Glutamate dehydrogenase (GDH) has been encapsulated into mouse erythrocytes by a hypotonic dialysis/isotonic resealing method. Although a low GDH entrapment yield was achieved (3.8%), this percentage appeared sufficient enough to metabolize high quantities of ammonia. Carrier cell recovery yield was 56%. Due to the decrease in cell volume and haemoglobin content, constant mean cell haemoglobin concentration (MCHC) values were obtained. The osmotic fragility curves (OFC) indicated that dialyzed/resealed-RBCs are more resistant to hypotonic haemolysis than native-RBCs. The successful in vitro ammonia degradation by GDH-RBCs was reflected in its total disappearance from the incubation medium at around 48 h. In contrast, initial ammonia levels were not affected during the incubation in the presence of native-RBCs and remained constant. Two different methods were used for the preparation of hyperammonaemic mice model. Since the intraperitoneal (i.p.) administration of ammonium acetate produced high ammonia levels that lasted only a few minutes, the i.p. administration of urease was chosen, given that it generated elevated ammonia levels for longer periods of time. Hyperammonaemic mice quickly removed high levels of circulating ammonia in the presence of GDH-RBCs, whereas in the presence of native-RBCs ammonia was slowly metabolized. These results suggest that loaded GDH-erythrocytes can be used as a potential carrier systems for the in vivo removal of high levels of ammonia from blood.

The role of molecular crowding and viscosity on the apparent translational diffusion coefficient (ADC) of small metabolites was investigated in different subcellular organelles using the pulse-field gradient spin-echo 1H NMR technique. ADCs of metabolites with increasing radius of gyration (0.7 A < RG < 4.5 A) were measured in the cytoplasm of rat or chicken erythrocytes, in the nucleus of chicken erythrocytes, and in isolated rat liver mitochondria. Metabolite ADCs in these systems were compared with the corresponding ADCs determined in model solutions of increasing bulk viscosity but different molecular crowding. For solutions having the same viscosity, metabolite ADCs decreased with increasing concentration of cosolutes. This effect is adequately described by the modified Stokes-Einstein relationship, ADC = k/RG (1 + 2.5Phi), where k is a constant for a given temperature and Phi is an obstruction factor reporting the fractional volume of solution occupied by cosolutes, a measure of the molecular crowding in the solution. Cytoplasmic values of Phi for metabolites of different sizes did not depend exclusively on metabolite RG but on additional factors including the chemical nature of the metabolite, the presence of diffusional barriers, and metabolite-specific binding sites. In the case of water, nuclear Phi values approached those of the extracellular space while mitochondrial Phi values were significantly higher than those of the cytoplasm. Taken together, these results reveal important differences in molecular crowding within the different subcellular compartments, suggesting considerable diffusional heterogeneity for small metabolites within the different intracellular organelles.

The isozymes of three glycolytic regulatory kinases: hexokinase, phosphofructokinase and pyruvate kinase are fractionated by a single ion exchange chromatographic procedure on DEAE-cellulose. Enriched-erythroblast bone marrow cells showed two heterogeneous peaks, each consisting of two overlapping peaks: one major and one minor peak, but only two isozymes were observed in reticulocytes and erythrocytes. Phosphofructokinase showed multiple isozymic forms in the three cell populations, but while in erythroblasts the main one eluted in the last fractions, in reticulocytes and erythrocytes it eluted in the early fractions. Pyruvate kinase showed a main early activity peak with a shoulder in erythroblasts, reticulocytes and erythrocytes but the response to the allosteric effectors (fructose-1,6-bisphosphate and ATP) suggests the presence of different pyruvate kinase isozymes in reticulocytes and erythrocytes.

Rat erythrocytes subjected to hypotonic-isotonic dialysis, or crosslinking with bifunctional reagents (glutaraldehyde and dimethyl suberimidate hydrochloride) show a high percentage of methemoglobin and decreased oxyhemoglobin content which implies a low oxygen carrying capacity. Such modified cells maintain reversible oxygen binding properties although, they present a high hemoglobin oxygen affinity (low P50) and a diminished cooperativity in binding oxygen to hemoglobin (low n). These results suggest a reduced capacity of liberating oxygen to tissues under low PO2. Changes produced in erythrocytes can not be restored even in the presence of energy (ATP), reduced glutathione and 2,3-bisphosphoglyceric acid during the dialysis process or after crosslinking/permeabilizing treatment.

