Reversal of hyperventilation induced hyperlactatemia by acetazolamide

Respiration Physiology (1970171) 11,127-134;forth-~oZ/a~

REVERSAL INDUCED

D.

Pub~jshing Company, Amsterdam

OF HYPERVENTILATION

HYPERLACTATEMIA

BY ACETAZOLAMIDE

T. ZBOROWSKA-SLUIS, R. I. OGILWE AND G. A. KLASSEN

The department of Cardiology, ~cGi1~ University C&tic, Royal Victoria ~ospitai, Montreal 112, Quebec, Canada

Abstract. Acetazolamide administration to hyperventilating dogs was found to both reverse and to prevent the associated hyperlactatemia. This inhibition of glycolysis was associated with a reversal of the hyperventilation induced alkalosis, an increase in Pco2, and an increase in blood CO2 content. A unique effect of these three variables on the control of the glycolytic rate as measured by a rise in arterial lactate concentration could not be identified. Acetazolamide Carbonic anhydrase

Glycolysis Lactate

Previous work from this laboratory (ZBOROWSKA-SILJIS and DOSSETOR,1967) has shown that hyperlactatemia induced by hyperventilation with room air could be reversed by hyperventilation with air containing 5% carbon dioxide. It was also established that a signi~cant source of the increased lactate was from erythrocytes. The present study was undertaken to explore the possible mechanisms by which changes in carbon dioxide influence the concentration of lactic acid. Carbonic anhydrase inhibition by acetazolamide was selected as an intervention because it blocked the catalyzed dehydration reaction (ROUGHTON, 1964). This results in a temporary unsteady state with whole body carbon dioxide storage (MITHOEFER, 1959). The pool size (CHINARD, ENN~ and NOLAN, 1962), form of carbon dioxide (CAIN and OTIS, 1961), and rate at which each form of carbon dioxide contributes to expired COZ (CHINARD,ENNS and NOLAN, 1960; CAIN and OTIS, 1961) are also altered by acetazolamide. ENNS (1967) has shown that the diffusion characteristics for gaseous COZ across the red cell membrane are slowed by inhibition of carbonic anhydrase. It seemed reasonable that the use of this agent might alter the cellular pH, Accepted for publication 4 July 1970. 127

128

D. T. ZBOROWSKA-SLUIS,R. I. OGILVIEAND G. A. KLASSEN

P COZY and carbon dioxide content in a unique fashion with a resultant alteration in the glycolytic rate as measured by an increase in lactate concentration. The relationship between stimulated glycolysis and carbon dioxide, pH, or Pco2 might then be identified. Our results indicate vented or reversed

that the hyperlactatemia

by acetazolamide

of hyperventilation

could be pre-

administration.

