Division of Clinical Chemistry, Clinical Research Centre, Watford Road, Harrow, Middlesex, England

THE IN VIVO METABOLISM OF CORTISOL AND CORTICOSTERONE BY THE

MACAQUE

MONKEY

(Macaca fascicularis) By K. D. R. Setchell and C. H. L. Shackleton

ABSTRACT

[4-14C] Cortisol was administered intramuscularly to one adult female monkey, MF3 (Macaca fascicularis). To adult female macaque monkey, MF4, [4-14C]corticosterone was administered intramuscularly. Urine samples were collected and the metabolites excreted identified using gas chromatography, radio-gas chromatography and gas chromatography-mass spectrometry. The principal metabolites of cortisol were identified as glucuronide conjugates of 11-oxygenated-17-oxosteroids. The excretion of tetrahydrocortisol and tetrahydrocortisone relative to the other corticosteroid metabolites was low compared with that of man. Two compounds, 3\g=b\-cortol and 3\g=b\-cortolone not normally present in human urine were identified in the urine from this species. The principal metabolites of corticosterone were glucuronide conjugates of hexahydroCompound A and hexahydrocorticosterone. Two unidentified radioactive compounds were also present. macaque

and apes, due to their phylogenetic similarity to man, have become of great importance as models with which to find the solution to many problems in human biology and pathology. Experimental work in such fields as infec¬ tious pathology, oncology, haematology, neuroendocrinology and reproduction

Monkeys

the last few decades has illustrated that monkeys physiologically and pathologically are considerably closer to man than most classical laboratory animals. Surprisingly little is known of the in vivo metabolism of corticosteroids in primates, although a considerable amount of literature has been published on aspects of in vitro metabolism. The metabolism of cortisol, corticosterone and aldosterone in the chimpanzee has been reported, and the metabolism of these steroids in this species has been shown to be similar to that of man (Kirdani et al. 1963; Layne et al. 1964). Chimpanzees are expensive, larely breed in captivity and are generally impractical to keep as animal models. Very little data has been reported on the metabolism of corticosteroids in species of Old World monkey, although considerable interest has centered on the aspects of progesterone and oestrogen biosynthesis and metabolism (Solo¬ over

Leung 1972). Having shown that the macaque monkey is a suitable model for man in which to study aspects of corticosteroid secretion and its control by the hypo¬ thalamus and pituitary glands (Setchell 8c Himsworth 1973; Setchell et al. 1974, in prep.), the metabolism of the principal corticosteroids, cortisol and corticosterone was investigated. The advent of combined gas chromatography and mass spectrometry has enabled specific and relatively accurate determinations of many steroids in biological material. It was this technique combined with radio-gas chromato¬ graphy that was therefore chosen for the study of steroid metabolism. mon

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MATERIALS AND METHODS

Refecence steroids and reagents Reference steroids were obtained from three principal sources: Medical Research Council Steroid Reference Collection, Curator Dr. D. N. Kirk, Westfield College,

Hampstead, London; Koch-Light Laboratories, Bucks., England; British Drug Houses, (BDH) Poole, Dorset, England. [4-"C]Cortisol (54 mCi/mmol) and [4-"C] corti¬ costerone (61 mCi/mmol) were obtained from The Radiochemical Centre, Amersham, England. Ketodase® (/?-glucuronidase enzyme) was obtained from Warner-Chilcott Labora¬

tories, Morris Plains, New Jersey, USA; chemicals for the preparation of methyloximetrimethylsilyl ethers of steroids from Applied Science Laboratories, P. O. Box 440, State College, Penn., USA and Sephadex LH-20 from Pharmacia AB, Uppsala, Swe¬ den. Analar solvents were obtained from B.D.H., Poole, Dorset, England and, with the exception of hexane, were not purified. Hexane used for dissolving steroid trime-

thylsilyl

ethers

was

washed with

sulphuric

acid and redistilled

prior

to

use.

Steroid abbreviations Aeticcholanolcne: 3a-hydroxy-5,#-androstan-17-one, aldosterone: 1 l/>',21-dihydroxy3,20-dioxy-4-pregnen-18-aI, a/7o-tetrahydrocortisone: 3 ,17 ,21- trihydroxy-5a-pregnane-ll,20-dione, androstanediol: 5a-androstane-3a,17a-diol, androsterone: 3a-hydroxy5a-andrcstan-17-one, cholanediol: 5/i-cholane-3a,24-diol, cholesteryl buÍyrate: 5-cholestene-3/)'-yl-butyrate, corticosterone: 1 l/ï,21-dihydroxy-4-pregn-ene-3,20-dione, cor¬ tisol: ll/S,17a,21-trihydroxy-4-pregnenc-3,20-dione, cortisone: 17a,21-dihydroxy-4pregnene-3,ll,20-trione, a(/íí)-cortol: 5/y-pregnane-3a,ll/¿,17a,20a(/i),21-pentol, (ß)cortolone: 3a,17a,20a(/i),21-tetrahydroxy-5/i-pregnan-11 -one, hexahydrocorticosterone: 5/?- ^ 3 -3 ,11/ ,20//,21- 1, hexahydroCompound : 3 ,20/j,21 -trihydroxy-5/fpregnan-11-one, 11-hydroxyaetiocholanolone: 3a,l l/J'-dihydroxy-5/J-androstan-17-one, 11-hydroxyandrosterone: 3a, 1 l/i-dihydroxy-5a-androstan-17-one, 11-oxoandrosterone: 3a-hydroxy-5a-androstane-l 1,17-dione, pregnanetriol: 5//-pregnane-3a, 17a,20a-triol, tetrahydroaldosterone: 3a, 1 Iß,21 trihydroxy- 5ß pregnan 20- on-18 al, tetrahy drocorticosterone: 3a,l l/?,21-trihydroxy-5/)'-pregnan-20-one, tetrahydroCompound A: 3a,21dihydroxy~5/3'-pregnane-ll,20-dione, tetrahydrocortisol (THF): 3 , 11/?,17 ,21 -tetrahydroxy-5/?-pregnan-20-one, tetrahydrocortisone (THE): 3 , 17 ,21-trihydroxy-5/ípregnane-11,20-dione. -

