Effects of inhaled corticosteroids with different lung deposition on exhaled hydrogen peroxide in stable COPD patients.

BACKGROUND: The effects of inhaled corticosteroids (ICS) on markers of oxidative stress in patients with stable COPD are unclear. OBJECTIVES: The aim was to investigate the effect of ICS on exhaled H(2)O(2) in stable COPD patients and to compare ICS with different lung deposition. METHODS: Forty-one stable patients with moderate COPD (FEV(1) approximately 60% predicted) were randomized to sequence 1; first HFA-134a beclomethasone dipropionate (HFA-BDP, an ICS with more peripheral deposition) 400 microg b.i.d., then fluticasone propionate (FP, an ICS with more central deposition) 375 microg b.i.d. (n = 20) or sequence 2; first FP, then HFA-BDP (n = 21). Both 4-week treatment periods were preceded by a 4-week washout period. After each period, the concentration of H(2)O(2) in exhaled breath condensate was measured. RESULTS: The H(2)O(2) concentration decreased significantly after the first treatment period in both sequence 1 and 2 (p < 0.05, p = 0.01, respectively). In neither sequence was there a return to baseline values after the second washout, indicating a carry-over effect. The concentrations remained low in both sequences during the second treatment period. CONCLUSIONS: Both ICS appeared to reduce exhaled H(2)O(2) in stable COPD patients. However, this study showed no difference between ICS with different deposition patterns, which in part may be due to the carry-over effect.

Markers of inflammation and oxidative stress during lower respiratory tract infections in COPD patients.

BACKGROUND: Lower respiratory tract infections (LRTI) occur frequently in patients with Chronic Obstructive Pulmonary Disease (COPD), and are a major cause of morbidity, mortality and health care utilization. The aim of this study was to investigate if non- or less invasive markers of inflammation and oxidative stress can predict the course of the infections. METHODS: Twenty-five COPD patients who were admitted to hospital with a LRTI were included. Within 24 hours after admittance, spirometry (FEV1, FVC, MEF50), measurement of hydrogen peroxide (H2O2) in exhaled breath condensate (EBC), symptom scores and analyses of ESR, CRP, ECP, and MPO in serum were performed. All patients were treated with intravenous dexamethasone, nebulised salbutamol/ipratropium and, if needed, antibiotics. The tests were repeated at day 2, 3, 7 and 30. RESULTS: Complete data of the first four visits were collected in 19 patients. The H2O2 concentration and spirometry parameters did not change significantly during the study period. CRP, ESR and MPO levels decreased significantly during treatment, while the other serum inflammatory parameters did not change. There were no significant correlations between H2O2 concentration, spirometry and serum inflammatory parameters. CONCLUSIONS: In conclusion, this study showed no significant changes in H2O2 concentration in EBC, or spirometry during treatment of a LRTI in COPD patients. In contrast, several serum inflammatory markers did decrease during hospitalization, thus providing a simple tool to monitor exacerbations.

Variability of exhaled hydrogen peroxide in stable COPD patients and matched healthy controls.

BACKGROUND: Because inflammation induces oxidative stress, exhaled hydrogen peroxide (H(2)O(2)), which is a marker of oxidative stress, may be used as a non-invasive marker of airway inflammation in chronic obstructive pulmonary disease (COPD). There are no data on the circadian variability of exhaled H(2)O(2) in COPD patients. OBJECTIVE: The aim of this study was to investigate the variability of the H(2)O(2) concentration in breath condensate of stable COPD patients and of matched healthy control subjects. METHODS: We included 20 patients with stable mild COPD (forced expiratory volume in 1 s approximately 70% of predicted) and 20 healthy subjects, matched for age, sex and pack-years, all smokers or ex-smokers. Breath condensate was collected and its H(2)O(2) concentration determined fluorometrically three times on day 0 (9 and 12 a.m., and 3 p.m.) and once on days 1, 2, 3, 8 and 21. RESULTS: The mean H(2)O(2) concentration increased significantly during the day in both the patient and control groups (p = 0.02 and p < 0.01, respectively). Over a longer period up to 21 days, the mean concentration did not change in both groups. There was no significant difference between patients and controls. The mean coefficient of variation over 21 days was 45% in the patient group and 43% in the control group (p = 0.8). CONCLUSIONS: The exhaled H(2)O(2) concentration increased significantly during the day in both stable COPD patients and controls. Over a period of 3 weeks, the mean H(2)O(2) concentration did not change and the variability within the subjects was similar in both groups.

