Aggregation of red cells in disease: some deductions and speculations based on results of "ARC" experiment on the space shuttle "Discovery" STS 51-C

Experiment on STS 51-C in January 1985, carried out on blood samples obtained from patients with heart disease, diabetes, hyperlipidaemia and cancer showed that, under zero gravity, the morphology of red cell aggregates aggregates was normal, in contradistinction to the parallel and simultaneous observations under 1 g, which showed large and unorientated clumps of red cells. As such clumps could be considered of disadvantage in the microcirculation and tissue perfusion, the zero gravity observations were significant in a number of ways. In particular, a preliminary deduction (subject to further zero g experimentation) was that cell-cell interaction and adhesion are affected by zero gravity, and that most likely the microarchitecture of the cell membrane is modified; and that probably the receptors, their position and/or activity, are affected by zero gravity. Of particular interest could be a possible change in the properties of the discrete surface areas which respond preferentially to specific macromolecules (or ligands). There is a dissonance between these in vitro results and theoretical deductions on flow in the microcirculations by Oka, and as well of deductions on space sickness by Dintenfass, both assuming a disabling effect of zero g on the in vivo microcirculation. This dissonance should be explored, as effect of zero g might be different on blood flow in vivo and in vitro. However, the data available from the in vitro experiment suggest that studies in immunology and oncology might be enriched by zero gravity findings; and that studies under zero gravity might open a new avenue of research in these important fields.     

A quenched-flow technique for the measurement of glucose influx into human red blood cells

A quenched-flow apparatus is described and applied to measurements of the hydrolysis of 2,4-dinitrophenyl acetate by sodium hydroxide and the entry of D-[U-14C]glucose into human red blood cells at 37 degrees C. Glucose influx into red cells was a saturable process obeying Michaelis-Menten kinetics with a Km for glucose of 6.6 +/- 0.61 mM and a maximum rate for glucose entry under "zero trans" conditions of 20.7 +/- 0.76 mmol (L cell water)-1 s-1. The technique used requires only readily available laboratory equipment and should be easily adaptable to the study of other rapid transport processes.     

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.     

Kinetics of nucleoside transport in human erythrocytes. Alterations during blood preservation

The transmembrane equilibration of radiolabeled uridine was measured by rapid kinetic techniques in human erythrocytes from freshly drawn blood and in the same cells during conventional storage of the blood as well as in cells from outdated blood. Our results confirm earlier reports that the maximum velocity of uridine equilibrium exchange (Vee) at 25 degrees C is about 30% lower in outdated than fresh red cells, whereas the opposite is the case for the Michaelis-Menten constant for equilibrium exchange (Kee), and that maximum zero-trans efflux (Vzt21) is about 4-times greater than maximum zero-trans influx (Vzt12) in outdated cells (directional asymmetry), whereas they are about the same in fresh red cells. At 25 degrees C, the nucleoside-loaded carrier of fresh cells moves on the average 6-times more rapidly than the empty carrier, whereas the differential mobility of loaded and empty carrier from outdated cells is about 15-fold. Our results also show that greater efflux than influx in outdated cells is not due to a general leakiness of outdated cells, that the differences in kinetic properties of the transporter developed during the first two weeks of blood storage and that the differences are greatly amplified when transport is measured at 5 degrees C rather than 25 degrees C. At 5 degrees C, the loaded carrier from outdated red cells moves about 325-times more rapidly than the empty carrier and maximum zero-trans efflux exceeds maximum zero-trans influx about 14-times, whereas the transport of fresh cells exhibits directional symmetry just as at 25 degrees C. The changes in kinetic properties of transport induced by temperature and storage are probably related to structural alterations in the plasma membrane and suggest that the operation of carrier is subject to modification by the membrane environment. Other results show that the kinetics of the sugar transport of human red cells is not affected in the same manner by blood storage as those of the nucleoside transporter.     

