Some Biochemical Effects seen after the Administration of Synthetic Adrenal Cortex Hormones to Normal Sheep

Br. vet. J. (1968), 124> 259

SOME BIOCHEMICAL EFFECTS SEEN AFTER THE ADMINISTRATION OF SYNTHETIC ADRENAL CORTEX HORMONES TO NORMAL SHEEP By D. M.

PUGH

Department of Pharmacology, School of Veterinary Medicine, Trinity College, Dublin

SUMMARY

The effects of single intramuscular injections of either prednisolone acetate or desoxycorticosterone acetate were investigated in normal sheep. Following prednisolone administration plasma glucose concentration was significantly increased, and plasma potassium, total circulating eosinophils and packed cell volume were significantly decreased. The rate of removal of an intravenously administered glucose load was significantly reduced in a sheep pretreated with prednisolone. Following both prednisolone and DOCA administrations, significant increases in daily water intakes were recorded. Urine outputs were not significantly increased (P = 0'05)' The only other statistically significant effects detected following DOCA administration were an increase in total circulating eosinophils and a decrease in packed cell volume. INTRODUCTION

While preparing for an investigation into the effects of adrenalectomy and subsequent hormonal maintenance on selected normal values in sheep (Pugh, 1961), it became apparent that there was little information available in the literature either on normal adrenocortical function or on the effects produced following the administration of synthetic corticosteriods to intact sheep. This made it necessary to determine whether or not these drugs produced effects in the sheep comparable to their known actions in other species, before proceeding to study their efficacy when used as replacement hormones in adrenalectomized sheep. METHODS

The methods used in determining total circulating eosinophils, packed cell volume and plasma glucose, sodium, potassium and chloride concentration have been described previously (Pugh, 1966). The intravenous glucose tolerance test was carried out according to Holmes (195 1 ). The fluid balance studies, in which only water consumption and urine output were measured, were performed on sheep confined in a metabolism cage constructed according to Duthie (1959). The experiments were carried out on sheep accustomed to handling and daily

BRITISH VETERINARY JOURNAL, 124,6

blood sampling and which were allowed feed and water throughout. Husbandry details were as described in an earlier paper in this series (Pugh, 1966). RESULTS

On four separate occasions ewes received a single intramuscular injection of 20 mg. prednisolone acetate. Jugular blood samples were collected immediately before and for varying periods after the injection. In a fifth experiment, an ewe was sampled at half-hourly intervals after injection in order to define the time of onset of effects in this sheep. Following prednisolone administration, there was an increase in plasma glucose concentration accompanied by a simultaneous decline in packed cell volume (peV) and an almost complete disappearance of circulating eosinophils. TABLE I THE EFFECTS OF A SINGLE INTRAMUSCULAR INJECTION OF PREDNISOLONE ACETATE IN INTACT SHEEP

Hours SHEEP NO. 712 (A) 0 20 mg. P 2 4t 6 7t SHEEP NO. 712 (B) Control 0 20 mg. P 2 4 6 8 9! 24 SHEEP NO. 704 Control ° 20 mg. P It 3 4 5 SHEEP NO. 665 Control ° 20 mg. P I! 3t 517t

Glucose (mg . %)

Eosinophils permm 3

Na (m-equiv.JI. )

5°·3

981

135

4·5

96.8

34

66·7 84·7 88·7 80·0

500 11 3 47 50

137 133 137 138

4. 12 3·94 5"00 4. 2

95"5 95·7 97.0 97·5

31.5 29·5 29 31

34·7 36. 1

806 9 62

147 145

4·4 4.56

96.3 93·7

32 31.5

5 1.4 78.6 1°9. 0 99.0 72.4 49·5

4 16 69 14 34 69 7 18

139 147 148 146 146 148

4·44 4.67 3·53 3·75 4. 12 4.25

90.0 94.0 96.5 96 .5 97.0 97"4

31.5 30.0 28·0 3°·5 3°·0 3°·0

36.7 32.5

63 56

148 15 2

4·5 4.6

92.4 93.0

42.0 61·0 56.0 46.0

41 31 26 9

152 152 148 152

4.6 4·5 4.6 4·4

92.9 93. 1 92.9 92.9

36.0 4 1.3

156 13 1

149 150

4·44. 2

98 .8 99.0

46.0 58.0 69.0 61·0

122 106 50 25

153 146 150 146

4·3 4. 2 4·3 4.2

98 .8 99.2 98 .9 99·5

K (m -equiv.JI. )

The "P" signifies the injection time of 20 mg. prednisolone acetate.

