Diurnal and episodic variations of plasma hydrocortisone concentrations in horses

DOMESTIC

ANIMAL

ENDOCRINOLOGY

Vol. 5(l):%-59,

DIURNAL AND EPISODIC VARIATIONS HYDROCORTISONE CONCENTRATIONS P.L. Toutain*,

M. Oukessou’,

A Autefage”

1988

OF PLASMA IN HORSES and M. Alvinerie*

‘INRA, Station de Pharmacologic 180 chemin de Tournefeuille 31300 Toulouse, France

l *ENVT, 23 chemin des Capelles 31076 Toulouse, Received

July

France 1, 1987

ABSTRACT Using a specific high-performance liquid chromatographic technique, plasma hydrocortisone values were measured hourly in 6 horses and every 10 minutes in 4 horses over 24 hours. Both circadian and episodic variation was observed. The mean plasma hydrocortisone concentration was a maximum of 58.8 f 9.54 ng/mI at 9.19 & 0.59 hr and a minimum of 27.85 -t 6.85 g/ml at 21.19 2 0.59 hr. The number of episodes of secretion was 10.0 ? 1.41; the mean amplitude and duration of a peak were 26.21 -t 3.71 rig/ml and 105.25 f 21.24 min respectively. INTRODUCTION

Circadian variations in plasma hydrocortisone have been demonstrated in many species, including horses (l-5). With horses, relatively large differences between morning and evening hydrocortisone concentrations are generally reported. However, there are differences of opinion about the times when peaks and troughs occurrence. Maximal/minimal plasma concentrations have been observed at 0800/1600 hr (4), about 0900/2100 hr (3), 900-llOO/ 2100-2300 hr (2) and 0600/1800 hr (1,5). For these various observations, only a few samples have been obtained over an entire 24 hr span (generally at 2 or 4 hour intervals). Such designs do not allow an acurate estimation of circadian rhythms; in addition, they render it difficult or impossible to detect and quantify episodic secretions (6) as described in other domestic species, including dogs (7) bulls (8) and sheep (9). The absence of accurate information on ultradian and circadian variations in plasma hydrocortisone concentrations makes it difficult to interpret plasma hydrocortisone values in clinical situations, sports medicine, and pharmacology (e.g., assessment of the suppressive effect of exogenous corticosteroids). The present study was undertaken in order to describe, with an appropriate sampling schedule, the diurnal variations in plasma hydrocortisone concentrations in normal horses and to determine whether the secretions follow episodic patterns. MATERIAL

AND

METHODS

Six healthy horses (saddlebred, 5 males and 1 female) from 5 to 17 years old and weighing between 5 10 and 720 kg were used. Each horse was kept in an individual stall and fed a complete diet (about 6 kg of food per day) in three equal meals per day (0800-0900, 1200, and 1900 hr). Straw and water were given ad libitum. Except on the day of sampling, horses were exercised Copyright

0 1988 by DOMENDO.

INC.

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0739-7240/88/$3.00

56

TOUTAIN,

OUKESSOU,

AUTEFAGE

AND ALVINERIE

for 2-3 hr each afternoon. All horses were accustomed to handling and sampling sessions. The experiment was conducted in two periods: November, 1986 and January, 1987 at Toulouse. In order to minimize any stress resulting from frequent venipuncture, an indwelling catheter was inserted into one jugular vein 24 hr before the beginning of sampling. For the first period, all 6 horses were used, and blood samples were obtained at hourly intervals beginning at 0900 hr. For the second period, only 4 of the 6 horses were used and blood samples were taken every 10 minutes for 24 hr. This second experiment was started at 1330 hr. During the period of darkness, samples were collected without light. Blood samples were collected in heparinized tubes and centriIuged. Plasma was stored at -20C until analysed. The horses apparently were not disturbed by the sampling procedure and generally remained lying down when samples were obtained during periods of sleep. Plasma hydrocortisone was determined using the HPLC method previously described (10). The level of sensitivity was approximately 2-3 rig/ml and the interassay coefficient of variation was approximately 5%. The 24 hr hormonal profiles were analysed for episodic peaks using PULSAR, a program designed for identifying ultradian pulsation in time series (11). A constant assay coefficient of variation (5%) was selected to describe the relationship between concentrations of hormone and intraassay standard deviation. The level of sensitivity was fixed at 3 rig/ml. For characterization of the circadian rhythm, each 24 hr profile obtained in the first period was fitted to equation 1 using a program for non-linear regression analysis (12). cp = co f c ’ Cos(ot

+ cp)

