- Email: [email protected]
The Suitability of the antimony electrode for pH determinations in mammalian heart
The Suitability of the Antimony Electrode for pH Determinations in Mammalian Heart
N. 5. DHALLA, J. C. YATES, I. KLEINBERG, J. C. KHATTER,
AND
R. J.
HOESCHEN
Antimony electrode provides a stable and reproducible means for the determination of intramuscular pH. large changes in bicarbonate or phosphate concentrations are required before a significant alteration in electrode response is observed and thus is quite suitable for physiological measurements. However, dramatic shift in mv-pH relationship for this electrode was seen upon changing pOz of the medium. When the electrode was employed to monitor intramuscular pi-l in isolated perfused rat heart, it was found that Na+ or K+ free medium produced a severe acidosis @H < 6.5) along with loss of contractility. Ca2+ free perfusion, on the other hand, resulted in a failure of contractility within 30 set without any change in interstitial pH. Upon stretching the heart when the perfusion rate was maintained at constant level, an increase in both contractile force andpH occurred. Norepinephrine along with increased perfusion flow produced an increase in both rate and force of contraction without any significant change in interstitial pH. However, at constant perfusion rate, the interstitial~H increased dramatically. Similarly, global ischemia resulted in a decline in contractile force and initial increase and subsequent decrease in interstitial pH. No decrease in pH was noted when coronary flow was reduced gradually. These results suggest that the antimony electrode is only suitable in situations which are not associated with larger changes in ~0,. Key Words:
Antimony electrode;
Intramuscular pH; Contractile force
INTRODUCTION Continuous and accurate measurement of pH changes in intact muscle and body fluids is of considerable value in physiological and pharmacological research. Antimony-antimony oxide-type electrode has considerable potential in such applications as this electrode is quite robust and can easily be made in a variety of shapes and sizes for specific applications (Kleinberg, 1958). Earlier, we reported that the antimony electrode is quite stable in the heart tissue and does not suffer from isolation problems resulting in “memory” (Dhalla et al., 1973; Yates et al., 1979). Furthermore, whereas the response time of glass pH electrodes can often be a From the Division of ExperimentalCardiology, Departments of Physiology, Medicine and Oral Biology, University of Manitoba, Winnipeg, Mant., Canada. Address reprint requests to: Dr. J. C. Khatter, Department of Physiology, University of Manitoba, Winnipeg, Mant. R3E OW3 Canada. Received May 29, 1979; revised July 76, 1979.
fournal of Pharmacological Methods 3, 221-234 (1930~ @19&l Elsevier North Holland, Inc., 52 Vanderbilt Avenue, New York, NY 10017
221
owmo2mom3o22~i4w2.25
222
N. S. Dhatfa et al.
limiting factor, the speed of response of the antimony-antimony oxide system is apparently limited by the time constants of the measuring instruments (Bishop and Short, 1964). The subject of construction of a suitable antimony electrode for pH measurements has been reviewed by Stock et al. (1958)‘ and the construction of useful electrodes of quite small dimensions has been reported subsequently (Vieira and Malnic, ‘1968; Bicher and Ohki, 1972). Applications to biological situations thus far have included pH measurement in blood (Buytendiyk, 1927; Brinkman and Buytendiyk, 1928), gastric contents (Erb and Senior, 1938; Haggard and Greenberg, 1941; Amann and Kronberger, 1958; ,Kobayashi, 1960), teeth and contents of the oraf cavity (Moore and Fosdick, 1952; Thompson and Brudevold, 1951,1954; Brudevold and Thompson, 1954; Kleinberg, 1961, 1967; Kleinberg and Jenkins, 1964), living cells (Kurella and Popov, 1960; Vorobev et al., 1961; Bicher and Ohki, 1972), renal cortical tubules (Vieira and Malnic, 1968) and heart muscle (Dhalla et al., 1973; Yates et al., 1979). In spite of a considerable amount of information gained by its use, there has been no attempt to systematically evaluate the suitability of the antimony electrode for such applications. The purpose of the present study was fourfold. First, to establish the stability of the electrode in an environment simulating mammalian tissue or body fluids; second, to confirm the lineari~ of the mv-pH relationship within a range of pH values likely to be encountered in biological systems; third, to determine whether any of the substances normally present in such an environment interfere, chemically or otherwise, with the function of the electrode; and last, to determine the changes in myocardial pH in isolated perfused rat heart when contractile force is modified through various inte~entions. METHODS The antimony-antimony oxide electrodes used in this study consisted of short antimony rods, approximately 0.5 mm in diameter, fused to the end of a silver wire and insulated, except at the tip, by a thin coating of epoxy resin. The reference electrode consisted of a saturated KCI bridge and a standard saturated calomel electrode. The antimony electrode and reference electrode were coupled to a highimpedance digital voltmeter (Data Technology DT-360-B) from which the potential difference was read. Two or three different antimony electrodes were used in each experiment and compared to the reading from a glass-calomel reference combination electrode coupled to a radiometer pH meter. The buffer solutions used as standards in this study were made by mixing various proportions of equimolar solutions of NaH2P04 and Na,HPO,. The perfusion solution used in certain experiments was a modified Krebs-Henseleit solution containing 120 mM NaCI; 25.4 mM NaHCO,; 8 mM glucose 4.8 mM KCI; 1.2 mM KH,PO,; 0.86 mM MgSO *; and 1.25 mM CaCI,. This solution was also employed to simulate
the
chemical
environment
of mammalian
tissues,
and the
stability,
consistency, and linearity of thepH response of the antimony electrode was studied in modifications of this solution in which each constituent was systematically varied
Suitability of the AntimonypH
Electrode
between pH 5.0 and 9.0 by the addition of HCI or KOH. The CaCI, was omitted from most solutions to avoid precipitation at the higher pH values. The influence of CaCI, on the pH response of the antimony electrode was studied in solutions from which NaHCO, and KH,PO, had been omitted to avoid precipitation at higher pH values. The effect of p0, on the mv-pH relationship of the antimony electrode was studied in 0.1 M phosphate buffer saturated with 95% Oz, air, or in solutions gassed with 100% nitrogen as they became equilibrated with air. The oxygen tension of the solution was monitored by a Clark electrode coupled to a Giison Oxygraph. Tissue pH measurements were made in the spontaneously beating, isolated, perfused rat hearts prepared according to the procedures described previously (Yates and Dhalla, 1975). In experiments in which the composition of the perfusion medium was altered, the osmolarity was maintained by the addition of equimolar amounts of sucrose. All the perfusion media were gassed with 95% O2 and 5% COz mixture and maintained at 37°C. The pH of these solutions, unless indicated otherwise in the text, was in the range of 7.3-7.4. The coronary flow was controlled at 8 ml/min by using a Harvard peristaltic pump and the perfusion pressure varied between 70-75 mm Hg. In some experiments, ventricles were electrically driven using a square-wave stimulator to apply pulses just above the threshold between two platinum electrodes located in the apex of the heart and the intraventricular septum at” the base of the heart. The results with paced hearts were found to be similar to those obtained with spontaneously beating hearts. All these hearts were punctured with the tip of fine scissors at the beginning of the perfusion to avoid fluid accumulation in the ventricles. The intramuscular pH of myocardium was measured by inserting the electrode in the ventricular wall. The continuity between the reference electrode and the tissue was achieved by means of a saturated KCI bridge. Diffusion of KCI through the plug in the measuring end of the salt bridge was very slight but was sufficient for electrical continuity (Kleinberg, 1967). Antimony and calomel electrodes were connected to a radiometer pH meter (model 26) and the signal recorded on the Crass polygraph. Simultaneous recording of contractile force and surface electrical activity were made using a 3-channel Grass polygraph. A resting tension of 5 g was applied to all the hearts used in this study on starting the perfusion and the contractile force was monitored via a force displacement transducer (FT 0.03). The surface electrical activity was recorded as a difference in potential between two platinum electrodes, one connected to the right atrium and the other to the base of the left ventricle. The hearts were perfused with normal medium for 15 min to allow equilibration before any experimental intervention was made. Global ischemia was simulated by clamping the tube supplying perfusion medium to the cannula. For the sodiumfree perfusion, NaCl and NaHCO, were replaced by an equivalent amount of sucrose and thepH was adjusted to 7.4 with KOH. In the K+ free medium KCI was replaced by an equivalent amount of sucrose and KH2P04 was replaced by an equivalent amount of NaH,PO,. No adjustment was made for the omission of CaCI, from the calcium-free medium. Where norepinephrine was used, 0.1 pg was injected into the flow of perfusion medium just before it entered the cannula. The
223
224
N. S. Dhalla et al.
=03--I
PH
FIGURE 1. Effectsof different phosphate buffer concentrationson mv-pH relationships of an~~ny electrodesystem.A- A 0.05 M phosphate, 8-m 0.10 M phosphate, a4 0.20 A4 phosphate, --best fit lines for pH < 7.0 and pti > 7.0 in 0.20 M phosphate.
