Passive cigarette smoking increases isoprostane formation

Passive smoking has been demonstrated to exert a variety of deleterious effects eventually resulting in vascular damage. Isoprostanes, a reliable marker of in vivo oxidation injury, have been shown to increase in active cigarette smoking. Data for passive smoking are lacking. We were examining the isoprostane 8-epi-PGF2alpha in 12 smokers and non-smokers exposed daily to passive cigarette smoke for 12 days. Plasma samples stored at liquid nitrogen from people having been examined earlier were used. Prevalues of 8-epi-PGF2alpha are higher in cigarette smokers. Exposure to passive smoking causes a significant increase in 8-epi-PGF2alpha in non-smokers, while in smokers there is only a trendwise increase. After repeated passive smoke exposure, 8-epi-PGF2alpha in non-smokers approaches the respective values of smokers. There is a significant correlation of 8-epi-PGF2alpha to the thromboxane (plasma, serum, conversion from exogenous precursor, 11-dehydro-TXB2) parameters (MDA, HHT- conversion) examined in these patients before. The findings document a significant temporary increase in in vivo oxidation injury due to passive smoke favouring development and/or progression of vascular disease.     

Cigarette smoking reduces human salivary eicosanoids.

The effect of cigarette smoking on salivary eicosanoid levels was investigated in 10 smoker and 10 non-smoker volunteers. The smokers consumed an average of 20 cigarettes/day for the past 5 years or longer. The smoking status was validated by salivary cotinine level. Eicosanoids were extracted from saliva with ethanol, and the radioimmunoassay was performed to determine the concentrations of four major eicosanoids, i.e. prostaglandin E2 (PGE2), PGF2 alpha, 6-sulphidopeptide-containing leukotrienes (LTs) and 12-hydroxyeicosatetraenoic acid (12-HETE). The levels of PGE2, PGF2 alpha, and LTs were significantly lower in the saliva of smokers as compared to that of the non-smokers (1.74 +/- 0.32 vs 2.41 +/- 0.64, p = 0.006; 0.36 +/- 0.12 vs 0.54 +/- 0.18, p = 0.04; 2.24 +/- 0.96 vs 4.92 +/- 1.29, p = 0.006; mean +/- SD, ng/ml saliva). No significant differences were found in the levels of 12-HETE between the two groups. The results suggest that cigarette smoking reduces the concentrations of both the cyclooxygenase and 5-lipoxygenase products in saliva.     

6-oxo-PGF(1 alpha)and 8-epi-PGF(2 alpha)in the arterial wall layers of various species: a comparison between intact and atherosclerotic areas

PGI(2)and 8-epi-prostaglandin(PG)F(2 alpha)are antagonizing compounds. For both a key role in vascular pathology has been hypothesized. The isoprostane 8-epi-PGF(2 alpha)and the stable derivative of PGI(2), 6-oxo-PGF(1 alpha)were determined immunologically in the arterial wall of various species including humans. Human arterial tissue contained the highest amounts of 8-epi-PGF(2 alpha)and synthesized the lowest PGI(2). A significant negative correlation between 8-epi-PGF(2 alpha)and 6-oxo-PGF(1 alpha)was observed. Atherosclerotic segments showed significantly higher 8-epi-PGF(2 alpha)and lower 6-oxo-PGF(1 alpha). 8-epi-PGF(2 alpha)in the intima was higher than in the media, the highest amounts being found in foam-cell rich areas. Synthetic (activated) smooth muscle cells were associated with an enhanced 8-epi-PGF(2 alpha)as well as 6-oxo-PGF(1 alpha). Tissue samples derived from smokers contained more 8-epi-PGF(2 alpha)and produced less PGI(2). The by far highest 8-epi-PGF(2 alpha)/6-oxo-PGF(1 alpha)ratio was found in foam cell rich areas. Similar findings were obtained in rabbit and in minipig arteries. The total 8-epi-PGF(2 alpha)/6-oxo-PGF(1 alpha)ratio is low in normal tissue, increases significantly in an active atherosclerotic process and seems to be even further increased in an inactive atherosclerotic process. These findings are providing an information on the extent of oxidation injury at various sites of different types of atherosclerotic process.     

