8-isoprostane in exhaled breath condensate after experimental exposure to wood smoke in humans.

Wood smoke, a well-known indoor and outdoor air pollutant, may cause adverse health effects through oxidative stress. In this study 8-isoprostane, a biomarker of oxidative stress, was measured in exhaled breath condensate (EBC) and urine before and after experimental exposure to wood smoke. The results were compared with measurements of other biomarkers of oxidative stress and inflammation. Thirteen subjects were exposed first to clean air and then, after 1 week, to wood smoke in an exposure chamber during 4-hour sessions. Exhaled breath condensate, exhaled nitric oxide, blood and urine were sampled before and at various intervals after exposure to wood smoke and clean air. Exhaled breath condensate was examined for 8-isoprostane and malondialdehyde (MDA), while exhaled air was examined for nitric oxide, serum for Clara cell protein (CC16) and urine for 8-isoprostane. 8-isoprostane in EBC did not increase after wood smoke exposure and its net change immediately after exposure was inversely correlated with net changes in MDA (r(s)= -0.57, p= 0.041) and serum CC16 (S-CC16) (r(p)= -0.64, p= 0.020) immediately after the exposure. No correlation was found between 8-isoprostane in urine and 8-isoprostane in EBC. In this study controlled wood smoke exposure in healthy subjects did not increase 8-isoprostane in EBC.

Exhaled breath condensate, nasal eosinophil cationic protein level and nasal cytology during immunotherapy for cypress allergy.

Interest in cypress allergy is widely rising: an increasing number of studies have pointed out the efficacy of immunotherapy to reduce cypress-related symptoms and drug use. Cypress immunotherapy is well tolerated, but there are few studies dealing with its sub-clinical effects on the airways. The aim of this investigation is to assess the effects of immunotherapy on airways by the analysis of exhaled breath condensate (EBC), nasal lavage fluid (NAL) and nasal cytology. Fifteen mono-sensitized to cypress pollen patients have been observed, among them 9 have been treated with sub-cutaneous immunotherapy (SCIT), 3 with sub-lingual immunotherapy (SLIT) and 3 which were not treated underwent EBC, NAL and nasal cytology out of the pollen season. 8-isoprostane in EBC, Eosinophil cationic protein (ECP) and inflammatory cells in nasal cytology were also evaluated. The median value of 8-isoprostane in EBC was 18.58 pg/ml in patients who did not undergo immunotherapy, 49.38 pg/ml in SCIT patients and 13.41 pg/ml in SLIT subjects. The median value of ECP in nasal lavage was higher in non- treated subjects (27.3 mg/l) than in those treated with SCIT (1 mg/l)(p less than 0,05) or SLIT (2.6 mg/l). All nasal cytology specimens did not show any sign of inflammation. In conclusion SLIT seems to be well tolerated and to reduce significantly the levels of ECP in nasal lavage. In addition the levels of 8-isoprostane in EBC among SCIT patients were unexpectedly high and need to be further evaluated.

Thromboxane and prostacyclin biosynthesis in heart failure of ischemic origin: effects of disease severity and aspirin treatment.

SUMMARY BACKGROUND: Thromboembolism is a relatively common complication of chronic heart failure (HF) and the place of antiplatelet therapy is uncertain. OBJECTIVES: We characterized the rate of thromboxane and prostacyclin biosynthesis in chronic HF of ischemic origin, with the aim of separating the influence of HF on platelet activation from that of the underlying ischemic heart disease (IHD). PATIENTS AND METHODS: We compared urinary 11-dehydro-thromboxane (TX)B(2), 2,3 dinor 6-keto-PGF(1alpha,) 8-iso-prostaglandin (PG)F(2alpha), and plasma N-terminal pro-brain natriuretic peptide (NT-pro-BNP), asymmetric dimethylarginine (ADMA), and soluble CD40 ligand (sCD40L), in 84 patients with HF secondary to IHD, 61 patients with IHD without HF and 42 healthy subjects. RESULTS: HF patients not on aspirin had significantly higher urinary 11-dehydro-TXB(2) as compared with healthy subjects (P < 0.0001) and IHD patients not on aspirin (P = 0.028). They also showed significantly higher 8-iso-PGF(2alpha) (P = 0.018), NT-pro-BNP (P = 0.021) and ADMA (P < 0.0001) than IHD patients not on aspirin. HF patients on low-dose aspirin had significantly lower 11-dehydro-TXB(2) (P < 0.0001), sCD40L (P = 0.007) and 2,3-dinor-6-keto-PGF(1alpha) (P = 0.005) than HF patients not treated with aspirin. HF patients in NYHA classes III and IV had significantly higher urinary 11-dehydro-TXB(2) than patients in classes I and II, independently of aspirin treatment (P < 0.05). On multiple linear regression analysis, higher NT-pro-BNP levels, lack of aspirin therapy and sCD40L, predicted 11-dehydro-TXB(2) excretion rate in HF patients (R(2) = 0.771). CONCLUSIONS: Persistent platelet activation characterizes HF patients. This phenomenon is related to disease severity and is largely suppressable by low-dose aspirin. The homeostatic increase in prostacyclin biosynthesis is impaired, possibly contributing to enhanced thrombotic risk in this setting.

