Biochemical and Biophysical Research Communications
Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase
Human crythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded crythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the β anomer of glucose was more rapidly utilized than the α anomer in intact and unloaded crythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are α- and β-preferential, respectively, in glucose phosphorylation.
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Cited by (18)
Factors associated with the performance of carrier erythrocytes obtained by hypotonic dialysis
2004, Blood Cells, Molecules, and DiseasesCarrier erythrocytes containing drugs, enzymes or peptides can be used as a delivery system that allows changes in the kinetic behaviour and selective biodistribution of the substances encapsulated. Hypotonic dialysis is the method most commonly used in the preparation of carrier erythrocytes, but many factors affect the yield and characteristics of the ghost erythrocytes obtained using this method. This review analyses the factors that affect the performance of carrier erythrocytes prepared by hypotonic dialysis. Factors such as the composition and osmolality range of the hypotonic buffer used, the duration of the hypotonic dialysis, temperature, the volume ratio between the erythrocyte suspension and the dialysis buffer, the inclusion in the process of an annealing phase, the composition and osmolality of the resealing buffer, and the conditions under which the final washing of the erythrocytes is carried out may all affect the morphological properties and the later in vivo behaviour of the ghost erythrocytes obtained. Changes in the yield of the encapsulation process, the in vitro drug or enzyme controlled delivery, the pharmacokinetic properties or the in vivo tissue targeting may be modified depending on the conditions under which the preparation of carrier erythrocytes by hypotonic dialysis is carried out. Chemical alterations to the membrane of carrier erythrocytes obtained by hypotonic dialysis with substances such as glutaraldehyde, band 3 cross-linking reagents, trypsin or NHS-biotin, among others, may affect the release rate of the substances encapsulated and may increase the uptake of cells by macrophages both in vitro and in vivo.
Drug, enzyme and peptide delivery using erythrocytes as carriers
2004, Journal of Controlled ReleaseErythrocytes are potential biocompatible vectors for different bioactive substances, including drugs. These can be used successfully as biological carriers of drugs, enzymes and peptides. There are currently diverse methods that permit drug encapsulation in erythrocytes with an appropriate yield. The methods most commonly employed are based on a high-haematocrit dialysis procedure, mainly hypo-osmotic dialysis.
Erythrocytes loaded with drugs and other substances allow for different release rates to be obtained. Encapsulation in erythrocytes significantly changes the pharmacokinetic properties of drugs in both animals and humans, enhancing liver and spleen uptake and targeting the reticulo-endothelial system (RES).
Amongst other applications, erythrocytes have been used for drug-targeting the RES with aminoglycoside antibiotics; the selective transport to certain organs and tissues of certain antineoplastic drugs, such as methotrexate, doxorubicine, etoposide, carboplatin, etc.; the encapsulation of angiotensin-converting enzyme (ACE) inhibitors, systemic corticosteroids, the encapsulation of new prodrugs with increased duration of action, etc.
Erythrocytes are also attractive systems in the sense of their potential ability to deliver proteins and therapeutic peptides. Thus, erythrocytes have been used for the transport of enzymes destined for the correction of metabolic alterations as l-asparaginase, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH) among others.
Erythrocytes have been used successfully as carriers of anti-HIV peptides, such as AZT, nucleoside analogues, antisense oligonucleotides, antineoplastic peptides, erythropoietin, interleukin 3, etc.
Amongst other applications, mention may be made of paramagnetic erythrocytes, encapsulation of MRI contrast agents or the study of the metabolism of the red cell.
Although erythrocytes have been applied with different uses in human medicine, their deployment is still very limited due to difficulties involving storage, its exposure to contamination and the absence of a validated industrial procedure for its preparation.
