Erythrocyte ATP Research Articles

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.

The vascularly and luminally perfused small intestine in vitro: dissection technique and model system

The quantitative study in vitro of a wide range of intestinal functions is best achieved by perfusing the intestine simultaneously by the luminal and vascular routes. A comprehensive description is given of the dissections involved in preparing a selected segment of jejunum for such simultaneous perfusions. Attention is drawn to a number of occasional aberrant vascular structures which require special attention if successful perfusions are to be established. It is essential to restore the ATP and diphosphoglycerate concentrations of erythrocytes, and to assess their deformability, before incorporating them in the vascular perfusion medium. The perfusion equipment, the perfusion media and the general conduct of a perfusion experiment, are described.

S-adenosylmethionine increases erythrocyte ATP in vitro by a route independent of adenosine kinase

The mechanism by which S-adenosylmethionine (SAM) and adenosine (Ado) increase ATP levels in intact human erythrocytes in vitro has been compared. The use of erythrocytes from healthy controls and from subjects totally deficient in adenine phosphoribosyltransferase (APRT), plus inhibitors of adenosine kinase (AK) and adenosine deaminase (ADA) separately and together, has enabled us to demonstrate that this increment in ATP levels occurred via totally different metabolic routes. The results show that: (i) whilst the Ado-induced increment in ATP was AK dependent, that produced by SAM was independent of AK; and (ii) the SAM-induced increment in ATP was totally dependent on APRT and that some of the increment produced by Ado might also be APRT dependent. The above data are consistent with the metabolism of SAM to ATP by a route recently identified by us whereby ATP is formed from deoxyadenosine: namely binding to the enzyme S-adenosylhomocysteine hydrolase with subsequent release of adenine and further conversion to ATP via APRT.

31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg 2+ in human erythrocytes

Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly tha oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg 2+ ([Mg 2+](infree) in human erythrocytes are 225 μmol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 μmol/l at an oxygen saturation of 38.1% based on the separation between the α- and β-ATP peaks. [Mg 2+] free in the erythrocytes decreased to 104 μmol/l at high 2,3-DPG concentration of 25.4 mmol/l re blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg 2+] free to 61 μmol/l. These results indicate, that the 31P-NMR determined [Mg 2+] free in huma erythrocytes, based solely on the separation of the α-n and β-ATP peaks, does not give a true measure of intracellular free Mg 2+ changes with different oxygen saturation level. Furthermore the measurement is influenced by the concentration of the Mg 2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discprepancies in the literature regarding [Mg 2+] free.

Effects of exercise and adrenaline on equine erythrocyte atp content

To investigate the claim that equine erythrocytes released from the spleen are older cells than those found at rest in the circulation, the 2,3, diphosphoglycerate (2,3 DPG), adenosine triphosphate (ATP) and creatine concentration of erythrocytes before and following splenic emptying were examined. Normal values for thoroughbreds (43) and ponies (10) at rest were established. Following either exercise or intravenous injection of adrenaline in six thoroughbreds, there was an increase in erythrocyte creatine content and a decrease in ATP concentration. Exercise produced a slight increase in 2,3 DPG while no change occurred with adrenaline. In two splenectomised ponies adrenaline only produced a decrease in erythrocyte ATP, with no change in creatine or 2,3 DPG content. It was concluded that erythrocytes released from the spleen are not aged cells. However, the stimuli used produced a decrease in ATP content which may affect the properties of the erythrocytes.

Studies on the energy metabolism of opossum ( didelphis virginiana) erythrocytes—VI. De novo purine nucleotide biosynthesis is limited to the final steps of the pathway in vitro

1. 1. High pressure liquid radiochromatography was used to show the incorporation of [ 14C] formate with Z-compounds into ATP and GTP in opossum erythrocytes. 2. 2. The use of Z-riboside with [ 14C] formate resulted in more extensive labeling of ATP than the Z-base/[ 14C] formate combination as substrates for nucleotide biosynthesis 3. 3. Substantial accumulation of ZMP and ZTP, but no ZDP was detected in the chromatograms. 4. 4. ATP was unstable in red cells metabolizing in the presence of Z-compounds under an atmosphere of air as gas phase in these experiments.

