U.J. Dumaswala

Protein and lipid oxidation of banked human erythrocytes:: Role of glutathione

In banked human erythrocytes (RBCs), biochemical and functional changes are accompanied with vesiculation and reduced in vivo survival. We hypothesized that some of these changes might have resulted from oxidative modification of membrane lipids, proteins, or both as a result of atrophy of the antioxidant defense system(s). In banked RBCs, we observed a time-dependent increase in protein clustering, especially band 3; carbonyl modification of band 4.1; and malondialdehyde, a lipid peroxidation product. Examination of the antioxidative defense system showed a time-dependent decline in glutathione (GSH) concentration and glutathione-peroxidase (GSH-PX) activity, with a concomitant increase in extracellular GSH, cysteine, and homocysteine, and unchanged catalase activity. When subjected to acute oxidant stress by exposure to ferric/ascorbic acid or tert-butylhydroperoxide (tert-BHT), catalase activity showed a steeper decline compared with GSH-PX. The results demonstrate that GSH and GSH-PX appear to provide the primary antioxidant defense in stored RBCs, and their decline, concurrent with an increase in oxidative modifications of membrane lipids and proteins, may destabilize the membrane skeleton, thereby compromising RBC survival.

Erythrocyte Membrane Vesiculation and Changes in Membrane Composition during Storage in Citrate-Phosphate-Dextrose-Adenine-1

Abstract. Serial studies were made of the membranes of the erythrocytes and the vesicles shed during storage of blood in polyvinyl chloride containers for 35 days in citrate‐phosphate‐dextrose‐adenine anticoagulant. Special precautions were taken to eliminate artifacts created by contaminating leukocytes, platelets and red blood cell ghosts. A total of 15.6% of the cholesterol and 5.2% of the phospholipids of the membranes was lost with no gross change in the gel electrophoretic patterns. The quantity of vesicles found in the supernatant plasma increased during storage and their membranes were characterized by the absence of spectrin, ankyrin, and periodic acid Schiff bands 2 and 3. The ratio of lipids to protein in the vesicles increased as they accumulated perhaps reflecting a rearrangement of the erythrocyte membrane constituents during prolonged maintenance at 4°C.

Studies in Red Blood Cell Preservation: 4. Plasma Vesicle Hemoglobin Exceeds Free Hemoglobin

Abstract. Studies were designed to find out how much of the plasma hemoglobin (Hb) in whole blood was in microvesicles and how much was free Hb after 21 days of storage in citrate‐phosphate‐dextrose anticoagulant and to determine the effect of the plasticizer, di‐(2‐ethylhexyl)phthalate (DEHP). The total plasma Hb in polyolefin (PO) containers without DEHP was much higher than in polyvinyl chloride (PVC) with the plasticizer (p=0.004). Less than 30% of the Hb was in free solution in either type of container. The addition of 300 μg/ml of DEHP to the plasma in the PO containers resulted in marked reduction in the microvesiculation (p<0.01) but did not affect the level of free Hb. RBC hypotonic fragility and morphology scores were significantly improved. It is concluded that microvesiculation contributes more to plasma Hb concentration than free Hb during storage.

Glutathione loading prevents free radical injury in red blood cells after storage.

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1–6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4–6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (14C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (∼ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.

Studies in Red Blood Cell Preservation

The purpose of this study was to examine whether vesiculation of RBC plays a significant role in their rejuvenation. Outdated units of Adsol® blood, were divided into two aliquots and incubated with equal volumes of a solution of 100 mM pyruvate and inosine, 103 mM phosphate and 5 mM adenine (PIPA) or 0.9% saline. Following 1 h incubation, vesicles were isolated from the supernatants and quantitated for hemoglobin content. Restoration of RBC ATP, 2,3‐DPG, morphology, and osmotic fragility after rejuvenation was satisfactory. The postrejuvenation mean corpuscular volumes (88.2+6.9 fl) were significantly lower (p<0.001) than the prerejuvenation (94.6+6.8 fl) and control (104.0+7.3 fl) volumes. The hemoglobin shed in vesicles during rejuvenation was significantly greater than in the saline controls (0.44+0.31 vs. 0.18+0.10 mg/dl RBCs; p = 0.026). These data suggest that the decreased MCV following rejuvenation is in part due to membrane loss in exocytic vesiculation.