Postprandial hyperglycemia is a determinant of platelet activation in early type 2 diabetes mellitus.

BACKGROUND: Chronic hyperglycemia is a major contributor to in vivo platelet activation in diabetes mellitus. OBJECTIVES: To evaluate the effects of acarbose, an alpha-glucosidase inhibitor, on platelet activation and its determinants in newly diagnosed type 2 diabetic patients. METHODS: Forty-eight subjects (26 males, aged 61 +/- 8 years) with early type 2 diabetes (baseline hemoglobin A(1c) < or = 7% and no previous hypoglycemic treatment) were randomly assigned to acarbose up to 100 mg three times a day or placebo, and evaluated every 4 weeks for 20 weeks. The main outcome measures were urinary 11-dehydro-thromboxane (TX)B(2) (marker of in vivo platelet activation) and 8-iso-prostaglandin (PG)F(2alpha) (marker of in vivo lipid peroxidation) excretion rate, 2-h postprandial plasma glucose (PPG) after a test meal, and assessment of glucose fluctuations by mean amplitude of glycemic excursions (MAGE). RESULTS: Baseline measurements revealed biochemical evidence of enhanced lipid peroxidation and platelet activation. As compared with the placebo group, patients treated with acarbose had statistically significant reductions in urinary 11-dehydro-TXB(2) and 8-iso-PGF(2alpha) excretion rate as early as after 8 weeks and at each subsequent time point (between-group P < 0.0001 at 12, 16 and 20 weeks), following earlier decreases in PPG and MAGE. Multiple regression analyses in the acarbose group revealed that PPG was the only significant predictor of 11-dehydro-TXB(2) urinary excretion rate (beta = 0.39, P = 0.002) and MAGE the only predictor of 8-iso-PGF(2alpha) urinary excretion rate (beta = 0.42, P = 0.001). CONCLUSIONS: Postprandial hyperglycemia is associated with enhanced lipid peroxidation and platelet activation in early type 2 diabetes. A moderate decrease in PPG achieved with acarbose causes time-dependent downregulation of these phenomena, suggesting a causal link between early metabolic abnormalities and platelet activation in this setting.

Novel perspectives in the detection of oral and nasal oxidative stress and inflammation in pediatric united airway diseases.

United airway disease (UAD) concept proposed that asthma and rhinitis are both different clinical manifestation of a single inflammatory process. The aim of this study is to assess in upper and lower airways the level of inflammation and oxidative stress and to investigate the relationship between biomarkers in persistent allergic rhinitis (PER) and in concomitant asthma with PER. By a crosssectional study we measured oral and nasal (FENO) and oral and nasal EBC 8-isoprostane, LTB4 and PGE2 in children with PER (n=14) and with PER and concomitant intermittent asthma (IA; n=25), mild persistent asthma (mA; n=28), moderate persistent asthma (MA; n=13) and in Healthy Controls (HCs; n=13). Oral and nasal FENO concentrations were increased in children with PER, IA, mA and MA when compared with HCs. Nasal 8-isoprostane was higher in EBC of children with PER and asthma than in HCs. Oral and nasal LTB4 were higher in EBC of children with PER and mA than in HCs. Oral and nasal PGE2 concentrations were higher in EBC of children with PER than in HCs. Positive correlations between oral and nasal biomarkers were found in IA for LTB4 and PGE2, in mA for FENO, 8-isoprostane, LTB4 and PGE2, and in MA for PGE2. No correlations were observed in children with PER and HCs. Our results suggest that non-invasive markers of inflammation and oxidative stress might be useful to study the relationships between oral and nasal compartments in allergic children with PER and concomitant asthma with the aim of defining the UAD.