Cl (m -equiv.JI.)

PCV (%)

EFFECTS OF ADRENAL CORTEX HORMONES IN SHEEP

261

TABLE II ADMINISTRATION

OF

17'5

MG, PREDNISOLONE INTACT EWE (82 9)

ACETATE TO AN

Minutes after injection of prednisolone

0

30

60

90

120

15 0

180

2 10

Plasma glucose concentration (mg, % ) 60'3 534 59'8 55'0 50 '0

5 2 '5 60'0 54'7

50 '5 55'4

5 2 '5 60'4 54'3

67'9 56 '4

60'3

61'9

Total circulating eosinophils per mm 3 706 794 38 7 45 6 3 13

59 1 4 06 434

487 33 8

362 28 7 188

147 173

106

87

''''hen compared with the within-I-day variation of the same blood constituents in a group of untreated sheep from the same flock (Pugh, 1966), the within-sheep variation of the following constituents was significantly greater in the prednisolone treated animals, The relevant data was extracted by an analysis of variance and was examined for significance by an F-test at the I per cent level of probability, The experimentally obtained Fvalues were as follows: plasma glucose, F = 74'2 with 16 and IS degrees of freedom; eosinophils, 110 100 90

1000

80 Q)

V>

0 U :J

t')

70 60 50

V>

.<:

a.

40

500

0

30

" V>

0

W

Q)

E

32

:J

0

0

31

Q)

30

>

u -0

'"'"u 0

29 28

20mg, pred nisolone

I:l-

o

2

4

I

I

I

4 I

6

8

10

22

Hours

Fig,

I,

The effect of a single intramuscular injection of 20 mg, prednisolone on total circulating eosinophils, packed cell volume and plasma glucose in an intact ewe (712),

BRITISH VETERI ARY JOURNAL, 1'24, 6

F = 12 ·62 with 16 and 18 degrees of freedom; PCV, F = I I ·07 with 4 and 4 degrees of freedom; plasma potassium, F = 5.69 with 14 and 16 degrees of freedom. Detailed results are given in Tables I and II and an example is illustrated in Fig. I. Glucose tolerance tests were performed on one ewe before and after the intramuscular injection of 25 mg. prednisolone. The effect of the steroid in delaying the rate of fall of plasma glucose concentration is shown in Fig. 2, where the results are expressed as the log of the glucose increment plotted against time. Polynomial regression analysis applied to the data relevant to curve A shows that the variation in log glucose concentration with time is fitted by a function of time of at least the second order. The reduction in the residual sum of squares by including a quadratic term in the equation describing the curve is very highly significant (F = 2.71 X 10 3 ). This means that the experimentally obtained curve A differs significantly from the normal straight line relationship exemplified by C.

100

....,

60

'"E .,on

40

0

20

<.)

:>

(3

10

.~. 90

120

Minutes

Fig. 2. The rate of removal of an intravenously administered glucose load from the plasma ofa normal sheep. C is a control value and A and B were obtained following an intramuscular injection of 20 mg. prednisolone acetate.

The increases in water consumption and in urinary output following the intramuscular injection of 30 mg. prednisolone acetate and, later, 30 and 50 mg. DOCA are illustrated in Fig. 3. Although urinary output was increased, the net result in each instance was a retention of water, a typical mineralo-corticoid effect. When compared with the control period, daily water intake was significantly increased in the treatment period (P = 0·001, n = 18, t = 9.287). There was no significant difference at the 0·05 level in urine output between the same two periods.

EFFECTS OF ADRENAL CORTEX HORMONES IN SHEEP

263

2500-

~

20 00

E

"

~

1000

~

E

mg. DOCA

mg. prednisolone

I division; I day

30

30

30

")0

50

200 400

"e

600 -

OJ

800

Fig. 3. Daily water consumption and urin e ou tput record of a sheep showing the effects of treatment with prednisolone acetate and DOCA.

One sheep was injected with 20 mg. DOCA intramuscularly and the analytical results from serial blood samples are given in Table III. The only effects detected were an increase in circulating eosinophils and a decrease in packed cell volume. TABLE ADMINISTRATION

Hours 0 20 mg. DO CA 2 3;f 5 6!