Cigarette smoking: profiles of thromboxane- and prostacyclin-derived products in human urine

Thromboxane (TX) B2, 2,3-dinor-TXB2, 11-dehydro-TXB2, 6-oxoprostaglandin (PG)F1 alpha and 2,3-dinor-6-oxo-PGF1 alpha were measured in 24 h urine samples obtained from 30 apparently healthy chronic cigarette smokers and 37 closely matched non-smoking control subjects. Samples were analysed using a newly developed assay based on immunoaffinity chromatography and capillary column gas chromatography/electron capture negative ion chemical ionisation mass spectrometry. There were significant and comparable increases in the excretion rates of both 2,3-dinor-TXB2 and 11-dehydro-TXB2 in the smoking compared with the non-smoking group (2P less than 0.001). Excretion rates of 2,3-dinor-TXB2 were 418 +/- 35 and 265 +/- 26 pg/mg creatinine in the two groups, respectively. 11-Dehydro-TXB2 excretion rates were 440 +/- 54 and 221 +/- 18 pg/mg creatinine, respectively (mean +/- S.E.). There were significant (2P less than 0.05) positive correlations between average reported cigarette consumption and excretion of both thromboxane metabolites. There were small but significant (2P less than 0.02) increases in the excretion rates of both 6-oxo-PGF1 alpha and 2,3-dinor-6-oxo-PGF1 alpha in the smoking compared with the non-smoking group. There was no significant difference in the rates of excretion of TXB2 in the two groups. The effects of acute cigarette smoke exposure (five cigarettes in 2 h) was also studied in four normally non-smoking healthy volunteers. There was no significant change in the excretion rate of any of the eicosanoids measured during control and smoking periods (at least 2 weeks apart), indicating that increased TXA2 biosynthesis in chronic smokers is unlikely to be a consequence of acute platelet activation.     

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.     

6-Oxo-PGF1alpha and 8-epi-PGF2alpha in human atherosclerotic vascular tissue.

Isoprostanes are a new family of compounds generated by the free radical catalyzed action on arachidonic acid. Formed during oxidation they have been claimed to be a reliable indicator of in vivo oxidation injury. We assessed the amount of 8-epi-PGF2alpha in human surgical specimens as compared to PGI2 (via its stable metabolite 6-oxo-PGF1alpha), the major compound generated by vascular tissue. 8-epi-PGF2alpha is low in normal vascular tissue as is the 8-epi-PGF2alpha/6-oxo-PGF1alpha ratio. The vessels of smokers in general exhibited an increased 8-epi-PGF2alpha (r=0.82) and a decreased 6-oxo-PGF1alpha (r=0.71). The 8-epi-PGF2alpha/6-oxo-PGF1alpha ratio is, not significantly, increased in vessels derived from hyperlipidemics and hypertensives. These findings indicate that lipid peroxidation occurs within the human arterial wall as evidenced by 8-epi-PGF2alpha, probably further decreasing the synthesis of PGI2 and promoting atherogenic mechanisms.     

Prostaglandin release from the guinea-pig uterus superfused in vitro. Effect of stage of estrous cycle, progesterone, estradiol, oxytocin and A23187

Prostaglandin (PG) E2 was the major PG released from the superfused guinea-pig uterus on Day 7, followed by in descending order 6-oxo-PGF1 alpha, thromboxane (TX) B2 and PGF2 alpha. However, the outputs of all four substances were low and were very similar. By Day 15, PGF2 alpha output from the superfused uterus had increased 21.9-fold, whereas the outputs of PGE2, 6-oxo-PGF1 alpha and TXB2 had increased only 1.8-, 2.9- and 1.2-fold, respectively. A mechanism is apparently "switched on" between Days 7 and 15 which causes a fairly specific increase in the release of PGF2 alpha from the uterus. Progesterone and/or estradiol had no effect on PG or TX release when superfused over the uterus on Day 7, nor did they have any effect on PG and TX release from the Day 15 uterus when administered separately. When administered together, however, they significantly inhibited PGF2 alpha, PGE2 and 6-oxo-PGF1 alpha, but not TXB2, release from the Day 15 uterus. Oxytocin had no effect on PG release from the Day 7 or Day 15 uterus, while A23187 stimulated PGF2 alpha, 6-oxo-PGF1 alpha and, to a lesser extent, PGE2 release from the uterus on both Days 7 and 15. Oxytocin is apparently not important for stimulating PGF2 alpha release from the guinea-pig uterus in relation to luteolysis, whereas increasing intracellular free Ca++ levels may be part of the mechanism for "switching on" uterine PG synthesis. Furthermore, changes in intracellular free Ca++ levels in the endometrium may be responsible for the pulsatile nature of PGF2 alpha release from the uterus.     

