5 Rapid Ion Exchanges across the Red Cell
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Membrane E D W A R D D . C R A N D A L L and R O B E R T E . FORSTER Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, Pa. 19104
A stopped-flow rapid-reaction apparatus was used to measure the time course of pH changes in human erythrocyte suspensions. In one set of experiments a red cell suspension at pH 7.2 was mixed with an isotonic saline solution whose pH had been adjusted to a value between 2.1 and 10.4. Analysis of the results enabled computation of erythrocyte hydroxyl ion permeability as a function of pH. Further experiments were then performed in which erythrocyte suspensions at low pCO2 were mixed with bicarbonate solutions at high pCO2. Analysis revealed that CO equilibrium in the mixture was reached quickly, but pH equilibrium was delayed. Evaluation of the results indicates that variation in red cell OH- permeability with pH is not compatible with a simple fixed-charge hypothesis of membrane permselectivity, and the uncatalyzed hydration-dehydration of CO in extracellular fluid is required to produce pH equilibration after blood-gas exchange. 2
2
T N i f f u s i o n across the red blood cell membrane may play an important, if not limiting, role i n blood-gas exchanges, regulatory mechanisms, and other physiological processes. Accordingly, great efforts have been made to try to elucidate the determinants of these exchanges. The litera ture is filled with studies of the transmembrane movements of many different substances. I n this work we have limited study to several ionic exchanges under special in vitro conditions, hoping to shed light on the movements of the ions, on properties of the membrane, and on some of the accepted notions of blood-gas exchanges. W e have directed our attention to, in particular, the fixed-charge hypothesis of membrane perm65 Reneau; Chemical Engineering in Medicine Advances in Chemistry; American Chemical Society: Washington, DC, 1973.
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selectivity and carbon dioxide-bicarbonate exchanges between red cells and plasma with their concurrent reaction phenomena. W e present first the equipment developed for the study of rapid p H changes i n cell suspensions, and we then discuss the use of this equipment i n studies of the hydroxyl ion permeabihty (POH-) of the human erythrocyte membrane and i n studies of p H equilibration between red cells and their environment during blood-gas exchanges. Finally, the implications of these studies, relative to previously presented hypotheses about anionic and blood-gas exchanges, are critically examined. Rapid-Reaction
Apparatus
Previous workers used continuous-flow ( I ) and stopped-flow (2) rapid-reaction instruments to follow fast p H changes by means of spectrophotometric measurements of indicator dyes. A pH-sensitive glass electrode was used successfully i n a continuous-flow rapid-reaction ap paratus to follow rapid p H changes (3) and rapid N H exchanges (4) in red cell suspensions. The fastest device of the pH-electrode, stoppedflow type previously reported seems to have had a response time of less than 0.3 sec (5). A n undocumented report of a system with a 60 msec response time has also appeared i n the literature (6). W e developed a stopped-flow rapid-reaction apparatus, using a commercially available pH-sensitive glass electrode, which can follow changes of ± 0 . 0 2 p H units i n less than 0.005 sec i n a fluid volume of 0.1 m l and requires reaction solution volumes on the order of 1 m l . 3
Description. A schematic diagram of the stopped-flow equipment is shown i n Figure 1. Reacting solutions i n a pair of 3-ml syringes, A and B, are forced into a four-tangential-jet mixing chamber (^-0.004 m l ) , out of which the mixture flows against the p H electrode in a 0.1 m l chamber and then out into the stop syringe (or a bypass). The mixing chamber and electrode assembly are attached to a Gibson-Milnes type instru ment (S). The A g - A g C l reference electrode and the glass electrode have maximal diameters of about 0.3 cm ( commercially available as Leeds and Northrup #117233) with the pH-sensitive glass forming the conical tip of the glass electrode. The outputs from the glass and reference elec trodes go directly into a high input impedance amplifier. Several different devices were tried, the best being a Transidyne General ( A n n Arbor, M i c h . ) M P A - 6 dc preamplifier (solid state) with its own power source. The output from this impedance matching device is fed directly into a storage oscilloscope (Tektronix Type 564). It was possible to read the oscilloscope output to about ± 0 . 0 1 p H unit, limited by 60 cps noise. Response Characteristics. The p H electrode in the assembled appa ratus was calibrated with standard buffer solutions. The electrode output
Reneau; Chemical Engineering in Medicine Advances in Chemistry; American Chemical Society: Washington, DC, 1973.
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Rapid Ion Exchange
C R A N D A L L A N D FORSTER
REFERENCE ELECTRODE „ STOP /> SYRINGE KCL WELL BRIDGED TEFLON PLUG
MEASURING CHAMBER
1
x
= \ DRIVING BLOCK Journal of General Physiology
Figure 1.
Schematic diagram of the stopped-flow rapid-reaction apparatus (7)
was linear with p H up to about 10.0 above which alkaline error became significant. The sensitivity of the electrode was usually about 56 m v / p H unit at 37°C. To determine the speed of response of the instrument, a step change in the p H of the fluid around the electrode would have been ideal. It was not possible to do this experimentally i n less than 0.030 sec, owing to the time required to wash out the electrode chamber. However, it was possible to estimate this speed using a ramp input, namely, the linear increase i n p H produced by the dehydration of carbonic acid (1,2,5,9): k H+ + H C 0 - - H C 0 ^ H 0 + C 0 Κ ku v
3
2
3
2
2
(1)
a
K is the true equilibrium constant for the first reaction, a neutralization which for practical purposes can be considered instantaneous (10). T h e forward (dissociation) and backward (association) rate constants for the second reaction are k and k . a
v
u
Reneau; Chemical Engineering in Medicine Advances in Chemistry; American Chemical Society: Washington, DC, 1973.
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C H E M I C A L ENGINEERING I N M E D I C I N E
W h e n the reacting solutions are 0.01 Ν HC1 and 0.02 M N a H C O , it can be shown ( I I ) that s
* „
(
H C0 2
3
becomes important. A representative curve is shown i n Figure 2. The rate constant k at 26 °C was calculated from the initial slopes of this curve and others like it to be 24.1 sec" , comparable with the values of 25.5 sec" at 25 °C reported by Rossi-Bernardi and Berger (3) and 20.0 sec" at 25°C reported by Khalifah (9). v
1
1
1
Reneau; Chemical Engineering in Medicine Advances in Chemistry; American Chemical Society: Washington, DC, 1973.
5.
C R A N D A L L A N D FORSTER
Rapid Ion Exchange
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The response time of the electrode system was estimated from Figure 2 by extrapolating the linear slope backward. It was estimated that the electrode system responds to a ramp change in p H i n