The in-vivo survival of51Cr-labelled murine red blood cells (RBCs) loaded with recombinant human erythropoietin (rhEpo-RBCs) was slightly lower than that of normal RBCs. Intravenous administration to normal mice of the encapsulated rhEpo shows the pharmacokinetic bicompartmental profile typical of the free rhEpo. Distribution and elimination half-life values for the RBC-entrapped rhEpo were no longer than those for the free protein. The area under the curve value was significantly increased for rhEpo-RBCs. Hypertransfused polycythaemic mice were evaluated as an adequate animal model to study the in vivo biological activity of encapsulated rhEpo-RBCs stimulate the erythropoiesis of polycythaemic mice in a linear dose-radio-iron incorporation response relationship. These results suggest that rhEpo-RBCs may behave as an alternative to the administration of free rhEpo in the clinical field.

The stability against time (up to 170h) of encapsulated enzymes were studied in ADH- and GDH-carrier RBCs, at 4 degrees-C and 37 degrees-C, in comparison with that of free enzyme solutions. Encapsulation into RBCs suggest a protective effect of both enzyme activities. An efflux of the encapsulated enzymes from the carrier RBCs was observed during a similar incubation. The continuous degradation of ethanol and the simultaneous appearance/disappearance of acetaldehyde by ADH-RBCs, as a function of time (up to 72h), suggest the use of these carrier RBCs to fully metabolize ethanol. The rapid utilization of ammonia in the presence of GDH-RBCs suggest the use of these RBCs as carrier systems. These properties open the possibility of using ADH- and GDH-RBCs as carrier systems under in vivo situations.

The optimal conditions for electroporated/resealed loading of alcohol dehydrogenase (ADH) and/or acetaldehyde dehydrogenase (ALDH) into human erythrocytes were established prior to the study, with the following characteristics: 300 V, 1 ms pulse time, eight pulses every 15 min and 1 h resealing at 37 degreesC. High encapsulation yield and carrier cell recoveries were achieved. Cell volumes increase while hemoglobin contents decrease; in consequence a decrease in cell hemoglobin concentration was observed. A lower hypotonic resistance of loading erythrocytes (throughout osmotic fragility curves) and unaltered oxygen transport capability (as given by oxygen equilibrium curves) were observed. The stability against time (up to 168 h-7 days) of encapsulated individual enzymes, either ADH- or ALDH-red blood cells (RBCs), was studied at 4 degreesC and 37 degreesC, in comparison with that of free enzyme solutions. Both enzymes were released from carrier RBCs to the incubation medium. The stability of carrier RBCs was studied under similar conditions. Non-significant variations in hematological parameters were observed. However, the hemoglobin derivative forms showed modifications. The continuous degradation of ethanol by ADH-RBCs and coencapsulated ADH- and ALDH-RBCs, as a function of time (up to 70 h) suggests the use of these carrier RBCs as agents for complete metabolization of ethanol. The mentioned properties bare the possibility of using ADH and ALDH as carrier systems in in vivo situations.

Chemical conditions of crosslinking mouse erythrocytes with BS3 and DTSSP have been studied. These two crosslinking reagents seem to react with band 3 protein in mouse erythrocytes membrane. Extent of crosslinking is dependent on the concentration of the reagent used. Similar cell volumes were observed in crosslinked erythrocytes with respect to control erythrocytes. In vivo behaviour of these modified erythrocytes revealed prominent targeting of crosslinked erythrocytes to liver. This effect is clearly evident when concentrations of 5 mM BS3 or DTSSP were used and can be dependent of reagent concentration. Consequently, from our results BS3 and DTSSP can be considered as very useful tools to control and modulate targeting of crosslinked erythrocytes.

