Molecular heterogeneity of the glucose-6-phosphate dehydrogenase deficiency in the Hellenic population.

We report results from a systematic study to identify the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on a sample of 299 male subjects from the Hellenic population. Our stepwise approach involved partial biochemical characterization and quantitation of the enzyme's activity, MboII restriction endonuclease digestion to identify the G6PD Mediterranean variant, which represents the most frequent G6PD variant in our population and a nonradioactive polymerase chain reaction-single-strand conformation polymorphism methodology for the detection of the underlying molecular defect(s) in the rest of the non-Mediterranean G6PD-deficient individuals. Through this approach, six different G6PD variants were identified (G6PD Mediterranean, G6PD Hermoupolis, G6PD Cassano, G6PD Seattle, G6PD Ierapetra and G6PD Acrokorinthos), two of which were new (G6PD Hermoupolis, G6PD Acrokorinthos). In essence, this study underlines the remarkable genetic heterogeneity of the G6PD deficiency in the Hellenic population, while the finding of the double mutant, G6PD Hermoupolis, may help to outline the relationship and evolution of mutations in the human G6PD locus.

Benign osteopetrosis, in a patient with sickle-cell beta+ (beta+) thalassaemia.

We describe a case of a patient suffering from benign osteopetrosis and sickle-cell beta+ thalassaemia. This case allows us to study the combined action of various pathogenetic mechanism involved in both diseases. The coexistence of osteopetrosis with sickle-cell beta+ thalassaemia seems to intensify the anaemia and sickling, but does not appear to modify the course of the osteopetrosis.

Biochemical characterization of four new erythrocyte pyruvate kinase variants.

Pyruvate kinase (PK) from four patients with moderate to severe congenital non-spherocytic haemolytic anaemia was characterized by methods recommended by the ICSH. The possibility that two of the patients are true homozygotes cannot be ruled out, while the other two apparently represent double heterozygotes. All but one had levels of PK activity between 44 and 65% of normal. The variant enzymes were designed 'PK Pontos', 'PK Macedonia', 'PK Athens' and 'PK Larisa'. Multiple physicochemical as well as kinetic aberrations were detected in the above variants. Their altered kinetic behaviour is discussed in terms of the concerted transition model for allosteric enzymes and their abnormal properties are compared with other known variants, while it is also attempted to correlate them with possible mechanisms resulting in chronic haemolytic anaemia.

T24 h-ras gene-expression increases the activity of phosphoglycerate kinase, enolase and pyruvate-kinase and decreases the activity of adenosine-deaminase in fibroblast cells.

We examined the possible implication of ras in the regulation of the activity of several metabolic enzymes by employing an inducible H-ras expression system (RFLSVrasLAP cell line), in which the addition of IPTG decreases the levels of ras p21 3-fold. We measured the activity of hexokinase (E.C. 2.7.1.1.), glucose phosphate isomerase (E.C. 5.3.1.9), phospho-fructokinase (E.C. 2.7.1.11), aldolase (E.C. 4.1.2.13), phosphoglycerate kinase (E.C. 2.7.2.3), enolase (E.C. 4.2.1.11), pyruvate kinase (E.C. 2.7.1.40), lactate dehydrogenase (E.C. 1.1.1.27), adenosine deaminase (E.C. 3.5.4.4) and purine nucleoside phosphorylase (E.C. 2.4.2.1) from cells grown in the presence and absence of IPTG. We found that the addition of IPTG to RFLSVrasLAP cells led to lower activity of phosphoglycerate kinase (p=0.004), enolase (p=0.027) and pyruvate kinase (p=0.031). Enolase mRNA levels were found to be increased in cells overexpressing either the normal or mutant H-ras. The total rate of glycolysis was not affected by H-ras expression indicating that the implication of H-ras in the activity of phosphoglycerate kinase, enolase and pyruvate kinase may be associated with glycolysis-independent functions of these enzymes. Adenosine deaminase activity was found to increase after IPTG addition (P=0.009), indicating also a possible role for H-ras in the control of the purine nucleotide salvage pathway.

Furosemide-induced hyperglycaemia: the implication of glycolytic kinases.

