Red Blood Cell Adenosine Triphosphate Research Articles

Pyruvate kinase activators: targeting red cell metabolism in sickle cell disease.

Hemoglobin S (HbS) polymerization, red blood cell (RBC) sickling, chronic anemia, and vaso-occlusion are core to sickle cell disease (SCD) pathophysiology. Pyruvate kinase (PK) activators are a novel class of drugs that target RBC metabolism by reducing the buildup of the glycolytic intermediate 2,3-diphosphoglycerate (2,3-DPG) and increasing production of adenosine triphosphate (ATP). Lower 2,3-DPG level is associated with an increase in oxygen affinity and reduction in HbS polymerization, while increased RBC ATP may improve RBC membrane integrity and survival. There are currently 3 PK activators in clinical development for SCD: mitapivat (AG-348), etavopivat (FT-4202), and the second-generation molecule AG-946. Preclinical and clinical data from these 3 molecules demonstrate the ability of PK activators to lower 2,3-DPG levels and increase ATP levels in animal models and patients with SCD, as well as influence a number of potential pathways in SCD, including hemoglobin oxygen affinity, RBC sickling, RBC deformability, RBC hydration, inflammation, oxidative stress, hypercoagulability, and adhesion. Furthermore, early-phase clinical trials of mitapivat and etavopivat have demonstrated the safety and tolerability of PK activators in patients with SCD, and phase 2/3 trials for both drugs are ongoing. Additional considerations for this novel therapeutic approach include the balance between increasing hemoglobin oxygen affinity and tissue oxygen delivery, the cost and accessibility of these drugs, and the potential of multimodal therapy with existing and novel therapies targeting different disease mechanisms in SCD.

Rho-kinase inhibition improves haemodynamic responses and circulating ATP during hypoxia and moderate intensity handgrip exercise in healthy older adults.

Skeletal muscle haemodynamics and circulating adenosine triphosphate (ATP) responses during hypoxia and exercise are blunted in older (OA) vs. young (YA) adults, which may be associated with impaired red blood cell (RBC) ATP release. Rho‐kinase inhibition improves deoxygenation‐induced ATP release from OA isolated RBCs. We tested the hypothesis that Rho‐kinase inhibition (via fasudil) in vivo would improve local haemodynamic and ATP responses during hypoxia and exercise in OA. Healthy YA (25 ± 3 years; n = 12) and OA (65 ± 5 years; n = 13) participated in a randomized, double‐blind, placebo‐controlled, crossover study on two days (≥5 days between visits). A forearm deep venous catheter was used to administer saline/fasudil and sample venous plasma ATP ([ATP]V). Forearm vascular conductance (FVC) and [ATP]V were measured at rest, during isocapnic hypoxia (80% SpO2), and during graded rhythmic handgrip exercise that was similar between groups (5, 15 and 25% maximum voluntary contraction (MVC)). Isolated RBC ATP release was measured during normoxia/hypoxia. With saline, ΔFVC was lower (P < 0.05) in OA vs. YA during hypoxia (∼60%) and during 15 and 25% MVC (∼25–30%), and these impairments were abolished with fasudil. Similarly, [ATP]V and ATP effluent responses from normoxia to hypoxia and rest to 25% MVC were lower in OA vs. YA and improved with fasudil (P < 0.05). Isolated RBC ATP release during hypoxia was impaired in OA vs. YA (∼75%; P < 0.05), which tended to improve with fasudil in OA (P = 0.082). These data suggest Rho‐kinase inhibition improves haemodynamic responses to hypoxia and moderate intensity exercise in OA, which may be due in part to improved circulating ATP. Key points Skeletal muscle blood flow responses to hypoxia and exercise are impaired with age. Blunted increases in circulating ATP, a vasodilator, in older adults may contribute to age‐related impairments in haemodynamics.Red blood cells (RBCs) are a primary source of circulating ATP, and treating isolated RBCs with a Rho‐kinase inhibitor improves age‐related impairments in deoxygenation‐induced RBC ATP release.In this study, treating healthy older adults systemically with the Rho‐kinase inhibitor fasudil improved blood flow and circulating ATP responses during hypoxia and moderate intensity handgrip exercise compared to young adults, and also tended to improve isolated RBC ATP release.Improved blood flow regulation with fasudil was also associated with increased skeletal muscle oxygen delivery during hypoxia and exercise in older adults.This is the first study to demonstrate that Rho‐kinase inhibition can significantly improve age‐related impairments in haemodynamic and circulating ATP responses to physiological stimuli, which may have therapeutic implications.

