Glutathione in the red blood cells of embryonic mice

A micro-method for the determination of red blood cell glutathione levels has been developed. It has been shown that, in order to obtain true values of glutathione concentrations within red blood cells, it is necessary to correct for loss of labelled glutathione and also to take account of the time taken to complete the analytical procedure. Glutathione is the major small molecular weight thiol present in embryonic red blood cells. The glutathione to haemoglobin ratio is maintained at 0.6 from 13 days gestation to adulthood in the mouse. Decay of glutathione in both adult and embryonic red blood cells can be avoided by incubation of the red blood cells in glucose containing buffers.     

Diurnal changes in the haemoglobin level, red blood cell number and mean corpuscular haemoglobin in foals during the first 13 weeks of life and in their lactating mothers

1. In six foals and their mothers (of Standard breed) diurnal changes in the haemoglobin level, red blood cell number and mean corpuscular haemoglobin during the first 13 weeks of foal life were studied. 2. Studies begun when a foal reached 7 days of age and were repeated every two weeks till 13 weeks of foal life. Blood was taken every 6 hr in foals and every 4 hr in mares. 3. No diurnal rhythmicity in parameters studied either in foals or in mares was found. 4. Decrease of haemoglobin level in lactating mares (14.0-12.5 g/100 ml) without changes in red blood cell number were observed. Mean corpuscular haemoglobin changed distinctly from one study day to another. 5. In foals were stated: significantly higher haemoglobin level and erythrocyte number and lower mean corpuscular haemoglobin than in mares.     

Deoxyuridine triphosphate nucleotidohydrolase activity and its correlation with multiplication of erythroid cells in rat spleen

The administration of phenylhydrazine to rats brought about a marked increase in the dUTPase activity in the cytosol fractions of spleen and red blood cells; the activity began to increase with a two-day lag and reached the maximum at the 5th or 6th day of the phenylhydrazine treatment (13 and 5 times the control values in total activity in the spleen and red blood cells, respectively), and then the activity decreased. The activities of thymidine kinase and sigma-aminolevulinate synthase in the spleen and red blood cells also changed in parallel with that of dUTPase. The increases of these activities were suppressed completely by methotrexate, an inhibitor of DNA synthesis. The time courses of the enzyme activity changes in the red blood cells, however, were slightly behind those in the spleen. Thus, a close correlation was assumed between the dUTPase activity and the multiplication of erythroid cells in rat spleen.     

Osmotic fragility of rat embryo red blood cells collected during intrauterine growth

Whole blood was tapped from the embryos of anaesthetized pregnant New Zealand rats using a micropipette of approximately 250 microm diameter, inserted through the uterine wall via a 5-mm incision in the anterior abdominal wall. The procedure was repeated 3 times (days 15, 17 and 19 of fetal life) in the same pregnant rats, and thereafter, twice in the surviving newborns (days 14 and 30 of neonatal life). Osmotic fragility of the red blood cells (RBC), reticulocyte counts and haemoglobin values of each sample were analysed. The fragility curve of the fetuses showed the presence of 2 types of cell population: a fragile and a more resistant type. This was correlated with the number of immature RBC (reticulocytes) in the samples. Starch gel electrophoresis of the haemoglobin revealed 4 fractions in both the fetuses and newborn animals.     

Changes in enzyme activities involved in the degradation of 1,3-bisphosphoglycerate during erythropoiesis in rat bone marrow

Rat bone marrow cells have been fractionated by density gradient in Percoll. Differential counting of erythroid cells, haemoglobin concentration and bisphosphoglycerate mutase and phosphoglycerate kinase activities have been determined in cellular fractions. As shown by means of a statistical approach, an increase in bisphosphoglycerate mutase activity and a slight decrease in phosphoglycerate kinase activity is found in erythroid cells as their haemoglobin content increases. Our results suggest that there is a synthesis of 2,3-bisphosphoglycerate during the erythropoietic process which parallels the synthesis of haemoglobin.     

