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J Appl Physiol 91: 33-38, 2001;
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Vol. 91, Issue 1, 33-38, July 2001

Physiological consequences of oxygen-dependent chloride binding to hemoglobin

H. D. Prange1, J. L. Shoemaker Jr.1, E. A. Westen1, D. G. Horstkotte1, and B. Pinshow2

1 Medical Sciences Program, Indiana University, Bloomington, Indiana 47405-7005; and 2 Jacob Blaustein Institute for Desert Research and Department of Life Sciences, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, 84990 Israel


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The PO2-dependent binding of chloride to Hb decreases the Cl- concentration of the red blood cell (RBC) intracellular fluid in venous blood to ~1-3 mmol/l less than that in arterial blood. This change is physiologically important because 1) Cl- is a negative heterotropic allosteric effector of Hb that competes for binding sites with 2,3-bisphosphoglycerate and CO2 and decreases oxyhemoglobin affinity in several species; 2) it may help reconcile several longstanding problems with measured values of the Donnan ratios for Cl-, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and H+ across the RBC membrane that are used to calculate total CO2 carriage, ion flux rates, and membrane potentials; 3) it is a factor in the change in the dissociation constant for the combined nonvolatile weak acids of Hb associated with the Haldane effect; and 4) it diminishes the decrease in strong ion difference in the RBC intracellular fluid that would otherwise occur from the chloride shift and prevent the known increase of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration in that compartment.

blood-gas transport; Donnan ratio; Bohr effect; Haldane effect; strong ion difference; bicarbonate; erythrocyte; chloride shift


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DURING GAS EXCHANGE, CHLORIDE ions move between the plasma and red blood cell intracellular fluid (RBC-ICF). The discovery of this phenomenon, known as the chloride shift, is generally attributed to Hamburger (18). The chloride shift is linked to the transport of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the opposite direction as part of the generally accepted model of CO2 transport originally formulated by Roughton (34). Studies of Hb structure using the technique of NMR and other techniques indicate that chloride in the RBC-ICF exists in a bound form along with the free pool (4, 5, 22, 23, 28, 29, 31-33, 39). More importantly, several of these studies showed that the affinity for Hb to bind chloride varies inversely with O2 saturation. Because these studies often compared the effects of exposure to 100% O2 with that to 0% O2, the physiological relevance of this phenomenon was unclear. Some authors (30) have, as an adjunct to other studies, concluded that chloride binds to Hb, but the effects of Cl- binding are too small to be of physiological consequence. The focus of the research presented here has been to investigate the binding of Cl- to Hb, both qualitatively and quantitatively, to determine the existence and extent of its physiological consequences.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To test the existence and importance of O2-dependent binding of chloride to Hb under physiologically relevant conditions, we measured the changes in the ratio of bound to free Cl- with 35Cl-NMR in Hb solutions, in whole blood, and in isolated plasma equilibrated with either venous or arterial blood gases. To quantify the changes in bound and free chloride pools, we used a chloride electrode to measure the changes in free [Cl-] (where brackets denote concentration) in Hb solutions under the same conditions.

Whole blood samples were drawn from volunteers who had signed informed consent documents. Packed cells were obtained from recently expired units of blood. Gas mixtures used either were mixed with a Wosthoff gas-mixing pump or were purchased as premixed tanks. The dry composition of simulated arterial gas was 15.2% O2-5.1% CO2-balance N2. Simulated venous gas was 3.1% O2-7.2% CO2-balance N2. Partial pressures varied slightly but not importantly with changes in barometric pressure. Samples of whole blood, plasma, or Hb solutions were equilibrated with humidified gases in a tonometer (IL 237, Instrumentation Laboratories) before a measurement was made. The PO2, PCO2, pH, [Cl-], [Na+], [K+], [Hb], and O2 saturation were measured using a Radiometer ABL-505 blood gas/electrolyte analyzer and a Radiometer OSM 3 hemoximeter.