An efficient and reproducible method for measuring hydrogen peroxide in exhaled breath condensate.

We investigated the sensitivity and reproducibility of a test procedure for measuring hydrogen peroxide (H202) in exhaled breath condensate and the effect of storage of the condensate on the H2O2 concentration, and compared the results to previous studies. Twenty stable COPD patients breathed into our collecting device twice for a period of 10 min. The total exhaled air volume (EAV) and condensate volume were measured both times and the H2O2 concentration of the condensate was determined fluorimetrically. The concentration was measured again after freezing the reaction product at -70 degrees C for a period of 10, 20 and 40 days. We collected 2-5 ml condensate in 10 min. The EAV and condensate volumes were strongly correlated. There was no significant difference between the mean H2O2 concentration of the first and second test. We obtained a detect on limit for the H2O2 concentration of 0.02 micromoll(-1). The H2O2 concentration appeared to remain stable for a period up to 40 days of freezing. Compared to previous studies we developed a more efficient breath condensate collecting device and obtained a lower H2O2 detection limit. The measurement of exhaled H2O2 was reproducible. In addition, storage of the samples up to 40 days showed no changes in H2O2 concentration.

Influence of mammary artery as a bypass vessel on the results of seven biochemical assays after coronary artery bypass surgery.

We compared the changes in troponin T, creatine MB isoenzyme mass concentration (CK-MB mass), creatine kinase MB isoenzyme activity (CK-MB activity), creatine kinase (CK), alpha-hydroxybutyrate dehydrogenase (HBD), lactate dehydrogenase (LD) and aspartate aminotransferase (AST) concentrations after coronary artery grafting with saphenous vein grafts, without or in combination with uni- or bilateral internal mammary artery(ies) as bypass vessels in 73 patients. An increase in CK concentration after surgery was highest for the bilateral internal mammary artery bypass patient group and lowest for the group who received only saphenous vein grafts. We present 90th percentile values for the seven tests.

Differentiation between transmural perioperative myocardial infarction and subendocardial injury after coronary artery bypass grafting using biochemical tests, elaborated by cluster and discriminant analysis.

The aim of this study is to differentiate between transmural perioperative myocardial infarction (T-PMI) and subendocardial perioperative myocardial injury (S-PMI) as a complication of coronary artery bypass grafting (CABG). Seventy-three patients undergoing CABG were followed post operatively by measuring troponin T, CK-MB isoenzyme mass concentration (CK-MB mass), creatine kinase MB isoenzyme activity (CK-MB activity), creatine kinase (CK), alpha hydroxybutyrate dehydrogenase (HBD), and aspartate aminotransferase (AST) at five sampling times. Lacking a proper definition of the gold standard for the diagnosis of perioperative myocardial infarction, a statistical procedure was used. Supported by the cluster analysis method of Ward, patients were assigned to a patient group with a perioperative myocardial infarction (PMI) or a patient group without a PMI (non-PMI) as a confirmation of interpretation of the biochemical results. Using the results of electrocardiogram (ECG) and echocardiography, the PMI patient group was split into a T-PMI patient group and a S-PMI patient group. With discriminant analysis, two canonic discriminant functions were drawn up to differentiate between patients suffering from a T-PMI or S-PMI and non-PMI patients.

Effects of four different methods of sampling arterial blood and storage time on gas tensions and shunt calculation in the 100% oxygen test.

