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Urinary catecholamines, flow rate and tobacco smoking
Biological Psychology, I, 1974, 229-236. @ ~ort~-~~l~a~d Publishing Conzpany
URINARY CATECHOLAMINES, TOBACCO
FLOW RATE AND
SMOKING
C. AGU6 Medical Research, Sandoz Ltd., Basle, Switzerland Accepted for publication
17 August 1973
Twenty-four habitual smokers smoked one nicotine-free and three tobacco cigarettes with a known content of nicotine in the course of four successive sessions. Urine samples were collected hourly and under a predetermined water load. A specially devised fluorimetric method was used to determine adrenaline and nor-adrenaline. Flow rate and osmolality were also calculated. No changes were observed in noradrenaline excretion. Adrenaline excretion rose si~ifi~ntly after smoking, although no major differences were found between the treatments, returning to baseline levels one hour after the experimental sessions. Similarly, increased flow rates and low osmolality were observed. The implication of these findings are discussed. It is suggested that the excretion of adrenaline may indicate an ‘adaptive’ mechanism to ‘stress’.
Doubts as to whether habitual tobacco smoking is sufficient to produce an increased release of catecholamines from the adrenal medulla have been formulated by Rehder and Roth (1959), Silvette, Larson and Haag (1961), and Armitage and Milton (1965). Conclusive evidence for rises in the urinary output of amines, especially in the adrenaline fraction, has only been found after the administration of relatively large doses of the drug obtained through heavy smoking (Westfall and Watts, 1964; Kershbaum, Bellet, Hirabayashi and Feinberg, 1967) or after smoking at least two cigarettes (Frankenhaeuser, Myrsten, Waszak, Neri and Post, 1968; Frankenhaeuser, Myrsten and Post, 1970). The level of plasma catecholamines found after smoking a single cigarette has been related to factors other than direct drug effects (Unghvary, Csomai, Hovanyi and Farka, 1962). An experiment was designed especially to evaluate the effects of small doses of nicotine through tobacco smoking upon various psychophysiological parameters (Ague, 1971). This paper deals with the effects produced in some urinary variables supposed to give a reliable index of sympathetic stimulation.
2. rvIethod 2.1. Subjects Twenty-four male volunteers were studied. Their age ranged from 17-24 yr (mean=20.71 yr), and their weight from 53.53-90.27 kg (mean=7144 kg). 229
C. AguP
230
All were habitual smokers of at least 5 cigarettes/day. Tobacco, coffee, alcohol, etc., had been suspended for a minimum of 8 hr prior to the sessions and the liquid intake was restricted to a prescribed water load. 2.2 Design of the experiment Basically, the design of the experiment corresponded to a 4 (cigarettes) x 2 (time of day) x 2 (rates of smoking) factorial, with repeated measures on the first factor (Winer, 1962). Both the order of treatments and level of factors were randomized separately. 2.2.1. Cigarettes. The cigarettes used were one control non-nicotine (lettuceleaf) and three specially manufactured filter-tip cigarettes, containing 0.75, 1.02 and 2.11 mg of nicotine in the mainstream smoke, respectively’. From thenicotine recovered in the filter tips, total mean estimated doses of nicotine inhaled were calculated. These were: 0, 0.2912*0.0222, 0.9037&0.0609, and 1.3454kO.0750 mg, respectively (Ague, 1973a). None could be visually identified by the subjects. They were smoked up to 2 mm from the special holder, with a maximum puff and inhalation, at each corresponding rate. Each subject smoked the four experimental cigarettes. 2.2.2. Time of day. Each subject was studied at the same time throughout the experiment. The times were 8.00 and 10.30 a.m. (‘morning’) and 2.00 and 4.30 p.m. (‘afternoon’). The subjects were allocated at random to each time of day. Each sub-group was thus constituted by six subjects. ‘t test’ comparisons between the various parameters obtained at each two times failed to reveal any significant differences. Thus, the corresponding 12 subjects were pooled in the morning condition and the other 12 in the afternoon. 2.2.3. Rates of smoking. Two rates were arbitrarily chosen. These were automatically signalled to the subjects and were: (1) a ‘fast’ rate (1 puff e/30 set), and (2) a ‘slow’ rate (1 puff e/60 set). Each puff had a maximum duration of 5 sec. The subjects were distributed at random to each rate of smoking. 2.3. Procedure The subjects were informed of the overall purpose of the experiment during an initial interview. Throughout the sessions, each lasting a minimum of 90 and a maximum of 120 min, the subjects sat on a comfortable chair, whilst attached to the various monitoring devices for the psychophysiological variables (Ague, 1974). Minimal verbal interaction was maintained and the ‘These analyses, as well as the calculations of the estimated doses, were performed by Mr. C. A. Grant, Harrogate, U.K.