To prepare the Hb solutions, packed human RBCs were washed at least twice with a 0.9% NaCl solution. After each wash, the cells were centrifuged at 10,000 rpm for 20 min, and the supernatant and buffy coat were removed. A lysing buffer (5 mM Na2HPO4, pH 8) was added to the washed cells and stirred for 1 h. The mixture was then centrifuged at 10,000 rpm for 40 min to separate the ghosts from the supernatant. The supernatant was concentrated until the [Hb] in the solution was 26 g/dl by using a Millipore Minitan ultrafiltration system with a retentate cutoff of 10 kDa. The Hb solution was then equilibrated in the tonometer for 1 h at 37°C with either a venous or arterial gas mixture. The preparation of Hb solutions removed Cl- from the retentate; therefore, it was necessary to restore Cl- to the preparation before analysis. During equilibration, a calculated amount of 0.9 mol/l KCl was added to the solution to bring the [Cl-] to 150 mmol/l. This concentration was somewhat higher than presumed intracellular levels but was necessary to ensure a detectable signal for NMR analysis. Hb solutions were adjusted to a pH of 7.15 using 0.1 mol/l KOH, and 2,3-bisphosphoglycerate (2,3-BPG) was added to achieve a concentration that was 25% of the measured [Hb] to simulate the environment of the RBC-ICF (1).

Whole blood samples were taken from human volunteers by venipuncture and were immediately placed on ice to minimize 2,3-BPG degradation. The whole blood samples were subjected to the same treatments and equilibration as the Hb samples except that the whole blood samples did not have 2,3-BPG added. A 10-ml sample of the subject's blood was also centrifuged at 10,000 rpm for 20 min at 4°C to obtain packed RBCs to add to the blood samples after they were diluted with the KCl standard and 2H2O to bring the hematocrit to a value between 0.41 and 0.46.

A small volume of 100% 2H2O was added to each mixture for increased NMR stability. The PO2, PCO2, [Hb], and [Cl-] were measured with the blood-gas analyzer and hemoximeter. Samples were placed in gastight J. Young NMR tubes that were flushed with either arterial or venous gas mixtures and sealed. At least four tubes were analyzed for each gas mixture. 35Cl-NMR spectra were acquired at 37°C by using a Varian UNITY Inova NMR spectrometer operating at 9.4 T. One-dimensional spectra were acquired with 512 scans with an interscan delay of 0.5 s to ensure complete relaxation recovery between scans. Chemical shifts and line widths were measured from spectra processed with 5 Hz of line-broadening apodization. Immediately after the NMR experiments, the samples were reanalyzed to ensure that pH, PO2, PCO2, and [Hb] had not significantly changed during the NMR analysis.

To quantify the reversible binding of chloride to Hb, Hb was isolated and purified as in the NMR studies. As the isolated Hb was being equilibrated to a venous gas mixture, we added 0.9 M KCl, 0.1 M KOH, and 2,3-BPG to create a Hb solution in which [Cl-] = 80 mmol/l, pH = 7.15, [Hb] = 24 gm/dl, and [2,3-BPG] = 25% of the molar [Hb]. These parameters, with the exception of [Hb], which was low because of dilution, correspond to the internal environment of a RBC. A given sample was then alternately equilibrated to arterial and venous gas mixtures to demonstrate the on and off binding of chloride. Each sample was cycled between arterial and venous gases at least four times. The [Cl-] of the free pool was measured with the Cl- electrode of the blood-gas analyzer. The accuracy of this measurement was constrained by the blood-gas analyzer, which reports [Cl-] to only the nearest millimoles per liter.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NMR data. Results of the 35Cl-NMR studies on plasma, Hb solutions, and whole blood are presented in Tables 1-3, respectively. Included in Tables 1-3 are the associated blood chemistry values for the samples. The arteriovenous differences in standard values of blood chemistry are as expected.