At the present time, plastic syringes are most commonly used for collecting arterial blood. The oxygen tension of the arterial blood (Pa,O2) in these syringes may fall. We studied the effect of the type of syringe, metabolism, and storage time on the arterial oxygen pressures measured and on the pulmonary shunt calculated. In 10 patients, 2-3 h after aortacoronary bypass surgery, a 100% oxygen test was performed. Four arterial blood gas samples were withdrawn from each patient in random order, two in glass syringes and two in plastic syringes. One glass and one plastic syringe were stored at room temperature (RT), and the others were stored in ice-water (IW). Each sample was analysed as soon as possible, and repeated 15, 30, 60 and 120 min after sampling. The Pa,O2 measurement in blood in the glass syringe in IW measured as soon as possible after sampling was considered the "gold standard". Pulmonary shunt calculations were performed using the results of the various blood gas analyses. Compared with the "gold standard", all of the other methods showed significant deterioration in the Pa,O2 measurement. The effect due to diffusion was 0.05 kPa x min(-1), and that due to metabolism 0.11 kPa x min(-1). The Pa,O2 in the glass syringes stored in IW remained stable with time. The pulmonary shunt was significantly overestimated when the "gold standard" blood gas results were not used (range 0.8-9.9%). Glass (not plastic) syringes should be used in the 100% oxygen test. The syringe should be cooled immediately, even when the sample is analysed as soon as possible.

Influence of changes in ionized calcium on cardiovascular reactivity during hemodialysis.

In order to prevent hypercalcemia due to the treatment of secondary hyperparathyroidism the use of low calcium dialysate is advocated. However, as calcium ions play a pivotal role in both myocardial and vascular smooth muscle contraction, lowering the dialysate calcium concentration might result in a further impairment of the cardiovascular response during dialysis. Therefore, arterial blood pressure, forearm vascular resistance (FVR) and venous tone (VT) (straing-gauge plethysmography) as well as cardiac dimensions and output (echocardiography) were measured in 10 hemodynamically stable dialysis patients (ejection fraction > 30%) during two standardized sessions of three-hour combined ultrafiltration-hemodialysis (UF + HD) at two different dialysate calcium concentrations: 1.25 and 1.75 mmol/l. High calcium UF + HD resulted in a significant increase in plasma ionized calcium (+0.19 +/- 0.11 mmol/l; p < 0.01) while ionized calcium remained unchanged during low calcium UF + HD (-0.02 +/- 0.07 mmol/l). As a result, systolic, diastolic and mean arterial blood pressure were respectively 14 +/- 10, 5 +/- 7 and 9 +/- 9 mmHg higher during high calcium UF + HD as compared to low calcium UF +/- HD (p < 0.05). There were no significant differences in FVR and VT between the two treatments. During both treatments FVR increased while VT decreased. In addition, there were no differences in calculated systemic vascular resistance. However, with comparable end-diastolic dimensions, stroke volume (-18 +/- 13 ml) and cardiac output (-1.3 +/- 1.5 l/min) decreased significantly (p < 0.05) only during low calcium UF + HD. We conclude that even in hemodynamically stable patients changes in plasma ionized calcium are an important determinant of the blood pressure response during dialysis therapy. Whereas peripheral vascular reactivity is unaffected by changes in ionized calcium, myocardial contractility is improved with higher dialysate calcium concentrations.

Evaluation of the Radiometer whole blood glucose measuring system, EML 105.

The performance of a new glucose electrode system from Radiometer was tested using two EML 105 analyzers (Radiometer Medical A/S, Copenhagen, Denmark). Results were very precise (both analyzers reported CV = 1.0% at a glucose concentration of 13.4 mmol/l). Comparison of methods was performed according to the NCCLS EP9-T guideline. Patients glucose results from both analyzers were lower compared with the results obtained with a Hitachi 911 (Boehringer Mannheim, Mannheim, Germany). There was no haematocrit dependency of relevance.

Radial immunodiffusion and immunoelectrophoresis compared for identifying autoantibodies to lactate dehydrogenase in human serum.