Responses of juvenile and sexually mature two-summer-old salmon ( Salmo salar L.) to prolonged swimming

Two-summer-old juvenile and sexually mature Atlantic salmon were forced to swim for 0.5, 1, 2, 4, 8 or 12 h at 50 cm/s (ca. 2.5 body length/s). Collapsed fish and fish swimming the whole test time were sampled for physiological analyses. Both the juvenile and mature fish exhibited an initial stress response to increased flow, as shown by the elevated plasma cortisol and lactate concentrations and by the changes in erythrocytic ATP and chloride concentrations. The physiological state of the mature males recovered well within 2–4 h of swimming. In contrast, the juveniles had continuously high plasma cortisol and lactate values with a large individual variation, indicating that the energy production for swimming was partially anaerobic. The collapsed fish showed clear signs of anaerobic metabolism. The results indicate that the aerobic capacity for prolonged swimming is lowered in juvenile salmon also beyond the time of smolt migration, whereas the mature males have a swimming capacity similar to wild parr. However, the responses of juvenile salmon to swimming in autumn are not similar to those observed on migrating smolt.

NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell

During the maturation process reticulocytes lose their intracellular organelles and undergo changes in membrane lipid composition and ion transport properties. While several reports indicate differences in the levels of magnesium, sodium and calcium in reticulocytes and erythrocytes, controversy remains concerning the actual magnitude and direction of ionic alterations during reticulocyte maturation. One problem with all of these studies is that the techniques used are invasive and are limited to measuring only the total cell ion content. We have used 31P, 23Na and 19F nuclear magnetic resonance (NMR) spectroscopy to compare the intracellular free ion and phosphometabolite levels in guinea pig reticulocytes and mature red blood cells. In contrast to a sharply decreased concentration of ATP in erythrocytes in comparison to reticulocytes, the intracellular free magnesium, measured using 31P-NMR, was increased by about 65% upon maturation (150 mumol/l cell water in reticulocytes in comparison to 250 mumol/l cell water in erythrocytes). Sizeable but opposite changes in intracellular sodium (5.5 mumol/ml cells in reticulocytes vs. 8.5 mumol/ml cells in erythrocytes) and intracellular free calcium (99 nM vs. 31 nM in reticulocytes and mature red cells, respectively) were also observed, suggesting that alterations in the kinetics of membrane ion transport systems, accompanying changes in phospholipid and cholesterol content, occur during the process of red cell maturation. However, in contrast to dog red blood cells, there was no evidence for the presence of a Na+/Ca2+ exchanger in guinea pig reticulocytes or erythrocytes.

Erythrocyte O2 transport and metabolism and effects of vitamin B6 therapy in type II diabetes mellitus.

The effects of vitamin B6 on erythrocyte metabolism, erythrocyte hemoglobin O2 affinity (P50), and nonenzymatic glycosylation were studied in 15 Caucasian men with type II (non-insulin-dependent) diabetes mellitus. A control group of 13 healthy Caucasian men was also evaluated. Before treatment, diabetic subjects had low mean cell hemoglobin concentration values and increases in both erythrocyte 2,3-diphosphoglycerate (2,3-DPG) levels and erythrocyte hexokinase activities. Although all three of these changes are associated with a decrease in hemoglobin O2 (Hb-O2) affinity, P50 values were normal in diabetic subjects. Moreover, P50 values normalized to pH 7.4 (P50(7.4] were inversely related to the level of glycosylated hemoglobin (HbA1c). Both erythrocyte 2,3-DPG and erythrocyte ATP were also inversely related to HbA1c. Vitamin B6 nutriture, as determined by erythrocyte aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, was normal in all diabetic subjects before vitamin B6 therapy. Nonetheless, HbA1c levels decreased after 6 wk of treatment with 150 mg/day pyridoxine and increased again during placebo administration. These changes were not explained by changes in fasting blood glucose. Pyridoxine therapy also decreased P50(7.4) values and increased erythrocyte AST and ALT activities but had no effect on 2,3-DPG, ATP, or the activities of hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. These observations suggest that 1) nonenzymatic glycosylation may play a role in regulating both erythrocyte metabolism and Hb-O2 affinity in diabetic subjects, and 2) vitamin B6 therapy may modify nonenzymatic glycosylation of hemoglobin in this population.

