Specific analysis in plasma and urine of 2,3-dinor-5, 6-dihydro-isoprostane F(2alpha)-III, a metabolite of isoprostane F(2alpha)-III and an oxidation product of gamma-linolenic acid

F(2)-isoprostanes (iPs) are free radical-catalyzed isomers of prostaglandin F(2alpha). Circulating and urinary iPs have been used as indices of lipid peroxidation in vivo. Utilizing an (18)O-labeled homologous internal standard, we developed a gas chromatography/mass spectrometry assay for the 2,3-dinor-5,6-dihydro (dinor-dihydro) metabolite of iPF(2alpha)-III. Although urinary excretion of iPF(2alpha)-III reflects systemic lipid peroxidation, the metabolite is more abundant (median of 877 (range of 351-1831) versus 174 (range of 56-321) pg/mg of creatinine; p < 0.01) than the parent iP in urine and can be measured in plasma. Metabolite analysis may be preferable in plasma due to the abundance of arachidonic acid as a source of ex vivo lipid peroxidation. Also, iPF(2alpha)-III may be formed in blood samples in a cyclooxygenase-dependent manner by platelets ex vivo. By contrast, the metabolite is not formed by aggregated platelets (0.71 +/- 0.08 versus 0.65 +/- 0.09 pg/ml). Although the metabolite/parent ratio is altered in cirrhosis, urinary dinor-dihydro-iPF(2alpha)-III is elevated and increases further during reperfusion following orthoptic liver transplantation. In addition to its formation as an iPF(2) metabolite, analysis of gamma-linolenic acid autooxidation products and the compound present in freeze-thawed plasma suggests that gamma-linolenic acid may also be an important source of dinor-dihydro-iPF(2alpha)-III.     

Control of matrix effects in the analysis of urinary F2-isoprostanes using novel multidimensional solid-phase extraction and LC-MS/MS

F(2)-isoprostanes (F(2)-iPs), established markers of oxidative stress, exist as four sets of regioisomers. Simultaneous and specific determination of F(2)-iPs can be achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed novel methods for urine sample preparation and HPLC to control matrix-related ion suppression effects in the LC-MS/MS analysis of F(2)-iPs. A selective solid-phase extraction (SPE) wash protocol was developed with an Oasis HLB (hydrophilic-lipophilic balance) SPE cartridge using an elution profile of [(3)H]8-iso-prostaglandin (PG)F(2alpha) (iPF(2alpha)-III) when the methanol concentration was increased under acidic, neutral, and base wash conditions. A multidimensional (MD)-SPE method that incorporated size exclusion chromatography [corrected] reverse-phase chromatography, and normal-phase chromatography was developed using an Oasis HLB SPE cartridge and an HLB microElution SPE plate. Average extraction recoveries of the deuterated internal standards of iPF(2alpha)-III and iPF(2alpha)-VI were 62 +/- 8% and 60 +/- 10%. A buffer-free HPLC method for the separation of F(2)-iP isomers was developed on base-deactivated C8 columns. Average matrix effects for iPF(2alpha)-III and iPF(2alpha)-VI were 95 +/- 6% and 103 +/- 5%. The clean extraction of urine F(2)-iPs using MD-SPE and the separation of F(2)-iP isomers using a novel HPLC method did not cause apparent ion suppression in the analysis of iPF(2alpha)-III and iPF(2alpha)-VI using LC-MS/MS. These findings should be useful for establishing a routine LC-MS/MS method for the analysis of F(2)-iPs.     

Quantification of 8-iso-prostaglandin-F(2alpha) and 2,3-dinor-8-iso-prostaglandin-F(2alpha) in human urine using liquid chromatography-tandem mass spectrometry

