Red Blood Cell Water Research Articles

Water content for clot composition prediction in acute ischemic stroke.

Mechanical thrombectomy (MT) has become the gold standard care for treating acute ischemic stroke (AIS) due to large vessel occlusion. Emerging evidence suggests that understanding the composition of clots prior to intervention could be useful for the selection of neuroendovascular techniques, potentially improving the efficacy of treatments. However, current imaging modalities lack the ability to distinguish clot composition accurately and reliably. Since water content can influence signal intensity on CT and MRI scans, its assessment may provide indirect clues about clot composition. This study aimed to elucidate the correlation between water content and clot composition using human clots retrieved from stroke patients and experimentally generated ovine clots. This study involved an analysis of ten clots retrieved from patients with AIS undergoing MT. Additionally, we created ten red blood cells (RBC)-rich and ten fibrin-rich ovine blood clots, which were placed in a human intracranial vascular model under realistic flow conditions. The water content and compositions of these clots were evaluated, and linear regression analyses were performed to determine the relationship between clot composition and water content. The regression analysis in human stroke clots revealed a significant negative association between RBC concentration and water content. We also observed a positive correlation between water content and both fibrin and platelets in ovine blood clots. Conclusion.

Red blood cell membrane water permeability increases with length of ex vivo storage

Water transport across the red blood cell (RBC) membrane is an essential cell function that needs to be preserved during ex vivo storage. Progressive biochemical depletion during storage can result in significant conformational and compositional changes to the membrane. Characterizing the changes to RBC water permeability can help in evaluating the quality of stored blood products and aid in the development of improved methods for the cryopreservation of red blood cells. This study aimed to characterize the water permeability (Lp), osmotically inactive fraction (b), and Arrhenius activation energy (Ea) at defined storage time-points throughout storage and to correlate the observed results with other in vitro RBC quality parameters. RBCs were collected from age- and sex-matched blood donors. A stopped flow spectrophotometer was used to determine Lp and b by monitoring changes in hemoglobin autofluorescence when RBCs were exposed to anisotonic solutions. Experimental values of Lp were characterized at three different temperatures (4, 20 and 37 °C) to determine the Ea. Results showed that Lp, b, and Ea of stored RBCs significantly increase by day 21 of storage. Degradation of the RBC membrane with length of storage was seen as an increase in hemolysis and supernatant potassium, and a decrease in deformability, mean corpuscular hemoglobin concentration and supernatant sodium. RBC osmotic characteristics were shown to change with storage and correlate with changes in RBC membrane quality metrics. Monitoring water parameters is a predictor of membrane damage and loss of membrane integrity in ex vivo stored RBCs.

Investigating the Osmotic Component in Mechanisms of Malaria Parasite Plasmodium Falciparum Egress and Invasion of Human Red Blood Cells

Human red blood cells (RBC) experience a transient echinocytic shape change immediately after parasite invasion and another within the last minutes before parasite egress when infected RBC seems to round up. The rapidity of shape changes in both events makes an osmotic mechanism a likely candidate. Indeed, it was shown that the rate of parasite egress is influenced by the osmolarity of the culture medium, suggesting the involvement of ion channels in the egress program and Aquaporin 1, a RBC water channel, was implicated in the initiation of the intraerythrocytic development of Plasmodium falciparum. Currently available methods are able to resolve infected red blood cell volume at the various stages of the 48h plasmodium developmental cycle, yet lack time resolution to capture fast-progressing events such as parasite egress and invasion of RBC. To gain insight into the putative osmotic component of these processes, we tested the suitability of a recently developed method for volume measurements of live cells, based on fluorescence exclusion, for volume evaluation in RBC during fast egress-invasion events in vitro. We show that volumes can be captured with video rate speed, high throughput, and sensitivity to relative changes of 0.3 µm3. We conclude that the fluorescence exclusion method is well suited for testing parasite egress-invasion events in real time and studying multiple individual events in the same cell suspension. Preliminary volume measurements of infected erythrocytes approaching parasite egress will be presented.

Morphology and water permeability of red blood cells from green sea turtle (Chelonia mydas).