Defective platelet responsiveness to thrombin and protease-activated receptors agonists in a novel case of gray platelet syndrome: correlation between the platelet defect and the alpha-granule content in the patient and four relatives.

BACKGROUND: We report a novel case of gray platelet syndrome (GPS). A 14-year-old boy had bleeding diathesis, mild thrombocytopenia, giant platelets with severe defect of alpha-granule secretory proteins, myelofibrosis and splenomegaly. METHODS AND RESULTS: Platelet function studies showed a marked reduction of aggregation and Ca(2+) mobilization by thrombin, protease-activated receptor 1 (PAR1)-activating peptide (AP) and PAR4-AP, PAR1 expression at 55% of normal levels, and a more than two hundred fold reduction of in vitro whole-blood thromboxane B(2) (TXB(2)) production. Sequencing of coding regions of the PAR1 gene failed to show abnormalities. This patient was initially classified as a sporadic case of GPS, as electron microscopy failed to identify giant platelets and/or alpha-granule deficiency in his relatives. However, further studies on the father and three other relatives showed a relative lack of platelet alpha-granule proteins by immunofluorescence microscopy, a defective platelet response to PAR4-AP, and severely reduced in vitro whole-blood TXB(2) production. On this basis, we suggest that in this family, GPS was transmitted in a dominant fashion with highly variable penetrance. CONCLUSIONS: Our study suggests that current diagnostic criteria fail to identify some patients with a mild GPS phenotype and that such patients might be identified by the methods cited above. It also better characterizes the pathogenesis of defective platelet responses to thrombin, and raises interesting questions on the correlation between abnormal PAR function and the lack of alpha-granule content in GPS.

Induced sputum, exhaled breath condensate and nasal lavage fluid in electroplating workers exposed to chromium.

Occupational exposure to chromium may cause airway inflammation and bronchial asthma. In this study we investigated the effect of chromium on the respiratory tract of exposed and non-exposed electroplating workers using spirometry and analysis of induced sputum (IS), exhaled breath condensate (EBC) and nasal lavage fluid (NLF). In both groups spirometry was normal; chromium in induced sputum was higher in exposed workers (7.90 +/- 0.855 microg/L, vs 1.78 +/- 0.075 microg/L; p<0.001); no significant difference was found in induced sputum cellularity. Median nitrite concentration in EBC was significantly higher in exposed subjects (4.35 micromol/L, 5 degrees -95 degrees percentile: 1.88-10.13 vs 0.11 micromol/L, 5-95 percentile: 0-0.72) (p<0.001). IL-6 and TNF-alpha were not detectable in EBC. Median IL-6 concentration in nasal lavage fluid was higher in exposed workers (5.72 pg/ml, 5-95 percentile: 0-65.25 pg/ml vs 0.28 pg/ml, 5-95 percentile: 0-1.7 pg/ml) (p<0.01). No differences in Eosinophil Cationic Protein concentration were found. TNF-alpha was not detectable in NLF. Chromium in induced sputum correlated with nitrites in EBC. For the first time three non-invasive methods were used to assess changes in respiratory tract in workers exposed to chromium. The results suggest chromium exerts an inflammatory/irritative action on airways.

Inhibition of thromboxane biosynthesis by triflusal in type 2 diabetes mellitus.

Triflusal is an antiplatelet drug related to aspirin, with different pharmacological properties and a lower haemorrhagic risk. We aimed at comparing their effects on platelet and endothelial activation in type 2 diabetes mellitus (T2DM). In a randomized, double-blind, parallel group study, we compared the effects of three daily regimens (300, 600, and 900 mg) of triflusal, and aspirin (100mg/day) on urinary 11-dehydro-thromboxane (TX)B(2), index of in vivo platelet activation, ex vivo platelet function using the analyzer PFA-100, plasma von Willebrand factor (vWF), P-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and serum nitrite and nitrate (NO(2)(-)+NO(3)(-)) in 60 T2DM patients. Triflusal induced a dose-dependent reduction in 11-dehydro-TXB(2) and a prolongation of closure time in the presence of collagen plus epinephrine (Coll/Epi-CT). The effects of the highest triflusal dose were not different from those of aspirin. The closure time in the presence of collagen plus ADP (Coll/ADP-CT), ICAM-1, VCAM-1, and NO(2)(-)+NO(3)(-) were not modified either by triflusal or aspirin. Plasma P-selectin and vWF were reduced by triflusal but not by aspirin. In T2DM triflusal causes a profound inhibition of platelet TXA(2) biosynthesis in vivo, acting on different targets involved in the platelet-endothelial cell interactions.