Albumin Glycation Affects the Delivery of C-Peptide to the Red Blood Cells.

Serum albumin is a prominent plasma protein that becomes modified in hyperglycemic conditions. In a process known as glycation, these modifications can change the structure and function of proteins, which decrease ligand binding capabilities and alter the bioavailability of ligands. C-peptide is a molecule that binds to the red blood cell (RBC) and stimulates the release of adenosine triphosphate (ATP), which is known to participate in the regulation of blood flow. C-peptide binding to the RBC only occurs in the presence of albumin, and downstream signaling cascades only occur when the albumin and C-peptide complex contains Zn2+. Here, we measure the binding of glycated bovine serum albumin (gBSA) to the RBC in conditions with or without C-peptide and Zn2+. Key to these studies is the analytical sample preparation involving separation of BSA fractions with boronate affinity chromatography and characterization of the varying glycation levels with mass spectrometry. Results from this study show an increase in binding for higher % glycation of gBSA to the RBCs, but a decrease in ability to deliver C-peptide (0.75 ± 0.11 nM for 22% gBSA) compared to samples with less glycation (1.22 ± 0.16 nM for 13% gBSA). A similar trend was measured for Zn2+ delivery to the RBC as a function of glycation percentage. When 15% gBSA or 18% gBSA was combined with C-peptide/Zn2+, the derived ATP release from the RBCs significantly increased to 113% or 36%, respectively. However, 26% gBSA with C-peptide/Zn2+ had no significant increase in ATP release from RBCs. These results indicate that glycation of BSA interferes in C-peptide and Zn2+ binding to the RBC and subsequent RBC ATP release, which may have implications in C-peptide therapy for people with type 1 diabetes.

Red blood cell ATP release correlates with red blood cell hemolysis.

The precise matching of blood flow to skeletal muscle during exercise remains an important area of investigation. Release of adenosine triphosphate (ATP) from red blood cells (RBCs) is postulated as a mediator of peripheral vascular tone in response to shear stress, hypoxia, and mechanical deformation. We tested the following hypotheses: 1) RBCs of different densities contain different quantities of ATP; 2) hypoxia is a stimulus for ATP release from RBCs; and 3) hypoxic ATP release from RBCs is related to RBC lysis. Human blood was drawn from male and female volunteers (n = 11); the RBCs were isolated and washed. A Percoll gradient was used to separate RBCs based on cellular density. Density groups were then resuspended to 4% hematocrit and exposed to normoxia or hypoxia in a tonometer. Equilibrated samples were drawn and centrifuged; paired analyses of ATP (luminescence via a luciferase-catalyzed reaction) and hemolysis (Harboe spectrophotometric absorbance assay) were measured in the supernatant. ATP release was not different among low-density cells versus middle-density versus high-density cells. Similarly, hemoglobin (Hb) release was not different among the red blood cell subsets. No difference was found for either ATP release or Hb release following matched exposure to normoxic or hypoxic gas. The concentrations of ATP and Hb for all subsets combined were linearly correlated (r = 0.59, P ≤ 0.001). With simultaneous probing for Hb and ATP in the supernatant of each sample, we conclude that ATP release from RBCs can be explained by hemolysis and that hypoxia per se does not stimulate either ATP release or Hb release from RBCs.

Regulation of skeletal muscle blood flow during exercise in ageing humans.

The regulation of skeletal muscle blood flow and oxygen delivery to contracting skeletal muscle is complex and involves the mechanical effects of muscle contraction; local metabolic, red blood cell and endothelium-derived substances; and the sympathetic nervous system (SNS). With advancing age in humans, skeletal muscle blood flow is typically reduced during dynamic exercise and this is due to a lower vascular conductance, which could ultimately contribute to age-associated reductions in aerobic exercise capacity, a primary predictor of mortality in both healthy and diseased ageing populations. Recent findings have highlighted the contribution of endothelium-derived substances to blood flow control in contracting muscle of older adults. With advancing age, impaired nitric oxide availability due to scavenging by reactive oxygen species, in conjunction with elevated vasoconstrictor signalling via endothelin-1, reduces the local vasodilatory response to muscle contraction. Additionally, ageing impairs the ability of contracting skeletal muscle to blunt sympathetic vasoconstriction (i.e. 'functional sympatholysis'), which is critical for the proper regulation of tissue blood flow distribution and oxygen delivery, and could further reduce skeletal muscle perfusion during high intensity and/or large muscle mass exercise in older adults. We propose that initiation of endothelium-dependent hyperpolarization is the underlying signalling event necessary to properly modulate sympathetic vasoconstriction in contracting muscle, and that age-associated impairments in red blood cell adenosine triphosphate release and stimulation of endothelium-dependent vasodilatation may explain impairments in both local vasodilatation and functional sympatholysis with advancing age in humans.