LDL-apheresis decreases plasma levels and urinary excretion of 8-epi-PGF2alpha

Isoprostanes (IP) generated during free radical catalyzed oxidation injury have been claimed as a reliable indicator of oxidative stress in vivo. In particular, they are formed during LDL-oxidation. Vascular content, plasma levels and urinary excretion of IP were reported to be elevated in hypercholesterolemia. We therefore assessed the values of the IP 8-epi-PGF2alpha in plasma and urine in nine patients (7 males, 2 females) suffering from severe heterozygous hypercholesterolemia before and after LDL-apheresis as well as during the interval. LDL-apheresis caused a significant (P<0.01) drop in 8-epi-PGF2alpha in plasma and urine. The respective values in smokers (n = 4) were significantly (P<0.01) higher as compared to non-smokers. No sex difference was seen. Together with the findings of a parallel decrease in oxidized LDL, these data show a significant benefit of LDL-apheresis reducing in vivo oxidation injury. This benefit may at least partly contribute to the clinical improvement seen in the patients treated.     

Cigarette smokers have decreased lymphocyte and platelet alpha-tocopherol levels and increased excretion of the gamma-tocopherol metabolite gamma-carboxyethyl-hydroxychroman (gamma-CEHC)

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet alpha- and gamma-tocopherol, as well as the specific urinary vitamin E metabolites alpha- and gamma-carboxyethyl-hydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte alpha- and gamma-tocopherol in smokers and non-smokers. However, smokers had significantly lower alpha-tocopherol (mean+/-SD, 1.34+/-0.31 micromol/g protein compared with 1.94+/-0.54, P = 0.001) and gamma-tocopherol (0.19+/-0.04 micromol/g protein compared with 0.26+/-0.08, P = 0.026) levels in their lymphocytes, as well as significantly lower alpha-tocopherol levels in platelets (1.09+/-0.49 micromol/g protein compared with 1.60+/-0.55, P = 0.014; gamma-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary gamma-tocopherol metabolite, gamma-CEHC (0.49+/-0.25mg/g creatinine compared with 0.32+/-0.16, P = 0.036) compared to non-smokers, while their alpha-CEHC (metabolite of alpha-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.     

Enhanced accumulation of the isoprostane, 8-epi-PGF2 alpha,in human aortic and pulmonary valves of patients with coronary heart disease

The aim of the present study was to investigate whether the isoprostane 8-epi-PGF2 alpha differently accumulates in semilunar valves of patients suffering from coronary heart disease (CHD, n = 19) as compared to valves from healthy heart donors (controls, n = 6). Sections from isolated aortic and pulmonary valves were analyzed by semiquantitative immunohistochemistry. The 8-epi-PGF2 alpha-content was determined by using a specific radioimmunoassay. The accumulation of 8-epi-PGF2 alpha in both valves was higher in CHD-patients in comparison to controls (Aortic valves: 36.49 +/- 11.26% vs. 15.78 +/- 3.04%; pulmonary valves: 46.79 +/- 9.80% vs. 14.99 +/- 3.57%). The results from the radioimmunoassay revealed comparable findings in both groups (CHD vs. controls: 395.95 +/- 86.09 vs. 139.50 +/- 47.46 pg/mg protein in the aortic valves and 430.47 +/- 76.30 vs. 147.33 +/- 53.84 pg/mg protein in pulmonary valves). Pulmonary valves seem to be more susceptible to oxidative stress than aortic valves as evidenced by a higher accumulation of 8-epi-PGF2 alpha in CHD patients. Considering the data presented in this study, we suggest that 8-epi-PGF2 alpha is a valuable indicator of oxidative injury in human semilunar valves.     

