Glutathione, vitamin E and oxidative stress in coronary artery disease: relevance of age and gender.

BACKGROUND: Observational studies suggest that low levels of antioxidants are associated with high risk for coronary artery disease (CAD). We investigated whether the biomarkers of oxidative balance undergo the same modifications in all CAD patient groups, regardless of gender and age. MATERIALS AND METHODS: One hundred sixty-eight CAD patients and 107 healthy controls were assayed for plasma levels of reduced glutathione (GSH), alpha- and gamma-tocopherol (alpha- and gamma-T) as endogenous antioxidants. A damage score (DS), representative of oxidative stress status, was calculated. ANCOVA models were used to test the association between antioxidants, DS and CAD and its modulation by age and gender. RESULTS: The DS was higher in CAD than in controls. GSH levels, were lower in CAD patients (mean +/- SEM: 57.61 +/- 1.87 micromol 10 g(-1) haemoglobin vs. 68.55 +/- 2.23 in controls, P < 0.0006) in males and in older subjects. Levels of other antioxidants exhibited a complex pattern. Overall, no difference was found in alpha- and gamma-T contents between CAD and controls, but lower alpha-T values were observed in CAD females. A significant interaction between CAD status and gender was observed (P = 0.003). CONCLUSIONS: Our study shows that the involvement of antioxidants in CAD is related to patients' characteristics. These findings may be relevant in planning antioxidant therapies.

Age- and gender-related oxidative status determined in healthy subjects by means of OXY-SCORE, a potential new comprehensive index.

Oxidative stress has been related to various diseases, gender and ageing, and has been measured by various markers. The authors developed a procedure to compute a global oxidative stress index (OXY-SCORE), reflecting both oxidative and antioxidant markers in healthy subjects. Its performance was tested in relation to age and gender and in coronary artery disease (CAD) patients. Eighty-two healthy subjects and 20 CAD patients were enrolled. Plasma free and total malondialdehyde (F- and T-MDA), glutathione disulphide/reduced form ratio (GSSG/GSH) and urine isoprostanes (iPF2alpha-III) levels were combined as oxidative damage markers (damage score). GSH, alpha- and gamma-tocopherol (TH) levels, and individual antioxidant capacity were combined as antioxidant defence indexes (protection score). The OXY-SCORE was computed by subtracting the protection score from the damage score. Among single parameters, T-MDA and iPF2alpha-III significantly correlated with age; only GSH and both tocopherols correlated with male gender in healthy subjects. The OXY-SCORE was positively associated with age (p=0.004) and male gender (p=0.03). As expected, the OXY-SCORE was higher in CAD with a very significant p-value (<0.0001), after adjusting for age, gender and smoking. Combining different markers can potentially provide a powerful index in the evaluation of oxidative stress related to age, gender and CAD status.

Development of a multianalyte method for the determination of anabolic hormones in bovine urine by isotope-dilution GC-MS/MS.

A sensitive, specific and selective multianalyte GC-MS/MS method has been developed for the determination of 11 anabolic hormones in bovine urine. After adjusting the urine pH to 4.8, the samples were spiked with deuterated internal standards and submitted to enzymatic hydrolysis with beta-glucuronidase/arylsulfatase. Hormones were eluted with methanol through a C18 solid phase cartridge and submitted to a liquid-liquid extraction. Analytes were derivatized by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane and GC-MS data were obtained in the positive electron impact tandem mass mode. Under these conditions, no matrix effects were observed and limit of detection values were in the range of 0.005 ng/mL (diethylstilbestrol) to 0.38 ng/mL (17alpha-methyltestosterone and 17alpha-ethynylestradiol). Recoveries from 81% (alpha-zeranol) to 149% (17alpha-methyltestosterone) were found under the selected conditions. These results were better than those found using heptafluorobutyric anhydride (HFBA) as derivative reagent and those measured in full scan and selective ion monitoring modes.

Determination of asymmetric and symmetric dimethylarginines in plasma of hyperhomocysteinemic subjects.

