Erythrocyte Phosphofructokinase Research Articles

Effect of acid citrate dextrose during plateletpheresis on first-time healthy donors: Between safety and toxicity

Introduction: During apheresis, anticoagulation is accomplished by use of acid citrate dextrose (ACD). Acid citrate dextrose returns to the donor with the remainder of blood after separation of the targeted blood component. Objectives: This study was conducted to assess the potential toxic effects of ACD in first-time blood donors through clinical and biochemical assessment. Methodology: Seventy -five healthy plateletpheresis donors were recruited in the study where they were subjected to clinical and laboratory assessment just before the procedure (baseline), at 30 min. & at 60 min. during the procedure and 30 min. post procedure. Clinical evaluation included vital signs evaluation and reporting of any clinical manifestation developed. Biochemical assessment included ABG, serum ionized Ca (iCa), ionized Mg (iMg) and parathyroid hormone (PTH) levels. Also activity of erythrocyte phosphofructokinase (PFK) and hematological parameters including RBCs (million/ul), hemoglobin level (Hb g/dl) and hematocrite (Hct %) were evaluated. Results: Apheresis using citrate anticoagulant resulted in changes in mineral homeostasis in the form of hypocalcemia and hypomagnesemia with subsequent tremors (9.3%) and tetany (5.3%). Also, some of the donors exhibited tachycardia and hypotension. Parathyroid hormone (PTH) level increased during apheresis which nearly returned to normal level 30 min. after the procedure while, no significant changes in ABG were detected at different time intervals of the procedure. When RBCs PFK activity was measured, a significant decrease was found during the procedure with significant decrease in RBCs at 60 min. and post 30 min. But no significant changes were detected as regard Hb level and Hct % in comparison to baseline ones. Conclusion: Citrate toxicity may occur even with first time exposure in platelet apheresis donors in form of possible developing tremors, tetany, tachycardia and hypotension with decreased calcium & magnesium levels, increased PTH, decreased erythrocyte PFK activity with subsequent decreased RBCs integrity and possible hemolysis.

Hereditary Phosphofructokinase Deficiency in Wachtelhunds

Hereditary phosphofructokinase (PFK) deficiency was diagnosed in two Wachtelhund dogs and suspected in three related Wachtelhund dogs with exercise intolerance, hemolytic anemia, and pigmenturia. Severe, persistent reticulocytosis in light of only mild anemia together with hemoglobinuria after strenuous exercise suggested PFK deficiency. Low erythrocyte PFK activity together with low 2,3-diphosphoglycerate concentrations and a high hemoglobin-oxygen affinity confirmed the diagnosis. The PFK deficiency is due to a single missense mutation in the muscle-type PFK M-PFK gene in English springer and American cocker spaniels, whippets, and mixed-breed dogs; however, these PFK-deficient Wachtelhunds do not have the same PFK mutation.

Hemolysis, myopathy, and cardiac disease associated with hereditary phosphofructokinase deficiency in two Whippets

Two male castrated Whippet littermates were presented at 1 year of age for pallor, tachycardia, systolic heart murmur, dark yellow to orange feces, intermittent lethargy, pigmenturia, and muscle shivering or cramping after exercise. Persistent macrocytic hypochromic anemia with marked reticulocytosis and metarubricytosis was found when CBC results were compared with reference values for Whippets. Increased serum creatine kinase activity and hyperkalemia also were sometimes present over the 4-year period of evaluation. Progressively increasing serum concentrations of N-terminal prohormone brain natriuretic peptide suggested cardiac disease. Erythrocytes from the whippets were less osmotically fragile but more alkaline fragile than those from control dogs. Erythrocyte phosphofructokinase (PFK) activities and 2,3-diphosphoglycerate concentrations were decreased. Restriction enzyme-based DNA test screening and DNA sequencing revealed the same mutation in the muscle-PFK gene of the Whippets as seen in English Springer Spaniel dogs with PFK deficiency. This is the first report of PFK deficiency in Whippet dogs. In addition to causing hemolysis and exertional myopathy, heart disease may be a prominent clinical component of PFK deficiency in this breed and has not been previously recognized in PFK-deficient English Springer Spaniels.

