A Hundred-Year Researching History on the Low Ionic Strength in Red Blood Cells: Literature Review

This review article provides a critical survey of work from 1904 to 2003 on the effects of low ionic strength in Red Blood Cells (RBCs) incubated in media with impermeable sugars such as sucrose. In 1904 Gürber A washed RBCs of different species with isotonic sucrose solution to eliminate the outside ions in order to better analyse their intracellular ionic composition; however, this approach was not feasible because of a substantial salt efflux from the cells. A prominent feature of the salt loss is the shrinking of the RBCs. A central role in the understanding of the ionic movements is thereby the new Donnan equilibrium of the anions. Experimental evidence has been given by Jacobs MH and Parpart AK in 1933. In the sucrose medium two phases could be predicted: 1) a very rapid anionic shift resulting in an unequal distribution of chloride and hydroxyl anions on both sides of the membrane and 2) a leakage of salts from the RBCs. In 1940 Wilbrandt W assumed that a positive membrane potential is in line with the salt loss at low ionic strength in RBCs. In 1977 Knauf PA, Fuhrmann GF, Rothstein S and Rothstein A observed in RBCs an inhibition of both, anion exchange and also of net anion efflux, by incubation with disulfonic stilbene derivates. At low ionic strength the Donnan equilibrium is immediately obtained by the Anion Exchanger Protein (AEP). The resulting positive membrane potential opens at least two new types of cation pores or channels. Thereby is the conductivity pathway for the anions, namely the AEP, in charge of the net anion loss at low ionic strength. The AEP pathway is extensively blocked by disulfonic stilbene compounds. The permeability ways for cations through these pores or channels are not yet explored.


Differences in Red Cell Species by Sucrose Treatment
It seemed such a simple idea of August Gürber [1] to use an isotonic sucrose solution, to wash RBCs free of their adhering serum with ions and to analyse thereafter the cells for their salt content. Sucrose is a disaccharide consisting of two sugars, a glucose and fructose residue, connected by a -glycosidic linkage. This molecule is not taken up by any of the red blood cell species. Washing of the red blood cells in isotonic sucrose solution was performed with the help of a centrifuge with about 2500 rpm, driven by water pressure. The following RBCs from: Cattle or ox, mutton, pig, rabbit, dog, cat and horse were examined. After the fi rst centrifugation On hand of the behaviour of the diff erent RBCs during the washing procedure three types of cells could be diff erentiated: Best suited were the RBCs from cattle or ox, mutton, pig and rabbit. After the fi fth and sixth centrifugation no serum could be detected anymore in the washing solution, but there was chloride, sodium and potassium, the latter very prominent presented from pig RBCs. The second type contained the RBCs of the dog, which were completely unsuited, because of strong haemolysis already after the fi rst wash. The third type was horse and cat RBCs. Horse RBCs change already by contact with sucrose the colour from pink to dark red and start clotting.
In horse RBCs agglutination and shape change with spicules at their surface, giving rise to crenated cells (Stomatocytes) with slow sedimentation rates of those cells, and cloudiness of the washing solution was noticed. By the fi fth and sixth centrifugation a tight agglutination of the cells occurred, which could not anymore be dissolved, as with cattle RBCs.
Thus, the simple intention of August Gürber to analyse the salt content of RBCs by washing them with isotonic sucrose solution, could not be realized. The RBCs lost anions and cations during this washing procedure and demonstrated osmotic fragility, agglutination with shape changes and subsequent fi nal haemolysis. Best suited were the RBCs of cattle or ox, but they still showed sodium and potassium loss by semi-quantitative fl ame analysis.
The eff ect of the non-permeable non-electrolytes on cation effl ux in RBCs proved to be very interesting in future science. With a further physico-chemical analysis Ivor Bang [2] confi rmed the loss of electrolytes in sucrose solutions and showed that washing of RBCs increased their resistance against hypotonic media. He explained the dark red colour of ox blood as taken from the vena subclavia by the slaughter and the light red cattle blood as taken from the carotis.

Using Cation Permeable RBCs of the Ox after Treatment with Sucrose as Model Cells for Conductivity Measurements
Rudolf Höber [3] used the washing procedure of cattle or ox RBCs with the isotonic sucrose solution of August Gürber to induce an electrolyte loss. The effl ux was measured by conductance change of the red cell suspension.
He demonstrated the intactness of the RBC membrane by saponine treatment, which immediately increased the conductivity. Saponines are amphipathic glycosides, which act by complexation with cholesterol of the RBC membranes by forming pores and subsequent haemolysis.
A young scientist Arthur Joel [4] was advised by Rudolf Höber to use the method of August Gürber to produce in cattle or ox RBCs an electrolyte loss by sucrose and test the eff ects of narcotics. In contrast to intact RBCs, sucrose treated cells