Chemistry
Department,
Tobacco
Research
Council
Laboratories,
Urinary catechoiamines,
flow rare and tobacco smoking
231
various instructions were read via an intercom from the adjacent recording room. The experimental room was air-conditioned and maintained at a temperature of about 24°C. During each session, the following sequence of events took place: (1) -9 hr: last cigarette, tea, coffee, etc; (2) -2 hr: voiding of bladder, 200 cc of tap water; (3) - 1 hr : arrival at the laboratory, voiding of bladder: sample 1 (occasion l)*, 200 cc of tap water, basal recording of psychophysiological variables; (4) 0 hr: voiding of bladder: sample 2 (occasion 2)3, 200 cc of tap water, SMOKING (approximately 10 min), post-smoking recording of psychophysiological variables; (5) + 1 hr: voiding of bladder: sample 3 (occasion 3), 200 cc of tap water, departure from the laboratory, habitual activities4; and (6) +2 hr: return to the laboratory, voiding of bladder: sample 4 (occasion 4)5. 2.4. Determination of urinary catecholamines (except dopamine) The differential estimation of adrenaline and noradrenaline was carried out fluorimetrically using the trihydroxyindole reaction. A semi-automated method combining the advantages of earlier procedures (Merrils, 1963 ; McCullough, 1968) was developed especially for this study (De Souza, 1973), this being necessary because of the widely varying ‘blank’ fluorescence encountered when working with urine rather than with plasma, as in the original methods. Recovery with the present technique ranged from 95-100 % for adrenaline and 97-100x for noradrenaline. The reproducibility was satisfactory. 2.5. Collection, storage and treatment of samples Sample containers were prepared as follows: polypropylene screw cap jars (Henley’s Medical Supplies) and 2 oz medical flat bottles (after routine washing) were soaked overnight in 0.4N HCl, rinsed with water and then with distilled water. The bottles were dried in an oven at 110 “C. The jars were dried in a dust-free atmosphere at room temperature. The volume of urine was obtained by weighing the jars empty and then containing the samples and calculating the difference. Each sample was mixed well before aliquots were taken for: (1) creatinine and osmolality?Self-rating questionnaires of mood were obtained immediately before each urine sample, except for occasion 2 (Ague, 1973b). “This sample was obtained in the sitting position, as the subject had to remain connected to the recording apparatus. Five subjects who failed to urinate in this position were discarded from the experiment in a preliminary session. *During this period, the abstinence from psychoactive substances was maintained. ‘A return to baseline measurements was hypothesized in this period, thus partialling out the possible effects of the experimental sessions.
232
C. AguP
these were stored at 4°C in numbered 10 ml tall plastic contamers; and (2) catecholamines-20 ml or more of measured urine were put into numbered acid-washed bottles, to which was added 1 ml of 0.5 HCl per 10 ml of urine to bring the pH to 2-3, and then stored in the deep freeze. The volume of urine was again ascertained by weighing. For the extraction of catecholamines, all 16 samples from each subject were extracted and analysed ‘blind’ in one single batch. Urine osmolality was measured using an advanced osmometer. All specimens were assayed twice. The average of both readings constituted the final value. Parametric statistics were used throughout. Separate analyses of variance were calculated clerically for data corresponding to each variable. Thus, main effects were obtained for cigarettes, rate of smoking, time of day and for occasions, and their corresponding interactions. Scheffe’s tests for multiple comparisons between treatment means at each different occasions were also computed (Edwards, 1968). All tests of significance were conservative (with 1, 20 df), with a=0.05. 3. Results 3.1. AdrenaIine The mean values obtained ranged from 7.46-6.94 ng/min, due to the lettuceleaf and highest nicotine cigarettes, respectively. As expected, the main effects of these failed to reach significance. Neither the time of day nor rate of smoking produced significant differences in excretion. These were, however, significantly increased from pre-smoking values (about 6.5 ng/min) during occasions 1 and 2, to 9.2 ng/min one hour after smoking (occasion 3) and returning to base levels at occasion 4. This is presented in fig. 1. These effects yielded a significant F ratio (61.5964; p
No significant
3.3. Flow rate Progressive increases in mean flow rate were seen until I hr after smoking (occasion 3), returning thereafter to base levels. The flow rate ranged from 1.6-4.7 ml/min. These effects were highly significant (F=27.2833; p
Urinary
catecholamines,
flow rate and tobacco
smoking
233
ng/min
10
Occasions:
ADRENALINE
Fig. 1. Mean values of adrenaline
4
3
2
1
excretion for each occasion.