Comparisons of different methods of anesthesia and analgesia on the levels of glycometabolism rate-limiting enzymes in erythrocytes and plasma glucose and stress hormones in patients undergoing esophagus surgery

Objective To compare the effects of different methods of anesthesia and analgesia on the activities of phosphofructokinase(PFK), glucose-6-phosphate dehydrogenase(G-6PD) and aldose reductase(AR) in erythrocytes and levels of plasma glucose and stress hormones in patients undergoing esophagus surgery. Methods Sixty-two patients scheduled for esophagus surgery were randomly divided into three groups: group I( n = 20) receiving only general anesthesia(GA) followed by intravenous patient controlled analgesia(PCA) with fentanyl 15μg/kg. The other two groups receiving both general anesthesia combined with thoracic epidural anesthesia (GEA) and either intravenous PCA with fentanyl 15μg/kg (group II, n = 21) or thoracic epidural analgesia(TEA) with 0.125% ropivacaine and 0.0002% fentanyl mixture(group III, n = 21) after the operation. Venous blood samples were collected for measurements of PFK, G-6PD and AR activities in erythrocytes and plasma glucose, cortisol, epinephrine and norepinephrine before induction (T 1), 60 min following the incision (T 2), 60 min(T 3) after operation, on the 1st(T 4) and 2nd postoperative day(T 5). Results The activities of PFK decreased( P < 0.01 or P = 0.004) and the activities of G-6PD and AR increased( P < 0. 01) in groups I and II on T 4 compared with those on T 1. Between the two groups, the activities of these enzymes in group II changed less than those of group I( P < 0.01 or P < 0.05). These enzymes activities changed slightly in group III on T 4( P > 0.05). There were significant differences between group III and the other two groups( P < 0.01 or P < 0.05). The levels of plasma glucose increased significantly on T 2( P < 0.01), reached peak values on T 4( P < 0.01) and fell on T 5 in the three groups. Compared to those of groups I and II, the values of plasma glucose in group III were lower on T 4 and T 5( P < 0.05 or P < 0.01). The cortisol concentration in each group increased significantly at T 2( P < 0.01 or P < 0.05), and remained elevated on T 5( P < 0.01 or P < 0.05), while on T 2 and T 3 the cortisol levels of group'were higher than that of groups II and III ( P < 0.05). The levels of group III were lower than those of the other groups on T 4 and T 5( P < 0.01 or P < 0.05). The levels of epinephrine and norepinephrine were also significantly higher in group I than those of the other two groups on T 2( P < 0.01 or P < 0.05), and their levels in group I and II were higher than that of group III on T 4. The patients of the three groups obtained satisfactory pain relief, with all Vidual Analogue Scale(VAS) scores less than 3. VAS scores of group I were much greater 4h after operation. Group III VAS scores were the lowest 24h after operation. However, the number of times patients pressed the bolus switch was higher in group II ( P < 0.01). Conclusion Compared with GA and intravenous PCA, general anesthesia combined with thoracic epidural anesthesia and analgesia obtain better pain relief and could markedly alleviate the stress response and improve these erythrocyte glucose metabolism changes after esophagus surgery.

Toxic effect of Aβ25–35 and fullerene C60 on erythrocytes

Using light microscopy and spectrophotometry, it has been shown that amyloid beta-peptide Abeta25-35 and water-soluble fullerene C60 cause lysis of human and rat erythrocytes. Both fullerene C60 and Abeta25-35 partly inhibited the activities of membrane-associated phosphofructokinase and plasmalemmal lactate dehydrogenase in erythrocytes.

