Myeloperoxidase as serum marker for detection of CMV infections and rejections in patients after liver or heart transplantation.

Rejection episodes and infections are common problems after organ transplantations (TX). Rejection can be diagnosed in liver-transplant (LTX) patients when liver-specific enzymes in the serum are elevated. As endomyocardial biopsy (EMB) is the gold standard for detecting heart transplant (HTX) rejection, serum parameters would permit more selective use of this invasive procedure. Cytomegalovirus (CMV) infections can have serious consequences for TX patients and so should be diagnosed and treated timely. At present, there are no suitable diagnostic methods other than CMV antigen pp65 and CMV polymerase chain reaction (PCR). Our study aimed to test the sensitivity of myeloperoxidase (MPO), an enzyme of neutrophilic granulocytes, as a new serum parameter in addition to established serum parameters and EMB for diagnosis of infection and rejection episodes after LTX and HTX. MPO in plasma from 246 blood samples (103 used for statistical analysis) from 27 patients (18 LTX and 9 HTX) was determined using ELISA; C-reactive protein (CRP), gamma-glutamyl-transpeptidase (GGT), white blood count and CMV pp65 antigen were monitored routinely. EMBs were performed at defined intervals after HTX. Results were analyzed with descriptive statistics, T-test, Wilcoxon test and Cox regression analysis, whereby a p<0.05 was viewed as significant. MPO values in TX patients with an infection (7 LTX, 2 HTX) were significantly higher than in TX patients without complications (control group) (253.9 microg/l vs. 116.6 microg/l, p=0.0194). In TX patients with rejections (6 LTX, 6 HTX), there is also a significant increase in comparison to controls (429.7 microg/l vs. 116.6 microg/l, p=0.0001). Data from individual TX patients, however, indicate that MPO levels rise distinctly earlier with infection (CMV) than with rejection, enabling earlier detection of the complication and initiation of suitable treatment. Our findings suggest that a larger and prospective study should be designed to evaluate the usefulness of MPO levels in assessing organ transplant recipients.

Antioxidant status of patients on peritoneal dialysis: associations with inflammation and glycoxidative stress.

BACKGROUND: Patients on peritoneal dialysis (PD) frequently exhibit oxidant-antioxidant imbalance, advanced glycation end-product overload, and subclinical inflammation but the interrelations between these pathophysiological changes have not been fully elucidated. SUBJECTS AND METHODS: To study possible associations, a cross-sectional study of antioxidant status, glycoxidative stress, and inflammation, using HPLC and ELISA methods, was undertaken in 37 PD patients and age- and sex-matched healthy controls. RESULTS: Plasma ascorbate concentrations were low in patients not taking at least low-dose vitamin C supplements. In patients taking vitamin C supplements, there was a positive relation between ascorbate and pentosidine concentrations. Vitamin E and carotenoid concentrations were comparable between patients and controls, while lycopene and lutein/zeaxanthin concentrations were lower. Interleukin-6, C-reactive protein (CRP), and pentosidine concentrations were elevated in PD patients. beta-Cryptoxanthin, lycopene, and lutein/zeaxanthin concentrations were inversely related to interleukin-6 concentrations. beta-Cryptoxanthin concentrations were also inversely related to CRP concentrations. Pentosidine showed a low dialysate-to-plasma ratio, indicating low peritoneal clearance. Pentosidine concentrations increased with duration of PD therapy, while alpha- and beta-carotene concentrations decreased. Malondialdehyde concentrations were elevated compared to controls but remained within the normal range. Retinol concentrations decreased with PD therapy and were inversely related to interleukin-6 and CRP concentrations. CONCLUSIONS: Low-dose vitamin C supplements and a carotenoid-rich diet should be recommended for PD patients to maintain normal antioxidant status and efficiently counteract the chronic inflammatory response, rather than high doses of vitamin C, which could play a role as a precursor of pentosidine.