Quantification of 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) has been suggested to be a reliable indicator of lipid peroxidation that may be related to in vivo free radical generation, oxidative damage, and antioxidant deficiency. We have developed a LC-MS/MS method to quantify 8-iso- PGF(2alpha) and its dinor metabolite, 2,3-dinor-8-iso-prostaglandin F(2alpha) (2,3-dinor-8-iso-PGF(2alpha)), in human urine samples. After an initial purification step using an automated C18 solid phase extraction procedure, the urine sample was injected directly into a liquid chromatography (LC) system and detected with tandem mass spectrometry. The detection limit of the assay was 9 pg for 8-iso-PGF(2alpha) and 3 pg for 2,3-dinor-8-iso-PGF(2alpha) with both inter- and intraday variations of less than 12%. The inaccuracies were less than 3% for both analytes at three different levels. The urinary excretion rate of 2,3-dinor-8-iso-PGF(2alpha) was higher than that of 8-iso-PGF(2alpha), and changed in proportion to the parent compound (R = 0.70, n = 60). Values obtained with this method showed good linear correlation to duplicate 8-iso-PGF(2alpha) measurements performed with GCMS (R = 0.97, n = 15). The mean excretion rates of 8-iso-PGF(2alpha) and 2,3-dinor-8-iso-PGF(2alpha) were significantly higher in smokers than in nonsmokers (0.53 +/- 0.37 vs. 0.25 +/- 0.15 microg/g creatinine, p = 0.002 for 8-iso-PGF(2alpha) and 8.9 +/- 3.8 vs. 4.6 +/- 2.6 microg/g creatinine, p = 0.003 for 2,3-dinor-8-iso-PGF(2alpha), respectively). The excellent accuracy, reproducibility, and high throughput of this method should permit it to be used in large clinical studies and standard clinical laboratories.     

Urinary F₂-isoprostanes, obesity, and weight gain in the IRAS cohort

Obesity has been associated with increased F(2)-isoprostane (F(2)-IsoP) levels cross-sectionally. However, the prospective association may be inverse, based on our earlier finding that elevated urinary F(2)-IsoP levels predict lower risk of diabetes. This earlier finding led us to hypothesize that urinary F(2)-IsoPs reflect the intensity of oxidative metabolism and as such predict lower risk of both diabetes and weight gain. We examined cross-sectional relationships with obesity and prospective relationships with weight gain using the data from 299 participants of the Insulin Resistance Atherosclerosis Study (IRAS), all of whom were free of diabetes at baseline. Four urinary F(2)-IsoPs were assayed in stored baseline urine samples using liquid chromatography with tandem mass spectrometry: iPF(2α)-III, 2,3-dinor-iPF(2α)-III, iPF(2α)-VI, and 8,12-iso-iPF(2α)-VI (F(2)-IsoP 1-4, respectively). Baseline F(2)-IsoPs were positively associated with baseline measures of obesity; the strongest associations were found with two F(2)-IsoPs: odds ratios (95% confidence intervals) for overall and abdominal obesity were 1.74 (1.26-2.40) and 1.63 (1.18-2.24) for F(2)-IsoP2 and 1.47 (1.12-1.94) and 1.64 (1.22-2.20) for F(2)-IsoP4. F(2)-IsoP2 showed the strongest and significant inverse association with weight gain during the 5-year follow-up period: increase in F(2)-IsoP2 equal to 1 s.d. was associated with 0.90 kg lower weight gain (P = 0.02) and the odds ratios for relative (≥5%) and absolute (≥5 kg) weight gain were 0.67 (0.47-0.96) and 0.57 (0.37-0.87), respectively. The other three F(2)-IsoPs were consistently inversely associated with weight gain, although not significantly, suggesting that different F(2)-IsoPs vary in their ability to detect the association with weight gain.     

Determination of isoprostanes in urine samples from Alzheimer patients using porous graphitic carbon liquid chromatography-tandem mass spectrometry

F2-isoprostanes (F2-iPs) comprise four classes of isomers produced non-enzymatically by free radical attack on arachidonic acid, a component of the cell membrane. This paper describes a new method for the quantification of F2-isoprostanes in urine samples from thoroughly diagnosed Alzheimer's disease (AD) patients. The sample pretreatment consisted of liquid extraction of 900 microl urine with diethyl ether, its subsequent evaporation, and finally, reconstitution in 50 microl water. Of this, 20 microl was injected into a HPLC system with a 15 mm x 1 mm porous graphitic carbon column coupled to a triple quadrupole mass spectrometer running in negative electrospray ionization mode. The F2-isoprostanes were separated in 15 min using a linear solvent gradient comprising water, methanol, acetonitrile and ammonium hydroxide at a pH of 9.5. The average recovery obtained was approximately 75%. The limit of detection (3S/N) was calculated for iPF2alpha-III to be 0.7 pg injected on column, corresponding to 0.1 nM. The average level of iPF2alpha was 241 +/- 163 pg/mg creatinine in the urine samples from AD patients (average +/- standard deviation). The corresponding control values were 216 +/- 101 pg/mg creatinine, i.e. no statistically significant difference was noticed. No correlation pattern specific to Alzheimer's disease was revealed by principal component analysis of the isoprostane peaks obtained either. The results from this study support earlier findings that levels of peripheral isoprostanes are not increased in patients with Alzheimer's disease.     