The Enzyme Activity Allosteric Regulation Model Based on the Composite Nature of Catalytic and Regulatory Sites Concept

A new kinetic model of enzymatic catalysis is proposed, which postulates that enzyme solutions are equilibrium systems of oligomers differing in the number of subunits and in the mode of their assembly. It is suggested that the catalytic and regulatory sites of allosteric enzymes are of composite nature and appear as a result of subunits joining. Two possible joining modes are postulated at each oligomerization step. Catalytic site may arise on oligomer formed only by one of these modes. Effector acts by fastening together components of certain oligomeric form and increases the life time of this form. It leads to a shift of oligomer equilibrium and increases a proportion of effector-binding oligomers. Effectors-activators bind the oligomers carrying composite catalytic sites and effectors-inhibitors bind the oligomers, which do not carry active catalytic sites. Thus, catalytic activity control in such system is explained by effector-induced changes of a catalytic sites number, but not of a catalytic site activity caused by changes of subunit's tertiary structure. The postulates of the model do not contradict available experimental data and lead to a new type of general rate equation, which allows to describe and understand the specific kinetic behavior of allosteric enzymes as well as Michaelis type enzymes. All known rate equations of allosteric The equation was tested by modeling the kinetics of human erythrocyte phosphofructokinase. It enabled to reproduce quantitatively the 66 kinetic curves experimentally obtained for this enzyme under different reaction conditions.

Activation of Membrane Skeleton-Bound Phosphofructokinase in Erythrocytes Induced by Serotonin

We show here that serotonin, bothin vivoandin vitro,induced a marked activation of phosphofructokinase, the rate-limiting enzyme in glycolysis, in the membrane-skeleton fraction from erythrocytes. Concomitantly, the hormone induced a striking increase in lactate content, reflecting stimulation of glycolysis. The enzyme's activity in the cytosolic (soluble) fraction remained unchanged. These results suggest a defense mechanism in the erythrocytes against the damaging effects of serotonin, whose concentration in plasma increases in many diseases and is implicated as playing an important role in circulation disturbances.

Effects of potassium antimony tartrate on rat erythrocyte phosphofructokinase activity.

In an earlier study, we observed a marked accumulation of antimony in erythrocytes of rats administered potassium antimony tartrate (Sb) in drinking water. This observation has raised concerns of possible adverse effects on the hematological systems. A study was therefore carried out to investigate the effects of Sb on phosphofructokinase (PFK), a rate-limiting enzyme of erythrocyte glycolysis. Preincubation of PFK with Sb caused a marked inhibition of the enzyme with 95% loss of activity at 5 mM. In comparison, 5 mM sodium arsenite, a known enzyme inhibitor, reduced PFK activity by only 38%. Increasing the concentrations of fructose-6-phosphate (F6P) or magnesium had no effects on the inhibitory potency of Sb. Varying the concentrations of ATP and Sb produced a complex effect on PFK activity. At 1 mM ATP, 0.2 mM Sb was required for 50% inhibition (IC50) of PFK but only 0.05 mM Sb was required for the same inhibition when the concentration of ATP was reduced to 0.2 mM. Glutathione (2-10 mM) and hemoglobin (8-40 micronM partially protected the enzyme from the Sb effect, with the protection being more effective at low antimony concentrations. When Sb was added to assay mixtures after initiation of a PFK reaction with physiological concentrations of ATP (0.2 mM) and F6P (0.1 mM), PFK activity was approximately 50% inhibited by 0.5 mM Sb and completely inhibited by 5 mM Sb. In contrast, glucose utilization in whole blood was only 16% lower over an 8 hour incubation period in the presence of 5 mM Sb. It is concluded that while PFK is markedly inhibited by Sb under in vitro assay conditions, glycolysis in erythrocytes is not significantly affected except at very high Sb concentrations. The weak effect of Sb on glycolysis in erythrocytes may be due in part to the protective effect of hemoglobin and, to a lesser extent, glutathione on PFK.

Characterization of phosphofructokinase-deficient canine erythrocytes.

Dogs homozygously affected with muscle-type phosphofructokinase (PFK) deficiency had about 20% of normal erythrocyte PFK activity and exhibited a compensated haemolytic anaemia. Erythrocyte glucose-6-phosphate and fructose-6-phosphate concentrations were increased and dihydroxyacetone phosphate and 2,3-bisphosphoglycerate values were below normal in affected dogs. Other intermediates distal to the PFK step were not significantly below normal and fructose-1,6-bisphosphate was even above normal. Erythrocyte ATP was higher than normal in affected dogs owing to the reticulocytes present. Abnormal adenylate metabolism was demonstrated by low ATP/AMP and ADP/AMP ratios and the inability to maintain ATP content when affected erythrocytes were incubated with cyanide. Glucose-1,6-bisphosphate content was normal, and fructose-2,6-bisphosphate content in affected canine erythrocytes was higher than normal. Studies of erythrocyte PFK isozymes revealed altered enzyme kinetic properties in affected dogs which appeared to be due to the loss of the M-type subunit.

