THE INFLUENCE OF CARBON DIOXIDE ON THE NEUROMUSCULAR BLOCK CAUSED BY TUBOCURARINE CHLORIDE EN THE HUMAN SUBJECT

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1 Brit. J. Anaesth. (1964), 36, 272 THE INFLUENCE OF CARBON DIOXIDE ON THE NEUROMUSCULAR BLOCK CAUSED BY TUBOCURARINE CHLORIDE EN THE HUMAN SUBJECT BY ANIS BARAKA Department of Anaesthesia, University of Liverpool, England SUMMARY The influence of carbon dioxide changes on the neuromuscular blocking effect of tubocurarine in the anaesthetized human subject has been investigated. To assess neuromuscular transmission a simple spring myograph attached to a finger was used and the motor nerve stimulated with supramaximal tetanic stimuli. The plasma levels of tubocurarine were estimated using extraction into ethyl dichloride and a spectrophotometric method of assay. Respiratory acidaemia in one group of patients was associated with high plasma levels of the drug and prolonged neuromuscular block; respiratory alkalaemia in a further group resulted in low plasma levels of the drug after the same initial dose and rapid recovery from the block. It is suggested that a likely explanation of these findings is related to the fact that the tubocurarine molecule contains, besides its quaternary ammonium groups, two phenolic hydroxyl groups which have pk m values of 8.1 and 9.1 and so vary their degree of ionization within the range of ph changes associated with variations in pulmonary ventilation. The influence of carbon dioxide on the neuromuscular blocking effect of tubocurarine chloride has been studied in the cat by Payne (1958). He showed that carbon dioxide inhalation potentiates tubocurarine block. This has also been shown in the albino rabbit by Gamstorp and Vinnars (1961a, b). It was still necessary to investigate this effect in the human subject, because of the great species variation characteristic of the action of relaxant agents. If, in the human, the activity of the drug varied with the carbon dioxide tension of the blood, it would also be of interest to investigate whether this variation could be correlated with blood levels of the drug. It was known that in the dog the blood levels of tubocurarine as estimated spectrophotometrically at a given time after injection are higher in conditions of respiratory acidaemia than in alkalaemia (Utting, 1963). METHOD The observations were carried out in twenty healthy adult patients undergoing herniorrhaphy or stripping of varicose veins. Present address: University. Department of Anaesthesia, Cairo 272 Anaesthesia. The patients were all premedicated with promethazine 50 mg given by mouth before retiring on the night before operation and atropine 0.6 mg given intramuscularly at the usual time before operation. Sleep was induced with thiopentone 250 mg intravenously. This was followed by the administration of a 50 per cent mixture of cyclopropane with oxygen and by topical laryngeal anaesthesia with 4 per cent lignocaine to facilitate intubation. Anaesthesia was then maintained using a mixture of nitrous oxide (3 l./min) and oxygen (1 L/min). The pulmonary ventilation was controlled throughout using a Blease Pulmoflator to deliver a constant minute volume, usually of 25 l./min as checked by a Wright anemometer. Blood carbon dioxide levels. The range of carbon dioxide tensions aimed for was within that observed commonly with different techniques of anaesthesia. In two groups of patients, each of ten patients, the carbon dioxide tensions of the blood were varied. In one group, the Boyle Mark HI soda lime canister was included in the circuit This