Effects of cyclo-oxygenase inhibition on exhaled eicosanoids in patients with COPD.

BACKGROUND: Leukotriene (LT) B4 concentrations are increased and prostaglandin (PG) E2 concentrations are decreased in exhaled breath condensate (EBC) in patients with chronic obstructive pulmonary disease (COPD). A study was undertaken to investigate the short term effects of cyclo-oxygenase (COX) inhibition on exhaled LTB4 and PGE2 concentrations in patients with COPD and to identify the COX isoform responsible for exhaled PGE2 production. METHODS: Two studies were performed. A double blind, crossover, randomised, placebo controlled study with ibuprofen (400 mg qid for 2 days), a non-selective COX inhibitor, was undertaken in 14 patients with stable COPD, and an open label study with oral rofecoxib (25 mg once a day for 5 days), a selective COX-2 inhibitor, was undertaken in a different group of 16 COPD patients. EBC was collected before and after drug treatment. Exhaled LTB4 and PGE2 concentrations were measured with specific immunoassays. RESULTS: All patients complied with treatment as indicated by a reduction in ex vivo serum thromboxane B2 concentrations (ibuprofen) and a reduction in lipopolysaccharide induced increase in ex vivo plasma PGE2 values (rofecoxib) of more than 80%. Exhaled LTB4 was increased after ibuprofen (median 175.5 (interquartile range 128.8-231.5) pg/ml v 84.0 (70.0-98.5) pg/ml, p < 0.001) and exhaled PGE2 was reduced (93.5 (84.0-105-5) pg/ml v 22.0 (15.0-25.5) pg/ml, p < 0.0001). Rofecoxib had no effect on exhaled LTB4 (p = 0.53) or PGE2 (p = 0.23). CONCLUSIONS: Non-selective COX inhibition decreases PGE2 and increases LTB4 in EBC, whereas selective COX-2 inhibition has no effect on these eicosanoids. PGE2 in EBC is primarily derived from COX-1 activity, and COX inhibition may redirect arachidonic acid metabolism towards the 5-lipoxygenase pathway.

Cyclooxygenase-1, but not -2, is upregulated in NB4 leukemic cells and human primary promyelocytic blasts during differentiation.

Cyclooxygenase (COX)-1 or -2 and specific prostaglandin (PG) synthases catalyze the formation of various PGs. We investigated the expression and activity of COX-1 and -2 during granulocyte-oriented maturation induced by all-trans-retinoic acid (ATRA) of NB4 cells, originated from a human acute promyelocytic leukemia (APL), and in blasts from APL patients. The expression of COX isoenzymes or prostaglandin synthases was also investigated in circulating granulocytes and human bone marrow. COX-1 was expressed and enzymatically active in NB4 cells and primary blasts. COX-1 mRNA and protein were induced by ATRA. COX-1 protein increased approximately 2-3.5-fold by culture day 3 in NB4 cells and primary blasts, while basal COX-2 expression was very low and unaffected by ATRA. COX-1-dependent PGE(2) biosynthesis increased during differentiation approx. 5-fold. Indomethacin and the selective COX-1 inhibitor SC-560, but not selective COX-2 inhibition, impaired NB4 differentiation, reducing NADPH-oxidase activity, CD11b and CD11c expression. The immunohistochemistry of granulocytes and myeloid precursors in the bone marrow showed a large prevalence of COX-1 as compared to COX-2. In conclusion, COX-1 is induced during ATRA-dependent maturation and appears to contribute to myeloid differentiation both in vitro and ex vivo, and COX-1 activity may potentiate the differentiation of human APL.Leukemia (2004) 18, 1373-1379. doi:10.1038/sj.leu.2403407 Published online 10 June 2004

Exhaled leukotrienes and prostaglandins in COPD.

