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Urinary excretion of testosterone and epitestosterone in men, women and children, in health and disease
CLINICA
CHIMICA ACTA
URINARY
EXCRETION
MEN, WOMEN
215
OF TESTOSTERONE
AND CHILDREN,
IN HEALTH
AND EPITESTOSTERONE
IN
AND DISEASE
S. B. PAL
Universitiit Ulm, Abteilung Endokrinologie und Stoffwechsel, Zentrum fiir innere Medizin und Kinderheilkunde, D 79 Ulm (Donau) (D.B.R.) (Received
December
zg, 1970)
SUMMARY
A fluorimetric method for the determination of urinary conjugated testosterone and epitestosterone in human subjects is described. According to this method a Io-zo-ml aliquot of a 24-h urine collection was hydrolysed with #I-glucuronidase and sulphatase at pH 5.2 for 72 h, and the hydrolysed urine was filtered and fractionated on a Sephadex column. After the extraction of the liberated steroids, testosterone was separated from epitestosterone by paper chromatography and they were then further purified in the same way. Testosterone and epitestosterone were eluted from paper chromatograms and, after further purification, were measured fluorimetrically with a sulphuric acid-ethanol-induced reaction. After the addition of testosterone glucuronide and epitestosterone alcohol (0.1 pg/ml of each steroid) to give a concentration of 0.1 ,ug-0.4 ,ug to 20 ml water and to a zo-ml aliquot of normal urine, taken through the entire procedure, the mean recoveries of testosterone and epitestosterone from 20 experiments, in each case were: 70.6% and 73.2% from water and 68.8% and 71% from urine. By the application of this method the average testosterone and epitestosterone excretion in IOO normal women (age 18-61 years) was 4.7 pg/z4 h (range 0.8-13.8) and 2.9 pg/z4 h (range 0.6-12.4) respectively; in IOO normal men (age 20-63 years) 47.6 pug/z4 h (range 13-176.4) and 31.4 pg/z4 h (range 8.2-100.1) respectively; and out of 21 normal children, 9 girls (age 7-12 years) 0.8 pg/z4 h (range 0.2-1.6) and 0.5 rug/z4 h (range 0.2-1.1) respectively; 12 boys (age 6-13 year-s) 1.3 pg/z4 h (range 0.4-3.2) and 0.9 ,ug/z4 h (range 0.3-2.6 ) respectively. Results from patients with various endocrine disorders are presented. This method was compared with a gas-liquid chromatographic procedure and good agreements were found. The method appeared to be a clinically useful one.
INTRODUCTION
It is generally considered that the quantitative determination of free testosterone (17/Chydroxyandrost-4-en-3-one) in plasma is the best available parameter of androgen secretion. Testosterone is the most powerful naturally occurring androCliPZ.Chim. A&Z. 33
(1971)
215-227
216
PAL
gen-it is secreted largely by the Leydig cells of the testis in men, and small amounts are also produced by the adrenal cortex in both sexes and by the ovary in women. However, Murphy1 reported that of all the steroids tested so far by the competition of various steroids with tritiated testosterone in late pregnancy plasma, dihydrotestosterone was found to be the most potent, being about three times as effective as testosterone itself. Tissues of non-endocrine origin can also convert steroid precursors, especially d4-androstenedione (androst-4-ene-3,r7-dione) and dehydroepiandrosterone (@hydroxyandrost-5-en-r7-one) to testosterone. A number of methods for the determination of free testosterone in plasma have been published in recent years but all these methods are very time consuming and are not always suitable for diagnostic work. The determination of urinary testosterone or urinary production rate&” are very useful in some clinical investigations such as in women with Cushing’s syndrome, SteinLeventhal syndrome, virilizing ovarian neoplasm and idiopathic hirsutism, children with congenital adrenal hyperplasia, virilizing adrenocortical tumours and men with hypogonadism secondary to hypopituitarism or primary hypogonadism, eunuchoidism and impotence. Testosterone was first isolated from urine after parenteral administration of the steroid in large doses*. It was extracted from an adrenal carcinoma5. The isolation and determination of testosterone glucuronide from the mine of normal men and women was first reported by Schubert and WehrbergeP. Epitestosterone (r7a-hydroxyandrost-4-en-j-one) which is the r7-hydroxy epimer of testosterone, was first isolated by Korenman et aZ.7 from the urine of both normal men and women; isolation from normal human urine was also reported by Brooks and Giuliani* in 1964. As our knowledge of testosterone metabolism is becoming increasingly important, it was found necessary to develop a suitable method for the estimation of urinary testosterone and epitestosterone. In certain disease states, such as hirsutism