Hyperglycaemia is a well known adverse effect of therapy with diuretics. In adipose tissue, hydrochlorothiazide and furosemide inhibit the rate of glucose transport. In skeletal muscle, furosemide decreases the rate of glucose phosphorylation and glycolysis. However, whether furosemide has any direct effect on the activities of any of the glycolytic enzymes is not known. In the present study, the effects of furosemide on the activities of the hexokinase, phosphofructokinase and pyruvate kinase were examined. Pieces of skeletal muscle (quadriceps) and liver were obtained from 10 non-diabetic subjects during surgery. Tissues were homogenized and the activities of the enzymes were measured in the presence or absence of furosemide (0-1.5 mM). Furosemide inhibited the activity of all three key glycolytic enzymes. The concentration of furosemide required to inhibit phosphofructokinase in muscle was lower than that required to inhibit the activity of this enzyme in the liver or to inhibit the activities of hexokinase and pyruvate kinase in both muscle and liver. These direct effects of furosemide may contribute to the decrease in glucose utilisation following therapy with this and similar agents in man.

Diagnostic strategy for thalassemias and other hemoglobinopathies: a program applied to the Hellenic Army recruits.

A program for the detection of thalassemias and other hemoglobinopathies in high-risk populations is described. This program, based on two screening tests, was applied to the Hellenic Army recruits and was found to work well. Red cell one-point osmotic fragility was used for the detection of thalassemic samples and hemoglobin electrophoresis for screening of other hemoglobinopathies. Samples with decreased red cell osmotic fragility and/or abnormal electrophoretic pattern were submitted for further detailed investigation. Following this program, 64,814 recruits, representing 0.651% of the total Greek population and 9.917% of the 20-year-old Greek male population, were tested. beta-Thalassemia was found with an average incidence of 5.476% and alpha-Thalassemia with an incidence of 0.201%. Hemoglobinopathy Lepore was detected in 51 samples (0.079%) and hemoglobinopathy-S in 352 samples (0.543%).

Massive bilateral pleural effusion as the only first presentation of systemic lupus erythematosus.

A rare case of systemic lupus erythematosus (SLE), with massive bilateral pleural effusions as the first manifestation, is described. The patient was a previously healthy 20-year-old soldier. Initial investigations were unrevealing, but after 3 months the patient developed the full-blown syndrome. He responded well to corticosteroids and cyclophosphamide with resolution of the pleural effusions and improvement of the clinical picture. SLE should always be considered in cases of massive pleural effusions, even in the absence of other overt stigmata of the disease.

Thalassemia minor: routine erythrocyte measurements and differentiation from iron deficiency.

The clinical differentiation of the causes of microcytosis is difficult because of the lack of a method for the diagnosis of alpha thalassemia. A number of laboratory tests have been proposed for the differentiation of alpha thalassemia from iron deficiency, including decision functions based on the red blood cell indices generated by electronic cell counters. The accuracy of these screening methods was assessed in 93 patients with microcytosis known to be secondary to either iron deficiency or beta thalassemia minor and, prospectively, in 26 patients with microcytosis in whom globin chain synthesis ratio was used to diagnose thalassemia. The functions evaluated were: RBC volume distribution curve; osmotic fragility; erythrocyte count; discriminant function = MCV - (5 X Hgb) - RBC - 8.4; ratio of MCH/RBC; ratio of MCV/RBC; and 0.01 X MCH X (MCV)2. A simplified method of measuring anisocytosis using the RBC volume distribution curve was significantly more accurate (P less than 0.01) in distinguishing iron deficiency from thalassemia than any of the other decision functions. Analysis of red blood cell volume distribution, although not sufficiently accurate for definitive diagnosis, appears to be a useful technic in the initial screening of patients with microcytosis and in determining which additional testing should be done.

alpha-Thalassemia: prevalence and hematologic findings in American Blacks.

alpha-Thalassemia is common in southeast Asia and the Mediterranean, where the predominant lesion seems to be a deletion of one or more of the four gene loci responsible for alpha-globin chain production. In the United States, the prevalence of alpha-thalassemia in blacks was once thought to be low, but more recent studies show that the prevalence of alpha-thalassemia is high. We measured the globin chain synthetic rations in 144 black Americans to determine the prevalence and hematologic manifestations of alpha-thalassemia in this population. There were 120 subjects with a mean synthetic ration of 0.986 +/- 0.04, with a range of 0.90 to 1.06; these were classified as normal. Five subjects were found to have beta-thalassemia; 19 subjects had mild alpha-thalassemia. The overall gene frequency for alpha-thalassemia was estimated to be 0.07 in this population. The hemoglobin values of subjects with mild alpha-thalassemia were not statistically significantly different from normal black or white control subjects of the same sex, but there was a significant decrease in the mean corpuscular volume and in the mean corpuscular hemoglobin value.

