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THE EFFECTS OF INTRAPERITONEAL INJECTION OF LIGNOCAINE ON PLASMA CATECHOLAMINE LEVELS IN UNSTRESSED AND STRESSED RATS
Brit. J. Anaesth. (1972), 44, 460
THE EFFECTS OF INTRAPERITONEAL INJECTION OF LIGNOCAINE ON PLASMA CATECHOLAMINE LEVELS IN UNSTRESSED AND STRESSED RATS M. B. TURTON, T. DEEGAN AND I. C. GEDDES SUMMARY
The suppressant action of lignocaine in ventricular arrhythmias, first reported by Southworth and coworkers (1950) in patients undergoing cardiac catheterization, was applied by Lown and co-workers (1967) to the same condition in patients suffering acute myocardial infarction. Investigations by Likoff (1959) and Harrison, Sprouse and Morrow (1963) demonstrated that the drug was relatively free from haemodynamic and other side effects when administered at therapeutic levels. The possible involvement of increased levels of circulating catecholamines in the production of arrhythmias in the period immediately following myocardial infarction has been suggested by several investigators (Valori, Thomas and Shillingford, 1967; Jewitt et al., 1969; Gutpa et al., 1969; McDonald et al., 1969). This raises the question whether the action of lignocaine is associated with an effect upon the levels of these metabolites. This paper reports the effect of lignocaine on such levels when given intraperitoneally to rats, some of which were subjected to a controlled stress stimulus, which, in common with a similar reaction in man, provoked an increase in catecholamine secretion (Carlson, Levi and Oro, 1968). METHODS
Design of animal experiments. (i) Unstressed groups. Groups of male rats (c. 250 g body weight) were injected intraperitoneally with either 1 ml physiological saline or 1 ml ligno-
caine solution, 250 ng/ml, in saline (Lidothesin; Pharmaceutical Manufacturing Co., Bolton, Lanes.). The amount of lignocaine administered was equivalent on a body weight basis to the therapeutic dose given to humans. The intraperitoneal route combined uniform release of the drug with easier application, compared with alternative types of injection.
FIG. 1. The rat holder; the three holes to the rear of the container allowed rods to be inserted to secure the rat.
After treatment, the rats were placed in triangular transparent plastic holders, depicted in figure 1, with their heads projecting through the narrow aperture. This was to facilitate handling and allowed decapitation 10 or 30 min later, using a specially constructed M.
B. TURTON, B.SC.; T.
GEDDES, M.D., F.F.A.R.C.S., Centre and Department Liverpool. Requests for reprints Regional Cardiac Centre, pool LI 5 2HE.
DEEGAN, M.A., PH.D.; I.
C
D.A.; Liverpool Regional Cardiac of Anaesthesia, University of to M. B. Turton, Liverpool Sefton General Hospital, Liver-
Downloaded from http://bja.oxfordjournals.org/ by guest on August 11, 2015
Groups of rats were injected intraperitoneally with either saline or lignocaine solutions. Following decapitation 10 or 30 min later, blood was collected and analyzed for plasma adrenaline and noradrenaline content. Controlled stress by means of amplified audio-signals was applied to some groups for 5 min before sampling. Results showed that a rise in adrenaline level was produced by stress as well as by both saline and lignocaine injection 10 min before decapitation. This response had disappeared 30 min after injection. Application of stress following injections of either saline or lignocaine produced greatly raised adrenaline levels after 10 min. After 30 min the increases were much less. It is concluded that lignocaine is unlikely to influence plasma catecholamine levels.
EFFECTS OF INTRAPERITONEAL INJECTION OF LIGNOCAINE
Catecholamine assay. The method used for the extraction of plasma catecholamines was based upon that of Vendsalu (1960) and the final fluorescence analysis upon that of Haggendal (1963). Immediately after collection, the blood sample was centrifuged, and the plasma separated and deproteinized with 1/10 volume of perchloric acid (4N). The clear supernatant obtained on centrifugation was adjusted to pH 4.0 with potassium carbonate (5N) and applied to a micro-column of Dowex 50 W-X8 ion-exchange resin (16 mm2 x 20 mm; Na+ form). After washing the column with 0.1% EDTA and 0.1 M phosphate buffer (pH 6.5), the catecholamines were eluted with 4 ml hydrochloric acid (IN). Oxidation with potassium ferricyanide in the presence of phosphate buffer and Cu++ ions was followed after 5 min by the addition of 1/10 vol of 2% ascorbic acid in 5N sodium hydroxide solution. The fluorescence of the lutines produced in this way was measured in an Aminco-Bowman spectrophotofluorimeter, at a single emission wavelength of 540 nm with excitation wavelengths of 390 and 450 nm. Recovery experiments, involving the addition of each catecholamine separately, over a range of
10-100 ng, to a pooled plasma sample gave values of 90.2±3.7% and 89.6±3.7% for adrenaline and noradrenaline respectively (n=40). Addition of mixed catecholamine standards over the range 5-100 ng gave similar results, viz. 88.8 + 3.8% and 87.6 + 3.6% (n=32). Recoveries of this order were also obtained when the amount of added standards was reduced to give total concentrations within the upper physiological limits (1-2 ng/ml). In order to investigate possible fluorescence quenching effects arising from factors co-eluted with the amines, small quantities of adrenaline and noradrenaline were added to aliquots of various eluates. Recoveries in these instances were 98.2 + 1.8% for adrenaline and 98.4+1.7% for noradrenaline, indicating that interference of this form was minimal. All values of catecholamines are reported in ng/ml plasma, as mean ± standard deviation; results were analyzed by Student's (-test with application of a 5 % level of significance. RESULTS
The levels of plasma adrenaline and noradrenaline obtained for the various groups of animals are presented in table I; individual values are depicted in figure 2. Comparisons of the group values for the two amines with their associated levels of significance are given in table II. The baseline values for the two amines, derived from the group of untreated rats, were 0.31+0.22 ng/ml for adrenaline, and 0.58 + 0.19 ng/ml for noradrenaline. These values are lower than those reported by Anton and Sayre (1962), but lack of TABLE I.