and the Stein-Leventhal syndrome, the determination of epitestosterone level becomes meaningful in spite of the fact that little is known about its biological significance. It has been suggested that the origin of epitestosterone can be the resultant of both peripheral and endocrine gland secretions. Although testosterone glucuronide is not a unique metabolite of testosterone, the measurement of urinary testosterone is more closely related to testosterone production than is the determination of urinary 17ketosteroids under certain clinical conditions. There are various published methods for the estimation of urinary testosterone, testosterone and epitestosterone, separately or combined, but only a few are suitable for performing estimations in children’s urine. Testosterone is primarily excreted in urine as the glucuronide and a very minute amount of testosterone sulphate and free testosterone are also excreted. Therefore, the determination of urinary testosterone glucuronide appears to be a useful method to study androgen metabolism. The aim of the present study was to develop a clinically useful method to determine urinary testosterone and epitestosterone in men, women and particularly in children, and patients with various endocrine disorders. This method is in many ways similar to one which was previously publishedlO. After a preliminary study, good agreement was found when the present method was compared with a gas-liquid chromatographic procedure.
Clin. Chim.
Acta,
33 (1971) 215-227
URINARYTESTOSTERONE
AND
217
EPITESTOSTERONE
MATERIALS
All chemicals
used were of analytical
grade (Hopkin
& Williams
Ltd., U.K. and
E. Merck AG., Darmstadt). Sue &Helix pomatia. I ml contains IOOOOO units of /?-glucuronidase (Fishman), 50000 units sulphatase (Whitehead). (Industrie Biologique Francaise S.A., Quai du Moulin de Cage-Genevilliers, Seine, France). These quantities of enzymes, contained in a r-ml ampoule, were dissolved into 9 ml of 2 pvlsodium acetate-acetic acid buffer of pH 5.2 before use. Ketodase (/?-glucuronidase) 5000 units/ml (Warner Chilcott Laboratories, New Jersey, U.S.A.) was also used on several occasions. All solvents were of general purpose reagent grade (Hopkin & Williams Ltd.) and were further purified before use according to standard methods. Later on, all solvents used were of pro analysi grade (E. Merck AG). Glass-distilled water was used throughout the work. Glass-stoppered test tubes 15 x 2.5 cm approximate capacity 54 ml and 12.5 x I .5 cm approximate capacity 15 ml were used (Quickfit & Quartz Ltd, U.K.). A fro-cm long glass column (1.5 cm internal diam.) with a stopcock and sintered glass filter was packed with Sephadex G-25 fine for gel filtration (Pharmacia, Uppsala, Sweden) to a height of 30 cm, which was previously soaked in distilled water (changed on alternate days) for 14 days. The column had a reservoir of 50-ml capacity at its top and a ro-ml syphon for collecting the fractions. Testosterone, epitestosterone and other Yeference steroids used were obtained from Steroid Reference Collection (Medical Research Council, Chemistry Department, Westfield College, Hampstead, London, U.K.). Some of these steroids were also available from different sources as gifts, which, when needed, were further purified by paper chromatography and repeated crystallisation from methanol, ethanol and acetone. Melting points were determined on a Kofler hot stage and agreed with published figures. Testosterone glucuronide was purchased from Ikapharm Ltd., Ramat-Gan, 31, Jabotinski Road, Israel. [I+-3H]Testoste~one (N)-3,!-D-glucuronide (specific activity 30-50 C/mmole was purchased from New England Nuclear Corporation, Boston, Massachusetts, U.S.A. [r-%]n-Hexadecane was used as an internal standard for liquid scintillation counting which was purchased from Radiochemical Centre, Amersham, U.K. Toluene scintillator was prepared by adding 3.0 g of 2.5-diphenyloxazole (PPO) and 0.3 g of r,4-bis-z,4-methyl-5-phenyloxozolyl benzene (POPOP) both scintillator grade in I litre of purified toluene and was stored in an amber glass bottle at 4O. Toluene of scintillation grade from E. Merck was also used for preparing scintillator solution. Liquid scintillation countings were made in a Packard Tri-Carb Liquid Scintillation Spectrometer (Model 314 EX), automatic dual channel. Packard Radiochromatogram Scanner (Model 7201) was used for measuring and recording radioactivity on paper chromatograms. a&Diphenyloxazole (PPO), r,4-his-2,4-methyl-5-phenyloxozolyl (dimethyl-POPOP) and high quality glass vials (Low-Potassium-I) were all purchased from Packard Instrument Company, Inc., Box 429, La Grange, Ill, U.S.A. All the extracts for paper chromatography were evaporated to dryness throughout this work in a water bath at 40~ under a stream of nitrogen; similarly, the radioczin. Chim. Acta, 33
