CAN THE P50 OF RED BLOOD CELLS BE MODULATED WHILE PRESERVING THE BOHR EFFECT?

Abstract Most of the 1,300 human hemoglobin (Hb) variants are characterized by normal O2 binding, including here the highly prevalent Hbs S [HBB:c.20A>T; p.Glu6Val] and C [HBB:c.19G>A; p.Glu6Lys]. Hb S and Hb C carriers are generally asymptomatic and might be admitted as blood donors. However, the functional efficiency of these variants in standard blood bags - red blood cells (RBC) concentrates - under the interference of anticoagulant and storage is not well established. In this prospective study, three samples of RBC concentrates (phenotypes AA, AS and AC), with citrate phosphate dextrose adenine (CPDA-1) as anticoagulant, were analyzed for equilibrium tests and compared to fresh Hb A without anticoagulant. The Hb samples were extracted after 48 hours and 35 days of storage and purified by exclusion and ion exchange chromatography (Sephadex G-25 and Amberlite MB-3 columns in Hepes buffer without NaCl). O2 affinity was evaluated by spectrophotometry-tonometry method, by determining the p50 in stripped lysates ([Hb]=70 μM/Heme), in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) in all studied pHs. The activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. The p50 of Hb A after 35 days in stored blood bag was decreased in all pHs, in both presence and absence of IHP, when compared to fresh Hb A, suggesting that the anticoagulant and the storage might affect HbA-O2 binding, increasing its affinity for the ligand. No other differences were observed: heme-heme cooperativity and Bohr effect were similar to fresh Hb A without anticoagulant. The same was found when stored Hb AS was compared to Hb A from blood bag (35 days storage, in the stripped state and under the interference of IHP). However, the stripped lysate containing Hb AC revealed an important reduction of the O2 affinity after both 48 hours and 35 days of storage, mainly at pHs ³ 7, thus interfering in the Bohr effect. The heme-heme cooperativity resulted in normal n coefficients, indicating that the Hb-O2 ligation remained cooperative, in spite of the significant reduction of O2 affinity. Similar results were observed in the presence of IHP (48 hours and 35 days), although Bohr effect was reestablished, indicating that the supplementation with a 2,3-BPG-like allosteric effector might restore some Hb-ion interactions, and therefore, Hb C function. As the 2,3-BPG is consumed with storage of RBC concentrates, Hb AC showed an even lower affinity for O2 after 35 days of storage. Although still preliminary, our results indicate a significant functional reduction of Hb C in RBC concentrates in standard blood bag (CPDA-1 anticoagulant), proportional to the time of storage. Considering that the screening for hemoglobinopathies in the blood banks routine is mainly focused in the detection of Hb S, a greater understanding about the properties of native Hb and its most frequent variants in stored RBC concentrates could also contribute with the determination of safe parameters in hemotherapy based on the functional efficiency of each hemoglobin. Financial Support: Fapesp, CNPq, Capes, Faepex-Unicamp. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Enhancement of the O2 Release Capacity and of the Bohr-Effect of Human Red Blood Cells after Incorporation of Inositol Hexaphosphate by Fusion with Effector-Containing Lipid Vesicles

Hemoglobin and Oxygen Binding ◽

10.1007/978-1-349-06488-5_39 ◽

1982 ◽

pp. 277-282 ◽

Cited By ~ 1

Author(s):

Klaus Gersonde ◽

Claude Nicolau

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Lipid Vesicles ◽

Bohr Effect ◽

Inositol Hexaphosphate ◽

Human Red Blood Cells ◽

Release Capacity

Download Full-text

Respiratory characteristics of Hoplostenum littorale (Siluriformes, Callichthyidae)

Acta Amazonica ◽

10.1590/1809-43922001312262 ◽

2001 ◽

Vol 31 (2) ◽

pp. 249-249 ◽

Cited By ~ 7

Author(s):

Elizabeth Gusmão AFFONSO

Keyword(s):

Red Blood Cells ◽

Whole Blood ◽

Blood Cells ◽

Hematological Parameters ◽

Bohr Effect ◽

South American ◽

Starch Gel Electrophoresis ◽

Different Seasons ◽

Starch Gel ◽

2,3 Dpg

Hematological parameters, intraerythrocytic phosphates, hemoglobin, and whole blood Bohr effect of the South American armored catfish Hoplostenum littorale were studied during different seasons of the year. In addition, the degree of dependence on air breathing was determined for this species. The hematological parameters presented seasonal variations, which were not correlated to oxygen, temperature, and water level oscillations. Five anodic hemoglobin fractions were detected in starch gel electrophoresis. In addition to ATP, GTP and Fe-GTP being detected, 2,3-DPG was also detected in red blood cells of H. littorale. The latter is an intraerythrocytic phosphate characteristic to red blood cells of mammalians. The increased production of 2,3-DPG could be associated with decreasing Hb-O2 affinity and both features could be related to environmental temperature increase. Whole blood Bohr effect was influenced by water temperature. This study confirms H. littorale to be continuous and not obligate air breather, under all dissolved oxygen level conditions.