Methods Twelve mongrel dogs (average weight 12.5 kg) were anesthetized with intravenous sodium pentobarbital (30 mg/kg). Blood sampling catheters were placed in the femoral artery and pulmonary artery. Muscle paralysis was obtained by the use of succinylcholine, 20 mg as a priming dose, and repeated 10 mg doses to maintain respiratory cooperation. Ventilation was through a cuffed endotrachial tube by a Harvard piston-pump positive pressure respirator. Following a control period, with minute ventilation set to maintain normal arterial was carried out at five times control ventilation. This PO, and Pco,, hyperventilation was achieved by doubling tidal volume and increasing frequency. Arterial Pco2 rapidly fell to IO-15 mm of mercury. Two experimental protocols were followed: 1. Reversal of hyperventilation induced hyperlactatemia: (n= 9). Following a control period, animals were hyperventilated for 3+ hr on room air. At the end of the second hour a priming dose of acetazolamide 10 mg/kg body weight was given, followed by a constant infusion of 20 mg/kg/hour. This dose of acetazolamide results in 99% inhibition of carbonic anhydrase activity in the canine erythrocyte within 30 min (BRODIE, 1965). 2. Prevention of hyperventilation induced hyperlactatemia : (n = 3). The acetazolamide was given 30 min prior to and during 3 hr of hyperventilation. Arterial samples were taken anaerobically during the control period and at 30 minute intervals during hyperventilation for pH, Pco,, Po2, lactate and pyruvate. In four studies mixed venous and arterial samples were taken for 0, and CO, content. Mixed expired air was collected at the exit of a mixing box. Minute ventilation was measured by a Wright respirometer. Body temperature was monitored and remained at 37 ‘. Blood Pco2, PO,, and pH, and expired Pcol and PO, were measured using an IL electrode system (Model 113-S2 Instrument Laboratories, Lexington, Mass.) at 37 ‘. All blood gas measurements were completed within two minutes of sampling. Plasma bicarbonate was derived from pH and Pco2 using the Siggaard-Anderson nomogram, Blood gas content was measured by the method of VAN SLYKE and NEILL (1924). Lactic and pyruvic acid were measured in protein free filtrates of whole blood obtained by directly pipetting blood into cold trichloracetic acid. Lactate was measured enzymatically (H~~RN and BRUNS, 1956), and pyruvate by the method of FRIEDEMAN and HAUGEN (1943). In four studies cardiac output was measured by dye dilution during the procedure. All results are expressed as means and standard error of means (SEM). Significance of differences was assigned using the Students’ t test.

ACETAZOLAMIDE

REVERSAL

129

OF HYPERLACTATEMIA

Results 1.

REVERSAL

OF HYPERVENTILATION

INDUCED

HYPERLACTATEMIA

(FIGS.

1,

2)

Figure 1 indicates the effect of hyperventilation on arterial pH, Pco,, CO, content, HCO;, mixed venous Po2, mixed expired PO,, and change in arterial lactate and pyruvate during hyperventilation and following acetazolamide. A significant increase in pH of 0.32 f 0.2 occurred after 15 min of hyperventilation.

I” E 160

r

<

E

25

w” 20 i zl Fig.

:; 5

60

0 0

60

120

1. Hyperventilation

vertical bars represent *

120

160210

180 210 for 34 hr, with acetazolamide infusion from SEM. Legend indicates time of hyperventilation

8

A-V

ml 02/

60

120

100 ml

CARDIAC

OUTPUT

‘. I

0

60

120

180210

the end of the 2nd hour. The

and acetazolamide

infusion.

L/min

6 5 4 3 2 I 0

Fig. 2. Arteriovenous

0

180 210

0

60

120

180 210

difference for oxygen and cardiac output during study. All notations as in fig. 1.

130

D. T. ZBOROWSKA-SLUIS,

R. I. OGILVIE AND G. A. KLASSEN

This decreased to control values or below following acetazolamide. Arterial PcoZ was steady at 12 mm Hg during hyperventilation and increased to 30 f 2 mm Hg following acetazolamide, in spite of continuing hyperventilation. Bicarbonate and CO, content both decreased during hyperventilation. The increase in bicarbonate following acetazolamide was not significant but a significant increase in CO2 content was observed (P < 0.01). Arterial Po, increased during hyperventilation and increased even further following the intervention. Mixed venous P,, fell associated with a fall in cardiac output, (fig. 2) and then increased to control following acetazolamide, even though cardiac output remained depressed. The mean resting lactate was 1.085 +O. 148 ,uM/ml and pyruvate 0.130~0.14 FM/ml. An increase in lactate and pyruvate concentration occurred during hy~rventilation. At the end of the acetazo~amide infusion period arterial con~ntration of both acids were not significantly different from control concentrations.

*40

...... ....... ~ZZZ~ACETA~~L~~I~E m HYPERVENTILATION

@

.30F

I40 I3ol

@

120 p 110 !i100 g 90 t;;: 60 a 70 60

0

60

120

0

60

120

180210

0

60

120

I80210

180 210

z ;i a t.10 v ii 3

0

h:

a -10 0 Fig. 3. Ace&z&amide

infusion 30 min prior to and throughout in fig. 1,

hyperventilation.

