Activation of K+/Cl- cotransport in human erythrocytes exposed to oxidative agents.

Activation of K+/Cl- cotransport was studied after exposure of normal human erythrocytes to the oxidative action of acetylphenylhydrazine (APH), menadione sodium bisulfite (MSB), hydrogen peroxide (H2O2) or phenazine metasulfate (PMS). In order to better define the relative contributions of K+/Cl- cotransport on ouabain and bumetanide-resistant (OBR) K+ efflux induced by oxidation, we used (dihydroindenyl)oxyalkanoic acid (DIOA) and carbocyanine as specific inhibitors, respectively, of cotransport system and Ca(2+)-activated K+ channel. APH, MSB and - to much less extent - H2O2 promoted a K+ efflux pathway with features corresponding to those of K+/Cl- cotransport. This pathway showed: (i) kinetics of efflux compatible with a specific cation transport system; (ii) requirement for chloride anion; (iii) resistance to ouabain, bumetanide and carbocyanine inhibition; (iv) stimulation by hypotonic challenge; (v) susceptibility to inhibition by DIOA. Dithiothreitol (DTT) or 2-mercaptoethanol (2-ME) decreased K+/Cl- cotransport activation, suggesting that oxidative mechanisms affected crucial SH groups of the transporter. These data suggest that oxidation represents a factor capable of modulating activation of K+/Cl- cotransport. Its possible contribution in situations with high oxidative risk, such as sickle-cell anaemia or beta thalassemia, is discussed.

The canine erythroid cell system: a new model for study of regulatory mechanism in hemoglobin synthesis.

The total methodology for a new mammalian erythroid cell system which permits direct investigation of the terminal stages of hemoglobin synthesis and assembly is described. The canine system allows quantitative separation of native heme containing alpha and beta chains which recombine to for tetrameric hemoglobin with normal functional properties (n = 2.17). These chains can be utilized for investigation in a cell-free system in which the rates of synthesis of alpha and beta chains are equal (alpha/beta = 0.96 +/- 0.05). Methodology for the quantitative separation of globin allows study of the effects of balanced and unbalanced globin chain synthesis in both the intact cells and the cell-free system.

Standardization of a simple method for the estimation of haemoglobin A2.

An improved method for the estimation of haemoglobin A2, at the same time precise, simple and cheap, is proposed. Haemoglobin A is separated from haemoglobin A2 by electrophoresis on Cellogel in discontinuous buffer at alkaline pH. The strips of cellulose acetate containing the haemoglobin fractions are completely dissolved in 80% acetic acid. The percentage of haemoglobin A2 present in each sample is calculated from the values for the spectrophotometric absorbance at 396 nm. The average percentage of haemoglobin A2 (+/- standard deviation) determined by this method was 2.31 +/- 0.37 in 51 normal subjects, and 4.64 +/- 0.53 in 29 subjects with heterozygous beta-thalassaemia.

Micro-chromatographic measurement of hemoglobin A1 in uremia.

In an investigation of the reliability of the measurement of HbA1 by microcolumn chromatography for monitoring glucose metabolism in chronic renal failure, measurements were made in 96 uremic patients. Thirty-one patients were undialyzed, 42 patients including 10 with primary diabetes mellitus were on hemodialysis, and 23 patients were on continuous ambulatory peritoneal dialysis (6 with primary diabetes mellitus). Significantly raised HbA1 values were observed in all groups, whether their glucose tolerance was normal or decreased. Azotemia was not statistically correlated either with HbA1 values, or with glucose tolerance. Dialyzed primary diabetic patients showed HbA1 levels which were significantly higher than those in non-diabetics, but some overlap was evident. The results suggest that the increased values of HbA1 in uremic patients depend on the plasma concentration of either glucose which leads to the formation of glycosylated Hb or of urea which leads to the formation of carbamylated Hb. These are indistinguishable by microcolumn chromatography. Therefore this method cannot be recommended for evaluation of glucose metabolism in uremic patients.

The diagnostic value of serum copper levels and other hematochemical parameters in malignancies.

An increase in the serum copper (Cu++) level has been described as a sensitive index of disease activity in several hematologic and nonhematologic malignancies. In order to explore the diagnostic value of Cu++ compared to other hematochemical parameters frequently abnormal in malignancies, Cu++, serum alpha2 globulin (alpha2), plasmatic fibrinogen (Fibr), the erythrocyte sedimentation rate (ESR), and serum iron (Fe++) have been detected and evaluated in 267 patients affected with the following diseases: Hodgkin's lymphoma (HL), non-Hodgkin's Lymphomas (NHL), Acute Leukemias (AL), Chronic Myeloid Leukemia (CML), Chronic Lymphocytic Leukemia (CLL), Myeloma (MM), and Breast Cancer (BC). The best correlation between Cu++ increase and disease activity has been found in HL, NHL, AL, and BC. In these diseases, when the considered parameters were compared, Cu++ and ESR showed a similar pattern, i.e., a high frequency of abnormalities in active disease. It is concluded that Cu++ represents a good complement to some other aspecific parameters in evaluating the activity and diffusion of neoplasias and the therapeutic results, particularly in HL, NHL, AL and BC.

Evaluation of the scintigraphic technique for detecting bone metastases in 100 breast cancer patients.