Values of plasma adrenaline and noradrenaline in groups of rats.
Group
Adrenaline (ng/ml)
Noradrenaline (ng/ml)
Control Control*
0.31 ±0.22 0.79 ±0.17
0.58 ±0.19 0.64 + 0.31
Animals decapitated 10 min after injection Saline 5 0.61 ±0.17 Saline* 5 1.17 + 0.22 Lignocaine 9 0.79 ±0.22 Lignocaine* 11 1.81 ±0.60
0.58 ±0.17 0.96 ±0.21 0.69 ±0.16 0.85±0.34
Animals decapitated 30 min after injection Saline 5 0.20 ±0.14 Saline* 5 0.55 ±0.25 Lignocaine 5 0.34 ±0.16 Lignocaine* 5 0.67 + 0.35
0.41 ±0.21 0.59 ±0.18 0.37 ±0.12 0.76 ±0.14
Mean±SD. *=stress applied prior to sampling. n=number of rats per group.
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guillotine. Approximately 5 ml blood was collected, via a funnel, in a 20 ml universal bottle containing 2 ml 1% EDTA/saline solution as anticoagulant. (ii) Stressed groups. A standardized stress stimulus was applied by placing a loudspeaker at a fixed height over the rat holder and emitting an amplified signal of 4.25 kHz from an audiogenerator for exactly 5 min prior to decapitation. This treatment was given to groups of saline- and lignocaine-treated animals. (iii) Control groups. Baseline values were .provided by a group who were not injected but were placed in the holders for 10 min before decapitation. The effect of the stress stimulus was assessed by its application for 5 min before decapitation in a second similar group. In the initial experimental series, all groups were sacrificed 10 min after injection. Observation, however, suggested that definite signs of confusion and unusual movement were apparent in the lignocainetreated rats at this time. Accordingly, further groups were studied 30 min after injection when these effects had disappeared. In this way it was hoped that any observed change in catecholamine levels would reflect in this series the direct metabolic effects of the drug.
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TABLE II. Statistical comparisons of the levels of adrenaline and noradrenaline found in the groups of rats. N Group Group Adrenaline Noradrenaline Control Control* 10 Saline Control 30 Saline Control 30 10 Saline Saline 10 Lignocaine Control 30 Lignocaine Control 30 10 Lignocaine Lignocaine 10 Saline* Control 30 Saline* Control 10 30 Saline* Saline* Lignocaine* 10 Control Lignocaine* 30 Control Lignocaine* 30 Lignocaine* 10 10 10 Saline Saline* 30 30 Saline Saline* 10 Lignocaine Lignocaine* 10 30 Lignocaine Lignocaine* 30 10 10 Saline Lignocaine 30 30 Saline Lignocaine 10 Saline* Lignocaine* 10 30 Saline* Lignocaine* 30 * = stress applied prior to sampling. t=time of sample after injection (min). information from this source about methods of animal handling and blood sampling makes direct comparison impossible. In untreated animals, the standardized stress stimulus provoked an increase in adrenaline concentration (P<0.01), but had no effect on the noradrenaline concentration. An identical unilateral effect upon plasma adrenaline level was observed in
P<0.01 P<0.05 n.s. P<0.01 P<0.01 n.s. P<0.01 P<0.001 n.s. P<0.01 P<0.001 n.s. P<0.01 P<0.01 P<0.05 P<0.001 n.s. n.s. n.s. P<0.05 n.s. N=degrees of freedom. n.s.=not significant (P>0.05). 9 8 g g 12 g 12 g 8 8 14 8 14 8 8 18 8 12 8 14 8
n.s. n.s. n.s. n.s. n.s. n.s. P<0.01 P<0.02 n.s. P<0.02 n.s. n.s. n.s. P<0.02 n.s. n.s. P<0.01 n.s. n.s. n.s. n.s.
rats sacrificed 10 min after either saline or lignocaine injection alone (saline, P<0.05; lignocaine, P<0.01). The effect was transitory, for 30 min after injection the levels of both amines were slightly depressed but directly comparable with those of the baseline group, irrespective of the substance given. The difference between the levels of adrenaline at 10 and 30 min was significant in both saline-treated and lignocaine-
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A A A
A
i
FIG. 2. Individual values of plasma adrenaline (A) and noradrenaline ( # ) levels (ng/ml) found in the various groups of rats. *=application of stress to a group. 10'/30'=time of sampling after injection (min). Com =control groups Sal = saline injection groups. Lig =lignocaine injection groups.
