Circadian variations of plasma catecholamine, cortisol and immunoreactive insulin concentrations in supine subjects

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Clinica Chimica Acta, 55 (1974) 389-397 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CCA 6620

CIRCADIAN VARIATIONS OF PLASMA CATECHOLAMINE, CORTISOL AND IMMUNOREACTIVE INSULIN CONCENTRATIONS IN SUPINE SUBJECTS

M.B. TURTON AND T. DEEGAN Liverpool Regional Cardiac Centre, Sefton General Hospital, Liverpool L15 2HE (U.K.) (Received April 19, 1974)

Summary

Circulating levels of adrenalin, noradrenalin, cortisol and immunoreactive insulin were assayed in a group of supine, convalescent patients, at 2-h intervals during a period of 24 h. Circadian variation was demonstrated in both catecholamines and in cortisol. In all three factors, levels were higher during the day than during the night, but each showed a different periodicity. The amplitudes of the individual responses showed a positive correlation with the individual 24-h mean values in both catecholamines, but not in cortisol. The concentrations of adrenalin and noradrenalin recorded were consistent with the presence of some stress in the patients. Immunoreactive insulin responses to food stimulus were greater in the morning than in the evening, but the latter response was more prolonged. The implications of these results in continuous assessment of ill patients is discussed.

Introduction

Circadian variations have been reported in several metabolic parameters, in particular the urinary excretions of 17-ketosteroids [l] and catecholamines [2] and the plasma concentrations of cortisol [3] and immunoreactive insulin (IRI), the latter in both fasting [4] and non-fasting subjects [5]. The degree and the nature of these variations are dependent upon secondary effects, such as working habits [ 61, physical activity [ 71 and stress [ 81. The presence of circadian variations, modified or otherwise, should be considered when such parameters are studied continuously over periods of 24 h or longer. The advent of intensive therapy units has increased interest in the roles played in clinical sequelae by some of the factors subject to circadian variation, for example, in those of cortisol and the catecholamines in arrhythmic states following acute myocardial infarction. In attempting such associations, it is necessary to define the patterns of change in non-affected individuals

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in circumstances which, as far as possible, simulate those applicable to affected individuals. This paper describes a 24-h study of plasma concentrations of adrenalin, noradrenalin, cortisol and immunoreactive insulin in a group of supine, convalescent patients, in order to provide such a basis for comparison. Subjects and Methods Subjects Twelve male patients (mean age: 51 t 9 years), charge from hospital following cardiac catheterization nary artery disease, agreed to participate in the study. monal, renal or gastro-intestinal disorders and none foodstuffs known to influence the metabolism or the eters studied. The catheterization findings showed that were minimal.

who were awaiting disfor assessment of coroNone had hepatic, horwere receiving drugs or estimation of the paramcirculatory disturbances

Procedure The patients were confined to bed throughout the period of the study and smoking was forbidden. At 0900 h a venous catheter (1 mm internal diameter; Abbott Laboratories, N. Chicago, Ill., U.S.A.) was introduced into the antecubital vein to a distance of approximately 15 cm and was kept patent with a slow infusion of 5% dextrose (40 ml per h). (A separate investigation has established that infusion at this rate has no detectable effect upon plasma glucose concentration - Turton, unpublished observation.) Food intake was limited to three meals; a mid-day meal at 1200 h, an evening meal at 1730 h and breakfast at 0800 h. Drinks were provided at other times on request. The average calorific content of the meals, together with the contributions of protein, carbohydrate and fat, are listed in Table I. Records of fluid balance and food intake were kept and an assessment was made of the demeanour of each patient. In general, all the patients were relaxed throughout the study; the daytime was spent in activities such as reading, conversation and listening to the radio, and, with one exception, all slept well, usually from 2200 h until 0600 h. Blood specimens were withdrawn at 2-h intervals from 1100 h until 0900 h the following day. Extreme care was taken to avoid disturbing the patients while sleeping. The specimens were transferred to cooled bottles containing 25 mg EDTA as anticoagulant. Plasma was separated immediately by centrifugation at 1700 X g for 15 min at 5”. TABLE

I

CALORIFIC

CONTENT

OF

Total

MEALS

calories

Protein

(%)

Carbohydrate

(%)

Fat

Mid-day

meal

600

12

46

Evening

meal

650

14

29

57

550

12

46

42

13

40

41

Breakfast Total

1800

42

(W)

391

Analyses Duplicate aliquots (0.1 ml) of plasma were taken for IRI assay and a single aliquot (1.0 ml) was taken for cortisol estimation. After measurement of the volume, the remaining plasma was deproteinized with 4 M perchloric acid and the concentrations of adrenalin and noradrenalin were determined in the protein-free supernatant. Details of the methods used for the separate estimations and the associated accuracies have been reported previously [ 91. Statistics Results are expressed as mean ? standard deviation; data were analysed by the appropriate Student’s t tests or by chi-squared analysis, with a 5% level of significance. Results The mean values of cortisol and IRI at each the overall means of the change in mean levels are

the plasma concentrations of adrenalin, noradrenalin, sampling time are presented in Table II, together with four parameters for the 24-h period. The patterns of depicted in Fig. 1.

