[Possibilities for pathogenetic clarification of hemolytic anemias using incubation hemolysis]. / Möglichkeiten zur pathogenetischen Klärung hämolytischer Anämien mit Hilfe der Inkubationshämolyse.

The haemolysis is the uniform answer of red blood cells to detrimental effects. The method of incubation haemolysis was controlled for its evidence concerning certain groups of causes. The determination of the free haemoglobin in the plasma before incubation may be regarded as measure of haemolysis in vivo. Compensation of the Hb-release under addition of glucose indicates to such causes which are accompanied by an increased expenditure of energy, e.g. transports of ions, protein kinases, spectrin phosphorylations. The incubation with ATP is effective above all in such cases of a haemolysis in which by enzyme defects the red blood cell itself is not able to the sufficient syntheses of connections rich in energy. Some instances for causes of an increased membrane permeability for Hb suggest that the Dacie-test allows only a superficial orientation to groups of diseases.

[Drug-induced hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency]. / Medikamentös bedingte Hämolysen bei Patienten mit Glukose-6-Phosphat-Dehydrogenase-Mangel.

9 carriers of G6PD-defect with a drug induced haemolysis are reported. From a pathogenic point of view various medicaments and their degrading and detoxicating products will cause an accumulation of radically effective compounds. With the damaged red blood cell being unable to eliminate these compounds because of its limited provision of NADPH, cell destruction cannot be avoided. Therefore, the exposition prophylaxis, which also embraces medicaments having a depressive effect on the bone-marrow, can be considered as a necessary and efficient measure.

[Formate incubation of red blood cells from glucose-6-phosphate-dehydrogenase-deficient patients]. / Formiatinkubation roter Blutzellen von Glukose-6-Phosphat-dehydrogenase-Defektträgern.

Attempts of incubating red blood cells of G6PD-defect carriers in 10 from 25 patients showed format in these blood samples is able under the elected conditions to suppress haemolysis, to reduce the numbers of Heinz's bodies and to enhance GSH-concentration. A more distinct efficiency could be observed in enzyme variants with instable temperatures and if kinetic properties had changed. Prevention of haemolysis is particularly suitable as a parameter because it will reflect all damages connected with an enhanced membrane permeability. The increase of GSH-concentration as an important equivalent of reduction in the metabolism of red blood cells may diminish the toxicity of damaging factors having an effect on oxidation. The number of Heinz's bodies is essential in those cases where oxidation products of haemoglobin are increasingly formed only under certain conditions of incubation. The findings allow a format therapy to be recommended for selected G6PD defects.

[Effect of formate on mouse erythrocytes in vivo and in vitro]. / Der Einfluss von Formiat auf Mäuseerythrozyten in vivo und in vitro.

The effect of formiat was tested in erythrocytes of mice damaged by oxidisation. Decrease of Heinz's bodies, stabilisation of reduced glutathion, and increase of enzyme activities by referring to an example of superoxidismutase could be identified as a positive effect. Possibilities of formiat reactions in the metabolism of red blood cells are discussed.

[Determination of superoxide dismutase (sod) in erythrocytes in glucose-6-phosphate dehydrogenase deficiency (G6PD-deficiency)]. / Bestimmung der Superoxiddismutase (SOD) in den Erythrozyten beim Glukose-6-Phosphatdehydrogenase-Mangel (G6PD-Mangel).

Determining SOD in red blood cells was made in 26 carriers of G6PD defect 9 haemicygotic mark carriers and 16 heterocytic mark carriers had a diminished SOD enzyme activity. This is probably due to an inactivation of SOD by diminished reductive equivalents in the metabolism of defect cells. In this way, more oxidatively effective radicals will appear, even OH. among them. The latter may render the enzyme ineffective by binding it to the imidazol group in the catabolic centre. Consequently, an accumulation of the substrate O2-. may lead to a further cell damage, thus causing an increased haemolysis.

[Oxidative damage of mice erythrocytes infected with Plasmodium berghei]. / Oxidative Schädigung an Erythrozyten der Maus unter einer Infektion mit Plasmodium berghei.

