scholarly journals Enzymes and membrane proteins of ADSOL-preserved red blood cells

2000 ◽  
Vol 118 (2) ◽  
pp. 41-45 ◽  
Author(s):  
Maria Sueli Soares Leonart ◽  
Aguinaldo José Nascimento ◽  
Kimiyo Nonoyama ◽  
Cinthia Barbosa Pelissari ◽  
Orlando Cesar de Oliveira Barretto
Keyword(s):  
Membrane Proteins ◽  
Biochemical Parameters ◽  
Good Condition ◽  
Red Cells ◽  
University Hospital ◽  
Plasma Sodium ◽  
Red Cell ◽  
Cell Enzyme ◽  
Cell Preservation ◽  
2,3 Dpg

CONTEXT: The preservative solution ADSOL (adenine, dextrose, sorbitol, sodium chloride and mannitol) maintains red cell viability for blood trans-fusion for 6 weeks. It would be useful to know about its preservation qualities over longer periods. OBJECTIVE: To determine some red cell biochemical parameters for peri-ods of up to 14 weeks in order to determine whether the red cell metabo-lism integrity would justify further studies aiming at increasing red cell preservation and viability. DESIGN: Biochemical evaluation designed to study red cell preservation. SETTING: São Paulo University erythrocyte metabolism referral center. SAMPLE: Six normal blood donors from the University Hospital of the Universidade Federal do Paraná, Curitiba, Brazil. MAIN MEASUREMENTS: Weekly assay of erythrocyte adenosine-5´-triphosphate (ATP), 2,3-diphosphoglycerate (2,3DPG), hexokinase (HX), phosphofructokinase (PFK), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconic dehydrogenase (6-PGD), glyceraldehyde-3-phosphate dehydrogenase (GAPD), glutathione reduc-tase (GR), glutathione peroxidase (GSHPx), plasma sodium and potas-sium, blood pH, and membrane proteins of red cells preserved in ADSOL were studied during storage for 14 weeks storage. RESULTS: During ADSOL preservation, erythrocyte ATP concentration decreased 60% after 5 weeks, and 90% after 10 weeks; the pH fell from 6.8 to 6.4 by the 14th week. 2,3-DPG concentration was stable during the first week, but fell 90% after 3 weeks and was exhausted after 5 weeks. By the end of the 5th week, an activity decrease of 16-30% for Hx, GAPD, GR, G-6-PD and 6-PGD, 35% for PFK and GSHPx, and 45% for PK were observed. Thereafter, a uniform 10% decay was observed for all enzymes up to the 14th week. The red blood cell membrane pro-teins did not show significant alterations in polyacrylamide gel electro-phoresis (SDS-PAGE) during the 14 weeks. CONCLUSION: Although the blood viability was shown to be poor from the 6th week up to the 14th week of storage due to ATP and 2,3-DPG depletion, the other biochemical parameters remained in fairly good condition for longer storage. As there is a gradual and uniform decay in activity throughout these 14 weeks, it seems that ADSOL-preserved red cells may be used as red cell enzyme standards and membrane proteins as well.

scholarly journals Effect of oxalate and malonate on red cell metabolism

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1389-1393
Author(s):  
E Beutler ◽  
L Forman ◽  
C West
Keyword(s):  
Pyruvate Kinase ◽  
Cell Metabolism ◽  
Inhibitory Effect ◽  
Red Cells ◽  
Red Cell ◽  
Acid Analogue ◽  
2,3 Dpg

The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

scholarly journals A Red Cell Enzyme Method for the Diagnosis of Acute Intermittent Porphyria

Blood ◽  
1974 ◽  
Vol 44 (6) ◽  
pp. 857-868 ◽  
Author(s):  
C. Richard Magnussen ◽  
Joel B. Levine ◽  
Joyce M. Doherty ◽  
Judy O. Cheesman ◽  
Donald P. Tschudy
Keyword(s):  
Aminolevulinic Acid ◽  
Acute Intermittent Porphyria ◽  
Mean Value ◽  
Red Cells ◽  
Red Cell ◽  
Enzyme Preparations ◽  
Cell Enzyme ◽  
Enzyme Method ◽  
Latent Disease ◽  
The Mean

Abstract A method has been devised for the measurement of uroporphyrinogen I synthetase ih red cells. By using trichloroacetic acid as a protein precipitant, heme is removed from the final solution, allowing accurate measurement of porphyrins. The method is highly reproducible and adaptable to varying incubation volumes and enzyme preparations. It is of great value as an enzyme diagnostic method for acute intermittent porphyria and appears capable of detecting patients with the latent disease who have normal urinary δ-aminolevulinic acid and porphobilinogen excretion. It also appears to distinguish other types of porphyria from acute intermittent porphyria. The mean value of the enzyme in red cells of patients with acute intermittent porphyria was approximately 50% that of normals, indicating that the mutation causes complete lack of catalytic activity in the mutant enzyme.