EFFECTS OF INTRAPERITONEAL INJECTION OF LIGNOCAINE
Differential effect of the stress stimulus. The extent to which the stress procedure contributed to the changes was assessed at both sampling times by comparison of the levels of similarly injected, stressed and unstressed groups. In the saline-treated animals, stress increased the adrenaline level at both times (P<0.0l, P<0.05) and the noradrenaline level at 10 min (P<0.02). The h'gnocainetreated animals showed a marked reaction at 10 min with a greatly increased adrenaline level (P<0.001) but no change in noradrenaline level. This was supplanted, at 30 min, by an unchanged adrenaline level in the presence of a raised noradrenaline level
Differential effect of lignocaine. Saline was a factor in both injections; the extent to which lignocaine contributed in excess of the effect of saline was assessed under the various circumstances by comparison of saline-treated groups with the corresponding lignocaine-treated groups. The only circumstances in which a difference was apparent was in the stressed groups sampled at 10 min. At this rime the lignocaine-treated rats had a
higher level of adrenaline than their saline-treated counterparts (P<0.05). Elsewhere, the identity of the sets of levels was most striking. DISCUSSION
Carlson, Levi and Oro (1968) have reported increased excretion of catecholamines in human volunteers after subjection to stressful episodes. In addition, Turton (unpublished observation) found considerably higher levels of plasma adrenaline and noradrenaline in patients about to undergo cardiac catheterization compared with those obtained subsequently from the same patients when resting. The controlled stress stimulus in the present study provoked increases in plasma adrenaline only, suggesting that the response to stress in rats is more specific in nature than in humans. Simple injection of either saline or lignocaine also increased plasma adrenaline levels transitorily, and to the same extent as the stress stimulus. It is probable that this change was equally a stress response and arose from the short period between handling for injection and decapitation. The values obtained after the animals had rested in the holders for 30 min following injection, which tended to be lower than those of the baseline group, support this premise. The reaction to stress of these groups after 25 min was also relevant; in both instances the changes were similar to those induced in the untreated group. Throughout the various circumstances above, no differences were apparent between, the saline-treated and lignocaine-treated groups, A condition approximating to that observed frf stressed humans was obtained when the salinetreated group was stressed soon after injection; in this single instance both amines were raised above the corresponding baseline levels. In the lignocainetreated group, however, only a massive increase in adrenaline concentration was recorded in the same circumstances. At this time these animals were showing unusual signs of restlessness and movement and it is possible that the unilateral change observed may have been associated with this secondary effect of the drug. Throughout the study it was apparent that lignocaine exerted no direct effect upon plasma adrenaline and noradrenaline levels. In most instances the changes observed paralleled those induced by saline injection. Even in the single difference in reaction noted in the previous paragraph, lignocaine did not lower catecholamine levels. Provided that the mode of action of lignocaine is similar in humans, it is
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treated animals (P<0.01), and in the latter group was accompanied by an equally significant difference in noradrenaline (P<0.01). Similar differences in effect with respect to time were apparent when the stress procedure was applied 5 and 25 min after injection, with sacrificing of the rats at 10 and 30 min respectively. In animals receiving saline, the amine levels at 10 min were both raised compared with those of the baseline group (adrenaline, P<0.001; noradrenaline, P<0.02). After 30 min, however, the changes were similar to those induced by stress in normal animals; only the adrenaline level was increased and to a marginal degree (0.05
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doubtful whether its anti-arrhythmic properties are associated with direct action upon plasma adrenaline and noradrenaline levels. ACKNOWLEDGEMENT
We wish to acknowledge financial assistance from the Medical Research Committee of the United Liverpool Hospitals towards the cost of this investigation. REFERENCES
EFFETS EXERCES SUR LES TAUX PLASMATIQUES DE CATECHOLAMINES PAR UNE INJECTION INTRAPERITONEALE DE LIGNOCAINE CHES DES RATS SOUMIS OU NON A UN STRESS SOMMAIRE
Differentes series de rats ont fait l'object d'une injection intraperitoneale de solutions, soit a base de lignocaine, soit a base de solute physiologique chlorure. A la suite