ml/min
6
:: 5,
3
1
z
2
--
Cigarettes: OCCASIONS:
T
1234
1
P
12
1234
Fig. 2. Mean flow rates for the cigarette
3 4 3
2 x
1234 4
occasion interaction.
ing cigarettes ranged from 1.3-3.2 ml/min. These differential effects yielded a significant F ratio (5.7528; p
234
C. Aguh
r=0.049, 0.034, -0.008 and -0.389, none of them significant. although a negative trend is clearly observable. Similarly, regression analyses on these data showed no significant effects for cigarettes 1, 2 and 3. while reaching almost significance with the highest nicotine cigarette (F=3.9313; ~(0.10). 4. Discussion The results presented above indicate that nicotine, at least under the experimental conditions of this study and at the range of doses inhaled, does not appear to affect the urinary output of catecholamines, more than the smoking of nicotine-free cigarettes. In this respect, data are not available in the literature, except for those reported by Kershbaum et al. (1967) who found similar levels of ‘total catecholamines’ (expressed as noradrenaline equivalents) in a non-smoking control and after smoking lettuce-leaf cigarettes. However, the method used in their determinations (Crout, 1961) is somewhat inaccurate and does not permit comparisons with the present data6. it is also significant that the rate of smoking, which produced marked differences in the amount of nicotine inhaled (Ague, 1973a) and in some cardiovascular variables related to sympathetic activity (Ague, 1974). failed to reveal significant effects. The absence in this study of a non-smoking condition may suggest the interpretation of these results as due perhaps to the smoking behaviour per se, creating a determined expectation in the subjects, or mediated through tobacco components other than nicotine. In this respect, a pilot study, although limited in its possible generalization due to the small number of subjects, performed utilizing similar methodology but without the measurement of psychophysiological variables, revealed significant increases in the output of adrenaline, as compared to a non-smoking control, whilst no significant differences could be demonstrated after smoking lettuce-leaf cigarettes (Ague, 1971). In this sense and under ‘basal’ conditions, it may be accepted that some of the hormonal actions of nicotine are dependent on maximal effective doses situated in the lower range (Kershbaum et al., 1967) and not falling below 1.5 mg administered in about IO min. Despite the antidiuretic effect of the highest nicotine cigarette and its tenuous relationship to the excretion of adrenaline, the concomitant occurence of high adrenaline levels, high flow rates and diluted urine at the end of the experimental sessions, gives some support to the suggestion that the process of ADH inhibition and catecholamine release have a functional relationship (Hathaway, Brehm, Clapp and Bogdonoff, 1969). If our findings are viewed as evidence for increased plasma catecholamines and hence of greater 6Expressed as ‘noradrenaline equivalents’, no differentiation can be made between the recovered amines. Also, in this method, the eluates read only at pH 6.5, and although both amines are oxidized to aminochrome at this pH, the fluoresaence/unit of adrenaline is only 53 %/unit of noradrenaline. No correction in the results is provided, and hence its results are rather inaccurate, especiaify when changes in adrenaline are considered.
Urinary
carecholamines,
j7ow rate and tobacco
smoking
235
autonomic activity, they could also reflect an overall reaction to the conditions of the experimental procedure, inasmuch as significant changes in self-rated mood were obtained at the end of the sessions, whilst somewhat lower scores in ‘anxiety’ and ‘aggression’ appeared related to the highest nicotine cigarette (Ague, 1973b), to the imposed water load, or both (Hathaway et al., 1969). The fact that responses purported to measure a state of subjective ‘activation’ were also found significantly dose-related, stresses once more the difficulty of considering the excretion of catecholamines and its associated changes univocally, suggesting that it may be plausible to envisage it, as part of an ‘adaptive’ mechanism to unspecific ‘stress’, which could vary considerably in terms of autonomic responses (Bridges, Jones and Leak, 1968; Levi, 1969). In this respect, the absence of significant differences in excretion attributable to times of day, especially when overall higher forearm blood flow and skin conductance levels (Ague, 1974), as well as higher self-rated ‘anxiety’ (Ague, 1973b) were observed during the afternoon, seem to support this suggestion, insofar as this parameter, at least under the conditions of this experiment, appeared as an indicator of environmental high ‘noiselevel’, capable of obscuring possible drug effects (Jonsson, 1969). In conclusion, the excretion of urinary catecholamines has been shown to be differentially unaffected by smoking cigarettes containing small doses of nicotine, or nicotine-free, showing itself as an extremely sensitive index of reactivity to environmental stimuli. It is not possible to ascertain from our data whether these changes were independent or secondary to mood influences. In either case, some caution is deemed necessary if they are to be interpreted in the absence of other psychophysiological parameters. Acknowledgements This work was carried out at the Department of Psychology, Institute of Psychiatry, London and was supported by the Tobacco Research Council. The author is greatly indebted to Dr. M. Buckell, Consultant Chemical Pathologist of the Maudsley Hospital, whose full co-operation and advice made it possible to overcome the difficulties encountered. The author also wishes to thank the staff, particularly Mr. V. F. A. DeSouza, who conducted all the biochemical aspects of this‘work. References Ague, C. (1971). Nicotine and smoking: an evaluation of its psychological, psychopharmacological and biochemical effects in habitual smokers. Unpublished Ph.D. thesis, University of London. Ague, C. (1973a). Smoking patterns, nicotine intake at different times of day and changes in two cardiovascular variables while smoking cigarettes. Psychopharmacologia (Berlin), 30, 135-144. Ague, C. (1973b). Nicotine and smoking: effects upon subjective changes in mood. Psychopharmacologia
(Berlin),
30,323-328.