Effect of giving up cigarette smoking and restarting in patients with clinically manifested atherosclerosis

Cigarette smoking, a key risk factor for the development of vascular disease, is associated with an increased 8-epi-prostaglandin (PG) F(2alpha). Elevated 8-epi-PGF(2alpha) has been found in vascular tissue, blood and urine as well. We examined the influence of quitting cigarette smoking in 71 patients (38 males, 33 females; aged 32-67 a) with clinically manifested atherosclerosis and various risk factors. In addition, in eight patients with hypercholesterolemia without clinical manifestation of atherosclerosis quitting smoking was monitored as well. Twenty-six of the patients with manifested atherosclerosis and five with hypercholesterolemia restarted and the isoprostanes in plasma, serum and urine were monitored in these patients as well. Quitting cigarette smoking induces an immediate decline becoming significant after 1 or 2 weeks. Restarting smoking results in an increase in 8-epi-PGF(2alpha) reaching prevalues within almost 1 week. These findings indicate that the in vivo oxidation injury associated with cigarette smoking quickly decreases after quitting but increases soon after restarting immediately.     

Short-term passive smoking causes endothelial dysfunction via oxidative stress in nonsmokers

Recent studies have shown that passive smoking impairs vascular endothelial function and induces oxidative stress in humans. However, in most of the previous human data regarding tobacco-induced pathophysiology, vascular endothelial dysfunction and oxidative stress have been separately assessed. This study was designed to determine the association between the acute effect of passive smoking on vascular endothelial function and in-vivo oxidative stress status. We studied 30 healthy male Japanese volunteers (32 +/- 7 years) including 15 habitual smokers and 15 nonsmokers. After baseline echocardiographic, hemodynamic recording, and blood sampling, subjects were exposed to passive smoking for 30 min. Endothelium-dependent vasodilation was measured by using % flow-mediated vasodilation (%FMD) of the brachial artery and plasma levels of 8-isoprostane was measured by enzyme immunoassay before and after the passive smoking exposure. Baseline %FMD was lower (4.3% +/- 1.2% vs. 10.9% +/- 3.1%, p < 0.001) and baseline plasma 8-isoprostane level was higher (41.5 +/- 5.8 pg/mL vs. 26.9 +/- 5.4 pg/mL, p < 0.001) in smokers than those in nonsmokers. The %FMD and 8-isoprostane level did not change after passive smoking in smokers. In nonsmokers, however, the %FMD decreased (to 5.0% +/- 1.9%, p < 0.001) and the 8-isoprostane level increased (to 37.8 +/- 9.6 pg/mL, p < 0.001) significantly after 30 min passive smoking exposure, equivalently to the levels of smokers. Sixty corrected samples before and after passive smoking exposure in all patients showed a significant negative correlation between the % FMD and the plasma 8-isoprostane levels (n = 60, r = -0.69, p < 0.001). Even 30 min of passive smoking rapidly impairs vascular endothelial function, which is associated with oxidative stress. Our data provide the pathophysiological insight for the recent epidemiological evidence about the increased risk of coronary heart disease among nonsmokers exposed to passive smoking.     

Salivary DNA,lipid, and protein oxidation in nonsmokers with periodontal disease

Reactive oxygen species (ROS) are implicated in the destruction of the periodontium during periodontitis. The imbalance in oxidant activity may be a key factor.The aim of this paper is to determine whether periodontitis is associated with increased oxidative damage to DNA, lipids, and proteins and modification of total antioxidant capacity (TAC) in saliva. Saliva was collected from 58 periodontitis patients and 234 healthy controls, all nonsmokers. Periodontal disease status was characterized using the Community Periodontal Index of Treatment Needs (CPITN). Assays for 8-OHdG (ELISA), 8-epi-PGF2α (ELISA), and total protein carbonyls (ELISA), and oxy-blotting (Western)/mass spectrometry were performed to quantify oxidative damage to nucleic acids, lipids, total and individual proteins, respectively, in whole nonstimulated saliva. Salivary TAC was measured by inhibition of ABTS oxidation by metmyoglobin. We observed (i) significantly higher levels of 8-OHdG, 8-epi-PGF2α, and carbonylated proteins in saliva of periodontal patients as compared with controls (P = 0.0003, < 0.0001 and < 0.0001); (ii) 8-OHdG, 8-epi-PGF2α, and carbonylated proteins were independently negatively associated with CPITN (P = 0.004, 0.02, and < 0.0001); (iii) a positive correlation between salivary TAC and periodontal disease status in the study group (P < 0.0001); and (iv) specific oxidation of transferrin, human IgG1 heavy chain fragment, and salivary amylase in periodontitis. Periodontal disease is associated with increased oxidative modification of salivary DNA, lipids, and proteins. Augmented salivary total antioxidant capacity may represent an adaptive response to oxidative stress. Salivary amylase, transferrin, and human IgG1 heavy chain fragments are particularly prone to enhanced oxidation in periodontitis.     