2 INFLUENCE OF CARBON DIOXIDE ON THE NEUROMUSCULAR BLOCK 273 produced blood carbon dioxide tensions varying between 14.5 and 22.9 mm Hg and ph values varying between 7.49 and In the other group, excluding the soda lime canister and adding carbon dioxide to form per cent of the fresh gas flow resulted in blood carbon dioxide tensions of between 45 and 90 mm Hg and ph values between 7.12 and Blood taken for the Pco 2 and ph estimations was "arterialized venous" (Brooks and Wynn, 1955). Each sample was taken into a syringe, the deadspace of which had been filled with neutral mineral oil, to which one drop of heparin was added before collection. The blood ph and Pco 3 values were then estimated with the capillary electrode following the method described by Robinson and Utting (1961). Observations on neuromnscular transmission by the "twitch response" to tetanic stimuli. When the patient was placed on the operation table, the arm was extended laterally on an armboard and fixed to it by adhesive strapping. After preparation of the skin with electrode jelly, a surface-stimulating electrode was placed over the ulnar nerve immediately posterior and lateral to the medial epicondyle. The indifferent electrode was placed on the arm. Supramaximal (with reference to the resultant twitch) impulses each of 0.1 m.sec duration were delivered in bursts of 0.3 sec duration and 20/sec frequency. The bursts were applied at intervals of 10 sec. This form of stimulation was used because muscles are normally activated not by single shocks, but by bursts of tetanic activity. The ring finger was connected by a linen thread to a strong flat steel spring myograph recording with ink on a rotating drum (fig. 1). It is recognized that this method of recording is insensitive and the twitch amplitude is a function of the lever system. Once recording constantly, however, the changes were qualitatively of significance and the times necessary for a return of a recordable twitch as between the two series were so strikingly different and so closely similar within each group that there was no doubt as to their significance. General procedure. A period of at least 10 minutes was allowed to elapse between the start of anaesthesia and the start of investigation in order to ensure exhalation of cyclopropane used during induction and to reach a steady state of both the carbon dioxide tension and the twitch response. Surgery commenced during this time. Fio. 1 Diagrammatic representation of the recording system used. The ring finger is attached by a linen thread to a flat steel spring myograph. The effect of 5 per cent carbon dioxide on the twitch response without the intervention of muscle relaxants was investigated. The neuromuscular blocking effect of tubocurarine 4 mg/stone (0.64 mg/kg) on the twitch response in ten patients in respiratory alkalosis was observed and compared with the effect of the same dose in a further ten patients during hypercarbia. Determinations of plasma concentrations of tubocurarine. The method used to determine plasma concentrations of tubocurarine was that described by Elert (1956) and is based on estimation of the drug by ultraviolet spectrophotometry. Tubocurarine is extracted from plasma into ethylene dichloride at alkaline ph in the presence of iodide (Quinn and Woislawski, 1950). The tubocurarine is subsequently re-extracted into 0.01 N hydrochloric acid and is estimated in the spectrophotometer at m/j. Some minor modifications of this method have been described by Utting (1963) and the experimental procedures used by him have been fol-

3 274 BRITISH JOURNAL OF ANAESTHESIA Hype rvcntllat ton Carbon Dioxide 5% 'mmiiiimiiiiiiiiiimmi^ Tlm«InUrv«U 10 llnluhiiiinihiiiiiiiiimimiiiiiiiiiiiiiiiiiiiiiiiiininillllllluiiimiiiiiinnmiiiimiimiiiihiiimiiiiiiihimimiiiimimiiiiiiiim FIG. 2 A kymograph tracing showing the influence of the addition of carbon dioxide 5 per cent together with removal of the absorption canister, on the twitch response to ulnar nerve stimulation in an uncurarized patient Slight depression of the twitch response was observed which was readily reversed by hyperventilation, omitting carbon dioxide and re-inserting the canister. A. ALKALAEMIA Tubocurflrine 4my/fiton B. ACIDAEMIA 8 Tubocurarlne. 4 mg/fltone 20 mln Time Intervals 5 min Fio. 3 Kymographs obtained in patients during (A) respiratory alkalaemia and (B) respiratory acidaemia after injection of tubocurarine 4 mg/ stone (0.64 mg/kg). It can be seen that there is evidence of recovery after 20 minutes in tracing (A) but no twitch is observed after 60 minutes in tracing (B). The gap in the tracing represents 20 minutes.