effects
of stretching
the heart were tested
by increasing
the resting
tension
on the
myocardium. RESULTS Comparison phate
buffer
of the mv-pH revealed
relationship
that the calibration
with
different
curves
concentrations
obtained
in this
of phos-
manner
were
affected in two distinct ways by the buffer concentrations. As can be seen in Fig. 1, the mv-pH relationship is shifted upward as the buffer concentration is increased, and furthermore, that with higher buffer concentrations is no longer a straight line, but is better fit by two linear segments slopes
and intercepts.
regression
Table
1 gives the values
the relationship having different
for the slope and intercept
of the
lines obtained
using two different electrodes in 0.05 M, 0.1 M, and 0.2 coefficient) M phosphate buffers. As can be seen, the RZ value (R - correlation obtained for the sets of data points above and below pH 7.0 using a buffer concentration of 0.2 M indicate a better linearty than the R2 value for the set of data points obtained over the entire range of pH values. Table 2 provides the values for the slope and intercept of the mv-pH relationship obtained in Krebs-Henseleit solutions and in modified Krebs-Henseleit solution in which the concentration of individual components have systematically been varied above and below their normal values. As indicated, the slope and intercept of the
Suitabili~ of the Antimony~H Electrode TABLE1 Effectsof Different Concentrationsof Phosphate Buffer on Slope and Intercept of mv-pH relationship Obtained with Two SeparateAntimon~Antimony Oxide Electrodes
0.05 M Phosphate
SLOPE C S.E. INTERCEPT % S.E.
9 0.998
0.10 M Phosphate
10 0.997
0.20 M Phosphate
IO 0.985
0.20 M Subset 0.20 M Subset
4 1.000
RZ
N
24.302 “1.744 26.1% 22.338 89.615 25.228 17.866 t1.257 150.218 ko.872
49.628 -to.249 50.534 20.285 45.440 -to.709 56.477 to.607 37.824 20.398
6 0.997
Phosphate < pH 7.0 Phosphate 1 pH 7.0
ELECTRODE 2
ELECTRODE 1
__I-_ N R2
SLOPE k S.E. INTERCEPT i
9 0.998
29,367
48.991 10.258 45.401 ‘0.249 46.030 20.639 55.471 20.454 38.826 kO.010
10 0.998 IO 0.9a8 6 0.998 4 1.000
S.E.
-co.801
35.401 k2.047 83.974 c4.714 22.746 kO.941 141.377 20.022
N = is the number of experiments. R = is the correlation coefficient.
mv-pH
relationship
differs
significantly
(p < 0.05) from that obtained
Krebs-Henseleit solution in those solutions increased sodium bicarbonate or potassium none of, the effect. These similar
result
The
effect
electrodes
in the normai
having reduced sodium chloride, or phosphate concentrations, but that
other modifications of the test solutions produced any significant experiments were performed using two different electrodes, with a in each case. of oxygen
tension
in a solution
on the mv reading
at constant
pH is shown
from
two different
in Fig. 2. The influence
antimony of oxygen
tension was quite similar and very dramatic with both electrodes, and was most pronounced in the range of oxygen tension (IS-20%) most likely to be encountered in
mammalian
electrode
tissues.
reading
the antimony
In as much
is potentially
oxide-type
as the
influence
the most serious
electrode
for
biological
of oxygen
factor affecting pH
tension
on the
the suitability
measurements,
a series
of of
simple experiments were carried out to confirm the in vitro findings. When the flow of perfusion medium to isolated perfused rat hearts was abruptly discontinued (Fig. 3) an upward shift in the mv reading of an antimony electrode inserted into the ventricular wall was seen to coincide well with a 40-50% decline in the contractile
force developed
the decline
of oxygen
by the isolated tension
heart which
of the tissue.
may be considered
Subsequently
to reflect
the mv reading
of the
electrode feil steadily, as would be expected to occur with the accumulation of lactic acid in the anaerobic muscle. When the flow of perfusion medium to the heart was resumed,
the mv reading of the electrode
equal
rise,
to its initial
presumably
in response
dropped to the
abruptly
return
by an amount
of tissue
oxygen
tension to its original value. Following a short period of time necessary to wash out the accumulated lactic acid from the tissue, the electrode reading stabilized near its initial value. In a similar
experiment,
in which the flow of perfusion
medium
was significantly
225
226
N. S. Dhalla et al. TABLE 2 Effects of Changes in Different Components in Krebs-Henseieit Solutions on the Slope and Intercept of the mv-pH Relationship Obtained Two Separate An~mon~Antimony Oxide Electrodes
with
ELECTRODE 2
ELECTRODE 1 N
R2
SLOPE 4 S.E.
INTERCEPT
R2
Sk.op~ C S.E.
INTERCEPT
Control
20
0.998
15
0.997
30.123 k2.938 23.881 23.313
0.997
735 mM NaCl 5mM NaCI 45 mM NaHCO, SmM NaHCO, 2.4 mM KH,PO, 0.6mM KH,PO, 100 mM KCI 2.4 mM KCI 2.4 mM
13
0.998
12
1.000
11
0.999
11
1.ooo
8
0.999
47.353 rto.121 47.304 20.187 S4.375* 20.204 50.4OP lto.097 47.370 ~0.150 49.656* 20.352 47.681 rtO.207 47.437 +0.171 47.710 to.294 47.333 20.263 47.775 kO.197 47.395 20.426 47.483 co.534
49.375 20.137 49,090 kO.190 56.410* kO.220 51.292* I?rO.lll 49.427 20.152 49.933’ zkO.098 49.209 20.174 49.691 20.118 49.179 rtO.189 49.546 20.176 49.746 1-0.779 49.640 20.265 49.483 20.218
21.372 k3.326 18.631 23.361 -46.4421 23.028 4.269* +I .406 22.859 21.834 78.036 k1.222 18.512 ?‘I .516 24.884 k1.032 20.149 21.156 24.388 50.985 24.007 ‘1.08-l 22.450 21.842 21 .I56 ?I .421
MgSO, 0.6 mM MgSO, 2.5 mM CaCI, 0.6 mM CaCl 2
9
0.999
6
0.999
6
0.999
6
1 .ooo
6
0.998
6
1.000
-32.607* 22.809 9.529’ 21.230 28.544 +f .815 18.047* 21.318 25.868
0.997 0.938 0.999 0.999 I.000 0.999
21.806 28.331 -tl.501 26.263 21.797 29.852 21.473 26.972 +I.188 29.840 22.773 27.730 5-l .423
1.000 1.000 1 .ooo 1.000 0.999 1.000
* Indicates that value differs significantly @ < 0.05) from control. N = number of experiments in each case. R = is correlation coefficient.
reduced but not stopped entirely (Fig. 4), an increase in the mv reading of the electrode proportional to the decrease in coronary perfusion rate was seen to occur. The upper panel (Fig. 4A) shows the decrease in contractile force and increase in mv reading which follows upon the reduction of rate of perfusion from 5.5 ml/min to 1.7 mllmin. A greater reduction of the rate of perfusion from 5.5 ml/ min to 0.7 mllmin (Fig. 4B) results in a significant increase in mv reading. Furthermore, the gradual decline in the electrode reading observed in the experiment shown in (Fig. 3) and attributed to lactic acid accumulation was not observed in this situation where some degree of coronary flow persists. In another experiment, it was observed that the increased oxygen demand due to increase in rate and force of contraction (Fig. SA) resulting from an infusion 0.1 ,ug of norepinephrine into the isolated rat heart preparation was met by a 30 to
Suitabilityof the Antimony pH Electrode
300
0
20
*~"Satur~~ion
SO
100
%
FIGURE 2, Effectof oxygen saturationon mv reading of antimony electrode in sob tion of constant pH e-0 electrode #l, m-8 electrude #2.