BACKGROUND: The role of eicosanoids, including leukotrienes (LTs) and prostaglandins (PGs), in chronic obstructive pulmonary disease (COPD) is uncertain. The aim of this study was to investigate whether eicosanoids are measurable in exhaled breath condensate (EBC), a non-invasive method of collecting airway secretions, in patients with stable mild to moderate COPD, and to show possible differences in their concentrations compared with control subjects. METHODS: LTB(4), LTE(4), PGE(2), PGD(2)-methoxime, PGF(2alpha), and thromboxane B(2) (TxB(2)) were measured in EBC in 15 healthy ex-smokers, 20 steroid naïve patients with COPD who were ex-smokers, and in 25 patients with COPD who were ex-smokers and who were treated with inhaled corticosteroids. The study was of cross sectional design and all subjects were matched for age and smoking habit. RESULTS: LTB(4) and PGE(2) concentrations were increased in steroid naïve (LTB(4): median 100.6 (range 73.5-145.0) pg/ml, p<0.001; PGE(2): 98.0 (range 57.0-128.4) pg/ml, p<0.001) and steroid treated patients with COPD (LTB(4): 99.0 (range 57.9-170.5) pg/ml, p<0.001; PGE(2): 93.6 (range 52.8-157.0) pg/ml, p<0.001) compared with control subjects (LTB(4): 38.1 (range 31.2-53.6) pg/ml; PGE(2): 44.3 (range 30.2-52.1) pg/ml). Both groups of patients had similar concentrations of exhaled LTB(4) (p=0.43) and PGE(2) (p=0.59). When measurable, LTE(4) and PGD(2)-methoxime concentrations were similar in COPD patients and controls, whereas PGF(2alpha) concentrations were increased in the former. TxB(2)-LI was undetectable in any of the subjects. CONCLUSIONS: There is a selective increase in exhaled LTB(4) and PGE(2) in patients with COPD which may be relatively resistant to inhaled corticosteroid therapy.

Lipid and protein oxidation contribute to a prothrombotic state in patients with type 2 diabetes mellitus.

Diabetes mellitus (DM) is associated with enhanced lipid oxidation and persistent platelet activation. We investigated whether oxidant stress (OS) also affects circulating proteins and is associated with an abnormal coagulative pattern. In 72 type 2 DM (T2DM) patients, urinary 8-iso-prostaglandin (PG) F2alpha and 11-dehydro-thromboxane B2 (TXM) were measured as markers of lipid peroxidation and platelet activation, respectively. The carbonyl content of plasma proteins (PCARB) was measured as global index of protein oxidation. 8-Iso-PGF2alpha and PCARB levels were higher in DM patients than in controls (P < 0.05). Likewise, both TXM and prothrombin F1+2 levels were higher in diabetics (P < 0.05). By contrast, anticoagulant markers, such as activated protein C, protein C activation peptide, and soluble thrombomodulin (TM) were depressed in T2DM (P < 0.05). In conclusion, OS in T2DM involves circulating proteins and is associated with an unbalanced promotion of procoagulant reactions. These effects in concert with platelet activation may contribute to atherothrombotic complications in T2DM.

Validation of leukotriene B4 measurements in exhaled breath condensate.

OBJECTIVE: To qualitatively validate an enzyme immunoassay to measure leukotriene B4 in exhaled breath condensate. Exhaled breath condensate is a new non-invasive method to monitor airway inflammation. SUBJECTS: Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection. METHODS: Samples were pooled together and purified by reverse-phase high-performance liquid chromatography. The fractions eluted were assayed for leukotriene B4 by enzyme immunoassay. RESULTS: A single peak of leukotriene B4-like immunoreactivity co-eluting with leukotriene B4 standard (retention time: 24 min) was identified by enzyme immunoassay. Reverse phase-high performance liquid chromatography peak of leukotriene B4 was clearly separated from those of 6-trans-leukotriene B4 (retention time: 14 min) and leukotriene B5 (retention time: 18 min) for which the antiserum used in the enzyme immunoassay had the highest cross-reactivity. Leukotriene B4 recovery was 64%. CONCLUSIONS: This study provides evidence for the presence of leukotriene B4 in the exhaled breath condensate and the specificity of the enzyme immunoassay used.

Validation of 8-isoprostane and prostaglandin E(2) measurements in exhaled breath condensate.