Two Phases of Loss of Osmotic Content in Ox RBCs After Sucrose Exposition
In contrast to Arthur Joel, who used cattle or ox RBCs after pre-treatment with 3 times washing on a centrifuge in 10% sucrose solution for his conductivity measurements, BLOOD DISORDERS | HEMATOLOGY the authors Jacobs MH and Parpart AK [5] investigated RBCs from ox right after mixing of two drops of defi brinated blood in 50 ml hypotonic 200 mM sucrose solution at 20°C to test their osmotic content by measuring their haemolysis in an even more hypotonic sucrose solution of only 125 mM. This method has the advantage that the time for haemolysis of 75% of the RBCs can be measured within seconds by a convenient opacimetric method. This is absolutely necessary because in the experiments with sucrose already after one second of time a dramatic loss in osmotic content occurred, which could be deduced from the fast prevention of haemolysis in 125 mM sucrose. Within 20 second the change was greater than ever obtained in a day with a hypotonic NaCl solution. This process lasted for about 2 minutes after which a slower loss of osmotic content was following.
The authors asked the important question, if such results would indicate an equally great diff erence in the rate of escape of salts from the RBs in sucrose solution.
Their answer was that the change, which occurs in RBs exposed to non-electrolyte solutions, consists of two phases: a very rapid ionic shift, whose eff ect is apparent within a few seconds, and is complete within a minute or two. Following this, usually after a distinct pause, which in the presented experiment amounted to approximately 10 or 15 minutes, a much slower leakage of salts from these cells occurs.
In respect to the slow second change the authors are in agreement with Ivor Bang [2] and with Eric Ponder and George Saslow [6], who reported a salt loss of both anions and cations from RBCs suspended in sucrose solution. However, Jacobs and Parpart had a diff erent explanation for the fi rst and more striking change, which had apparently not previously been recognized as a diff erent phenomenon. Jacobs MH and Parpart AK considered the newly recognized rapid initial changes as being due to an ionic exchange rather than to a leakage of salts. Its rapidity and its reversibility suggest an ionic exchange modus, while the slower salt loss is not reversible.
The reversibility was shown in an experiment in which ox RBCs at 20°C were exposed to 200 mM sucrose plus 2 mM NaCl. Therefore, the cells were after 3 minutes exposure to 200 mM sucrose placed in 200 mM sucrose plus 2 mM NaCl for varying times and the resulting suspension was suddenly diluted to 125 mM sucrose plus 1 mM NaCl. By the presence of 2 mM NaCl the percentage of haemolysis returned to about its initial high value. In other experiments it was found that CaCl 2 is roughly twice as eff ective as NaCl in reversing the non-electrolyte eff ect.
For the fi rst fast eff ect in sucrose the authors proposed an exchange of OHoutside against Clinside the RBCs in respect to the Donnan equilibrium [7]. The usual cause is the presence of a diff erent charged substance (For example negative charged haemoglobin) that is unable to pass through the membrane and thus creates an uneven electrical charge.
[Cl] inside / [Cl]  Donnan equilibrium as above in accordance with the general theory given by Hans Netter [8]. For attainment of the equilibrium condition there must be an exchange of OHfrom the external solution for Clfrom the RBCs. Such an exchange leads to an increase of negative charge of haemoglobin and binding of potassium, causing a decreased osmotic pressure within the RBCs.
Jacobs's MH and Parpart's AK calculations showed that the expected osmotic eff ect of such an exchange is in the order of magnitude actually observed. So, the fi rst fast decrease in osmotic content seemed only be related to the OH -/Clexchange, while the second slow process was explained by a salt loss.

Cation Loss of Human RBCs in 7% Glucose Solution
Montague Maizels [9] Table 1: Q is the ratio of K + present in unit of volume of "original" RBCs resulting from suspension in glucose solution. to that present before suspension. Thus Q = K f x V/K 0 . where K f is the K + content per litre of suspended RBCs. K 0 is the K + content per litre original RBCs and V is the ratio of the volume after suspension to that before [9]

The Effect of Non-Electrolytes on Cation Loss in Different RBC Species
Another study on the permeability of erythrocytes was presented by Hugh Davson [10]. He investigated RBCs of human, guinea-pig, rat, pig, rabbit, ox and cat. The non-electrolyte solution used was 320 mM glucose for all species except human for which 320 mM sucrose was used, since human RBCs are comparatively permeable to glucose. The whole blood was added to the non-electrolyte solution, contained in a centrifuge tube immersed in a thermostat at 25°C; the ratio of volumes to suspension medium was 1:10. Samples were taken every hour by centrifugation, the supernatant fl uid removed and the K + and Na + content of the RBCs determined chemically. pH changes in the suspension medium were determined colorimetrically.
Addition of NaCl or KCl to the sucrose solution decreased the salt losses.

Are the two Phases of Osmotic loss in RBCs by Non-Electrolytes Causally Related?
In 1940 Walther Wilbrandt [11]  BLOOD DISORDERS | HEMATOLOGY Figure 3a: Change in the pH of the outside isotonic sucrose (Saccharose = sucrose) solution at 20°C, which includes the 1. Phase of the OH-/Cl-exchange in comparison to an effect in isotonic NaCl solution. In fi gures 3b the changes of pH in sucrose solution are also compared with a control in isotonic NaCl solution over a long time period of 400 minutes. This represents the 2 phase of salt loss in isotonic sucrose solution [11] fi gure 1. Figure 3a shows a sharp change from above of pH 7.5 in the outside pH of the unbuff ered isotonic sucrose solution to less than a pH-value of 6.5, which is most probably the expression of an exchange of OHions from the outside against Clfrom the inside of the cells. It can be assumed that the cell interior will become by OH -/Clor OH -/HCO 3 exchange inside alkaline.
This will change the haemoglobin buff er capacity and haemoglobin itself has more negative charges (polyanion) leading to additional potassium binding and reduction of osmotic pressure.
At the other hand the decrease in outside pH will generate from HCO 3 a CO 2 increase (pK 1 = 6.4), which easily penetrates the cell membrane and reducing inside pH by combining reversible to N-terminal amino groups of haemoglobin to form carbamates (Bohr eff ect, 1904) or with carboanhydrase and H 2 O, to produce a proton and HCO 3 -. Wilbrandt has also tried to measure inside pH ( Figure 3b). However, his fi rst time point is taken at 100 minutes, which might not be responsible for the 1. phase of OH -/Clexchange. Walther Wilbrandt further demonstrated in the next fi gure 4, that the pH changes as shown in fi gure 3b might be connected with the observed osmotic change, which is directly related to the salt loss by an equivalent change in the outside pH. Wilbrandt pointed out that the driving force acting on the cation was not only its concentration gradient, but also the electrical gradient contributed by the anion equilibrium.
The last part was devoted to the relationship between the above results and the proposed change of anion in elective cation permeability in pig and ox RBCs by Rudolf Mond [13] in 1927.

Wilbrandt's Criticism about the Experiments of Rudolf Mond on the Reversion of Anion in Cation Permeability in RBCs
Rudolf Mond [13] proposed very enthusiastically that the question about the cause of elective ion permeability has been solved, since the famous experiments of Leonor Michaelis [14] in artifi cially charged collodium membranes.  RBCs are not very sensitive to non-electrolyte treatment. Walther Wilbrandt [11] showed in his experiments with human RBCs that by changing to alkaline pH the Donnan equilibrium for OHand Clanions is drastically changed, leading to a considerably increased osmotic loss of salts in isotonic sucrose solution. These results are in agreement with Hugh Davson [10], who also demonstrated that the loss of potassium in human RBCs is increased in alkaline solution. The 25 minutes of incubation in sucrose solution in Rudolf Mond's experiments [13] at diff erent alkaline pH values result in experimental data, which are not directly comparable, because there is only one time point obtained at very diff erent equilibrium conditions.