scholarly journals Red cell diphosphoglycerate mutase. Immunochemical studies in vertebrate red cells, including a human variant lacking 2,3-DPG

Blood ◽  
1978 ◽  
Vol 52 (5) ◽  
pp. 953-958 ◽  
Author(s):  
LL Peterson
Keyword(s):  
Enzyme Activity ◽  
Genetic Variant ◽  
Neonatal Period ◽  
Human Erythrocytes ◽  
Red Cells ◽  
The Other ◽  
Radial Immunodiffusion ◽  
Red Cell ◽  
Human Adult ◽  
2,3 Dpg

Abstract Diphosphoglycerate mutase (DPGM) was purified to homogeneity from human erythrocytes. The enzyme and Freund adjuvant were injected into chickens and yielded a monospecific precipitating antibody. Radial immunodiffusion with this antibody was used to measure the amount of DPGM in hemolysates from human adult and cord red cells. Dog, rabbit, rat, chicken, and goat red cells all had DPGM during the neonatal period, but goat adult red cells had no detectable enzyme. Single bands with no spurs were present on Ouchterlony plates in which human hemolysate was placed adjacent to hemolysates from the other species tested. The amount of human red cell DPGM did not differ between young and old cells separated by centrifugation. Red cells from a patient with a DPGM genetic variant who had erythrocytosis and no detectable enzyme activity contained a reduced amount of DPGM as determined by radial immunodiffusion. The abnormal DPGM differed from normal by immunoelectrophoresis and in stability as measured by the amount of crossreacting material in young versus old erythrocytes.

scholarly journals Effect of oxalate and malonate on red cell metabolism

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1389-1393 ◽  
Author(s):  
E Beutler ◽  
L Forman ◽  
C West
Keyword(s):  
Pyruvate Kinase ◽  
Cell Metabolism ◽  
Inhibitory Effect ◽  
Red Cells ◽  
Red Cell ◽  
Acid Analogue ◽  
2,3 Dpg

Abstract The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

scholarly journals Inappropriately low red cell 2,3-diphosphoglycerate and p50 in transfused beta-thalassemia

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 803-806
Author(s):  
A Correra ◽  
JH Graziano ◽  
C Seaman ◽  
S Piomelli
Keyword(s):  
Cell Function ◽  
Hemoglobin Concentration ◽  
Thalassemia Major ◽  
Red Cells ◽  
Deficiency Anemia ◽  
Beta Thalassemia ◽  
Control Group ◽  
Red Cell ◽  
Normal Individuals ◽  
2,3 Dpg

The relationships among hemoglobin concentration (Hb), red cell 2,3- diphosphoglycerate (2,3-DPG), and p50 were studied in 20 chronically hypertransfused patients with thalassemia major. In the nontransfused control group, which included normal individuals as well as patients with sickle cell disease or iron deficiency anemia, the Hb correlated inversely with both 2,3-DPG concentration and p50, as is well established. In contrast, however, prior to transfusion, at the nadir of Hb, patients with thalassemia major had inappropriately low 2,3-DPG concentrations and p50s. These findings occurred in all patients, regardless of whether they had received packed, leukocyte-poor, or frozen-thawed red cells. The hypothesis that the time of blood storage was a factor was excluded by repeatedly transfusing one patient with packed red cells administered within 4 hr of collection in CPDA-1. A second hypothesis, that red cell function might be impaired by the iron- overloaded thalassemic environment, was excluded by studying a newly diagnosed, newly transfused patient with aplastic anemia. In both cases, the same inability to appropriately increase 2,3-DPG and p50 as the Hb fell during the intertransfusion interval was noticed. These data suggest that red cells of chronically transfused patients are unable to adapt to the decline in Hb that occurs during the intertransfusion interval.

scholarly journals Red cell diphosphoglycerate mutase. Immunochemical studies in vertebrate red cells, including a human variant lacking 2,3-DPG

Blood ◽  
1978 ◽  
Vol 52 (5) ◽  
pp. 953-958
Author(s):  
LL Peterson
Keyword(s):  
Enzyme Activity ◽  
Genetic Variant ◽  
Neonatal Period ◽  
Human Erythrocytes ◽  
Red Cells ◽  
The Other ◽  
Radial Immunodiffusion ◽  
Red Cell ◽  
Human Adult ◽  
2,3 Dpg