OBJECTIVE: To qualitatively validate radioimmunoassays for 8-isoprostane and prostaglandin (PG) E(2) in exhaled breath condensate. SUBJECTS: Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection. METHODS: Samples were pooled together and purified by reverse phase high performance liquid chromatography (RP-HPLC). The eluted fractions were assayed for 8-isoprostane-like immunoreactivity and PGE(2)-like immunoreactivity by radioimmunoassays. In addition, simultaneous measurements of exhaled breath condensate unextracted samples with two anti-8-isoprostane and anti-PGE(2) sera with different cross-reactivity were performed. RESULTS: A single peak of 8-isoprostane-like immunoreactivity and PGE(2)-like immunoreactivity co-eluting with 8-isoprostane (retention time: 13 min) and PGE(2) (retention time: 21 min) standards, respectively, was identified by radioimmunoassays. Testing with two different antisera showed similar results for both 8-isoprostane-like immunoreactivity (limits of agreement = 4.5 pg/ml and - 4.1 pg/ml, n = 12) and PGE(2)-like immunoreactivity (limits of agreement = 6.1 pg/ ml and - 6.1 pg/ml, n = 12). CONCLUSION: This study provides evidence for the specificity of the radioimmunoassays for 8-isoprostane and PGE(2) in exhaled breath condensate. This is critical for proposing these markers as a non-invasive way for monitoring airway inflammation.

Effects of cocaine- and amphetamine-regulated transcript peptide, leptin and orexins on hypothalamic serotonin release.

We have studied the effects of cocaine- and amphetamine-regulated transcript (CART) peptide-(55-102), leptin, orexin-A and orexin-B on basal and depolarization (K(+) 15 mM)-induced serotonin (5-hydroxytryptamine, 5-HT) release from rat hypothalamic neuronal endings (synaptosomes) in vitro. We have found that leptin and CART peptide-(55-102) have no effect on 5-HT release, while orexin-A and orexin-B inhibit depolarization-stimulated serotonin release. We can conclude that leptin and CART peptide-(55-102), which play a physiological role as feeding inhibitors, do not acutely affect 5-HT release from hypothalamic synaptosomes; on the other hand, feeding induced by orexin-A and orexin-B could be partially explained by decreased 5-HT release.

Prognostic significance of cyclooxygenase-2 in laryngeal squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) overexpression is an unfavorable prognostic marker in laryngeal squamous cell carcinoma (SCC). EGFR stimulates cyclooxygenase-2 (COX-2) expression in normal human keratinocytes and squamous carcinoma cells. Based on these observations a prognostic role of COX-2 expression in laryngeal SCC can be hypothesized. Consequently, COX-2 expression was studied in laryngeal SCC (median follow-up = 47 months; range: 2-87 months) by quantitative immunohistochemistry (n = 61) and EGFR by binding assay (n = 51). Well-differentiated regions of laryngeal SCC revealed strong COX-2 immunostaining, whereas histologically normal areas neighboring tumor as well as poorly-differentiated tumors were negative. Immunohistochemical results were confirmed by Western blot analyses. Cox's regression analysis showed that the combination of low levels of COX-2 integrated density and high levels of EGFR covariates provided strong prediction, at 5-year follow-up, of both poor overall survival (chi(2) = 12.905; p = 0.0016) and relapse-free survival (chi(2) = 9.209; p = 0.01). In vitro studies on CO-K3 cell line, obtained from an EGFR positive, COX-2 negative poorly-differentiated laryngeal SCC, revealed that EGF stimulation failed to induce COX-2 expression and PGE2 production suggesting a change in EGFR signaling pathway. These findings indicate that COX-2 is overexpressed in less aggressive, low grade laryngeal SCC, whereas its expression is lost when tumors progress to a more malignant phenotype.

Oxidative stress and platelet activation in homozygous homocystinuria.

BACKGROUND: Severe hyperhomocysteinemia due to cystathionine beta-synthase deficiency (CbetaSD) is associated with early atherothrombotic vascular disease. Homocysteine may exert its effects by promoting oxidative damage. In the present study, we investigated whether in vivo formation of 8-iso-prostaglandin (PG) F(2alpha), a platelet-active product of arachidonic acid peroxidation, is enhanced in CbetaSD and whether it correlates with in vivo platelet activation, as reflected by thromboxane (TX) metabolite excretion. METHODS AND RESULTS: Urine and blood samples were obtained from patients with homozygous CbetaSD (n=13) and age-matched healthy subjects. Urinary 8-iso-PGF(2alpha) excretion was significantly higher in CbetaSD patients than in control subjects (640+/-384 versus 213+/-43 pg/mg creatinine; P=0.0015) and correlated with plasma homocysteine (rho=0.398, P=0.0076). Similarly, urinary 11-dehydro-TXB(2) excretion was enhanced in CbetaSD (1166+/-415 versus 324+/-72 pg/mg creatinine; P=0.0015) and correlated with urinary 8-iso-PGF(2alpha) (rho=0.362, P=0.0153). Vitamin E supplementation (600 mg/d for 2 weeks) was associated with a statistically significant increase in its plasma levels (from 16.6+/-4.6 to 40.4+/-8.7 micromol/L, P=0.0002) and with reductions in 8-iso-PGF(2alpha) (from 790+/-159 to 559+/-111 pg/mg creatinine, P=0.018) and 11-dehydro-TXB(2) (from 1273+/-383 to 913+/-336 pg/mg creatinine, P=0.028). A statistically significant inverse correlation was found between urinary 8-iso-PGF(2alpha) and plasma vitamin E levels (rho=-0.745, P=0.0135). CONCLUSIONS: The results of the present study suggest that enhanced peroxidation of arachidonic acid to form bioactive F(2)-isoprostanes may represent an important mechanism linking hyperhomocysteinemia and platelet activation in CbetaSD patients. Moreover, they provide a rationale for dose-finding studies of vitamin E supplementation in this setting.