BLOOD DISORDERS | HEMATOLOGY
In his experiments Rudolf Mond discussed an exchange of Clwith OHanions by the high concentration of added NaOH of 14.3 to 28.6 mM below pH 8, the isoelectric point for the pore proteins. Above pH 8 by addition of 35.7 to 57.1 mM NaOH he postulated a passive cation exchange of K + against Na + , which to our present knowledge does not exist.
It is not surprising, that in view of the unusual treatment of RBCs with 1 M NaOH the survival of RBCs is threatened, which would drastically change pH, ion content, osmolality and cause membrane damage. Therefore, the results of Rudolf Mond remain diffi cult to interpret.

Discovery of the Na + -K + ATPase in Human RBCs
The human RBC has a high concentration of about 150 mM K + in cell water against 4.5 mM in serum and about 10 mM Na + in cell water against 145 mM Na + in serum. These ionic gradients are generated by a specifi c transport system, the Na + -K + ATPase, discovered by Jens Skou in 1957 from the shore crab [16] and 3 years later found by Post RL, et al. [17] in the human RBC.
The accuracy of measuring fl uxes of Na + and K + was greatly increased by using the radioactive isotopes 24 Na and 42 K [18].
Three Na + out and two K + in the human RBC are transported per ATP hydrolysed, which shows that the ATPase is working electrogenic.
These results clearly demonstrate that the RBC is not at all selectively permeable for anions. However, the chloride permeability is about 2 million-fold higher than the corresponding permeability for sodium and potassium (4 x 10 -4 cm/sec against 2 x 10 -10 cm/sec) [19]. Fuhrmann

The Role of the Potential across the RBC Membrane in a Medium of Low Ionic Strength
In 1960 Walther Wilbrandt and Hans Jörg Schatzmann [20] asked the important question, whether the cation loss in low electrolyte media yielded results that can be interpreted as due     [20] have demonstrated that the fl ux of cation loss is in the right direction of a positive membrane potential. By convention, the sign of the membrane potential is designated as the voltage inside relative to ground outside the cell.
The dependence of the rate of cation loss and the outside concentration of Clare in the right direction for an action of a positive membrane potential either as driving force or on the membrane permeability. However, the rate of cation loss is at high potential (> + 100 mV) and at low potential (< + 50 mV) not directly proportional to the membrane potential. This is  The second component shows a steep rise within seconds, which has been tentatively attributed to the OH -/Clexchange as discussed by Jacobs and Parpart [5], but the conductivity change here is undoubtedly due to electrolyte loss. The Further, urethane, low concentration of cocaine and calcium chloride decreases the induced cation permeability. K + and Na + effl ux is approximately equal.

Effl ux of Salt from Human RBCs Suspended in Isotonic Sucrose Plus Low Concentration of Salt Measured under Steady State Conditions
In 1968 Paul L LaCelle and Aser Rothstein [22]  Parpart [5] used with two drops about 100μl of ox RBCs in 50 ml suspension, so that the contribution of electrolyte outfl ow becomes negligible at the used outside concentration. The second would be to use initial rates as also Jacobs and Parpart [5] by haemolysis technique and Wilbrandt and Schatzmann [20] in their conductivity measurements performed.
A new method has been introduced by the above authors In the fi rst minute a readjustment of pH was observed (Inserted fi gure), representing the Cl --OHshift noted by Jacobs and Parpart [5] and studied in detail by Wilbrandt [11].
In the second phase the loss of salt proceeded at a virtually constant rate until about 25 to 30% of the total salt content of the RBCs remained. Thereafter the rate diminished gradually and fi nally ceased. All following measurements of rates reported in the above paper were within the second linear phase of the curve excluding the fi rst immediate equilibrium period, the usual observation period being 10 to 30 minutes with a salt effl ux of less than 10%.
With no control exerted upon the pH, the initial value of the suspension averaged pH 6.8 with an extreme range of 6.5     In summary the fi rst steep slope at high potentials is nearly independent in steepness of temperature between 14° and 37 °C and behaves also independent to pH changes between pH 4 and pH 9. In contrast the second low slope at lower potentials increases as the temperature is raised and the infl ection points are shifted to the right and upward. It is dependent on pH changes between pH 4 and pH 9. In general the salt effl ux is increased at higher pH values. At higher salt concentrations the eff ect of pH is proportionately higher and it tends to occur at higher pH values.

The Passive Cation Effl ux from Human RBCs under Different Driving Forces and Tonicities
The previous experiments by Paul LaCelle and Aser Rothstein [22] were extended to higher outside concentrations of NaCl. In addition to the statting method at NaCl concentrations below 20 mM the corresponding cation effl ux was measured under non-steady state conditions by periodic sampling. In addition to 300 mM sucrose the tonicity was increased to 400, 500 and 600 mM sucrose plus diff erent salt concentrations. Measurements of chloride (Cl 36 ) distribution, of osmotic response and a titration of RBC haemolysate were necessary to obtain the parameters used in calculation of the electrochemical driving force to fi nally try to apply the Goldman equation [24,25].     36 Cl distribution and the potential calculated at 4 outside concentrations of 5. 10. 20 and 50 mM chloride in 300. 400. 500 and 600 mM sucrose solutions. The estimation has been made that electroneutrality exists inside the RBC. and that chloride is the major internal anion Jerry Donlon [25]   For the above example: (57/60) + i.p. 6.9 = pH 7.85 chloride inside and by the Donnan ratio a pH outside of pH of 6.6 can be calculated. This is so, when an average i.p. of haemoglobin of pH 6.9 and an average internal buff er capacity from the two values given by Jerome Donlon of 58 and 62 meq/liter RBCs pro pH unit was chosen.
Whereas at the high outside concentrations for example of 20 mM chloride the above calculations of the inside and outside pH are in good agreement with the presented values by Jerome Donlon, the values at low chloride concentrations, for example of 5 and 10 mM chloride, disagree for more than one pH unit. This discrepancy exists also between Paul LaCelle and Aser Rothstein [22] and Jerome Donlon's data. The initial outside pH values of the fi rst authors averaged at pH 6.8 with an extreme range of 6.5 and 7.0 and with only small changes (0.1 pH unit) during the course of a 10 to 30 min. experiment. This is in complete agreement with an outside pH, calculated from the above data, for example, with 5 mM chloride outside and 300, 400 and 500 mM sucrose of pH's calculated outside of 6.58, 6.73 and 6.92, but not with pH's outside of 5.5, 5.42 and 5.35 given under comparable conditions by Jerome Donlon [24].
The main result, however, is the confi rmation of the two regions and the detection of a third region of passive cation effl ux and that at diff erent Osmolarities. All the curves are linear in respect to the logarithm of the external salt concentration. This means that the second phase of salt effl ux according to Jacobs and Parpart [5] is not only dependent on the chloride potential, but can be divided into three cation permeability zones according to a limiting chloride potential of +170 mV and +45 mV in 300 mOsm sucrose solution. It was discussed that the infl ection points probably refl ect a molecular confi gurational change in the membrane due to the electric fi eld. The Goldman equation predicts a straightline relationship between electrochemical driving force and fl ux. By applying the Goldman equation to the data the permeability from 0 to +45 mV is not signifi cantly changed. From +45 to +170 mV the permeability increases signifi cantly with a tendency to a maximum and plateau above +100 mV. Above +170 mV a second steep increase in permeability is observed, but the data do not extent far enough to ascertain whether or not a new stable state is reached. This increase in permeability may represent the behaviour of normal channels or the opening of new channels. The gradual increase in permeability from +45 to +170 mV could be explained by diff erent populations of channels or the gradual opening of channels under the force exerted by the potential.