Smoking alters thromboxane metabolism in man

Chronic smoking is a major risk factor of atherosclerosis and coronary heart disease. The measurement of three major thromboxane A2 metabolites, 11-dehydrothromboxane B2, 2,3-dinorthromboxane B2 and thromboxane B2, in the urines of 13 apparently healthy smokers (average 39 years, range 27-56 years) showed significantly elevated excretion rates for all thromboxane A2 metabolites as compared to 10 apparently healthy age-matched non-smokers (average 37 years, range 26-56 years). Importantly, characteristic alterations in the thromboxane A2 metabolite pattern were found in the urines of smokers. The contribution of 2,3-dinorthromboxane B2 to total measured excretion of thromboxane A2 metabolites was 59.2% in smokers (404.0 +/- 53.0 pg/mg creatinine) versus 19.4% in non-smokers (85.2 +/- 8.3 pg/mg creatinine), that of 11-dehydrothromboxane B2 35.7% in smokers (673.2 +/- 88.9 pg/mg creatinine) as compared to 75.5% in non-smokers (332.6 +/- 30.9 pg/mg creatinine). The contribution of thromboxane B2 (57.5 +/- 7.7 pg/mg creatinine in smokers versus 21.9 +/- 1.5 pg/mg creatinine in non-smokers) was similar at 5.1%. The excretion of cotinine, the major urinary metabolite of nicotine that correlates well with the reported daily cigarette consumption (r = 0.97, P less than 0.0001), showed a good correlation to thromboxane A2 metabolite excretion (2,3-dinorthromboxane B2: r = 0.92, P less than 0.0001; 11-dehydrothromboxane B2; r = 0.87, P less than 0.0001).     

Divergence in urinary 8-iso-PGF(2alpha) (iPF(2alpha)-III, 15-F(2t)-IsoP) levels from gas chromatography-tandem mass spectrometry quantification after thin-layer chromatography and immunoaffinity column chromatography reveals heterogeneity of 8-iso-PGF(2alpha). Possible methodological,mechanistic and clinical implications

Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F(2)-isoprostane family which may theoretically comprise up to 64 isomers. We have previously shown that the combination of TLC and GC-tandem MS (referred to as method A) allows for the accurate and highly specific quantification of 8-iso-PGF(2alpha) (iPF(2alpha)-III, 15-F(2t)-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF(2alpha) (i.e. 15(S)-8-iso-PGF(2alpha)) has been shown by others to be highly selective and specific for this 8-iso-PGF(2alpha) isomer when quantified by GC-MS. In the present study we established IAC for urinary 8-iso-PGF(2alpha) for subsequent quantification by GC-tandem MS (referred to as method B). This method was fully validated and found to be highly accurate and precise for urinary 15(S)-8-iso-PGF(2alpha). 8-iso-PGF(2alpha) was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF(2alpha) was determined to be 291+/-102 pg/mg creatinine by method A and 141+/-41 pg/mg creatinine by method B. Analysis of the combined through and wash phases of the IAC step, i.e. of the unretained compounds, by method A showed the presence of non-immunoreactive 8-iso-PGF(2alpha) at 128+/-55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF(2alpha) is heterogenous, with 15(S)-8-iso-PGF(2alpha) contributing by approximately 50%. PGF(2alpha) and other 8-iso-PGF(2alpha) isomers including 15(R)-8-iso-PGF(2alpha) are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF(2alpha). We assume that ent-15(S)-8-iso-PGF(2alpha) is also contributing by approximately 50% to urinary 8-iso-PGF(2alpha). This finding may have methodological, mechanistic and clinical implications.     

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.     