                              
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  		Table 1.   35Cl-NMR data and related blood chemistry for plasma


                              
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  		Table 2.   35Cl-NMR data and related blood chemistry for Hb solutions


                              
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  		Table 3.   35Cl-NMR data and related blood chemistry for whole blood

We drew our conclusions regarding chloride binding based primarily on analysis of changes in line width of the NMR spectra rather than the longitudinal (T1) and transverse relaxation times (T2). Line width is a good measure of the relative amount of chloride binding to Hb over time (over several milliseconds) and is inversely related to T2. As such, it has been commonly used in studies on anion binding to Hb (22). Cl- free in solution has a line width of ~17 Hz, and chloride bound to Hb has a very broad line width (too broad to detect) (4, 22). When Cl- is exchanging rapidly (faster than 1 exchange/ms), the line width is a time-weighted average of the line width of free and bound chloride. An increase in line width, therefore, indicates an increase in the amount of chloride that is bound.

T1 and T2 do not show the same significance as line width because of the very slow tumbling of RBCs, which enhances the anisotropic portion of the quadripolar relaxation effect in the line width. For a viscous sample, where anisotropic effects have to be considered, such as RBC tumbling in whole blood, a nucleus like 35Cl has a quadripolar moment in addition to the standard dipole moment. For this reason, line width is a more sensitive measurement in structurally complex solutions like whole blood (26).

In plasma, the strong ions were unchanged, whereas those variables dependent on PCO2 changed appropriately. The NMR values for isolated plasma (Table 1) were not statistically significantly different for arterial and venous samples. This finding indicates that there is no important binding of chloride in the plasma and no change in plasma [Cl-] due directly to changes in gas equilibration. Hence, in whole blood samples, whatever changes are observed in chloride binding must reflect events that occur in the RBC-ICF.

In venous Hb solutions (Table 2), there were decreases in [Cl-] and increases in [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] and [H+], whereas [Na+] and [K+] were unchanged. The NMR line widths of arterial and venous equilibrated Hb solutions show that statistically significantly more chloride is bound at venous than at arterial PO2. The average chemical shift between arterial and venous samples was statistically significantly different only for Hb solutions and not for whole blood samples, because of the greater inhomogeneity of those samples.

In venous whole blood samples (Table 3), there were, relative to arterial whole blood, decreases in [Cl-], increases in [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] and [H+], and no change in [Na+] and [K+]. The arterial line width was significantly less than the venous line width. These data concur with our observations from Hb solutions and further support our hypothesis that, in whole blood, Hb binds more chloride in the oxygen-depleted state.

Cl- electrode data. The concentration of free Cl- in Hb solutions decreased by a consistent 1-2 mmol/l from arterial to venous blood-gas partial pressures. This change was reversed by subsequent equilibration to arterial blood gas. The measures of changes in the amount of chloride bound to Hb that occur between arterial and venous conditions are appropriate in both their quantity and direction. For a given experiment, which consisted of no fewer than four cycles between gas mixtures, the change was always identical in direction and magnitude. In three replications of the experiment, the reported change was measured invariably as either 1 or 2 mmol/l for a given set. Because the resulting variance of a given set of data was zero, no statistical comparison of the data is necessary.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The binding of chloride to Hb, as indicated by NMR studies of Hb solutions and whole blood and by tonometry studies of Hb solutions, is greater in venous than in arterial blood. Thus the phenomenon of oxygen-dependent binding of chloride to Hb, previously studied with large changes in PO2, operates in whole blood with physiologically relevant changes in PO2. Furthermore, this reversible binding, which is in the millimolar range, is of sufficient magnitude to be of importance to several phenomena of the RBC. Although further research is necessary to elucidate the degree to which chloride binding to Hb influences each of the following examples, we will briefly discuss four areas wherein understanding may be enhanced by incorporation of the reversible binding of chloride to Hb: Donnan ratios, Haldane effect, allostery, and acid-base balance of the RBC-ICF.