DIE WIRKUNG EINER INTRAPERITONEALEN LIGNOCAIN-INJEKTION AUF DEN PLASMACATECHOLAMIN-SPIEGEL VON RATTEN IM UND OHNE STRESS ZUSAMMENFASSUNG
Mehrere Gruppen von Ratten erhielten intraperitoneale Injektionen von Kochsalz oder Lignocain. Zehn bzw. dreifiig Minuten nach Injektion wurden die Tiere dekapitiert, und ihr Blut auf den Plasma-Adrenalin und -Noradrenalingehalt untersucht. Einige Gruppen wurden 5 Minuten vor der Blutuntersuchung mit yerstarkten Audio-Signalen gestresst. Die Ergebnisse zeigten, dafi sowohl Stress als auch Kochsalz sowie Lignocain 10 Minuten vor der Dekapitierung den Adrenalinspiegel erhohten. Diese Reaktion war 30 Minuten nach der Injektion verschwunden. Die Applikation von Kochsalz oder Lignocain mit nachfolgendem Stress ftihrte zu einer deutlichen Adrenalinspiegel-Erhohung nach 10 Minuten. Nach 30 Minuten waren die Steigerungen bereits viel weniger ausgepragt. Daraus wird geschlossen, da6 Lignocain die Plasma-Catecholaminspiegel kaum beeinflufit. LOS EFECTOS INTRAPERITONEAL LOS NIVELES CATECOLAMINAS EN
DE LA INYECCION DE LIGNOCAINA SOBRE PLASMATICOS DE RATAS CON Y SIN STRESS
RESUMEN
Grupos de ratas fueron inyectados intraperitonealmente con soluciones salinas o de lignocaina. Fueron decapitadas 10 6 30 min mas tarde y se recogio y analizo la sangre para su contenido plasmatico en adrenalina y noradrenalina. Fue aplicado a algunos grupos un stress controlado por medio de sefiales auditivas amplificadas durante 5 min antes de obtener las muestras. Los resultados mostraron que el stress, asf como la injeccion de solucion salina o de lignocaina 10 minutos antes de la decapitacidn, producian una elevacion en el nivel de adrenalina. Esta respuesta habia desaparecido 30 min despu^s de la inyeccion. La aplicacion de stress despues de inyecciones de solucion salina o de lignocaina producia niveles muy elevados de adrenalina despues de 10 min. Los aumentos eran mucho menores despues de 30 min. Se concluye que es poco probable que la lignocaina influya sobre los niveles de catecolaminas en el plasma.
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Anton, A. H., and Sayre, D. F. (1962). A study of the factors affecting the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol, exp. Ther., 138, 360. Carlson, J. A., Levi, L., and Oro, L. (1968). Plasma lipids and urinary excretion of catecholamines in man during experimentally induced emotional stress, and their subsequent modification by nicotinic acid. J. din. invest., 47, 1795. Gupta, D. K., Jewitt, D. E., Young, R., Hartog, M., and Opie, L. H. (1969). Increased plasma free-fatty-acid concentrations and their significance in patients with acute myocardial infarction. Lancet, 2, 1209. Haggendal, J. (1963). An improved method for fluorimetric determination of small amounts of adrenaline and noradrenaline in plasma and tissues. Acta physiol. scand., 59, 242. Harrison, D. C , Sprouse, J. H., and Morrow, A. G. (1963). The antiarrhythmic properties of Lidocaine and procainamide. Circulation, 28, 486. Jewitt, D. E., Reid, D., Thomas, M., Mercer, C. J., Valori, C , and Shillingford, J. P. (1969). Free noradrenaline and adrenaline excretion in relation to the development of cardiac arrhythmias and heart failure in patients with acute myocardial infarction. Lancet, 1, 635. Likoff, W. (1959). Cardiac arrhythmias complicating surgery. (Editorial.) Amer. J. cardiol, 3, 427. Lown, B., Fakhro, A. M., Hood, W. B., and Thorn, G. W. (1967). The coronary care unit. J. Amer. med. Ass., 199, 188. McDonald, L., Baker, C , Bray, C , McDonald, A., and Restieaux, N. (1969). Plasma catecholamines after cardiac infarction. Lancet, 2, 1021. Southworth, J. L., McKusick, V. A., Peirce, E. C., and Rawson, F. L. (1950). Ventricular fibrillation precipitated by cardiac catheterization. J. Amer. med. Ass., 143, 717. Valori, C , Thomas, M., and Shillingford, J. P. (1967). Free noradrenaline and adrenaline excretion in relation to clinical syndromes following myocardial infarction. Amer. J. Cardiol, 20, 605. Vendsalu, A. (1960). Studies on adrenaline and noradrenaline in human plasma. Acta physiol. scand., 49, suppl. 173, 23.
d'une decapitation des animaux effectuee 10 ou 30 minutes plus tard, le sang a ete preleve et analyse en vue d'en etudier les concentrations plasmatiques en adrenaline et noradrenaline. Avant que le sang ne soit recueilli, certaines series ont subi, pendant une duree de 5 minutes un stress controle au moyen de signaux auditifs amplifies. Les resultats de cet essai ont montre qu'une augmentation du taux d'adrenaline a ete engendree aussi bien par le stress que par l'injection de serum physiologique et de lignocaine effectuee 10 minutes avant la decapitation. La mise en oeuvre du stress a la suite d'une injection de serum physiologique ou de lignocaine, a entraine au bout de 10 minutes, une augmentation marquee des taux d'adrenaline. Au bout de 30 minutes, les augmentations notees ont ete bien moindres. On en conclut qu'il est peu vraisemblable que la lignocaine exerce un effet sur les concentrations plasmatiques en catecholamines.
