Biomedical subjects
Publications and source records attributed to G Rothe.
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In 58 unselected necropsies, zinc (Zn) in specimens of liver (mean +/- s = 280 +/- 178 micrograms/g d.w./4.28 +/- 2.72 mumol/g d.w.), kidney (170 +/- 57 micrograms/g d.w./2.60 +/- 0.87 mumol/g d.w.), spleen (69 +/- 13 micrograms/g d.w./1.05 +/- 0.20 mumol/g d.w.), and lung (64 +/- 16 micrograms/g d.w./0.98 +/- 0.24 mumol/g d.w.) were analysed by means of flameless atomic absorption spectrometry. In the examined range from 25 to 92 years of age, the Zn concentrations in all tissues measured did not show age or sex-dependency. There was a high correlation between the tissue concentration of Zn and Mg in lung, liver, and spleen, between Zn, Mg, and Pb in liver as well as between Zn and Pb and Zn and Cd in kidney. All hepatic cirrhosis cases had very low Zn conc. in liver tissue, the tumor cases generally increasing Zn conc. in liver tissue with progressive stage of regional invasion and distant metastasis.
The dipeptidyl rhodamine diamide substrates (Z-Phe-Arg)2-R110 and (Z-Arg-Arg)2-R110 are 820- and 360-fold more selective for cathepsin L than for cathepsin B allowing a sensitive determination of cathepsin L activity in the presence of high activity of cathepsin B. The results obtained with cell lysates suggest that the cysteine proteinase activity of vital macrophages detected by flow cytometry with these substrates is mainly due to cathepsin L.
The analysis of lysosomal proteases in cell lysates is complicated by pH-dependent and oxidative changes of their activity and complex formation with cytosolic inhibitors. Therefore, new flow cytometric methods were developed for the intracellular measurement of protease activities in viable cells. Intracellular cleavage of substrates such as Z-Arg-Arg-4-trifluoromethylcoumarinyl-7-amide to green fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) in viable neutrophils and monocytes was only detected following phagocytosis of Escherichia coli. A measurement of the cysteine or serine proteinase activities in resting human leukocytes was, however, not possible with AFC derivatives as the overlapping blue fluorescence of the substrates reduces sensitivity. Nonfluorescent bis-substituted peptide derivatives of rhodamine 110 (R110), which are intracellularly cleaved to green fluorescent mono-substituted R110 and free R110 proved to be more sensitive substrates. The activity of the lysosomal cysteine proteinases of human monocytes or rat macrophages, i.e. cathepsin B and L, was specifically measured with (Z-Arg-Arg)2-R110, (Z-Phe-Arg)2-R110, or (Z-Ala-Arg-Arg)2-R110. Fluorescence generation was completely inhibited by Z-Phe-Ala-diazomethane or E-64. The serine proteinases of human neutrophils were analyzed with Elastase-substrates such as (Z-Ala-Ala)2-R110 or (Z-Ala-Ala-Ala)2-R110. Specificity was shown by inhibition with diisopropylfluorophosphate.
Strong binding of the acute phase protein serum amyloid-A (SAA) to human neutrophils was found using flow cytometry. This binding was shown to be functionally relevant with respect to the oxidative burst reaction assayed on N-formyl peptide-stimulated neutrophils by the intracellular oxidation of non-fluorescent dihydrorhodamine to fluorescent rhodamine 123. The results show reduction of the oxidative burst response by isolated SAA (and recombinant SAA2). Inhibition was also demonstrated by acute phase as compared to normal human serum. This inhibitory effect was abolished by the purified monoclonal anti-amyloid A antibody mc29, strongly suggesting that SAA counteracts neutrophil responses to cytokines or bacterial products. This newly recognized function of SAA may help to prevent oxidative tissue destruction.
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A new flow cytometric method for the investigation of the respiratory burst of macrophages/microglia isolated from neonatal rat brain has been established. Respiratory burst activity was measured quantitatively in single viable cells by the intracellular oxidation of non-fluorescent dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123. Cultured microglia exhibited high spontaneous respiratory burst activity already before stimulation. After maximal stimulation with phorbol myristate acetate, DHR oxidation rose by 40-95%. The respiratory burst activity in resident or inflammatory, i.e. thioglycolate elicited, peritoneal macrophages was significantly lower than in cultured brain macrophages suggesting a high potential of microglia for oxidative tissue destruction.