Postnatal hematologic development in phosphofructokinase-deficient dogs

Adult dogs with phosphofructokinase (PFK) deficiency have compensated hemolytic anemia, due to an absolute or functional deficiency of the muscle-type (M-type) subunit that normally accounts for a majority of total erythrocyte PFK activity in dogs. Potential effects of PFK deficiency on hematologic development were evaluated in dogs. Routine hematologic parameters were similar in normal and affected dogs when 1 day old, because all newborn dogs had erythrocyte PFK activities about three times that of normal adult dogs. Based on chromatographic separation of PFK isozymes and enzyme immunoprecipitation studies, the high PFK activity at birth was attributed to the predominance of the liver-type (L-type) subunit of PFK, which is negligible or absent in normal adult dog erythrocytes. Both total PFK activities and the amounts of L-type subunit present decreased dramatically during the first 6 to 8 weeks of life. The muscle-type subunit was negligible or absent at birth, but appeared and increased as the L-type decreased in normal dogs. These changes may result from the replacement of erythrocytes formed in the fetus with those formed after birth. A postnatal physiologic anemia developed to a similar degree in both affected and normal dogs because of decreases in both mean corpuscular volume and erythrocyte numbers. Reticulocyte counts were high in all dogs at birth and remained high in affected dogs, but decreased from 2 months of age onward in normal dogs. Erythrocyte 2,3-diphosphoglycerate (DPG) values were very low in all newborn pups and increased to values expected for adults in the respective groups by 2 to 4 weeks of age. A low 2,3-DPG concentration occurs in affected dogs because PFK deficiency inhibits glycolysis above the side shunt that forms 2,3-DPG.

75 EXERCISE-INDUCED ALTERATION OF ERYTHROCYTE GLYCOLYSIS ASSOCIATED WITH HYOGENIC HYPERURICEMIA

Myogenic hyperuricemia is caused by increased production of uric acid secondary to enhanced release of inosine and hypoxanthine from exercising muscles into blood. This novel mechanism is evident in glycogenosis types VII (muscle phosphofructokinase deficiency), III (debranching enzyme deficiency), and V. In this study, we examined the effect of muscular exercise on the glycolysis of circulating erythrocytes in glycogenosis types VII and III. The concentration of erythrocyte 2,3-bisphosphoglycerate, which had decreased because of genetic partial deficiency of erythrocyte phosphofructokinase, was further decreased after prolonged bed rest in patients with type VII. Ergometric exercise rapidly increased fructose-1,6-P2, dihydroxyacetone-P plus glyceraldehyde-3-P, and 2,3-bisphosphoglycerate in circulating erythrocytes. Similar changes were observed after exercise in patients with type III. The exercise-induced metabolic alteration of erythrocytes was reproduced in vitro by incubating normal erythrocytes in the presence of inosine. We conclude that physical activity affects glycolysis in erythrocytes in glycogenosis types VII and III, and that myogenic factors including inosine are responsible for this change.

Erythrocyte glycolysis and its marked alterations by muscular exercise in type VII glycogenosis