THE EFFECTS OF INTRAPERITONEAL INJECTION OF LIGNOCAINE ON PLASMA CATECHOLAMINE LEVELS IN UNSTRESSED AND STRESSED RATS M. B. TURTON, T. DEEGAN AND I. C. GEDDES SUMMARY
The suppressant action of lignocaine in ventricular arrhythmias, first reported by Southworth and coworkers (1950) in patients undergoing cardiac catheterization, was applied by Lown and co-workers (1967) to the same condition in patients suffering acute myocardial infarction. Investigations by Likoff (1959) and Harrison, Sprouse and Morrow (1963) demonstrated that the drug was relatively free from haemodynamic and other side effects when administered at therapeutic levels. The possible involvement of increased levels of circulating catecholamines in the production of arrhythmias in the period immediately following myocardial infarction has been suggested by several investigators (Valori, Thomas and Shillingford, 1967; Jewitt et al., 1969; Gutpa et al., 1969; McDonald et al., 1969). This raises the question whether the action of lignocaine is associated with an effect upon the levels of these metabolites. This paper reports the effect of lignocaine on such levels when given intraperitoneally to rats, some of which were subjected to a controlled stress stimulus, which, in common with a similar reaction in man, provoked an increase in catecholamine secretion (Carlson, Levi and Oro, 1968). METHODS
Design of animal experiments. (i) Unstressed groups. Groups of male rats (c. 250 g body weight) were injected intraperitoneally with either 1 ml physiological saline or 1 ml ligno-
caine solution, 250 ng/ml, in saline (Lidothesin; Pharmaceutical Manufacturing Co., Bolton, Lanes.). The amount of lignocaine administered was equivalent on a body weight basis to the therapeutic dose given to humans. The intraperitoneal route combined uniform release of the drug with easier application, compared with alternative types of injection.
FIG. 1. The rat holder; the three holes to the rear of the container allowed rods to be inserted to secure the rat.
After treatment, the rats were placed in triangular transparent plastic holders, depicted in figure 1, with their heads projecting through the narrow aperture. This was to facilitate handling and allowed decapitation 10 or 30 min later, using a specially constructed M.
B. TURTON, B.SC.; T.
GEDDES, M.D., F.F.A.R.C.S., Centre and Department Liverpool. Requests for reprints Regional Cardiac Centre, pool LI 5 2HE.
DEEGAN, M.A., PH.D.; I.
C
D.A.; Liverpool Regional Cardiac of Anaesthesia, University of to M. B. Turton, Liverpool Sefton General Hospital, Liver-
Downloaded from http://bja.oxfordjournals.org/ by guest on August 11, 2015
Groups of rats were injected intraperitoneally with either saline or lignocaine solutions. Following decapitation 10 or 30 min later, blood was collected and analyzed for plasma adrenaline and noradrenaline content. Controlled stress by means of amplified audio-signals was applied to some groups for 5 min before sampling. Results showed that a rise in adrenaline level was produced by stress as well as by both saline and lignocaine injection 10 min before decapitation. This response had disappeared 30 min after injection. Application of stress following injections of either saline or lignocaine produced greatly raised adrenaline levels after 10 min. After 30 min the increases were much less. It is concluded that lignocaine is unlikely to influence plasma catecholamine levels.
EFFECTS OF INTRAPERITONEAL INJECTION OF LIGNOCAINE
Catecholamine assay. The method used for the extraction of plasma catecholamines was based upon that of Vendsalu (1960) and the final fluorescence analysis upon that of Haggendal (1963). Immediately after collection, the blood sample was centrifuged, and the plasma separated and deproteinized with 1/10 volume of perchloric acid (4N). The clear supernatant obtained on centrifugation was adjusted to pH 4.0 with potassium carbonate (5N) and applied to a micro-column of Dowex 50 W-X8 ion-exchange resin (16 mm2 x 20 mm; Na+ form). After washing the column with 0.1% EDTA and 0.1 M phosphate buffer (pH 6.5), the catecholamines were eluted with 4 ml hydrochloric acid (IN). Oxidation with potassium ferricyanide in the presence of phosphate buffer and Cu++ ions was followed after 5 min by the addition of 1/10 vol of 2% ascorbic acid in 5N sodium hydroxide solution. The fluorescence of the lutines produced in this way was measured in an Aminco-Bowman spectrophotofluorimeter, at a single emission wavelength of 540 nm with excitation wavelengths of 390 and 450 nm. Recovery experiments, involving the addition of each catecholamine separately, over a range of
10-100 ng, to a pooled plasma sample gave values of 90.2±3.7% and 89.6±3.7% for adrenaline and noradrenaline respectively (n=40). Addition of mixed catecholamine standards over the range 5-100 ng gave similar results, viz. 88.8 + 3.8% and 87.6 + 3.6% (n=32). Recoveries of this order were also obtained when the amount of added standards was reduced to give total concentrations within the upper physiological limits (1-2 ng/ml). In order to investigate possible fluorescence quenching effects arising from factors co-eluted with the amines, small quantities of adrenaline and noradrenaline were added to aliquots of various eluates. Recoveries in these instances were 98.2 + 1.8% for adrenaline and 98.4+1.7% for noradrenaline, indicating that interference of this form was minimal. All values of catecholamines are reported in ng/ml plasma, as mean ± standard deviation; results were analyzed by Student's (-test with application of a 5 % level of significance. RESULTS
The levels of plasma adrenaline and noradrenaline obtained for the various groups of animals are presented in table I; individual values are depicted in figure 2. Comparisons of the group values for the two amines with their associated levels of significance are given in table II. The baseline values for the two amines, derived from the group of untreated rats, were 0.31+0.22 ng/ml for adrenaline, and 0.58 + 0.19 ng/ml for noradrenaline. These values are lower than those reported by Anton and Sayre (1962), but lack of TABLE I.