The morphology and diffusional water permeability (P d) of red blood cells (RBCs) from green sea turtle (GST) (Chelonia mydas) are presented for the first time. The RBCs had an ellipsoidal shape with full-axis lengths (diameters): D = 14.4μm; d = 10.2μm; h = 2.8μm. The values of P d (cms(-1)) were 5.1 × 10(-3) at 15°C, 5.7 × 10(-3) at 20°C, 6.3 × 10(-3) at 25°C, 6.8 × 10(-3) at 30°C, and 7.9 × 10(-3) at 37°C (i.e., significantly higher than in human RBCs in which it was measured to be 4.2 × 10(-3) at 25°C, 5.0 × 10(-3) at 30°C, and 6.2 × 10(-3) at 37°C). There was a lack of inhibition of P d of GST RBCs by p-chloromercuribenzoate (PCMB), a well-known inhibitor of the RBC water channel proteins (WCPs). The activation energy of water diffusion (E a,d) in GST RBCs was 15.0 ± 1.6kJmol(-1) which is lower than the E a,d for human RBCs (~25kJmol(-1)). These results indicate that in the membrane of GST RBCs, there were no WCPs that were inhibited by the mercurial reagent, while the lipid bilayer of this membrane is unusually permeable to water. This is likely to be a phylogenetically old trait, like that found in amphibians and even the later birds, all of which have nucleated erythrocytes; and it is also likely to be a result of the animal's adaptation to a herbivorous diet (algae and seagrasses).

Effects of 5-hydroxymethyl-2-furfural on the volume and membrane permeability of red blood cells from patients with sickle cell disease.

The heterocyclic aldehyde 5-hydroxymethyl-2-furfural (5HMF) interacts allosterically with the abnormal form of haemoglobin (Hb), HbS, in red blood cells (RBCs) from patients with sickle cell disease (SCD), thereby increasing oxygen affinity and decreasing HbS polymerization and RBC sickling during hypoxia. We hypothesized that should 5HMF also inhibit the main cation pathways implicated in the dehydration of RBCs from SCD patients - the deoxygenation-induced cation pathway (Psickle), the Ca(2+)-activated K(+) channel (the Gardos channel) and the K(+)-Cl(-) cotransporter (KCC) - it would have a synergistic effect in protection against sickling, directly through interacting with HbS, and indirectly through maintaining hydration and reducing [HbS]. This study was therefore designed to investigate the effects of 5HMF on RBC volume and K(+) permeability in vitro. 5HMF markedly reduced the deoxygenation-induced dehydration of RBCs whether in response to maintained deoxygenation or to cyclical deoxygenation/re-oxygenation. 5HMF was found to inhibit Psickle, an effect which correlated with its effects on sickling. Deoxygenation-induced activation of the Gardos channel and exposure of phosphatidylserine were also inhibited, probably indirectly via reduced entry of Ca(2+) through the Psickle pathway. Effects of 5HMF on KCC were more modest with a slight inhibition in N-ethylmaleimide (NEM, 1 mm)-treated RBCs and stimulation in RBCs untreated with NEM. These findings support the hypothesis that 5HMF may also be beneficial through effects on RBC ion and water homeostasis.

High cholesterol impairs water and gas transport in red blood cells and is ameliorated by the PLA2 inhibitor ONO-RS-082

Elevated levels of plasma cholesterol pose significant health risks and are causal to diseases including angina, cardiovascular disorders and diabetes. Although the long-term effect of elevated plasma cholesterol is known for its accretion in arterial walls, little is understood of its immediate effects. Since cholesterol levels in red blood cell (RBC) membranes are inversely associated with O 2 transport, and the RBC water channel AQP1 is also capable of transporting CO 2 , the detrimental effect of cholesterol on active water transport, and likely on the CO 2 and O 2 transport in RBC, was hypothesized and tested in the present study. The rapid gating of water into isolated rat RBC and the corresponding increase in their volume and surface area was determined using dynamic light scattering. Similarly, the entry of CO 2 into the RBC was determined using zeta potential measurements, as increase in intracellular acidity reduces the negative charge at the RBC membrane. Results from our study demonstrate for the first time that the immediate effect of elevated plasma cholesterol is dysfunction of active water and CO 2 transport, possibly via the AQP1 channel at the RBC membrane. Since there is growing evidence that PLA2 may play a key role in atherogenesis, and statins significantly reduce blood PLA2 levels, the effect of the PLA2 inhibitor on ameliorating the detrimental effects of cholesterol was investigated. Interestingly, the PLA2 inhibitor ONO-RS-082 was found to reverse the cholesterol effect on RBC function.