50% increase in coronary perfusion
rate, and that the interstial pH was not noticeably altered. When the experiment was repeated under circumstances where the coronary perfusion rate of the isolated heart was prevented from increasing, an increase in the pH of 0.3-0.6 units was observed (Fig. 53) and the return of the interstitial pH to the original level coincided with the disappearance of inotropic and chronotropic effects of the drug. In a different set of experiments isolated hearts were subjected to stretch by large increases, in the order of 5 to 8 g, in resting tension applied at the apex of the heart (Fig. 6). When the perfusion rate was maintained constant, stepwise
Ebttrlca?
Activity
--__
FIGURE 3. Recording of pfi, eontractifeforce, and surface electrical activity from an experiment in which the perfusion was stopped completely ( 4 ) and restarted after 5 min ( t 1. These results are typical of 6 experiments.
227
228
N. S. Dhalla et al.
EIactticnI
Ccntractite
Activity
Farce
B
FIGURE 4. (A) Recording of pH, contractile force and surface electrical activity from a typical experiment in which the perfusion rate was abruptly decreased from 5.5 ml/ min to 1.7 milmin ( 4 ) by partially clamping the tube carrying the perfusion medium to the heart. After 5 min the clamp was released f t 1. (B) Recording from another experiment in which the perfusion rate was reduced by clamps from 5.5 mlfmin to 0.7 mllmin ( J ) for a period of 5 min, after which the clamp was released ( t 1.
increase in resting tension resulted in a parallel stepwise increase in both contractile force and interstitial pti. Figure 6 is a recording of such an experiment which was begun with a resting tension of about 5 g and a perfusion rate of 5 ml/mm while the resting tension was increased from 5 to 13, 20, 28, and finally to 36 g. The contractile force developed from 5.5 to 9.4, 11, 11.4, and finally to 12 g while the interstitial pH increased from 7.1 to 7.25, 7.5, 7.55, and finally to 7.9 pH units. At the time indicated by the arrow, the clamp limiting the rate of flow was released. As the perfusion rate rapidly increased to over 7 mllmin the pti began to gradually decline, Perfusion with a sodium-free medium resulted in a rapid decline and disappear-
Suitability of the Antimony pH Electrode
FIGURE 5. (A) Recording of pH, contractile force and electrical activity from a typical experiment in which 0.1 mg of norepinephrine was infused into the heart via the perfusion cannula ( J ) while the rate of perfusion was not restricted. The rate of perfusion prior to the ~repinephdne infusion was 4.5 mllmin, increasing to 7 ml/min after the drug was administered. The perfusion rate returned to the previous level as the chronotropic and inotropic effects of the drug disappeared. (6) Recording from an experiment similar to that shown in (A) with the difference that the perfusion rate, which was 5 ml/min before infusion of 0.1 mg of norepinephrine was maintained at this same level following administration of the drug.
ante of contractile force which was associated with the development of a severe acidotic state. By the time contractility fails, the interstitial pH is 6.5 and falls still lower over the next few minutes (Fig. 7). The state of arrest produced by sodiumfree perfusion is completely reversible if reperfusion with sodium is begun within 3 to 5 min. As can be seen in the lower panel (J3) of Fig. 7, recovery of contractile force after 3 min of sodium-free perfusion is associated with an almost parallel increase in interstitial pH, temporarily rising 0.2-0.3 pH units higher than the initial
22!3
236
N. S. Dhalla et al.
Contractile
Force
Electrical
Activity
FIGURE 6. Recording of pH, contractile force, and electrical activity from a typical experiment in which the heart is subjected to stretching by large increments in tension upon the hook located in the apex of the heart. The perfusion rate was maintained at 5 mllmin until the time indicated by the arrow (t ) when restrictive clamps were removed. At this time the perfusion rate increased to more than 7 mllmin. As the resting tension is increased from 5 to X%,29,26, and finally 36 g, the contractile force developed increases from 5.5 to 9.4,11,11.4, and finally to 12 g while the intramuscular pH increases from 7.1 to 7.25, 7.55, and finally to 7.9 pH units. Note that when the perfusion rate is allowed to increase ( t ) the intramuscular pH begins to fali again.
value and stabilizing thereafter to sodium-free perfusion. During
the course
of potassium-free
to vary in a consistent rises above nating pattern
control
strong
at a level of pH comparable
pattern.
level at about
2 min,
and weak
contractions,
changes
corresponds
of pH
perfusion
The contractile
(Fig. 8) interstitial force
becomes
and then rather
to that recorded
usually
irregular
fails abruptly
loosely
to the
prior
pH was found
declines
somewhat,
thereafter at about
with
alter-
IO min. The
contractility
changes
decreasing by 0.1 to 0.2 pH units during the first minute, rising above the control level for the next 2 to 3 min (Fig. 8A) then dropping to about pH 7.0 during the period of irregular contractions (Figs. 8B). Upon failure of contractility, stitial pH decreases steadily to a pl-l somewhat below 6.5. Upon
perfusion
of the
isolated
heart
with
calcium-free
medium
the inter(not
shown)
complete uncoupling of excitation and contraction was apparent within 30 sec. All contractile activity had ceased by this time, whereas, the surface electrical activity was not altered. occur.
In this set of experiments
no change
in interstitial
pH was seen to
The results presented here indicate that careful consideration must be given to the possibility of erroneous measurements due to ionic shifts and changes in tissue p0, when employing the antimony electrode forpH measurements in physiological research. interference due to changes in bicarbonate or phosphate are not likely to be critical factors in most experimental situations
concentrations since the influ-
Suitability of the Antimony pH Electrode *+
pH
l--l--x 7-5
7.0
I 1~ I 04
A
Tirne.3Pl't.
Cohactila
Force
----
RGURE 7. (A) Recording of pH, contractileforce, and surfaceelectricalactivity from a typical experiment in which the isolated heart was abruptly switchedfrom perfusion with normal Krebs-Henseleit medium to perfusion with a medium from which all sodium was omitted ( 4 ). (8) After a period of about 3 min, perfusion with sodiumcontaining medium was resumed ( f 1. Note the dramaticdecreasein intramuscularpH which occurs during the period of sodium-free perfusion and increase in pH to above baseline levels which occurs following return to perfusion with sodium containing medium.
ence of the electrode reading is quite small when compared to the changes in concentrations of these substances that are likely to be encountered in most circumstances. With regard to the influence of Na+ on the electrode, the location of the electrode in a typical high Na+ extracellular plasma environment will of course be an important factor and must be taken into account when calibrating the electrode. Much more serious is the influence of tissue O2 tension on the electrodes accuracy since tissue ~0, values fall in a range where very small changes in OS content produce large variations in the mv-pH relationship characteristics of the electrode. The sensitivity of the electrode to changes in p0, was recognized by other investigators in their experiments on the squid giant axon (Bicher and Ohki, 19721, and great care was taken to avoid this influence by performing all experiments and calibration in solutions saturated with air, Vieira and Malnic (1968), on the other hand, have stated that the influence of oxygen on the pH measurements in the range of p0, 34-128 was not important and could be safely disregarded. Although our experiments indicate otherwise, it is possible that some details of construction of their electrode renders it less sensitive to oxygen than ours. In any case, careful consideration must be given in any experimental model employing the antimony electrode for pH measurement to the possibility that the tissue pOZ
231
232
N. S. Dhalla et al.
Contractile
force
FIGURE 8. (A) Recording of RH and contractile force from a typical experiment in which the isolated heart is abruptly switched from perfusion with normal Krebs-Henseleit medium to perfusion with a medium from which all potassium has been omitted ( & ). (B) Continuation of the recording in (A) showing the pH changes that occur after about IO-min perfusion with potassium-free medium at a time when contractile activity ceases and the heart goes into contracture.