Donnan ratios. The distribution of ions between the RBC-ICF and plasma takes on the appearance of a Donnan equilibrium (27). The exact ratios of intracellular-to-extracellular concentrations of Cl-, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and H+ have been the subject of research going back many decades (15). Calculation of the Donnan ratios has been used to analyze RBC membrane potential (15), water movement between plasma and RBCs (10, 11), and the band 3 anion exchanger (2, 6, 41). However, we know of no cases in which intracellular chloride activity was measured. Rather, total intracellular chloride concentration was measured, and it was assumed that the fraction of the total chloride that was ionized was the same inside the cell as it was in the plasma (15, 19, 40).

This assumption has long been recognized as problematic and has been postulated to account for aberrations between calculated and measured Donnan ratios as well as differences between the ratios for [H+] and [Cl-] by way of a difference either of the activity coefficient for chloride (15, 19, 40) or of protein binding of chloride (15). We feel that the findings of our research demonstrate that the fraction of total chloride that is ionized is, in fact, different between plasma and RBC-ICF because of Hb binding, which may help to explain the aforementioned aberrations between calculated and experimental data.

As an example of the effect of binding of chloride, consider that Funder and Wieth (15) reported an inequality between the Donnan ratios for Cl- and H+ (rCl- = 0.707; rH+ = 0.676). If, of the 82 mmol/l they report for intracellular [Cl-], the concentration were reduced by 3.5 mmol/l as a result of binding to Hb, the concentration of ionized Cl- would be sufficiently reduced such that rCl- would equal rH+. The chloride binding in the preceding example is greater than the binding found in our study. However, we measured only the change of oxylabile chloride and not total bound chloride. It is well established that Hb has both oxylabile and nonoxylabile chloride binding sites (4, 5); therefore, the quantity bound may be greater than our measurements indicate. The binding of chloride is, therefore, an important factor in the reconciliation of the discrepancies in the Donnan ratios for Cl-, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and H+.

Haldane effect. Deoxyhemoglobin is a weaker acid than oxyhemoglobin. This fact was first demonstrated in 1914 (7) and has become known as the Haldane effect. The change in the dissociation constant for the combined nonvolatile weak acids (KA) of Hb associated with the Haldane effect causes more H+ to bind to Hb and allows more HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to be carried in the RBC-ICF than would otherwise be possible. The Haldane effect is caused by a conformational change of Hb secondary to deoxygenation, which exposes relatively more weak acid amino acids than are exposed in oxyhemoglobin.

It has been shown several times that the reversible binding of chloride to Hb in deoxygenated blood occurs primarily at those sites that have been newly protonated by the Haldane effect (5, 29, 39). It seems reasonable to conclude then that the binding of chloride is at least partially dependent on the Haldane effect. The reverse is also true, i.e., the binding of chloride enhances the Haldane effect (20, 24, 25). Intuitively, one might guess that the binding of chloride would facilitate the binding of H+ because it increases the negativity of the Hb molecule. In fact, Perutz et al. (32) have shown that chloride binding electrostatically stabilizes the deoxygenated state. Although this may be true, the effect is apparently not as simple as an electroneutral binding of H+ and Cl- in deoxyhemoglobin. Rather, Cl- is a chaotropic effector of Hb in that it disrupts the protein molecule in such a way that 1 meq of effector alters the charge of the protein by more or less than 1 meq (30). In the case of Cl- and Hb, 1 meq of bound chloride increases the negativity of Hb by 3-5 meq (30). How and why this occurs is not completely understood nor is the exact role of the chaotropic effect of chloride binding in facilitating H+ binding. Unfortunately, the definitive work that quantified the Haldane coefficient (35) did not report [Cl-] or changes in [Cl-] associated with the Haldane effect.

Allostery. Chloride decreases the O2 affinity of Hb and increases the alkaline Bohr effect through allosteric interaction (33). It has been estimated that ~30% of the Bohr effect is eliminated when chloride binding is inhibited (29). Relative to 2,3-BPG, the negative allosteric effect of chloride is of lesser importance in healthy humans. However, information on the importance of Cl- binding in human disease is lacking. Inorganic phosphate, for example, binds to Hb, and, although it occurs to only a limited extent, knowledge of its mechanism has led to successful therapeutic treatment of at least one human condition, diabetic ketoacidosis (8). In bovines, where Cl- is the major allosteric effector, hyperchloremia has been implicated in altering the O2 half-saturation pressure of Hb (3, 14, 16, 17). We believe that the chloride binding found in the present study, as well as the importance of chloride binding for the Bohr effect, suggests that similar clinical benefits may be uncovered through further study of chloride binding in humans.

