3 weeks of pacing. NPR-B gene expression was found in all cardiac regions analyzed, and a down-regulation was observed in ventricles after HF. The co-localization of the CNP system and NPR-B suggests a possible role of CNP in HF and may prompt novel therapeutical strategies.     

Alterations in atrial and plasma atrial natriuretic factor (ANF) content during development of hypoxia-induced pulmonary hypertension in the rat

Distension of the atrial wall has been proposed as a signal for the increased release of atrial natriuretic factor (ANF) from atrial myocytes in response to perceived volume overload. To determine whether pressure changes resulting from hypertension in the pulmonary circulation may stimulate release of ANF, rats were exposed to chronic hypobaric hypoxia for 3 or 21 days and the ANF concentration in the atria and plasma were determined by specific radioimmunoassay. Exposure to chronic hypoxia resulted in significant increases in hematocrit at both 3 (p less than 0.025) and 21 days (p less than 0.005) and in the development of right ventricular hypertrophy (RVH) expressed as the ratio of the weight of the right ventricle to the weight of the left ventricle and septum (RV/LV+S) at both 3 (RV/LV+S = 0.278 +/- 0.005) and 21 days (RV/LV+S = 0.536 +/- 0.021). After 21 days, left atrial (LA) ANF content was significantly increased in hypoxic rats compared to controls (508 +/- 70 ng/mg tissue vs 302 +/- 37 ng/mg), while right atrial (RA) ANF content was significantly reduced (440 +/- 45 vs 601 +/- 58 ng/mg). At this time, plasma ANF concentration was significantly elevated compared to controls (238 +/- 107 pg/ml vs 101 +/- 10 pg/ml). These results suggest that the development of pulmonary hypertension following chronic hypobaric exposure induces altered atrial ANF content and increased plasma ANF concentration as a result of altered distension of the atrial wall.     

Norepinephrine-stimulated vascular prostacyclin synthesis. Receptor-dependent calcium channels control prostaglandin synthesis

Norepinephrine-stimulated prostacyclin synthesis was studied in rat aortic rings by measuring 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay. Norepinephrine (10(-6) M) results in a 10- to 20-fold increase in 6-keto-PGF1 alpha synthesis by rat aortic rings (54 +/- 11 to 437 +/- 260 pg X mg wet weight-1 X 20 min-1). The maximal stimulation of 6-keto-PGF1 alpha synthesis was observed with a norepinephrine concentration of 10(-5) M at a mean effective concentration (EC50) of 9.5 +/- 3.2 X 10(-7) M which is similar to the contractile response (Emax = 10(-5) M, EC50 = 6.5 +/- 1.8 X 10(-7) M). Potassium chloride (30 mM), although causing a similar maximal contractile response as 10(-6) M norepinephrine, did not increase 6-keto-PGF1 alpha synthesis. Norepinephrine-stimulated 6-keto-PGF1 alpha synthesis was dependent upon extracellular calcium. Norepinephrine stimulation in Ca2+-free medium did not lead to a significant increase in 6-keto-PGF1 alpha synthesis. However, on the introduction of Ca2+, 6-keto-PGF1 alpha synthesis was restored to its initial level. Phentolamine (10(-6) M) (an alpha-adrenergic antagonist) and trifluroperazine (2.5 X 10(-4) M) (a calmodulin inhibitor) completely inhibited norepinephrine-stimulated 6-keto-PGF1 alpha synthesis, whereas verapamil 3 X 10(-6) M (a calcium channel blocking drug) only partially inhibited synthesis (control, 74 +/- 12; norepinephrine, 437 +/- 260; norepinephrine + verapamil, 123 +/- 8 pg X mg wet weight-1 X 20 min-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomarkers of exposure and potential harm in adult smokers of 3-7 mg tar yield (Federal Trade Commission) cigarettes and in adult non-smokers.