The aim of this study was to investigate the possible relationship among dimethylarginines (asymmetric, ADMA; symmetric, SDMA) and homocysteine (Hcy) levels in subjects affected by chronic, mild to intermediate, hyperhomocysteinemia. ADMA and SDMA were assayed by an optimised HPLC method in 75 patients (Hcy = 20.8 micromol/L, 17.1-30.2; median and percentile range) and, for comparison, in 85 healthy subjects (Hcy = 8.0 micromol/L, 7.0-9.1). In controls, the cut-off values were set at 0.61 micromol/L for ADMA and 0.56 or 0.48 micromol/L for male and female SDMA, respectively. In patients, ADMA and SDMA levels were increased (p < 0.001) with respect to controls, but no correlation with Hcy was observed. Hyperhomocysteinemic subjects showed a different behaviour in respect to ADMA and SDMA levels and this allowed their stratification in 3 subgroups characterized by ADMA and SDMA in the normal range, only SDMA, or both ADMA and SDMA over the cut-off values. A lack of correlation with Hcy was again observed, thus minimizing the direct role of Hcy on ADMA and SDMA metabolism and suggesting the need for further studies on this issue.

Effect of local hyperthermia of the bladder on mitomycin C pharmacokinetics during intravesical chemotherapy for the treatment of superficial transitional cell carcinoma.

AIMS: To assess the effect of local hyperthermia on the systemic absorption of mitomycin C (MMC) during intravesical chemotherapy for the treatment of superficial transitional cell carcinoma of the bladder, and to establish the likely safety of this procedure. METHODS: Group 1 (n = 12) received 20 mg intravesical MMC plus local hyperthermia, group 2 (n = 13) 20 mg MMC alone, group 3 (n = 16) 40 mg MMC plus local hyperthermia and group 4 (n = 10) 40 mg MMC alone. Patients in groups 1, 2, and 4 underwent post-tumour resection adjuvant treatment, whereas those in group 3 still had tumour present and were treated to eradicate it. Intravesical instillation lasted 60 min, with the solution (50 ml) being replaced after the first 30 min. Blood samples were taken before, and every 15 min during instillation. MMC concentrations in plasma and in urine were determined by h.p.l.c. RESULTS: The highest MMC plasma concentration (67.9 ng ml(-1)) occurred in a patient in group 3. This value was well below the threshold concentration (400 ng ml-1) for myelosuppression. Local hyperthermia associated with the intravesical chemotherapy enhanced plasma MMC concentrations at 30, 45 and 60 min compared with chemotherapy alone (Group 1 vs 2, P < or = 0.008). Systemic exposure to MMC was not significantly increased by doubling the intravesical dose when intravesical chemotherapy alone was administered. Patients in group 3 displayed the highest degree of MMC absorption and the greatest variability in pharmacokinetics between patients. CONCLUSIONS: Local hyperthermia enhances the systemic absorption of MMC during intravesical chemotherapy for bladder cancer. In the doses used, plasma MMC concentrations were always more than six times lower than those shown to cause toxicity.

Hyperhomocysteinemia in myelodysplastic syndromes: specific association with autoimmunity and cardiovascular disease.

Hyperhomocysteinemia (HH) has been associated with cardiovascular and autoimmune diseases and oxidative cell damage. Myelodysplastic syndromes (MDS) are associated with autoimmunity (AI) and increased oxidative stress. We tested the association of HH and oxidative stress in 33 MDS patients, by measuring plasma homocysteine and malondialdehyde (MDA). HH was found in 42% of cases, (4/5) cases with associated cardiovascular events (CVE)(80%), and 9/15 cases with associated AI (60%). Thus in MDS, HH was significantly associated with AI/CVE (chi(2) : p=0.0011), and this association seems to be specific, as demonstrated by the comparison of MDS presenting AI/CVE with the ischemic cardiopathy/rheumatoid arthritis control group (13/20, 65% vs 19/69, 27%; chi(2) : p=0.0021). The levels of MDA indicated increased oxidative stress. Our data may suggest that in a subset of MDS, HH may simultaneously contribute to bone marrow myelodysplasia, CVE and AI pathogenesis, possibly through oxidative cell damage.

Mechanisms of action of malondialdehyde and 4-hydroxynonenal on xanthine oxidoreductase.

Studies have been made on the possible involvement of malondialdehyde (MDA) and (E)-4-hydroxynon-2-enal (HNE), two terminal compounds of lipid peroxidation, in modifying xanthine oxidoreductase activity through interaction with the oxidase (XO) and/or dehydrogenase (XDH) forms. The effect of the two aldehydes on XO (reversible, XO(rev), and irreversible, XO(irr)) and XDH was studied using xanthine oxidase from milk and xanthine oxidoreductase partially purified from rat liver. The incubation of milk xanthine oxidase with these aldehydes resulted in the inactivation of the enzyme following pseudo-first-order kinetics: enzyme activity was completely abolished by MDA (0.5-4 mM), while residual activity (5% of the starting value) associated with an XO(irr) form was always observed when the enzyme was incubated in the presence of HNE (0.5-4 mM). The addition of glutathione to the incubation mixtures prevented enzyme inactivation by HNE. The study on the xanthine oxidoreductase partially purified from rat liver showed that MDA decreases the total enzyme activity, acting only with the XO forms. On the contrary HNE leaves the same level of total activity but causes the conversion of XDH into an XO(irr) form.

