Mechanisms of ascorbic acid recycling in human erythrocytes (original) (raw)
Related papers
Journal of Biological Chemistry, 2004
Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD-and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.
Recycling of the ascorbate free radical by human erythrocyte membranes
Free Radical Biology and Medicine, 2001
Reduction of the ascorbate free radical (AFR) at the plasma membrane provides an efficient mechanism to preserve the vitamin in a location where it can recycle ␣-tocopherol and thus prevent lipid peroxidation. Erythrocyte ghost membranes have been shown to oxidize NADH in the presence of the AFR. We report that this activity derives from an AFR reductase because it spares ascorbate from oxidation by ascorbate oxidase, and because ghost membranes decrease steady-state concentrations of the AFR in a protein-and NADH-dependent manner. The AFR reductase has a high apparent affinity for both NADH and the AFR (Ͻ 2 M). When measured in open ghosts, the reductase is comprised of an inner membrane activity (both substrate sites on the cytosolic membrane face) and a trans-membrane activity that mediates extracellular AFR reduction using intracellular NADH. However, the trans-membrane activity constitutes only about 12% of the total measured in ghosts. Ghost AFR reductase activity can also be differentiated from NADH-dependent ferricyanide reductase(s) by its sensitivity to the detergent Triton X-100 and insensitivity to enzymatic digestion with cathepsin D. This NADH-dependent AFR reductase could serve to recycle ascorbic acid at a crucial site on the inner face of the plasma membrane.
Erythrocyte defenses against hydrogen peroxide: the role of ascorbic acid
Biochimica et Biophysica Acta (BBA) - General Subjects, 1998
. Ascorbate has been reported to increase intracellular hydrogen peroxide H O generation in human erythrocytes. In the 2 2 present work, the basis for this prooxidant effect of the vitamin was investigated in the context of erythrocyte defenses against H O . Ascorbate added to erythrocytes caused a dose-dependent increase in intracellular H O , which was 2 2 2 2 Ž . measured as inactivation of endogenous catalase in the presence of 3-amino-1,2,4-triazole aminotriazole . Ascorbate-induced catalase inactivation was not observed when only the intracellular ascorbate concentration was increased, when cells were incubated with ascorbate in plasma, or when extracellular Fe 3q was chelated. Together, these results suggest that the observed ascorbate-induced H O generation is due to Fe 3q -catalyzed oxidation of extracellular, as opposed to intracellular, 2 2 ascorbate by molecular oxygen. Rather than generate an oxidant stress in erythrocytes, ascorbate was one of the most sensitive intracellular antioxidants to H O coming from outside the cells. On the other hand, intracellular ascorbate 2 2 contributed little to the detoxification of H O , which was found to be mediated by both catalase and by the GSH system. 2 2 q 1998 Elsevier Science B.V. Ž .
Journal of Biological Chemistry, 2006
Human erythrocytes contain an unidentified plasma membrane redox system that can reduce extracellular monodehydroascorbate by using intracellular ascorbate (Asc) as an electron donor. Here we show that human erythrocyte membranes contain a cytochrome b 561 (Cyt b 561 ) and hypothesize that it may be responsible for this activity. Of three evolutionarily closely related Cyts b 561 , immunoblots of human erythrocyte membranes showed only the duodenal cytochrome b 561 (DCytb) isoform. DCytb was also found in guinea pig erythrocyte membranes but not in erythrocyte membranes from the mouse or rat. Mouse erythrocytes lost a majority of the DCytb in the late erythroblast stage during erythropoiesis. Absorption spectroscopy showed that human erythrocyte membranes contain an Asc-reducible b-type Cyt having the same spectral characteristics as recombinant DCytb and biphasic reduction kinetics, similar to those of the chromaffin granule Cyt b 561 . In contrast, mouse erythrocytes did not exhibit Asc-reducible b-type Cyt activity. Furthermore, in contrast to mouse erythrocytes, human erythrocytes much more effectively preserved extracellular Asc and transferred electrons from intracellular Asc to extracellular ferricyanide. These results suggest that the DCytb present in human erythrocytes may contribute to their ability to reduce extracellular monodehydroascorbate.
Vox Sanguinis, 2017
Background and Objectives Red blood cells (RBCs) suffer from lesions during cold storage, depending in part on their ability to counterbalance oxidative stress by activating their antioxidant defence. The aim of this study was to monitor the antioxidant power (AOP) in erythrocyte concentrates (ECs) during cold storage. Materials and Methods Six ECs were prepared in saline-adenine-glucose-mannitol (SAGM) additive solution and followed during 43 days. The AOP was quantified electrochemically using disposable electrode strips and compared with results obtained from a colorimetric assay. Haematological data, data on haemolysis and the extracellular concentration of uric acid were also recorded. Additionally, a kinetic model was developed to extract quantitative kinetic data on the AOP behaviour. Results The AOP of total ECs and their extracellular samples attained a maximum after 1 week of storage prior to decaying and reaching a plateau, as shown by the electrochemical measurements. The observed trend was confirmed with a colorimetric assay. Uric acid had a major contribution to the extracellular AOP. Interestingly, the AOP and uric acid levels were linked to the sex of the donors. Conclusion The marked increase in AOP during the first week of storage suggests that RBCs are impacted early by the modification of their environment. The AOP behaviour reflects the changes in metabolism activity following the adjustment of the extracellular uric acid level. Knowing the origin, interdonor variability and the effects of the AOP on the RBCs could be beneficial for the storage quality, which will have to be further studied.