Stimulatory effect of fatty acid treatment on glucose utilization in human erythrocytes
1997, Biochimica et Biophysica Acta - General SubjectsWe previously reported that treatment of human erythrocytes with bee venom phospholipase A2 increased the rate of lactate production from glucose. This increase was suggested to be mediated through liberation of free fatty acids from membrane phospholipids. So, in the present study we examined the mechanism of stimulation of glycolysis by fatty acids. Treatment of intact erythrocytes with most of the 15 fatty acids tested resulted in stimulation of lactate production from glucose. Among the fatty acids tested, myristoleic acid showed the highest stimulatory activity. The ratio of moles of lactate produced to those of glucose utilized was about 1.9 in both myristoleic acid-treated and untreated cells. Treatment of erythrocytes with myristoleic acid did not affect the amount of 2,3-bisphosphoglycerate. Lactate production from d-glyceraldehyde, which is thought to be phosphorylated to d-glyceraldehyde 3-phosphate and then metabolized in the glycolytic pathway, was not at all affected by treatment of cells with myristoleic acid. The cross-over plot of glycolytic intermediates suggested that the enhancement of glycolysis was induced by activation of the 6-phosphofructokinase (PFK) step. Fatty acids incorporated into erythrocytes were found to be present predominantly in the cytoplasm rather than in the plasma membrane. The PFK activity, but not the hexokinase activity, in hemolysates was clearly increased by a set of fatty acids, and myristoleic acid was again the most potent. However, partially purified human erythrocyte PFK was not activated by the acid. We conclude that fatty acids stimulate glycolysis through activation of PFK in cooperation with some other component(s) in erythrocytes.
4-Hydroxy-2-nonenal hardly affects glycolysis
1997, Free Radical Biology and Medicine4-Hydroxy-2-nonenal (HNE), one of the major products of lipid peroxidation, inactivated the rate-limiting enzymes (from animal sources) of the glycolytic pathway and the pentose phosphate pathway when incubated at 37°C for 1 h in the absence of glutathione (GSH). The HNE concentration for half-maximal inactivation of 6-phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase was 3–10 μM; and that value for pyruvate kinase, glucose-6-phosphate dehydrogenase, and hexokinases I and II was 0.15-0.6 mM. In the presence of 5 mM GSH, however, only PFK, irrespective of the source (muscle, liver, or erythrocyte), was inactivated by 40–-50% when incubated with 0.1 mM HNE for 1 h. Even PFK was not inactivated in the presence of both GSH and its substrate, ATP (2 mM). Glycolysis in human erythrocytes was not affected by treatment of cells with 0.1 mM HNE at 37°C for 30 min. The results suggest that HNE, at concentrations observable under physiological and pathological conditions, hardly affects glycolysis in cells.
Hexose-6-kinases in germinating honey locust cotyledons: Substrate specificity of d-fructo-6-kinase
1994, PhytochemistryExtracts of the cotyledons of germinated honey locust (Gleditsia triacanthos) seeds, which contain galactomannan as a reserve polysaccharide in the endosperm, were fractionated by chromatography and the fractions examined for the presence of a specific manno-6-kinase which could phosphorylate the d-mannose released by hydrolysis of galactomannan. One particulate hexokinase (the major hexose-6-kinase fraction) and two soluble hexokinase fractions (the minor portion), as well as a soluble fructo-6-kinase fraction, were initially separated. From chromatography, electrophoresis and kinetic studies, no evidence for a specific manno-kinase was obtained. This and the level and kinetic behaviour of the particulate hexokinase implicated it as the enzyme catalysing the phosphorylation of released d-mannose. The fructo-kinase activity was further separated into three fractions. Kinetic studies on one of these with native and synthetic substrates indicated that the structural requirements for the monosaccharide substrate were a β-d-anomeric 2-OH in the furanose ring, a 4-OH trans to the d-5-CH2OH and a -CH2OH substituent on C2 (trans to the 5-CH2OH) which could be modified. The orientation of the hydroxyl on C-3 had only a limited effect.
cAMP, ethanol, and co<inf>2</inf> production with the addition of d-glucose anomer to starved yeast cells
1994, Biochemical and Biophysical Research CommunicationscAMP, ethanol, and CO2 production in starved yeast cells after the addition of D-glucose anomer was measured and compared over a wide range of anomer concentrations. At 1.0 g/l or higher concentrations, the addition of β-D-glucose resulted in a higher cAMP peak, β-D-glucose was more rapidly metabolized to ethanol and CO2 than α-D-glucose, although there was no notable difference in the uptake rates of the two anomers. At 0.4 g/l D-glucose anomer, the differences in cAMP and ethanol production rates for the two anomers were not significant. At 0.2 g/l D-glucose anomer or lower concentrations, ethanol production with α-D-glucose was higher than that with β-D-glucose. The uptake rate of α-D-glucose was higher than that of β-D-glucose at this low concentration.