Values of plasma adrenaline and noradrenaline in groups of rats.
Group
Adrenaline (ng/ml)
Noradrenaline (ng/ml)
Control Control*
0.31 ±0.22 0.79 ±0.17
0.58 ±0.19 0.64 + 0.31
Animals decapitated 10 min after injection Saline 5 0.61 ±0.17 Saline* 5 1.17 + 0.22 Lignocaine 9 0.79 ±0.22 Lignocaine* 11 1.81 ±0.60
0.58 ±0.17 0.96 ±0.21 0.69 ±0.16 0.85±0.34
Animals decapitated 30 min after injection Saline 5 0.20 ±0.14 Saline* 5 0.55 ±0.25 Lignocaine 5 0.34 ±0.16 Lignocaine* 5 0.67 + 0.35
0.41 ±0.21 0.59 ±0.18 0.37 ±0.12 0.76 ±0.14
Mean±SD. *=stress applied prior to sampling. n=number of rats per group.
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guillotine. Approximately 5 ml blood was collected, via a funnel, in a 20 ml universal bottle containing 2 ml 1% EDTA/saline solution as anticoagulant. (ii) Stressed groups. A standardized stress stimulus was applied by placing a loudspeaker at a fixed height over the rat holder and emitting an amplified signal of 4.25 kHz from an audiogenerator for exactly 5 min prior to decapitation. This treatment was given to groups of saline- and lignocaine-treated animals. (iii) Control groups. Baseline values were .provided by a group who were not injected but were placed in the holders for 10 min before decapitation. The effect of the stress stimulus was assessed by its application for 5 min before decapitation in a second similar group. In the initial experimental series, all groups were sacrificed 10 min after injection. Observation, however, suggested that definite signs of confusion and unusual movement were apparent in the lignocainetreated rats at this time. Accordingly, further groups were studied 30 min after injection when these effects had disappeared. In this way it was hoped that any observed change in catecholamine levels would reflect in this series the direct metabolic effects of the drug.
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TABLE II. Statistical comparisons of the levels of adrenaline and noradrenaline found in the groups of rats. N Group Group Adrenaline Noradrenaline Control Control* 10 Saline Control 30 Saline Control 30 10 Saline Saline 10 Lignocaine Control 30 Lignocaine Control 30 10 Lignocaine Lignocaine 10 Saline* Control 30 Saline* Control 10 30 Saline* Saline* Lignocaine* 10 Control Lignocaine* 30 Control Lignocaine* 30 Lignocaine* 10 10 10 Saline Saline* 30 30 Saline Saline* 10 Lignocaine Lignocaine* 10 30 Lignocaine Lignocaine* 30 10 10 Saline Lignocaine 30 30 Saline Lignocaine 10 Saline* Lignocaine* 10 30 Saline* Lignocaine* 30 * = stress applied prior to sampling. t=time of sample after injection (min). information from this source about methods of animal handling and blood sampling makes direct comparison impossible. In untreated animals, the standardized stress stimulus provoked an increase in adrenaline concentration (P<0.01), but had no effect on the noradrenaline concentration. An identical unilateral effect upon plasma adrenaline level was observed in
P<0.01 P<0.05 n.s. P<0.01 P<0.01 n.s. P<0.01 P<0.001 n.s. P<0.01 P<0.001 n.s. P<0.01 P<0.01 P<0.05 P<0.001 n.s. n.s. n.s. P<0.05 n.s. N=degrees of freedom. n.s.=not significant (P>0.05). 9 8 g g 12 g 12 g 8 8 14 8 14 8 8 18 8 12 8 14 8
n.s. n.s. n.s. n.s. n.s. n.s. P<0.01 P<0.02 n.s. P<0.02 n.s. n.s. n.s. P<0.02 n.s. n.s. P<0.01 n.s. n.s. n.s. n.s.
rats sacrificed 10 min after either saline or lignocaine injection alone (saline, P<0.05; lignocaine, P<0.01). The effect was transitory, for 30 min after injection the levels of both amines were slightly depressed but directly comparable with those of the baseline group, irrespective of the substance given. The difference between the levels of adrenaline at 10 and 30 min was significant in both saline-treated and lignocaine-
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A A A
A
i
FIG. 2. Individual values of plasma adrenaline (A) and noradrenaline ( # ) levels (ng/ml) found in the various groups of rats. *=application of stress to a group. 10'/30'=time of sampling after injection (min). Com =control groups Sal = saline injection groups. Lig =lignocaine injection groups.
