Medizin - Open Access LMU - Teil 13/22 Archiv

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. An increasing body of evidence has implicated oxidative stress as being contributory to homocysteines deleterious effects on the vasculature. Elevated levels of homocysteine may lead to increased generation of superoxide by a biochemical mechanism involving nitric oxide synthase, and, to a lesser extent, by an increase in the chemical oxidation of homocysteine and other aminothiols in the circulation. The resultant increase in superoxide levels is further amplified by homocysteinedependent alterations in the function of cellular antioxidant enzymes such as cellular glutathione peroxidase or extracellular superoxide dismutase. One direct clinical consequence of elevated vascular superoxide levels is the inactivation of the vasorelaxant messenger nitric oxide, leading to endothelial dysfunction. Scavenging of superoxide anion by either superoxide dismutase or 4,5-dihydroxybenzene 1,3-disulfonate (Tiron) reverses endothelial dysfunction in hyperhomocysteinemic animal models and in isolated aortic rings incubated with homocysteine. Similarly, homocysteineinduced endothelial dysfunction is also reversed by increasing the concentration of the endogenous antioxidant glutathione or overexpressing cellular glutathione peroxidase in animal models of mild hyperhomocysteinemia. Taken together, these findings strongly suggest that the adverse vascular effects of homocysteine are at least partly mediated by oxidative inactivation of nitric oxide.

In severely ill patients low concentrations of the corticosteroid binding globulin are typically found; the aim of this study was to quantify directly free bioactive cortisol concentrations in the sera of postoperative cardiosurgical patients. Serum samples of 12 consecutive patients undergoing aortocoronary bypass surgery taken preoperatively and on the postoperative days 1 to 4 were analyzed. Total serum cortisol was quantified using liquid chromatographytandem mass spectrometry with an online sample extraction system and trideuterated cortisol as the internal standard, and free serum cortisol was measured after overnight equilibrium dialysis. Whereas on the first postoperative day, the median total serum cortisol concentration was approximately twofold increased compared to preoperative samples (preoperatively, 245 nmol/l (interquartile range (IQR) 203293 nmol/l); first postoperative day, 512 nmol/l (IQR 410611 nmol/l)), median dialyzable free cortisol concentration was almost sevenfold increased (preoperatively, 14.2 nmol/l (IQR 10.920.7 nmol/l); first postoperative day, 98.3 nmol/l (IQR 81.3134 nmol/l)). On the fourth postoperative day, median free cortisol was still significantly increased compared to baseline sampling (p < 0.05), whereas median total cortisol was not. A median of 5.7% (IQR 5.47.0%) of total cortisol was found as free cortisol on the preoperative day, 21.2% (IQR 18.9 23.5%) on the first postoperative day and 10.5% (IQR 9.814.0%) on the fourth postoperative day. It is concluded that during the postoperative period the freeto bound ratio of cortisol is highly variable and that during the acute phase response direct quantification of free bioactive cortisol concentrations seems to be biologically more appropriate than the measurement of total cortisol concentrations.

This review provides a concise survey of liquid chromatography tandem mass spectrometry (LCTMS) as an emerging technology in clinical chemistry. The combination of two mass spectrometers with an interposed collision cell characterizes LCTMS as an analytical technology on its own and not just as a more specific detector for HPLC compared with conventional techniques. In LCTMS, liquid chromatography is rather used for sample preparation but not for complete resolution of compounds of interest. The instrument technology of LCTMS is complex and comparatively expensive; however, in routine use, methods are far more rugged compared to conventional chromatographic techniques and enable highthroughput analyses with very limited manual handling steps. Moreover, compared to both gas chromatographymass spectrometry (GCMS) and conventional HPLC techniques, LCTMS is substantially more versatile with respect to the spectrum of analyzable compounds. For these reasons it is likely that LCTMS will gain far more widespread use in the clinical laboratory than HPLC and GCMS ever did. In this article, the key features of LCTMS are described, method development is explained, typical fields of application are discussed, and personal experiences are related.