Ague, C. (1974). Responses of cardiovascular variables, estimation to small doses of nicotine. To be published.
skin conductance
and time
236
C. Ague’
Armitage, A. K. and Milton, A. S. (1965). The release of adrenaline by nicotine from the adrenal medulla. In: v.Euler, U.S. (Ed.) Tobacco Alkaloids and Related Compounds. Pergamon Press: Oxford, 205-212. Bridges, P. K., Jones. M. T. and Leak. D. (1968). A comparative study of four physiological concomitants of anxiety. Archives of General Ps~~chiatr,v. 19, 141-145. Crout, J. R. (I 961). Catechol amines in urine. In: Standard Methods of Clinical Chemistry. 3. Academic Press: New York, 62-80. DeSouza, V. F. A. (1973). Private communication. Edwards, A. L. (1968). Experimental Design in Psychological Research. Holt, Rinehart and Winston: New York. Frankenhaeuser, M., Myrsten, A. L., Waszak, M., Neri. A. and Post. B. (1968). Dosage and time effects of cigarette smoking. Psychopharnmcologia (Berlin). 13, 3 I l-3 19. Frankenhaeuser, M., Myrsten. A. L. and Post, B. (1970). Psychophysiological reactions to cigarette smoking. Scandinavian Journal of Psychology, 11, 237-245. Hathaway, P. W., Brehm, M. L., Clapp, J. R. and Bogdonoff, M. D. (1969). Urine flow, catecholamines, and blood pressure. The variability of response of normal subjects in a relaxed laboratory setting. Psychosotnafic Medicine, XXXI, 20-30. Jonsson, C. 0. (1969). Behavioural studies of diethylpropion in man. In: Ejqwist, F. and Tallie, M. (Eds.) Abuse of Central Stimulants. Stockholm, 71-80. Kershbaum, A., Bellet, S., Hirabayashi, M. and Feinberg, L. (1967). Regular, filter-tip, and modified cigarettes : nicotine excretion. Journal of the American Medical Association, 201, 135-l 36. Levi, L. (1969). Sympatho-adrenomedullary activity. diuresis, and emotional reactions during visual sexual stimulation in human females and males. Psychosomatic Medicine, XxX1,
251-268.
McCullough, H. (1968). Semi-automated method for the differential determination of plasma catecholamines. Journal of Clinical Parhology, 21, 7.59-763. Merrils, R. S. (1963). A semiautomatic method for the determination of catecholamines. Analytical
Biochemistry,
6, 272-282.
Redher, K. and Roth, G. M. (1959). Effect of smoking on the fasting blood sugar and pressor amines. Circulation, 20, 224-228. Silvette, H., Larson, P. S. and Haag, H. B. (1961). Action of nicotine and tobacco smoking on the adrenal medulla. Archives of Infernal Medicine. 107, 915-931. Unghvary, L., Csomai, I., Hovanyi. M. and Farkas, F. (1962). Wirkung der Konzentration der Gesamtkatecholaminstoffe des Vollblutes and deren Veraanderungen under der Wirkung des Rauchens. Cardiologia (Base/), 41, 316-323. Westfall, T. C. and Watts, D. T. (1964). Catecholamine excretion in smokers and nonsmokers. Journal of Applied Physiology. 19, 40-42. Winer, B. J. (1962). SraristicalPrinciplesin E~~perimenraiDesigu. McGraw-Hill: New York.