Vascular endothelial growth factor: an angiogenic factor reflecting airway inflammation in healthy smokers and in patients with bronchitis type of chronic obstructive pulmonary disease?

BackgroundPatients with bronchitis type of chronic obstructive pulmonary disease (COPD) have raised vascular endothelial growth factor (VEGF) levels in induced sputum. This has been associated with the pathogenesis of COPD through apoptotic and oxidative stress mechanisms. Since, chronic airway inflammation is an important pathological feature of COPD mainly initiated by cigarette smoking, aim of this study was to assess smoking as a potential cause of raised airway VEGF levels in bronchitis type COPD and to test the association between VEGF levels in induced sputum and airway inflammation in these patients.Methods14 current smokers with bronchitis type COPD, 17 asymptomatic current smokers with normal spirometry and 16 non-smokers were included in the study. VEGF, IL-8, and TNF-α levels in induced sputum were measured and the correlations between these markers, as well as between VEGF levels and pulmonary function were assessed.ResultsThe median concentrations of VEGF, IL-8, and TNF-α were significantly higher in induced sputum of COPD patients (1,070 pg/ml, 5.6 ng/ml and 50 pg/ml, respectively) compared to nonsmokers (260 pg/ml, 0.73 ng/ml, and 15.4 pg/ml, respectively, p < 0.05) and asymptomatic smokers (421 pg/ml, 1.27 ng/ml, p < 0.05, and 18.6 pg/ml, p > 0.05, respectively). Significant correlations were found between VEGF levels and pack years (r = 0.56, p = 0.046), IL-8 (r = 0.64, p = 0.026) and TNF-α (r = 0.62, p = 0.031) levels both in asymptomatic and COPD smokers (r = 0.66, p = 0.027, r = 0.67, p = 0.023, and r = 0.82, p = 0.002, respectively). No correlation was found between VEGF levels in sputum and pulmonary function parameters.ConclusionVEGF levels are raised in the airways of both asymptomatic and COPD smokers. The close correlation observed between VEGF levels in the airways and markers of airway inflammation in healthy smokers and in smokers with bronchitis type of COPD is suggestive of VEGF as a marker reflecting the inflammatory process that occurs in smoking subjects without alveolar destruction.     

Thromboxane synthase inhibition: implications for prostaglandin endoperoxide metabolism. II. Testing the 'redirection hypothesis' in an acute intravenous challenge model

In the preceding paper we described the characterisation of an acute intravenous challenge model for the evaluation of the effects of thromboxane synthase inhibition (TXSI) on eicosanoid metabolism. Herein we describe the biochemical pharmacology of two TXSI and aspirin in this model. Both TXSI caused significant inhibition of plasma TXB2 in vivo without elevation of 6-oxo-PGF1 alpha levels. Similar results were obtained when combined levels of 6-oxo-PGF1 alpha,13,14 dihydro 6-oxo-PGF1 alpha,13,14 dihydro 6,15-dioxo-PGF1 alpha and 6-oxo-PGE1 were measured as an index of PGI2 biosynthesis (PGI2m). Thus no evidence of in vivo redirection of PGH2 to PGI2 was found. Ex vivo experiments performed in serum gave an apparent stimulation of immunoreactive 6-oxo-PGF1 alpha following TXSI but RPHPLC analysis of extracted serum showed that this stimulation was accounted for by increase in a product co-eluting with [3H]PGF2 alpha. The implications of these findings in relation to TXSI and receptor antagonists are discussed.     