Between 1997 and 2000 we investigated in a prospective study the voided urine samples of all consecutive patients undergoing cystoscopy independent from their clinical background (n=705) with the BTA-TRAK(TM) assay (Bard Diagnostics, Redmont, USA) detecting complement factor H-related protein (CFHrP) and the NMP22 assay (Matritech, Newton, USA) measuring nuclear matrix protein, which is supposed to be specific for bladder cancer. The individuals were divided into three groups concerning the clinical background: 233 patients had urological diseases, 268 patients had urinary bladder cancer and 150 patients had other urological malignancies. Based on the clinical findings we compared our results with well established diagnostic methods for urinary bladder cancer such as cytology and the detection of hematuria. In addition, we investigated urine samples from 30 healthy individuals and 24 patients with urinary tract infection without performing cystoscopy. Following the recommendations of the European Group on Tumor Markers we used 95% specificity for benign urological diseases and urinary tract infections, which resulted in a sensitivity of 17% for active bladder cancer for the BTA-TRAK(TM) assay and 31% for NMP22. We compared these results with the detection of hematuria (specificity: 72%) and cytology, which had a sensitivity of 64% and 89%, respectively. Subsequently, we calculated sensitivity and specificity for the detection of relapse of the disease. Again using 95% specificity, in this case for patients with no evidence of disease (NED), in patients with recurrent disease the BTA-TRAK(TM) assay showed % sensitivity as compared to 12% for the NMP22 assay. Due to an insufficient specificity and sensitivity, both tests can neither be clinically useful in screening of high risk patients, nor in primary diagnosis of bladder cancer. They cannot replace neither cystoscopy nor cytology. In the follow-up care more investigations may be necessary to prove the benefit of existing diagnostic strategies for the discrimination between active and inactive bladder cancer.

This international multicenter study was designed to evaluate the technical performance of the new double-monoclonal, single-step Elecsys neuron-specific enolase (NSE) enzyme immunoassay (EIA) and to assess its utility as a sensitive and specific test for the diagnosis of small-cell lung cancer (SCLC). Intra and interassay coefficients of variation, determined in five control or serum specimens in six laboratories, ranged from 0.7 to 5.3 (interlaboratory median: 1.3%) and from 1.3 to 8.5 (interlaboratory median: 3.4%), respectively. Laboratory-to-laboratory comparability was excellent with respect to recovery and interassay coefficients of variation. The test was linear between 0.0 and 320 ng/ml (highest measured concentration). There was a significant correlation between NSE concentrations measured using the Elecsys NSE and the established Cobas Core NSE EIA II in all subjects (n=723) and in patients with lung cancer (n=333). However, NSE concentrations were systematically lower (approximately 9%) with the Elecsys NSE than with the comparison test. Based on a specificity of 95% in comparison with the group suffering from benign lung diseases (n=183), the cutoff value for the discrimination between malignant and benign conditions was set at 21.6 ng/ml. NSE was raised in 73.4% of SCLC patients (n=188) and was significantly higher (p

Nonequimolar-response assays for prostate-specific antigen (PSA) are criticized for overestimating total PSA in some men without prostate cancer (PCA), and underestimating total PSA in some men with PCA. We recently studied three nonequimolar-response PSA assays that had undergone modifications. While two of the studied assays achieved equimolar-response characteristics with improved areas under receiver operating characteristic (ROC) curves (AUC), the modification of the Chiron ACS PSA assay (ACS PSA2, Chiron) failed to achieve this. Recently, the ACS assay underwent another modification (ACS PSA, Bayer), which we investigated. Sera from 305 men (155 without and 150 with PCA, PSA greater than or equal to2 and less than or equal to30 mug/l, TandemE) were measured using both modifications of the ACS assay and equimolar-response reference methods (TandemR free and Tandem E, Hybritech). Molar response relative to the reference method and clinical performance (comparison of AUCs) between the previous and new ACS assay modifications were studied. The new modification of the ACS assay (ACS PSA, Bayer) achieved equimolar-response characteristics but reported lower values (average 10%) than the Tandem E assay. Compared to the previous modification (ACS PSA2, Chiron), a 3% improvement in AUC (p=0.01) was found. Using results of the redesigned equimolar-response assay (ACS PSA, Bayer), we calculated that 6 of 155 men without PCA in this sample set could be spared unnecessary biopsy compared with the previous nonequimolar-response assay (ACS PSA2, Chiron) without missing additional PCA (90% sensitivity). These data provide additional evidence for clinical advantages of equimolar-response over nonequimolar-response PSA assay formats.