The Anion Exchanger Band 3 and the Effect of Low Ionic Strength in Salt Effl ux
The fi rst prediction of anion exchange being involved in the eff ect of low ionic strength was given by Jacobs and Parpart [5]. The precise technique developed by Jacobs in 1930 to measure the osmotic properties of erythrocytes in approximately 1.2 seconds for the study of haemolysis permitted experiments of fast processes. The involvement of an anion exchange at low ionic strength was stimulated by Jacobs from the theoretical work of Netter H [8], who pointed out that anions from erythrocytes would tend to be exchanged for OHions from the aqueous solutions in such way as to make the interior of cells more alkaline. The subsequent work of Jacobs and Parpart [5] with highly diluted blood of the ox lead to the prediction of two phases of RBCs in sucrose solution: at fi rst a very rapid OH -/Clexchange, whose eff ects are apparent within a few seconds and complete within a minute or two, followed by the second phase of salt effl ux.
At that time, however, it was believed that the anion transport was mediated by pores lined with fi xed positive charges to account for the ion selectivity of the membrane.
On the basis of labelling experiments with disulfonic stilbene inhibitors of anion transport it appears that a prominent component of the membrane, a 95 000 Dalton polypeptide known as band 3, was characterized to mediate anion transport [26]. This protein represents over 25% of the total membrane protein. From ultra-structural data band 3 protein corresponds to intramembrane particle covering nearly the whole membrane by 1.2 x 10 6 copies [27]. By virtue of its high content of band 3 the erythrocyte membrane appears to be specialized for anion transport. CO 2 rapidly diff uses into the red cells where it is catalysed by Carbonic Anhydrase (CA) into H + and HCO 3 anion, which is quickly equilibrated between serum and red cells by Clexchanger (A). In the short time of less of a second it has to leave the lungs as CO 2 . Thus HCO 3 of the serum has to enter the red cells again by the anion exchanger, where it is cleaved by carbonic anhydrase into a proton and CO 2 to be exhaled.
Thus, at low ionic strength the fast physiological HCO 3 -/Clexchange mechanism for CO 2 exhalation is used to ascertain in times less than a second the new Donnan equilibration between anions. This is obvious from the many experiments cited in this review. The rapidity of reaching a positive Nernst potential, which is dependent on the chloride ratio in RBCs, determines the salt effl ux. In contrast, the equilibrium is also immediately interrupted by addition of an anion, such as chloride to the outside medium, which also quickly stopped the salt effl ux.

The Role of the Chloride Gradient across the RBC Membranes on Sodium and Potassium Movements
By Cotterrell D and Whittam R [30] an investigation was made to explore whether active and passive movements of sodium and potassium in human red blood cells are infl uenced by changing the chloride gradient and hence the membrane potential. Because of alkalization by the Nernst equilibrium the active transport could only be investigated from -9 to + 30 mV and compared with passive movement of sodium and potassium.
Chloride distribution was measured between red cells and isotonic solutions with a range of concentrations of chloride and non-penetrating anions such as EDTA, citrate or gluconate. The chloride ratio from internal/external was approximately equal to the inverse of the hydrogen ion ratio at normal and low external chloride, and inversely proportional to external pH. The results demonstrate that chloride is passively distributed, making it valid to calculate the membrane potential from the chloride ratio.
By changing the membrane potential from -9 to +30 mV the quabain-sensitive active potassium infl ux and sodium effl ux were decreased by not more than 20 and 40%. In contrast, the passive movements were reversibly altered, potassium infl ux was decreased to about 60% and the effl ux increased some tenfold. Passive sodium infl ux was unaff ected by the nature of the anion and dependent only on the external sodium concentration, whereas passive sodium effl ux increased about threefold. The results suggest that reversing the chloride gradient and the potential had little eff ect on the sodium-potassium pump, but increased the passive outward fl uxes of sodium and potassium.
External chloride was reduced to 10-30 mM by replacement with citrate or EDTA or gluconate, there was more chloride in the cells (90 -110 mmoles/litres RBCs) than in the medium giving a distribution ratio of about 3.0. Normal cell permeability could be restored by returning cells to chloride medium, demonstrating that the foreign anions used to alter the chloride gradient did not irreparably damage RBCs. Similar permeability changes were induced by EDTA, citrate and gluconate. The net potassium loss and small sodium gain was found to be in line with the past work on the relation between potassium loss in non-electrolyte media and membrane potential, although the eff ects with the impermeant polyvalent anions are not so dramatic as those in very low ionic strength sucrose media.

BLOOD DISORDERS | HEMATOLOGY
Clfor example as well as the net effl ux of anions, which is in contrast to the silent exchange, a conductance pathway. The question, whether the RBC chloride conductance in low ionic strength might be rate limiting for the net loss of salts from RBCs in a sucrose medium has been investigated by a new technique of fast sampling [31].

