Effect of androgens on maturation and metabolism of erythroid tissue.

Specific changes taking place in the erythroid tissue following depletion or replacement of androgens were studied in rats. The reduction of testosterone levels in blood of orchiectomized animals did occur in conjunction with a decline of erythrocyte glucose-6-phosphate (G6P) and lactate levels. No evidence of anemia was observed. The subcutaneous administration of testosterone propionate (16.0 mg/kg) to orchiectomized rats restored, within 12 hours, blood testosterone levels as well as erythrocyte G6P levels and lactate production. The in vitro incorporation of glucose-1-14C into rat erythrocytes incubated with testosterone was comparable to that of control cells. A radioautographic study of rat erythroid marrow pulsed with glucose-1-14C showed a lower labeling when testosterone propionate was administered. The authors conclude that testosterone does directly affect glucose metabolism of erythroid cells, via the pentose shunt pathway. The possible role of the androgen-dependent enhancement of erythroid glycolysis is discussed in relation to the function of testosterone receptocytes present in marrow cells and a 17beta-hydroxysteroid dehydrogenase present in erythrocytes.

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate in erythrocytes during chicken development.

In contrast to mammalian erythrocytes, chicken erythrocytes contain fructose 2,6-bisphosphate at levels (0.5 nmol/10(9) cells) similar to those of 2,3-bisphosphoglycerate (1.2 nmol/10(9) cells) and slightly lower than those of glucose 1,6-bisphosphate (5.2 nmol/10(9) cells). In chick embryo erythrocytes the levels of both fructose 2,6-bisphosphate and glucose 1,6-bisphosphate are much lower. They begin to increase at hatching and reach the levels in chicken in a few days.

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate in rabbit erythroid cells during differentiation.

Fructose 2,6-bisphosphate concentration and 6-phosphofructo-2-kinase activity markedly decrease during differentiation of rabbit erythroid cells, being higher in erythroblasts (654 +/- 97 pmol/10(9) cells; 238 +/- 81 U mu/10(9) cells) than in reticulocytes (40 +/- 15 pmol/10(9) cells; 11 +/- 3 U mu/10(9) cells) and much higher than in mature erythrocytes (10 +/- 0.8 pmol/10(9) cells; 2 +/- 1 U mu/10(9) cells). The enzymatic activities involved in glucose 1,6-bisphosphate metabolism also decrease, but the levels of aldohexose 1,6-bisphosphates remain essentially constant during differentiation of erythroid cells.

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate levels in erythrocytes with high and low 2,3-bisphosphoglycerate content during postnatal development.

In rabbit and sheep erythrocytes the concentrations of 2,3-bisphosphoglycerate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate suffer important changes after birth, which differ in both species. The changes of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate correlate with the changes in the levels of the enzymatic activities involved in their synthesis. The change of 2,3-bisphosphoglycerate levels in rabbit but not in sheep erythrocytes could be explained by the changes of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios.

Determination of erythrocyte glucose 1,6-bisphosphate--a comparison of two methods using a centrifugal analyzer.

Glucose 1,6-bisphosphate is a key effector of human erythrocyte glycolysis, yet to date its assay has been problematical. Two methods for measuring glucose 1,6-bisphosphate were modified and adapted to a centrifugal analyser and the inaccuracy and imprecision of each method were compared. One assay, based on stimulation of phosphoglucomutase, was shown to underestimate the erythrocyte levels by approximately 5% due to inhibition of the mutase by endogenous 2,3-bisphosphoglycerate. An alternative chemical/enzymic method, consisting of acid hydrolysis of glucose 1,6-bisphosphate to glucose 6-phosphate and subsequent determination of the monophosphate was modified by omitting an initial alkaline hydrolysis step and by increasing the duration of acid hydrolysis. The modified method also enabled the determination of erythrocyte glucose 6-phosphate. The normal concentration of glucose 1,6-bisphosphate in whole blood and in washed human erythrocytes, determined using the more accurate chemical/enzymic method was 83 +/- 5 mumol/l cells and 86 +/- 4 mumol/l cells, respectively; the corresponding concentrations of glucose 6-phosphate were 26 +/- 2 mumol/l cells and 15 +/- 3 mumol/l cells.

An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation.

An improved quantitative method for measuring galactose-1-phosphate uridyltransferase (EC 2.7.7.12, Gal-PUT) activity in erythrocytes was developed based on the detection of glucose 1-phosphate generated under the catalytic influence of the enzyme. This is achieved by incubating the enzyme with galactose 1-phosphate and uridyldiphosphoglucose during 15 min, followed by deproteinisation. The glucose 1-phosphate generated is quantitated subsequently by measuring NADPH formation from added NADP+ in a second incubation step with added phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Gal-PUT activity is calculated from the increment in absorption at 340 nm. Because it is technically a simple assay that is sensitive, specific and not affected by UDPgalactose-4-epimerase (EC 5.1.3.2) activity in the erythrocytal lysates it is suggested to be the method of choice for measuring Gal-PUT activity. Activities in erythrocytes of controls varied from 264 to 556 U/kg hemoglobin; in obligate heterozygotes from 53 to 190 U/kg Hb and in homozygous deficient patients less than 5 U/kg Hb was measured at 37 degrees C.