Red blood cells stored for increasing periods produce progressive impairments in nitric oxide–mediated vasodilation

Clinical outcomes in transfused patients may be affected by the duration of blood storage, possibly due to red blood cell (RBC)-mediated disruption of nitric oxide (NO) signaling, a key regulator of vascular tone and blood flow. AS-1 RBC units stored up to 42 days were sampled at selected storage times. Samples were added to aortic rings ex vivo, a system where NO-mediated vasodilation could be experimentally controlled. RBC units showed storage-dependent changes in plasma hemoglobin (Hb), RBC 2,3-diphosphoglycerate acid, and RBC adenosine triphosphate conforming to expected profiles. When freshly collected (Day 0) blood was added to rat aortic rings, methacholine (MCh) stimulated substantial NO-mediated vasodilation. In contrast, MCh produced no vasodilation in the presence of blood stored for 42 days. Surprisingly, the vasoinhibitory effects of stored RBCs were almost totally mediated by RBCs themselves: removal of the supernatant did not attenuate the inhibitory effects, while addition of supernatant alone to the aortic rings only minimally inhibited MCh-stimulated relaxation. Stored RBCs did not inhibit vasodilation by a direct NO donor, demonstrating that the RBC-mediated vasoinhibitory mechanism did not work by NO scavenging. These studies have revealed a previously unrecognized vasoinhibitory activity of stored RBCs, which is more potent than the described effects of free Hb and works through a different mechanism that does not involve NO scavenging but may function by reducing endothelial NO production. Through this novel mechanism, transfusion of small volumes of stored blood may be able to disrupt physiologic vasodilatory responses and thereby possibly cause adverse clinical outcomes.

In vivo viability of stored red blood cells derived from riboflavin plus ultraviolet light–treated whole blood

A novel system using ultraviolet (UV) light and riboflavin (Mirasol System, CaridianBCT Biotechnologies) to fragment nucleic acids has been developed to treat whole blood (WB), aiming at the reduction of potential pathogen load and white blood cell inactivation. We evaluated stored red blood cell (RBC) metabolic status and viability, in vitro and in vivo, of riboflavin/UV light-treated WB (IMPROVE study). The study compared recovery and survival of RBCs obtained from nonleukoreduced WB treated using three different UV light energies (22, 33, or 44 J/mL(RBC)). After treatment, WB from 12 subjects was separated into components and tested at the beginning and end of component storage. After 42 days of storage, an aliquot of RBCs was radiolabeled and autologously reinfused into subjects for analysis of 24-hour recovery and survival of RBCs. Eleven subjects completed the in vivo study. No device-related adverse events were observed. By Day 42 of storage, a significant change in the concentrations of sodium and potassium was observed. Five subjects had a 24-hour RBC recovery of 75% or more with no significant differences among the energy groups. RBC t(1/2) was 24 ± 9 days for the combined three groups. Significant correlations between 24-hour RBC recovery and survival, hemolysis, adenosine triphosphate (ATP), and CO(2) levels were observed. This study shows that key RBC quality variables, hemolysis, and ATP concentration may be predictive of their 24-hour recovery and t(1/2) survival. These variables will now be used to assess modifications to the system including storage duration, storage temperature, and appropriate energy dose for treatment.

Red blood cell storage lesion.

The past two decades have witnessed increased scrutiny regarding efficacy and risk of the once unquestioned therapy of red blood cell (RBC) transfusion. Simultaneously, a variety of changes have been identified within the RBC and storage media during RBC preservation that are correlated with reduced tissue oxygenation and transfusion-associated adverse effects. These alterations are collectively termed the storage lesion and include extensive biochemical, biomechanical, and immunologic changes involving cells of diverse origin. Time-dependent falls is 2,3-diphosphoglycerate, intracellular RBC adenosine triphosphate, and nitric oxide have been shown to impact RBC deformability and delivery of oxygen to the end-organ. The accumulation of biologic response modifiers such as soluble CD40 ligand (sCD40L), lysophosphatidylcholine (lyso-PC), and Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) have been associated with altered recipient immune function as well. This review will address the alterations occurring within the RBC and storage media during RBC preservation and will address the potential clinical consequence thereof.