Effect of ascorbic acid on storage of Greyhound erythrocytes
American journal of veterinary research, 2015
OBJECTIVE To assess changes in biochemical and biophysical properties of canine RBCs during cold (1° to 6°C) storage in a licensed RBC additive solution (the RBC preservation solution designated AS-1) supplemented with ascorbic acid. SAMPLE Blood samples from 7 neutered male Greyhounds; all dogs had negative results when tested for dog erythrocyte antigen 1.1. PROCEDURES Blood was collected into citrate-phosphate-dextrose and stored in AS-1. Stored RBCs were supplemented with 7.1mM ascorbic acid or with saline (0.9% NaCl) solution (control samples). Several biochemical and biophysical properties of RBCs were measured, including percentage hemolysis, oxygen-hemoglobin equilibrium, and the kinetic rate constants for O2 dissociation, carbon monoxide association, and nitric oxide dioxygenation. RESULTS Greyhound RBCs stored in AS-1 supplemented with ascorbic acid did not have significantly decreased hemolysis, compared with results for the control samples, during the storage period. CON...
Ascorbate Recycling by Erythrocytes During Aging in Humans
Rejuvenation Research, 2009
Erythrocytes play a crucial role in recycling ascorbate in blood plasma. The erythrocyte ascorbate free radical (AFR) reductase is involved in the reduction of AFR to ascorbic acid (ASC) in the plasma. In the present study, we report an age-dependent increase in the activity of erythrocyte AFR reductase in humans that shows a significant positive correlation with the activity of plasma membrane redox system (PMRS). We explain the agedependent increase in erythrocyte ASC recycling on the basis of a compensatory/protective mechanism that operates to maintain the ASC level in plasma and thereby minimize oxidative stress during aging.
Vitamin C in mouse and human red blood cells: An HPLC assay
Analytical Biochemistry, 2012
Although vitamin C (ascorbate) is present in whole blood, measurements in red blood cells (RBCs) are problematic because of interference, instability, limited sensitivity, and sample volume requirements. We describe a new technique using HPLC with coulometric electrochemical detection for ascorbate measurement in RBCs of humans, wild-type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below the detection threshold (25 nM). Twenty microliters of whole blood or 10 μl of packed RBCs was sufficient for assay. RBC ascorbate was stable for 24 h from wholeblood samples at 4 °C. Processed, stored samples were stable for >1 month at −80 °C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R = 0.8 and 0.9, respectively). In healthy humans, RBC ascorbate concentrations were 9-57 μM, corresponding to ascorbate plasma concentrations of 15-90 μM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately sevenfold and decreased 50% after 6 weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in RBCs. Keywords Ascorbic acid; Vitamin C; Red blood cells; Electrochemical detection; High-pressure liquid chromatography Ascorbate (ascorbic acid, vitamin C) was first described to be present in whole blood more than 70 years ago [1,2]. However, spectrophotometric assays for ascorbate in red blood cells (RBCs) 2 were subject to multiple types of interference [3-5]. As a consequence of inconsistency and lack of reliability, ascorbate concentrations in RBCs were controversial and considered inaccurate [3,6,7]. Despite the uncertainties, based on estimates from several investigators a fixed value for RBC ascorbate was taken as either as 19.9 [2,8,9] or 28.9 μM [10]. Although the former value appeared in older hematology textbooks [11], the *
Human Physiology, 2014
Effects of strict 105 day isolation on the blood antioxidant status, processes in erythrocyte mem branes, and oxygen binding properties of hemoglobin were studied in six male volunteers (25 to 40 years old) in ground based simulation of a mission to Mars (Mars 105 experiment). The study was performed with venous blood samples and red blood cells isolated from them, which were collected during the baseline data collection period, on days 35, 70, and 105 of the experiment, and on days 7 and 14-15 after its completion. Biochemical (determination of enzyme activity and thin layer chromatography) and biophysical (laser inter ference microscopy and Raman spectroscopy) methods showed changes in the relative content of lipid and phospholipid fractions, suggesting an increase in the membrane microviscosity and the content of TBA RP (active lipid peroxidation products interacting with thiobarbituric acid). A significant increase in the activity of glucose 6 phosphate dehydrogenase and superoxide dismutase and a reduction in the catalase activity was found, which indicates both reparative processes in red blood cells and imbalance between the amount of generated reactive oxygen species and antioxidant protection mechanisms in cells. The hemoglobin affinity for oxygen and the blood level of oxyhemoglobin also increased. It is assumed that the adaptation of the body to stresses experienced during and after the experiment can disturb the balance between the antioxidant defense systems. The latter, in turn, leads to peroxidation of membrane phospholipids, alteration in their con tent, increase in membrane microviscosity, and eventual disturbance of the gas exchange function of red blood cells.