2H 2O quadrupolar splitting used to measure water exchange in erythrocytes

The 2H NMR resonance from HDO (D = 2H) in human red blood cells (RBCs) suspended in gelatin that was held stretched in a special apparatus was distinct from the two signals that were symmetrically arranged on either side of it, which were assigned to extracellular HDO. The large extracellular splitting is due to the interaction of the electric quadrupole moment of the 2H nuclei with the electric field gradient tensor of the stretched, partially aligned gelatin. Lack of resolved splitting of the intracellular resonance indicated greatly diminished or absent ordering of the HDO inside RBCs. The separate resonances enabled the application of a saturation transfer method to estimate the rate constants of transmembrane exchange of water in RBCs. However both the theory and the practical applications needed modifications because even in the absence of RBCs the HDO resonances were maximally suppressed when the saturating radio-frequency radiation was applied exactly at the central frequency between the two resonances of the quadrupolar HDO doublet. More statistically robust estimates of the exchange rate constants were obtained by applying two-dimensional exchange spectroscopy (2D EXSY), with back-transformation analysis. A monotonic dependence of the estimates of the efflux rate constants on the mixing time, t mix, used in the 2D EXSY experiment were seen. Extrapolation to t mix = 0, gave an estimate of the efflux rate constant at 15 °C of 31.5 ± 2.2 s −1 while at 25 °C it was ∼50 s −1. These values are close to, but less than, those estimated by an NMR relaxation-enhancement method that uses Mn 2+ doping of the extracellular medium. The basis for this difference is thought to include the high viscosity of the extracellular gel. At the abstract level of quantum mechanics we have used the quadrupolar Hamiltonian to provide chemical shift separation between signals from spin populations across cell membranes; this is the first time, to our knowledge, that this has been achieved.

Phase I Study of Combination Treatment with Hydroxyurea and Magnesium Pidolate in Children with Sickle Cell Anemia.

Red blood cell (RBC) sickling is promoted by increased intracellular deoxygenated Hb S concentration and via cell dehydration. Magnesium (Mg) can reduce RBC water loss through its negative regulation of K-Cl cotransport, a pathway which mediates cell dehydration. Hydroxyurea (HU) reduces sickling in part by increasing intracellular fetal hemoglobin (Hb F). The combination of HU and Mg pidolate, two drugs with distinct mechanisms of action, could offer additive or synergistic effects. We performed a phase I study of treatment with HU and Mg pidolate in children with sickle cell anemia (SCA), to estimate the maximum tolerated dose (MTD) and toxicity of this drug combination, and obtained preliminary data regarding cellular effects of this treatment. Children with SCA who had been treated with HU on a stable dose for a minimum of 6 months, received 6 months of treatment with Mg pidolate in addition to HU. The Mg dose was 0.6 mEq/kg/day for the first cohort of patients and was escalated by 50 % in each subsequent cohort to a maximum of 1.9 mEq/kg/d. HU was continued at the patients' pre-study dose. Compliance was measured using returned bottles and pills counts for both HU and Mg pidolate. Hematologic studies were performed at baseline, 3, and 6 months, and 3 months after Mg pidolate discontinuation (9 months). In vitro RBC adhesion to immobilized thrombospondin was measured under low shear flow conditions. Sixteen patients participated in the study; 9 were boys, and the median age was 7 years (range 4 – 12 years). All were African American and had Hb SS. The median HU dose was 28.5 mg/kg/day (range 25 to 30 mg/kg/d) and the median pre-study HU treatment duration was 1.9 years (range 0.7 – 8.4 years). The MTD for Mg pidolate used in combination with HU was 0.9 mEq/kg/day. The dose limiting toxicity (DLT) of Mg pidolate was diarrhea. There were 4 cases of grade III toxicity (all in the first 4 weeks of the study), 2 cases of grade II and one case of grade I diarrhea. There also were grade III (2), and grade II (3) abdominal pain events. There were no cases of hematologic toxicity, and no patients required HU dose reduction or interruption. Compliance was above 85 % for both drugs in 95 % of the visits. At baseline, high density RBC and KCl co-transport activity were much lower, and intracellular K was much higher than in untreated Hb SS RBC; these values remained relatively stable on Mg pidolate therapy. Mean RBC adhesion to thrombospondin at baseline was low (323 ± 286 RBCs/mm2) and decreased even further to 245 ± 161 RBCs/mm2 during Mg pidolate use. No significant changes in Hb, WBC, platelets, reticulocytes, Hb F, and MCV occurred. We conclude:The MTD for Mg pidolate when used in combination with HU for children with SCA is 0.9 mEq/kg/d,The most common dose-limiting toxicities from Mg pidolate are diarrhea and abdominal pain,There is no additional hematologic toxicity from HU therapy when Mg pidolate is added,RBC adhesion to endothelium may be further decreased in SCA patients treated with HU when Mg pidolate is added,HU therapy when used at 30 mg/kg/day in fully compliant patients may promote decreased RBC adhesion, higher intracellular K, and lower KCl co-transport activity.