Anticancer activities of adenine nucleotides in mice are mediated through expansion of erythrocyte ATP pools.

ATP and AMP exhibit significant anticancer activities against established footpad CT26 colon adenocarcinoma in CB6F1 mice. Adenosine, inorganic phosphate, and inorganic pyrophosphate were without such effects under identical conditions. Daily intraperitoneal injections of adenine nucleotides in large volumes of saline, starting after the tumors became palpable, resulted in inhibition of tumor growth and a few "cures." The treatment was not toxic to the host as determined by changes in body weights. Weight loss observed in animals upon progression of the fast-growing CT26 tumors was slowed markedly in adenine nucleotide-treated mice. The inhibition of weight loss in tumor-bearing mice was shown to be neither the cause nor the effect of the inhibition of tumor growth. Intraperitoneal injections of AMP or ATP but not of adenosine yielded expansions of erythrocyte ATP pools in host animals. The expanded erythrocyte ATP pools are stable over a period of hours, while slowly releasing micromolar amounts of ATP into the blood plasma compartment, leading to several-fold increases in plasma (extracellular) ATP levels. Based on previous studies in which 1-5 microM extracellular ATP effectively inhibited the growth of a variety of tumor cells in several in vitro systems, it is suggested that similar levels of ATP in blood plasma account for the anticancer activities observed in a murine host.

Competition between Li + and Mg 2+ for ATP in human erythrocytes A 31P NMR and optical spectroscopy study

We have investigated the influence of Li + on free intracellular Mg 2+ concentration in human erythrocytes by 31P NMR and optical absorbance spectroscopies. In red cells loaded with 3 mM intracellular Li +, the chemical shift separation between the α- and β-phosphate resonances of MgATP 2− was approx. 0.9 ppm larger than that observed in Li +-free red cells. By analyzing the interaction of each red cell component with Mg 2+ and Li +, we found that Mg 2+ is displaced in part from MgATP 2− upon addition of Li + and that the released Mg 2+ is bound to the red cell membrane causing an overall decrease in free intracellular Mg 2+ concentration.

Erythrocyte ATP (iATP) as an indicator of neonatal hypoxia.

Neonatal hypoxia is a major cause of morbidity and mortality in new-borns (NB). Fetal asphyxia has traditionally been diagnosed and graded using clinical signs (meconium-staining, fetal bradycardia, Apgar score [1]) and biochemical parameters (pH, lactate [2]). However, the sensitivity and specificity of these indices are not optimal and more accurate methods for assessing hypoxia are needed.KeywordsUmbilical Cord BloodFetal DistressPerinatal AsphyxiaNeonatal HypoxiaFetal AsphyxiaThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Hypoxic Responses in a Fish From a Stable Environment: Blood Oxygen Transport in the Antarctic Fish Pagothenia Borchgrevinki