The erythrocyte sedimentation rates: some model experiments

In order to obtain a better understanding of the erythrocyte sedimentation rate (ESR), several models are presented. The first directs attention to the importance of geometrical models to represent the structure of mixtures. Here it is our intention to understand the effect of the structure on the packing of red blood cells. In this part of the study, "Cheerios" (trademark General Mills) are used as a macroscopic model. It is interesting that a random sampling of "Cheerios" has the same volume distribution curve that is found for erythrocytes with a Coulter Sizing Apparatus. In order to examine the effect of rouleaux formation, the "Cheerios" are stacked one on top of another and then glued. Rouleaux of 2,3,4,5, 7 and 10 discs were used. In order to examine a more realistic biological model, the experiments of Dintenfass were used. These investigations were performed in a split-capillary photo viscometer using whole blood from patients with a variety of diseases. The novel part of this research is the fact that the work was performed at 1g and at near zero gravity in the space shuttle "Discovery." The size of the aggregates and/or rouleaux clearly showed a dependence upon the gravity of the experiment. The purpose of this model was to examine the condition of self-similarity and fractal behavior. Calculations are reported which clearly indicate that there is general agreement in the magnitude of the fractal dimension from the "Cheerios" model, the "Discovery" experiment with those determined with the automatic sedimentimeter. The final aspect of this work examines the surface texture of the sedimention tube. A series of tubes were designed with "roughened" interiors. A comparison of the sedimentation rates clearly indicates a more rapid settling in "roughened" tubes than in ones with a smooth interior surface.     

Partial requirements forin vitro survival of human red blood cells

Some of the requirements for survival of human red blood cells were studied in vitro at 25 and 37 degrees C for 1--2 weeks. During the first week at 25 degrees C in Krebs-Ringer bicarbonate medium with glucose, the cells at 2--5% hematocrit (HCT) maintained normal K+, Na+, and water contents with negligible hemolysis. After six days ion gradients decreased, preceded by decline of ATP. With adenosine, ATP was maintained for 1--2 weeks. Sustained in vitro survival of human red blood cells at 25 or 37 degrees C requires constant pHo and sufficient substrates to support a glycolytic carbon flux as well as a nitrogen flux via nucleotide turnover. In Earle's salts buffered with HEPES and supplemented with glucose, Eagle's essential vitamins, albumin, and antibiotics, suspensions at 0.1% HCT exhibited constant pH at 7.39 +/- 0.03 for at least two weeks at 37 degrees C. With glucose alone, ATP declined steadily to negligible levels despite constant pHo, but 0.1 mM adenine supported ATP for one week. Also, several amino acids partially prevented the decline of reduced glutathione during the first week at 37 degrees C. These results and current knowledge of red cell metabolism suggest a new defined medium for experiments requiring long term incubations, and extend the characterization of human red cell in vitro survival to a time period not previously studied.     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.     

Preservation of red blood cells with purines and nucleosides. III. Synthesis of adenine, guanine, and hypoxanthine nucleotides

Purine nucleotides of fresh human red cells and of red cells during storage at 4 degrees and 25 degrees C with additions of adenine, guanine, guanosine and inosine were estimated by HPLC. Six nucleotides were found in red cells: ATP, ADP, AMP, GTP, GDP, and IMP. The adenine nucleotides represented 92 per cent of the total purine nucleotides, guanine nucleotides 7 per cent and IMP less than 1 per cent. In red cells stored with adenine the total concentration of purine nucleotides increased to 125 per cent of the normal value. An adenine-free but guanine and guanine + inosine containing medium caused a decrease of the concentration of purine nucleotides by 10 to 20 per cent. When red cells were stored without adding guanine or guanosine the content of the guanine nucleotides decreased from 0.32 to 0.17 mumol/g Hb due to the decrease in the GTP content, but the GDP concentration increased slightly. In CPD-AG blood, however, the concentration of guanine nucleotides increased considerably up to 0.6 mumol/g Hb. IMP was estimated in all investigated stored red cells. In CPD-A and in CPD-AG blood 0.4 mumol/g Hb were produced during 3 weeks of storage, but twice of that in CPD-AI blood. The principles of the synthesis and the degradation of purine nucleotides in stored red cells are discussed in detail.     