All notations

as

ACETAZOLAMIDEREVERSALOF HYPERLACTATEMIA

131

2. PREVENTIONOF HYPERVENTILATIONINDUCED HYPERLACTATEMIA(FIG. 3) During the period of 30 min, while normal ventilation was maintained and acetazolamide administered, pH fell, Pcoz increased, CO, content did not change (one animal), and bicarbonate decreased. The arterial PO, increased, and there was an insignificant decrease in pyruvate and lactate (control arterial lactate = 1.429 + 0.330 PM/ml, pyruvate=0.161 k.037 PM/ml). Following the onset of hyperventilation pH increased, then remained constant in two studies and returned to control in one. Arterial Pcol decreased and then remained constant, CO, content gradually decreased to a level comparable to that observed at the end of the reversal study. Bicarbonate was also decreased. Arterial PO, increased in a comparable manner to the previous study. Lactate and pyruvate concentrations did not change in one animal and fell slightly in two. Discussion The administration of acetazolamide was found to both reverse and to prevent the hyperlactatemia of hyperventilation. In this respect the response of the hyperventilated dog was similar to that previously reported for 5 % CO, administration during hyperventilation (ZBOROWSKA-SLUIS and DOSSETOR, 1967). The mechanism by which acetazolamide achieved this is less clear. As many of the enzymes of the glycolytic cycle have optimal activity at an alkaline pH it has been proposed that the alkaline pH per se is the stimulant for the increased glycolysis observed during hyperventilation (HUCKABEE, 1958). Because Pco, and pH are directly related in uiuo as indicated by the whole body titration line (COHEN, BRACHETT and SCHWARTZ, 1964), less attention has been paid to CO* and its possible direct link to the glycolytic cycle. Evidence in vivo (BICZ, 1960; KIERLER, 1960; CRAW et al., 1963) and in vitro (EICHENHOLZ et al., 1962) has suggested that a low CO, concentration may stimulate glycolysis independent of pH. Studies which have proposed that alkalosis was the sole stimulant for glycolysis have achieved this by the removal of C02, so that either or both mechanisms could be involved (UI, 1966). As acetazolamide retarded the stimulation of glycolysis induced by hyperventilation it was necessary to examine the effect of this agent on both CO, and pH. P co2 Both MITHOEFER (1959) and MAREN (1967) have investigated

the difficulties

associated

with measurement of Pcol and pH in arterial blood following acetazolamide, as the uncatalyzed dehydration reaction continues in the syringe. ROUGHTON (1964) has estimated that time for 90% completion of the dehydration reaction, as occurs in the lung, as 254 sec. As MAREN (1967) has indicated, inhibition of carbonic anhydrase will result at equilibrium, in arterial blood, having a higher Pco2 and a lower pH than occurred in vivo, and venous blood a lower Pco2 and a higher pH. As our samples of arterial blood were measured before equilibrium could occur for Pcol and pH, they represent values which are closer to the in vivo situation.

D. T. ZBOROWSKA-SLUIS,

132 Co,

R. I. OGILVIE AND G. A. KLASSEN

CONTENT

MITHOEFER (1959) has measured the changes in CO, output following acetazolamide administration to hyperventilated dog. During the initial unsteady state he observed less CO, output so that when total expired CO, output equalled metabolically

produced COZ, in an animal which had received acetazolamide and hyperventilated, there was an increase in CO, stores compared to control. This increase was reflected in our measurement of whole blood CO2 content, following acetazolamide during constant hyperventilation; or, the prevention of a fall in CO2 content when the acetazolamide was given before a comparable period of hyperventilation. Bicarbonate did not increase significantly. This probably reflects both an error in the measurement of bicarbonate from pH and PcoZ as previously discussed, and also a shift in the form of CO, present. CAIN and OTIS (1961) suggested that there would be an increase in carbamino CO2 following acetazolamide. As this form of CO, is entirely within the erythrocyte this suggests that following acetazolamide there was a greater portion of CO, being transported by erythrocytes than in the presence of carbonic anhydrase. Thus the normal ratio for CO, stores, plasma to erythrocyte was shifted. PH