ABSTRACT The effects of hypoxic exposure on whole-blood oxygen-affinity were examined in the antarctic fish Pagothenia borchgrevinki. Fish exposed to for 11–14 days at −1·5°C had a P50 value of 20·6 ± 4·8 mmHg (S.D., N =13) at pH8-16, compared with 31·1 ± 4·3mmHg (N = 10) at pH8-00 for normoxic fish. Exposure to low oxygen levels resulted in a significant (66 %) rise in haemoglobin concentration, and erythrocyte [ATP] decreased by approximately 27%. There was no evidence for erythrocyte swelling. An aberrant gill morphology was observed in six fish and these showed unexpectedly high erythrocyte ATP levels. Oxygen-carrying capacity increased by approximately 40% in hypoxic fish and was correlated with a 34 % decrease in spleen mass. Despite the fact that antarctic fish have exceptionally low demands for oxygen and are unlikely ever to encounter environmental hypoxia, this antarctic fish has the necessary machinery to respond to hypoxia in a way that is typical of teleosts that naturally inhabit oxylabile environments. The ability to make short-term adaptive changes in the O2 delivery system in response to hypoxic exposure may be typical for vertebrates in general, rather than a feature seen only in those organisms which encounter environmental hypoxia on a regular basis.

Erythrocyte osmotic fragility and cation concentrations during experimentally induced bovine anaplasmosis

1. 1. The osmotic fragility, the concentrations of Na, K and Ca, the osmolality and the total ATPase activity of bovine erythrocytes from uninfected and Anaplasma marginale-infected bovines were studied in an attempt to correlate these parameters with the decrease in the cellular ATP concentration reported during bovine anaplasmosis. 2. 2. The osmotic fragility found in infected bovine erythrocytes, at 0.52% NaCl, was about two times greater than that observed in non-infected bovines. The increase in osmotic fragility was directly related to the increase in intra-erythrocytic parasitemia. 3. 3. The decrease in ATP concentration reported during bovine anaplasmosis could not be directly related to the increased fragility of these cells. The artificial depletion of erythrocytic ATP did not reproduce the same alteration in the osmotic response to NaCl. 4. 4. The plasmatic and cytoplasmatic concentrations of Na, K and Ca did not change significantly during bovine anaplasmosis, whereas the interior of the erythrocytes became hyperosmolal. 5. 5. A. marginale-infected bovine erythrocyte membranes showed an increased ATPase activity when compared to control bovines. Parasite-enriched fractions also presented ATPase activity.

Riboflavin Deficiency and Glutathione Metabolism in Rats: Possible Mechanisms Underlying Altered Responses to Hemolytic Stimuli

Riboflavin deficiency suppresses parasitic growth in malaria. Three possible mechanisms have been proposed previously to explain the survival advantage of riboflavin-deficient hosts: a) enhanced fragility of red blood cells (RBC), b) decreased formation of reticulocytes and/or c) decreased concentrations of reduced glutathione (GSH) and ATP. The validity of these proposed mechanisms was tested by investigating whether riboflavin deficiency alters the hemolytic response to three stimuli: hydrogen peroxide (H2O2), a hypotonic medium or ferriprotoporphyrin IX (FP). Reticulocyte counts and concentrations of ATP and GSH were also determined. The percentage of hemolysis induced by H2O2 or FP was significantly less in riboflavin-deficient than in control animals. By contrast, hemolytic response to a hypotonic medium was enhanced during riboflavin deficiency. Despite diminished activity of glutathione reductase and normal glutathione peroxidase activity during riboflavin deficiency, the erythrocyte concentration of GSH was increased over that in control animals. Concentrations of ATP and hemoglobin in erythrocytes as well as the reticulocyte count were unaltered during riboflavin deficiency. Thus, diminished malarial parasitemia in riboflavin-deficient animals occurs despite greater resistance of RBC to either H2O2- or FP-induced hemolysis, and in the presence of a normal reticulocyte count and erythrocytes ATP concentration. Results of this study raise the possibility that Plasmodium parasites have greater requirements for flavin coenzymes, GSH or ATP than those of host erythrocytes, which may explain the apparent protection of the riboflavin-deficient host from malaria.