URINARY CATECHOLAMINES, TOBACCO
FLOW RATE AND
SMOKING
C. AGU6 Medical Research, Sandoz Ltd., Basle, Switzerland Accepted for publication
17 August 1973
Twenty-four habitual smokers smoked one nicotine-free and three tobacco cigarettes with a known content of nicotine in the course of four successive sessions. Urine samples were collected hourly and under a predetermined water load. A specially devised fluorimetric method was used to determine adrenaline and nor-adrenaline. Flow rate and osmolality were also calculated. No changes were observed in noradrenaline excretion. Adrenaline excretion rose si~ifi~ntly after smoking, although no major differences were found between the treatments, returning to baseline levels one hour after the experimental sessions. Similarly, increased flow rates and low osmolality were observed. The implication of these findings are discussed. It is suggested that the excretion of adrenaline may indicate an ‘adaptive’ mechanism to ‘stress’.
Doubts as to whether habitual tobacco smoking is sufficient to produce an increased release of catecholamines from the adrenal medulla have been formulated by Rehder and Roth (1959), Silvette, Larson and Haag (1961), and Armitage and Milton (1965). Conclusive evidence for rises in the urinary output of amines, especially in the adrenaline fraction, has only been found after the administration of relatively large doses of the drug obtained through heavy smoking (Westfall and Watts, 1964; Kershbaum, Bellet, Hirabayashi and Feinberg, 1967) or after smoking at least two cigarettes (Frankenhaeuser, Myrsten, Waszak, Neri and Post, 1968; Frankenhaeuser, Myrsten and Post, 1970). The level of plasma catecholamines found after smoking a single cigarette has been related to factors other than direct drug effects (Unghvary, Csomai, Hovanyi and Farka, 1962). An experiment was designed especially to evaluate the effects of small doses of nicotine through tobacco smoking upon various psychophysiological parameters (Ague, 1971). This paper deals with the effects produced in some urinary variables supposed to give a reliable index of sympathetic stimulation.
2. rvIethod 2.1. Subjects Twenty-four male volunteers were studied. Their age ranged from 17-24 yr (mean=20.71 yr), and their weight from 53.53-90.27 kg (mean=7144 kg). 229
C. AguP
230
All were habitual smokers of at least 5 cigarettes/day. Tobacco, coffee, alcohol, etc., had been suspended for a minimum of 8 hr prior to the sessions and the liquid intake was restricted to a prescribed water load. 2.2 Design of the experiment Basically, the design of the experiment corresponded to a 4 (cigarettes) x 2 (time of day) x 2 (rates of smoking) factorial, with repeated measures on the first factor (Winer, 1962). Both the order of treatments and level of factors were randomized separately. 2.2.1. Cigarettes. The cigarettes used were one control non-nicotine (lettuceleaf) and three specially manufactured filter-tip cigarettes, containing 0.75, 1.02 and 2.11 mg of nicotine in the mainstream smoke, respectively’. From thenicotine recovered in the filter tips, total mean estimated doses of nicotine inhaled were calculated. These were: 0, 0.2912*0.0222, 0.9037&0.0609, and 1.3454kO.0750 mg, respectively (Ague, 1973a). None could be visually identified by the subjects. They were smoked up to 2 mm from the special holder, with a maximum puff and inhalation, at each corresponding rate. Each subject smoked the four experimental cigarettes. 2.2.2. Time of day. Each subject was studied at the same time throughout the experiment. The times were 8.00 and 10.30 a.m. (‘morning’) and 2.00 and 4.30 p.m. (‘afternoon’). The subjects were allocated at random to each time of day. Each sub-group was thus constituted by six subjects. ‘t test’ comparisons between the various parameters obtained at each two times failed to reveal any significant differences. Thus, the corresponding 12 subjects were pooled in the morning condition and the other 12 in the afternoon. 2.2.3. Rates of smoking. Two rates were arbitrarily chosen. These were automatically signalled to the subjects and were: (1) a ‘fast’ rate (1 puff e/30 set), and (2) a ‘slow’ rate (1 puff e/60 set). Each puff had a maximum duration of 5 sec. The subjects were distributed at random to each rate of smoking. 2.3. Procedure The subjects were informed of the overall purpose of the experiment during an initial interview. Throughout the sessions, each lasting a minimum of 90 and a maximum of 120 min, the subjects sat on a comfortable chair, whilst attached to the various monitoring devices for the psychophysiological variables (Ague, 1974). Minimal verbal interaction was maintained and the ‘These analyses, as well as the calculations of the estimated doses, were performed by Mr. C. A. Grant, Harrogate, U.K.