OF

III

DOCA TO

Na

AN INTA C T

EWE

Glucose (mg. 'Yo)

Eosinophils jJer mm 3

(m-equil'·II. )

K (m -equiv·11. )

48 '3

369

147

5'3

54'2 54. 6 52 '9 52 '9

57 2 70 9 60 3 560

147 147 143 147

5'0 5' 15 5' 65 4.8

(82 5) Cl (m-equiv· ll. ) 101 98 '3 96 '7 97'0 97'5

PCV ('Yo) 37 33 32 30 31

DIS CUSS IO N

Prednisolone is principally a glucocorticoid, i.e. administration causes farreaching effects on intermediary metabolism. A convenient measure of its effects on carbohydrate metabolism is the rise in blood sugar concentration which follows its administration. This investigation has demonstrated that, in common with all other species investigated, a significant increase in plasma glucose concentration occurs in the sheep, the maximum effect occurring about 6 hours after intramuscular injection. Bassett, Mills & Reid (1966) recorded a moderate hyperglycaemia in fed sheep following cortisol administration. As the delay in the onset of effect of prednisolone on plasma glucose levels in sheep 829 was 3 hours (Table II), it is suggested that the reduced rate of removal of the glucose load from the plasma in this sheep after prednisolone

BRITISH VETERINARY JOURNAL, 124, 6

indicates impaired utilization of glucose, although it is not possible to exclude the possible interfering effects of a prednisolone-stimulated gluconeogenesis. In this context it is interesting to note that Bassett et at. (1966) found support for the thesis that the initial effect of cortisol acetate on glucose in the sheep was inhibition of utilization. Statistical treatment of the data in this paper demonstrates that following prednisolone administration, the glucose tolerance curve is significantly changed in a manner which suggests the operation of a factor which affects both the rate and pattern of glucose removal from the blood. The marked decrease in the number of circulating eosinophils which occurred following prednisolone is, again, a typical glucocorticoid response. This effect was used as the basis of an adrenal cortex function test in man (Thorn et at., 1951 ), and exemplifies the depressant action of a glucocorticoid on mesenchymal tissues. The actions of corticosteroids on electrolyte and fluid balance are known as the mineralo-corticoid effects and most so-called glucocorticoids possess some mineralo-corticoid potency. Although no significant changes in sodium and chloride concentration occurred in this investigation, the fall in PCV and plasma potassium concentration, and the stimulation of increased drinking and increased urine production demonstrate that prednisolone is capable of affecting fluid balance. It is assumed that the haemodilution, indicated by fall in PCV, prevented the potential changes in electrolyte concentration from becoming apparent. DOCA is principally a mineralo-corticoid and would be expected to cause an increase in plasma sodium and chloride concentrations, a decrease in PCV and a decrease in plasma potassium concentrations. The only significant effects recorded in this experiment were a decrease in PCV and an unexpected increase in total circulating eosinophils. The absence of significant electrolyte effects was probably due to the reasons given above and to the fact that 20 mg. of DOC A was an inadequate dose in the sense that the sheep could compensate for its presence by reducing its endogenous mineralo-corticoid production. This conclusion is reinforced by the results of the water-balance study where 50 mg. DOCA were required to produce an obvious effect. The diuretic effect of a water-load is another criterion used in man as a presumptive test of adrenocortical function (Robinson, Power & Kepler, 194 I). In order to demonstrate plasma electrolyte change in a normal sheep, Scott ( 1962 ) infused aldosterone intravenously for 3-4 days. A C KNOWLEDGMENTS

This paper is based on material extracted from a thesis presented for the degree of M.Sc. at Bristol in 196I. I am grateful to all the members of the academic and lay staff at Bristol who helped me in so many ways and especially to Professor R. J. Fitzpatrick, my supervisor. The work was in part supported by a grant from Glaxo Laboratories Ltd, who also supplied the prednisolone acetate. I am indebted to Organon Laboratories Ltd for repeated gifts of DOCA.