Levels of erythrocyte glycolytic intermediates after the phosphofructokinase (PFK) step, including 2,3-bisphosphoglycerate (2,3-DPG), were decreased at rest in patients from separate families with type VII glycogenosis. The concentration of 2,3-DPG was about half of the normal control value during a period of unrestricted daily activity but was further decreased to one third of normal after a one-day bed rest. Mild ergometric exercise rapidly increased the levels of fructose-1,6-bisphosphate, dihydroxyacetone phosphate plus glyceraldehyde-3-phosphate, and 2,3-DPG in patients' circulating erythrocytes but did not in those of normal subjects. This indicated that a crossover point at the PFK step in glycolysis disappeared after physical exercise and, consequently, the 2,3-DPG concentration, which had decreased because of blockage of the PFK step, was restored considerably. This apparently exercise-related alteration in intermediary metabolism at the beginning of glycolysis was reproduced in vitro by incubating normal erythrocytes in the presence of inosine or ammonia, both of which have increased levels in circulating blood during and after exercise in this disorder. We conclude that physical activity in addition to a genetic deficiency in erythrocyte PFK affects glycolysis in erythrocytes in type VII glycogenosis and that myogenic factors released from exercising muscles may be responsible for this change.

Erythrocyte Glycolysis and Its Marked Alteration by Muscular Exercise in Type VII Glycogenosis

Abstract Levels of erythrocyte glycolytic intermediates after the phosphofructokinase (PFK) step, including 2,3-bisphosphoglycerate (2,3- DPG), were decreased at rest in patients from separate families with type VII glycogenosis. The concentration of 2,3-DPG was about half of the normal control value during a period of unrestricted daily activity but was further decreased to one third of normal after a one-day bed rest. Mild ergometric exercise rapidly increased the levels of fructose- 1,6-bisphosphate, dihydroxyacetone phosphate plus glyceraldehyde-3- phosphate, and 2,3-DPG in patients' circulating erythrocytes but did not in those of normal subjects. This indicated that a crossover point at the PFK step in glycolysis disappeared after physical exercise and, consequently, the 2,3-DPG concentration, which had decreased because of blockage of the PFK step, was restored considerably. This apparently exercise-related alteration in intermediary metabolism at the beginning of glycolysis was reproduced in vitro by incubating normal erythrocytes in the presence of inosine or ammonia, both of which have increased levels in circulating blood during and after exercise in this disorder. We conclude that physical activity in addition to a genetic deficiency in erythrocyte PFK affects glycolysis in erythrocytes in type VII glycogenosis and that myogenic factors released from exercising muscles may be responsible for this change.

Biochemical studies of canine muscle phosphofructokinase deficiency.

Skeletal muscle from four dogs with erythrocyte phosphofructokinase (PFK; EC 2.7.1.11) deficiency were studied in vitro. Muscle PFK activities were severely decreased to 1% of the normal mean. The residual activities had a high Km for fructose-6-phosphate (F6P). Anaerobic lactate production of PFK-deficient muscle was minimal with the addition of glycogen and hexose-monophosphates, but was normal with fructose-1,6-diphosphate (FDP). Muscle glycogen concentration was twice normal, indicating a glycogen storage disorder. Histochemical studies for muscle PFK activity showed no enzymatic staining with F6P as substrate. In two muscle biopsies from asymptomatic related dogs, intermediate PFK activities were found. These data characterize canine muscle PFK deficiency in vitro.

Nonspherocytic haemolytic anaemia due to phosphofructokinase deficiency in an English Springer Spaniel

ABSTRACTA five‐year‐old female English Springer Spaniel was evaluated for chronic episodic anaemia, pigmenturia and icterus. There was increased erythrocyte pyruvate kinase activity, increased in vitro erythrocyte alkaline fragility, decreased erythrocyte 2,3‐diphosphoglycerate activity and severely decreased erythrocyte phosphofructokinase activity. The clinical features and haematological values were identical with erythrocyte phosphofructokinase deficiency and resulting nonspherocytic haemolytic anaemia recently described in this breed.

Autosomal recessive inherited phosphofructokinase deficiency in English springer spaniel dogs

Three families of English springer spaniel dogs with phosphofructokinase (PFK) deficiency causing haemolysis were studied. Four male dogs and one female dog with chronic haemolysis and haemolytic crises were found to have markedly reduced PFK activity in erythrocytes (8-20% of control English springer spaniels). PFK-deficient erythrocytes exhibited an extreme alkaline and sucrose lysis. The oxygen dissociation curve of erythrocyte suspensions was shifted to the left with a 50% saturation of haemoglobin at a partial oxygen pressure of 16-17 mmHg (normal 26-31 mmHg). Muscle wasting and mildly increased serum creatine phosphokinase activity were also noted. Six clinically normal first degree relatives of affected dogs had erythrocyte PFK activities that were 38-51% of controls. In these family members, there was an erythrocytosis and mild reticulocytosis probably due to a mildly enhanced haemoglobin-oxygen affinity but no increase in serum creatine phosphokinase. These studies confirm the familial nature of muscle-type PFK deficiency in English springer spaniels and support the conclusion that this animal model of the human glycogen storage disease type VII is inherited as an autosomal recessive trait.