High plasma buffering and the absence of a red blood cell β-NHE response in brown bullhead ( Ameiurus nebulosus)

The high non-bicarbonate buffer capacity of brown bullhead ( Ameiurus nebulosus) plasma was postulated to function as an alternative mechanism for the protection of red blood cell (RBC) intracellular pH (pHi) in the absence or attenuation of a RBC adrenergic response. The requirement for protecting RBC pHi arises from the presence of a Root effect haemoglobin in bullhead. In support of this hypothesis, bullhead RBCs incubated in vitro with isoproterenol (10 −8–10 −5 mol l −1) or forskolin (10 −4 mol l −1) exhibited significant cyclic AMP accumulation, but failed to exhibit cell swelling or significant Na + or Cl − accumulation; plasma pH (pHe) was also unaffected. Similarly, no significant effect on RBC water content, Na + or Cl − concentration, or pHe was detected in bullhead blood incubated with 8-bromo cyclic AMP (10 −4–10 −2 mol l −1) in vitro. These results suggest that while bullhead RBCs possess a β-adrenoreceptor linked to cyclic AMP formation, stimulation of this adrenergic receptor does not result in measurable activation of a Na +/H + exchanger.

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant (Elephas maximus) and Man (Homo sapiens)

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P d of water in RBC was measured by using a Mn2+-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P d were, in the case of elephant RBC, 3.2 × 10−3 cm/s at 25 °C, 3.9 × 10−3 cm/s at 30 °C, 5.2 × 10−3 cm/s at 37 °C and 6.5 × 10−3 cm/s at 42 °C; all values were significantly lower than the corresponding values of P d for human RBC, namely 4.3 × 10−3 cm/s at 25 °C, 5.2 × 10−3 cm/s at 30 °C, 6.1 × 10−3 cm/s at 37 °C, 7.8 × 10−3 cm/s at 42°C. The maximal inhibition of P d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10−3 cm/s for elephant and 2.6 × 10−3 cm/s for human RBC. The values of the activation energy for water permeability (E a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. IX. Australian feral chicken and domestic chicken (Gallus domesticus)

The diffusional water permeability (Pd) of Australian feral chicken and Australian and European domestic chicken red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique. The values of Pd were around 1.7 × 10−3 cm/s at 15°C, 2.0 × 10−3 cm/s at 20°C, 2.5 × 10−3 cm/s at 25°C, 3.7 × 10−3 cm/s at 30°C, 4.3 × 10−3 cm/s at 37°C, and 6.1 × 10−3 cm/s at 42°C, with no significant differences between the three strains of chicken. There was no effect of p-chloromercuribenzene sulphonate on water diffusion. The activation energy of water diffusion was around 37 kJ/mol for all strains of chicken. These results suggest that no changes in the RBC water permeability are correlated with marked alterations in the habitat of chicken introduced to Australia (and that membrane proteins play little role in the diffusion of water across chicken RBC membrane).

Distribution of lactate in plasma anderythrocytes during and after exercise in horses

The distribution of lactate between red blood cells (RBC) and plasma was examined at rest, during exercise and 30 min after exercise in six Standardbred horses. Lactate and water concentrations were measured in blood and plasma samples collected prior to exercise, during the last 15 s of each step of an incremental exercise test and at 5 min intervals during the first 30 min after exercise. The mean ratio of RBC lactate concentration (RBCLa) to plasma lactate concentration (PLa) prior to exercise was 1.02 +/- 0.34. Haemoconcentration during exercise was associated with more rapid accumulation of lactate in plasma than in RBCs. Mean whole blood lactate concentration (BLa) was only 59% of PLa in samples collected during exercise. BLa was highly correlated with PLa during exercise (r = 0.98; P < 0.001), but individual PLa values differed from predicted BLa values by up to 2.1 mmol l-1 when PLa exceeded 8 mmol l-1. At each exercise speed and time after exercise there was a large variation between horses in the ratio of BLa/PLa. During exercise at 11 m s-1 the ratio ranged from 0.46-0.73. The BLa/PLa ratio was significantly correlated with increasing exercise intensity, (r = -0.68, P < 0.001) and with haematocrit, (r = -0.69, P < 0.001). The effects of PLa and haematocrit on the BLa/PLa ratio during exercise and the post-exercise period varied greatly between horses. The ratio of lactate concentrations in water of RBCs and plasma varied greatly during and after exercise (0.61-1.22). The ratio also varied considerably between horses, with coefficients of variation ranging from 14-34%. Lactate concentrations in RBC and plasma water vary greatly between horses during and after exercise. We concluded that lactate is unevenly distributed between red blood cells and plasma in blood samples centrifuged immediately after collection, and the variability depends on haematocrit.