Ca techolamines The mean concentrations of both amines were higher during the day than during the night. Consequently a greater number of values in excess of the 24-h mean value for adrenalin were recorded between 1100 h and 2100 h than between 2300 h and 0500 h (x2 = 14.00; p < 0.001). A similar relationship also applied to the noradrenalin values (x2 = 26.52; p < 0.001). The patterns of change, although similar for both amines, differed with respect to time. The diurnal increase in adrenalin values commenced immediately upon waking, whereas that in noradrenalin values was delayed by 4 h. The difference persisted throughout the day and led to an earlier return to lower nocturnal values in the case of adrenalin. TABLE11 MEANLEVELSATDIFFERENTTIMESOFDAY

Time(h)

Adrenalin(wg/l)

Noradrenalin(fig/l)

Cortisol(fig/l)

Insulin(mu/l)

1100 1300 1500 1700 1900 2100 2300 0100 0300 0500 0700 0900

0.36 + 0.24 0.30 r 0.15 0.34 + 0.15 0.25? 0.12 0.25 f 0.18 0.20 k 0.12 0.15 k 0.01 0.15 f 0.10 0.16 + 0.09 0.19 + 0.10 0.26 + 0.17 0.21 + 0.08

0.66 ?z0.19 0.69 f 0.25 0.60 k 0.21 0.58 zk0.19 0.54 * 0.15 0.47 f 0.18 0.46 + 0.14 0.35 i.0.12 0.36 i 0.15 0.36 It0.19 0.36 f 0.14 0.34 r 0.14

112+ 68 105+ 56 98r59 84-r 43 79r 40 62i 51 68+ 48 87t97 85i86 119? 65 132+ 65 122? 67

34t14 79*40 46f 21 24?13 44t31 322 22 29+18 16+4 15% 4 15+5 28C19 61+ 32

24hmean

0.24

0.48

96

35

140 120

2

100

z

80 60

11

13

15

17

19

21

23

01

03

05

07

51 ‘Z b 0

09

Time

Fig. 1. Relationship

of mean levels to time of day. (A, Adrenalin;

A, noradrenalin;

l, cortisol;

;, IRI.)

Assessment of the significance of differences between mean values for the two amines at the various times supported this finding (Table III). The period of the day during which adrenalin values were significantly raised when compared with the lower nocturnal values (p> 0.05) was earlier than the corresponding period for noradrenalin. Comparison of the number of values elevated in excess of the 24-h mean values of each amine during the period of sleep and during the waking period also reflected the difference in behaviour. A greater number of raised values, although not significant at the 5% level, was associated with the adrenalin distribution in the period from 0700 h to 0900 h (x2 = 3.00; p < 0.08). In contrast, the distribution of noradrenalin values in the same period was identical to that during the period of sleep (x2 = 0.016). Normal values of 0.15 + 0.08 pg/l for adrenalin and 0.30 + 0.13 pg/l for noradrenalin have been established previously in antecubital vein specimens taken from 20 healthy staff members, who had rested in the supine position for 15 min and who were not apprehensive of venepuncture [9] . All specimens

393 TABLE

III

SIGNIFICANCE Where those

OF

difference at times

was

DIFFERENCES significant,

on horizontal 1100

1300

BETWEEN p <

value

MEAN

shown;