We describe the modification of an existing method of ligament strain measurement at the knee joint in detail. At ten fresh joint specimens we used that technique where strain gauges are attached to the ligamentous insertions and origins. We both improved the preparation of the attachment site and the application of the strain gauges. In a special apparatus the specimens were moved from 0degrees extension to 100degrees flexion while simulating muscle strength and axial force. Testing was performed at the posterior cruciate ligament with both intact and transsected anterior cruciate ligament. In contrast to other existing techniques it does not affect the motion of the joint or the integrity and the function of the ligaments. Unlike the original description of that method we could register a loading behaviour of the posterior cruciate ligament that is similar to those reported in the literature.

In this paper a method for the simultaneous quantification of the anti-fungal drug itraconazole and its co-active metabolite hydroxyitraconazole in plasma employing liquid chromatography tandem-mass spectrometry and automated solid-phase extraction is described. The method proved rugged, enables short turn-around times and is highly specific. Since there is growing evidence for the importance of therapeutic drug monitoring of itraconazole in the prophylaxis and treatment of invasive fungal infections, the method described here is of interest for a large number of tertiary care hospital laboratories.

Background/Aim: N-6 fatty acids are considered to promote diseases prevalent in industrialized countries and characterized by an increased eicosanoid biosynthesis from arachidonic acid (AA). We investigated the impact of the linoleic acid (LA) intake on AA levels in humans. Methods: Six healthy female volunteers (age range 2334 years) were given liquid formula diets (LFD) devoid of AA for 6 weeks, providing a constant intake of zero energy% (LFD 0: protein 15%, carbohydrates 85%) or 20 energy% (LFD 20: protein 15%, carbohydrates 55%, fat 30%) LA, for 3 weeks each. Fatty acids of plasma cholesteryl esters and platelet lipids were determined each week, and the prostaglandin biosynthesis was measured in 24-hour urine samples. Results: LFD 0 increased (+31% of initial value) and LFD 20 lowered (-30% of initial value) the percentage of AA in plasma cholesteryl esters and platelet lipids. Moreover, absence of dietary AA lowered the percentages of AA in plasma (-31% week 0 vs. week 6) and platelet (-11%) lipids, indicating a low transformation of LA. LFD 0 reduced urinary metabolite levels of prostaglandins D, E, and F in 24-hour urine samples (-48%, p < 0.001) within 24 h, but did not significantly affect platelet aggregation (-10%) and thromboxane formation (-25%). LFD 20 significantly lowered platelet aggregation (-25%) and thromboxane formation (-43%). The prostaglandin metabolite levels increased during the first 10 days, declined thereafter, and were lower than the preexperimental values at the end of the 3-week period. Conclusions: The results show that dietary LA does not increase the AA levels in plasma or platelet lipids and does not persistently contribute to prostaglandin biosynthesis which is increased by AA intake with Western diets.