4 INFLUENCE OF CARBON DIOXIDE ON THE NEUROMUSCULAR BLOCK 275 lowed in the present work. Under these circumstances the error in the estimations of plasma concentrations of the drug should be less than 0.7 fig/ml. Undoubtedly the main source of error is the introduction of contaminants, and sedulous attention to cleaning glassware and meticulous technique in collecting samples for estimation of the drug are the most important prerequisites for satisfactory results. Samples of blood were taken before induction of anaesthesia as a blank, and 20 and 60 minutes after the injection of tubocurarine while the twitch was being recorded. RESULTS The influence of carbon dioxide on neuromuscular transmission. Pulmonary hyperventilation in the uncurarized patient increased the twitch response to supramaximal nerve stimulation and sometimes resulted in spontaneous activity; two out of twenty patients who were passively hyperventilated manifested carpal spasm and Chvostek's sign, which disappeared on curarization. On the other hand, the addition of 5 per cent carbon dioxide to the inhaled mixture and removal of the absorption canister from the circuit produced slight depression of the twitch; the stimulation throughout was supramaximal. This slight depression was readily reversed by pulmonary hyperventilation omitting the carbon dioxide and re-inserting the canister (fig. 2). The influence of carbon dioxide on the neuromuscular block caused by tubocurarine. When tubocurarine 4 mg/stone (0-64 mg/kg) was injected intravenously into ten patients during pulmonary hyperventilation, the twitch response disappeared for min only, after which there was rapid recovery (fig. 3A). When the same dose was injected into a further ten patients during hypercarbia, there was no evidence of recovery for min (fig. 3B). If carbon dioxide were added to the inhaled mixture of a hyperventilated patient who was already showing recovery from neuromuscular block, the recovery was seen to be arrested until the carbon dioxide was removed and pulmonary hyperventilation resumed (fig. 4). The influence of carbon dioxide on tubocurarine plasma levels. The estimation of plasma levels of tubocurarine while the twitch was being recorded presented evidence of the interdependence of the plasma level of tubocurarine on the one hand and its neuromuscular blocking effect on the other. During respiratory acidaemia the prolonged neuromuscular block of tubocurarine was associated with high plasma levels; the mean value was 3.9 /ig/ml (SD 0.66) after 20 min and 1.96 /<g/ml (SD 0.57) after 60 min (table I). TABLE I Plasma levels of tubocurarine estimated in nine patients during respiratory acidaemia. Og/ml) Time following injection: 20 min 30 min 45 min 60 min Average 3.9 (SD 0.66) Average 1.96 (SD 0.57) During alkalaemia, the rapid rate of recovery from tubocurarine block was associated with low plasma levels; the mean value was only 1.88 /ig/ml (SD 0.61) after 20 min, and less than 1 ilg after 60 min (table II). The limits of the method of estimation do not permit accurate assessments at levels lower than 1/ig/ml. Hype r\entl Lotion Carbon Dlcide Hyper ventilation Fio. 4 Kymograph tracing showing recovery from tubocurarine 4 mg/stone (0.64 mg/kg) in a patient during pulmonary hyperventilation. During the period when soda lime was excluded and carbon dioxide 10 per cent added to the inhaled mixture, it was observed that the recovery was arrested until hyperventilation was resumed.

5 276 BRITISH JOURNAL OF ANAESTHESIA TABLE II Plasma levels of lubocurarine estimated in eight patients during respiratory alkalaemia. 0-g/ml) Time following injection: 20 min 30 min 60 min Average 1.88 (SD 0.16) < *** These values being less than one are outside the accuracy of the method. DISCUSSION The ability of carbon dioxide to depress the twitch response could be attributed to its depressant effect on nerve excitability which has been shown by Lehmann (1937) Lorente de N6 (1946) and by Gamstorp and Vinnars (1961a, b). Maintaining the nerve stimulation as supramaximal did not prevent the occurrence of depression; it is therefore possible that carbon dioxide, either primarily or secondarily to an increase of ionized calcium following upon the change in ph, can depress tissue excitability not only at the nerve but also at the myoneural junction. Fatt and Katz (1952) have shown that calcium increases the threshold point at which an endplate potential gives rise to an action potential. Ths effect of carbon dioxide on the neuromuscular block of tubocurarine had been considered by Payne (1960) to be a specific effect on tubocurarine differing from that of ph. However, the findings of Kalow (1954) and Gamstorp and Vinnars (1961) show that this effect is related to the changes in ph, and hence similar effects could be produced by metabolic disturbances. The tubocurarine molecules have, beside their cationic quaternary ammonium groups which combine with the anionic endplate receptors, two ionizable phenolic hydroxyl groups. The pk. value of the quaternary ammonium groups is above 13 (Albert, 1952), and these groups are, therefore, completely ionized within the range of ph changes of these observations. On the other hand, Kalow (1954) has shown that the pk, values of the hydroxyl groups are 8.1 and 9.1, and these are, therefore, liable to vary thendegree of ionization when the ph of their surroundings is altered within the relevant range. The effect of ph changes on the extent of ionization has been shown by Albert (1952) and it is from his tables that the percentage ionization of the two phenolic hydroxyl groups of tubocurarine at different ph values has been calculated (fig. 5) C o c,30 c V ph FIG. 5 A graph showing the effect of ph changes and the percentage ionization of the two phenolic hydroxyl groups of tubocurarine chloride. The upper one represents the hydroxyl group with a pk» value 8.1, and the lower one represents the group with a pk» value 9.1. Data from Albert (1952). Introduction of anionic charges as a result of increased ionization of these hydroxyl groups in alkalaemia could hypothetically result in two effects. Firstly, there would be decreased attachment of the tubocurarine molecule to the endplate receptors, because the increasingly negatively charged part of the molecule would be repelled by the negatively charged cholinergic receptors (Kalow, 1954). This could diminish the "occupancy" achieved by the tubocurarine which, according to Paton and Waud (1962), would diminish competition block. The second effect could be increased disappearance at sites of loss.