Effects of brazilin on erythrocyte deformability and its related biochemical factors in streptozotocin induced diabetic rats

Impaired erythrocyte deformability was considered to play an important role in microcirculatory disturbances. We recently confirmed that brazilin, the main active principle ofCaesalpinia sappan, enhanced activity of erythrocyte deformability and reduced blood viscosity. In this study, we examined the effects of brazilin on three biochemical parameters (ATP, 2,3-diphosphoglycerate, and calcium) which influenced erythrocyte deformability. Treatment with brazilin increased erythrocyte deformability and ATP concentrations in streptozotocin-induced diabetic rats. Concentrations of 2,3-diphosphoglycerate and calcium in diabetic rats following brazilin administration were decreased significantly compared to those of diabetic control rats. The results suggest that brazilin have a potential effect to improve rheological abnormalities in diabetes.

A human cell beryllium acute toxicity assay.

A rapid, inexpensive beryllium acute toxicity assay using human erythrocyte ATP levels has been developed. The assay uses a photometric measurement of the luciferin-luciferase reaction in erythrocytes incubated in HEPES buffer with the tested toxicant. Incubation in HEPES significantly increases the sensitivity of erythrocytes to beryllium when compared to incubation in either plasma or physiological saline. After a one-hour incubation period in HEPES buffer and beryllium there is a loss of 50% of the erythrocyte ATP at 3 micrograms/ml of beryllium, and an 80% loss of ATP at 5 micrograms/ml of beryllium. The source of human erythrocytes does not appear to influence the test. Erythrocytes from 10 individuals, one with chronic beryllium disease and another with an acute sensitivity to beryllium, all gave similar biphasic dose-response curves to beryllium.

Acute exposure to graded levels of hypoxia in rainbow trout ( Salmo gairdneri): metabolic and respiratory adaptations

We have studied the mechanisms of acute hypoxia tolerance in rainbow trout ( Salmo gairdneri). Fish held at 9°C were exposed to various levels of hypoxia for 24 h. At an environmental P O 2 of 30 Torr, the fish showed an initial plasma acidosis probably of metabolic origin which was subsequently offset such that blood pH returned to normal within about 4h. Over this time period, red cell pH was maintained constant. Comparing the effects of different levels of hypoxia following 24 h exposure, oxygen consumption of the animal remained unchanged over a broad range of inspired oxygen tensions but declined by over 30% of normoxic values at inspired water P O 2 levels of 80 Torr. This appeared to be a true metabolic depression becaused signs of increased anaerobic metabolism did not occur until there was a further reduction in water oxygen levels. Rainbow trout appear to be able to maintain a relatively high energy status in their white muscle during 24 h exposure to severe hypoxia (water P O 2 = 30 Torr). As the level of hypoxia was intensified, there was a reduction in the oxygen gradient across the gills, probably facilitated in part by the release of catecholamines into the blood. The erythrocytic ATP:Hb 4 molar ratios declined with increasing hypoxic stress as did the pH gradient between the erythrocyte and plasma. The overall effect was no change in Hb O 2-affinity after 24 h exposure to severe hypoxia.

Adaptations in the capacity of ionic and osmotic regulation in young baltic salmon ( salmo salar l.) in brackish water

1. 1. One-year-old Baltic salmon were reared in fresh water (FW) and brackish water (BW, salinity 6 ppt) for their second year of life. 2. 2. After 2.5 months of adaptation, the plasma ionic and osmotic concentrations of FW and BW fish responded similarly to 48 hr exposure to deionized water (DW), FW, BW and artificial sea-water (SW, salinity 17 ppt), but there were some differences in the intracellular ion and erythrocytic haemoglobin and ATP concentrations. 3. 3. After 10 months of adaptation, the ionic and osmotic balance of SW-exposed fish was similar in FW and BW fish, but the BW fish lost more ions in DW exposure than the FW ones. 4. 4. The results indicate that the overall capacity of young salmon to regulate hyper and hypoosmotically is only slightly affected by adaptation to BW. The differences in intracellular ion and ATP concentrations may partly be related to differences in the respiratory metabolism.

Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes.

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.