Red Cell ATP Levels Research Articles

Erythrocyte phosphoglycerate kinase deficiency: enzymatic and oxygen binding studies.

Enzyme and oxygen transport studies have been made on seven members of a kindred with red cell deficiency of phosphoglycerate kinase (PGK), an enzyme known to be linked to the X-chromosome. The two affected males were children, had little detectable PGK activity and had severe neurological damage. Both had moderate haemolytic anaemia complicated by the occurrence of haemolytic crises. Red cell ATP levels were substantially decreased in both subjects. However, they had more than double the normal level of 2,3-diphosphoglycerate (2,3-DPG) in their red cells, associated with a rightward shift in their oxygen-haemoglobin equilibrium curves. This shift in the curve was sufficient to permit oxygen delivery to most body tissues, including the brain, at better tensions than normal, except during a haemolytic crisis. Two of the three known heterozygotes were aged over 70. They had moderate haemolytic anaemia, considerable reduction of red cell PGK activity (19% and 35% of normal, respectively), and considerable increase in red cell 2,3-DPG with rightward shifting of the oxygen-haemoglobin equilibrium curve. The other known heterozygote was aged 22 and showed little abnormality in her blood oxygen transport.

Hexokinase Deficiency in Erythrocytes: A New Variant in 5 Members of a Finnish Family

15 cases of congenital haemolytic anaemia have thus far been attributed to hexokinase (HK) deficiency in erythrocytes. We report some clinical, biochemical and genetic findings from 5 members of a Finnish family with this deficiency. The proband, a 1-year-old girl, was the only patient with anaemia. All subjects had either mild or marked reticulocytosis. Red cell ATP levels were at the lower range of normal in all subjects and 2,3-DPG was abnormally low in one. The activities of red cell enzymes, other than HK, were within or above the normal range, respectively. The Km-values for glucose and fructose were elevated (ATP normal) in the subjects with HK deficiency. We speculate that the family represents heterozygosity of a mutant allele and that there is phenotypic variation associated with the HK mutant. The locus might be subject to mutations which lead to a variety of HK variants and to a spectrum of diseases. This point of view is in accordance with the overwhelming variation of reaction kinetics and metabolic effects of this and other reported cases.

Reduced blood O2 affinity associated with air breathing in osteoglossid fishes

Respiratory properties of blood in two closely related species of tropical freshwater teleosts, the osteoglossids Arapaima gigas, an obligatory air breather, and Osteoglossum bieirrhosum, an exclusive water breather, have been compared. Additionally, the O2 uptake rates (VO2) and breathing responses to hypoxic water were compared.The two species had similar values of hematocrit, hemoglobin (Hb) concentration, and O2, capacity, these being 28%, 7.5 g/100 ml, and 11.0 ml/100 ml for Osteoglossum and 30.8%, 7.5 g/100 ml, and 10.4 ml/100 ml for Arapaima. Red cell ATP and GTP levels were also similar in both species.A striking difference existed in the O2 affinity of red cell suspensions with a P50 value of 6.1 mmHg (1 mmHg = 133.322 Pa) at pH 7.4 (28 °C) for Osteoglossum against 21.0 mmHg for Arapaima. The difference persisted after purification and stripping of the hemoglobins of their red cell cofactors. We conclude that the O2-binding properties of the hemoglobin molecules in the two species are intrinsically different. The low O2 affinity in the air-breathing Arapaima permits O2 unloading at relatively high [Formula: see text]'s thus supporting a high [Formula: see text]. The high O2 affinity in Osteoglossum is necessary for Hb to function in O2 transport from a very hypoxic medium.

Metabolic Dependence of Protein Arrangement in Human Erythrocyte Membranes. I. Analysis of Spectrin-rich Complexes in ATP-depleted Red Cells