OBJECTIVE: In a randomized 5-yr multi-intervention trial, we tested the efficacy of intensified health education (IHE) in improving metabolic control and reducing the level of coronary risk factors and incidence of ischemic heart disease (IHD). RESEARCH DESIGN AND METHODS: Within the intervention group, the benefit of clofibric acid was evaluated in a double-blind study. One thousand one hundred thirty-nine newly diagnosed middle-aged (30- to 55-yr-old) patients with non-insulin-dependent diabetes mellitus (NIDDM) entered the study. They were classified as diet controlled after a 6-wk screening phase with conventional dietary treatment. During the follow-up, the control group (n = 378) was cared for at different diabetes outpatient clinics with a standardized surveillance. The intervention group (n = 761) had a structured IHE that included dietary advice, antismoking and antialcohol education, and ways to enhance physical activity. RESULTS: Randomly, 379 of the IHE patients received 1.6 g clofibric acid/day, and the others received placebo. IHE resulted in improved glucose control (adjusted fasting blood glucose) levels after 5 yr (control subjects 9.27 mM, IHE group 8.71 mM, and IHE plus clofibric acid group 8.60 mM, P less than 0.01). The better glycemic control was achieved with fewer antidiabetic drugs. After 5 yr, antidiabetic drugs were prescribed to 47% of the control subjects, 28% of the IHE group, and 34% of the IHE plus clofibric acid group (cutoff limit for drug application was postprandial blood glucose of greater than or equal to 13.87 mM). The ratio of polyunsaturated to saturated fatty acids (0.26 vs. 0.40, P less than 0.01) and physical activity (174 vs. 327 scores, P less than 0.01) were increased, and blood pressure, tobacco, and alcohol consumption were significantly reduced by IHE. However, IHE had no effect on calorie intake, percentage of fat in the diet (45%), and body weight. The most important finding was the significant increase of blood cholesterol in all three groups (+0.47, +0.36, and +0.34 mM, respectively). Clofibric acid only prevented the increase of triglyceride levels (+0.56, +0.24, and +0.05 mM, respectively). The incidence rate per 1000 for myocardial infarction was 30.3 for control subjects, 53.6 for the IHE group, and 55.6 for the IHE plus clofibric acid group. The corresponding rates for IHD incidence were 90.9, 97.8, and 98.8, respectively. Men suffered more frequently from myocardial infarction, whereas women developed ECG criteria for IHD more frequently. Among the 35 cases of death, besides cardiovascular diseases, liver cirrhosis and neoplasia were the predominant causes. The death rate per 1000 in control subjects was 46.2, 30.6 in the IHE group, and 27 among patients with IHE plus clofibric acid. CONCLUSIONS: IHE was of substantial benefit for the control of glycemia, significantly diminished the need for antidiabetic drugs, and reduced a cluster of risk factors but had no effect on the control of blood lipids. This could be one major reason for the failure of IHE, effective lowering of blood pressure, and clofibric acid to prevent cardiovascular complications. Clofibric acid was only effective in reducing triglycerides.
Acute paraplegia caused by an epidural hematoma developed in a patient following the removal of an epidural catheter. This catheter had been used for 3 days for postoperative pain relief with no apparent complications. Heparin (10,000 units/day) had been infused for thrombosis prophylaxis and was associated with a normal activated partial thromboplastin time (aPTT) for the first two postoperative days. However, test results from blood drawn prior to catheter removal revealed, in retrospect, an unexpected prolongation of the aPTT (75 s) and PT (56%, Quick's method). An epidural hematoma extending from T12 to L4 was evacuated during emergency laminectomy and neurologic deficits resolved completely over the next days. Thus, the removal of an epidural catheter has the potential for inducing formation of an epidural hematoma. Accordingly, it may be safest to leave epidural catheters in place if test results demonstrate a bleeding diathesis or if a potential for bleeding is suspected on clinical grounds.
In 101 unselected autopsies, cadmium (Cd) in lung tissue was analyzed by means of flameless atomic absorption spectrometry (AAS). The subjects originated from Bochum (BO), Dortmund (DO), and neighbouring cities in the Ruhr District (BO/DO) as well as from Muenster (MS) and vicinity. The sample included eight persons who had died from bronchial carcinoma. The Cd concentration in lung tissue (CdL) did not show a significant age-dependency. There was only a slight increase of CdL in the age around 50. For males, the median CdL value was found to be 1.32 micrograms/g dry wt in the age group from 20-45, 1.48 micrograms/g dry wt from 45-65, and 0.64 micrograms/g dry wt greater than 65 (the corresponding means and standard deviations were 1.48 +/- 1.22, 1.73 +/- 1.42, and 1.18 +/- 1.27 micrograms/g dry wt). CdL in men was twice that in women. There were no differences between the two regions (BO/DO:MS) examined. CdL of the bronchial carcinoma cases were mostly clearly above the expected level, often similar to the Cr and Ni concentration of the same specimens. Besides the amount of inhaled metal aerosols, insufficient lung clearance may play a leading role for their retention in lung parenchyma. However, with regard to the determination of causality between uptake and disease the data are difficult to interpret, in our study due to a lack of enough consistent and reliable data on occupation, environment, and smoking habits, and in general due to a lot of variables which, until now, cannot be sufficiently quantified.