Cardiomyocytes cease to divide shortly after birth and an irreversible cell cycle arrest is evident accompanied by the downregulation of cyclin-dependent kinase activities. To get a better understanding of the cardiac cell cycle and its regulation, the effect of functional recovery of the mitosis-promoting factor (MPF) consisting of cyclin B1 and the cyclin-dependent kinase Cdc2 was assessed in primary cultures of postmitotic ventricular adult rat cardiomyocytes ( ARC). Gene transfer into ARC was achieved using the adenovirus-enhanced transferrinfection system that was characterized by the absence of cytotoxic events. Simultaneous ectopic expression of wild-type versions of cyclin B1 and Cdc2 was sufficient to induce MPF activity. Reestablished MPF resulted in a mitotic phenotype, marked by an abnormal condensation of the nuclei, histone H3 phosphorylation and variable degree of decay of the contractile apparatus. Although a complete cell division was not observed, the results provided conclusive evidence that cell cycle-related events in postmitotic cardiomyocytes could be triggered by genetic intervention downstream of the G1/S checkpoint. This will be of importance to design novel strategies to overcome the proliferation arrest in adult cardiomyocytes.

Only very limited data are available concerning patient adherence to antithrombotic medication intended to prevent a recurrent stroke. Reduced adherence and compliance could significantly influence the effects of any stroke prevention strategies. This study from a large stroke data bank provides representative data concerning the rate of stroke victims adhering to their recommended preventive medication. During a 2-year period beginning January 1, 1998, all patients with acute stroke or TIA in 23 neurological departments with an acute stroke unit were included in the German Stroke Data Bank. Data were collected prospectively, reviewed, validated and processed in a central data management unit. Only 12 centers with a follow-up rate of 80% or higher were included in this evaluation. 3,420 patients were followed up after 3 months, and 2,640 patients were followed up one year after their stroke. After one year, 96% of all patients reported still adhere to at least one medical stroke prevention strategy. Of the patients receiving aspirin at discharge, 92.6% reported to use that medication after 3 months and 84% after one year, while 81.6 and 61.6% were the respective figures for clopidogrel, and 85.2 and 77.4% for oral anticoagulation. Most patients who changed medication switched from aspirin to clopidogrel. Under the conditions of this observational study, adherence to stroke prevention strategies is excellent. The highest adherence rate is noticed for aspirin and oral anticoagulation. After one year, very few patients stopped taking stroke preventive medication. Copyright (C) 2003 S. Karger AG, Basel.

Combined cerebellar and spinal ischemic stroke is a rare, critical condition. We report a patient with combined cerebellar and bilateral posterolateral cervical spinal cord infarction due to bilateral stenosis of the vertebral arteries. MRI is the method of choice for imaging this condition; diffusion-weighted imaging of the spinal cord gives reliable results.

The mouse has evolved to be the primary mammalian genetic model organism. Important applications include the modeling of human cancer and cloning experiments. In both settings, a detailed analysis of the mouse genome is essential. Multicolor karyotyping technologies have emerged to be invaluable tools for the identification of mouse chromosomes and for the deciphering of complex rearrangements. With the increasing use of these multicolor technologies resolution limits are critical. However, the traditionally used probe sets, which employ 5 different fluorochromes, have significant limitations. Here, we introduce an improved labeling strategy. Using 7 fluorochromes we increased the sensitivity for the detection of small interchromosomal rearrangements (700 kb or less) to virtually 100%. Our approach should be important to unravel small interchromosomal rearrangements in mouse models for DNA repair defects and chromosomal instability. Copyright (C) 2003 S. Karger AG, Basel.

A variety of studies have demonstrated that motor disorders, parkinsonism and extrapyramidal motor symptoms (EPMS) are common in patients with Alzheimer's disease (AD). Several studies have reported an association of EPMS with severity, progression and poor prognosis of AD. The majority of these studies used clinical assessments for the rating of EPMS. In this study, kinematic handwriting analysis was used to quantify differences in fine hand motor function in patients with probable AD and mild cognitive impairment (MCl, as an assumed initial stage of AD) compared to depressed patients and healthy controls. Both patients with MCl and patients with probable AD exhibited loss of fine motor performance. Movements of AD patients were significantly less regular than those of healthy controls. Copyright (C) 2003 S. Karger AG, Basel.