EFFECTS OF INTRAPERITONEAL INJECTION OF LIGNOCAINE
Differential effect of the stress stimulus. The extent to which the stress procedure contributed to the changes was assessed at both sampling times by comparison of the levels of similarly injected, stressed and unstressed groups. In the saline-treated animals, stress increased the adrenaline level at both times (P<0.0l, P<0.05) and the noradrenaline level at 10 min (P<0.02). The h'gnocainetreated animals showed a marked reaction at 10 min with a greatly increased adrenaline level (P<0.001) but no change in noradrenaline level. This was supplanted, at 30 min, by an unchanged adrenaline level in the presence of a raised noradrenaline level
Differential effect of lignocaine. Saline was a factor in both injections; the extent to which lignocaine contributed in excess of the effect of saline was assessed under the various circumstances by comparison of saline-treated groups with the corresponding lignocaine-treated groups. The only circumstances in which a difference was apparent was in the stressed groups sampled at 10 min. At this rime the lignocaine-treated rats had a
higher level of adrenaline than their saline-treated counterparts (P<0.05). Elsewhere, the identity of the sets of levels was most striking. DISCUSSION
Carlson, Levi and Oro (1968) have reported increased excretion of catecholamines in human volunteers after subjection to stressful episodes. In addition, Turton (unpublished observation) found considerably higher levels of plasma adrenaline and noradrenaline in patients about to undergo cardiac catheterization compared with those obtained subsequently from the same patients when resting. The controlled stress stimulus in the present study provoked increases in plasma adrenaline only, suggesting that the response to stress in rats is more specific in nature than in humans. Simple injection of either saline or lignocaine also increased plasma adrenaline levels transitorily, and to the same extent as the stress stimulus. It is probable that this change was equally a stress response and arose from the short period between handling for injection and decapitation. The values obtained after the animals had rested in the holders for 30 min following injection, which tended to be lower than those of the baseline group, support this premise. The reaction to stress of these groups after 25 min was also relevant; in both instances the changes were similar to those induced in the untreated group. Throughout the various circumstances above, no differences were apparent between, the saline-treated and lignocaine-treated groups, A condition approximating to that observed frf stressed humans was obtained when the salinetreated group was stressed soon after injection; in this single instance both amines were raised above the corresponding baseline levels. In the lignocainetreated group, however, only a massive increase in adrenaline concentration was recorded in the same circumstances. At this time these animals were showing unusual signs of restlessness and movement and it is possible that the unilateral change observed may have been associated with this secondary effect of the drug. Throughout the study it was apparent that lignocaine exerted no direct effect upon plasma adrenaline and noradrenaline levels. In most instances the changes observed paralleled those induced by saline injection. Even in the single difference in reaction noted in the previous paragraph, lignocaine did not lower catecholamine levels. Provided that the mode of action of lignocaine is similar in humans, it is
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treated animals (P<0.01), and in the latter group was accompanied by an equally significant difference in noradrenaline (P<0.01). Similar differences in effect with respect to time were apparent when the stress procedure was applied 5 and 25 min after injection, with sacrificing of the rats at 10 and 30 min respectively. In animals receiving saline, the amine levels at 10 min were both raised compared with those of the baseline group (adrenaline, P<0.001; noradrenaline, P<0.02). After 30 min, however, the changes were similar to those induced by stress in normal animals; only the adrenaline level was increased and to a marginal degree (0.05
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doubtful whether its anti-arrhythmic properties are associated with direct action upon plasma adrenaline and noradrenaline levels. ACKNOWLEDGEMENT
We wish to acknowledge financial assistance from the Medical Research Committee of the United Liverpool Hospitals towards the cost of this investigation. REFERENCES
EFFETS EXERCES SUR LES TAUX PLASMATIQUES DE CATECHOLAMINES PAR UNE INJECTION INTRAPERITONEALE DE LIGNOCAINE CHES DES RATS SOUMIS OU NON A UN STRESS SOMMAIRE
Differentes series de rats ont fait l'object d'une injection intraperitoneale de solutions, soit a base de lignocaine, soit a base de solute physiologique chlorure. A la suite