(1971)
*q--227
218
PAL
active extracts were evaporated directly in glass vials. The radioactive samples were counted after using 15 ml of scintillator solutions at an average efficiency of 35% for 3H with a background of 14 counts/min. Sample measurements were corrected for quenching by the internal standardisation method. The background and radioactive samples were counted long enough to collect IOOOO counts. The paper chromatograms were prepared from a sheet of Whatman No. 2 filter paper without any previous washing and r-cm wide lanes were cut out between the strips. Chromatograms were equilibrated for at least 3 h and developed at room temperature, between zo-22', keeping the tanks in a wooden draft-proof box, later on in a thermostatically controlled hot box at 25". The following solvent systems of Bush were used throughout the work: (A) IOOO ml. pet. ether (b.p. 100-120“) 800 ml methanol, 200 ml water (v/v). (B) 667 ml toluene, 333 ml pet. ether (b.p. 1oo-12o~), 600 ml methanol, 400 ml water (v/v). Ultraviolet lamps “Chromatolite” (Hanovia Ltd., Slough, U.K.) and Uvis (Desaga, Heidelberg) were used throughout this work. A rotary-$lm evaporator (Rotavapor) R was supplied by Biichi (Flawil, Switzerland). The fluorescence reagent was prepared by slowly adding 8 vol. concentrated sulphuric acid from a burette to 2 vol. ice-cold 90% ethanol, the mixture being cooled in ice water. This reagent was made freshly every day. Fluorescence measurements were performed in an Aminco-Bowman Spectrophotofluorometer (American Instrument Company Inc., Silver Spring, Md., U.S.A.) at zero sensitivity and meter multiplier set at 0.01 using silica cuvettes of r-cm light path (external dimensions: 1.2 cmzx4.8 cm high, internal volume 4.8 ml) at fluorescent wavelength 530 nm, activating wavelength 480 nm. No filters were incorporated in the light path. The slit system was as follows: No. I, I/S in., No. 2, r/16 in., No. 3, I/S in., No. 4, I/S in., No. 5,1/16 in., No. 6, I/S in., photomultiplier slit, 1/16 in. A IP 21 photomultiplier tube was used. In certain experiments a Moseley Autograph Model I X-Y recorder (F. 0. Moseley Co., Pasadena, Calif., U.S.A.) was coupled to the fluorimeter. A Pye series 104 gas chromatograph Model 64 fitted with dual flame ionization detectors was used (Pye Unicam Instruments, Cambridge, U.K.). All samples were injected on to the column in solution (benzene or acetone) with a Hamilton I-$ syringe fitted with a rr.5-cm long needle. The gas-liquid chromatography columns 3% SE-30 on 100/120 mesh Gas Chrom 2 in 7-ft. long glass columns, 0.4 cm internal diam., 3% QF-I on 100/120 mesh Gas Chrom Q in 9-ft. long glass columns, o.4-cm internal diam. were purchased ready packed from Pye Unicam Instruments. METHODS Collection of urine specimens Urine was collected over a period of 24 h without any preservative and stored at 4’ until processed. When the volume was less than 500 ml (in case of children) it was made up to this volume with distilled water; urine collections of over 500 ml but of less than I litre were made up to I litre, and those of over I litre were made
URINARYTESTOSTERONEAND
219
EPITESTOSTERONE
up to I litre, and those of over I litre were made made up to 2 litres; distilled water was used in each case. Extractio?z of free q-ketosteroids
and free testosterone
A 5o-ml aliquot of urine from a 24 h collection was extracted in a 2oo-ml capacity separating funnel 3 times with equal volume of diethyl ether. This ether extract was rejected. Enzymatic
hydrolysis of the urine
A zo-ml aliquot of urine was taken in duplicate in glass-stoppered tubes, the pH was adjusted to 5.2 with 2 N hydrochloric acid. To each urine sample, approximately 3000 counts/min of [1,2-H3]testosterone (N)-3-D-glucuronide was added. The contents of the tubes were mixed well and Helix pomatia in buffer (I ml containing IOOOO units of ,%glucuronidase and 5000 units of sulphatase) at pH 5.2 was added to each tube. This was incubated at 37” for 24 h. After this incubation period, another r-ml portion of Helix pomatia (of same concentration as above) was added and incubated for a further period of 48 h. of hydrolysed urine on a Sephadex columlz The hydrolysed urine samples were filtered through a fluted Whatman filter paper No. I of 7.5-cm width and the urines were percolated through a Sephadex column and eventually eluted with distilled water and 6x IO-ml fractions of the eluate were collected. The radioactivity was always found in the second and third fractions. They were pooled together.