Changes in the chemical composition and the enzymic activities of hepatic microsomes of the chick embryo during development

1. The values of the protein, RNA and phospholipid concentrations within the total microsomal fractions obtained from different stages of embryonic chick liver are compared. 2. Only the phospholipid content increases significantly with increasing developmental age. 3. The lack of membranes in the early stages of development and the relative constancy of RNA values during development suggests that some of the protein present at the early developmental stages is of a non-membranous non-ribosomal nature. 4. Glucose 6-phosphatase, adenosine triphosphatase, NADH(2)-cytochrome c reductase and diaphorase all increased in activity as development progressed. 5. Comparisons of submicrosomal fractions with respect to their protein, RNA and phospholipid content showed that in all embryonic stages fraction II (rough-membrane fraction) contained more than 60% of the proteins, RNA and phospholipid of the microsomal fraction. 6. Glucose 6-phosphatase was shown to be present predominantly in fraction II, whereas adenosine triphosphatase was present predominantly in fraction Iab (smooth-membrane fraction). 7. The significance of the differences between the smooth- and rough-microsomal fractions is discussed.     

Total body haematocrit iron kinetics and erythrocyte life span in pigeons (Columba livia)

1. The mean pigeon erythrocyte life span was found to be 17-25 days by Cr51-labeled erythrocytes and 21 +/- 3.4 days by iron kinetics. 2. Total red blood cell volume has been calculated by Cr51-labeled erythrocytes while total plasma volume was determined both by a dye method and iron kinetic data. From these results total blood volume and total body haematocrit were found to be 0.090 +/- 0.002 ml/g body wt and 36 +/- 4.3%, respectively. 3. Venous haematocrit, haemoglobin concentration, erythrocyte count, mean corpuscular haemoglobin concentration, plasma iron and red blood cell iron have also been measured. 4. A significant difference between total body and venous haematocrit and a short mean red blood cell life span, due to ageing and to random destruction of erythrocytes were shown. 5. The above observations are compared with analogous available data for human beings and their physiological significance is discussed.     

Separation of glycosylated haemoglobins using immobilized phenylboronic acid. Effect of ligand concentration, column operating conditions, and comparison with ion-exchange and isoelectric-focusing

Haemoglobins from diabetic and non-diabetic individuals have been separated by affinity chromatography using immobilized phenylboronate, which interacts specifically with diol-containing compounds such as glycosylated haemoglobin. The effects of ligand concentration, flow rate, column geometry, preincubation of sample, buffer composition and temperature have been investigated. Significant correlation was found between results from affinity-chromatography and ion-exchange and isoelectric-focusing methods. Isoelectric-focusing of the haemoglobin fractions obtained from affinity chromatography indicate that, in addition to haemoglobin A1c, some haemoglobin A is also bound to immobilized phenylboronic acid. Assays of haemolysates obtained from red blood cells incubated in glucose solutions suggest that unstable pre-(haemoglobin A1c) does not interfere. The assay is not affected by the presence of haemoglobin F.     

Scanning cytophotometry of carp, Cyprinm carpio L., erythrocyte populations: the influence of short-term hypoxic environment and the recovery period following severe bleeding

Scanning stage absorbance cytophotometry was used to examine haemoglobin contents in individual cells or carp erythrocyte populations. Changes in frequency distributional profiles in response to respiratory stress caused experimentally by hypoxia and/or bleeding were studied at intervals.
Histograms were drawn of haemoglobin contents of individual red blood cells in populations obtained from the same carp on four consecutive days after the fish was temporarily exposed to hypoxic environment. The modes and means of the haemoglobin values increased during the 3 days following to the stimulus, whereas a decline was measured on the 4th day. Erythrocyte counts showed the total number of red blood cells to be approximately constant during the period of investigation. On the basis of these findings, it is concluded that the mature, nucleated red blood cells of carp are capable of resuming haemoglobin synthesis after stimuli such as reducing ambient oxygen concentration.
Frequency distributional profiles covering a period of 4 weeks following severe loss of blood showed that it took 10–12 days after bleeding until masses of immature erythrocytes appeared in the peripheral blood and that there were then two distinct populations of red blood cells. In the course of about 2 weeks the precursor cells developed into mature erythrocytes.     