Acid-base balance of the RBC-ICF. Analysis and understanding by means of the independent variable approach, first proposed by Stewart (36, 37), has gained acceptance among physiologists as a useful framework for the study of acid-base balance in the body (9, 13, 20, 21, 38). It combines the laws of mass action, conservation of mass, and electroneutrality into a summary equation (Eq. 1) that describes the electrochemical environment of a solution
<AR><R><C>[SID]<IT>+</IT>[H<SUP>+</SUP>]<IT>−</IT><FR><NU><IT>K</IT><SUB>C</SUB><IT>×</IT>P<SC>co</SC><SUB>2</SUB></NU><DE>[H<SUP>+</SUP>]</DE></FR></C></R><R><C>       [HCO<SUP>−</SUP><SUB>3</SUB>]</C></R></AR><AR><R><C>−<FR><NU>K<SUB>A</SUB><IT>×</IT>[A]<SUB>tot</SUB></NU><DE>(<IT>K</IT><SUB>A</SUB><IT>+</IT>[H<SUP>+</SUP>])</DE></FR></C></R><R><C>[A<SUP>−</SUP>]</C></R></AR> (1)

<AR><R><C>−<FR><NU>K<SUB>3</SUB><IT>×K</IT><SUB>C</SUB><IT>×</IT>P<SC>co</SC><SUB>2</SUB></NU><DE>[H<SUP>+</SUP>]<SUP>2</SUP></DE></FR></C></R><R><C>[CO<SUP>2−</SUP><SUB>3</SUB>]</C></R></AR><AR><R><C>−<FR><NU>K′<SUB>W</SUB></NU><DE>[H<SUP>+</SUP>]</DE></FR></C></R><R><C>[OH<SUP>−</SUP>]</C></R></AR><IT>=</IT>0
The utility of this approach lies in its distinction between those variables that can be directly or independently controlled and those that are dependent or can be only indirectly controlled.

The three independent variables are, first, the strong ion difference ([SID]), which is the difference between the combined concentrations of the strong (completely dissociated) cations and strong anions; second, the PCO2; and, third, the sum of the concentrations of the associated and dissociated nonvolatile weak acids including albumin and other proteins ([A]tot). The dependent variables are [H+], [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>], [CO<UP><SUB>3</SUB><SUP>2−</SUP></UP>], and [OH-]. KA and the dissociation constants for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, carbonate, and water [KC, K3, and K'W, respectively (37)] complete the terms of Eq. 1.

In biological solutions, [SID] is always positive and with the second term, [H+], makes up the total positive charge in the solution. The remaining terms are the weak anion concentrations expressed in terms of their dissociation constants and independent variables. They are HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, combined nonvolatile weak acid anions ([A-]), carbonate ([CO<UP><SUB>3</SUB><SUP>2−</SUP></UP>]), and hydroxyl ions ([OH-]), respectively. The magnitudes of the [H+], [CO<UP><SUB>3</SUB><SUP>2−</SUP></UP>], and [OH-] are vanishingly small relative to the other terms (9) but are presented here for mathematical completeness.

When the terms of the equation are converted to a common denominator for mathematical solution, the individual chemical components lose their identity, and the equation takes on a quartic form (Eq. 2), which is practically insoluble by hand but can be solved readily on a desktop computer with the appropriate software (we used MathSoft's MathCad 4.0)
[H<SUP>+</SUP>]<SUP>4</SUP><IT>+</IT>(<IT>K</IT><SUB>A</SUB><IT>+</IT>[SID])<IT>×</IT>[H<SUP>+</SUP>]<SUP>3</SUP><IT>+</IT>{<IT>K</IT><SUB>A</SUB><IT>×</IT>([SID]<IT>−</IT>[A]<SUB>tot</SUB>) (2)