Fractionation
Extraction
of steroids
The pooled eluates were extracted 3 times with IO ml of ether and the combined ether extract was washed 3 times with 5 ml N sodium hydroxide solution and 3 times with 5 ml of water and then evaporated to dryness in a rotary-film evaporator. For blank, 20 ml of water was taken, 0.2 ,ug and 0.4 pg of testosterone glucuronide in 20 ml of water were treated as standards. These were also taken in duplicate and were processed as mentioned above. Paper chromatography
The extract was quantitatively transferred to a centrifuge tube with methylene dichloride and this was evaporated to dryness. The residue was dissolved in 0.02 ml of methanol and was spotted on a z.5-cm wide Whatman No. 2 filter paper. Similarly, a mixture of reference testosterone and epitestosterone (2 ,ug of each) was spotted as a guide strip (in duplicate), also, a chromatogram was prepared from an extract of a pooled urine. Chromatograms were developed in a Bush A system, solvent being run for 16 h after overnight equilibration and the chromatograms were dried at room temperature. The guide strips containing reference testosterone and epitestosterone, the chromatogram containing extract from pooled urine, were examined under a UV lamp. The positions of testosterone and epitestosterone were located on the chromatograms. Parts of the chromatograms containing steroids were cut out and eluted with methylene dichloride and were evaporated to dryness. Testosterone and epitestosterone were spotted separately on a 2.5-cm wide Whatman No. z filter paper C&?t. Chim. Acta, 33
(1971)
215-227
220
PAL
and were chromatographed in a Bush Bz system under the same conditions as before. Testosterone, epitestosterone and the guide strips were located and eluted as above. The eluates were evaporated to dryness and dissolved into IO ml of 85% methanol in water and partitioned against 2 ml petroleum ether (b.p. 80-100”).The petroleum ether layer was removed by suction and the methanol was evaporated to dryness under reduced pressure in a rotary film evaporator. Fluorimetry The fluorimetric determination was carried out by adding I ml sulphuric acidethanol reagent from a burette, at r-min intervals, to all the tubes containing steroid residues including blanks and standards. The contents were mixed well and the tubes were warmed up at 60” in a water bath for 15 min and cooled immediately in ice water. The samples were diluted with 1.5ml 95% ethanol, and were mixed well. Fluorescence was read after 15 min at I-min intervals. The activation wavelength and fluorecent wavelength
were 480 nm and 530 nm respectively.
Calculation The fluorescence of the extracts was examined in terms of fluorescence intensity (F.I.) given by multiplying the galvanometer reading by the meter multiplier setting of the spectrophotofluorimeter. Urinary testosterone and epitestosterone per 24 h is calculated as follows : F.I.
of sample F.I.
testosterone of standard
in standard
vol. of 24-h urine
IO or 20 ml urine
Wavelength On various occasions the activation and fluorescence wavelengths were checked for solutions of authentic testosterone and epitestosterone which were used as reference steroids. In both cases, maximum fluorescence was obtained at 530 nm when the activation wavelength was 480 nm at 15 min in 60”. The activation fluorescence spectra of urinary extracts from normal human individuals and pathological urines (at high and low concentrations) were parallel as observed with authentic testosterone and epitestosterone and extracts from testosterone and epitestosterone added to urines. Fluovimetric
examination
of a number of steroids relative to testosterone
and epitestosterone
in sul~huric acid-ethanol reagent The fluorescence of steroids available during the course of this work, some of which had chromatographic mobilities similar to those of testosterone and epitestosterone, were estimated according to the fluorimetric procedure mentioned in this text. It is well established that the presence of a double bond in a steroid molecule is a necessary requirement for fluorescence but not essential. Various workers have reported the absence of a characteristic structure in a steroid molecule responsible for sulphuric acid-ethanol-induced fluorescence. 0.4 ,ug of each steroid was taken for estimation. The fluorescence of testosterone and epitestosterone was set at = 100% (see Table I). From this it was found that these steroids did not interfere with the fluorescence of testosterone and epitestosterone.