A simple and rapid method for determination of G gamma:A gamma globin chain synthetic ratio.

A simple and rapid method for the determination of the G gamma- to A gamma-globin chain relative synthetic ratios is described. This technic uses 35S-methionine as the labeled amino acid and introduces a simple procedure for Hb-F purification. The chain separation is based on slab gel isoelectric focusing, and the gel after visualization of protein bands without staining is cut and solubilized in periodic acid, and the radioactivity is counted. This method can be applied in blood samples with low Hb-F levels, and as many as 20 samples can be analyzed at one time. The method was found to yield the expected synthetic ratio, and its reproducibility and repeatability were found to be high.

Glycosylated hemoglobin A2 components.

Partially purified hemoglobin A2 has been examined for the existence of glycosylated components by isoelectric focusing and by acid agar gel electrophoresis. Bands analogous to the glycohemoglobin derivatives of hemoglobin A, hemoglobin-A1.a.b.c, were readily detected. Evidence that these minor bands are in fact glycohemoglobins was obtained by showing that 14C-glucose bound to hemoglobin A2 moved with these minor bands. The amounts of glycohemoglobin derivatives of hemoglobin A2 were increased in the blood of diabetic patients.

Red cell glycolytic intermediates in diabetic patients.

The glycolytic intermediates of the red cells of 11 adult diabetic patients were compared with those of eight normal controls. A statistically significant elevation of glucose-6-P and fructose-6-P levels were correlated with the blood sugar levels and 2,3-DPG levels. The absence of a metabolic crossover point suggests that the effect of high levels of glucose may be exerted through regulation of the hexokinase step, itself, but the nature of the regulation cannot be deduced from the complex situation which exists in vivo.

A simplified method for studies of haemoglobin biosynthesis.

A simplified method for studies of haemoglobin chain biosynthesis using 35S-methionine as the labeled amino acid and Cellogel electrophoresis for globin chain separation is described. This technique simplifies the procedure globin preparation, improves the electrophoretic separation of globin chains and may be applied to samples with normal reticulocyte counts. As many as 20 samples can be prepared simultaneously and 10 samples can be subjected to electrophoretic separation at one time. The method was fund to yield the expected synthetic ratios and to be highly reproducible when applied to normal and abnormal samples from peripheral blood or bone marrow.

Platelet glycolysis in platelet storage. III. The inability of platelets to utilize exogenous citrate.

The metabolism of exogenous citrate by freshly collected human platelets has been investigated by measuring the disappearance of citrate from platelet suspensions and by measuring the formation of CO2 and glycogen from purified radioactive citrate. These studies indicate that platelets do not have the capacity to metabolize exogenous citrate.

Platelet glycolysis in platelet storage. II. Levels and turnover of metabolic intermediates.

The metabolism of freshly collected platelets and platelets stored for three or four days at room temperature has been investigated. The levels of glycolytic intermediates were measured in platelets before and after incubation for 15 minutes in a buffered aerobic medium. Platelet intermediates were relatively well maintained, and levels of intermediates that were partially depleted, such as ATP, were restored toward normal after short term incubation even after storage for four days. Incorporation of 32PO4 into fresh and stored platelets during incubation in aerobic buffered medium was also studied. The incorporation pattern was only very modestly influenced by storage. These studies suggest that changes in glycolytic capacity do not play a major role in the loss of viability of platelets during storage at room temperature.

Platelet glycolysis in platelet storage I. The glycolytic enzymes.

Sixteen glycolytic enzymes were measured in essentially leukocyte-free platelets before storage and daily after room temperature storage or platelets as concentrates. Most of the glycolytic enzymes proved to be very stable to storage. Even the most severely affected enzymes, enolase, triosephosphate isomerase, and lactate dehydrogenase lost less than one-quarter of their enzyme activity. Depletion of glycolytic enzymes does not seem to be a major factor in the storage lesion of platelets.