Variant electrophoretic patterns of lactate dehydrogenase isoenzymes were studied. By radial immunodiffusion and immunoelectrophoresis, immunoglobulin and light chain class of autoantibodies to lactate dehydrogenase were identified in nine sera: seven of these sera demonstrated IgG (5 lambda, 2 kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase, the other two demonstrated IgA (both kappa) autoantibodies to lactate dehydrogenase. We conclude that radial immunodiffusion and immunoelectrophoresis are equally effective for identifying auto-antibodies to lactate dehydrogenase in serum. Radial immunodiffusion, however, is easier to perform than immunoelectrophoresis.

Autoantibodies to lactate dehydrogenase in serum identified by use of immobilized protein G and immobilized jacalin, a jackfruit lectin.

In this method for identifying autoantibodies to lactate dehydrogenase (anti-LDs) in serum, we used immobilized Protein G to bind IgG-complexed LD and immobilized jacalin to bind IgA-complexed LD, leaving non-complexed LD in solution. The non-complexed LD and total LD were kinetically measured. We report results as LD bound to immobilized Protein G and LD bound to immobilized jacalin. Using sera demonstrating IgG and IgA anti-LDs by immunoelectrophoresis (IEP), respectively, we optimized the method for incubation time and concentration of binding agents. We demonstrated concomitant binding of LD and greater than or equal to 98% of IgG and of LD and greater than or equal to 92% of IgA. For LD bound to immunobilized Protein G the detection limit was 10 U/L, within- and between-run CVs ranged from 2.9% to 9.1%, and values for normal sera were less than or equal to 3% of total LD. Results for LD bound to immobilized jacalin were similar. We tested 10 sera displaying aberrant LD electrophoretograms: In seven, LD bound to immobilized Protein G was increased (range: 26-99% of total LD), indicating IgG-complexed LD. This was confirmed by IEP, demonstrating IgG1,2, or IgG3 anti-LDs in these sera. In the other three sera, LD bound to immobilized jacalin was increased (range: 38-72% of total LD), indicating IgA-complexed LD. This was confirmed by IEP, demonstrating IgA anti-LDs in these sera. Evidently this method is an alternative to IEP for identifying anti-LDs in serum.

Evaluation of the Hitachi 704 automatic analyzer.

We evaluated the analytical performance of the Hitachi 704 automatic analyzer. The spectrophotometer showed a linearity of response at 340 nm up to 2.8 A. Photometric imprecision measured bichromatically at 340 and 376 nm was 0.49% at 0.16 A, 0.14% at 0.46 A, and 0.17% at 0.76 A. Imprecision of the sample probe was 0.4% for 5, 10, and 20 microL, and the volume delivered deviated -2.4%, -4.4%, and -4.2% from these preset volumes, respectively. Imprecision of the reagent probe over the range 50 to 500 microL ranged from 0.14% to 0.29%; volume delivered deviated from +1.7% to +4.4%). At equilibrium, the temperature in the cuvets was 29.8 (SD 0.05) degree C as measured by cresol red spectrophotometry. No sample carryover was detected. Reagent carryover was detected when a bilirubin assay was preceded by a total protein assay and when lactate dehydrogenase was measured after alanine aminotransferase. Imprecision for nine tests at three concentrations ranged from 1.1% to 4.4%. Comparison of methods with the SMAC II as reference method showed good results. Precision was better than reported for the Hitachi 705 automatic analyzer.

Rate-nephelometric determination of fibronectin in plasma.

We describe a kinetic immunonephelometric method for the determination of fibronectin in human plasma, used with the Beckman ICS rate nephelometer. The method is rapid and cost-effective. Two commercially available controls stated by the manufacturer to contain 200 and 295 mg/L were found to contain 198 and 290 mg/L, respectively. Mean analytical recovery was 104%. Within-run precision (CV) for normal samples was 3.8%, between-day precision 5.1%. For samples containing subnormal concentrations of fibronectin, these figures were 3.8% and 6.7%, respectively. Results by the method described here agreed and correlated well with those by a commercially available turbidimetric assay. With appropriately diluted samples, the range of measurement is 40 to 1000 mg/L. Normal values for women and men were 286 (SD 84) and 340 (SD 55) mg/L, respectively, in good agreement with values published by others.