Chemistry
Department,
Tobacco
Research
Council
Laboratories,
Urinary catechoiamines,
flow rare and tobacco smoking
231
various instructions were read via an intercom from the adjacent recording room. The experimental room was air-conditioned and maintained at a temperature of about 24°C. During each session, the following sequence of events took place: (1) -9 hr: last cigarette, tea, coffee, etc; (2) -2 hr: voiding of bladder, 200 cc of tap water; (3) - 1 hr : arrival at the laboratory, voiding of bladder: sample 1 (occasion l)*, 200 cc of tap water, basal recording of psychophysiological variables; (4) 0 hr: voiding of bladder: sample 2 (occasion 2)3, 200 cc of tap water, SMOKING (approximately 10 min), post-smoking recording of psychophysiological variables; (5) + 1 hr: voiding of bladder: sample 3 (occasion 3), 200 cc of tap water, departure from the laboratory, habitual activities4; and (6) +2 hr: return to the laboratory, voiding of bladder: sample 4 (occasion 4)5. 2.4. Determination of urinary catecholamines (except dopamine) The differential estimation of adrenaline and noradrenaline was carried out fluorimetrically using the trihydroxyindole reaction. A semi-automated method combining the advantages of earlier procedures (Merrils, 1963 ; McCullough, 1968) was developed especially for this study (De Souza, 1973), this being necessary because of the widely varying ‘blank’ fluorescence encountered when working with urine rather than with plasma, as in the original methods. Recovery with the present technique ranged from 95-100 % for adrenaline and 97-100x for noradrenaline. The reproducibility was satisfactory. 2.5. Collection, storage and treatment of samples Sample containers were prepared as follows: polypropylene screw cap jars (Henley’s Medical Supplies) and 2 oz medical flat bottles (after routine washing) were soaked overnight in 0.4N HCl, rinsed with water and then with distilled water. The bottles were dried in an oven at 110 “C. The jars were dried in a dust-free atmosphere at room temperature. The volume of urine was obtained by weighing the jars empty and then containing the samples and calculating the difference. Each sample was mixed well before aliquots were taken for: (1) creatinine and osmolality?Self-rating questionnaires of mood were obtained immediately before each urine sample, except for occasion 2 (Ague, 1973b). “This sample was obtained in the sitting position, as the subject had to remain connected to the recording apparatus. Five subjects who failed to urinate in this position were discarded from the experiment in a preliminary session. *During this period, the abstinence from psychoactive substances was maintained. ‘A return to baseline measurements was hypothesized in this period, thus partialling out the possible effects of the experimental sessions.
232
C. AguP
these were stored at 4°C in numbered 10 ml tall plastic contamers; and (2) catecholamines-20 ml or more of measured urine were put into numbered acid-washed bottles, to which was added 1 ml of 0.5 HCl per 10 ml of urine to bring the pH to 2-3, and then stored in the deep freeze. The volume of urine was again ascertained by weighing. For the extraction of catecholamines, all 16 samples from each subject were extracted and analysed ‘blind’ in one single batch. Urine osmolality was measured using an advanced osmometer. All specimens were assayed twice. The average of both readings constituted the final value. Parametric statistics were used throughout. Separate analyses of variance were calculated clerically for data corresponding to each variable. Thus, main effects were obtained for cigarettes, rate of smoking, time of day and for occasions, and their corresponding interactions. Scheffe’s tests for multiple comparisons between treatment means at each different occasions were also computed (Edwards, 1968). All tests of significance were conservative (with 1, 20 df), with a=0.05. 3. Results 3.1. AdrenaIine The mean values obtained ranged from 7.46-6.94 ng/min, due to the lettuceleaf and highest nicotine cigarettes, respectively. As expected, the main effects of these failed to reach significance. Neither the time of day nor rate of smoking produced significant differences in excretion. These were, however, significantly increased from pre-smoking values (about 6.5 ng/min) during occasions 1 and 2, to 9.2 ng/min one hour after smoking (occasion 3) and returning to base levels at occasion 4. This is presented in fig. 1. These effects yielded a significant F ratio (61.5964; p
No significant
3.3. Flow rate Progressive increases in mean flow rate were seen until I hr after smoking (occasion 3), returning thereafter to base levels. The flow rate ranged from 1.6-4.7 ml/min. These effects were highly significant (F=27.2833; p
Urinary
catecholamines,
flow rate and tobacco
smoking
233
ng/min
10
Occasions:
ADRENALINE
Fig. 1. Mean values of adrenaline
4
3
2
1
excretion for each occasion.