Pyruvate kinase and phosphofructokinase in maternal erythrocytes in pelation to newborn size

The enzymatic activity of pyruvate kinase (PK) and phosphofructokinase (PFK) was determined in maternal erythrocytes at term. The activity of the two enzymes was positively correlated to the weight, but not to the crown to heel length. Only PK was positively correlated to the head circumfererce of the newborn infants. The results agree with the hypothesis of impaired energy metabolism in fetal malnutrition and suggest the use of the maternal erythrocytes as markers for fetal cells.

Inherited phosphofructokinase deficiency in dogs with hyperventilation- induced hemolysis: increased in vitro and in vivo alkaline fragility of erythrocytes

Two male English springer spaniel dogs with a chronic hemolytic anemia and sporadic hemolytic crises, historically related to “stress” situations, were studied. Although canine erythrocytes are in general known to be more alkaline fragile, erythrocytes from both patients began to lyse earlier, at significantly lower pH values (near pH 7.4 at 37 degrees C), than erythrocytes from control dogs. Hyperventilation induced by 30 minutes of exercise, placement in a 39 degrees C water bath, or intravenous doxapram increased venous blood pH in dog 1 and control dogs, but transient hemoglobinemia, hemoglobinuria, and severe bilirubinuria occurred only in the studied patient. The erythrocyte phosphofructokinase (PFK) activity was severely decreased in both dogs (10% of controls). The erythrocyte 2,3-diphosphoglycerate content was markedly reduced and the cell chloride content was consequently increased. This change in cell chloride content is related to an increase in the erythrocyte pH, which may partially explain the pathogenesis of hemolysis in canine PFK deficiency. Thus, these studies demonstrate a presumably inherited erythrocyte PFK deficiency in English springer spaniels, which causes an increased in vitro and in vivo erythrocyte alkaline fragility. Dogs with PFK deficiency and inducible hemolytic crises may become a valuable genetic animal model in which to study the pathophysiology of hemolysis.

Inherited Phosphofructokinase Deficiency in Dogs With Hyperventilation-Induced Hemolysis: Increased In Vitro and In Vivo Alkaline Fragility of Erythrocytes

Two male English springer spaniel dogs with a chronic hemolytic anemia and sporadic hemolytic crises, historically related to "stress" situations, were studied. Although canine erythrocytes are in general known to be more alkaline fragile, erythrocytes from both patients began to lyse earlier, at significantly lower pH values (near pH 7.4 at 37 degrees C), than erythrocytes from control dogs. Hyperventilation induced by 30 minutes of exercise, placement in a 39 degrees C water bath, or intravenous doxapram increased venous blood pH in dog 1 and control dogs, but transient hemoglobinemia, hemoglobinuria, and severe bilirubinuria occurred only in the studied patient. The erythrocyte phosphofructokinase (PFK) activity was severely decreased in both dogs (10% of controls). The erythrocyte 2,3-diphosphoglycerate content was markedly reduced and the cell chloride content was consequently increased. This change in cell chloride content is related to an increase in the erythrocyte pH, which may partially explain the pathogenesis of hemolysis in canine PFK deficiency. Thus, these studies demonstrate a presumably inherited erythrocyte PFK deficiency in English springer spaniels, which causes an increased in vitro and in vivo erythrocyte alkaline fragility. Dogs with PFK deficiency and inducible hemolytic crises may become a valuable genetic animal model in which to study the pathophysiology of hemolysis.

Rapid purification of human erythrocyte phosphofructokinase

Rapid purification of human erythrocyte phosphofructokinase