Abstract The discocyte-echinocyte transformation and the decrease in deformability associated with red cell ATP depletion have been attributed to changes in the physical properties of spectrin and actin, membrane proteins located at the membrane-cytosol interface. We investigated the spontaneous formation of spectrin-rich complexes in human erythrocyte membranes, employing two-dimensional SDS- polyacrylamide gel electrophoresis. Membranes of red cells depleted in ATP under aerobic conditions exhibited (1) an increase in components 4.5 and 8 and globin subunits, (2) a spontaneous formation of heterodimers of spectrin 1 + 2 and spectrin 2 + component 4.9, and (3) a large molecular weight (greater than 10(6) daltons) protein complex with a high spectrin to band 3 ratio. These complexes were dissociated with dithiothreitol and were prevented by anaerobic incubation or the maintenance of red cell ATP and GSH levels with glucose, adenine, and inosine. The complexes 1 + 2 and 2 + 4.9 were also seen in acetylphenylhydrazine-treated, glucose-6-phosphate dehydrogenase- deficient fresh erythrocytes that showed marked GSH depletion but preserved greater than 70% of the original ATP level. However, membranes of these cells did not contain the greater 10(6) dalton aggregate with a high spectrin to band 3 ratio. We concluded that the formation of the latter complex results from rearrangement of spectrin and other polypeptides in membranes of ATP-depleted red cells. Under aerobic conditions, the rearranged proteins undergo spontaneous intermolecular crosslinkings through disulfide couplings.

Metabolic dependence of protein arrangement in human erythrocyte membranes. I. Analysis of spectrin-rich complexes in ATP-depleted red cells

The discocyte-echinocyte transformation and the decrease in deformability associated with red cell ATP depletion have been attributed to changes in the physical properties of spectrin and actin, membrane proteins located at the membrane-cytosol interface. We investigated the spontaneous formation of spectrin-rich complexes in human erythrocyte membranes, employing two-dimensional SDS-polyacrylamide gel electrophoresis. Membranes of red cells depleted in ATP under aerobic conditions exhibited (1) an increase in components 4.5 and 8 and globin subunits, (2) a spontaneous formation of heterodimers of spectrin 1 + 2 and spectrin 2 + component 4.9, and (3) a large molecular weight (greater than 10(6) daltons) protein complex with a high spectrin to band 3 ratio. These complexes were dissociated with dithiothreitol and were prevented by anaerobic incubation or the maintenance of red cell ATP and GSH levels with glucose, adenine, and inosine. The complexes 1 + 2 and 2 + 4.9 were also seen in acetylphenylhydrazine-treated, glucose-6-phosphate dehydrogenase-deficient fresh erythrocytes that showed marked GSH depletion but preserved greater than 70% of the original ATP level. However, membranes of these cells did not contain the greater 10(6) dalton aggregate with a high spectrin to band 3 ratio. We concluded that the formation of the latter complex results from rearrangement of spectrin and other polypeptides in membranes of ATP-depleted red cells. Under aerobic conditions, the rearranged proteins undergo spontaneous intermolecular crosslinkings through disulfide couplings.

Physiologic effects of hyperventilation and phlebotomy in baboons: systemic and cerebral oxygen extraction.

Eighteen anesthetized baboons were studied to determine the effects of passive hyperventilation and phlebotomy on oxygen transport. After 1 hour of hyperventilation a significant increase in the red cell affinity for oxygen occurred in vivo. This was not associated with any significant changes in cardiac output, oxygen consumption, or in lactic acid production. There was a 40% decrease in cerebral blood flow, a 10 mm Hg decrease in the pulmonary artery Po2 level, and a 17 mm Hg decrease in the jugular venous Po2 level. After 1 hour of hyperventilation, the plasma inorganic phosphorus level decreased significantly, the red cell ATP level decreased slightly, and the red cell 2. 3 DPG level increased significantly, indicating that inorganic phosphorus had been removed from the blood during hyperventilation. Passive hyperventilation was maintained, and the baboons were bled 32% of their red cell volume. The blood volume was partially restored with nonbuffered isotonic saline. One hour after the phlebotomy and volume restoration (2 hours of hyperventilation) there were no changes in oxygen consumption, cardiac output, cerebral blood flow, or blood lactate levels, but the pulmonary artery Po2 level was decreased by 15 mm Hg, and the jugular venous Po2 level was decreased by 20 mm Hg. Systemic oxygen consumption was not affected by the significant decrease in pulmonary artery Po2.

Preservation of red blood cell 2,3-diphosphoglycerate in stored blood containing dihydrozyacetone.