DIE WIRKUNG EINER INTRAPERITONEALEN LIGNOCAIN-INJEKTION AUF DEN PLASMACATECHOLAMIN-SPIEGEL VON RATTEN IM UND OHNE STRESS ZUSAMMENFASSUNG
Mehrere Gruppen von Ratten erhielten intraperitoneale Injektionen von Kochsalz oder Lignocain. Zehn bzw. dreifiig Minuten nach Injektion wurden die Tiere dekapitiert, und ihr Blut auf den Plasma-Adrenalin und -Noradrenalingehalt untersucht. Einige Gruppen wurden 5 Minuten vor der Blutuntersuchung mit yerstarkten Audio-Signalen gestresst. Die Ergebnisse zeigten, dafi sowohl Stress als auch Kochsalz sowie Lignocain 10 Minuten vor der Dekapitierung den Adrenalinspiegel erhohten. Diese Reaktion war 30 Minuten nach der Injektion verschwunden. Die Applikation von Kochsalz oder Lignocain mit nachfolgendem Stress ftihrte zu einer deutlichen Adrenalinspiegel-Erhohung nach 10 Minuten. Nach 30 Minuten waren die Steigerungen bereits viel weniger ausgepragt. Daraus wird geschlossen, da6 Lignocain die Plasma-Catecholaminspiegel kaum beeinflufit. LOS EFECTOS INTRAPERITONEAL LOS NIVELES CATECOLAMINAS EN
DE LA INYECCION DE LIGNOCAINA SOBRE PLASMATICOS DE RATAS CON Y SIN STRESS
RESUMEN
Grupos de ratas fueron inyectados intraperitonealmente con soluciones salinas o de lignocaina. Fueron decapitadas 10 6 30 min mas tarde y se recogio y analizo la sangre para su contenido plasmatico en adrenalina y noradrenalina. Fue aplicado a algunos grupos un stress controlado por medio de sefiales auditivas amplificadas durante 5 min antes de obtener las muestras. Los resultados mostraron que el stress, asf como la injeccion de solucion salina o de lignocaina 10 minutos antes de la decapitacidn, producian una elevacion en el nivel de adrenalina. Esta respuesta habia desaparecido 30 min despu^s de la inyeccion. La aplicacion de stress despues de inyecciones de solucion salina o de lignocaina producia niveles muy elevados de adrenalina despues de 10 min. Los aumentos eran mucho menores despues de 30 min. Se concluye que es poco probable que la lignocaina influya sobre los niveles de catecolaminas en el plasma.
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Anton, A. H., and Sayre, D. F. (1962). A study of the factors affecting the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol, exp. Ther., 138, 360. Carlson, J. A., Levi, L., and Oro, L. (1968). Plasma lipids and urinary excretion of catecholamines in man during experimentally induced emotional stress, and their subsequent modification by nicotinic acid. J. din. invest., 47, 1795. Gupta, D. K., Jewitt, D. E., Young, R., Hartog, M., and Opie, L. H. (1969). Increased plasma free-fatty-acid concentrations and their significance in patients with acute myocardial infarction. Lancet, 2, 1209. Haggendal, J. (1963). An improved method for fluorimetric determination of small amounts of adrenaline and noradrenaline in plasma and tissues. Acta physiol. scand., 59, 242. Harrison, D. C , Sprouse, J. H., and Morrow, A. G. (1963). The antiarrhythmic properties of Lidocaine and procainamide. Circulation, 28, 486. Jewitt, D. E., Reid, D., Thomas, M., Mercer, C. J., Valori, C , and Shillingford, J. P. (1969). Free noradrenaline and adrenaline excretion in relation to the development of cardiac arrhythmias and heart failure in patients with acute myocardial infarction. Lancet, 1, 635. Likoff, W. (1959). Cardiac arrhythmias complicating surgery. (Editorial.) Amer. J. cardiol, 3, 427. Lown, B., Fakhro, A. M., Hood, W. B., and Thorn, G. W. (1967). The coronary care unit. J. Amer. med. Ass., 199, 188. McDonald, L., Baker, C , Bray, C , McDonald, A., and Restieaux, N. (1969). Plasma catecholamines after cardiac infarction. Lancet, 2, 1021. Southworth, J. L., McKusick, V. A., Peirce, E. C., and Rawson, F. L. (1950). Ventricular fibrillation precipitated by cardiac catheterization. J. Amer. med. Ass., 143, 717. Valori, C , Thomas, M., and Shillingford, J. P. (1967). Free noradrenaline and adrenaline excretion in relation to clinical syndromes following myocardial infarction. Amer. J. Cardiol, 20, 605. Vendsalu, A. (1960). Studies on adrenaline and noradrenaline in human plasma. Acta physiol. scand., 49, suppl. 173, 23.
d'une decapitation des animaux effectuee 10 ou 30 minutes plus tard, le sang a ete preleve et analyse en vue d'en etudier les concentrations plasmatiques en adrenaline et noradrenaline. Avant que le sang ne soit recueilli, certaines series ont subi, pendant une duree de 5 minutes un stress controle au moyen de signaux auditifs amplifies. Les resultats de cet essai ont montre qu'une augmentation du taux d'adrenaline a ete engendree aussi bien par le stress que par l'injection de serum physiologique et de lignocaine effectuee 10 minutes avant la decapitation. La mise en oeuvre du stress a la suite d'une injection de serum physiologique ou de lignocaine, a entraine au bout de 10 minutes, une augmentation marquee des taux d'adrenaline. Au bout de 30 minutes, les augmentations notees ont ete bien moindres. On en conclut qu'il est peu vraisemblable que la lignocaine exerce un effet sur les concentrations plasmatiques en catecholamines.