Chromium (Cr) and nickel (Ni) concentrations were measured in lung tissue from 110 random necropsies by means of atomic absorption spectrometry. The subjects originated from the Ruhr district (Bochum (71 cases) and Dortmund (16 cases) areas), which has been defined as a particular pollution area with locally high Cr and Ni emissions, and from Münster and vicinity (23 cases). The Cr and Ni concentrations in lung tissue of the subjects from the Ruhr district (3.09 (SD 2.99) micrograms Cr/g, 0.65 (SD 0.94) micrograms Ni/g dry weight of lung) were 4.8 and 2.8 times higher than those from Münster (0.66 (SD 0.49) micrograms Cr/g, 0.17 (SD 0.11) micrograms Ni/g dry weight of lung). Concentrations of Cr and Ni in men were twice those in women. All data showed an age dependent increase of Cr and Ni in the lung (about 2.4% a year for Cr and 3% a year for Ni) and Cr and Ni values showed a high correlation (r greater than or equal to 0.9). Thus it was possible to calculate age, sex, and region adjusted expected values of pulmonary Cr and Ni concentrations, and to identify the difference between expected and observed values. This might be helpful to interpret measurements in individual cases and in epidemiological studies. With this procedure the six cases of bronchial carcinoma in the series were shown to have pulmonary Cr and Ni concentrations that were mostly well above the predicted values, and it was possible to give a rough estimate of the degree of deviation.
Flow cytometric parameters of neutrophil function, such as phagocytosis and degradation of Escherichia coli, intracellular pH value, esterase activity, and cell volume, were evaluated as risk indicators for sepsis- and trauma-related pulmonary and cardiovascular organ failure in intensive care patients. Serial blood samples (n = 201) were obtained from 47 prospectively identified patients. Each patient's condition was classified daily within four categories: post-traumatic (n = 22) or septic (n = 28) organ failure, transition state (n = 119), and stable organ function after recovery (n = 27). Thirty-two parameters of neutrophil function were automatically calculated for each blood sample from several flow cytometric list mode measurements of cell samples vitally stained with acridine orange for intact and denatured DNA or with 1,4-diacetoxy-2,3-dicyanobenzene for intracellular pH and esterase activity. The DNA of dead cells was simultaneously counterstained with propidium iodide. The cell biochemical parameter pattern was significantly different among samples of patients from the four clinical categories (p less than 0.05). Hyperergic phagocytosis was observed after trauma, in contrast to hypoergic phagocytosis, increased neutrophil cell volume, and elevated intracellular pH during sepsis. The clinical categories were correctly identified in 82% of the samples by automated classification with the DIAGNOS1/SPSS program system from the flow cytometrically determined cell functions. The course of the disease was correctly predicted 3 days in advance to the clinical manifestation of pulmonary or cardiovascular organ failure in 92% of the samples. The multifunctional analysis of neutrophils by flow cytometry seems of interest for early medical intervention in preseptic and preshock patients.
Hydroethidine (HE) and 2',7'-dichlorofluorescin (DCFH) were used for the flow cytometric measurement of reactive oxygen metabolites in leukocytes. Hydroethidine and DCFH were both rapidly oxidized in a cell-free cuvette assay to ethidium bromide (EB) and 2',7'-dichlorofluorescein (DCF) by H2O2 and peroxidase, but not by H2O2 alone, while only HE was oxidized by KO2, a source of O2-. Quiescent lymphocytes, monocytes, and neutrophils spontaneously oxidized HE to EB, while DCFH was only oxidized to a low degree. Neutrophils increased 6.9-fold in EB red fluorescence and 12.5-fold in DCF green fluorescence during the respiratory burst induced by phorbol 12-myristate 13-acetate or 6.1-fold and 4.7-fold, respectively, during the respiratory burst induced by Escherichia coli bacteria. The HE or DCFH oxidation during the respiratory burst, unlike the spontaneous HE oxidation, was not inhibitable by 10 mM NaNe indicating a non-mitochondrial source of cellular oxidants during the respiratory burst such as NADPH oxidase, which produces O2-. The oxidation of DCFH, but not of HE, was decreased in stimulated neutrophils, which were simultaneously loaded with HE and DCFH. Intracellular DCFH oxidation induced by incubation of resting neutrophils with extracellular H2O2 was not influenced by the presence of HE. This indicates that HE is oxidized at an earlier step in the reactive oxygen metabolism of neutrophils than DCFH, i.e., by early oxygen metabolites like O2-, while DCFH is oxidized in part by H2O2 and phagosomal peroxidases. The differential oxidation of HE and DCFH during simultaneous cellular staining permits the analysis of up to three functionally different neutrophil populations in septic patients. This is of interest for the determination of disease-related alterations of oxygen metabolism in quiescent and stimulated leukocytes.
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Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations. In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis. In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4-diacetoxy-2,3-dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria. The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays. The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement. The calculated mean values were transferred into the self-learning database of the DIAGNOS1-program system. The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states. The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.
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