In a search for red blood cell metabolites which would preserve 2,3‐DPG during storage of blood, it was discovered that dihydroxyacetone (DHA) prolonged the maintenance of 2,3‐DPG levels for up to four weeks of storage, compared to about one week for presently used preservatives such as CPD. Four‐week preservation of 2,3‐DPG at normal levels was desired. CPD‐adenine was used as the starting point and a formulation having a pH of 7.0 and a DHA concentration of 20 millimoles per liter of blood was developed. The 2,3‐DPG level at four weeks of storage was proportional to DHA concentration in the 5 to 20 mM range. The osmotic fragility, red blood cell ATP levels, and plasma sodium, potassium, and hemoglobin during four weeks of storage in CPD‐adenine‐DHA were similar to those in blood stored in CPD‐adenine.

Haematological changes in heat-acclimated golden hamsters.

Summary. Haematological changes were investigated in heat‐acclimated hamsters. Haemoglobin, packed‐cell volume, and red‐cell counts were not affected by heat acclimation. Glucose uptake and osmotic fragility in the red blood cells of these animals were increased. The red cell ATP level was lower in heat‐acclimated than in control hamsters. No change was observed in the density distribution of the red cell population in heat‐acclimated hamsters. The resemblance of these cells to the red cells of hereditary spherocytosis and pyruvate‐kinase deficiency is discussed.

Some effects of pH and temperature upon normal human red cell adenosine triphosphate.

Red cell ATP levels were determined on the fresh blood of 58 normal subjects using an enzymic method. Values were obtained in the range 80 to 140 μmoles of ATP per 100 ml. of packed red cells.Conditions for obtaining reliable fresh blood ATP values are reported together with the effects of different anticoagulants.It was confirmed that red cell ATP is pH‐sensitive. Thus, at pH values below 7.6–7.7 (25° C.) it was stable but at values above this range it disappeared from the red cells. Kinetic studies of red cell ATP at different temperatures and pH values are described.

Red cell ATP and malaria infection.

SEVERAL genetic polymorphisms involving haemoglobin and glucose-6-phosphate dehydrogenase are thought to provide a measure of protection against human malarias1. These are all single gene traits, but quantitative variables within the human red cell may also be of importance. Thus Brewer and Powell2 have demonstrated a strong positive correlation between the level of erythrocyte ATP and the rapidity with which a parasitaemia of 1,000 per mm3 is reached during experimental infections of humans with falciparum malaria. The higher the level of red cell ATP, the more rapidly parasitaemia develops, at least in the early stages of infection. This study was designed to investigate the relationship between red cell ATP level and the development of parasitaemia in a different species infected with a different form of malaria.

A Comparison of Normal Red Cell ATP Levels as Measured by the Firefly System and the Hexokinase System

Abstract Widely divergent normal red cell ATP levels have been reported by investigators using different methods. In order to clarify the cause of these discrepancies and to establish correct normal values for red cell ATP, the firefly technic for measuring ATP levels was compared with other methods. The ATP content of TCA filtrates of red cells was the same when determined by the firefly method as by the hexokinase-linked technic. Relatively low concentrations of protein were found to stimulate light output when lyophilyzed firefly extract, but not freshly prepared firefly extract, was used. Thus, falsely, high values were obtained when red cell extracts were examined, unless protein was also added to the standard. Storage of heparinized blood for as little as 1 hour resulted in a substantial decrease in red cell ATP levels. The loss with ACD blood was less, and could be obviated entirely by using an ACD solution with a pH adjusted to between 3.5 and 4.0. Removing the buffy coat or washing cells in saline resulted in no further loss of red cell ATP. Extraction of washed red cells with TCA resulted in an average loss of 4.6 per cent of ATP, while extraction of whole blood with TCA resulted in a 14 per cent loss of ATP. In contrast, perchloric acid extraction resulted in no ATP loss. If ATP determinations are carried out using the firefly method, protein should be added to the standard. If red cells must be stored for any period of time prior to extraction of ATP, an ACD solution with a pH of 3.5 to 4.0 should be used. If extracts of red cells are made, perchloric acid appears significantly superior to trichloroacetic acid.

Damage to Red Cells Due to Their Collection and Storage in ACD

Experiments were performed using aliquots of the same blood incubated at 37 C. in spinner flasks under 95%N2 – 5% CO2. Various anticoagulant systems and pH's were compared, using the rate of glycolysis and ATP levels as criteria of metabolic activity. Citrate and lower pH's were detrimental to glycolysis and the maintenance of red cell ATP levels. Non‐citrate systems that were closer to normal blood pH were superior to ACD, by the criteria used in this in vitro study. The addition of phosphate, as in the citrate‐phosphate‐dextrose system, appeared beneficial.