Role of membrane thermotropic properties on hypotonic hemolysis and hypertonic cryohemolysis of human red blood cells

The hypothesis of a correlation between the effects of temperature on red blood cells hypotonic hemolysis and hypertonic cryohemolysis and two thermotropic structural transitions evidenced by EPR studies has been tested. Hypertonic cryohemolysis of red blood cells shows critical temperatures at 7 degrees C and 19 degrees C. In hypotonic solution, the osmotic resistance increases near 10 degrees C and levels off above 20 degrees C. EPR studies of red blood cell membrane of a 16-dinyloxyl stearic acid spin label show, in the 0-50 degrees C range, the presence of three thermotropic transitions at 8, 20, and 40 degrees C. Treatments of red blood cells with acidic or alkaline pH, glutaraldehyde, and chlorpromazine abolish hypertonic cryohemolysis and reduce the effect of temperature on hypotonic hemolysis. 16-Dinyloxyl stearic acid spectra of red blood cells treated with glutaraldehyde and chlorpromazine show the disappearance of the 8 degrees C transition. Both the 8 degrees C and the 20 degrees C transitions were abolished by acidic pH treatment. The correlation between the temperature dependence of red blood cell lysis and thermotropic breaks might be indicative of the presence of structural transitions producing areas of mismatching between differently ordered membrane components where the osmotic resistance is decreased.     

A mechanism conjugating cellular and individual adaptations could be produced from space biology]

To know a basic mechanism of biological organism on the earth, we can have a standard point to space. An example is hindlimb suspension model that could induce muscle atrophy. This model mimics adaptational changes under zero gravity; in turn the effect of gravity on the biological system developing on the earth. We can understand gravity is a stress from the specific changes of stress protein induced by mechanical stimuli depending on gravity. Recent development of fluorescent microscopy and time-lapse visual system brought us a possibility of analysis to see visualization of dynamic properties of molecular and cellular events in living cells. Especially dynamic fluctuation of cytoskeleton may include new ideas of biological strategy of living organism on the earth and possibly may suggest subtle changes in space.     

Effect of blood admixture on in vitro survival of chilled and frozen-thawed canine spermatozoa

Hematospermia in the dog usually occurs secondary to benign prostatic hypertrophy or trauma of the penis or prepuce during semen collection. Regarding the difficulty of removing blood cells from a hematospermic sample, the present study was performed to determine whether blood contaminated ejaculates can still be chilled (4 degrees C) or frozen (-196 degrees C) without an additional decrease in sperm quality. In the first experiment, blood additions of up to 10% exerted no negative effects on the functional characteristics of canine spermatozoa cooled (4 degrees C) and stored for 4 days in an egg-yolk-Tris extender. In contrast, in experiment 2, blood admixtures of 4% or more clearly caused negative effects on cryopreserved (-196 degrees C) spermatozoa, mainly on the motility parameters, on the membrane integrity and on the acrosomal status of the spermatozoa. In experiment 3, we showed that these negative effects of blood admixture on cryopreserved spermatozoa were mainly associated with the red blood cells (RBCs) whereas the addition of plasma, serum or inactivated serum exerted little or no negative effect. Moreover, in experiment 4, we showed that 58.3+/-11.6% of the RBCs hemolysed after a freeze-thaw process. In experiment 5, a clear and negative effect of hemoglobin on cryopreserved canine spermatozoa was observed. We conclude that the presence of up to 10% blood is not detrimental for the storage of chilled canine spermatozoa and that the detrimental effects of blood on cryopreserved spermatozoa are at least partly attributable to the high amount of hemoglobin originating from the RBC hemolysis observed after freezing and thawing.     

The effect of temperature on oxygen equilibrium curves of trout blood (Salmo gairdnerii)

• 1.1. Oxygen equilibrium curves were measured on trout red blood cell suspensions at pH 7.8 and 8.4 at 15, 20 and 25 C. Normal red cells and red cells that had been depleted of their ATP content were used.
• 2.2. The equilibrium data were fitted to the Adair's model and the enthalpy (ΔH) and entropy (ΔS) changes for the first and fourth steps of oxygenation and for overall oxygenation were calculated from the temperature dependencies of the Adair constants.
• 3.3. For normal red blood cells, the apparent heat for the first oxygenation step, δh₁, is close to zero.
• 4.4. Temperature insensitivity of this step at physiological pH, combined with a large pH dependence, probably denotes a property of Hb4, the Root effect Hb of trout blood.
• 5.5. At pH 7.8, ΔH₄ is about —4kcal/mol, a small value which may be attributed to the large release of Bohr protons that occurs at the last oxygenation step and corresponds to an endothermic process which opposes to the exothermic oxygenation of the haem.
• 6.6. The ΔH₄ value appears to have a large influence on the enthalpy for overall oxygenation.
• 7.7. Results for ATP-free red cells are consistent with a mere increase in the intracellular pH and suggest that ATP has no specific effect at and above pH_i ~ 7.7.
• 8.8. Effects of temperature and pH on trout red blood cell isotherms emphasize the primary importance of the major component of trout blood, namely Hb4, in trout blood functional properties.