may differ from that of the solution used in calibration of the electrode, and further, that tissue p0, may not remain constant during the course of the experiment. It is of interest to examine the cyclic variations in pH of ventricular myocardium during the contraction-relaxation cycle reported earlier (Rhalla et al., 1973, Yates et al., 1979), in light of the present findings. In these studies it was reported that a decline in pH occurred just prior to the onset of contraction and that the pH rose toward the baseline value during and following the relaxation phase. That these oscillations are an artefact due to changes in Na+ concentration appears unlikely in view of the magnitude of change in Na+ concentration required to produce such an effect and the fact that large shifts in sodium are not known to occur in the ourse of the cardiac cycle. Similarly, the possibility that these changes are an artifact of decreased bicarbonate or phosphate concentrations does not appear reasonable in view of the magnitude of change in concentrations required to produce such an artifact as well as the fact that splitting of high-energy phosphate compounds and increased metabolism during contraction would tend to increase rather than decrease the concentration of these substances. Reasoning along the same lines, increased metabolism during contraction would tend to decrease tissue pop, and if it were a predominant factor it would serve to raise rather than lower the mv reading of the electrode. Thus, it would appear that the cyclic fluctuations reported
previously
are not due to the influence
of substances
interfering
with the
Suitabilityof the AntfmonypH Electrode 233
pH sensitivity of the electrode, but rather that these influences, if effective at all, would tend to attenuate the decline in pH observed using an antimony electrode. The experiments described in this study reveal a definite pattern to pH changes elicited with regard to oxygen supply and demand. Interstitial pH increase dramattally when oxygen supply is limited by reducing the perfusion rate or when the contractile work is increased by stretching the heart as well as upon norepinephrine administration while the oxygen supply is kept constant by preventing an increase in perfusion rate. On the other hand, when oxygen supply is increased to the heart by increasing perfusion rate in response to the stretching or norepinephrine administration, there is no increase in pH. However, changes in pH do not seem to bear any direct relationship with changes in contractile force as seen upon reducing the flow rate, stretching of the heart, as well as upon norepinephrine administration. This is further supported by the fact that an initial increase in contractile force upon perfusion with K+-free medium was associated with an increase in intramuscularpH whereas perfusion with Na+-free medium produced decrease in contractile force as well as intramuscular pH. Complete contractile failure of heart upon perfusion with Ca 2+-free medium was seen in the absence of any changes in pH. The initial increase in pH seen upon complete interruption of the perfusion flow seems to be an artifact of a decrease in tissue pOzt since similar changes in mv readings of the antimony electrode were seen when the oxygen tension was decreased in the perfusion medium while standardizing the electrode. Although other investigators (Gebert et al., 1971) using glass electrode have also reported an initial increase in intramuscular pH upon occluding the coronary arteries in dog hearts, the sensitivity of their electrode to changes in pOZ has not been tested. Alternatively, there may be a slight initial increase in intramuscular pH due to hypoxia or ischemia with the formation of creatine and other basic substances in the myocardium (Dhalla et al., 19721, but this would be of transient nature due to the production of lactic acid. At any rate, this study has demonstrated that changes in tissue pH as measured with antimony electrode should take into account the interference due to changes in tissue ~0%. Thus, in order to take advantage of the rapid response and robust characteristics of the antimony electrode for pH measurements in physiological research, one must (a) calibrate the electrode in an environment similar to that in which experiments are performed, (6) take into consideration changes in Na+, bicarbonate, and phosphate concentrations that may influence the electrode, and (c) ensure that tissue pOz is not changing during the course of an experiment and thus producing spurious “pH changes.” This investigation was supported by a grant from the Manitoba Heart Foundation.
REFERENCES Amann W, Kronberger L (19SB) Die Bedentung der intragastraien pH-Messung in der mangenchirurgie. Medizinische 40: 1576-1581. Bicher iii, Ohki S (1972) Intracellu~arpH
electrode
experiments on the squid giant axon. Bioc~i~ Ejophys Acta 255: 900-904. Bishop E, Short CD (1964) Differential electrolytic potentiometry. XIV. An examination of elec-
234
N. S. Dhalla et al. trode parameters and electrode systems in acidbase titrimetry. Analyst 89: 415-420. Brinkmann R, Bu~endiyk FjJ (1928) Die Klinische Mikrobestimmung des ,oH des Blutes mit einer Antimonelecktrode. Biochem 2 199: 387-391. Brudeyold F, Thompson FC (1954) Evaluation of the microantimony electrode. 1 Dent Res 33: 854-858. Bu~endiyk FJJ(1927) The use of the antimony electrode in determination of pH in viva. Arch Neerland Physiol 12: 319-321. Dhalla NS, Yates JC, Waltz DA, McDonald VA, Olson RE (1972) Correlation between changes in the endogenous energy stores and myocardial function due to hypoxia in the isolated perfused rat heart. Canad J Physiof fbarmacol 50: 333345. Dhalla NS, Yates JC, Kleinberg I (1973) Oscillations of intramuscularpH in perfused rat heart. Canad J Physiol Pharmacol51: 234-238. Erb RC, Senior KL (1938) Preliminary report on a new method for gastric research. ] Am Osteopath Assoc 38: 95-96.
duction in human salivary sediment Arch Oral Bioi 72: 1457-1473.
mixtures.
Kleinberg I, Jenkins CN (1964) pH of dental plaques in the different areas of the mouth before and after meals and their relationship to the pH and rate of flow of resting saliva. Arch Oral Biol 9: 493-516. Kobayashi H (1960) Continuous potentiometric determination of pH of gastric contents. Osaka Diagaku Igaku Zasshi 12: 1399-1415. Kurella GA, Popov BA (1960) Determination of pH by means of the antimony micro-electrode. Biofizika 5: 373-375. Moore BN, Fosdick LS (1952) The micro-antimony electrode. 1 Dent Res 31: 512-513. Stock IT, Purdy WC, Garcia LM (1958) The antimony-antimony oxide electrode. Cbem Rev 58: 611-626. Thompson FC, Brudevold F (1951) A micro-antimony electrode suitable for intraoral pH measurements. 1 Dent Res 30: 523. Thompson FC, Brudevold F (1954) A micro-antimony electrode designed for intraoral pH measurements in man and small experimental animals. j Dent Res 33: 849-853.
Gebert G, Benzing H, Strohm M (1971) Changes in the interstitial pH of dog myocardium in response to local ischemia, hypoxia, hyper-, and hypocapnia, measured continuously by means of glass micro-electrodes. Pffiizers Arch 329: 7281.
Vieira FL, Malnic G (1968) Hydrogen ion secretion by rat renal cortical tubules as studied by an ntimony microelectrode. Am J P~ys~o~214: 710718.
Haggard HW, Greenberg LA (1941) Antimony electrode for continuous recording of acidity of human gastric contents. Science 93: 479-480.
Vorobev LN, Kurella GA, Popov GA (1961) Intracellular pH of Nitella flexillis at rest and after excitation. Biofizika 6: 582-589.
Kleinberg I (1958) The construction and evaluation of modified type of antimony micro-electrodes for intraoral use. Br Dent J 104: 197-204. Kleinberg 1 (1961) Studies on dental plaque I. The effect of different concentrations of glucose on the pH of dental plaque in viva. / Dent Res 40: 1087-1111. Kleinberg I (1967) Effect of varying sediment and glucose concentrations on the pH and acid pro-
Yates JC, Dhalla NS (1975) Introduction of necrosis and failure in the isolated perfused rat hearts with oxidized isoprotorenal. j MO/ Cell Cardioi 7: 807-816. Yates JC, Kleinberg I, Khatter JC, Hoeschen RJ, Das PK, Dhalla NS (1979) Modifications of intramuscular pH oscillations in the isolated perfused rat heart by different interventions. Can J Physiol P~afmacoi 57: 900-907.