URINARY TABLE
TESTOSTERONE I
FLUORIMETRIC -
EXAMIKATfOR
OF VARIOUS
STEROIDS
Retatwe 0;’ flUOYeSCf&
Relative y. j?ztcwescence of testosterone
Testosterone Epitestosterone Testosterone acetate Androstenedione Cortisol Corticosterone Desoxycorticosterone r7,GHydroxyprogesterone Cortisone Androsterone Etiocholanolone Epiandrosterone Dehydroepiandrosterone Androstanediol Etiocholanediol Pregnanediol Pregnanctriol I r~-~~~drox~androstelledione An amount METHODS.
221
AND EPITESTOSTERONE
=
100%
epitestostevone
100
IO0
TOO
100
65 35 28 20
15
of =
IOOa/~
66
35 29 21 ‘5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
of 0.4 pg of each steroid was estimated by the fluorimetric procedure described in The Auorescence of testosterone and epitestosterone were set at = IOO”/~in each case.
Accuracy. The accuracy of the method was assessed by determination of the recovery of testosterone and epitestosterone, when testosterone glucuronide and epitestosterone alcohol were added to normal urine and to distilled water respectively. An aqueous solution of testosterone glucuronide and epitestosterone alcohol (0.1 ,ug/ml of each steroid) were added to zo-ml aliquot of normal urine and to zo-ml distilled water to give a concentration of 0.1-0.4 pug,taken through the entire procedure; the mean recoveries from 20 experiments in each case, were 70.6% and 73.2o/o from water and 60.8% and 71% from urine. Similarly, from 20 experiments after adding 0.005 ,uC of pH]testosterone glucuronide, the mean recovery of testosterone was 74.6% and 68.3% from water and urine respectively. Precision. This was examined in terms of the degree of agreement of replicate estimations. The volume of a 24-h normal male urine collection was adjusted to 2 litres with distilled water and was divided into 20 equal parts. The concentration of testosterone and epitestosterone were determined on each sample. The mean value of testosterone was 73.4 ,ug/z4 h -& 5.8 (S.D.). The coefficient of variation was 7.9%. The mean value of epitestosterone was 26.3 ,ug/24 h & 2.6 (S.D.). The coefficient of variation was g,go/O.Similarly, from a normal female urine. The mean value of testosterone was 6.2 ,ug/24 h & 0.51 (SD.). The coefficient of variation was 8.2%. The mean value of epitestosterone was 3.4 ,ug/24 h f 0.35 (SD.). The coefficient of variation was ro.3%. S~ec~~c~~~}. The specificity of testosterone was investigated in terms of constant specific activity by the addition of a known amount of iPHItestosterone glucuronide (60000 disint./min in 0.5 pg) to an aliquot (400 ml) from a pooled male urine and was taken all through the procedure. A large Sephadex column (60 cm x 5 cm) was used for the fractionation of the hydrolysed urine sample. After the separation and purification of [3H]testosterone and epitestosterone, they were carried on further as follows. C&B.Chin%Acta, 33 (1971)
215--2’27
PAL
222
+
/z&h measured fluorimctrically I
/ 4 remainder acetylated chromatographed in Bush A system
Specific activity 702 disint. /min /pg
eluted [3H]Testosterone
J remainder saponified with sodium bicarbonate, chromatographed in Bush A system
4 I 150th counted
acetate
4, r Iroth measured fluorimetrically
J I /5oth counted
Specific activity 718 disint./min/pg
eluted [3H]Saponified $ r 15th measured fluorimetrically
testosterone 4, r /2oth counted Specific activity 686 disint./min//~g
During this experiment, pure testosterone was acetylated and saponified in parallel with [3H]testosterone. These derivatives were used as reference steroids for paper chromatography and as standards for the fluorimetric estimations respectively. The specificity of testosterone was also examined in terms of chromatographic mobility of derivative made out of testosterone isolated and pure testosterone at the same time. A portion of testosterone isolated was oxidised with chromic acid in parallel with pure testosterone. These oxidation products and 20 ,ug androstenedione were spotted on 3 separate chromatograms. When these chromatograms were developed in a Bush A system and were treated with Zimmermann reagent, single spots jn identical positions on each chromatogram positive to Zimmermann reaction were shown. Acetates, formates and propionates of testosterone isolated were also prepared at the same time with pure testosterone and the products formed were chromatographed on papers in parallel. When these chromatograms were treated with suitable reagents, single steroid spots in identical positions were shown. The specificity of epitestosterone was examined in various ways. After preparing derivatives such as acetates, formates, propionates and chromic acid oxidation product, and comparing their chromatographic mobilities against the derivatives prepared from pure epitestosterone, a single steroid spot from epitestosterone isolated was found in the identical position of the pure epitestosterone on the chromatogram in each case when developed in parallel. When pure epitestosterone was added to epitestosterone isolated and developed on paper chromatogram, a single steroid spot was found in each case. To dried sample of epitestosterone, 0.3 ml of 0.5% resorcylaldehyde-glacial acetic acid (v/v) were added. After heating for 8 min at 95” a blue colour developed Clin. Chim. Ada,
33
(1971)
215-227
URINARY
TESTOSTERONE
and the absorption testosterone
AND
EPITESTOSTERONE
spectra were determined.