mean

LEVELS levels

at times

on vertical

axis

were

greater

than

axis. 1500

1700

1900

2100

2300

0100

0300

0500

1100

0.01

1300

0.01

0.02

0.02

0.05

0.01

0.02

0700

0900

Adrenalin

1500

0.001

0.01

0.01

1700

0.02

0.02

0.05

0.05

0700

0.05

0900

0.05

Noradrenalin 1100

0.01

0.05

0.05

0.01

0.001

0.001

0.001

0.001

0.001

0.05

0.01

0.001

0.001

0.01

0.001

0.001

1500

0.01

0.01

0.01

0.01

0.01

1700

0.01

0.01

0.01

0.02

0.01

1900

0.01

0.01

0.02

0.01

0.01

1300

Cortisol 0500

0.05

0700

IRI

0.05

0.05

0900

0.01

0.02

0.05

0.05

0.01

0.001

1100

0.001

0.001

0.001

0.001

0.001

0.001

0.001

0.001

0.001

0.05

0.05

1900

0.01

0.01

0.01

2100

0.05

0.02

0.02

2300

0.05

0.02

0.02

0.05

0.05

0.05

0.001

0.001

0.001

1300

0.01

1500

0.05

0.001

0.05

0.02

1700

0700 0900

0.02

0.01

0.02

0.01

0.001

0.01

Cortisol The mean values at the various times followed a periodicity similar in form to that shown by the two catecholamines, but on a different time scale (Fig. 1). Values decreased continuously from 1100 h until the nadir was reached in the evening at 2100 h. During the early part of the period of sleep, little change was apparent, but from 0300 h the mean concentration increased steadily to reach a peak value at 0700 h, shortly after waking. The successive changes in individual values and the individual periodicities were less uniform than in the catecholamines and resulted in the large standard deviations associated with the mean values. In consequence, assessment of the significance of differences between these entities at the various times was less informative and merely emphasised the wide disparities between the mean values at the times of highest and lowest concentrations (Table III). In general terms, the 24-h period could be divided into two periods, each of 12 h, in one of which mean levels were lower than the overall 24-h mean value (1700 h-0300 h), whereas in the other (0500 h-1500 h) mean levels were of the same order or greater. In spite of the nature of the individual

394

ADRENALIN

NORADRENALIN

(k;-:_:

0

0.1

0.2

0.3

0

Mean Fig.

2.

Correlation

0.1 level

between

02

a3

0.4

0.5

0.6

0.7

rg/l individual

24-h

mean

levels

and

individual

ranges.

( A_ Adrenalin:

A, noradre-

nalin.)

changes, a greater number of individual values in excess of the 24-h mean value were recorded in the second period compared with the first period (x2 = 5.73; p < 0.02). Immunoreactive insulin During the day the circulating levels reflected the pattern of food intake, with two sharp increases following the mid-day meal and breakfast, and a lesser but more prolonged increase following the evening meal (Fig. 1). Assessment of the significance of differences between the mean values at the various times underlined these effects. The number of significant findings associated with the increased levels accompanying each of the meals at 1200 h and 0800 h was greater than that for the meal at 1730 h (Table III). The lowest concentrations were recorded between 0100 h and 0500 h. During this period, the concentrations at 0300 h and 0500 h were both lower than the previously obtained normal value of 20 + 8 mU/l [ 91, derived from specimens taken between 0900 h and 1100 h from 34 fasting, convalescent orthopaedic patients (in both cases, p < 0.05). Comparison of the normal value with those in the present study at 0900 h and 1100 h was not possible in view of the difference in the dietary state of the two groups. Correlations Significant positive correlations were found between the individual ranges and the individual 24-h mean levels of adrenalin (r = 0.95; p < 0.001) and noradrenalin (r = 0.69; p < 0.02) (Fig. 2). The comparable findings for cortisol were not significant at the 5% level (r = 0.44; 0.1 < p < 0.2), a finding which probably reflected the less uniform responses in this factor over the 24-h period. Similar amplitude-level relationships have been reported for other factors subject to circadian variation [ lO,ll] . Discussion Circadian variation in catecholamine metabolism was first suggested by the observation that urinary excretion was lower at night than during the day [2]. Subsequently, a relationship was proposed between sleep and low plasma catecholamine levels [ 121, which was supported when comparison of levels in blood specimens taken at 0700 h, 1500 h and 2400 h showed that the lowest