Oxidative stress and homocysteine in coronary artery disease.

BACKGROUND: Oxidative stress is present in cardiovascular diseases (CVDs), and hyperhomocysteinemia, an independent risk factor for these diseases, may play a role by inducing production of oxygen free radicals. METHODS: To evaluate the possible role of homocysteine (Hcy) in inducing oxidative stress in coronary artery disease (CAD), plasma Hcy was measured in 68 consecutive cardiovascular patients, and plasma malondialdehyde (MDA), both free and total (free + bound), was measured in 40 patients with CAD (18 with chronic stable angina and 22 with unstable angina). As controls, we tested 70 healthy volunteers. Hcy was measured by an immunoenzymatic method and MDA, an index of lipid peroxidation, by gas chromatography-mass spectrometry. RESULTS: Plasma Hcy concentrations were significantly higher in cardiovascular patients than in controls (10.2 vs 8.9 micromol/L; P <0.0002), with no significant difference between values in the stable and unstable angina subgroups. Similarly, total MDA was significantly higher in the CAD group than in the controls (2.6 vs 1.3 micromol/L; P <0.00001), again with no significant difference between stable and unstable angina patients. By contrast, free MDA, which was significantly higher in the CAD patients than the controls (0.4 vs 0.2 micromol/L; P < 0.00001), was also significantly higher in the unstable than in the stable angina group (0.5 vs 0.3 micromol/L; P <0.03). However, no correlation was observed among Hcy and free and total MDA. CONCLUSIONS: Our findings show that a moderate increase of Hcy is associated with CVD but that Hcy at the detected values cannot be considered completely responsible for oxidative damage. That lipid peroxidation is involved in CAD is shown by our observation of significantly increased plasma free and total MDA concentrations compared with controls. Moreover, free MDA values discriminated between unstable and chronic stable angina, and could thus represent a new diagnostic tool.

Free and total malondialdehyde assessment in biological matrices by gas chromatography-mass spectrometry: what is needed for an accurate detection.

A method to determine free and total (free and bound) malondialdehyde (MDA) in fresh human plasma, or in rat liver microsomes, using selected ion monitoring (SIM) gas chromatography-mass spectrometry in the electron impact mode was set up. The dideuterated internal standard, 3-hydroxy[1, 3-2H2]-2-propenal (dMDA), was added to the biological samples before their analytical manipulation. To detect free MDA the samples were reacted under mild conditions (25 degreesC, pH 4.0, 30 min) with phenylhydrazine (PH), affording 1-phenyl-1H-pyrazole and its 3, 5-dideuterated isotopomer. For the evaluation of total MDA level the plasma or microsomes were subjected, before the derivatization step, to hydrolysis in the presence of 1 M NaOH under preestablished conditions. This method offers several advantages such specificity, precision (within-day CV 2.0%, between-day CV 2.1%), linearity (0. 01-15 microM) and high sensitivity (5 pmol injected). The recovery of known added MDA amounts from plasma and microsomes, hydrolyzed or not, accounted for 98 +/- 0.6%. The free MDA levels found in the plasma and microsomes were 0.14 +/- 0.03 microM and 0.048 +/- 0.006 nmol/mg protein, respectively. The total MDA levels were 1.3 +/- 0. 07 microM in plasma and 0.36 +/- 0.04 nmol/mg protein in the microsomes.

Beta-ethoxyacrolein contamination increases malondialdehyde inhibition of milk xanthine oxidase activity.

beta-Ethoxyacrolein (BEA), a side product that forms during the preparation of malondialdehyde (MDA) by acidic hydrolysis of tetraethoxypropane (TEP), has been found to be an inhibitor of milk xanthine oxidase (XO) several times more potent than pure MDA (NaMDA). The incubation of XO with 10 microM BEA abolished 50% of the enzyme activity within 1 min; the inhibited enzyme was totally regenerated by dialysis and filtration through Sephadex. The BEA inhibition mode of the enzyme was mixed-type with the apparent inhibition constants (Ki) of 2.4 x 10(-6) M. An HPLC method for quantitation of BEA in the crude commonly used MDA preparation was set up.

pH sensitivity and plasma stability of liposomes containing N-stearoylcysteamine.