Acetazolamide has been recognised to produce both an intercellular and extracellular acidosis (MAREN et al., 1959). The effect of acetazolamide on the pH gradient of the red cell has not been established. Using the method of BATTAGLIA et al. (1965) over the pH range studied, an average pH gradient of -0.26 was measured for both control blood and blood following acetazolamide. It is apparent from our data that following acetazolamide the pH returned to control values thus reversing the hyperventilation induced alkalosis. As acetazolamide administration to the hyperventilating animal reversed the alkalosis and increased both the Pcol and CO2 content, this study has not allowed a separation of how these three may alter the glycolytic rate. Further studies on isolated erythrocyte systems are being undertaken in an attempt to determine if a unique CO, link to glycolysis exists. An increase in arterial PoZ following acetazolamide was observed by CARTER and CLARK

(1958) and TOMASHEFSKI, CHINN and CLARK

in ventilation.

As ventilation

was kept constant

(1954)

associated

with an increase

in our studies this mechanism

is not

applicable. MITHOEFER (1959) suggested that the rise in Po, resulted from a fall in alveolar Pco2 with a resultant increase in Po,. A second contributing factor would be a shift in the oxygen dissociation curve, (Bohr shift) as reported by CRAW et al. (1963). We have previously shown that cardiac output increased when 5 % CO, was used to reverse the hyperlactatemia of hyperventilation (ZBOROWSKA-SLUIS and DOSSETOR, 1967). In this study as shown in fig. 2 cardiac output remained depressed throughout. It is apparent that the reversal of the hyperlactatemia was not related to an improved cardiac output.

ACETAZOLAMIDEREVERSALOF HYPERLACTATEMIA

133

Acknowledgements The authors are indebted to Mrs. UMILLA STANFORD and Mrs. EDITH SUMMERSfor their expert technical assistance. Dr. D. T. Zborowska-Sluis was supported by a fellowship from the Muscular Dystrophy Association of Canada, Dr. R. 1. Ogilvie is a fellow of the Canadian Foundation for the Advancement of Therapeutics, and Dr. G. A. Klassen is a John and Mary R. Markle Scholar. This work was supported by grants from the Muscular Dystrophy Association of Canada and the Medical Research Council of Canada, Grant M.T. 3238. References BATTAGLIA,F. C., R. E. BEHRMAN,A. E. HELLEGERSand J. A. BATTAGLIA(1965). Intracellular hydrogen ion concentration changes during acute respiratory acidosis and alkalosis. J. Pediut.

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anhydrase:

chemistry,

physiology,

and inhibition.

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47: 595-781.

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its effect on carbon dioxide eliminated

134

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ROUGHTON,F. J. W. (1964). Transport

R. I. OGILVIE AND G. A. KLASSEN

of oxygen and carbon dioxide. In: Handbook of Physiology. Section 3. Respiration. Vol. I. Washington, DC., Am. Physiol. Sot., pp. 767-825. TOMASHEFSK~. J. F., H. I. CHINN and R. T. CLARK (1954). Effect of carbonic anhydrase inhibition on respiration. Am. J. P~~~j~~.177: 451-454. UI, MICHI~ (1966). The role of phosphofructokinase in pH-dependent regulation of glycolysis. Biorhim. Biophys. Acta 124: 310-322. VAN SLYKE, D. and J. M. NEILL (1924). Determination of gases in blood and other solutions by vacuum extraction and manometric measurement. J. Bid. Chem. 61: 523-573. ZBOROWSKA-SLUW,D. T. and J. B. DOSSETOR(1967). Hyperlactatemia of hyperventilation. .K Appl. Physioi. 22: 746-755.