An increase in the ratio of thromboxane A2 to prostacyclin in association with increased blood pressure in patients on cyclosporine A

The aim of this study was to determine the effect of two years of treatment with cyclosporine A on blood pressure and the rates of secretion into the circulation of the vasoconstrictor thromboxane A2 and the vasodilator prostacyclin. Seven patient suffering from multiple sclerosis took part. Their blood pressures and urinary concentrations of 2,3-dinor-thromboxane A2 (a major urinary metabolite of thromboxane A2) and of 2,3-dinor-6-keto-prostaglandin F1 alpha (the major urinary metabolite of prostacyclin) were determined at the end of two years of treatment with cyclosporine A, and once again three months after cessation of this treatment. No other drugs were given during or after cyclosporine A. Mean arterial blood pressure was 113 +/- 5 mmHg (mean +/- SEM) during the cyclosporine A treatment, but fell to 94 +/- 4 mmHg after the three-month's wash-out period. Urinary excretion of the thromboxane metabolite decreased slightly from 674 +/- 150 pg.mg-1 creatinine during cyclosporine A therapy to 503 +/- 90 pg.mg-1-creatinine after the end of therapy. At the same time the prostacyclin metabolite increased significantly from 82 +/- 17 pg.mg-1 creatinine to 113 +/- 23 pg.mg-1 creatinine (P less than 0.05). The ratio of 2,3-dinor-thromboxane B2 to 2,3-dinor-6-keto-prostaglandin F1 alpha (taken as a measure of vasoconstrictor prostanoid activity) fell significantly from 8.4 +/- 0.8 4.7 +/- 0.6 (P less than 0.005). The shift in prostanoid production observed during cyclosporine A treatment could be one causal factor for the hypertensive and thromboembolic events associated with the use of this drug.     

Quantification of the major urinary metabolite of 15-F2t-isoprostane (8-iso-PGF2alpha) by a stable isotope dilution mass spectrometric assay

The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane rings analogous to PGF2alpha. One F-ring IsoP, 15-F2t-IsoP (8-iso-PGF2alpha) has been shown to be formed in abundance in vivo and to exert potent biological activity. As a means to assess the endogenous production of this compound, we developed a method to quantify the major urinary metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t-IsoP (2,3-dinor-5, 6-dihydro-8-iso-PGF2alpha), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O2-labeled derivative for use as an internal standard. After purification, the compound was analyzed as a pentafluorobenzyl ester trimethylsilyl ether. Precision of the assay is +/-4% and accuracy is 97%. The lower limit of sensitivity is approximately 20 pg. Levels of the urinary excretion of this metabolite in 10 normal adults were found to be 0. 39 +/- 0.18 ng/mg creatinine (mean +/- 2 SD). Substantial elevations in the urinary excretion of the metabolite were found in situations in which IsoP generation is increased and antioxidants effectively suppressed metabolite excretion. Levels of 2,3-dinor-5, 6-dihydro-15-F2t-IsoP were not affected by cyclooxygenase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F2t-IsoP as a means to explore the pathophysiological role of this compound in human disease.     

Effects of 2 years of caloric restriction on oxidative status assessed by urinary F2‐isoprostanes: The CALERIE 2 randomized clinical trial

Calorie restriction (CR) without malnutrition slows aging in animal models. Oxidative stress reduction was proposed to mediate CR effects. CR effect on urinary F2‐isoprostanes, validated oxidative stress markers, was assessed in CALERIE, a two‐year randomized controlled trial. Healthy volunteers (n = 218) were randomized to prescribed 25% CR (n = 143) or ad libitum control (AL, n = 75) stratifying the randomization schedule by site, sex, and BMI. F2‐isoprostanes were quantified using LC‐MS/MS in morning, fasted urine specimens at baseline, at 12 and 24 months. The primary measure of oxidative status was creatinine‐adjusted 2,3‐dinor‐iPF(2α)‐III concentration, additional measured included iPF(2α)‐III, iPF2a‐VI, and 8,12‐iso‐iPF2a‐VI. Intention‐to‐treat analyses assessed change in 2,3‐dinor‐iPF(2α)‐III using mixed models assessing treatment, time, and treatment‐by‐time interaction effects, adjusted for blocking variables and baseline F2‐isoprostane value. Exploratory analyses examined changes in iPF(2α)‐III, iPF(2α)‐VI, and 8,12‐iso‐iPF(2α)‐VI. A factor analysis used aggregate information on F2‐isoprostane values. In CR group, 2,3‐dinor‐iPF(2α)‐III concentrations were reduced from baseline by 17% and 13% at 12 and 24 months, respectively; these changes were significantly different from AL group (p < .01). CR reduced iPF(2α)‐III concentrations by 20% and 27% at 12 and 24 months, respectively (p < .05). The effects were weaker on the VI‐species. CR caused statistically significant reduction in isoprostane factor at both time points, and mean (se) changes were ?0.36 (0.06) and ?0.31 (0.06). No significant changes in isoprostane factor were at either time point in AL group (p < .01 between‐group difference). We conclude that two‐year CR intervention in healthy, nonobese men and women reduced whole body oxidative stress as assessed by urinary concentrations of F2‐isoprostanes.     