In this study, we investigated the pH sensitivity of different liposomal formulations containing 10 mol% N-stearoylcysteamine, as pH sensitive molecule. Liposome stability was monitored by determining the release of different entrapped water soluble molecules, 5,6-carboxyfluorescein (CF) being the marker of leakage mainly used. Small unilamellar vesicles composed of egg phosphatidylcholine (EPC) and N-stearoylcysteamine (9:1 molar ratio) incubated at 20 degrees C in citrate phosphate buffer released, at pH 6.8, 2.5 fold the amount of CF released at pH 7.4. The addition of plasma to the incubation medium and an increase of temperature to 37 degrees C led to significantly increased the CF release from EPC/N-stearoylcysteamine SUV, both at pH 7.4 and 6.8. The addition of cholesterol had a stabilizing effect on liposomal vesicles with respect to both temperature and plasma, without affecting pH sensitivity. In fact, at 37 degrees C and in 25% plasma the ternary mixture showed the highest CF release, as a consequence of the moderate acidification of the medium from 7.4 to 6.8. Thus, these liposome formulations are potentially a useful tool for specific drug delivery to pathological tissues such as tumours, inflammation sites and ischemic areas where it is known that a lowering of the pH can occur.

Inhibition of in vitro lipid peroxidation by stable steroidic nitroxyl radicals.

4',4'-dimethylspiro (5 alpha-cholestane-3,2'-oxazolidin)-3'-yloxy (IK-1) and 7 alpha,12 alpha-dihydroxy-4',-4'-dimethylspiro (5 beta-cholan-24-oic-3,2'-oxazolidin)-3'-yloxy acid (IK-2), two stable steroidic nitroxyl radicals, were newly synthesized and tested as possible inhibitors of lipid peroxidation, induced by Fenton's reagent in both rat liver microsomes and egg phosphatidylcholine liposomes. The inhibitory activity, evaluated through the formation of thiobarbituric acid reactive substances (TBARS) and the conjugated diene, was compared with that of alpha-tocopherol and 2,2,6,6-tetramethylpiperidine-1-yloxy (TEMPO). In each model system IK-1 and IK-2 exhibited an IC50 of 8 microM and reduced the formation of TBARS and conjugated diene, showing IK-1 a potency comparable to alpha-tocopherol and higher than TEMPO. Moreover IK-1 and, to a lesser extent IK-2, reduced the lipid peroxidation induced in the microsomes by the water-soluble azo-initiator 2,2'-Azobis (2-methylpropionamidine) dihydrochloride (AMPH), indicating the IK-1 and IK-2 ability as chain-breaking antioxidants. The hydroxylamine 4',4'-dimethylspiro (5 alpha-cholestane-3,2'-oxazolidin)-3'-hydroxide (IK-3), obtained by chemical reduction of IK-1, was completely inactive as an inhibitor of lipid peroxidation in heat pre-treated microsomes and in liposomes. However in microsomes it was active since it was oxidized to the corresponding nitroxyl radical IK-1.

HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue.

This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathione in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined after reduction with dithiothreitol and protein precipitation with perchloric acid (PCA). A preliminary selective blockage of free sulfhydryl groups with N-ethylmaleimide was necessary to evaluate the different oxidized forms. The assay showed high sensitivity (< 0.05 pmol injected) and good precision (within-day CVs of 5.5% to 6.4%), recovery (101% +/- 4%), and linearity (r > 0.999). Samples, after PCA acidification, were stable at room temperature and 4 degrees C for 3 days, and at -20 degrees C and -80 degrees C for > 1 month. The method (involving automated derivatization) not only is very rapid and simple but also allows immediate processing of many different biological samples.

No documentable role for xanthine oxidase in the pathogenesis of hepatic in vivo ischaemia/reperfusion injury.