isolated
223 The epitestosterone
from the urine pool gave identical
standard
and epi-
spectra with an absorption
maximum of 585 nm and a peak at 560 nrn11p7. A known amount of pure epitestosterone (3 ,ug) and epitestosterone isolated were heated in 98% formic acid at 95” for 30 min, the fluorescence spectra were determined in the Aminco-Bowman spectrophotofluorometer. Pure epitestosterone and the epitestosterone isolated showed an excitation maximum of 400 nm and a fluorescence maximum of 555 nm. Testosterone didnot fluoresce under these conditions12’9. In three women who had undergone bilateral oophorectomy no testosterone or epitestosterone was detected in the urine.
and adrenalectomy,
Sensitivity. Urine extracts containing 0.01 ,ug of testosterone and 0.01 peg of epitestosterone gave readings on the fluorimeter 6 times above the reagent blank and 4.5-5 times above the paper blank. It was found that less than 0.8,ug/z4 h testosterone or epitestosterone was not significantly different from o, when the volume of 24-h urine collection was 2 litres. Practicability. A fully trained person, when well organised, should be able to perform IO analyses in duplicate per week consisting of 54 working days. From the criteria of reliability of the method examined, it is shown that this method is highly reproducible. Determination of testosterone and e$itestosterone by gas-liquid chromatogra@y The gas-chromatographic estimation of testosterone and epitestosterone was performed on 3% QF-I on 80/100 mesh celite glass columns 9 ft. x 0.4 cm at 235” with nitrogen flow rate 80 ml/min, 3% SE-30 on 100/120 mesh Gas Chrom Z glass columns 7 ft. x 0.4 cm at 230’ with nitrogen flow rate 50 ml/min using trimethylsilyl derivative which was prepared by trimethylchlorosilane. Sample of final testosterone and epitestosterone extracts containing 0.2-0.4 ,ug of each steroid as measured by fluorescence gave values in close agreement when analysed by gas chromatography. Moreover, in each instance, only the single testosterone and epitestosterone peaks were seen, suggesting purity of the final fraction. Under these conditions, 0.2 ,ug of T.4BLE II COMPARISON
OF
THE
PRESENT
FLUORIMETRIC
METHOD
WITH
A
GAS-LIQUID
CHROMATOGRAPHIC
PROCEDURE
Urine
Testosterone
Epitestosterone
Fluorimetry (~124 h) I
II.2
i
4.6 3.2 II.4 5.3 7.5 20.0 14.6
9 10
48.7 106.5
2
3 4 ii
All these estimations the
Gas chromatography (0~123 h)
duplicate
Fluorimetry (zwi24 h)
II.4 4.7 3.1
5.3
11.8
4.8 IO.1
5.0 10.3
5.5
7.4 14.9 20.3 49.0 106.2 were
estimations
done did
in duplicate not
Gas chromatography (N/24 h)
vary
by
and more
the
mean
than
2.1
5.5 2.4
1.8
2.0
8.3 2.1
8.4 2.2
10.2
IO.0
23.8
25.2
38.6
24.1
results
are
given.
The
difference
between
50/:. Clin.