ml/min
6
:: 5,
3
1
z
2
--
Cigarettes: OCCASIONS:
T
1234
1
P
12
1234
Fig. 2. Mean flow rates for the cigarette
3 4 3
2 x
1234 4
occasion interaction.
ing cigarettes ranged from 1.3-3.2 ml/min. These differential effects yielded a significant F ratio (5.7528; p
234
C. Aguh
r=0.049, 0.034, -0.008 and -0.389, none of them significant. although a negative trend is clearly observable. Similarly, regression analyses on these data showed no significant effects for cigarettes 1, 2 and 3. while reaching almost significance with the highest nicotine cigarette (F=3.9313; ~(0.10). 4. Discussion The results presented above indicate that nicotine, at least under the experimental conditions of this study and at the range of doses inhaled, does not appear to affect the urinary output of catecholamines, more than the smoking of nicotine-free cigarettes. In this respect, data are not available in the literature, except for those reported by Kershbaum et al. (1967) who found similar levels of ‘total catecholamines’ (expressed as noradrenaline equivalents) in a non-smoking control and after smoking lettuce-leaf cigarettes. However, the method used in their determinations (Crout, 1961) is somewhat inaccurate and does not permit comparisons with the present data6. it is also significant that the rate of smoking, which produced marked differences in the amount of nicotine inhaled (Ague, 1973a) and in some cardiovascular variables related to sympathetic activity (Ague, 1974). failed to reveal significant effects. The absence in this study of a non-smoking condition may suggest the interpretation of these results as due perhaps to the smoking behaviour per se, creating a determined expectation in the subjects, or mediated through tobacco components other than nicotine. In this respect, a pilot study, although limited in its possible generalization due to the small number of subjects, performed utilizing similar methodology but without the measurement of psychophysiological variables, revealed significant increases in the output of adrenaline, as compared to a non-smoking control, whilst no significant differences could be demonstrated after smoking lettuce-leaf cigarettes (Ague, 1971). In this sense and under ‘basal’ conditions, it may be accepted that some of the hormonal actions of nicotine are dependent on maximal effective doses situated in the lower range (Kershbaum et al., 1967) and not falling below 1.5 mg administered in about IO min. Despite the antidiuretic effect of the highest nicotine cigarette and its tenuous relationship to the excretion of adrenaline, the concomitant occurence of high adrenaline levels, high flow rates and diluted urine at the end of the experimental sessions, gives some support to the suggestion that the process of ADH inhibition and catecholamine release have a functional relationship (Hathaway, Brehm, Clapp and Bogdonoff, 1969). If our findings are viewed as evidence for increased plasma catecholamines and hence of greater 6Expressed as ‘noradrenaline equivalents’, no differentiation can be made between the recovered amines. Also, in this method, the eluates read only at pH 6.5, and although both amines are oxidized to aminochrome at this pH, the fluoresaence/unit of adrenaline is only 53 %/unit of noradrenaline. No correction in the results is provided, and hence its results are rather inaccurate, especiaify when changes in adrenaline are considered.
Urinary
carecholamines,
j7ow rate and tobacco
smoking
235
autonomic activity, they could also reflect an overall reaction to the conditions of the experimental procedure, inasmuch as significant changes in self-rated mood were obtained at the end of the sessions, whilst somewhat lower scores in ‘anxiety’ and ‘aggression’ appeared related to the highest nicotine cigarette (Ague, 1973b), to the imposed water load, or both (Hathaway et al., 1969). The fact that responses purported to measure a state of subjective ‘activation’ were also found significantly dose-related, stresses once more the difficulty of considering the excretion of catecholamines and its associated changes univocally, suggesting that it may be plausible to envisage it, as part of an ‘adaptive’ mechanism to unspecific ‘stress’, which could vary considerably in terms of autonomic responses (Bridges, Jones and Leak, 1968; Levi, 1969). In this respect, the absence of significant differences in excretion attributable to times of day, especially when overall higher forearm blood flow and skin conductance levels (Ague, 1974), as well as higher self-rated ‘anxiety’ (Ague, 1973b) were observed during the afternoon, seem to support this suggestion, insofar as this parameter, at least under the conditions of this experiment, appeared as an indicator of environmental high ‘noiselevel’, capable of obscuring possible drug effects (Jonsson, 1969). In conclusion, the excretion of urinary catecholamines has been shown to be differentially unaffected by smoking cigarettes containing small doses of nicotine, or nicotine-free, showing itself as an extremely sensitive index of reactivity to environmental stimuli. It is not possible to ascertain from our data whether these changes were independent or secondary to mood influences. In either case, some caution is deemed necessary if they are to be interpreted in the absence of other psychophysiological parameters. Acknowledgements This work was carried out at the Department of Psychology, Institute of Psychiatry, London and was supported by the Tobacco Research Council. The author is greatly indebted to Dr. M. Buckell, Consultant Chemical Pathologist of the Maudsley Hospital, whose full co-operation and advice made it possible to overcome the difficulties encountered. The author also wishes to thank the staff, particularly Mr. V. F. A. DeSouza, who conducted all the biochemical aspects of this‘work. References Ague, C. (1971). Nicotine and smoking: an evaluation of its psychological, psychopharmacological and biochemical effects in habitual smokers. Unpublished Ph.D. thesis, University of London. Ague, C. (1973a). Smoking patterns, nicotine intake at different times of day and changes in two cardiovascular variables while smoking cigarettes. Psychopharmacologia (Berlin), 30, 135-144. Ague, C. (1973b). Nicotine and smoking: effects upon subjective changes in mood. Psychopharmacologia
(Berlin),
30,323-328.