Extended storage of whole blood before the preparation of blood components: in vitro effects on red blood cells in Erythro‐Sol environment

Routine procedures for extended storage of whole blood (WB) before the preparation of blood components are of interest primarily for logistical reasons. We stored red cell units in either Erythro-Sol 2 (E-Sol 2, test units, 150 ml added) or in saline-adenine-glucose-mannitol (SAG-M) (reference units, 100 ml added) that were prepared after storage of WB at room temperature for 8, 12, 16 or 19 h after blood collection. Red blood cells were stored for 42 days. We measured pH, glucose, lactate, haemolysis, red blood cell adenosine triphosphate and 2,3-diphosphoglycerate on days 1, 7, 14, 21, 28, 35 and 42. Haematocrits were significantly lower in E-Sol 2 than in SAG-M due to the higher volume of E-Sol 2 added compared to SAG-M. Significantly reduced levels were found in E-Sol 2 of extracellular pH (throughout storage after 8-h hold and initially after 12-, 16- or 19-h hold), of lactate (initially after 8-h hold and throughout storage after 12-, 16- or 19-h hold), and of haemolysis from day 35 in the 8-h and on day 42 in the 12-h hold group. Significantly increased levels of adenosine triphosphate were seen in E-Sol 2 after 8-h hold (from day 14) and after 12-h hold (at days 21, 35 and 42) compared to SAG-M. Significantly higher concentrations of 2,3-diphosphoglycerate were noticed primarily after 8-h hold of WB. The use of E-Sol 2 as a replacement for SAG-M does not significantly improve in vitro data after extended storage of WB at room temperature before preparation of blood components. However, after 8-h hold in vitro characteristics similar to or better than in fresh blood will be maintained for several weeks in E-Sol 2, a situation that makes E-Sol 2 superior to SAG-M when storage of WB is limited to 8 h. Some improvement was noted after 12-h hold as well.

Postoperative hypophosphataemia

The possible mechanisms of postoperative hypophosphataemia were studied in women undergoing uncomplicated cholecystectomy. Six patients were allocated to each of three groups. Group I received no intravenous fluids, group II received dextrose/saline solution alone and group III received dextrose/saline solution with phosphorus supplementation. The serum concentration of inorganic phosphorus fell in all three groups, the greatest fall being in the group receiving dextrose/saline solution. However, the lowest level reached (0.82 mmol/l) was within the normal range for our laboratory. Concentrations of red blood cell adenosine triphosphate and 2,3-diphosphoglycerate showed similar changes. The operations were not associated with a large catabolic response nor with an increase in urinary phosphorus excretion. The postoperative reduction in serum concentration of inorganic phosphorus appeared to be due mainly to haemodilution. There is no indication for routine phosphorus supplementation following uncomplicated elective surgery.

Use of a Prestorage Leukoreduction Filter Effectively Removes Leukocytes from Canine Whole Blood While Preserving Red Blood Cell Viability

Leukoreduction of blood products is a technique used to prevent leukocyte‐induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4°C) for 4 hours. Filtration efficacy was determined by measurement of pre‐ and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin‐streptavidin‐phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24‐hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.

Use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability.

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4 degrees C) for 4 hours. Filtration efficacy was determined by measurement of pre- and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin-streptavidin-phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24-hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.