Urinary prostaglandin F2alpha is generated from the isoprostane pathway and not the cyclooxygenase in humans

Prostaglandins (PGs) derived from the enzymatic oxidation of arachidonic acid by the cyclooxygenases (COXs) are potent lipid mediators involved in human physiology and pathophysiology. Structurally similar compounds, the isoprostanes (IsoPs), are generated from the free radical-catalyzed oxidation of arachidonic acid independent of COX. IsoPs exhibit significant bioactivity and play a role in the pathogenesis of diseases associated with oxidant injury. As one of the major PGs, prostaglandin F(2alpha) (PGF(2alpha)) is present in human urine in significant concentrations and is presumed to be derived from COX activity. We determined, however, that levels of putative PGF(2alpha) in urine cannot be suppressed by nonsteroidal anti-inflammatory agents, suggesting that it is generated via another mechanism(s). An important difference between COX-derived PGF(2alpha) and the IsoPs is that the former is an optically pure compound, whereas IsoPs are racemic. Utilizing a rodent model of oxidative stress, we now show that significant amounts of compounds identical in all respects to PGF(2alpha) and its enantiomer, ent-PGF(2alpha), are formed in equal amounts esterified in tissue phospholipids, suggesting that these compounds are derived via the IsoP pathway. Further, employing liquid chromatography/mass spectrometry, the vast majority of putative PGF(2alpha) in human urine is derived from the free radical-initiated peroxidation of arachidonate independent of COX and is composed of PGF(2alpha) and its enantiomer, although the latter compound is approximately 2-fold more abundant. Thus, quantification of urinary PGF(2alpha) actually reflects oxidative stress status as opposed to COX activity. Indeed, levels of this compound are elevated in urine from cigarette smokers and in humans with hypercholesterolemia, two conditions associated with oxidant stress. The elucidation that urinary PGF(2alpha) in humans is derived from the IsoP pathway has implications regarding PG formation and inhibition in vivo.     

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.     

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.     

Altered salivary profile in heavy smokers and its possible connection to oral cancer

Saliva is the first biological fluid to encounter inhaled cigarette smoke, whose numerous carcinogens and oxidants are responsible for the oral cancer so prevalent among smokers. Whole saliva, collected from 25 consenting heavy smokers and from a control group of 25 age- and gender-matched non-smokers, was subjected to sialochemical, biochemical, immunological and oxidative analyses. The mean flow rate was significantly higher in smokers than in non-smokers, as were the median activity value of superoxide dismutase (SOD) and the total salivary antioxidant capacity (ImAnOx) (by 32% and 12%, respectively, p=0.05). The salivary carbonyl concentration (an oxidative stress indicator) was significantly higher by 126% (p=0.0006) among smokers, while lactate dehydrogenase, albumin, total immunoglobulin G, and the metalloproteinases MMP-2 and MMP-9 concentrations were significantly lower in the smokers, by 86% (p=0.003), 65% (p=0.003), 61% (p=0.048), 35% (p=0.005) and 55% (p=0.035), respectively. Apparently, the oral cavity's salivary antioxidant system fails to cope with the severe attack of reactive oxygen species originating in cigarette smoke. Moreover, various other salivary functional and protective parameters also decreased among the smokers. Hence, further research aimed at examining the possibility of administration of agents as antioxidants or saliva substitutes to the oral cavity of smokers should be considered.     

The effect of age and smoking on vascular prostaglandin production in men and women

6-Keto-PGF1 alpha, PGF2 alpha and PGE2 production by homogenates of aorta was unaffected by age, sex or smoking habits. Homogenates of saphenous vein from women aged 51-60 years produced greater and smaller amounts of 6-keto- PGF1 alpha and PGF2 alpha, respectively, than from women aged 41-50 and 61-70 years. In the 41-50 and 61-70 age groups, the amounts of 6-keto-PGF1 alpha and PGF2 alpha produced by homogenates of saphenous vein were smaller and greater, respectively, in women than in men. Cigarette smoking had no effect on PG production by homogenates of female saphenous vein. 6-Keto-PGF1 alpha production by homogenates of male saphenous vein was 20% lower in smokers and ex-smokers than in non-smokers, although this reduction was statistically significant only for ex-smokers. The amounts of PGE2 and PGF2 alpha produced by homogenates of male saphenous vein were smaller in smokers and ex-smokers, respectively, than in non-smokers. In spite of these changes in PG production by homogenates of saphenous vein, the basal outputs of PGs, particularly of 6-keto-PGF1 alpha, from the saphenous vein were little affected by age, sex or smoking habits.