Comparison of fluorescent polarization immunoassay (FPIA) versus HPLC to measure everolimus blood concentrations in clinical transplantation.

Clinical management of transplant patients depends on therapeutic drug monitoring (TDM) and regulation of immunosuppressive therapy. TDM of whole-blood concentrations is mandatory for everolimus (ERL) dosage individualisation. We compared the new semi-automated immunoassay (Innofluor Certican Assay System, Seradyn Inc) using FPIA technology on Abbott TDxFLx analyzers with established HPLC-UV as reference method. A total of 165 samples were analyzed from 52 transplant patients (40 kidney, 12 heart) receiving ERL or another immunosuppressive agent as part their routine care after organ transplantation. The correlation coefficient was r(2)=0.8229, and the regression equation (95% IC) yielded FPIA=1.111 x (HPLC)+0.378. FPIA compared to HPLC gave a positive bias of 1.19 ng/ml. The FPIA assay so appears to have a diagnostic efficacy comparable to HPLC for assessing the risk of acute rejection in transplant recipients. However, the values of the FPIA were higher than those calculated from HPLC measurements, because of the cross-reactivity of the antibody used in the FPIA assay with the ERL metabolite and/or with sirolimus; this cross-reactivity occurs frequently when transplant patients are switched from sirolimus to ERL.

Role of free radicals in aseptic loosening of hip arthroplasty.

Fibrous pseudocapsule around hip implants is an invariable finding at revision operations and is believed to release inflammatory mediators that stimulate bone resorption. Reactive oxygen species have been proposed to be causative factors in various disorders with tissue fibrosis. We were interested in investigating whether aseptic loosening is connected with high oxidative stress, and in showing the underlying mechanism of periprosthetic fibrosis and its role in loosening. Levels of oxidative stress markers reduced (GSH) and oxidized (GSSG) gluthatione and malondialdehyde (MDA) were assayed in 28 loose hips and in 12 stable hips revised for high rate of wear and osteolysis. Collagen in the periprosthetic tissues was measured as hydroxyproline content. Osteolysis and polyethylene wear were graded. Increased oxidative stress measured by low GSH/GSSG ratio as well as by increased MDA level was established in patients compared to controls. Oxidative stress markers intercorrelated significantly. MDA and both GSH and GSSG levels correlated significantly with hydroxyproline level. Levels of GSSG and MDA were higher in hips with greater polyethylene wear. The results suggest that high oxidative stress may play a role in formation of a fibrous membrane observed at revision of loose hips. The fibrous pseudocapsule is probably related to high intraarticular pressure and expansion of the effective joint space. This study may elicit some aspects of the pathogenesis of aseptic hip loosening and aid in future investigations aiming at prevention of this complication.

Contribution of nicotine to acute endothelial dysfunction in long-term smokers.

OBJECTIVES: The aim of this study was to determine whether nicotine, a constituent of cigarette smoke, contributes to acute endothelial dysfunction after smoking one cigarette. BACKGROUND: Animal studies suggest that nicotine might cause an impairment of endothelium-dependent vasodilation via an increase in oxidative stress. METHODS: Sixteen healthy smokers were entered into a randomized, observer-blinded crossover study comparing the effects of nicotine nasal spray (1-mg nicotine) and cigarette smoke (1-mg nicotine, 12 mg tar) on vascular reactivity in the brachial artery. Using high-resolution ultrasound, flow-mediated dilation (FMD) and endothelium-independent, nitroglycerin-induced dilation were assessed at baseline and 20 min after the administration of nicotine (spray or cigarette). RESULTS: In response to similar increases in nicotine serum levels, FMD values declined from 10.2 +/- 4.4% to 6.7 +/- 4.0% after the spray (mean difference: -3.6 +/- 2.0%, 95% confidence interval: -4.6; -2.5, p < 0.0001) and from 9.4 +/- 3.8% to 4.3 +/- 2.8% after the cigarette (-5.1 +/- 2.6%, -6.5; -3.7, p < 0.0001). Nitroglycerin-induced dilation remained similar within both periods. Performing a period effect analysis of variance, a significant influence on FMD was found for the mode of administration (p = 0.017) and the baseline value (p = 0.021). The effect on FMD was more pronounced after the cigarette than after the spray (estimated average effect difference: 1.9% FMD). Oxidation parameters did not increase significantly after nicotine spray or tobacco exposure. CONCLUSIONS: These results demonstrate that nicotine alone causes acute endothelial dysfunction, although to a lesser extent than smoking a cigarette of the same nicotine yield. However, the precise mechanisms by which nicotine leads to this altered vascular reactivity remain unclear.