395

values were recorded at midnight [ 131. To some extent, the validity of this finding was impaired by the high levels of adrenalin and noradrenalin which were reported. The present investigation has demonstrated the presence of a circadian periodicity in both catecholamines, at levels more comparable to those previously accepted for normal subjects [7,9] . Under the daily regime of the hospital ward, the highest levels were detected during the late morning and the afternoon and the lowest during the hours of sleep. The possibility that the raised concentrations in the late morning may have resulted from a degree of stress accompanying the insertion of the catheter is unlikely; the first specimens were taken not less than 2 h after this manipulation and all the patients were relaxed and admittedly comfortable at this time. However, some measure of overall stress was apparent in the patients since their catecholamine levels at similar times of day were higher than those of a group of normal subjects, who had followed similar patterns of waking and sleeping but were physically more active. Suppression of circadian variation in catecholamine excretion has been observed in conditions of sustained physical activity and of persistent sympathoadrenal function [ 141. However, in view of reported differences between plasma concentrations and excretion levels at times of prolonged increased function, e.g. immediately after an acute myocardial infarction [15], a similar suppression of the periodicity in plasma levels cannot be implied. Indeed the present findings suggest that, at least where the increases in function are only moderate yet persistent, a circadian rhythm is still evident. Although the mean cortisol levels of the group followed a pattern which mirrored previous findings [ 3,161, the individual levels showed marked fluctuations about the general trend. In an exhaustive study, involving monitoring of plasma levels at 20 min intervals for 24 h, Weitzman et al. [16] demonstrated that the control of circulating cortisol was governed not by the rate of secretion, which was found to be constant, but by the number and the duration of the secretory periods. Fluctuations in the latter accounted for the differences in individual patterns, and the acknowledged circadian variation was apparent only when the group mean level was considered over time intervals of 1 h duration. The extent to which such individual variations are controlled by fluxes in ACTH secretion following varying degrees of stress remains to be evaluated [3,16] . A further complication is the observation [ 171 that 70% of subjects in a large group had significant day to day differences in cortisol levels in specimens taken at the same time of day. Overall, the variability of individual responses makes it more difficult to assess the significance of an elevated or depressed circulating level in an acute clinical situation. The different responses in IRI to the stimulus of food in the morning and in the evening have been noted previously in normal subjects by Malherbe et al. [18] , but whereas in that study the response to breakfast was greater than to the mid-day meal, the reverse was the case in the present study. Differential intake of carbohydrate could not have provoked this difference, since in each investigation the amount consumed at both meals was the same. Perhaps the most likely cause was the difference in the physical activity of the two groups. The normal subjects of MaIherbe et al. [18] were allowed limited activity during the day whereas the participants in the present study were confined to bed throughout.

Evidence of an endogenous periodicity in insulin secretion has been provided by the morning elevation of the insulin:glucose ratio observed in fasting normal subjects [4], and has led to the proposition that hypersecretion is secondary to simultaneous increases in antagonistic compounds, in particular cortisol [4,11]. The waxing of glucocorticoid secretion is considered to have the dual role of increasing the resistance of peripheral tissues to insulin action, and of augmenting gluconeogenesis. Catecholamines, on the other hand, are known to suppress pancreatic release of insulin [ 191, and are presumed to promote the lowering of circulating levels during the later part of the day [4]. The patterns of change shown by cortisol and the separate catecholamines in the present study suggest that during the morning these two mechanisms would be in conflict, since all have maximal circulating levels at this time. The possibility exists that the gluconeogenetic action of adrenalin may override the suppressive action and supplement the similar action of cortisol, thereby promoting a need for enhanced insulin secretion. The earlier decline of adrenalin levels in the late afternoon, coupled with the rapid fall in cortisol, would diminish gluconeogenesis and promote the suppressive action of noradrenalin, which, owing to the time lag of its cycle, is still present in relatively large amounts. An alternative explanation suggests that the differences between the insulin responses in the morning and in the evening reflect the state of the tissue stores and the rate of pancreatic synthesis [ 181. On this basis, release from replete stores is maximal in the morning and diminishes towards the evening, when the rate limiting factor is the availability of newly synthesized insulin from islet cells. A similar balance between storage and de novo synthesis may account for the time lag between the patterns of the two catecholamines. The sharp increase in plasma adrenalin level on awakening [12] may require a primary diversion of noradrenalin for replenishment of adrenalin stores before its enhanced secretion into the circulation. The opposite situation would apply as the adrenalin level subsided in the late afternoon. The significance in overall physiological balance of the relationships between the differing circadian rhythms of the various factors must await further investigation. In terms of interpretation of sequential changes in clinical conditions, the problems are many. The position with regard to cortisol levels has already been noted; the possible influence of physical activity upon insulin responses complicates this area, and in the case of the catecholamines, the correlations between the ranges of the daily changes and the mean levels make difficult any assessment of the significance of individual elevated values in relation to group mean levels. In summary, caution seems necessary in assigning importance to elevations of circulating humoral factors when the clinical state may influence either the pattern or the degree of their circadian rhythm. Acknowledgements We wish to thank Drs N. Coulshed and E.J. Epstein for permission to study their patients, and the medical and nursing staff of the Centre for their assistance on the wards.

397

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