Figure 17:
The fi ltration technique as depicted above has been very successfully applied to permit rapid sampling of cell-free medium from a diluted suspension of red cells. Packed, radioactively labelled cells with a haematocrit of 1 to 2% were injected into the medium at the start of the experiment, and the rate of tracer effl ux was determined by following the increase of radioactivity in the initially non-radioactive extracellular medium [32].
With this fast-sampling method the following important properties of the anion exchange could be investigated: • The apparent high activation energy of the exchange for chloride, bromide, thiocyanate (CNS) and iodide is at low temperatures within the range of 29 to 37 kcal/ mole.
• Discrimination between the transport exchange system by a high specifi ty of the anions transport rates.
• The chloride exchange fl ux at 0°C has a rather fl at maximum between pH 7 and 8.5, and decreases steeply at both higher and lower pH.
• The hypothesis that chloride interacts with a limited number of transport sites in the membrane is corroborated by the observation that the self-exchange fl ux of chloride becomes saturated when intracellular chloride is above 120-150 meq/l cell water.
• Chloride exchange is inhibited by phloretin in contrast to the anion conductance pathway for example the KCl loss into sucrose media.
The next fi gure 18 shows the temperature dependence of chloride, bromide, thiocyanate and iodide in an Arrhenius plot. Figure 18: The rate coeffi cients were determined by following the effl ux of tracer anions from labelled RBCs into a medium containing 120 mM of either NaCl, NaBr, NaSCN, or NaI. The electrolyte medium was buffered with 22 mM NaHCO 3 and the pH was kept at 7.4 by titration with CO 2 [31] fi gure 2. By comparing the above effl ux of chloride in sucrose at 25°C with the data of LaCelle and Rothstein [22] at 23°C, it can be seen by their fi gure 9 that two diff erent phases of the effl ux have been compared. LaCelle and Rothstein measured with their statting method the second phase of salt effl ux in sucrose, whereas Wieth, et al. [31], by their rapid sampling, estimated the fi rst phase, described by Jacobs and Parpart [5] as OH -/Clexchange phase. Wilbrandt and Schatzmann [20] in fi gure 8 and LaCelle and Rothstein [22] in fi gure 9 showed by conductivity measurements that the so called OH -/Clexchange phase, lasting for only minutes, demonstrates also salt effl ux, but at a considerable higher rate as the salt effl ux in the second phase. By a rough estimation of the slopes in the fi rst and the second phases an increase by a factor 16 seems to be possible.
It would be more correct, therefore, to change the fi rst phase of OH -/Clexchange named by Jacobs and Parpart [5] into a "post OH -/Clexchange phase" of salt effl ux.
Wieth, et al. [31] concluded, from the result in fi gure 19, that the chloride conductance pathway is not rate limiting for the salt effl ux. Further information comes from the aglycon phloretin, which was present in the above experiment at 0.25 mM with no inhibition of the conductance chloride pathway. However, in chloride self exchange experiments the above concentration of phloretin inhibited the exchange pathway by 99.9% and increases valinomycin induced potassium self exchange. We observed for phloretin a keto-enol tautomerism, only the ketonic form of phloretin (pK value 7.26) at low pH was inhibitory in HCO 3 -/Clexchange and inhibited eff ective glucose effl ux but not the infl ux [33]. Thus, phloretin demonstrates an asymmetric behaviour in facilitated transporters.

The Effect of the Inhibitor DIDS's on the Anion Transporter Band 3 in Cation Permeability of Human RBCs in Low Chloride Media
A central role in anion transport, for example in inhibition and labelling the anion transporter band 3, has been the molecule DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), a potent inhibitor around the outside anion transport substrate site, known since 1974 by Cabantchik and Rothstein [26]. A precursor molecule SITS (4-acetamido-4'isothicyanostilbene-2.2'-disulfonic acid) was shown already by Philip A Knauf in 1970 [34] to inhibit the cation permeability increase in low chloride media. After this, a detailed investigation of the mechanism of DIDS in low chloride media in Phil Knauf's laboratory in Rochester was very consequent [35]. Fuhrmann Figure 20A at the left side shows the entire range of data in a semi-logarithmic plot from about 2 mM chloride outside to more than 100 mM chloride. The K + effl ux was measured by fl ame photometry from freshly prepared human RBCs at 2 to 2.5% hematocrit, incubated in sucrose solution with 2.5 mM HEPES at pH 7.6 and 37°C. Tetramethylamonium was used to replace Na + . Figure 20B is for clarity only with the log Cl o values from 10 mM and the corresponding fl ux rates up to 1.5 mmol/kg Hb x min [35] fi gure 1A+B.
Similar to LaCelle and Rothstein [22] and Donlon and Rothstein [24,25]  The inhibition mechanism by DIDS could involve the positive membrane potential as driving force for the salt effl ux in sucrose medium aff ected by the anion exchange or the net anion conductivity pathway. Indeed, inhibition is high with 91% at 2 mM chloride with about +105 mV membrane potential against no signifi cant inhibition at 40 mM chloride outside with only a slightly positive membrane potential of +25 mV. This potential might be too low for acting as an effi cient driving force. Donlon JA and Rothstein A [25] discussed that in the range of 0 to 45 mV the permeability at low ionic strength is independent of the potential. It is not clear why Jones GS and Knauf PhA [35] by knowing this fact, did their main experiments at 12.5 mM chloride outside in sucrose media with a membrane potential of +50.6 mV, which is at the border line to be eff ective as driving force for the salt effl ux in low ionic strength. From these investigations originated their main argument, that 10 μM DIDS did not aff ect signifi cantly the membrane potential, measured to be slightly reduced from +50.6 to + 47.4 mV. Even after DIDS treatment, ~16% of chloride permeability remains, so the membrane potential would still be largely controlled by the Clratio.
A mechanism of substrate and inhibitor binding, and ion translocation in band 3 is given by Philip A Knauf, et al. [36].
Already earlier measurements indicated that the transport of band 3 functions as a ping-pong mechanism. Fuhrmann  In contrast to DIDS, a further inhibitor of band 3, nifl umic acid, acting non-competitively by blocking the conformational change that mediates anion exchange with an apparent K ivalue of 0.6 μM was tested in sucrose media with 12.5 mM chloride, but did not show at the even high concentration of 50 μM nifl umic acid any eff ect on net KCl effl ux. From this it seemed that the exchange mechanism could be excluded.
Further experimental data suggested, that in low chloride media with DIDS, the selectivity for anion net fl ux might be so far decreased, that the conductivity channel of band 3 for anions could also be used for the transport of cations, for example by Na + and Rb + , as well as K + .