N. 5. DHALLA, J. C. YATES, I. KLEINBERG, J. C. KHATTER,
AND
R. J.
HOESCHEN
Antimony electrode provides a stable and reproducible means for the determination of intramuscular pH. large changes in bicarbonate or phosphate concentrations are required before a significant alteration in electrode response is observed and thus is quite suitable for physiological measurements. However, dramatic shift in mv-pH relationship for this electrode was seen upon changing pOz of the medium. When the electrode was employed to monitor intramuscular pi-l in isolated perfused rat heart, it was found that Na+ or K+ free medium produced a severe acidosis @H < 6.5) along with loss of contractility. Ca2+ free perfusion, on the other hand, resulted in a failure of contractility within 30 set without any change in interstitial pH. Upon stretching the heart when the perfusion rate was maintained at constant level, an increase in both contractile force andpH occurred. Norepinephrine along with increased perfusion flow produced an increase in both rate and force of contraction without any significant change in interstitial pH. However, at constant perfusion rate, the interstitial~H increased dramatically. Similarly, global ischemia resulted in a decline in contractile force and initial increase and subsequent decrease in interstitial pH. No decrease in pH was noted when coronary flow was reduced gradually. These results suggest that the antimony electrode is only suitable in situations which are not associated with larger changes in ~0,. Key Words:
Antimony electrode;
Intramuscular pH; Contractile force
INTRODUCTION Continuous and accurate measurement of pH changes in intact muscle and body fluids is of considerable value in physiological and pharmacological research. Antimony-antimony oxide-type electrode has considerable potential in such applications as this electrode is quite robust and can easily be made in a variety of shapes and sizes for specific applications (Kleinberg, 1958). Earlier, we reported that the antimony electrode is quite stable in the heart tissue and does not suffer from isolation problems resulting in “memory” (Dhalla et al., 1973; Yates et al., 1979). Furthermore, whereas the response time of glass pH electrodes can often be a From the Division of ExperimentalCardiology, Departments of Physiology, Medicine and Oral Biology, University of Manitoba, Winnipeg, Mant., Canada. Address reprint requests to: Dr. J. C. Khatter, Department of Physiology, University of Manitoba, Winnipeg, Mant. R3E OW3 Canada. Received May 29, 1979; revised July 76, 1979.
fournal of Pharmacological Methods 3, 221-234 (1930~ @19&l Elsevier North Holland, Inc., 52 Vanderbilt Avenue, New York, NY 10017
221
owmo2mom3o22~i4w2.25
222
N. S. Dhatfa et al.
limiting factor, the speed of response of the antimony-antimony oxide system is apparently limited by the time constants of the measuring instruments (Bishop and Short, 1964). The subject of construction of a suitable antimony electrode for pH measurements has been reviewed by Stock et al. (1958)‘ and the construction of useful electrodes of quite small dimensions has been reported subsequently (Vieira and Malnic, ‘1968; Bicher and Ohki, 1972). Applications to biological situations thus far have included pH measurement in blood (Buytendiyk, 1927; Brinkman and Buytendiyk, 1928), gastric contents (Erb and Senior, 1938; Haggard and Greenberg, 1941; Amann and Kronberger, 1958; ,Kobayashi, 1960), teeth and contents of the oraf cavity (Moore and Fosdick, 1952; Thompson and Brudevold, 1951,1954; Brudevold and Thompson, 1954; Kleinberg, 1961, 1967; Kleinberg and Jenkins, 1964), living cells (Kurella and Popov, 1960; Vorobev et al., 1961; Bicher and Ohki, 1972), renal cortical tubules (Vieira and Malnic, 1968) and heart muscle (Dhalla et al., 1973; Yates et al., 1979). In spite of a considerable amount of information gained by its use, there has been no attempt to systematically evaluate the suitability of the antimony electrode for such applications. The purpose of the present study was fourfold. First, to establish the stability of the electrode in an environment simulating mammalian tissue or body fluids; second, to confirm the lineari~ of the mv-pH relationship within a range of pH values likely to be encountered in biological systems; third, to determine whether any of the substances normally present in such an environment interfere, chemically or otherwise, with the function of the electrode; and last, to determine the changes in myocardial pH in isolated perfused rat heart when contractile force is modified through various inte~entions. METHODS The antimony-antimony oxide electrodes used in this study consisted of short antimony rods, approximately 0.5 mm in diameter, fused to the end of a silver wire and insulated, except at the tip, by a thin coating of epoxy resin. The reference electrode consisted of a saturated KCI bridge and a standard saturated calomel electrode. The antimony electrode and reference electrode were coupled to a highimpedance digital voltmeter (Data Technology DT-360-B) from which the potential difference was read. Two or three different antimony electrodes were used in each experiment and compared to the reading from a glass-calomel reference combination electrode coupled to a radiometer pH meter. The buffer solutions used as standards in this study were made by mixing various proportions of equimolar solutions of NaH2P04 and Na,HPO,. The perfusion solution used in certain experiments was a modified Krebs-Henseleit solution containing 120 mM NaCI; 25.4 mM NaHCO,; 8 mM glucose 4.8 mM KCI; 1.2 mM KH,PO,; 0.86 mM MgSO *; and 1.25 mM CaCI,. This solution was also employed to simulate
the
chemical
environment
of mammalian
tissues,
and the
stability,
consistency, and linearity of thepH response of the antimony electrode was studied in modifications of this solution in which each constituent was systematically varied
Suitability of the AntimonypH
Electrode
between pH 5.0 and 9.0 by the addition of HCI or KOH. The CaCI, was omitted from most solutions to avoid precipitation at the higher pH values. The influence of CaCI, on the pH response of the antimony electrode was studied in solutions from which NaHCO, and KH,PO, had been omitted to avoid precipitation at higher pH values. The effect of p0, on the mv-pH relationship of the antimony electrode was studied in 0.1 M phosphate buffer saturated with 95% Oz, air, or in solutions gassed with 100% nitrogen as they became equilibrated with air. The oxygen tension of the solution was monitored by a Clark electrode coupled to a Giison Oxygraph. Tissue pH measurements were made in the spontaneously beating, isolated, perfused rat hearts prepared according to the procedures described previously (Yates and Dhalla, 1975). In experiments in which the composition of the perfusion medium was altered, the osmolarity was maintained by the addition of equimolar amounts of sucrose. All the perfusion media were gassed with 95% O2 and 5% COz mixture and maintained at 37°C. The pH of these solutions, unless indicated otherwise in the text, was in the range of 7.3-7.4. The coronary flow was controlled at 8 ml/min by using a Harvard peristaltic pump and the perfusion pressure varied between 70-75 mm Hg. In some experiments, ventricles were electrically driven using a square-wave stimulator to apply pulses just above the threshold between two platinum electrodes located in the apex of the heart and the intraventricular septum at” the base of the heart. The results with paced hearts were found to be similar to those obtained with spontaneously beating hearts. All these hearts were punctured with the tip of fine scissors at the beginning of the perfusion to avoid fluid accumulation in the ventricles. The intramuscular pH of myocardium was measured by inserting the electrode in the ventricular wall. The continuity between the reference electrode and the tissue was achieved by means of a saturated KCI bridge. Diffusion of KCI through the plug in the measuring end of the salt bridge was very slight but was sufficient for electrical continuity (Kleinberg, 1967). Antimony and calomel electrodes were connected to a radiometer pH meter (model 26) and the signal recorded on the Crass polygraph. Simultaneous recording of contractile force and surface electrical activity were made using a 3-channel Grass polygraph. A resting tension of 5 g was applied to all the hearts used in this study on starting the perfusion and the contractile force was monitored via a force displacement transducer (FT 0.03). The surface electrical activity was recorded as a difference in potential between two platinum electrodes, one connected to the right atrium and the other to the base of the left ventricle. The hearts were perfused with normal medium for 15 min to allow equilibration before any experimental intervention was made. Global ischemia was simulated by clamping the tube supplying perfusion medium to the cannula. For the sodiumfree perfusion, NaCl and NaHCO, were replaced by an equivalent amount of sucrose and thepH was adjusted to 7.4 with KOH. In the K+ free medium KCI was replaced by an equivalent amount of sucrose and KH2P04 was replaced by an equivalent amount of NaH,PO,. No adjustment was made for the omission of CaCI, from the calcium-free medium. Where norepinephrine was used, 0.1 pg was injected into the flow of perfusion medium just before it entered the cannula. The
223
224
N. S. Dhalla et al.
=03--I
PH
FIGURE 1. Effectsof different phosphate buffer concentrationson mv-pH relationships of an~~ny electrodesystem.A- A 0.05 M phosphate, 8-m 0.10 M phosphate, a4 0.20 A4 phosphate, --best fit lines for pH < 7.0 and pti > 7.0 in 0.20 M phosphate.