Glutamate dehydrogenase (GDH) and alcohol dehydrogenase (ADH) have been encapsulated in sheep and human red blood cells (RBCs) by a hypotonic dialysis/isotonic resealing procedure. At a fixed enzyme level in the dialysis bag (100 units/ml of RBCs), a significant encapsulation yield was observed for ADH, both in human (17.2%) and sheep (47.9%) RBCs, whereas a very low entrapment of GDH was achieved (1-3%) in either species. Carrier cell recovery was 61-65% in humans and 30-34% in sheep. Because of the aggregation of GDH to large polymers at protein levels above 1 mg/ml, the yield of encapsulation and the specific activity in human carrier RBCs were compared at different enzyme concentrations. While entrapment was not affected by differences in ADH up to 13 000 units/ml of RBCs (38 mg/ml), the yield of GDH encapsulation significantly decreased as the enzyme level increased up to 750 units/ml of RBCs (15 mg/ml), thus demonstrating the importance of protein concentration in the encapsulation process for those enzymes that tend to aggregate.

The bifunctional imidoester dimethyl suberimidate hydrochloride can stabilize rat red blood cells (RBCs) by membrane protein crosslinking, and in that way they can be used as carrier systems for exogenous substances. Counter-current distribution fractionation in charge-sensitive dextran-polyethyleneglycol two-phase systems has been used to detect slight changes in surface charge in stabilized cells. A decrease in the surface charge of crosslinked RBCs and an apparent masking of the age-related cell surface properties have been found to result from the protein crosslinking. Digitonin treatment used to permeabilize crosslinked RBCs produces a significant decrease of the cell surface charge while the age-related surface properties do not seem to be modified by the treatment.

Rat bone marrow cell populations, containing different proportions of erythroid cells, have been fractionated by counter-current distribution in the non-charge-sensitive dextran/polyethyleneglycol two-phase systems on the basis of hydrophobic cell surface properties. Cell fractions with a low distribution coefficient, which contain non-erythroid cells and early erythoblasts, showed a low transferrin binding capacity and a low haemoglobin/cell ratio whereas cell fractions with a high distribution coefficient, which contain intermediate-late erythroblasts and mature red cells, showed an elevated transferrin binding capacity and the highest haemoglobin/cell ratio. These results support transferrin binding capacity as a good marker parameter for the erythroid bone marrow cell differentiation and maturation processes.

L-Asparaginase has been encapsulated in human red blood cells using a hypotonic dialysis process. Erythrocytes loaded with L-Asparaginase were separated into eight fractions using a discontinuous Percoll density gradient. A minor cell subpopulation of low density cells and a major subpopulation of denser erythrocytes was obtained after hypotonic dialysis treatment, in both the absence or presence of L-Asparaginase. The encapsulated L-Asparaginase activity per resealed erythrocyte was higher in low-density cells and decreased progressively with increasing in cellular density.

The fractionation of normal human erythrocytes by counter-current distribution (CCD) in charge-sensitive dextran-polyethylene glycol two-phase systems was confirmed and extended to red blood cells from heterozygous beta-thalassaemic patients. The differences between the distribution profiles of normal (homogeneous) and abnormal (heterogeneous) red blood cells reflect their different surface-charge properties. As suggested by the decline of membrane sialic acid released after neuraminidase treatment and the specific activities of two age-dependent enzymes (membrane acetylcholinesterase and intracellular pyruvate kinase) in the distribution profiles (from the left- to the right-hand side fractions), the fractionation seems to be according to red blood cell age. A constancy of the 2,3-bisphosphoglycerate level was observed in ageing red blood cells.

The transferrin-binding capacity of rat bone marrow cells and different erythroid-enriched populations from rat bone marrow has been studied and compared with that previously reported for pure reticulocyte populations. Two components with different transferrin-binding capacities were found. The one with higher affinity was present mainly in erythroid cells and showed an association constant similar to that observed in reticulocyte populations. The component with a lower transferrin affinity was observed mainly in bone marrow fractions containing low proportions of erythroid cells.

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of rat erythrocyte membrane sialoglycoproteins (glycophorins) has been developed. Samples of erythrocytes and reticulocytes were analysed using this assay, and response compared among them and purified glycophorins samples. A broadly homologous behaviour of glycophorins was found in both cell types, suggesting the presence in reticulocytes of glycophorin molecules closely similar to those on the erythrocyte. A quantitative evaluation of glycophorins on both cell types yielded comparable levels of these glycoproteins, but with significantly higher values (1.7-fold) for reticulocytes. It is suggested that the lower number of epitopes present on the erythrocyte membrane might be due to the disappearance of some glycophorin-associated antigenic determinants during the maturation of reticulocyte to erythrocyte.