By using the model of infection with plasmodium berghei in white mice the attempt was made to explain the oxidative damages of red blood cells as a cause for haemolysis. In addition to a diminished new formation of erythrocytes there was an increased cell lysis under the impact of infection. Without any changes of the Met-Hb-percentage and of Heinz bodies an increase of GSH and GSSG could be measured. The conclusion was drawn that a damage of red blood cells caused by oxidation may occur by changing the relation of reduced tripeptides to oxidised ones.

[Separation of red blood cells from G6PD-deficient patients in dextran density gradients]. / Trennung roter Blutzellen von G6PD-Mangelträgern im Dextrandichtegradienten.

Red blood cells of 30 patients with G6PD deficiency were separated and characterized by means of isopyknic dextran density gradient centrifugation. The simultaneous determination of G6PD activity and the percentage of NADPH deficiency cells in relation to the maturation parameters of density, reticulocyte share, GOT and PK activity made it possible to recognize differences in the maturation of red blood cells with G6PD deficiency in normal persons as well as within a group of patients. In each case the more or less diminished enzyme activity of the cell suspension was accompanied by a marked enzyme deficiency of the youngest fraction. It is possible that NADPH defect cells are being eliminated at first. In many cases a direct correlation between the percentage of "empty cells" and the in vitro stability tests with and without NADP+ addition could be identified. Decreased maximal speed, changed kinetic behaviour, and instability of these variants are stressed as being the decisive parameters for the life expectation of red blood cells in patients with G6PD deficiency.

New stable mutant (Gd(-) variants: G6PD Tashkent and G6PD Nucus. Molecular basis of hereditary enzyme deficiency.

This paper represents studies on the molecular basis of G6PD deficiency in erythrocytes of 4 hemizygote patients. In all cases G6PD deficiency was due to an abnormal kinetic and (or) physico-chemical characteristics of the mutant enzymes. Two of 4 variants were characterized as G6PD EL Fayoum like (unstable, class 2). The other two are new variants: G6PD Tashkent (low Ki by NADPH, class 3) and G6PD Nukus (Km for G6P 127 microM, class 2). For the new mutant Gd(-) stable variants the amount of G6PD in total red cell population found immunologically was similar to the normal level. Stimulation of the pentose phosphate pathway (PPP) of G6PD Nucus erythrocytes insignificantly increased the rate of glucose consumption while in the case of G6PD Tashkent methylene blue raised the rate of PPP to half of the maximal rate of stimulated normal red cells.

[Energy metabolism of the rat renal cortex, as measured by incubation in various substrates]. / Energiestoffwechsel der Rattennierenrinde, gemessen in Schnitten bei Inkubation in verschiedenen Substraten.

Different substrate mixtures were investigated for their effect on energy metabolism using sections of the rat renal cortex. Simultaneous determination of adenine nucleotide concentrations and determination of the damage quotient of oxidative phosphorylation proved to be appropriate parameters for selecting substrate mixtures that have a favorable effect on energy metabolism. A mixture of albumin (1 mM) and octanoate (5.6 mM) with electrolytes proved to be adequate. The extent of oxygen consumption (60%) and 14CO2 formation (75%) argues in favor of the metabolization of this mixture; a damage quotient of 23% and general constancy of the concentration of high-energy compounds render prospective their testing in animal experiments. Addition of dicarbosylic acids increase the antimycin A resistant oxygen consumption without any energy conservation being demonstrable. Therefore, these substrates should not be used for conservation.

[Energy metabolism of erythrocytes in pyruvate kinase enzymopathies]. / Energiestoffwechsel roter Blutzellen bei Pyruvatkinase-Knzymopathien.

Until now pyruvate kinase enzymopathies have been described only for red blood cells. On the basis of these results special structural properties of the erythrocyte PK was assumed, which are not yet totally established. PK defects may cause a nonspherocytic hemolytic anemia. This enzymopathy is characterized by a polymorphism, which is expressed in more than 5 different pathological variants. Up to now 16 cases of PK deficiency have ben diagnozed in the GDR. The following parameters are used for the characterization of the PK: the PEP-dependance, the inhibition by ATP and alanine, the specificity to nucleotides, the stability to temperature and urea and the maturation dependence. Two pathological variants of the PK with a decreased PEP-affinity are described. Furthermore the differences in the energy metabolism of the red blood cells of these two patients under aerobic and anaerobic conditions are discussed.

[Glucose-6-phosphate dehydrogenase deficiency of erythrocytes in the GDR]. / Glukose-6-phosphat-Dehydrogenase-Mangel roter Blutzellen in der DDR.