Effects of vitamin E depletion/repletion on biomarkers of oxidative stress in healthy aging.

The effects on ex vivo LDL resistance to oxidation and biomarkers of in vivo oxidative stress in response to 3-month dietary vitamin E restriction to 25% of recommended intake and 2-month unrestricted dietary intake and supplementation with 800 IU/d were studied in 100 healthy, nonsmoking 20-75-year-old volunteers. Significant changes in vitamin E status were associated with decreases and increases, respectively, in LDL resistance to oxidation in the depletion and supplementation period and with decreases in lipid peroxidation and oxidative DNA modification in the supplementation period. Healthy aging was not associated with enhanced susceptibility to oxidation in the depletion period.

Liquid chromatographic method for simultaneous determination of mycophenolic acid and its phenol- and acylglucuronide metabolites in plasma.

A simple, sensitive and reproducible HPLC method is presented for the simultaneous determination of mycophenolic acid (MPA) and its metabolites phenolic MPA-glucuronide (MPAG) and acyl glucuronide (AcMPAG) in human plasma. Sample purification requires protein precipitation with 0.1 M phosphoric acid/acetonitrile in the presence of Epilan D as an internal standard (IS). Separation was performed by reversed-phase HPLC, using a Zorbax SB-C18 column, 32% acetonitrile and a 40 mM phosphoric acid buffer at pH 3.0 as mobile phase; column temperature was 50 degrees C, flow rate 1.4 ml/min, and measurement by UV detection was at 215 nm (run time 12 min). The method requires only 50 microl plasma. Detection limits were 0.1 microg/ml for MPA and AcMPAG, and 2.0 microg/ml for MPAG, respectively. Mean absolute recovery of all three analytes was >95%. This analytical method for the determination of MPA and its metabolites is a reliable and convenient procedure that meets the criteria for application in routine clinical drug monitoring and pharmacokinetic studies.

Determination of myeloperoxidase in EDTA plasma: comparison of an enzyme-linked immunosorbent assay with a chemiluminescent automated immunoassay.

BACKGROUND: The increased demand in clinical chemistry laboratories for determination of myeloperoxidase (MPO) as a potential marker for cardiovascular diseases has led to the development of several analytical methods, especially enzyme-linked immunosorbent assay (ELISA, e. g. from Immundiagnostik AG), and recently a new chemiluminescent automated immunoassay (CMIA, Architect MPO assay, Abbott Diagnostics). METHODS: We compared data from 115 patients obtained from an ELISA reference method (Immundiagnostik AG) with data obtained from the automated Architect MPO assay. RESULTS: The new MPO assay from Abbott Diagnostics correlates well with the ELISA (y=0.767x+7.035; R(2)=0.9204). The difference plot according to Bland-Altman analysis shows a mean bias of -1.048 microg/l, with a probability of 95%. CONCLUSIONS: The Architect MPO assay can easily be adapted to the Architect instrument system and it is advantageous as far as technology and analysis time are concerned. This new automated MPO assay shows excellent analytical performance and provides a precise and convenient automated method for the measurement of MPO.

Increased concentrations of circulating vitamin E in carriers of the apolipoprotein A5 gene - 1131T>C variant and associations with plasma lipids and lipid peroxidation.