-

-

-

Methodology samples were collected over a period of 5 days from two adult female monkeys (Macaca fascicularis). One animal (MF3) was administered 10 ,«Ci of [4-14C] cortisol intramuscularly and the other (MF4) was administered 10 /¿Ci of [4-14C] corticosterone intramuscularly. The animals were housed in individual meta¬ bolism cages during the period of collection and the cages were washed with 250 ml water between each collection. The urine samples and water washes were collected in polystyrene bottles containing chloroform (10 ml) added as a preservative, and stored at -30°C until required lor analysis. The urine volume and amount of radioactivity in each collection was measured prior to pooling the 5 daily collections. Steroids were then extracted on large columns 7 cm i. d.) as described by Bradlow of Amberlite XAD-2 resin (bed size 40 cm was dissolved in water (10 ml) and the free steroids extract The XAD-2 (1968). removed by shaking three times with méthylène chloride (50 ml). Portions (2 ml) of the aqueous phase were then hydrolysed in acetate buffer (0.5 M, pH 4.8) with 12 500 units of /f-glucuronidase enzyme (Ketodase, 2 ml) for 48 h at 37°C. The steroids were then extracted on small Amberlite XAD-2 columns (bed size 15 cm 0.7 cm i. d.) as before, and then fractionated by Sephadex LH-20 column chromatography. Sephadex LH-20 (6 g) was soaked for 30 min in the solvent cyclohexane:ethanol (80:20, v/v) 1 cm i. d.). The steroid extract was and the slurry poured into glass columns (30 cm then applied to the column in 2 2 ml portions of the solvent and six fractions were collected (Setchell Sc Shackleton 1973). Following the addition of internal standard, cholesteryl butyrate (10 µg), to portions of the Sephadex LH-20 fractions, the extracts were converted to trimethylsilyl ethers and methyloxime-trimethylsilyl ethers. Trimethylsilyl ether derivatives were prepared of Sephadex LH-20 fractions 1, 2 and 3 using hexamethyldisilazane and trimethylchlorosilane in pyridine (Makita Sc Wells 1963). The methyloxime-trimethylsilyl ether derivatives of fractions 4, 5 and 6 were prepared with 2 % methoxylamine hydrochloride in pyridine (overnight, room temperature) followed by silylation (Fales Sc Luukkainen 1965). Daily

macaque

urine

Gas

chromatography

Gas

Chromatographie analyses were carried out by a Pye 104 gas Chromatograph equipped with flame ionisation detection, using a silanised glass column (2 metres 4 mm i. d.) packed with 1 °/o silicone OV-1 on Gas Chrom Q. The oven temperature was programmed from 200-300°C in increments of 1.5°C/min. Flash heater temperature was 270°C. Carrier gas flow rate was approximately 40 ml/min and the hydrogen and air flow rates were approximately 30 ml/min and 500 ml/min, respectively.

Identification of steroid metabolites Partial identification of silyl ether and oxime-silyl ether derivatives of urinary steroid metabolites was by their gas Chromatographie retention time as compared with reference steroids calculated using the méthylène unit (Horning 1968). Final identification was carried out by combined gas chromatography-mass spectrometry using a Varian-Aerograph 2700 gas Chromatograph coupled to a Varían MAT 731 double beam focussing mass spectrometer, operated at an ionisation voltage of 70 eV and an accelerating voltage of 8 kV. The carrier gas separator and transfer lines were maintained at a temperature of 250°C. The gas Chromatographie conditions were identical to those described above. Mass spectral scans were made over the mass range 50-1000 mass units at 2 seconds/decade and the data was processed by a Varían 100 MS SpectroSystem. Identification of the steroids was made by comparison of the mass spectra against reference compounds.

Identification of radioactive steroid metabolites Simultaneous gas chromatography radio-gas chromatography was used for the identification of [4-14C] radioactive steroid metabolites. A Panax-Radiogas Detector System (Panax Instruments, Redhill, Surrey) was coupled to a Pye 104 gas Chromato¬ graph

and using a twin pen recorder the mass peaks detected the radioactive peaks were recorded simultaneously.

by

flame ionisation and

Semi-quantitation Semi-quantitation was achieved by comparing the area under the peaks obtained for the steroids against the area obtained for known amounts of added internal standard. Cholesteryl butyrate was used as the internal standard, and a correction for the difference in responses obtained by equal amounts of steroid and standard (response factor) applied.

RESULTS

Recovery of radioactivity The urine volume and amount of radioactivity recovered in the collections are shown in Table 1. The average daily urine volume was 92 ml. Of the radiolabelled cortisol administered 67 °/o was recovered over the 5 days in monkey MF3, while 42 °/o of the radiolabelled corticosterone dose was re¬ covered in monkey MF4. The radioactivity associated with the unconjugated steroid fraction was

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The in vivo metabolism of cortisol and corticosterone by the macaque monkey (Macaca fascicularis).

(4-14C) Cortisol was administered intramuscularly to one adult female macaque monkey, MF3 (Macaca fascicularis). To adult female macaque monkey, MF4, ...
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