     

Effects of Roundup Herbicide and Increase in Water Temperature on the Parameters of Peripheral Blood Cells in Amur Sleeper <Emphasis Type="Italic">Perccottus glenii</Emphasis> Dybowski

The separate and combined effects of chronic 30-day exposure to the herbicide Roundup in a sublethal concentration of 2 μg/L and an increase in water temperature at a rate of 8°C/h on the parameters of red and white blood in juveniles of Amur sleeper Perccottus glenii Dybowski have been studied. The ratio of mature and immature erythrocytes in the peripheral blood do not change under the influence of the studied factors. An increase in temperature after chronic exposure to Roundup leads to a decrease in red blood cell sizes and increase in the share of abnormal cells. Exposure to the herbicide and the rise in water temperature have the opposite effect on the number of amitosis in erythrocytes and the ratio of leucocyte cells; an antagonistic effect is identified under the combined action of the factors. Changes in white blood correspond to a nonspecific stress response; changes in red blood indicate a reduction in compensatory responses to hypoxia.     

On measuring the diffusional water permeability of human red blood cells and ghosts by nuclear magnetic resonance

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes and ghosts by a doping nuclear magnetic resonance technique. In contrast to all previous investigations, systematic measurements were performed on blood samples obtained from a large group of donors. The mean values of P ranged from 2.2 X 10(-3) cm.s-1 at 5 degrees C to 8.1 X 10(-3) cm.s-1 at 42 degrees C. The reasons for some of the discrepancies in the permeability coefficients reported by various authors were found. In order to estimate the basal permeability, the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzenesulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 1.3 X 10(-3) cm.s-1 at 20 degrees C, 1.6 X 10(-3) cm.s-1 at 25 degrees C, 1.9 X 10(-3) cm.s-1 at 30 degrees C and 3.2 X 10(-3) cm.s-1 at 37 degrees C. The results reported here represent the largest series of determinations of water diffusional permeability of human red blood cells (without or with exposure to mercurials) available in the literature, and consequently the best estimates of the characteristics of this transport process. The values of P can be taken as references for the studies of water permeability in various cells or in pathological conditions.     

Measurement temperature plays a pivotal role in the distribution of erythrocyte deformability after LPS

Reductions in red blood cell membrane deformability (RBC(D)) may perturb microcirculatory blood flow and impair tissue O(2)-availability. We investigated the effect of assay temperature on the distribution of RBC(D) in endotoxin (LPS) incubated and control RBCs. Fresh blood from healthy rats was incubated with and without the presence of LPS for 6 hrs. An index of red blood cell membrane deformability, delta, was measured via the micropipette aspiration technique at 25 degrees C and 37 degrees C at 0, 2 and 6 hrs of incubation. The ATP content of RBC was measured by the luciferin-luciferase technique. At 25 degrees C, LPS caused a significant decrease in mean delta after 2 and 6 hours incubation compared to controls (-10.0%, p=0.03 and -24.0%, p=0.03, respectively) characterized by a left shift in the distribution (skewness: -1.4). However, at 37 degrees C a significant decrease in delta was only detected after 6 hrs of LPS incubation (-13.8%, p=0.01, compared to -5.1%, p=0.7 at 2 hours) and lacked the left shifted distribution (skewness: 0.2). No significant difference in ATP content of RBCs was observed between groups. We have shown that LPS incubation results in a significant decrease in RBC(D) and that room temperature measurement of physical membrane properties may exaggerate the differences between normal and perturbed RBCs.     

Suitable experimental design for determination of auxin polar transport in space using a spacecraft.