Download Full-text

Hemoglobin crystals of the red blood cells in the human renal cortex: electron microscopic observations of the renal lithiasis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083266 ◽

1978 ◽

Vol 36 (2) ◽

pp. 480-481

Author(s):

Kosuke Ueda ◽

Hiroto Washida ◽

Nakazo Watari

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Renal Cortex ◽

Uranyl Acetate ◽

Osmic Acid ◽

Renal Lithiasis ◽

Electron Microscopic ◽

Hemoglobin C ◽

First Case ◽

Ultrathin Sections

IntroductionHemoglobin crystals in the red blood cells were electronmicroscopically reported by Fawcett in the cat myocardium. In the human, Lessin revealed crystal-containing cells in the periphral blood of hemoglobin C disease patients. We found the hemoglobin crystals and its agglutination in the erythrocytes in the renal cortex of the human renal lithiasis, and these patients had no hematological abnormalities or other diseases out of the renal lithiasis. Hemoglobin crystals in the human erythrocytes were confirmed to be the first case in the kidney.Material and MethodsTen cases of the human renal biopsies were performed on the operations of the seven pyelolithotomies and three ureterolithotomies. The each specimens were primarily fixed in cacodylate buffered 3. 0% glutaraldehyde and post fixed in osmic acid, dehydrated in graded concentrations of ethanol, and then embedded in Epon 812. Ultrathin sections, cut on LKB microtome, were doubly stained with uranyl acetate and lead citrate.

Download Full-text

Densitometric Identification of Primitive Erythroblasts After Reaction With Diaminobenzidine

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072083 ◽

1973 ◽

Vol 31 ◽

pp. 328-329

Author(s):

John A. Trotter

Keyword(s):

Red Blood Cells ◽

Analytical Method ◽

Blood Cells ◽

Guinea Pigs ◽

Specific Protein ◽

Visual Examination ◽

Hemoglobin Synthesis ◽

Peroxidatic Activity ◽

Small Density

Hemoglobin is the specific protein of red blood cells. Those cells in which hemoglobin synthesis is initiated are the earliest cells that can presently be considered to be committed to erythropoiesis. In order to identify such early cells electron microscopically, we have made use of the peroxidatic activity of hemoglobin by reacting the marrow of erythropoietically stimulated guinea pigs with diaminobenzidine (DAB). The reaction product appeared as a diffuse and amorphous electron opacity throughout the cytoplasm of reactive cells. The detection of small density increases of such a diffuse nature required an analytical method more sensitive and reliable than the visual examination of micrographs. A procedure was therefore devised for the evaluation of micrographs (negatives) with a densitometer (Weston Photographic Analyzer).

Download Full-text

Scanning electron microscopy of erythrophagocytosis by Entamoeba histolytica trophozoites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012480x ◽

1992 ◽

Vol 50 (1) ◽

pp. 880-881

Author(s):

Victor Tsutsumi ◽

Adolfo Martinez-Palomo ◽

Kyuichi Tanikawa

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Red Blood Cells ◽

Entamoeba Histolytica ◽

Causative Agent ◽

Blood Cells ◽

Protozoan Parasite ◽

Complex Phenomenon ◽

Great Capacity ◽

Scanning Electron

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis in man. The trophozoite or motile form is a highly dynamic and pleomorphic cell with a great capacity to destroy tissues. Moreover, the parasite has the singular ability to phagocytize a variety of different live or death cells. Phagocytosis of red blood cells by E. histolytica trophozoites is a complex phenomenon related with amebic pathogenicity and nutrition.

Download Full-text

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162132 ◽

1990 ◽

Vol 48 (3) ◽

pp. 914-915

Author(s):

D.J.P. Ferguson ◽

A.R. Berendt ◽

J. Tansey ◽

K. Marsh ◽

C.I. Newbold

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Blood Cells ◽

Umbilical Vein ◽

Cell Types ◽

Amelanotic Melanoma ◽

Cytoplasmic Process ◽

Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

Download Full-text