The aim of this study was to investigate the effects of the apolipoprotein A5 (APOA5) 1131T>C gene variant on vitamin E status and lipid profile. The gene variant was determined in 297 healthy nonsmoking men aged 20-75 years and recruited in the VITAGE Project. Effects of the genotype on vitamin E in plasma, LDL, and buccal mucosa cells (BMC) as well as on cholesterol and triglyceride (TG) concentrations in plasma and apolipoprotein A-I (apoA-I), apoB, apoE, apoC-III, and plasma fatty acids were determined. Plasma malondialdehyde concentrations as a marker of in vivo lipid peroxidation were determined. C allele carriers showed significantly higher TG, VLDL, and LDL in plasma, higher cholesterol in VLDL and intermediate density lipoprotein, and higher plasma fatty acids. Plasma alpha-tocopherol (but not gamma-tocopherol, LDL alpha- and gamma-tocopherol, or BMC total vitamin E) was increased significantly in C allele carriers compared with homozygote T allele carriers (P = 0.02), but not after adjustment for cholesterol or TG. Plasma malondialdehyde concentrations did not differ between genotypes. In conclusion, higher plasma lipids in the TC+CC genotype are efficiently protected against lipid peroxidation by higher alpha-tocopherol concentrations. Lipid-standardized vitamin E should be used to reliably assess vitamin E status in genetic association studies.

Isocratic high-performance liquid chromatographic method with ultraviolet detection for simultaneous determination of levels of voriconazole and itraconazole and its hydroxy metabolite in human serum.

A simple, specific method is presented for simultaneous determination of voriconazole and itraconazole and its metabolite, hydroxyitraconazole, in human serum using one-step liquid-liquid extraction and high-performance liquid chromatography. Linearity tests ranged from 0.1 to 8.0 microg/ml; the minimum detectable concentration was 0.03 microg/ml.

Vitamin E attenuates oxidative stress induced by intravenous iron in patients on hemodialysis.

Intravenous iron application to anemic patients on hemodialysis leads to an "oversaturation" of transferrin. As a result, non-transferrin-bound, redox-active iron might induce lipid peroxidation. To test the hypothesis that vitamin E attenuates lipid peroxidation in patients receiving 100 mg of iron(III) hydroxide sucrose complex intravenously during a hemodialysis session, 22 patients were investigated in a randomized cross-over design, either with or without a single oral dose of 1200 IU of all-rac-alpha-tocopheryl acetate taken 6 h before the hemodialysis session. Blood was drawn before and 30, 60, 90, 135, and 180 min after the start of the iron infusion, and areas under the curve (AUC0-180 min) of ratios of plasma malondialdehyde (MDA) to cholesterol and plasma total peroxides to cholesterol (two markers of lipid peroxidation) were determined as the outcome variables. At baseline of the session without vitamin E supplementation, plasma alpha-tocopherol concentrations (27.6 +/- 1.8 micromol/L) and ratios of alpha-tocopherol to cholesterol (5.88 +/- 1.09 mmol/mol) were normal, plasma MDA concentrations were above normal (1.20 +/- 0.28 micromol/ L), and bleomycin-detectable iron (BDI), indicating the presence of redox-active iron, was not detectable. Upon iron infusion, BDI and MDA concentrations increased significantly (P < 0.001). BDI concentrations explained the increase over baseline in MDA concentrations (MDA = 1.29 +/- 0.075 x BDI). Vitamin E supplementation, leading to a 68% increase in plasma alpha-tocopherol concentrations, significantly reduced the AUC0-180 min of MDA to cholesterol (P = 0.004) and peroxides to cholesterol (P = 0.002). These data demonstrate that a single oral dose of vitamin E attenuates lipid peroxidation in patients on hemodialysis receiving intravenous iron. Given that intravenous iron is applied repeatedly to patients on hemodialysis, this therapeutic approach may protect against oxidative stress-related degenerative disease in the long term.