Organic phosphates increase the solubility of avian haemoglobin D and embryonic chicken haemoglobin.

The isolated minor haemoglobin fractions (haemoglobin D) of ostrich, chicken and duck haemoglobin, which constitute about 30% of total intracellular haemoglobin, form crystalline aggregates upon deoxygenation at physiological temperature, ionic strength and pH and at haemoglobin concentrations even well below those present in the red cell. The aggregation is reversed by oxygenation, and can be inhibited by addition of organic phosphates or the corresponding major haemoglobin fraction in a stoichiometric ratio of 1:1. Embryonic haemoglobin from chicken has similar characteristics with respect to its solubility. The results indicate close functional homology of alpha D and embryonic pi-chains as well as a novel role for organic phosphates in the regulation of haemoglobin function.     

2,3-Diphosphoglycerate Content of Human Arterial and Venous Blood

THE glycolytic intermediate, 2,3-diphosphoglycerate, is an intracellular regulator of the oxygen affinity of haemoglobin^1,2. At high altitudes there is a direct relationship between the decreased oxygen affinity of haemoglobin and the increased concentration of diphosphoglycerate in the blood³. This was explained by Benesch et al.⁴ and Chanutin et al.⁵, who found that the binding of diphosphoglycerate to haemoglobin reduces the oxygen affinity and by our finding that the concentration of diphosphoglycerate increases when the red cells are incubated under low oxygen tension^6,7, thereby releasing oxygen from haemoglobin. For the same reason, the oxygen tension is reduced during the circulation of blood from the pulmonary alveoli to the tissues; the decreased level of the diphosphoglycerate facilitates the binding of oxygen to haemoglobin in the pulmonary alveoli and the increased level of the diphosphoglycerate in the blood of the capillaries decreases the affinity of haemoglobin for oxygen. We have measured the amount of 2,3-diphosphoglycerate and other glycolytic intermediates in arterial and venous blood to test this supposition.     

Kinetics of the reaction of intraerythrocytic haemoglobin by single cell microspectroscopy: effect of shape and osmolarity

The kinetics of the reaction of CO with intraerythrocytic haemoglobin has been studied in single red blood cells (RBC) using a scanning microspectrophotometer and a photochemical perturbation method. Measurements have been carried out using red blood cells from man and camel (Camelus dromedarius), the latter at different osmotic pressures. Camel RBC, which are smaller and different in shape compared to human RBC, are known to remain intact even at an osmolarity 6-times lower than physiological (280–290 mosm/l), swelling up to twice their normal volume. The results show that the recombination time course is affected by diffusion of CO through a stagnant layer of solvent around the cell membrane, but that it is also influenced by other parameters such as intracellular diffusion of ligand and haemoglobin.

Microspectroscopy Camel hemoglobin Red blood cell Diffusion Osmolarity Cell volume     

Comparative studies on red blood cell glucose phosphorylating activities of mammals.

1. ATP-D-hexose-6-phosphotransferase activity was measured in red blood cells of man, rabbit, pig and cow. Mean values ranged from 0.60 to 1.06 units/g haemoglobin and no significant difference was obtained with different glucose concentrations. 2. The characteristics of glucose phosphorylating activities in red blood cells of the species studied were similar. 3. Chromatography on DEAE column revealed two different glucose phosphorylating activities in red cells of man, rabbit and pig, and only one in cow red cells. 4. The first hexokinase activity is the predominant form and is saturated with low glucose concentrations; the second is noticeably marked at high glucose concentrations.     