Comparative NMR studies of diffusional water permeability of erythrocytes from some animals introduced to Australia: Rat rabbit and sheep

The diffusional water permeability (P d) of the red blood cell (RBC) membrane of rat, rabbit and sheep, representing some animal species introduced to Australia, has been monitored, by a Mn2+-doping 1H nuclear magnetic resonance (NR) technique using a high-field spectometer operating at 400 MHz. In order to make comparisons with previous measurements on the same species (performed at 25 MHz) an analysis of the influence of instrumental parameters and of the state of blood oxygenation was performed on samples of rat and rabbit blood. It was found that by using a short interpulse delay (around 100 µs) in the Carr-Purcell-Meiboom-Gill sequence, and by performing the measurements of the transverse relaxation time of the water in the cell interior (T 2i) on packed cells (haematocrit >95%), the values of the water exchange time (T e) obtained with a high-field spectrometer are comparable with those obtained by using a low-field NMR spectrometer. The values of the diffusional water permeability (P d), calculated from the T e values, were, at 24.6°C around 10 × 10−3 cm/s in case of rat and rabbit RBC and around 5 × 10−3 cm/s for sheep RBC; at 37°C the P d values were around 16 × 10−3 cm/s for rat RBC, 14 × 10−3 cm/s for rabbit RBC and 7 × 10−3 cm/s for sheep RBC. These values are very close to the P d values previously reported for the European counterparts of these species. There were no significant differences in the P d values between laboratory rabbits and wild rabbits, or between castrated males, pregnant or nonpregnant females. These results suggest that no changes in the RBC water permeability are correlated with marked alterations in the habitat of animal species introduced to Australia or by sex hormone status. If the NMR instrument-parameters are carefully chosen then comparative studies of RBC water permeability in different laboratories, at separate locations and using different instruments are meaningful.

Nmr studies of diffusional water permeability of erythrocytes from eight species of marsupial

1. 1.The diffusional water permeability ( P d) of the red blood cell (RBC) membranes of eight species of marsupial were monitored by using a Mn 2+-doping 1H nuclear magnetic resonance (NMR) technique at 400 MHz (9.4T). 2. 2.There were differences in the water permeability of the RBC membranes among the species investigated. 3. 3.On the basis of parameters characterizing the RBC water permeability the animals could be divided in two groups. 4. 4.For the first group, including the large macropodid marsupials, eastern grey kangaroo ( Macropus giganteus) and red kangaroo ( M. rufus), the values of P d were 5.4–6.1 × 10 −3 cm/sec at 24.6°C; 6.6–7.2 × 10 −3 cm/sec at 30°C; 7.8–9.1 × 10 −3cm/sec at 37°C and 9.3°-11.1 × 10-3 cm/sec at 42.1°C. 5. 5.The values of the activation energy for the diffusion process (E a,d) ranged from 24–27 kJ/mol. 6. 6.For the second group, including bandicoot ( Isoodon macrourus), bilby ( Macrotis lagotis sagitta), Tasmanian devil ( Sarcophilus harrisii), koala ( Phascolarctis cinerens), brush tailed possum ( Trichosurus vulpecula), and Goodfellow's tree kangaroo ( Dendrolagus goodfellowi), the values of P d were higher, ranging from 8.1–10.4 × 10 −3 cm/sec at 24.6°C; 8.9–11.8 × 10 −3 cm/sec at 30°C; 10.3–13.7 × 10 −3cm/sec at 37°C and 11.9–15.3 × 10 −3cm/sec at 42.1°C. 7. 7.In parallel with the high water permeability the RBC of the marsupials in the second group exhibited lower values of E a,D ranging from 15–21 kJ/mol, except for Goodfellow's tree kangaroo, for which the E a,D was ~ 25 kJ/mol.