Eq.1

In equation 1, Cp (rig/ml) is the plasma hydrocortisone value at time t, Co (rig/ml) is the mean hydrocortisone level, C (rig/ml) the amplitude, o the frequency fixed at 27/24 radians, t, the time in hours, and cp the computative acrophase in radians (13). In order to evaluate the hydrocortisone half life, the average plasma concentration values obtained between 1500 hr and 2000 hr in the 4 horses during the second experiment were fitted to a monoexponential equation using a program for non-linear regression analysis (12). Statistical analysis was made using a correlation coefficient. P values <0.05 were taken to indicate statistical significance. RESULTS Similar trends for hormonal profiles were found in both experiments. Figure 1 shows a representative profile (horse F) obtained during the first experiment (1 hr interval sampling). Visual inspection of each proEle suggests an episodic pattern of hydrocortisone release with a succession of peaks and troughs. Typically, from 15.5 + 2.16 hr to 20.33 f 1.2 hr, plasma hydrocortisone values decreased regularly to reach minimums of 20.75 + 7.59 rig/ml. For all but one horse (B),a highly significant (P
PLASMA

HYDROCORTISONE

c 09

IN THE HORSE

I2

IS

18

57

21

TIME

Fig. M

1. 24.hour plasma hydrocortisone observed values obtained

0

03

8 09

06

( II,)

concentration (q/ml) profile every hour; O-+---O calculated

in a representative curve using cosine

horse (F). function.

Figure 2 shows a representative individual plasma hydrocortisone profile (horse F) as measured 3 months later on the basis of sampling at 10 min intervals. Visual inspection of the profile confirms the episodic nature of hydrocortisone secretion in the horse. Using the PULSAR program, 10.0 f 1.4 peaks were identified; the mean amplitude was 26.21 + 3.71 rig/ml and its mean duration was 105.25 & 2 1.24 min. The most important episodes of secretion were observed from about 0300 hr to 1150 hr. From 1830 hr to 2150 hr the mean values of hydrocortisone concentrations were lower than 15 rig/ml in all 4 horses. From 15.5 hr to 20.10 hr the mean cortisol values decreased progressively with an apparent half-life of 176.6 min. In all 4 horses a highly significant circadian rhythm was detected by fitting the 24 hr profiles using a cosine function (P
(units)

A

rig/ml

34.72

rig/ml (cp) radian rig/ml rig/ml

B’

D

E

F

Mean

2 SD

60.49

C 48.98

48.42

39.32

45.30

43.35

? 6.16

8.45 -2.217 43.17

4.69 -1.751 65.18

12.73 -2.319 61.71

14.72 -2.319 63.15

18.48 -2.631 57.80

23.08 -2.437 68.38

15.49 -2.406 58.85

+ 5.57 +- 0.154 + 9.54

26.27

55.80

36.25

33.69

20.84

22.22

27.85

+ 6.85

hr

8.47

6.69

9.27

8.86

10.05

9.19

k 0.59

hr

20.47

18.69

21.27

20.86

22.05

21.30

21.19

+ 0.59

hr

19

19

20

21

22

21

20.6

2

rig/ml

34.22

60.59

48.65

47.96

38.53

44.49

42.77

+ 6.24

acrophase calculated B was not considered

with 0O:OO hr as reference time. in the calculation of the means and

SD.

9.30

1.14

58

TOUTAIN,

Fig. 2. 24-hour plasma hydrocortisone concentration Observed values obtained every 10 minutes.

OUKESSOU,

(rig/ml)

AUTEFAGE

profile

AND ALVINERIE

in a representative

horse

(F).

DISCUSSION The circadian pattern of plasma hydrocortisone found in the present experiment is similar to that reported in man (14) and the rhesus monkey (15), with secretory episodes being superimposed on a marked basic circadian rhythm. This pattern is at variance with the secretory profile observed in dogs and cattle, for which no (7) or a very slight (8) circadian variation has been found. The most typical feature of hydrocortisone profiles in horses is a relatively regular decrease in plasma hydrocortisone concentrations during the afternoon and the beginning of the dark period. A half life of 176 min for hydrocortisone disappearance was calculated. This half life corresponds to the plasma half life of hydrocortisone (160.5 f 40.9 min), which was obtained in the same horse after administration of hydrocortisone (unpublished results). The correspondence strongly suggests that adrenal gland secretion is minimal or null in horses from about 1400 to 2000-2200 hr, i.e., at the time of their normal period of activity. Circadian variations have been detected previously in horses, but the reported time of their nadir varies greatly (1600 hr (4), 1800 hr (5), 2200 hr (l), 2100-2300 hr (2)). It is clear from the present experiment that the interpretation of the hydrocortisone plasma concentration values in horses must take into account the time at which samples are taken. Similarly, when a dexamethasone suppressive test is performed, the test must be concluded before the horse’s adrenal gland becomes inactive. For this reason, the schedule recommended by Eiler et al (16) (a control blood sample at 0900 hr followed by administration of dexamethasone, with collection of a second blood sample at 1300 hr) appears to be acceptable. Nevertheless a shift towards an early schedule (e.g., 0700-1100 hr) would seem advisable. The loss of a normal diurnal rhythm in hydrocortisone secretion is one of the typical symptoms of Cushing syndrome in different species, including horses (17). In men with Cushing syndrome, early morning hydrocortisone values may be normal while late evening hydrocortisone values may be higher than normal. Our results indicate the best time to detect abnormal increases in hydrocortisone levels in horses would be between 1800 and 2200 hr. It is well established that cortisol levels are lower between 2200 and 0400