EFFECTS OF ADRENAL CORTEX HORMONES IN SHEEP

265

REFERENCES

BASSETT, j. M., MILLS, S. C. & REID, R . L. (1966). Metabolism, 15, 9:.12. DUTHIE, I. F . (1959). L ab. Prac., 8, 408. HOLMES,j. R. (1951).J. comp o Path. Ther., 61, l. PUGH, D. M. ( 1961 ). Thesis, Bristol University. PUGH, D. M. ( 1966). Irish vet. J., 20, 142. ROBINSON, F.j., POWER, M. H. & KEPLER, E.j. (1941 ). Proc. Afa)'oCiin. , 16, 577. SCOTT, D . (1962). J. Physiol., Lond., 164, 27 P. THORN, G. W., FORSHAM, P. H., FRAWLEY, T. F., WILSON, D. D., RENOLD, A. E., FREDRICKSON, D. S. & J ENKINS, D. ( 1951). Am.J. Med., 10, 595. Certains effets biochitniques observes apres l'administration d ' hormones corti cosurrenales synthetiques a des moutons normaux (Pugb) Resume. Les effets d'une seule injection intramuseulaire, soit d'acetate de prednisolone, soit d'acetate de desoxycorticosterone, ont ete observes chez d es moutons norm aux. Apres l'injection de prednisolone, on a note une augmentation significative de la concentration de g lucose dans Ie plasma, ainsi qu'une diminution significative de potassium dans Ie plasma, des eosinophiles en circulation et du volume de ceHules agglomerees. La vitesse d'elimination d'une quantite de glucose administree par injection intraveineuse etait considerablement reduite chez les moutons prealablement traites au prednisolone. On a enregistre des absorptions d'eau journalieres considerablement accrues apres l'administration de prednisolone ainsi que d'acetate de desoxycorticosterone (DOCA). Les eliminations d'urine n'ont pas augmente de fac;:on significative (P = 0,05). Le seul autre dfet important du point de vue statitique enregistre a la suite d'administrations d 'acetate de desoxycorticosterone (DOC,\ ) consiste en une augmentation du nombre d'eosinophiles en circulation et une diminution du volume des ceHules agglomerees. Einige biochemische Effekte, die bei normalen Schafen nach Administration synthetischer Nebennierenrindenhormone auftreten (Pugh) Zusammenfassung. Die vVirkung nur einer intramuskularen Injektion von cntweder Prednisolonazetat oder Desoxykortikosteronazetat bei normalen Schafen wurde untersucht. Nach Prcd nisolongaben wurden bedeutend erhbhte Blutzuckerspiegcl, und eine erhebliche Erniedrigung des Plasmakalium, der Gesamtwertc der zirkulierenden Eosinophilen und der Hamatok ritwerte festgestellt. Die Normalisicrung des Bluzuckerspiegcls nach intravcnoser Glukosebelastung war bei mit Prednisolon vorbehandelten Schafen bcdeutend vcrlangsamt. Sowohl nach Prednisolon Administration als auch nach DOCA Adm inistration war ein bedeutendes Ansteigen der taglichen Wasscraufn ahme zu verzeichnen. Die Urinausscheidung war nicht wescntlich erhoht (P = 0,05). Die einzigen andercn, statistisch bedeutungsvollen Effekte naeh DOCA-Injektion waren ein Anstieg des Gcsamtwertes der zirkulierenden Eosinophilen und ein Absinken des Hamatokritwertes . Algunos efectos bioquimicos observados despue s de la administraci6n de hormonas de la corteza adrenal sinteticas a ovejas normales (Pugh) Resumen. Fueron investigados en ovejas nOl"males los efectos de inyecciones intramusculares lmicas de acetato de prednisolona 0 de acetato de desoxicorticosterona. D espues d e la adm inistraci6n de prednisolona, se habia aumentado de forma significante la concentraci6n de glucosa en el plasma, y habian disminuido de forma significante el potasio en el plasma, los eosin6filos circu lantes totales y el volumen de celulas aglomeradas. El ritmo de eliminaci6n de una carga de glucosa administrada intra\"enosamcnte se reducia de forma significativa en una oveja pretratada con prednisolona. Despues de la administraci6n de prednisolona 0 de DOCA, se registraron aumentos significativos en la absorci6n de agua diaria. La excreei6n de orina no aument6 de forma significativa (P = 0,05) . Los unieos demas efecLOs eS ladis ti camentc sign ificativos que se deteetaron despues de la administraci6n de DOCA fueron un aumento en el total de los eosin6filos circulantes y una disminuci6n en el volumen de celulas aglomeradas.