Circulatory water concentration in suckling and fasting northern elephant seal pups

This study examined circulatory water concentrations in the neonatal northern elephant seal (Mirounga angustirostris) in order to determine how suckling and fasting would alter the percentage of water in the whole blood, plasma, and red blood cells (RBC). Plasma water concentration dropped by about 3% during suckling and recovered about 1% during the fast (92.38 +/- 0.48% less than 1 week old, 90.15 +/- 0.36% weaning, 91.02 +/- 0.68% end of fast). RBC water values during this time were more variable than plasma values: RBC water increased about 1% during the first 2 weeks of suckling (from 67.80 +/- 0.28% to 68.68 +/- 0.51%) but dropped to slightly below original neonatal values by weaning (67.15 +/- 0.63%). The first several weeks of fasting were marked by wide variability in RBC water, but by the end of the fast RBC water was comparable to that at weaning. These results indicate: 1) Northern elephant seal pups do not exhibit circulatory dehydration during 10 weeks of fasting; 2) Measurements of plasma or RBC metabolites (such as plasma glucose or RBC hemoglobin) may show variations or trends due not to metabolic regulation but rather to changes in circulatory water concentration.

Water exchange through erythrocyte membranes: biochemical and nuclear magnetic resonance studies re-evaluating the effects of sulfhydryl reagents and of proteolytic enzymes on human membranes.

The water permeability of human red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on intact cells and resealed ghosts following exposure to various sulfhydryl-reacting (SH) reagents and proteolytic enzymes. The main conclusions are the following: (i) When appropriate conditions for exposure of erythrocytes or ghosts to mercury-containing SH reagents (concentration, temperature and duration of incubation) were found, the maximal inhibition of water diffusion could be obtained with all mercurials (including HgCl2 and mersalyl that failed to show their inhibitory action on RBC water permeability in some investigations). While previous studies claimed that long incubation times are required for the development of maximal inhibition of water diffusion by mercurials, the present results show that it can be induced in a much shorter time (5-15 min at 37 degrees C) if relatively high concentrations of PCMBS (2-4 mM) are used and no washings of the inhibitor are performed after incubation. Higher than optimal concentrations of mercurials and/or longer incubation times result in lower values of inhibition, sometimes a loss of inhibition, or can even lead to higher values of permeability compared to control RBCs. (ii) The conditions for inhibition by mercurials are drastically changed by preincubation of erythrocytes with noninhibitory SH reagents (such as NEM or IAM) or by exposure to proteolytic enzymes. If the cells are digested with papain, the duration of incubation with PCMBS should be decreased in order for inhibition to occur. This explains the lack of inhibition reported previously, when a relatively long duration of incubation with PCMBS was used subsequent to papain digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of the transfer coefficients of oxygen and carbon monoxide in the boundary of human and chicken red blood cells by a microphotometric method.

The reaction rates of O2 and CO with the human and chicken red blood cell (RBC) were measured by using a microphotometric apparatus. In the experiments on the human RBC, a small amount of RBCs were put in an air-tight reaction cuvette. Gas mixtures containing various concentrations of O2 and CO were sequentially injected into the cuvette and the change in O2 and CO saturation of hemoglobin was measured from the change in transmission of the RBCs at 402 and 416.5 nm. The reaction rate of CO with RBCs was significantly influenced by photodissociation of carboxyhemoglobin (COHb). To eliminate this, a short-pass filter (400 to 435 nm) and a sector (100 Hz) were used. By comparing the measured reaction rates of O2 and CO with the theoretical rates obtained from the numerical solutions of the partial differential equations of the diffusions of O2 and CO, the transfer coefficients of O2 and CO (eta O2 and eta CO) in the RBC boundary, including the RBC membrane and water layer around the RBC, were estimated. Both the values showed good agreement, ranging from 0.3 to 2.5 x 10(-6) cm.sec-1.Torr-1. Furthermore, the chorioallantoic capillary of chicken embryo was used for the measurements of the reaction rates of O2 and CO with RBC through the capillary membrane. The reaction rates of O2 and CO in the chorioallantoic capillary were slower than those obtained in the human RBC. By comparing the measured reaction rates and the numerical solutions, the eta O2 and eta CO in the boundary, including the capillary membrane, plasma, and RBC membrane, were estimated. These two values ranged from 0.1 to 0.4 x 10(-6) cm.sec-1.Torr-1 and showed good agreement. These results suggest that the diffusion rates for O2 and CO across the capillary and RBC membrane are similar.