In an attempt to evaluate the utility of bone scintigraphy for the diagnosis and follow-up of skeletal metastases, radioisotopic scan (total body) by 99mTc has been performed on 100 patients suffering from breast cancer in various stages. The radiographic control followed the scan on the basis of clinical or scintigraphic suspicion of bone lesions. Twenty-one patients showed metastases by scan, and in all but 2 the radiography was concordantly positive; in 22 subjects scintigraphy and radiology demonstrated non-malignant lesions. Both scan and radiology were used to explore 169 metastatic localizations, with concordant results in 45% of the sites; however, 7% of the sites with discordant results became positive for metastasis by radiology some months after the scan positivity. The false-negative results were more frequent in the radiographic study (27%) than with the scintigraphic scan (21%). The latter technique was unable to detect osteoblastic lesions in particular. The scintigraphic test also gave reliable results in the follow-up procedure, frequently anticipating the radiological modifications of metastatic lesions. In conclusion, bone scan often appears to be more accurate and preocious than the roentgengraphic test in the detection and the follow-up of skeletal metastatic lesions; it suggests the utility of the bone scan as a preliminary test, which allows a more rational use of the radiology.

[Criteria for choice of therapeutic procedures in lymphomas]. / Criteri di scelta delle procedure terapeutiche nei linfomi

Surgery, high-energy radiotherapy and cyclic polychemotherapy are best indicated for the treatment of Hodgkin's disease and other forms of lymphoma. Overall evaluation will determine the most suitable management in each case. Clinical andpathological stage, the histological picture, the degree of vascular invasion and the immunological reactivity of the patient are the most important factors in such an evaluation. Close cooperation between the haematologist, histologist, radiologist, surgeon, radiotherapist, pharmacologist and immunologist will obviously be required.

[Assessment of clinical competence of medical school graduates in Italy with standardized patients. The opinion of the examinees]. / Sperimentazione in Italia del metodo del paziente standardizzato per la valutazione della competenza clinica. L'opinione dei neolaureati.

An international collaborative project for the evaluation of clinical competence at the end of the Medical School curriculum using the ECFMG-CSA (Educational Commission for Foreign Medical Graduates--Clinical Skills Assessment) prototype was started in Italy in April 1996. Faculty representatives from Italian Medical Schools and experts from the ECFMG in Philadelphia participated in the Project. The CSA consists of integrated clinical encounters with 10 standardized patients during which the examinee is asked to obtain a focused history, perform a relevant physical examination and communicate initial diagnoses and management plan to the Standardized Patient (SP). The SP then completes checklists that are scored by Faculty members. The project was concluded in Spring 1998 and a total of 173 new graduates were examined. The data elaborated by the primary site in Chieti University will be available in the Fall 1998 by the ECFMG in Philadelphia. This preliminary communication reports the opinions of the examinees on the ECFMG-CSA, contained in the questionnaires administered after the test. Most of the examinees considered this new methodology as a valid tool for the assessment of clinical competence, especially history-taking and interpersonal skills and stated that the SP simulations were realistic. The 72% of examinees indicated that the Medical School curriculum does not adequately prepare for the CSA examination. Lastly, 68% was in favour of including the SP in the Medical Licensing Examination.

Increased potassium permeability induced in vitro by menadione in normal human red cells.

Normal red blood cells, preincubated for 75 min with 1.15 mM menadione sodium bisulfite lose potassium and water on subsequent incubation at 37 degrees C for 24 h without menadione. The potassium loss is increased by addition of calcium and prevented by addition of glucose. Since normal red cells treated with menadione behave like untreated hypochromic cells, both from beta-thalassaemia or iron deficiency anaemia in respect to membrane permeability to potassium, it may be supposed that menadione induces in normal red cells an abnormality similar to that naturally occurring in hypochromic cells.

[Erythrocyte deformability: critical evaluation of the study methods]. / La deformabilità dell'eritrocita: valutazione critica dei metodi di studio.

The rheological behavior of red cells in macrocirculation and in capillaries is briefly described. The following methods measuring red cell deformability are critically evaluated: a. degree of red cell 'packing' after centrifugation; b. single erythrocyte passage through microchannels (micropipette technique, rigidometer); c. filtration techniques of washed red cells; d. polymicroviscosimetry (paper filtration, filtrometer) and viscosimetry (ektacytometer). It is considered the adequacy of each method in assessing the different factors responsible for the variations of red cell deformability (internal viscosity, surface area/volume ratio, viscoelastic properties of the membrane.

Effects of dipyridamole on sodium and potassium content of human red blood cells.

The effects of dipyridamole on potassium and sodium content of human red blood cells from normals or from patients with hypochromic anaemia, and of normal red cells pretreated with menadione, have been studied. Dipyridamole added to the incubation medium decreases the loss of potassium and the accumulation of sodium in all three types of red cells incubated at 37 degrees C for 24 h, either with or without calcium. The possible mechanism underlying these effects of dipyridamole is discussed.

Increased potassium permeability by calcium in hypochromic red blood cells.

The red blood cell (RBC) content of Na+ and K+ were measured both on fresh cells from normal, heterozygous beta-thalassaemic and iron-deficiency-anaemic subjects, and on the same cells incubated for 24 h, at 37 degrees C, either in presence or in absence of Calcium (Ca2+). Ca2+ did not increase membrane permeability to Na+, but increased the K+ loss, both from normal cells and to a greater degree much more from hypochromic cells. Glucose largely prevented the K+ loss from hypochromic cells incubated either in absence or in presence of Ca2+, probably maintaining an adequate level of ATP during the incubation. EDTA only partially decreased the permeability to K+ in hypochromic cells incubated for 24 h at 37 degrees C, possibly removing Ca2+ bound to the cell membrane. The results suggest that Ca2+ does not represent the primary cause of K+ leak in hypochromic cells, but it is able to enhance a pre-existing peculiar abnormality of the cell membrane when the ATP level slows down.