Adenosine triphosphate release by osmotic shock and hemoglobin A 1C in diabetic subjects' erythrocytes

We investigated the significance of adenosine triphosphate (ATP) release from diabetic subjects' red blood cell (RBCs) following osmotic shock (OS) and its possible relationship with hemoglobin A 1c (HbA 1c) and with the RBC membrane protein skeleton. RBCs from type I (insulin-dependent [IDDM]) and type II (non-insulin-dependent [NIDDM]) diabetic subjects and age- and sex-matched control subjects were submitted to OS using NaCl solutions (from 0.9% to 0.045% final concentration). ATP release values were determined by the bioluminescent method. For pattern study, they were expressed both as absolute values and as percentages (%) of ATP maximum release (at 0.045% NaCl solution). Twenty-seven IDDM and 25 NIDDM subjects and two control groups were investigated. ATP content in RBCs was 2.08 ± 0.19 pmol/10 4 RBC in IDDM and 1.23 ± 0.20 pmol/10 4 RBC in NIDDM subjects. The ATP content of IDDM subjects' RBCs was significantly higher than that of the corresponding control group. ATP release at 0.49% NaCl OS, both as absolute value and as percentage value, was significantly lower in both diabetic groups, and ATP% was inversely correlated with HbA 1c (IDDM: r = −.489, P < .01; NIDDM: r = −.654, P < .01), suggesting a possible relationship between Hb glycation, RBC membrane protein skeleton glycation, and its influence on ATP release by OS. In conclusion, the proposed method seems useful for measuring RBC ATP content and, at the same time, for monitoring the leak effect of the RBC membrane before it bursts.

Changes in red blood cell transmembrane potential, electrolytes, and energy content in septic shock.

Septic shock was induced in adult baboons by the infusion of live Escherichia coli. A progressive derangement in skeletal muscle cell function was documented by the direct measurement of declining transmembrane potential difference (PD). A concurrent depolarization of the red blood cell (RBC) was characterized by cellular uptake of chloride, sodium, and water, and loss of potassium. The decrease in RBC PD was significantly greater than the change predicted to occur from acidosis alone. These findings are compatible with changes in membrane permeability and decreased active transport. The continuous accumulation of RBC adenosine triphosphate during shock suggests decreased energy utilization rather than decreased energy production as a factor leading to diminished active ion transport.

Storage and survival of red blood cells with elevated sodium levels.

Approximately 25 percent of black blood donors have an elevated red blood cell (RBC) sodium (Nai) level compared with white donors. This elevation results in a significant increase in the mean Nai from black (9.00 +/- 2.96 mmoles/l RBC) as compared to white blood donors (7.04 +/- 1.48 mmoles/l RBC, p less than 0.001). Red blood cells from four black donors with mean Nai levels of 15 +/- 2.8 mmoles/l RBC were stored for 35 days in citrate-phosphate-dextrose-adenine and compared to that of four donors with normal levels of Nai. Serial measurements of red blood cell adenosine triphosphate, diphosphoglycerate, glucose-6-phosphate dehydrogenase, pyruvic kinase, lactate production rates, and intracellular cations showed no differences between the two donor groups. Furthermore, the mean 24-hour posttransfusion survival was not significantly different for the high Nai group (83.2 +/- 5.6%) as compared with the control group (82.3 +/- 6.9%). Based on this study, it is not necessary to eliminate individuals with an elevated red blood cell Nai level as blood donors.

Red Blood Cell Alkalosis and Decreased Oxyhemoglobin Affinity

The behavior of the oxyhemoglobin curve was studied in ten patients with respiratory alkalosis (arterial[H+]≤ 37 nM, pCO2 ≤ 32mmHg and HCO3−≤22.0mEq/L) and ten patients with metabolic alkalosis ([H+] ≤ 34 nM, pCO2 ≥ 37mmHg and HCO3−≥28.0mEq/L) to determine whether different alkalotic states similarly affect the red blood cell [H+] and 2,3-diphosphoglycerate interaction and thus the oxygen affinity of hemoglobin. The findings were statistically indistinguishable in respiratory alkalosis and metabolic alkalosis: a) low plasma [H+], normal red blood cell [H+], and high transmembrane [H+] gradient; b) elevated red blood cell 2,3-diphosphoglycerate inversely proportional to low arterial plasma [H+]; c) decrease in oxygen affinity of hemoglobin when normalized for plasma [H+], but less decreased when normalized for red blood cell [H+]. Other factors capable of affecting the oxygen affinity of hemoglobin were: mean corpuscular hemoglobin concentration; red blood cell adenosine triphosphate; carboxyhemoglobin; and methemoglobin were not significantly different between groups. These results: 1) agree with data previously reported from this laboratory on patients with portal-systemic encephalopathy; 2) underscore the importance of RBC [H+] in defining the oxygen affinity of hemoglobin; 3) suggest the decrease in oxygen affinity of hemoglobin is mediated through the 2,3-diphosphoglycerate elevation; and 4) indicate the high transmembrane [H+] gradient is principally due to the cellular accumulation of [H+] (2,3-diphosphoglycerate ionization).