Erythrocyte glycolysis in protein-energy malnutrition.

Erythrocyte glycolysis has been studied in the anaemia associated with protein-energy malnutrition (PEM) in Kivu. Several results were compatible with a lowering of the mean age of the erythrocyte population, notably raised levels of glucose-6-phosphate, hexokinase, Na+-K+- adenosinetriphosphatases and potassium, and low sodium concentration. Non-significant differences were observed for glucose utilization, lactate formation, and for concentrations of fructose-6-phosphate, fructose-1,6-diphosphate, adenosine diphosphate and pyruvate kinase; there was no gross disturbance of cation transport. The level of adenosine triphosphate was slightly decreased and that of 2,3-bisphosphoglycerate was not elevated, in spite of anaemia. The latter could not be explained by an instability of this metabolite. It is concluded that slight erythrocyte glycolytic abnormalities may occur in the anaemia of Kivu PEM, but that they are not the main cause of the haemolysis observed in this syndrome.

Heterogeneity of erythrocyte pyruvate kinase deficiency and related metabolic disorders in patients with hematological diseases.

In several patients suffering from congenital non-spherocytic hemolytic anemia or from malignant hemotological disorder associated with erythrocyte pyruvate kinase (PK) deficiency, a metabolic study has been carried out involving the following biochemical determinations: assay of red cell enzyme activities; estimation of glucose consumption; measurement of the rate of glycolytic intermediates; and, in some cases, enzyme purification and characterization of the PK variant. Metabolic equilibrium most probably does not depend on kinetic characteristics of PK molecules. Furthermore, the data obtained allow separation of cases with congenital non-spherocytic hemolytic anemia (hereditary defect) and acquired PK deficiencies.

Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and 3H-deoxyglucose-6-phosphate release from human red blood cells.

At a low concentration of Naja naja kaouthia cardiotoxin (3 microM) Ca2+, Sr2+ and Ba2+ (2 mM), had little to no effect on 3H-deoxyglucose-6-phosphate (3H-dGlu-6-p) or hemoglobin release. At higher concentrations of N. n. kaouthia cardiotoxin (greater than or equal to 10 microM), Ca2+ (2 mM), but not Sr2+ or Ba2+, significantly enhanced 3H-dGlu-6-p and hemoglobin release. Mn2+ (2 mM) almost completely inhibited 3H-dGlu-6-p release and hemolysis at both the 3 microM and 10 microM concentrations of cardiotoxin. At a fixed concentration of N. n. kaouthia cardiotoxin (3 microM). Ca2+ at low concentrations (0.5 mM) enhanced 3H-dGlu-6-p and hemoglobin release, but at higher concentrations caused a dose-dependent inhibition of cardiotoxin action. The cardiotoxin from N. n. kaouthia venom (3 microM) induced 3H-dGlu-6-p release and hemolysis release with similar time courses and to similar extents. 3H-dGlu-6-p release induced by cardiotoxin was greatly enhanced as the pH of the medium was increased from 7.0 to 8.5. Similarities between 3H-dGlu-6-p and hemoglobin release do not support opening of pores in the plasmalemma of all red blood cells as the mode of action of cardiotoxins, but suggests that complete lysis of a subpopulation of cells occurs. Cardiotoxins have two components of lysis, only one of which is Ca2+-dependent. The Ca2+-dependent lysis is only evident at higher cardiotoxin concentrations and is likely due to trace phospholipase A2 contamination in the toxin fraction. Mn2+ is an effective antagonist of cardiotoxin action.

Usefulness of MC-540 fluorescent dye as probe versus scanning electron microscopy for assessing membrane changes.

The effect of primaquine enantiomers on cell membranes of glucose-6-phosphate (G-6PD)-deficient erythrocytes was studied in vitro. Staining with merocyanine (Mc-540) showed that exposure to primaquine enantiomers produces significant fluorescence in G-6PD-deficient erythrocytes, indicating marked drug-induced alterations in membrane fluidity. Scanning electron microscopy (SEM) studies confirmed that primaquine enantiomers altered membrane morphology (by producing stomatocytes) in both normal and G-6PD-deficient cells. The concentration-dependent effect, however, was more pronounced with MC-540, a lipophylic dye, than with SEM (an expensive technique).

Reduced anaerobic glycolysis in oral contraceptive users.

Oral contraceptives containing combinations of estrogens and progestogens are known to impair glucose tolerance. The biochemical mechanisms underlying this lesion are speculative. In the present study women treated with OC for periods exceeding 10 cycles showed significant reduction in the activity of the key glycolytic enzyme phosphofructokinase (40%) and the levels of lactate (42%) in the erythrocytes compared to controls. These observations in women are analogous to those made earlier in female rats.