6 INFLUENCE OF CARBON DIOXIDE ON THE NEUROMUSCULAR BLOCK 277 Kalow (1959) suggested that the permeation of cell membranes by tubocurarine might be facilitated by the formation of zwitterions. Therefore it is hypothetically possible that during pulmonary hyperventilation, the extracellular concentration of tubocurarine is lowered by the passage of the drug into cells. Rapid recovery would result. The liver is a probable site of disappearance. According to Schanker (1961) the membrane of the hepatic parenchymal cells acts as a lipoid membrane containing fairly large aqueous pores; the pores are large enough to admit a number of lipoid insoluble substances that do not penetrate many other cells. The method of assessment of tubocurarine levels in the plasma employed in this work determines both the free drug and that bound to protein. The high plasma level of the drug during acidaemia might be attributable to increased binding to blood proteins. Direct evidence for the occurrence of binding of tubocurarine has been presented by Aladjemoff, Dikstein and Shafrir (1958). A bound drug is not free to diffuse rapidly into the interstitial space (Goldstein, 1949). This would, however, limit the concentration of tubocurarine that is available to act at the endplate region and in turn would lead to a diminished neuromuscular block. Such a result contrasts with the finding here reported that the high plasma levels of tubocurarine under acidaemia are always associated with marked enhancement of its neuromuscular block. The most probable explanation again seems to be a change in ionization of the hydroxyl groups. In acidaemia, the percentage ionization of the phenolic hydroxyl groups of tubocurarine is small (fig. 5). Following the hypothesis suggested above, this would impede intracellular penetration of the cationic quaternary ammonium molecules and so maintain a high extracellular concentration at the endplate. ACKNOWLEDGMENTS This work has been undertaken at the suggestion of Professor Cecil Gray. I am greatly indebted to his constant advice and encouragement I am also grateful to all the members of the Departments of Anaesthesia, Surgery and Photography for their constant help. REFERENCES Aladjemoff, L., Dikstein, S., and Shafrir, E. (1958). Binding of d-tubocurarine chloride to plasma proteins. /. Pharm. Pharmacol., 123, 43. Albert, A. (1952). Ionization ph and biological activity. Pharmacol. Rev., 4, 136. Brooks, D., and Wynn, V. (1959). Use of venous blood for ph and carbon dioxide studies especially in respiratory failure and during anaesthesia. Lancet, 1, 227. Elert, B. (1956). A new ultraviolet spectrophotometric method for the determination of d-tubocurarine chloride in plasma. Amer. J. med. Technol, Fart, P., and Katz, B. (1952). The effect of sodium on neuromuscular transmission. /. Physiol. (Lond.), 118, 73. Gamstorp, I., and Vinnars, E. (1961a). Studies in neuromuscular transmission. 1: Influence on neuromuscular transmission of alkalosis and acidosis. Ada physiol. scand., 53, 142. (1961b). Studies in neuromuscular transmission. 2: Influence of changes in blood ph and carbon dioxide tension on the effect of tubocurarine and dimethyl tubocurarine. Ada physiol. scand., 53, 160. Goldstein, A. (1949). The interactions of drugs and plasma proteins. Pharmacol. Rev., 1, 102. Kalow, W. (1954). The influence of ph on the ionization and biological activity of d-tubocurarine. /. Pharm. Pharmacol., 110, 443. (1959). The distribution, destruction and elimination of muscle relaxants. Aneslhesiology, 20, 503. Lehmann, J. E. (1937). Effect of changes in ph on action of mammalian A