The paper reports levels of 24-h urine nicotine and five of its major metabolites (expressed as nicotine-equivalents) and blood carboxyhaemoglobin as biomarkers of exposure to particulate- and gas-phase cigarette smoke, respectively, from an exploratory pilot study of adult smokers of 3.0-6.9 mg tar delivery (Federal Trade Commission (FTC) method) cigarettes. On multiple occasions over 6 weeks, blood high-sensitivity C-reactive protein (hs-CRP), fibrinogen, HDL- and LDL-cholesterol, and 24-h urine 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) and 11-dehydro-thromboxane B2 (11-dehydro-TxB2) were also evaluated as biomarkers of potential harm. All the biomarkers examined, except for LDL-cholesterol, discriminated with high sensitivity and specificity between adult smokers and non-smokers overall. Except for HDL-cholesterol, all biomarker medians were greater in adult smokers than in non-smokers: urine nicotine-equivalents 64.514 versus < 0.034 nmol mg-1 creatinine (p<0.001), carboxyhaemoglobin 4.0 versus 0.4% saturation (p<0.001), hs-CRP 0.27 versus 0.12 mg dl-1 (p=0.05), fibrinogen 292 versus 248 mg dl-1 (p<0.001), HDL-cholesterol 46 versus 53 mg dl-1 (p=0.003), LDL-cholesterol 119 versus 109 mg dl-1 (p=0.18), urine 8-epi-PGF2alpha 1935 versus 1034 pg mg-1 creatinine (p<0.001) and urine 11-dehydro-TxB2 973 versus 710 pg mg-1 creatinine (p<0.001). All the biomarkers of exposure and most of the biomarkers of potential harm showed no time of sampling (by visit week) effect.     

Effect of overexpression of inhibin alpha (1-32) fragment on bovine granulosa cell proliferation, apoptosis, steroidogenesis, and development of co-cultured oocytes

The objective was to determine the effects of an inhibin alpha (1-32) fragment gene on proliferation, apoptosis, and steroidogenesis of bovine granulosa cells (GC) isolated from medium and small follicles (diameter >4-8 and 1-4mm, respectively), and the effect of GC, previously transfected with pEGISI, on oocyte maturation and in vitro embryo development. To enhance expression of the inhibin alpha (1-32) fragment, GC were transfected with pEGISI. Transfection inhibited (P<0.05) GC proliferation (88.8+/-2.1%; mean+/-S.E.M.) compared to the control and EGFP groups (100% and 97.5+/-2.1%) from medium follicles, with no significant effect on GC from small follicles. Apoptosis was higher (P<0.01) in transfected GC than in controls. Transfection increased (P<0.05) estradiol synthesis from both medium and small follicles (0.57+/-0.13 and 0.86+/-0.13 pg/mL vs. 0.19+/-0.05 and 0.35+/-0.09 pg/mL in controls) after culturing for 48 h, with suppression (P<0.05) in transfected GC after 96 h. Transfection reduced (P<0.05) progesterone synthesis in GC from both medium and small follicles (24.5+/-3.4 and 75.4+/-4.6 ng/mL vs. 45.42+/-5.33 and 117.32+/-11.99 ng/mL in controls) after culture for 48 h, with no significant difference after 96 h. Maturation rate of oocytes co-cultured with transfected GC from medium follicles was decreased relative to control (61.5+/-6.8% vs. 71.2+/-5.7%, P<0.05), with no significant effect on embryo development. In conclusion, overexpression of inhibin alpha (1-32) fragment regulated GC development; effects on subsequent oocyte maturation were both time- and stage-dependent.     

The concentrations of 6-keto-PGF1 alpha and TXB2 in plasma samples from patients with benign and malignant tumours of the breast

Peripheral plasma concentrations of 6-keto-PGF1 alpha and TXB2 were measured in patients with benign and malignant tumours of the breast, in patients with non-gynecological diseases, and in healthy female controls. The values were significantly higher in female patients with malignant tumours of the breast than in healthy controls (146 +/- 28 vs 13 +/- 2.5 pg/ml for 6-keto-PGF1 alpha p less than 0.01 and 78 +/- 17 vs 11 +/- 2 pg/ml for TXB2, p less than 0.01). Benign tumours of the breast were also associated with significantly raised plasma levels of 6-keto-PGF1 alpha and TXB2 compared to normal controls (52 +/- 5 vs 13 +/- 2.5 pg/ml for 6-keto-PGF1 alpha, p less than 0.01 and 26 +/- 5 vs 11 +/- 2 pg/ml for TXB2, p less than 0.05). The high levels of 6-keto-PGF1 alpha and TXB2 were not found to be correlated with clinical and histopathological data. The surgical removal of the primary tumour has apparently no effect on the plasma concentrations of 6-keto-PGF1 alpha and TXB2 over a follow-up period of 9 days after operation. The lack of alterations in the ratio of TXB2:6-keto-PGF1 alpha in the cancer patients and other subjects studied before and after surgery is indicative of the regulatory power of metabolic systems to preserve the homeostatic balance.