effects
of stretching
the heart were tested
by increasing
the resting
tension
on the
myocardium. RESULTS Comparison phate
buffer
of the mv-pH revealed
relationship
that the calibration
with
different
curves
concentrations
obtained
in this
of phos-
manner
were
affected in two distinct ways by the buffer concentrations. As can be seen in Fig. 1, the mv-pH relationship is shifted upward as the buffer concentration is increased, and furthermore, that with higher buffer concentrations is no longer a straight line, but is better fit by two linear segments slopes
and intercepts.
regression
Table
1 gives the values
the relationship having different
for the slope and intercept
of the
lines obtained
using two different electrodes in 0.05 M, 0.1 M, and 0.2 coefficient) M phosphate buffers. As can be seen, the RZ value (R - correlation obtained for the sets of data points above and below pH 7.0 using a buffer concentration of 0.2 M indicate a better linearty than the R2 value for the set of data points obtained over the entire range of pH values. Table 2 provides the values for the slope and intercept of the mv-pH relationship obtained in Krebs-Henseleit solutions and in modified Krebs-Henseleit solution in which the concentration of individual components have systematically been varied above and below their normal values. As indicated, the slope and intercept of the
Suitabili~ of the Antimony~H Electrode TABLE1 Effectsof Different Concentrationsof Phosphate Buffer on Slope and Intercept of mv-pH relationship Obtained with Two SeparateAntimon~Antimony Oxide Electrodes
0.05 M Phosphate
SLOPE C S.E. INTERCEPT % S.E.
9 0.998
0.10 M Phosphate
10 0.997
0.20 M Phosphate
IO 0.985
0.20 M Subset 0.20 M Subset
4 1.000
RZ
N
24.302 “1.744 26.1% 22.338 89.615 25.228 17.866 t1.257 150.218 ko.872
49.628 -to.249 50.534 20.285 45.440 -to.709 56.477 to.607 37.824 20.398
6 0.997
Phosphate < pH 7.0 Phosphate 1 pH 7.0
ELECTRODE 2
ELECTRODE 1
__I-_ N R2
SLOPE k S.E. INTERCEPT i
9 0.998
29,367
48.991 10.258 45.401 ‘0.249 46.030 20.639 55.471 20.454 38.826 kO.010
10 0.998 IO 0.9a8 6 0.998 4 1.000
S.E.
-co.801
35.401 k2.047 83.974 c4.714 22.746 kO.941 141.377 20.022
N = is the number of experiments. R = is the correlation coefficient.
mv-pH
relationship
differs
significantly
(p < 0.05) from that obtained
Krebs-Henseleit solution in those solutions increased sodium bicarbonate or potassium none of, the effect. These similar
result
The
effect
electrodes
in the normai
having reduced sodium chloride, or phosphate concentrations, but that
other modifications of the test solutions produced any significant experiments were performed using two different electrodes, with a in each case. of oxygen
tension
in a solution
on the mv reading
at constant
pH is shown
from
two different
in Fig. 2. The influence
antimony of oxygen
tension was quite similar and very dramatic with both electrodes, and was most pronounced in the range of oxygen tension (IS-20%) most likely to be encountered in
mammalian
electrode
tissues.
reading
the antimony
In as much
is potentially
oxide-type
as the
influence
the most serious
electrode
for
biological
of oxygen
factor affecting pH
tension
on the
the suitability
measurements,
a series
of of
simple experiments were carried out to confirm the in vitro findings. When the flow of perfusion medium to isolated perfused rat hearts was abruptly discontinued (Fig. 3) an upward shift in the mv reading of an antimony electrode inserted into the ventricular wall was seen to coincide well with a 40-50% decline in the contractile
force developed
the decline
of oxygen
by the isolated tension
heart which
of the tissue.
may be considered
Subsequently
to reflect
the mv reading
of the
electrode feil steadily, as would be expected to occur with the accumulation of lactic acid in the anaerobic muscle. When the flow of perfusion medium to the heart was resumed,
the mv reading of the electrode
equal
rise,
to its initial
presumably
in response
dropped to the
abruptly
return
by an amount
of tissue
oxygen
tension to its original value. Following a short period of time necessary to wash out the accumulated lactic acid from the tissue, the electrode reading stabilized near its initial value. In a similar
experiment,
in which the flow of perfusion
medium
was significantly
225
226
N. S. Dhalla et al. TABLE 2 Effects of Changes in Different Components in Krebs-Henseieit Solutions on the Slope and Intercept of the mv-pH Relationship Obtained Two Separate An~mon~Antimony Oxide Electrodes
with
ELECTRODE 2
ELECTRODE 1 N
R2
SLOPE 4 S.E.
INTERCEPT
R2
Sk.op~ C S.E.
INTERCEPT
Control
20
0.998
15
0.997
30.123 k2.938 23.881 23.313
0.997
735 mM NaCl 5mM NaCI 45 mM NaHCO, SmM NaHCO, 2.4 mM KH,PO, 0.6mM KH,PO, 100 mM KCI 2.4 mM KCI 2.4 mM
13
0.998
12
1.000
11
0.999
11
1.ooo
8
0.999
47.353 rto.121 47.304 20.187 S4.375* 20.204 50.4OP lto.097 47.370 ~0.150 49.656* 20.352 47.681 rtO.207 47.437 +0.171 47.710 to.294 47.333 20.263 47.775 kO.197 47.395 20.426 47.483 co.534
49.375 20.137 49,090 kO.190 56.410* kO.220 51.292* I?rO.lll 49.427 20.152 49.933’ zkO.098 49.209 20.174 49.691 20.118 49.179 rtO.189 49.546 20.176 49.746 1-0.779 49.640 20.265 49.483 20.218
21.372 k3.326 18.631 23.361 -46.4421 23.028 4.269* +I .406 22.859 21.834 78.036 k1.222 18.512 ?‘I .516 24.884 k1.032 20.149 21.156 24.388 50.985 24.007 ‘1.08-l 22.450 21.842 21 .I56 ?I .421
MgSO, 0.6 mM MgSO, 2.5 mM CaCI, 0.6 mM CaCl 2
9
0.999
6
0.999
6
0.999
6
1 .ooo
6
0.998
6
1.000
-32.607* 22.809 9.529’ 21.230 28.544 +f .815 18.047* 21.318 25.868
0.997 0.938 0.999 0.999 I.000 0.999
21.806 28.331 -tl.501 26.263 21.797 29.852 21.473 26.972 +I.188 29.840 22.773 27.730 5-l .423
1.000 1.000 1 .ooo 1.000 0.999 1.000
* Indicates that value differs significantly @ < 0.05) from control. N = number of experiments in each case. R = is correlation coefficient.
reduced but not stopped entirely (Fig. 4), an increase in the mv reading of the electrode proportional to the decrease in coronary perfusion rate was seen to occur. The upper panel (Fig. 4A) shows the decrease in contractile force and increase in mv reading which follows upon the reduction of rate of perfusion from 5.5 ml/min to 1.7 mllmin. A greater reduction of the rate of perfusion from 5.5 ml/ min to 0.7 mllmin (Fig. 4B) results in a significant increase in mv reading. Furthermore, the gradual decline in the electrode reading observed in the experiment shown in (Fig. 3) and attributed to lactic acid accumulation was not observed in this situation where some degree of coronary flow persists. In another experiment, it was observed that the increased oxygen demand due to increase in rate and force of contraction (Fig. SA) resulting from an infusion 0.1 ,ug of norepinephrine into the isolated rat heart preparation was met by a 30 to
Suitabilityof the Antimony pH Electrode
300
0
20
*~"Satur~~ion
SO
100
%
FIGURE 2, Effectof oxygen saturationon mv reading of antimony electrode in sob tion of constant pH e-0 electrode #l, m-8 electrude #2.