<IT>−</IT>(<IT>K</IT><SUB>C</SUB><IT>×</IT>P<SC>co</SC><SUB>2</SUB>)<IT>+K′</IT><SUB>W</SUB>}<IT>×</IT>[H<SUP>+</SUP>]<SUP>2</SUP><IT>−</IT>{<IT>K</IT><SUB>A</SUB><IT>×</IT>(<IT>K</IT><SUB>C</SUB><IT>×</IT>P<SC>co</SC><SUB>2</SUB><IT>+K′</IT><SUB>W</SUB>)

<IT>+K</IT><SUB>3</SUB><IT>×K</IT><SUB>C</SUB><IT>×</IT>P<SC>co</SC><SUB>2</SUB>}<IT>×</IT>[H<SUP>+</SUP>]<IT>−K</IT><SUB>A</SUB><IT>×K</IT><SUB>3</SUB><IT>×K</IT><SUB>C</SUB><IT>×</IT>P<SC>co</SC><SUB>2</SUB><IT>=</IT>0
Chloride is a strong ion. Its binding to Hb removes it from the RBC-ICF and thereby increases the [SID] of that compartment. The important effects of movements of Cl- on CO2 carriage in the plasma and RBC-ICF via their alteration of [SID] can be understood through manipulations of [SID] and subsequent solutions of the summary equation. It is conventionally assumed that the H+ liberated in the synthesis of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is increasingly bound to Hb as a result of a change in its KA that makes Hb a weaker acid (the Haldane effect, discussed above). However, the binding of chloride affects the production of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and change in pH independently of the Haldane effect through prevention of the change in [SID] that would otherwise occur as a result of the chloride shift. This phenomenon is usually not considered, and, because its effect is similar to a change in KA, its neglect may give the appearance of a change in KA that is larger than what actually occurs.

Manipulation of [SID] in Eq. 1 indicates that the binding of chloride can contribute a substantial amount to a decrease in the change in pH and an increase in the amount of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> that can exist in the RBC-ICF when the PCO2 changes from arterial to venous levels. The values we used for the constants and independent variables and resulting changes in dependent variables for the RBC-ICF are shown in Table 4. In the solutions to the equation that we have considered, we have calculated the changes in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and pH that occur in venous RBC-ICF if [SID] is maintained constant as a result of binding of the incoming chloride or if [SID] decreases as a result of the chloride shift that simply increases the concentration of that ion in the free pool in the RBC-ICF.

                              
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  		Table 4.   Values of constants and variables in the RBC-ICF used for calculations involving Eq. 1

By leaving KA constant to observe the effect of [SID], it can be seen that, relative to the arterial RBC-ICF, if the [SID] decreases by 2 mmol/l, as would be the case when no incoming Cl- is bound (Table 4, venousa), the pH decrease is 0.064, and, more importantly, the [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] actually decreases slightly. If the [SID] remains constant, i.e., if the incoming Cl- is bound (Table 4, venousb), the pH decrease is reduced by about one-half, and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] increases by 0.9 mmol/l. If the KA is allowed also to decrease while the [SID] is held constant (Table 4, Venousc), the change in pH is further reduced, and the [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] doubles to a value that closely matches measured values (12). These examples demonstrate that the binding of chloride has a substantial effect on CO2 carriage in the RBC-ICF via maintenance of the [SID], independent of the change of the KA of Hb.


    ACKNOWLEDGEMENTS

We thank M. Pagel and J. Frey for assistance with the NMR analysis, D. Daleke for assistance with Hb solution preparation, N. Marshall for technical and bibliographic assistance, and S. Rothenberger for units of expired blood.


    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grant GM-057372.

Address for reprint requests and other correspondence: H. D. Prange, Medical Sciences Program, Indiana Univ., Jordan Hall 104, 1001 East 3rd St., Bloomington, IN 47405-7005 (E-mail: prange{at}indiana.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 March 2000; accepted in final form 24 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 91(1):33-38
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