PLASMA

HYDROCORTISONE

IN THE HORSE

59

hr for people having a normal nocturnal sleep and normal day time activity ( 14), even though the exact relationship between secretory episodes and the basic rest-activity cycle remains unclear. The horse, like all other eutherian mammals, experiences different stages of sleep, including REM sleep (18). The major part of recumbency is seen after midnight, a time which coincides with the maximum hydrocortisone secretion in horses. In contrast, the minimum values of hydrocortisone correspond to the normal period of maximum activity for our horses. The fact that the basic rest-activity cycle and the hydrocortisone rhythm are out of phase in horses deserves attention in future basic investigations. REFERENCES

1. Bottoms GD, Roesel OF, Rausch FD. Akins AL. Circadian variation in plasma cortisol and corticosterone in pigs and mares. Am J Vet Res 33:785-790, 1972. 2. Kumar MSA, Liao TF, Chen CL. Diurnal variation in serum cortisol in ponies. J Anim Sci 42:1360. 1976. 3. James VHT, Horner MW, Moss MS, Rippon AE. Adrenocortical function in the horse. J Endocrinol 48:319-335, 1970. 4. Hoffsis GF, Murdik PW, Tharp VL, Ault K. Plasma concentrations of cortisol and corticosterone in the normal horse. Am J Vet Res 31:1379-1387, 1970. 5. Larsson M, Edqvist LE, Ekman L, Persson S. Plasma cortisol in the horse, diurnal rhythm and effects of exogenous ACTH. Acta Vet Stand 20:16-24, 1979. 6. Ross JL, Barnes KM, Brody S, Merriam GR, Loriaux DL, Cuter GB. A comparison of two methods for detecting hormone peaks: the effect of sampling interval on gonadotropin peak frequency. J Clin Endocrinol Metabol 59: 1159-l 163, 1984. 7. Kemppainen RJ,Sartin JL. Evidence for episodic but not circadian activity in plasma concentration of adrenocorticotrophin, cortisol and thyroxin in dogs. J Endocrinol 103:219-226, 1984. 8. Thun R, Eggenberger E, Zerobin K, Luscher T, Vetter W. Twenty-four hour secretory pattern of cortisol in the bull: evidence of episodic secretion and circadian rhythm. Endocrinology 109:2208-2212, 1981. 9. Fulkerson WJ, Tang BY. Ultradian and circadian rhythm in the plasma concentrations of cortisol in sheep. J Endocrinol 81:135-151, 1979. 10. Alvinerie M, Toutain PL. Simultaneous determination of corticosterone, hydrocortisone and dexamethasone in dog plasma using high performance liquid chromatography J Pharm Sci 71:816-818, 1982. 11. Merriam GR, Wachter KW. Algorithms for the study of episodic hormone secretion. Am J Physiol 243:310-318, 1982. 12. Koeppe P, Hamann C. A program for non-linear regression analysis to be used on desktop computers. Comp Programs Biomed 12: 12 l- 128, 1980. 13. Halberg F. Chronobiology. Ann Rev Physiol 31:675-725, 1969. 14. Atcheson JB, Tyler FH. Circadian rhythm: man and animals. In: Handbook of physiology Vol VI. Adrenal gland. Ed, Greep RO, Astwood EB. Am Physiol Sot., Washington 1975. 15. Quabbe HJ, Gregor M, Bumke-Vogt C, Hardel C. Pattern of plasma cortisol during the 24 hour sleep/wake cycle in rhesus monkey. Endocrinology 110: 164 l-1646, 1982. 16. Eiler H, Oliver J, Goble D. Adrenal gland function in the horse: effect of dexamethasone on hydrocortisone secretion and blood cellularity and plasma electrolyte concentrations. Am J Vet Res 210:727-729, 1979. 17. Orth DN, Holscher MA, Wilson MG, Nicholson NE, Plue RE, Mount CD. Equine cushing’s disease: plasma immunoreactive proopiomelanocortin peptide and cortisol levels basally and in response to diagnostic tests. Endocrinology 110: 14301441, 1982. 18. Dallaire A, Ruckebusch Y. Sleep and wakefulness with housed pony under different dietary conditions. Can J Comp Med 28:65-71, 1974.