Cation Loss and Haemolysis of RBCs of the High Potassium Type (HK + -Type) and Low Potassium Type (LK + -Type) Suspended in Isotonic Non-Electrolyte Media
The study of Zeidler RB and Kim HD [38] used diff erent mammals from which the following RBCs of the high potassium type (HK + -type) were derived: human, guinea pig, rat, rabbit, newborn calf, newborn piglet and pig, as well as mammals with RBCs of the low potassium type (LK + -type), which contain sodium as the predominant cation species, such us: dog, cat, sheep and cow. The governing factors were to investigate salt effl ux of those diff erent red blood cells at 5% hematocrit in 300 mM sucrose media, buff ered with 10 mM TRIS-HEPES pH 7.4 and 37°C. Besides the salt effl ux from the RBCs, measured by fl ame photometry and chloridometer, the haemolysis of the diff erent RBCs was investigated. For the fi rst type the order of cation response in RBC species agrees very well with the order given by Hugh Davson [15] with human > guinea-pig > rat < pig and rabbit. In addition, Zeidler and Kim [38] included the species of the calf and the newborn pig, in which the red cells of the calf produce haemolysis after about 10 minutes and then lose K + . However, the RBCs of the newborn pig show nearly no potassium loss, as is the case with the adult pig.
The order from the species of the LK + -type RBCs at the right side of the fi gure is: Cat < cow < dog and sheep. Dog RBCs are prone to early haemolysis and therefore cannot be observed longer than ten minutes. Sheep red cells show nearly no response of sodium loss. There is, however, no diff erence in potassium and sodium permeability in both species of RBCs. [38] is the diff erent behaviour of the initial K + loss in RBCs of human, rat and guinea pig (Figure 23). Fuhrmann  BLOOD DISORDERS | HEMATOLOGY Figure 23: Under A the effects of pH in isotonic sucrose medium for RBCs of the human, rat and guinea pig are shown. The main difference is the initial effect on K + loss in μmoles/ml RBC. Whereas human RBCs increase initial K + loss, there is a decrease in rat and guinea red blood cells noticeable. In B a summary of the initial fl uxes in μmoles K + / ml RBC x minutes for different RBC species is given. There is a decrease in fl ux seen in guinea pig and rat RBCs and no effect in RBCs of the adult and newborn pig and calf, while human RBCs continuously increase their K + fl ux [38], fi gure 2.

An interesting observation made by Zeidler and Kim
The salt initially in the sucrose medium was estimated by Zeidler and Kim with 0.2 -0.3 mM, whereas during the time of effl ux from 5% RBCs the salt concentration may increase to about 5 mM. It is especially the chloride concentration, which aff ects the cation effl ux in sucrose medium. The authors concluded, that their effl ux of cations must refl ect the permeability states of the low and high NaCl concentrations at the two slopes investigated by LaCelle and Rothstein [22] and Donlon and Rothstein [25]. However, these investigators started their steady state measurements after the fi rst fast initial K + effl ux at 23°C, which might not correspond to the two states mentioned by Zeidler and Kim in their effl ux experiments. An increase of K + effl ux in human RBCs by an increase in pH is in agreement with the reported data by Maizels [9] in 1935 and Davson [10] in 1939 as well by H. Passow [39] in 1965.
The eff ects of temperature on the K + and Na + fl ux from HK + -type (A) and LK + -type (B) RBCs are shown in fi gures 24A,B.

BLOOD DISORDERS | HEMATOLOGY
HK + -type RBCs increase their fl uxes with temperature, except for calf RBCs. The temperature dependence was similar in human and rat RBCs, but guinea pig cells showed a biphasic response. The apparent activation energy calculated from an Arrhenius-plot was 11,200 cal/mole for human RBCs and 15,000 cal/mole for rat RBCs.
The Na + fl ux from LK + -type RBCs of the dog and cat increased to a maximum at 37°C, after which the fl ux decreased. In this respect both types react diff erently.
Zeidler and Kim [38] found that RBCs of the newborn calf, because of the fetal type of haemoglobin are especially sensitive to haemolysis. Some mammalian RBCs haemolyzed when suspended in buff ered 300 mM sucrose, in a manner similar to RBCs of the newborn calves, which undergo a total haemolysis in a non-electrolyte medium. This is also documented from RBCs of the calf, dog and cat. Unexpectedly, there is a large variability in diff erent cats from no to total haemolysis.
In RBCs from the calf there is a good correlation between the process of haemolysis and intracellular alkalinisation produced by the anion exchange. This fact is also well known in dog RBCs. In the absence of a buff er haemolysis rarely occurs. This fact is explained by the authors to be due to the diffi culty to maintain the critical intracellular pH of 7.5.
The alkalinization process was directly related to the anion exchanger band 3, the authors found a good correlation between the extent of haemolysis and the integrity of the band 3 protein. When this protein was hydrolysed by proteolytic enzymes, haemolysis was progressively inhibited.
Haemolysis in these mammalian RBCs could be completely prevented by SITS, an inhibitor of anion exchange ( Figure   25). As expected, the inhibitor of the anion exchange SITS completely inhibits haemolysis in dog, calf and cat RBCs. It also inhibits the increase of internal pH and effl ux of chloride.

Our Investigation on Cation Permeability in RBCs at Low Ionic Strength from 1999 to 2003
We investigated by two doctor theses by Karen Dorothea Krüger [40] in 1999 and Florian Bruness [41]  The above experiment demonstrated an inhibition of glucose effl ux by low ionic strength (The addition of 150 + 75 mM = 225 mM NaCl or 300 + 150 = 450 mOsm sorbitol is for compensation of 150 mM glucose inside the RBCs in order to start effl ux at the original volume. By the Sen-Widdas procedure [42] an increase in the apparent Km-value (Intercept at the -x-axis) of glucose transport and a decrease of Vm (Intercept at the y-axes) by about 50% was noticed.
The next fi gure 27 shows the K+-effl ux in adult pig RBCs under comparable conditions as for human RBCs.