Deformability of stored red blood cells. Relationship to degree of packing.

An ektacytometer was used to measure red blood cell deformability during blood storage for 42 days at different degrees of cell packing in citrate-phosphate-dextrose preservative. Observed changes in deformability were studied in relationship to red blood cell adenosine triphosphate (ATP) levels, lipid content, osmotic fragility, and hematocrit during storage. Decrease in whole cell deformability as measured in isotonic medium did not occur until cellular ATP levels were reduced to less than 30 percent of initial values. The rate of deformability loss during storage increased with increased degrees of cell packing because of more rapid substrate depletion. The loss in isotonic deformability was further magnified when deformability measurements were carried out in hypotonic media, suggesting that a reduction in surface area-to-volume ratio was the dominant cause of the reduced deformability of stored red blood cells. Marked spherocytosis, decreased membrane lipid content and increased osmotic fragility confirmed loss of membrane surface area during storage. The significantly higher rate of deformability loss observed in packed red blood cells suggests that careful control of storage hematocrit may be necessary to avoid loss of cellular deformability and possibly posttransfusion viability.

Plasma Adenine and Cellular ATP in Red Cell Concentrates Collected and Stored in Modified CPD at 4 C

Eight units of blood were drawn into modified CPD containing 25 per cent higher glucose and 17.3 mg adenine (0.25 mM in blood). Red blood cell concentrates (RCC) were prepared to a mean hematocrit (Hct) of 70, the cells stored at 4 C, and plasma adenine and red blood cell adenosine triphosphate (ATP) were measured weekly for 42 days. The removal of plasma in the preparation of RCC reduced by 39 per cent the available adenine. As a result measurable plasma adenine was depleted by 21 days. The loss of ATP in RCC occurs at a significantly faster rate than in whole blood stored under the same conditions. When red blood cells are stored at higher HCT or for periods longer than 35 days, increased anticoagulant adenine levels are recommended.

Cellular regulation of an allosteric modifier of fish haemoglobin.

ADENOSINE TRIPHOSPHATE (ATP) is the major organic phosphate in the red blood cells of a wide variety of fish species1–4. Its role as an allosteric modifier of fish haemoglobins is similar to that of 2,3-diphosphoglycerate (2,3-DPG) in mammalian red blood cells, in that it decreases the affinity of haemoglobin for oxygen5,6. After exposure to the anoxia of high altitude or in certain conditions of anaemia, mammals increase red blood cell 2,3-DPG to facilitate oxygen unloading at the tissues7. By contrast, eels have been shown to decrease erythrocyte organic phosphate (that is, ATP) and increase haemoglobin–oxygen (Hb–O2) affinity when acclimated to low environmental oxygen8. To test if this phenomenon was expressed in another fish species, we acclimated Fundulus heteroclitus, a euryhaline minnow, to hypoxic conditions. Our finding that this fish also lowered red blood cell ATP by as much as 40% (Fig. 1) suggests that this is a general response to hypoxia among water-breathing vertebrates.

Pyruvate kinase in malaria host-parasite interaction.

THE mammalian malaria host–parasite systems seem particularly useful for studies aimed at elucidating the biochemical mechanisms of such interactions. During the vertebrate phase of their life cycles the malaria organisms are intracellular parasites of the red cell. The mature mammalian host red cell is also relatively simple metabolically; its energy metabolism is solely that of Embden–Meyerhof glycolysis and it also lacks the capacity for protein synthesis. We therefore studied several aspects of red cell glycolysis in monkeys heavily infected with Plasmodium knowlesi and mice heavily infected with P. berghei. In both these systems we have found an increase in red blood cell adenosine triphosphate (ATP) and a decrease in red cell 2,3-diphosphoglycerate (DPG). We also have evidence that these malaria parasites introduce a pyruvate kinase isozyme into their host red cells in amounts sufficient to alter red cell glycolysis. First, glycolytic intermediate data demonstrate an in vivo increase in pyruvate kinase activity in infected red cells; second, there is an increase in pyruvate kinase Vmax activity in infected cells; and third, gel electrophoretic patterns show a new pyruvate kinase isozyme in infected cells. We suggest that this alteration in red cell glycolysis is in a direction favourable to the parasite because it will increase red cell ATP at the expense of red cell DPG.