Diphosphoglycerate mutase (DPGM) was purified to homogeneity from human erythrocytes. The enzyme and Freund adjuvant were injected into chickens and yielded a monospecific precipitating antibody. Radial immunodiffusion with this antibody was used to measure the amount of DPGM in hemolysates from human adult and cord red cells. Dog, rabbit, rat, chicken, and goat red cells all had DPGM during the neonatal period, but goat adult red cells had no detectable enzyme. Single bands with no spurs were present on Ouchterlony plates in which human hemolysate was placed adjacent to hemolysates from the other species tested. The amount of human red cell DPGM did not differ between young and old cells separated by centrifugation. Red cells from a patient with a DPGM genetic variant who had erythrocytosis and no detectable enzyme activity contained a reduced amount of DPGM as determined by radial immunodiffusion. The abnormal DPGM differed from normal by immunoelectrophoresis and in stability as measured by the amount of crossreacting material in young versus old erythrocytes.

scholarly journals Effect of procaine HCLl on ATP: calcium-dependent alterations in red cell shape and deformability

Blood ◽  
1977 ◽  
Vol 50 (1) ◽  
pp. 155-164 ◽  
Author(s):  
J Palek ◽  
A Liu ◽  
D Liu ◽  
LM Snyder ◽  
NL Fortier ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Atp Depletion ◽  
Red Cells ◽  
Ionophore A23187 ◽  
Red Cell ◽  
Cation Composition ◽  
Red Cell Membrane ◽  
Membrane Bound

Abstract Procaine hydrochloric acid, a cationic anesthetic, although unable to prevent the effect of calcium ionophore A23187 on erythrocytes, inhibited the discocyte--echinocyte transformation, increased viscosity, and decreased filterability of red cells undergoing ATP depletion. The effects were abolished by washing ATP-depleted, procaine HCl-treated red cells prior to these determinations. Procaine HCl had no effects on volume, incubated osmotic fragility, or monovalent cation composition of ATP-depleted red cells. The drug increased 45Ca uptake by ATP-depleted red cells but did not change the fraction of membrane- bound calcium. Sodium dodecyl sulfate acrylamide gel electrophoresis of membrane proteins from ATP-depleted red cells revealed formation of high molecular weight protein complexes, which were not formed when biconcave shape and ATP content were maintained by incubation with adenine (0.54 mM) and inosine (12.7 mM); Formation of these complexes was not prevented when the biconcave shape was maintained by procaine HCl. It was concluded that the maintenance of the biconcave shape and normal deformability during ATP depletion by procaine HCl was not related to a displacement of membrane-bound calcium and inhibition of ATP-dependent rearrangement of red cell membrane proteins.

N.m.r. studies of red cells

1980 ◽  
Vol 289 (1037) ◽  
pp. 395-406 ◽  
Keyword(s):  
Small Molecules ◽  
Time Course ◽  
Pulse Sequence ◽  
Spin Echo ◽  
Cell System ◽  
Red Cells ◽  
Red Cell ◽  
Lactate And Pyruvate ◽  
Echo Pulse ◽  
2,3 Dpg

Recent n.m.r. studies of intact red cells are described. With 1 H n.m.r. the normal high resolution spectra of red cells, even at high fields, are relatively uninformative because the very large number of resonances from the cells merge into a broad envelope. If a simple 90- τ - 180° spin echo pulse sequence is used, however, many resonances can be resolved. These include signals from haemoglobin histidines, glutathione, lactate and pyruvate. 13 C and 31 P signals have also been seen with a spectrometer converted to observe these nuclei essentially simultaneously. N.m.r. is well suited to monitor the time course of events after a perturbation of the cell system. Lactate increase, glutathione recovery after oxidation and alkylation of glutathione by iodoacetate can all be observed directly in red cell suspensions by means of 1 H spin echo n.m.r. This method has also been used to measure isotope exchange ( 1 H - 2 H) of lactate and of pyruvate at both the C-3 and the C-2 positions, and some of these exchange rates can be interpreted in terms of the activity of specific enzymes in the cells. 1 H spin echo n.m.r. has also been used to obtain information about the transport rates of small molecules into cells. By means of the 13 C / 31 P spectrometer and [ 13 C-1] glucose, the 13 C enrichment of 2,3-diphosphoglycerate (2,3-DPG) can be monitored at the same time as the levels of 2,3-DPG, ATP and inorganic phosphate are observed by 31 P n.m.r.