nerve fibres. Amer. J. Physiol., 118, 600. Lorente, de N6, R. (1946). Correlation of nerve activity with polarization phenomena. Harvey Led., 42, 43. Paton, W. D. M., and Waud, D. R. (1962). Drugreceptor interactions at the neuromuscular junction. Ciba Foundation Study Group No. 12, Curare and Curare-like Agents, p. 34. London: Churchill. Payne, J. P. (1958). The influence of carbon dioxide on the neuromuscular blocking activity of relaxant drugs in the cat. Brit. J. Anaesth., 30, 206. (1960). The influence of changes in blood ph on the neuroblocking properties of tubocurarine and dimethyl tubocurarine in the cat. Ada anaesth. scand., 4, 83. Quinn, G. P., and Woislawski, S. (1950). A method for quantitative estimation of d-tubocurarine chloride in plasma. Proc. Soc. exp. Biol. (N.Y), 74, 365. Robinson, J. S., and Utting, J. E. (1961). A simple interpolation method for the estimation of Pcd in whole blood. Brit. J. Anaesth., 33, 327. Schanker, L. S. (1962). Passage of drugs across body membranes. Pharmacol. Rev., 14, 501. Utting, J. E. (1963). ph as a factor influencing plasma concentrations of d-tubocurarine. Brit. J. Anaesth., 35, 706.

7 278 BRITISH JOURNAL OF ANAESTHESIA L'INFLUENCE DE L'ACIDE CARBONIQUE SUR LE BLOQUAGE NEURO-MUSCULAIRE PAR CHLORURE DE TUBOCURARINE CHEZ L'HOMME SOMMAIRE L'auteur a e'tudie' l'influence exerce'e par l'acide carbonique sur le bloquage neuro-musculaire provoqud par la tubocurarine chez I'homme anesthe'sie'. Pour l'appre'ciation de la transmission neuro-musculaire il se seryit d'un simple myographe a ressort, attach^ a un doigt et du nerf moteur stimuli par des impulsions te'tanisantes de'passant la maximale. Les taux de tubocurarine plasmatique furent mesure's par extraction dans le dichlorure d'e'thylene et une mithode spectrophotome'trique. L'acide'mie respiratoire proyoque'e dans un groupe de patients s'accompagna de niveaux plasmatiques sieve's du medicament et de bloquage neuromusculaire prolong^, l'alcalie'mie respiratoire chez un autre groupe eut pour re'sultat des taux plasmatiques bas (apres administration de la meme dose initiale) et la fin rapide du bloquage. L'auteur pense que ces constatations peuvent tre expliqudes de fajon plausible par le fait que la molecule de tubocurarine contient en plus de ses groupes d'ammonium quaternaire deux groupes de pmnol-nydroxyl de pak a 8,1 et 9,1. Les degr^s d'ionisation varient ainsi dans les limites du ph assocides a des fluctuations de la ventilation pulmonaire. DER EINFLUSS VON KOHLENDIOXYD AUF DEN DURCH TUBOCURARIN-CHLORID ERZEUGTEN NEUROMUSKULXREN BLOCK BEIM MENSCHEN ZUSAMMENFASSUNG Es wurde der Einflufl von Kohlendioxydvera'nderungen auf die neuromuskuiare Blockbildung durch Tubocurarin bei anfisthesierten Menschen untersucht. Zur Feststelung der neuromuskularen Uberleitung wurde an einem Finger ein einfacher Federmyograph befestigt und der motorische Nerv durch einen supramaximalen tetanischen Reiz stimuliert. Die respiratorische Azidosis bei der einen Gruppe von Patienten flihrte zu hohen Plasmaspiegeln der Droge und langer anhaltendem neuromuskularen Block; respiratorische Alkalose in einer weiteren Gruppe resultierte in niedrigen Plasmaspiegeln der Droge nach der initialen Dosis und rascher RQckbildung des Blockes. Als mogliche Erk- ISrung fur diese Befunde wird behauptet, dao na'mlich die Tatsache, dao das TubocurarinmolekUl neben seinen quartaren Ammoniumgruppen zwei phenolische Hydroxylgruppen, die pak-werte von 8,1 und 9,1 haben, besitzen und so den Grad der Ionisierung im Rahmen von ph-vera'nderungen, wie sic sich aus den Veranderungen der Lungenbeatmung ergeben, variieren.