An investigation was made into the possible involvement of the enzyme xanthine oxidase (XO) (EC 1.1.3.22), both reversible (XOrev) and irreversible (XOirr), in damage observed after short-term in vivo hepatic ischaemia/reperfusion (60 or 120 min I and 15 min R) in fasted rats with: (i) a physiological content of XO (25%); and (ii) higher XO percentage (45%). In the latter the hepatic XO physiological percentage was increased by diethylmaleate treatment (300 mg kg-1) that depleted the cytosolic glutathione (GSH) to 14% of the controls. It was shown that, in animals with physiological content of XO, 60 and 120 min of hepatic ischaemia followed by 15 min reperfusion results in decreased GSH levels, and significantly increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels, without any modification of either the percentages of XO (XOirr and XOrev) or the hepatic thiobarbituric acid reactive substances (TBARS). Sixty minutes of ischaemia/reperfusion in rats with the higher XO level and lower hepatic GSH content led to further conversion of XDH to XOrev, with no increase in XOirr. In addition, the ALT and AST serum levels in these animals rose to the same extent as in normal rats after 120 min ischaemia and 15 min reperfusion, this extent being observed to be associated with a moderate increase in thiobarbituric acid reactive substances (TBARS). However, the administration of allopurinol, at a dose of 50 mg kg-1, which almost completely inhibits XO activity, did not lead to any decrease in liver damage or TBARS.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of glutathione depletion on the conversion of xanthine dehydrogenase to oxidase in rat liver.

The ability of endogenous glutathione (GSH) to modify the activity of the enzyme xanthine oxidase (XO) in rat liver was investigated. The effect of hepatic GSH depletion on the conversion of xanthine dehydrogenase (XDH) (EC 1.1.1.204) to XO (EC 1.1.3.22) was determined 10 min after i.p. administration of different amounts of diethylmaleate to fasted rats. After administration of 400 mg/kg, total hepatic non-protein GSH (reduced + oxidized GSH) decreased significantly to 14% of controls. In this condition the level of oxidized GSH was unchanged and no lipid peroxidation was observed, while a significant increase of reversible XO and a minor increase of the irreversible form of the enzyme was detected.

Determination of diclofenac in human plasma by selected ion monitoring.

A simple and rapid gas chromatographic/mass spectrometric method to determine plasma diclofenac was developed, which employs formation of the methyl ester with diazomethane. Methoxydiclofenac was used as the internal standard. Under the conditions used, the previously described partial cyclization of diclofenac to the indolone derivative was avoided. The limit of detection of plasma levels of diclofenac is 2 ng ml-1, which renders the method useful for clinical studies on oral, intravenous and rectal administration of the drug. The analysis is carried out by electron impact gas chromatography/mass spectrometry and can therefore be performed on the more common mass spectrometers. Linearity and reproducibility of the method were demonstrated by the high correlation coefficient of the calibration lines (r greater than 0.999) and from the low variation of their slopes (coefficient of variation 3%) determined on different days, respectively. Pharmacokinetic parameters (area under curve = 1.8 +/- 0.26 microgram h ml-1, tmax = 1.5 +/- 0.5 h, Cmax = 734 +/- 82 ng ml-1 and terminal half-life = 0.88 +/- 0.52 h) determined from the plasma decay of diclofenac in three healthy subjects given a single oral dose of diclofenac were in good agreement with those reported in the literature.

Lack of conversion of xanthine dehydrogenase to xanthine oxidase during warm renal ischemia.

Irreversible transformation of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) during ischemia was determined measuring XDH and total enzyme activity in kidneys before and after 60 min of clamp of the renal pedicle. Tissue levels of adenine nucleotides, xanthine and hypoxanthine were used as indicators of ischemia. After 60 min of clamping, ATP levels decreased by 72% with respect to controls whereas xanthine and hypoxanthine progressively reached tissue concentrations of 732 +/- 49 and 979 +/- 15 nmol.g tissue-1, respectively. Both total and XDH activities in ischemic kidneys (30 +/- 15 and 19 +/- 1 nmol.min-1.g tissue-1) were significantly lower than in controls when expressed on a tissue weight basis. The fraction of enzyme in the XDH form was however unchanged indicating that the reduction of the nucleotide pool is not accompanied by induction of the type-O activity of xanthine oxidase.

Xanthine oxidase activity: simultaneous HPLC evaluation of the "D" and "O" forms.

A new HPLC method was set up for the simultaneous evaluation of the amount of uric acid and NADH produced by incubation of tissue fractions containing xanthine oxidase, from which the activity of both type "O" (oxidase) and type "D" (dehydrogenase) xanthine oxidase can be calculated. After incubation of the enzyme fraction and ethanol extraction, HPLC analysis is directly carried out. Sensitivity of the method is high enough for the evaluation of xanthine oxidase activity at the lowest reported tissue values. The reliability of the method was tested measuring the enzyme activity in rat heart and kidney extracts.