Ague, C. (1974). Responses of cardiovascular variables, estimation to small doses of nicotine. To be published.
skin conductance
and time
236
C. Ague’
Armitage, A. K. and Milton, A. S. (1965). The release of adrenaline by nicotine from the adrenal medulla. In: v.Euler, U.S. (Ed.) Tobacco Alkaloids and Related Compounds. Pergamon Press: Oxford, 205-212. Bridges, P. K., Jones. M. T. and Leak. D. (1968). A comparative study of four physiological concomitants of anxiety. Archives of General Ps~~chiatr,v. 19, 141-145. Crout, J. R. (I 961). Catechol amines in urine. In: Standard Methods of Clinical Chemistry. 3. Academic Press: New York, 62-80. DeSouza, V. F. A. (1973). Private communication. Edwards, A. L. (1968). Experimental Design in Psychological Research. Holt, Rinehart and Winston: New York. Frankenhaeuser, M., Myrsten, A. L., Waszak, M., Neri. A. and Post. B. (1968). Dosage and time effects of cigarette smoking. Psychopharnmcologia (Berlin). 13, 3 I l-3 19. Frankenhaeuser, M., Myrsten. A. L. and Post, B. (1970). Psychophysiological reactions to cigarette smoking. Scandinavian Journal of Psychology, 11, 237-245. Hathaway, P. W., Brehm, M. L., Clapp, J. R. and Bogdonoff, M. D. (1969). Urine flow, catecholamines, and blood pressure. The variability of response of normal subjects in a relaxed laboratory setting. Psychosotnafic Medicine, XXXI, 20-30. Jonsson, C. 0. (1969). Behavioural studies of diethylpropion in man. In: Ejqwist, F. and Tallie, M. (Eds.) Abuse of Central Stimulants. Stockholm, 71-80. Kershbaum, A., Bellet, S., Hirabayashi, M. and Feinberg, L. (1967). Regular, filter-tip, and modified cigarettes : nicotine excretion. Journal of the American Medical Association, 201, 135-l 36. Levi, L. (1969). Sympatho-adrenomedullary activity. diuresis, and emotional reactions during visual sexual stimulation in human females and males. Psychosomatic Medicine, XxX1,
251-268.
McCullough, H. (1968). Semi-automated method for the differential determination of plasma catecholamines. Journal of Clinical Parhology, 21, 7.59-763. Merrils, R. S. (1963). A semiautomatic method for the determination of catecholamines. Analytical
Biochemistry,
6, 272-282.
Redher, K. and Roth, G. M. (1959). Effect of smoking on the fasting blood sugar and pressor amines. Circulation, 20, 224-228. Silvette, H., Larson, P. S. and Haag, H. B. (1961). Action of nicotine and tobacco smoking on the adrenal medulla. Archives of Infernal Medicine. 107, 915-931. Unghvary, L., Csomai, I., Hovanyi. M. and Farkas, F. (1962). Wirkung der Konzentration der Gesamtkatecholaminstoffe des Vollblutes and deren Veraanderungen under der Wirkung des Rauchens. Cardiologia (Base/), 41, 316-323. Westfall, T. C. and Watts, D. T. (1964). Catecholamine excretion in smokers and nonsmokers. Journal of Applied Physiology. 19, 40-42. Winer, B. J. (1962). SraristicalPrinciplesin E~~perimenraiDesigu. McGraw-Hill: New York.