BLOOD DISORDERS | HEMATOLOGY
In comparism to human RBCs effl ux of potassium in pig RBCs is nearly absent, which agrees very well with the results of Hugh Davson [10] on pig RBCs and with Zeidler and Kim [38] with adult and newborn pig RBCs.
Since pig RBCs, which are devoid of glucose transporters, do not show cation loss at low ionic strength, the hypothesis was framed, that glucose transporter at low chloride concentration might form a negatively charged channel, which is permeable for Na + and K + [43]. However, there are RBCs from diff erent animals which have no glucose transporter and show cation loss at low ionic strength as well as newborn pig RBCs probably with glucose transporter and no loss of cations [10] and [38].
In all the experiments heparinized freshly drawn human blood was used. RBCs were washed 3 times in 150 mM NaCl, 20 mM Hepes with Tris at pH 7.4. After the third wash RBCs were adjusted to 50% hematocrit. The experiment was started by addition of 500 μl 50% cells into 50 ml fl ux solution shaken at 37°C. By start of the experiment the 50% RBCs with their NaCl suspension were diluted 202 fold, thus 0.5% cells and 0.75 mM sodium chloride were obtained. During salt effl ux the chloride can increase by an additional 0.5 mM.  [41]. There is statistically no difference between 300 mM sorbitol or sucrose. Sorbitol is, however, easier to handle because of the lower viscosity. At zero time there are 276.9 ± 11.2 K + and 29.7 ± 1.8 Na + per kg haemoglobin (Mean ± SD, 16 experiments). 1 mM EDTA was added to remove contaminating calcium, which inhibits K + and Na + effl ux by about 15 to 20%.
The fl ux data were analysed by nonlinear regression by GraphPAD in Prism using a model with two exponential decays, which was signifi cantly better than a model with one exponential decay (p < 0.0001 for curves with 0.8, 2.0 and 5 mM chloride). Parameters table 6. Table 6: It shows the result for the parameters of the four curves at 0.8, 2.5 and 10 mM chloride. SPAN1 and SPAN2 are expressed in mmoles K + / kg Haemoglobin (Hb) for the two fl uxes with the rate constants K1 and K2 per minute. The two fl uxes are SPAN1 x K1 and SPAN2 x K2 in mmoles K + /kg Hb x minute. The plateau in mmoles K + / kg Hb is assumed to be the expression for a population of RBCs. which shows no permeability changes. SPAN1+ SPAN2 + plateau give the total mmoles K + /kg Hb for the zero time. For example at 0.8 mM chloride SPAN1+ SPAN2 + plateau = 274 mmoles K + /kg Hb. Since the sodium effl ux has statistically the same rate constants and behaves like potassium effl ux. Only the data for the latter are given.       [24], corrected for 37°C and Jones and Knauf [35] plotted against the logarithm of the NaCl concentrations.

Chloride
The data of fl ux B agree with the fl ux data of the second slope of Jerome Donlon [24] and the fl uxes given by Jones and Knauf [35]. Flux A is the initial fl ux which might be comparable with the fast fl ux measured by conductivity by Wilbrandt and Schatzmann [20], see fi gure 8, by statting [22], see insert of fi gure 9, by the fast sampling fi ltration technique with the fl ux measured by Wieth JO, et al. [31], and by the initial fl ux measurements in human RBCs by Zeidler and Kim  In order to test further properties of the possible channels we introduced the negatively charged sodium dodecylsulfate molecule, SDS, into the bilayer, which has been used to increase channel conductance [44]. SDS was shown to increase the currents through Na + and Ca ++ channels of cardiac myocytes. In the experiment of Turnheim, et al. [44] 20 μM SDS increased single channel conductance of rabbit colon BK Ca channels by 33%.   This result can only be explained by the presence of two diff erent channels for the cations, one has been activated by SDS, the other is inhibited.

BLOOD DISORDERS | HEMATOLOGY
The Dixon-plot shows intercepts at the x-axis at very low DIDS concentrations of 0.02 and 0.025 μM DIDS, which correspond to the apparent K i (Inhibitor concentrations required for 50% inhibition). The K i -values of fl ux A and fl ux B are not signifi cantly diff erent; rather they can be interpreted as an indication for the result of binding to increased outward facing anion exchanger in the unloaded E o form Furuaya W, et al. [45]. With H 2 DIDS at about equal concentrations of chloride inside and outside a K i -value of 0.119 μM has been observed against a K i -value of 0.052 μM Ki-value for cells with a large outwardly directed chloride gradient (10 mM chloride outside, temperature 0°C). According to Saul Ship, et al. [46] the rate of covalent reaction of DIDS was substantially faster than of H 2 DIDS, however, both reagents showed the same degree of inhibition of sulfate fl uxes. So, their K i -values might be comparable.
The slopes of the Dixon-plots for fl ux A and B are signifi cantly diff erent. This can be explained by the existence of two diff erent cation channels. The comparably low K ivalues of 0.02 and 0.025 μM DIDS might, however, be the expression of a DIDS binding and inhibition of the anion conductance channel.