34 persons with G-6-PD deficiency were diagnosed, and the pathological enzyme-variants of red blood cells were characterized according to the recommendations of WHO. We conclude from the differing residual G-6-PD-activities in red blood cells of the propositi and the differing reactivity of the enzyme in kinetic and physicochemical characterizations that a multiple variety of rare pathological G-6-PD variants exists in the GDR. Using the estimated enzymeparameters it was not possible in all cases to compare directly the newly demonstrated G-6-PD variants with cases already described in the literature. In addition, the differing combinations of parameters render a classification more difficult.

[Detection of female heterozygous glucose-6-phosphate dehydrogenase deficiency]. / Die Problematik der Erfassung heterozygoter Glukose-6-phosphat-Dehydrogenase-Mangelträger.

Diagnostics of heterozygotes are required for population studies, for the detection and consultation of persons with G-6-PD deficiency prone to hemolysis. The diagnostics of heterozygous females with the corresponding trait are problematic in families without hemizygous patients. 1. The determination of the activity is only applicable to the differentiation between heterozygotes and homozygotes if the activities are below the reference range. Heterozygous G-6-PD deficiency with normal activity cannot be identified by this method. 2. Existence of G-6-PD defects is demonstrated by mosaicism even in case of normactivity (Tönztest). 3. Incubation with and without NADP of stroma-free hemolysates involving heat labile enzyme mutants results in a marked decrease of activity within 20 min at 46 degrees C. 4. Electrophoresis on Cellogel demonstrates changes of charge in the mutated enzyme. 5. Family examination verifies suspicion of the heterozygous trait. A combination of parameters is recommended to obtain an improvement in the detection of persons with the heterozygous trait.

[O2-consumption and CO2 formation by kidney cortex sections incubated in an optimal substrate mixture]. / O2-Verbrauch und CO2-Bildung durch Nierenrindenschnitte bei Inkubation in einem optimierten Substratgemisch

Basing on the knowledge of renal metabolism a substrate mixture has been developed which appears to be promising for the preservation of this organ. Slices of rat kidney were incubated in different solutions, and the oxygen consumption and 14CO2 formation were determined at 6 degrees C after 4 and 9 hrs. In the substrate mixture the rat kidney cortex shows a continual O2 consumption, which could be increased by more than twice the amount compared with the values obtained after incubation without substrate. The proportion of fatty acid and amino acid mixture supplied amounts to 90% of the total CO2 formation by the renal cortex. Under the conditions selected, the incubation mixture allows one to spare endogenic substrates. The criteria of O2 consumption and CO2 formation alone are not sufficient to decide whether the functioning of the mitochondria and thus the energy supply are ensured under these conditions. Further studies are necessary to solve this problem.

[Heinz body formation and abnormal methemoglobin reduction. Expressions of damage to erythrocytes in chronic kidney failure]. / Heinzkörperbildung und gestörte Met Hb-reduktion Ausdruck der Schädigung von Erythrozyten bei chronischer Niereninsuffizienz

Erythrocytes of patients dialyzed due to chronic renal failure were investigated. An inhibition of the NADH-dependent methaemoglobin reductase, an insufficient methaemoglobin reduction by the NADPH-system, and Heinz-bodies as denaturation products, were found. The damage of erythrocytes was enhanced by incubation in both patient's plasma and dialysis medium. In erythrocytes of normal persons the damaging effect of incubation in plasma of chronic renal failure patients could also be demonstrated. The factors eliciting the oxidative injury are discussed. Factors that could further decrease the reduction capacity of red cells should be omitted.

Synchronization of rabbit bone-marrow cells in vivo.

The recovery of the rabbit bone-marrow from anaemia was investigated during an eight-day period of daily punctures of the tibiae after six days of phenylhydrazine treatment. A maximum of erythroid (range, 37.1 to 44.0%) and a minimum of leukoid cells (range, 8.4 to 13.4%) was observed on the fifth day of recovery. The rest, about 50% cells were reticulum cells. Signs of recovery were observed in peripheral blood as soon as on the first day after phenylhydrazine treatment. This led to the assumption that the tibiae became repopulated with active erythropoietic cells during anaemia, and that the reticulum cells might play a role as erythroid precursors in this process.