Indirect enhancement of neutrophil activity and adhesion to cultured human umbilical vein endothelial cells by isoprostanes (iPF2alpha-III and iPE2-III).

Isoprostanes are metabolites of arachidonic acid found in blood under various conditions of oxidative stress. Because arachidonic acid derivatives are major mediators of inflammation, we investigated the potential inflammatory effects of iPF2alpha-III (previously 8-isoPGF2alpha) and iPE2-III (8-isoPGE2) on human polymorphonuclear granulocytes (PMN), as well as on human umbilical vein endothelial cells (HUVECs). The early activation marker CD11b on PMN and the adhesion molecules ICAM-1, E-selectin, and P-selectin on HUVECs were quantified by flow cytometry. Levels of the cytokines interleukin (IL)-6 and IL-8 were measured in the culture supernatant by enzyme-linked immunosorbent assay. Furthermore, adhesion of PMN to HUVECs was assessed. Neither isoprostane showed any direct stimulatory effects on PMN or HUVECs at concentrations of 0.1 or 1 microM: there was no acute elevation in expression of CD11b or P-selectin and no change of ICAM-1 or E-selectin after 4 or 24 h of incubation, respectively. The levels of interleukin IL-6 and IL-8 were also unaltered. However, PMN adhesion was significantly enhanced both after 4 and 24 h of incubation of HUVECs with iPF2alpha-III, and CD11b expression on PMN was elevated by contact of these cells with the supernatant of pre-exposed HUVECs. Neither of these actions were inhibited by an endothelin receptor antagonist (bosentan) or a combined thromboxane A2/isoprostane-receptor antagonist (SQ29548). Thus, although not having a direct pro-inflammatory potential, isoprostanes might indirectly accentuate PMN stimulation. This seems to occur via a receptor-independent mechanism, perhaps the production of an active metabolite of isoprostanes by endothelial cells.     

Can serum amyloid A or macrophage colony stimulating factor serve as marker of amyloid formation process?

Amyloid formation depends on amyloid precursor production and is influenced by the activity of the underlying disorder and mediated by some proinflammatory cytokines. In this pilot study we tried to find some specific markers that could establish the activity of the disease. We investigated 45 samples of sera and 38 samples of urine from patients (pts) with secondary amyloidosis (AA), primary amyloidosis (AL), systemic autoimmune diseases with renal impairment (Vasc) and healthy controls (Co). Pts with AA had increased plasma levels of TNF alpha (9.97 +/- 4.22 vs. 2.63 +/- 1.34 pg/mL, p < 0.001) and SAA (43.14 +/- 16.0 vs. 3.42 +/- 0.7 ng/mL, p < 0.05) in comparison with Co. Plasma levels of M-CSF in the AA group were significantly increased in comparison with Co (1077.34 +/- 238.6 vs. 137.71 +/- 19.6, pg/mL, p < 0.001) and also in comparison with Vasc (482.24 +/- 86.7 pg/mL, p < 0.05). Urinary excretions of TNF alpha (8.92 +/- 8.1 vs. 0.17 +/- 0.11 microgram/mol creatinine, p < 0.01), sIL-6R (1.39 +/- 1.14 vs. 0.07 +/- 0.05 g/mol creatinine, p < 0.01) and M-CSF (650.2 +/- 153.7 vs. 33.3 +/- 8.6 micrograms/mol creatinine, p < 0.01) in AA were significantly increased in comparison with Co. Pts with AL had increased plasma levels of M-CSF (819.83 +/- 264.2 vs. 137.71 +/- 19.6 pg/mL, p < 0.05) and urinary excretion of M-CSF (865.0 +/- 188.4 vs. 33.3 +/- 8.6 micrograms/mol creatinine, p < 0.01) in comparison with Co. SAA has a low specificity for amyloidosis but is a sensitive acute phase reactant. TNF alpha, a proinflammatory cytokine, may reflect the activity of the underlying diseases in secondary amyloidosis. M-CSF was increased both in plasma and urine in amyloidosis groups and seems to be the most promising (possibly specific) marker of amyloidosis.     