It is necessary to establish a suitable experimental design for the determination of auxin (indole-3-acetic acid: IAA) polar transport in space using a spacecraft in concerning with the role of gravity. Problems in space experiments are as follows: I) Selection of suitable plant species; II) Preservation of integrity of plant segments for activities of auxin polar transport; III) Stop of auxin polar transport of the segments after the transport experiment in space. Segments of etiolated pea epicotyls and etiolated maize coleoptiles showed relatively high activities of auxin polar transport among dicotyledonous and monocotyledonous plants tested, respectively. The activities decreased dramatically when the segments were pre-stored at 25 degrees C only for 1 day. On the other hand, the storage at low temperature (5 degrees C) in the presence of antioxidants or chelating agents, especially EGTA, maintained relatively high activities of auxin polar transport in pea epicotyl segments. Low temperature (5 degrees C) substantially inhibited the activity of auxin polar transport. Based on the results in this study, a suitable experimental design for the space experiment of auxin polar transport using a spacecraft is also proposed.     

Inhibition of aspartate aminotransferase by glycation in vitro under various conditions

Incubation of 50 mM D-glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 x g supernatant) at 25 degrees C had no effect on enzyme activity. 50 mM D-fructose or D-ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM DL-glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM D-fructose or D-ribose was marked even at a temperature of 4 degrees C but it was less pronounced than at 25 degrees C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM L-aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by D-ribose or D-fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars.     

Improved preservation of human red blood cells by lyophilization

The lyophilization of human red blood cells has important implications for blood transfusion in clinical medicine. In this study, sugars, human serum albumin, polyvinylpyrrolidone, and dimethyl sulfoxide were used as protective reagents for the lyophilization of red blood cells. Freezing temperature, shelf temperature, and the rehydration conditions were optimized. The results showed that extracellular disaccharides, especially trehalose, did not increase the recovery of hemoglobin. However, when the concentration of human serum albumin was higher than 25%, it had a considerable protective effect on the recovery of lyophilized red blood cells; the cellular hemoglobin recovery was over 70%, which was significantly higher than that in the group without human serum albumin (P<0.01). As the concentration of polyvinylpyrrolidone was increased, the extent of vitrification also increased. But when the concentration of polyvinylpyrrolidone was over 40%, the resulting concentration of free hemoglobin was over 1g/L, which was significantly higher than that with 40% (P<0.01). When lyophilization was carried out after freezing at different temperatures, the recovery of cells and hemoglobin was 70-80% and there were no significant differences among the five groups. When the shelf temperature was higher than -30 degrees C, the samples were partly collapsed, but when the shelf temperature was lower than -30 degrees C, the recovery of cells in the -40 and -45 degrees C groups was significantly higher than in the -30 and -35 degrees C groups (P<0.05). The recovery of cells and hemoglobin after lyophilization and rehydration in solutions containing low concentrations of polymers was over 80%, which is significantly higher than the other groups (P<0.01). In addition, when the temperature was higher than 25 degrees C, the concentration of free hemoglobin was significantly lower than it was at 4 degrees C (P<0.01). In conclusion, our study showed the lyophilization of red blood cells is feasible. Disaccharides have no protective effect on lyophilized cells when they are only extracellular and extensive vitrification may be not beneficial. Although the recovery of cells after lyophilization and rehydration by our method was over 70%, the ultrastructure of the cells may be compromised and some hemolysis does still exist. Further research is required.     

The effect of ultrahigh frequency electromagnetic fields on the reduction of ferricyanide by human erythrocytes in the presence of methylene blue

Effect of microwave radiation with the frequency of 1000 +/- 10 MHz and specific absorption rate of 220-580 mV/g on the ferricyanide reduction by human red blood cells in the presence of methylene blue (carrier of oxidation-reduction equivalents through the membrane) was studied at different temperatures in the region of 23-34 degrees C. The temperature dependence of the ferricyanide reduction rate in Arrhenius plots shows two sharp "anomalous" sites with apparently negative activation energy at 26-27 and 29-30 degrees C. Broadness and expression of the "anomalous" sites increased with an increase of the blood storage time. The increase of the ferricyanide reduction rate under microwave irradiation was observed only in the temperature regions corresponding to the "anomalous" sites of the temperature dependence.