50% increase in coronary perfusion
rate, and that the interstial pH was not noticeably altered. When the experiment was repeated under circumstances where the coronary perfusion rate of the isolated heart was prevented from increasing, an increase in the pH of 0.3-0.6 units was observed (Fig. 53) and the return of the interstitial pH to the original level coincided with the disappearance of inotropic and chronotropic effects of the drug. In a different set of experiments isolated hearts were subjected to stretch by large increases, in the order of 5 to 8 g, in resting tension applied at the apex of the heart (Fig. 6). When the perfusion rate was maintained constant, stepwise
Ebttrlca?
Activity
--__
FIGURE 3. Recording of pfi, eontractifeforce, and surface electrical activity from an experiment in which the perfusion was stopped completely ( 4 ) and restarted after 5 min ( t 1. These results are typical of 6 experiments.
227
228
N. S. Dhalla et al.
EIactticnI
Ccntractite
Activity
Farce
B
FIGURE 4. (A) Recording of pH, contractile force and surface electrical activity from a typical experiment in which the perfusion rate was abruptly decreased from 5.5 ml/ min to 1.7 milmin ( 4 ) by partially clamping the tube carrying the perfusion medium to the heart. After 5 min the clamp was released f t 1. (B) Recording from another experiment in which the perfusion rate was reduced by clamps from 5.5 mlfmin to 0.7 mllmin ( J ) for a period of 5 min, after which the clamp was released ( t 1.
increase in resting tension resulted in a parallel stepwise increase in both contractile force and interstitial pti. Figure 6 is a recording of such an experiment which was begun with a resting tension of about 5 g and a perfusion rate of 5 ml/mm while the resting tension was increased from 5 to 13, 20, 28, and finally to 36 g. The contractile force developed from 5.5 to 9.4, 11, 11.4, and finally to 12 g while the interstitial pH increased from 7.1 to 7.25, 7.5, 7.55, and finally to 7.9 pH units. At the time indicated by the arrow, the clamp limiting the rate of flow was released. As the perfusion rate rapidly increased to over 7 mllmin the pti began to gradually decline, Perfusion with a sodium-free medium resulted in a rapid decline and disappear-
Suitability of the Antimony pH Electrode
FIGURE 5. (A) Recording of pH, contractile force and electrical activity from a typical experiment in which 0.1 mg of norepinephrine was infused into the heart via the perfusion cannula ( J ) while the rate of perfusion was not restricted. The rate of perfusion prior to the ~repinephdne infusion was 4.5 mllmin, increasing to 7 ml/min after the drug was administered. The perfusion rate returned to the previous level as the chronotropic and inotropic effects of the drug disappeared. (6) Recording from an experiment similar to that shown in (A) with the difference that the perfusion rate, which was 5 ml/min before infusion of 0.1 mg of norepinephrine was maintained at this same level following administration of the drug.
ante of contractile force which was associated with the development of a severe acidotic state. By the time contractility fails, the interstitial pH is 6.5 and falls still lower over the next few minutes (Fig. 7). The state of arrest produced by sodiumfree perfusion is completely reversible if reperfusion with sodium is begun within 3 to 5 min. As can be seen in the lower panel (J3) of Fig. 7, recovery of contractile force after 3 min of sodium-free perfusion is associated with an almost parallel increase in interstitial pH, temporarily rising 0.2-0.3 pH units higher than the initial
22!3
236
N. S. Dhalla et al.
Contractile
Force
Electrical
Activity
FIGURE 6. Recording of pH, contractile force, and electrical activity from a typical experiment in which the heart is subjected to stretching by large increments in tension upon the hook located in the apex of the heart. The perfusion rate was maintained at 5 mllmin until the time indicated by the arrow (t ) when restrictive clamps were removed. At this time the perfusion rate increased to more than 7 mllmin. As the resting tension is increased from 5 to X%,29,26, and finally 36 g, the contractile force developed increases from 5.5 to 9.4,11,11.4, and finally to 12 g while the intramuscular pH increases from 7.1 to 7.25, 7.55, and finally to 7.9 pH units. Note that when the perfusion rate is allowed to increase ( t ) the intramuscular pH begins to fali again.
value and stabilizing thereafter to sodium-free perfusion. During
the course
of potassium-free
to vary in a consistent rises above nating pattern
control
strong
at a level of pH comparable
pattern.
level at about
2 min,
and weak
contractions,
changes
corresponds
of pH
perfusion
The contractile
(Fig. 8) interstitial force
becomes
and then rather
to that recorded
usually
irregular
fails abruptly
loosely
to the
prior
pH was found
declines
somewhat,
thereafter at about
with
alter-
IO min. The
contractility
changes
decreasing by 0.1 to 0.2 pH units during the first minute, rising above the control level for the next 2 to 3 min (Fig. 8A) then dropping to about pH 7.0 during the period of irregular contractions (Figs. 8B). Upon failure of contractility, stitial pH decreases steadily to a pl-l somewhat below 6.5. Upon
perfusion
of the
isolated
heart
with
calcium-free
medium
the inter(not
shown)
complete uncoupling of excitation and contraction was apparent within 30 sec. All contractile activity had ceased by this time, whereas, the surface electrical activity was not altered. occur.
In this set of experiments
no change
in interstitial
pH was seen to
The results presented here indicate that careful consideration must be given to the possibility of erroneous measurements due to ionic shifts and changes in tissue p0, when employing the antimony electrode forpH measurements in physiological research. interference due to changes in bicarbonate or phosphate are not likely to be critical factors in most experimental situations
concentrations since the influ-
Suitability of the Antimony pH Electrode *+
pH
l--l--x 7-5
7.0
I 1~ I 04
A
Tirne.3Pl't.
Cohactila
Force
----
RGURE 7. (A) Recording of pH, contractileforce, and surfaceelectricalactivity from a typical experiment in which the isolated heart was abruptly switchedfrom perfusion with normal Krebs-Henseleit medium to perfusion with a medium from which all sodium was omitted ( 4 ). (8) After a period of about 3 min, perfusion with sodiumcontaining medium was resumed ( f 1. Note the dramaticdecreasein intramuscularpH which occurs during the period of sodium-free perfusion and increase in pH to above baseline levels which occurs following return to perfusion with sodium containing medium.
ence of the electrode reading is quite small when compared to the changes in concentrations of these substances that are likely to be encountered in most circumstances. With regard to the influence of Na+ on the electrode, the location of the electrode in a typical high Na+ extracellular plasma environment will of course be an important factor and must be taken into account when calibrating the electrode. Much more serious is the influence of tissue O2 tension on the electrodes accuracy since tissue ~0, values fall in a range where very small changes in OS content produce large variations in the mv-pH relationship characteristics of the electrode. The sensitivity of the electrode to changes in p0, was recognized by other investigators in their experiments on the squid giant axon (Bicher and Ohki, 19721, and great care was taken to avoid this influence by performing all experiments and calibration in solutions saturated with air, Vieira and Malnic (1968), on the other hand, have stated that the influence of oxygen on the pH measurements in the range of p0, 34-128 was not important and could be safely disregarded. Although our experiments indicate otherwise, it is possible that some details of construction of their electrode renders it less sensitive to oxygen than ours. In any case, careful consideration must be given in any experimental model employing the antimony electrode for pH measurement to the possibility that the tissue pOZ
231
232
N. S. Dhalla et al.
Contractile
force
FIGURE 8. (A) Recording of RH and contractile force from a typical experiment in which the isolated heart is abruptly switched from perfusion with normal Krebs-Henseleit medium to perfusion with a medium from which all potassium has been omitted ( & ). (B) Continuation of the recording in (A) showing the pH changes that occur after about IO-min perfusion with potassium-free medium at a time when contractile activity ceases and the heart goes into contracture.