iPF2alpha-III-17,18,19,20-d4: total synthesis and metabolism

The first total synthesis of 17,18,19,20-d4-iPF2alpha-III 32, a deuterated analog of iPF2alpha-III, is described. We have used this analog in some beta-oxidation studies with rat liver homogenates and have shown that 32 was metabolized to 17,18,19,20-tetradeutero-2,3-dinor-iPF2alpha-III 36 and 17,18,19,20-tetradeutero-2,3-dinor-5,6-dihydro-iPF2alpha-III 37.     

Eicosapentaenoic-acid-derived isoprostanes: synthesis and discovery of two major isoprostanes

The stereospecific synthesis of two all-syn-EPA-derived isoprostanes (iPs), 5-epi-8,12-iso-iPF(3alpha)-VI 17 and 8,12-iso-iPF(3alpha)-VI 18, has been accomplished. These two synthetic probes have been used to discover and identify their presence in human urine. The eventual quantitative measurement of these two iPs may be a valuable index of oxidative stress in people with eicosapentaenoic acid- (EPA) and docosahexaenoic acid- (DHA) enriched phospholipids.     

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.     

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.     

Paired analysis of thromboxane and prostacyclin metabolites in urine from healthy mothers and their children

Eicosanoids have been implicated in the adaptation of the fetal to the neonatal circulation, but biochemical support for an activation of their synthesis in relation to birth has not been presented. We addressed this by assessing in pairs the excretion of two metabolites of thromboxane A2, 11-dehydro-TxB2 (dTx) and 2,3-dinor-TxB2 (Tx-M), and one metabolite of prostacyclin, 2,3-dinor-6-keto-PGF1 a (PGI-M), in the first voided urine from 13 healthy term neonates and in the immediate pre-delivery urine from their respective mothers. The excretion of dTx was higher (p less than 0.01) in the neonates than in their mothers (7430 and 1330 pg/mg creatinine, respectively), and so was the excretion of Tx-M (3730 and 900 pg/mg creatinine, respectively, p less than 0.001). Also the excretion of PGI-M was higher (p less than 0.05) in the neonates than in their mothers (2550 vs. 1510 pg/mg creatinine). Amniotic fluid contained detectable levels of both Tx-M and PGI-M. These data indicate that activation of platelets takes place in the neonate after separation of the fetal and maternal vascular circuits. The possible physiological implication of such activation requires further studies.     

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.     

Urinary excretion of three nucleic acid oxidation adducts and isoprostane F(2)alpha measured by liquid chromatography-mass spectrometry in smokers, ex-smokers, and nonsmokers

To assess novel liquid chromatography/mass spectrometric methods for measuring oxidative damage to nucleic acids and lipids, we compared urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 5-hydroxymethyl-2'-deoxyuridine (5-OHmU), and 8-hydroxyguanosine (8-OxoG), and an isoprostane, 8-iso-prostaglandin F(2)alpha (IsopF(2)alpha) in 234 healthy men (n = 113) and women (n = 121), 80 current smokers, 96 never-smokers), and 58 ex-smokers (no tobacco use for 3 years). The 8-OHdG and 8-OxoG did not differ significantly by group; 5-OHmU was higher in smokers, compared with ex- (p <.003) and never- (p <.0001) smokers and in ex- vs. never-smokers (p =.014) at, respectively, 13.5 +/- 0.7, 11.3 +/- 1.0, and 8.7 +/- 0.3 microg/g creatinine. IsopF(2)alpha was higher in smokers, compared with ex- (p =.007) and never-smokers (p <.0001) and in ex- vs. never- smokers (p =.002) at, respectively, 1.1 +/- 0.10; 0.74 +/- 0.07, and 0.51 +/- 0.04 microg/g creatinine. There were significant correlations among all three nucleic acid adducts and between IsopF(2)alpha and both 5-OHmU and 8-OHdG. Many smokers and ex-smokers had high levels of either 5-OHmU excretion or IsopF(2)alpha excretion, but not both. We conclude that 5-OHmU and IsopF(2)alpha are more discriminating of oxidative stress from tobacco smoke than the other two compounds measured. Whether characteristic patterns of excretion of these indicators forecast differential disease risk should be explored in future research.