The effect of copper and zinc at neutral and acidic pH on the general haematology and osmoregulation of Oreochromis mossambicus

The objective of this study was to determine the effect of sublethal copper and zinc concentrations at a neutral and an acidic pH, on selected haematological parameters as well as on the total osmolality and electrolyte concentrations of Oreochromis mossambicus. In general, at neutral pH copper and zinc caused blood acidosis, increases in circulating white blood cell numbers, causing stimulation of the immune system, and a rapid release of red blood cells from haemopoietic tissue, as reflected in the decreases in mean corpuscular volume. Increases in red blood cells are attributed to an increase in the oxygen-carrying haemoglobin as an adaptation to altered respiratory homeostasis caused by copper and zinc. These increases are therefore a secondary reaction to the metals and not the result of direct stimulation of the haemopoietic tissue. In contrast, at an acidic pH copper and zinc concentrations usually caused blood alkalosis and decreases in white blood cell numbers, due to the bioconcentration of metals which blocks and suppresses the leucopoietic tissue. Decreases were also recorded in red blood cell counts, haemoglobin, haematocrit and mean corpuscular volume, which can be ascribed to anaemic and hypoxic conditions, gill damage and impaired osmoregulation.     

FV blood group and haemoglobin type versus haematological and blood chemical parameters in young Swiss bulls

A relationship between the FV blood group phenotype and 4 out of 45 haematological and blood chemical parameters — red cell number, mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), and serum iron — has been demonstrated in young bulls of three Swiss cattle breeds. There was also a relationship between haemoglobin type and 7 out of 45 haematological and blood chemical parameters (haemoglobin concentration, red cell number, MCV, MCHC and red cell concentrations of K⁺ and Na⁺ and their sum). In addition to expanding the species in which there is an effect of haemoglobin phenotype on MCV to include cattle, these data also demonstrate a significant correlation between their FV phenotype and MCV.     

FV blood group and haemoglobin type versus haematological and blood chemical parameters in young Swiss bulls

A relationship between the FV blood group phenotype and 4 out of 45 haematological and blood chemical parameters--red cell number, mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), and serum iron--has been demonstrated in young bulls of three Swiss cattle breeds. There was also a relationship between haemoglobin type and 7 out of 45 haematological and blood chemical parameters (haemoglobin concentration, red cell number, MCV, MCHC and red cell concentrations of K+ and Na+ and their sum). In addition to expanding the species in which there is an effect of haemoglobin phenotype on MCV to include cattle, these data also demonstrate a significant correlation between their FV phenotype and MCV.     

Alterations of glycosphingolipid-based blood group antigen expression on erythrocytes and in plasma studied on consecutive samples after a blood group O to A bone marrow transplantation.

A blood group A1Le(a-b+) individual with chronic myeloid leukaemia had received a bone marrow graft from an HLA-identical OLe(a+b-) donor. Twelve months after bone marrow transplantation (BMT), the red blood cells of the patient became agglutinable with anti-A blood group reagents. To elucidate whether the blood group A antigen expression was of plasma or of bone marrow origin, total non-acid glycosphingolipid fractions were prepared from red blood cells and plasma collected 17 months after BMT, and from plasma collected 13, 15 and 19 weeks after BMT. The glycolipid fractions were analysed by thin-layer chromatography and immunostained with monoclonal A-antibodies, and permethylated and permethylated-reduced derivatives of selected plasma samples were analysed by mass spectrometry. The results strongly indicate the presence of host bone marrow-produced blood group A red blood cells. Furthermore, the presence of a blood group H active pentaglycosylceramide type 1 (H-5-1) (Table I), characteristic for an OLe(a-b-) secretor, was seen in plasma 3-4 weeks before clinical chronic graft versus host disease (GVHD). After treatment of chronic GVHD, this expression disappeared. The blood group ALeb (A-7-1) antigen produced by the recipient seems to be present and to increase with time in all plasma samples. This also seems to be the case for the Leb and A-6-1 antigens.     

Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.