Common Pattern Found in Effects of Salt Effl ux at Low Ionic Strength
All investigators cited, with the exception of Rudolf Mond [13], reported a salt effl ux with shrinking of the volume of red cells incubated in an isotonic solution of non-electrolytes, like sucrose, glucose or sorbitol. There are species diff erences of red cells, best suited are human RBCs. Hugh Davson [10] came to the following order of RBC species: human > guinea-pig > rat > cat > ox (Cattle) > pig and rabbit.
A more recent and intensive study of Zeidler and Kim [38] obtained about the same order of RBC species, but divided the RBCs in high potassium and low potassium type RBCs (HK+and LK + -type RBCs) with the order for the fi rst type: human < guinea pig < rat < rabbit < newborn calf < newborn piglet and pig. In addition to Davson [10] are here the species of the calf and the newborn pig included, by which the red cells of the calf after about 10 minutes undergo haemolysis and then loose K + . The RBCs of the newborn pig show nearly no potassium loss at all, as does the adult pig.
The RBCs of the low potassium high sodium type (LK +type) in cat < dog < cow and sheep (Nearly no loss) loose sodium in low ionic strength and are very prone to haemolysis.
A central mechanism of the salt effl ux at low ionic strength in RBCs is the anion exchanger band 3 or more recently named as Anion Exchanger Protein (AEP). It so far is unique among biological transporters due to its potential to translocate a wide repertoire of inorganic and organic anions. Most important for respiration is the enormously fast AEP exchange rate of HCO 3 against Cl -, which takes place in the human red cells at 37°C with a very fast half time of about 0.3 seconds. The AEP is also the protein to equilibrate for the permeating anions according to the Donnan equilibrium.
On the basis of the thermodynamic theory of membrane equilibria Donnan [7] developed a prediction of distribution of non-permeating and permeating anions. An equation was set up by Van Slyke, et al. [47] describing the relationship between the composition of the medium and the ion distribution across a membrane permeable to H 2 O and small anions like Cl -, HCO 3 and OH -, and impermeable to cations and a limited number of anions, such as negatively charged proteins, mainly the polyanion haemoglobin. Calculations were done with three assumptions: • Electroneutrality on both sides of the membrane.
• Donnan equilibrium of diff usible anions: r = Cl in /Cl out = OH in /OH out etc.
• Osmotic equilibrium By changing human RBCs incubated in an isotonic NaCl solution to an isotonic sucrose medium with only 1 mM chloride the potential of the RBCs is drastically changed from -10 mV to about +122 mV at 37°C. A new Donnan equilibrium is immediately obtained, and an alkalinization of the cell interior is induced. Most probably the positive potential acts as driving force for opening one or more cation channels (K + and Na + ) in the red cells and the AEP is responsible for an equimolar net effl ux of anions to produce a salt effl ux as has been measured by an increase in conductivity of the medium, osmotic determinations, effl ux of K + and Na + by fl ame photometry, effl ux of Clby chlorometry and isotope effl ux and change of pH inside and outside of the red cells by all authors cited except Rudolf Mond [13].
The most drastic experiments to show the involvement of AEP come from Zeidler R and Kim HD in 1977 [38], who enzymatically lysed the anion exchanger in calf RBCs, which prevented alkalinization and haemolysis of the RBCs. A more gentle way to inhibit the anion exchanger is to use competitive inhibitors of the stilbene type, which do not permeate the red cells and compete with the binding site of the anion exchanger. It was Phil Knauf [34], who in his Ph. D. Thesis in 1970 was the fi rst to use SITS to inhibit the K + and Na + permeability increase in low chloride media.
The results of Zeidler and Kim [38] showed in low potassium high sodium type blood cells that the alkalinization process was directly related to the anion exchanger band 3, the authors found a good correlation between the extent of haemolysis and integrity of the band 3 protein. When this protein was hydrolysed by proteolytic enzymes, haemolysis was progressively inhibited. Haemolysis in these mammalian Fuhrmann

BLOOD DISORDERS | HEMATOLOGY
RBCs could be completely prevented by SITS, an inhibitor of anion exchange.
In a subsequent publication by Jones GS and Philip A Knauf [35] about the mechanism of the increase in cation permeability of human RBCs in low-chloride media the authors demonstrated the involvement of the anion transporter by the use of DIDS. They plotted according to LaCelle and Rothstein [22] and Donlon and Rothstein [25] the fl uxes of potassium against low chloride media on a logarithmic scale fi gure 42. More interesting is the fact, that a quasi-linear straight line can also be drawn through the DIDS treated data points (Open squared points). In the above fi gure 43 human RBCs washed in 150 mM chloride, 2.5 mM HEPES buff er pH 7.6 were exposed to a low chloride sucrose medium with 10 μM DIDS (Squares) at 37°C. DIDS inhibited the fl ux at the lowest 2 mM chloride concentrations with 91%, with 73% at 12.5 mM and no inhibition at 40 mM and higher chloride concentrations.
The linear slope might be the result of a not DIDS sensitive pathway, whose origin is unknown as also the slope at very high chloride concentrations, which is not infl uenced by DIDS.
Donlon A and Rothstein A [25] discussed in this respect at high chloride concentrations (45 mM and higher concentrations) that in this range of 0 to +45 mV the permeability at low ionic strength is independent of the potential.

For their very intensive investigations in sucrose media
Knauf and Jones used a high 12.5 mM chloride concentration, which is not very sensitive to the membrane potential.
Furthermore, they used a low pH of 6.5, in order to keep the pH inside constant and to prevent alkalization of the cell interior by the Donnan-equilibrium. Under these special conditions 10 μM DIDS inhibited the Clpermeability by 83.6% and changed the membrane potential slightly from +50.6 to +47.6 mV, so that the positive membrane potential is still dominant even after DIDS treatment. However, the K + fl ux after DIDS treatment was much lower than expected, by assuming that DIDS aff ects K + effl ux by changing the membrane potential only. In addition to its possible eff ects on membrane potential, DIDS might have a direct inhibitory eff ect on the K + permeability which accounts for most or all of the decrease in K + effl ux. The data suggested to the authors that in low chloride media the anion selectivity of the anion exchanger itself was so far decreased, so that it can act as a very low-conductivity channel for cations. Na + , Rb + as well as K + , could utilize this pathway.
In contrast to George S Jones and Philip A Knauf [35] Krüger BLOOD DISORDERS | HEMATOLOGY diff erent cation channels in human red cells, produced by the low ionic strength. In contrast, there is evidence for only one anion channel, namely the conductivity pathway of the anion exchanger AEP. This result is opposite to the predictions of Jones and Knauf [35], postulating an unselective anion/cation channel.
Already in the literature from 1990, Halperin JA, et al. [48], it was reported, that the cation permeability increased progressively as soon as the membrane potential in human red cells exceeds +20 mV inside positive. This result suggests that the voltage-activated cation transport pathway is not the result of non-specifi c dielectric breakdown of the lipid layer, but rather is related to some membrane components, presumably proteins, the identities of which have till to today not been resolved.
In summary, the cation permeability in low ionic strength can be explained by channels, which respond to a positive membrane potential in RBCs. There are at least more than two types of cation channels, and the conductivity pathway of the anion exchanger is in charge for the salt loss at low ionic strength. In addition, there is salt loss at high ionic strength at chloride concentrations more than 40 mM outside, the salt effl ux by which seemed not to be dependent on positive electrical potential. There might also be a lower concentration range of chloride producing salt loss, which is not blocked by DIDS, and whose way of salt effl ux is also unknown. The permeability ways for these salt losses are not yet determined.

ACKNOWLEDGMENT
It is a great pleasure to thank Professor Joseph Hoff man for his suggestion to write the above review and for support with literature.