AG-348 Activation of Pyruvate Kinase in Vivo Enhances Red Cell Glycolysis in Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4010-4010
Author(s):  
Charles Kung ◽  
Collin Hill ◽  
Yue Chen ◽  
Abhishek Jha ◽  
Penelope Kosinski ◽  
...  
Keyword(s):  
Single Dose ◽  
Pyruvate Kinase ◽  
Whole Blood ◽  
Red Cells ◽  
Red Cell ◽  
Atp Levels ◽  
Glycolytic Activity ◽  
Label Incorporation ◽  
2,3 Dpg

Abstract Pyruvate kinase deficiency (PKD) is an autosomal recessive enzymopathy that is the most common cause of hereditary nonspherocytic hemolytic anemia (HNSHA). PKD is a rare disease characterized by a life-long chronic hemolysis with severe co-morbidities. It is hypothesized that insufficient energy production to maintain red cell membrane homeostasis promotes the chronic hemolysis. Treatment is generally palliative, focusing on the resultant anemia, and there are no approved drugs that directly target mutated pyruvate kinase. AG-348 is an allosteric activator of the red cell isoform of pyruvate kinase (PKR) that has recently entered Phase I clinical trials in normal healthy volunteers. AG-348 increases the catalytic efficiency and enhances the protein stability of a spectrum of recombinantly expressed PKR mutant proteins that have been associated with PKD. PKD red cells are characterized by changes in metabolism associated with defective glycolysis, including a build-up of the upstream glycolytic intermediate 2,3-DPG and deficiency in the PKR product adenosine triphosphate (ATP). PKR flux, e.g. the rate of carbon flow through the PKR enzyme reaction, was examined in PKD patient or wild type (WT) donor blood samples by incubation of whole blood with a stable isotope tracer, [U-13C6]-glucose. At various time points after the addition of [U-13C6]-glucose, metabolism was quenched and metabolites were extracted. Metabolite pool sizes and 13C label incorporation into glycolytic intermediates were monitored by LC/MS. The rate of label incorporation was found to be significantly slower in PKD patient red cells, consistent with decreased glycolytic activity. Treatment of PKD red cells with AG-348 ex-vivo induces changes in metabolism consistent with increased glycolytic activity including reduced 2,3-DPG levels, increased ATP levels, and increased PKR enzyme activity levels. The effect of AG-348 on red cell metabolism in vivo was evaluated in mice. C57/BL6 mice were dosed by oral gavage either with a single dose, or with multiple doses (BID) of AG-348 for 7 days. Dose levels tested were 1 mpk, 10 mpk, 50 mpk, and 150 mpk. Following the last dose, mice were bled to evaluate drug exposure and pharmacodynamic markers including 2,3-DPG and ATP levels, and PKR activity. AG-348 was demonstrated to be a well-behaved compound, with dose-proportional increase in exposure, both in the single-dose and multiple dose studies. A single dose of AG-348 resulted in a dose-dependent increase in PKR activity levels, concomitant with reduction in 2,3-DPG levels. There were no significant changes in ATP levels after a single administration of AG-348. In the multiple-dose studies, similar changes in PKR activity and 2,3-DPG levels were observed. In contrast to the single-dose study, ATP levels were observed to be robustly increased in a dose-dependent manner. The effect of AG-348 on PKR flux was assessed in whole blood from mice treated with AG-348. C57BL/6 mice were dosed by oral gavage with AG-348 (150 mg/kg twice daily [BID]) for 3 days. Whole blood was incubated with [U-13C6]-glucose and the metabolite pool sizes and rate of 13C label incorporation into glycolytic intermediates were assessed. The data were subsequently analyzed using a mathematical model to quantify flux through the PKR reaction and it was determined that AG-348 treatment significantly increased flux through the PKR reaction. Collectively, these data demonstrate that AG-348 not only potently binds to and activates the PKR enzyme in vivo, but this enzyme activation induces enhanced glycolytic pathway activity in red cells that results in profound changes in cellular metabolism, as reflected in dramatically increased ATP levels and reduced 2,3-DPG levels. As AG-348 has similar potency against the WT PKR enzyme as against tested mutant PKR enzymes in vitro, these data support the hypothesis that AG-348 treatment may similarly enhance glycolytic activity in PKD patients and thus correct the underlying pathology of PKD. Figure 1 Figure 1. Disclosures Kung: Agios Pharmaceuticals: Employment, Stockholder Other. Hill:Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Jha:Agios Pharmaceuticals: Employment, Stockholder Other. Kosinski:Agios Pharmaceuticals: Employment, Stockholder Other. Clasquin:Agios Pharmaceuticals: Employment, Stockholder Other. Si:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Agios Pharmaceuticals: Employment, Stockholder Other. Hixon:Agios Pharmaceuticals: Employment, Stockholder Other. Dang:A: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. Silverman:Agios Pharmaceuticals: Employment, Stockholder Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other.

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