In vivo lipid peroxidation and platelet activation in cystic fibrosis.

F(2)-isoprostanes are bioactive peroxidation products of arachidonic acid whose urinary excretion provides an index of lipid peroxidation in vivo. We tested the hypothesis that formation of F(2)-isoprostanes is altered in patients with cystic fibrosis and contributes to platelet activation and pulmonary dysfunction in this setting. The urinary excretion of immunoreactive 8-iso-prostaglandin F(2alpha) (PGF(2alpha)) was significantly (p = 0.0001) higher in 36 patients with cystic fibrosis than in 36 age-matched healthy subjects: 618 +/- 406 versus 168 +/- 48 pg/mg creatinine. The urinary excretion of immunoreactive 11-dehydro-thromboxane B(2) (TXB(2)), an index of in vivo platelet activation, was also significantly (p = 0.0001) higher in patients than in control subjects: 2,440 +/- 1,453 versus 325 +/- 184 pg/mg creatinine. The excretion rate of 8-iso-PGF(2alpha) was correlated with that of 11-dehydro-TXB(2) (rho = 0.51; p = 0.0026) and inversely related to FEV(1) (rho = -0.40; p = 0.0195). Urinary 8-iso-PGF(2alpha) excretion was largely unaffected during cyclooxygenase inhibition with low-dose aspirin, nimesulide, or ibuprofen, consistent with a noncyclooxygenase mechanism of F(2)-isoprostane formation in cystic fibrosis. Increased vitamin E supplementation (from 200 to 600 mg/d) was associated with statistically significant (p = 0.005) reductions in urinary 8-iso-PGF(2alpha) and 11-dehydro-TXB(2) excretion, by 42% and 29%, respectively. We conclude that enhanced lipid peroxidation is an important feature of cystic fibrosis and may contribute to persistent platelet activation and pulmonary dysfunction via generation of bioactive isoeicosanoids. Our results provide a rationale for reassessing the adequacy of vitamin E supplementation in this setting.

Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers.

Most of the studies linking chronic obstructive pulmonary disease (COPD) with oxidative stress are in vitro, using invasive techniques, or measuring systemic oxidative stress. The aim of this study was to quantify oxidative stress in the lungs in patients with COPD and in healthy smokers, as reflected by 8-isoprostane concentrations in breath condensate. This is a noninvasive method to collect airway secretions. 8-Isoprostane is a prostaglandin-F(2alpha) isomer that is formed in vivo by free radical-catalyzed peroxidation of arachidonic acid. We also studied the acute effect of smoking on exhaled 8-isoprostane in healthy smokers. Exhaled 8-isoprostane was measured by a specific enzyme immunoassay in 10 healthy nonsmokers and 12 smokers, 25 COPD ex-smokers, and 15 COPD current smokers. 8-Isoprostane concentrations were similar in COPD ex-smokers (40 +/- 3.1 pg/ml) and current smokers (45 +/- 3.6 pg/ ml) and were increased about 1.8-fold compared with healthy smokers (24 +/- 2.6 pg/ml, p < 0.001), who had 2.2-fold higher 8-isoprostane than healthy nonsmokers (10.8 +/- 0.8 pg/ml, p < 0.05). Smoking caused an acute increase in exhaled 8-isoprostane by about 50%. Our study shows that free radical production is increased in patients with COPD and that smoking causes an acute increase in oxidative stress.