PIP: Combined oral contraceptives (30 mg ethinyl estradiol, 150 mg d-norgestrel) are known to reduce glucose tolerance. To study the possible mechanisms of this impairment, 20 women were divided into 3 groups based on length of contraceptive use -- 3-5 months, 6-11 months, and 12-36 months. Blood samples were analyzed for phosphofructokinase activity and levels of glycolytic metabolites. Changes in glucose tolerance are seen within 3-6 months of oral contraceptive use, but only the blood taken from the 3rd group (12-36 months) showed significantly lower levels of phosphofructokinase activity and lowered levels of fructose-1,6-diphosphate, lactate, and pyruvate. It is suggested that impaired glucose tolerance is due to reduced glycolysis due to lower levels of phosphofructokinase synthesis, which cannot be detected for at least a year in erythrocytes, since they do not synthesize the enzyme.

Photodynamic action of protoporphyrin on resealed erythrocyte membranes: mechanisms of release of trapped markers.

Photoactivation of protoporphyrin IX (PP) bound to resealed human erythrocyte (RBC) ghosts results in membrane damage which is manifested by the release of trapped markers Na+ and glucose-6-phosphate (G6P). Efflux of Na+ was rapid, continuous, and virtually complete before the onset of G6P efflux. The sugar phosphate emerged abruptly after a long lag. The antioxidant butylated hydroxytoluene (BHT) had no effect on the permeation of Na+, but greatly suppressed that of G6P. These results suggest that the markers are emitted via different mechanisms. For G6P, disruption of the bilayer by free radical lipid peroxidation appears to be necessary, inasmuch as BHT inhibited peroxidation as measured by thiobarbituric acid reactivity and appearance of phospholipid and cholesterol hydroperoxides on thin layer chromatograms. It is deduced that non-lipid damage is sufficient for Na+ release. This effect is manifested at low light intensities and low PP concentrations. Protein regulators of passive cation permeability may be the primary targets in this case. When sensitive sulfhydryl groups on these proteins were blocked with p-chloromercuri-benzene-sulfonate, Na+ leaked out rapidly, but G6P was unaffected, thereby mimicking the early stages of membrane photodamage.

Changes in glycolytic intermediates in rat erythrocytes during exposure to simulated high altitude.

Regulatory mechanisms of erythrocyte glycolysis and 2,3-diphosphoglycerate (2,3DPG) metabolism under hypoxia were studied in rats exposed to a simulated altitude of 18,000 ft (5,486 m) for 5 d. Changes in erythrocyte glycolytic intermediates were determined by enzymatic analysis. Marked alterations of glycolytic intermediates were found during 1 d of exposure which were quite different from those observed during exposure for 2, 3, and 5 d. Alterations of intermediates seem to be highly correlated with blood pH changes; however, pH alone cannot explain the overall changes in intermediates. Results suggested that overall intermediate changes are the results of the combined effect of alterations of cellular pH and hemoglobin desaturation. Increased 2,3DPG at initial stages of exposure (within 1 d) may be caused mainly by the increased cellular pH; sustained elevation of 2,3DPG at later stages could be attributed to the relief of product inhibition of diphosphoglycerate mutase by deoxygenation.

[Disordered glucose-6-phosphate transport as a possible cause of glycogenosis type Ib]. / O narushenii transporta gliukozo-6-fosfata kak vozmozhnoi prichine glikogenoza tipa Ib.

More than ten patients with glycogen-storage disease, which were classified as patients with glycogenosis of the I type--deficiency in glucose-6-phosphatase) on the basis of clinical data and biochemical analyses in vivo, were detected within the last few years. But activity of glucose-6-phosphatase was found to be normal in biopsy of samples of the liver tissue obtained from these patients. This disease was termed as glycogenosis of the Ib type. A hypothesis is advanced, according to which the discrepancy in data on biochemical study of the patients in vivo and in vitro is due to absence of a specific permease in liver tissue, which transfers glucose-6-phosphate from cytosol onto the innesurface of membranes of cytoplasmic network, where glucose-6-phosphatase is located.

[Effect of physical exercise on glycolysis in human red blood cells (author's transl)].

Untrained healthy male volunteers were studied to observe the effects of physical exercise (bicycle ergometer, 920 kpm/min for 10 min, 15 min and 30 min) upon glycolytic intermediates in red blood cells. The levels of glucose 6-phosphate, fructose 6-phosphate, pyruvate and lactate increased after each exercise. The levels of fructose 1,6-diphosphate increased and phosphoenolpyruvate decreased respectively only after 30 min of exercise. At the rest period of 30 min after 30 min of exercise the lactate level still remained unchanged, however all the other intermediates returned to the preexercise values. A negative crossover point seemed to exist between fructose 6-phosphate and fructose 1, 6-diphosphate after 15 min of exercise. A positive crossover point was observed between phosphoenolpyruvate and pyruvate after 30 min of exercise. There were significant increases in hexokinase and pyruvate kinase activities, but not in phosphofructokinase activity after 30 min of exercise. These facts suggested that the increase in pyruvate kinase activity was due to the elevated fructose 1,6-diphosphate level after 30 min of exercise. A significant increase in plasma dopamine-beta-hydroxylase activity was found after each exercise. A close positive correlation was observed between pyruvate-phosphoenolpyruvate ratio and dopamine-beta-hydroxylase activity after 30 min of exercise. It was suggested that pyruvate-phosphoenolpyruvate ratio provided a reliable index of physical exercise.