may differ from that of the solution used in calibration of the electrode, and further, that tissue p0, may not remain constant during the course of the experiment. It is of interest to examine the cyclic variations in pH of ventricular myocardium during the contraction-relaxation cycle reported earlier (Rhalla et al., 1973, Yates et al., 1979), in light of the present findings. In these studies it was reported that a decline in pH occurred just prior to the onset of contraction and that the pH rose toward the baseline value during and following the relaxation phase. That these oscillations are an artefact due to changes in Na+ concentration appears unlikely in view of the magnitude of change in Na+ concentration required to produce such an effect and the fact that large shifts in sodium are not known to occur in the ourse of the cardiac cycle. Similarly, the possibility that these changes are an artifact of decreased bicarbonate or phosphate concentrations does not appear reasonable in view of the magnitude of change in concentrations required to produce such an artifact as well as the fact that splitting of high-energy phosphate compounds and increased metabolism during contraction would tend to increase rather than decrease the concentration of these substances. Reasoning along the same lines, increased metabolism during contraction would tend to decrease tissue pop, and if it were a predominant factor it would serve to raise rather than lower the mv reading of the electrode. Thus, it would appear that the cyclic fluctuations reported
previously
are not due to the influence
of substances
interfering
with the
Suitabilityof the AntfmonypH Electrode 233
pH sensitivity of the electrode, but rather that these influences, if effective at all, would tend to attenuate the decline in pH observed using an antimony electrode. The experiments described in this study reveal a definite pattern to pH changes elicited with regard to oxygen supply and demand. Interstitial pH increase dramattally when oxygen supply is limited by reducing the perfusion rate or when the contractile work is increased by stretching the heart as well as upon norepinephrine administration while the oxygen supply is kept constant by preventing an increase in perfusion rate. On the other hand, when oxygen supply is increased to the heart by increasing perfusion rate in response to the stretching or norepinephrine administration, there is no increase in pH. However, changes in pH do not seem to bear any direct relationship with changes in contractile force as seen upon reducing the flow rate, stretching of the heart, as well as upon norepinephrine administration. This is further supported by the fact that an initial increase in contractile force upon perfusion with K+-free medium was associated with an increase in intramuscularpH whereas perfusion with Na+-free medium produced decrease in contractile force as well as intramuscular pH. Complete contractile failure of heart upon perfusion with Ca 2+-free medium was seen in the absence of any changes in pH. The initial increase in pH seen upon complete interruption of the perfusion flow seems to be an artifact of a decrease in tissue pOzt since similar changes in mv readings of the antimony electrode were seen when the oxygen tension was decreased in the perfusion medium while standardizing the electrode. Although other investigators (Gebert et al., 1971) using glass electrode have also reported an initial increase in intramuscular pH upon occluding the coronary arteries in dog hearts, the sensitivity of their electrode to changes in pOZ has not been tested. Alternatively, there may be a slight initial increase in intramuscular pH due to hypoxia or ischemia with the formation of creatine and other basic substances in the myocardium (Dhalla et al., 19721, but this would be of transient nature due to the production of lactic acid. At any rate, this study has demonstrated that changes in tissue pH as measured with antimony electrode should take into account the interference due to changes in tissue ~0%. Thus, in order to take advantage of the rapid response and robust characteristics of the antimony electrode for pH measurements in physiological research, one must (a) calibrate the electrode in an environment similar to that in which experiments are performed, (6) take into consideration changes in Na+, bicarbonate, and phosphate concentrations that may influence the electrode, and (c) ensure that tissue pOz is not changing during the course of an experiment and thus producing spurious “pH changes.” This investigation was supported by a grant from the Manitoba Heart Foundation.
REFERENCES Amann W, Kronberger L (19SB) Die Bedentung der intragastraien pH-Messung in der mangenchirurgie. Medizinische 40: 1576-1581. Bicher iii, Ohki S (1972) Intracellu~arpH
electrode
experiments on the squid giant axon. Bioc~i~ Ejophys Acta 255: 900-904. Bishop E, Short CD (1964) Differential electrolytic potentiometry. XIV. An examination of elec-
234
N. S. Dhalla et al. trode parameters and electrode systems in acidbase titrimetry. Analyst 89: 415-420. Brinkmann R, Bu~endiyk FjJ (1928) Die Klinische Mikrobestimmung des ,oH des Blutes mit einer Antimonelecktrode. Biochem 2 199: 387-391. Brudeyold F, Thompson FC (1954) Evaluation of the microantimony electrode. 1 Dent Res 33: 854-858. Bu~endiyk FJJ(1927) The use of the antimony electrode in determination of pH in viva. Arch Neerland Physiol 12: 319-321. Dhalla NS, Yates JC, Waltz DA, McDonald VA, Olson RE (1972) Correlation between changes in the endogenous energy stores and myocardial function due to hypoxia in the isolated perfused rat heart. Canad J Physiof fbarmacol 50: 333345. Dhalla NS, Yates JC, Kleinberg I (1973) Oscillations of intramuscularpH in perfused rat heart. Canad J Physiol Pharmacol51: 234-238. Erb RC, Senior KL (1938) Preliminary report on a new method for gastric research. ] Am Osteopath Assoc 38: 95-96.
duction in human salivary sediment Arch Oral Bioi 72: 1457-1473.
mixtures.
Kleinberg I, Jenkins CN (1964) pH of dental plaques in the different areas of the mouth before and after meals and their relationship to the pH and rate of flow of resting saliva. Arch Oral Biol 9: 493-516. Kobayashi H (1960) Continuous potentiometric determination of pH of gastric contents. Osaka Diagaku Igaku Zasshi 12: 1399-1415. Kurella GA, Popov BA (1960) Determination of pH by means of the antimony micro-electrode. Biofizika 5: 373-375. Moore BN, Fosdick LS (1952) The micro-antimony electrode. 1 Dent Res 31: 512-513. Stock IT, Purdy WC, Garcia LM (1958) The antimony-antimony oxide electrode. Cbem Rev 58: 611-626. Thompson FC, Brudevold F (1951) A micro-antimony electrode suitable for intraoral pH measurements. 1 Dent Res 30: 523. Thompson FC, Brudevold F (1954) A micro-antimony electrode designed for intraoral pH measurements in man and small experimental animals. j Dent Res 33: 849-853.
Gebert G, Benzing H, Strohm M (1971) Changes in the interstitial pH of dog myocardium in response to local ischemia, hypoxia, hyper-, and hypocapnia, measured continuously by means of glass micro-electrodes. Pffiizers Arch 329: 7281.
Vieira FL, Malnic G (1968) Hydrogen ion secretion by rat renal cortical tubules as studied by an ntimony microelectrode. Am J P~ys~o~214: 710718.
Haggard HW, Greenberg LA (1941) Antimony electrode for continuous recording of acidity of human gastric contents. Science 93: 479-480.
Vorobev LN, Kurella GA, Popov GA (1961) Intracellular pH of Nitella flexillis at rest and after excitation. Biofizika 6: 582-589.
Kleinberg I (1958) The construction and evaluation of modified type of antimony micro-electrodes for intraoral use. Br Dent J 104: 197-204. Kleinberg 1 (1961) Studies on dental plaque I. The effect of different concentrations of glucose on the pH of dental plaque in viva. / Dent Res 40: 1087-1111. Kleinberg I (1967) Effect of varying sediment and glucose concentrations on the pH and acid pro-
Yates JC, Dhalla NS (1975) Introduction of necrosis and failure in the isolated perfused rat hearts with oxidized isoprotorenal. j MO/ Cell Cardioi 7: 807-816. Yates JC, Kleinberg I, Khatter JC, Hoeschen RJ, Das PK, Dhalla NS (1979) Modifications of intramuscular pH oscillations in the isolated perfused rat heart by different interventions. Can J Physiol P~afmacoi 57: 900-907.