Studies in Red Blood Cell Preservation 1. Effect of the Other Formed Elements

Abstract. The purpose of this study was to determine if the removal of most of the leukocytes and platelets would affect the in vitro characteristics of stored red blood cells (RBC). Fresh RBC concentrates prepared by removing platelet‐rich plasma and filtration through Imugard IG 500 filters were compared with unmanipulated units after storage for up to 56 days. The filtered units were significantly better after 56 days storage for supernatant K+ (p = 0.001), hemolysis (p = 0.05), total vesicle membrane protein shed (p = 0.03), and RBC morphology score (p = 0.04). These differences occurred even though ATP levels were well maintained in both groups. The measurements that did not differ significantly were pH, hematocrit, ATP, 2,3‐DPG, glucose and supernatant Na+. It is suggested that enzymes, leukotrienes, catecholamines and eicosanoids released by degenerating leukocytes and platelets may be inimical to RBC. Some may act as agonists on a‐adrenergic and cholinergic muscarinic receptors present on RBC membranes.

Biochemical and structural changes in RBCs stored with different plasticizers: the role of hexanol

BACKGROUND: PVC containers are plasticized with di(2‐ethyl)hexylphthalate (DEHP) or a related phthalate. The toxicity of DEHP has been questioned. It has been proposed to use butyryltrihexylcitrate (BTHC) as the plasticizer. The purpose of this study was to determine if hexanol, a component of BTHC, plays a role in the preservation of RBCs stored in BTHC‐plasticized PVC bags.

Studies in Red Blood Cell Preservation 2. Comparison of Vesicle Formation, Morphology, and Membrane Lipids during Storage in AS-1 and CPDA-1

Abstract. The changes in morphology, the quantitative changes in membrane lipids and the shedding of exocytic vesicles by red blood cells (RBC) stored for 42 and 56 days in AS‐1 and CPDA‐1 were compared. RBC stored in AS‐1 shed significantly less vesicle membrane cholesterol, phospholipid and protein and maintained better morphology scores. RBC membrane cholesterol remained higher after 56 days in AS‐1 than in CPDA‐1. The data suggest that during the first weeks of storage cholesterol is lost from the RBC membrane followed by a larger release of phospholipids accompanied by alterations in the phosphoinositides. The shedding of exocytic vesicles appears to be secondary to the changes in morphology resulting from the perturbation of the membrane lipids.

Studies in red blood cell preservation. 3. A phosphate-ammonium-adenine additive solution.

Abstract. The maintenance of adenosine triphosphate (ATP) in red blood cells (RBC) during storage is largely dependent on the integrity of glycolytic metabolism in the Embden‐Meyerhof pathway. Meryman et al. [Transfusion 1986;26:500–505] hypothesized that a solution that increased the surface tension of the corpuscles through hypotonic swelling might retard the development of echinocytosis and membrane loss by the shedding of exocytic vesicles. Unexpectedly, maintenance of good ATP levels and satisfactory RBC survivals were found for as long as 18 weeks. The purpose of our study was to test their observations and to explore the possible mechanisms. Equal parts of units of packed RBC were stored in the experimental preservative and, for comparison, in ADSOL®. The most notable findings were ATP values at 4 weeks averaging 5.2 μmol/g Hb (130% of initial) and at 12 weeks 2.9 μmol/g Hb (73% of initial), whereas these values declined as expected in ADSOL. Mean RBC diameters and surface areas by morphometric analysis were not significantly different in the two preservatives indicating the absence of any hypotonic swelling. The morphology scores of the RBC were significantly better throughout (p<0.05) than in ADSOL. The shedding of exocytic hemoglobin‐containing vesicles was essentially the same in both preservatives. Our data confirm the observation that ATP levels are well maintained for at least 12 weeks, but do not show any evidence that hypotonic swelling was a part of the mechanism.

The effect of hypotonicity, glutamine, and glycine on red cell preservation.

BACKGROUND: Red cells (RBCs) stored in hypo‐os‐molar additive solutions with the same concentrations of adenine, dextrose, mannitol, and sodium chloride and varied amounts of ammonium, phosphate, glycerol, and glutamine were better preserved than RBCs in the standard additive solution (Adsol). Cell swelling occurred in all the experimental additives. This observation prompted the evaluation of glutamine and glycine alone, as well as a combination of glutamine and glycine, all of which have been described as producing swelling of rat liver cells.