Chim. Acta, 33
(1971)
215-227
PAL
224
testosterone gave a peak height of 20 mm with a relative retention time of 0.50 in relation to cholestane and same amount of epitestosterone gave a peak height of 15 mm with a relative retention time of 0.40. Samples of final testosterone and epitestosterone extracts containing o.z-0.4pg of each steroid as measured by fluorimetric method, as described, gave values in close agreement when analysed by gas chromatography, also, in each case, only the single testosterone and epitestosterone peaks were seen, implying purity of the final fractions (see Table II). RESULTS
The average excretion values of testosterone and epitestosterone from IOO normal women, IOO normal men and 21 normal children, 9 girls and 12 boys (Table III) are presented. All the adult subjects and children studied were in a normal healthy state without any known endocrine, kidney or liver disorders. TARIX III URIrhRYEXCRETION AND -I
CHILDREN
OF
OF
TESTOSTERONE
VARYING
AND
~-___I-.-
Numbcv, SPX
-.I.I-
Age
-. 30 w. 30 12'. 30 w.
18-28
jI-61
4’.
JJ.
m.
yl
In.
20-30 y, 31-41 y.
30 m 423.2 y. IO Ii,. 53-63 Y. 100 m. 9 girls 7-12 y. (2 girls post-pubertal) 12 boys 6 y. 6 m.-r3 (3 boys post-pubertal) _“..__..-
.__.
MES,
WOMEN
--
I.1
(1.4-13.8)
2.j
I.3
(0.8-2.1)
0.9
(oh1.2)
2.0
(ox-rz..*)
4.7 (0.8-13.8) 6z.6 (24.591II.j) 59.3 (22.5--176.-t) j7.s (23.X-55.7) 16.4 (13.0-23.7) 47.6 (13.0-176.4) 0.78 (0.2-1.6)
I00 xv. 30
XORMAL
T&OdWOW ~~~t~~t~st~~an~ (means -+ vmages in @w&hews) I~ -. ~ -.i-. 54 (I.&13.2) 3.3 (o.%r2.4) 5.x (I&1P.l) 3.2 (1.0-8.1)
29-39 Y. 40-50 y.
IO w.
(,UUg/24 h) IS
EPITESTOSTEROSE
AGES
4’.
1~3
-_-.
(0.4-3.2)
45.9
..~
(0.X-11.6)
(I6.2-93.2)
38.8 (20.2%1OO.I) 40.5 ('2.2-30.5) 11.-j (XL-17.6) 31.4 (8.c-Ioo.x) O.j0
(0.2-1.1)
0.90 (0.3-2.6) -
I.“._- -
From I00 normal women (age r8-61 years) the mean values of testosterone and epitestosterone were 4.7 pug/z4 h (range 0.8-13.8) and 2.9 pug/z4 h (range 0.6-12.4) respectively. These women were divided into 4 age groups at IO-yearly intervals, and 30 subjects from one group, aged between 29 and 39 years, gave the highest values of testosterone and epitestosterone, mean 5.8 pg/z4 11(range 1.0-12.1) mean 3.2 ,~q/‘z4 h (range r.o-S.1) respectively. From IOO normal men (age 20-63 years) the mean values of testosterone and epitestosterone were 47.6 ,,xgl24 h (range 13-176.4) and 31.4 ,~g/24 h (range 8.2-100.1) respectively. As before, from 4 age groups, 30 men aged between 42 and 52 years, showed a marked decline in testosterone and epitestosterone excretion, mean 57.8 ,ug/24 h (range 23.x--55,7) and mean 40.5 yg/z4 h (range 12.2-30.5) respectively. From 9 girls (age 7-12 years) (z girls were post-pubertal) the mean values of testosterone and epitestosterone were 0.7s pgj24 h (range 0.2-1.6) and 0.50 pug/24 h (range 0.2-x. I) respectively. Cli,n. Chim. Ada,
33
(1971)215~-227
URINARY
TESTOSTEROKE
AND
225
EPITESTOSTEKONE
From 12 boys (age 6 years 6 months-13 years) (3 boys were post-pubertal) mean values of testosterone and epitestosterone were 1.3 rug/24 h (range 0.4-3.2) 0.90 rug/24 h (range 0.3-2.6) respectively. TABLE
the and
IV
URINARY
EXCRETION
EKDOCRINE
OF TESTOSTERONE
AND
EPITESTOSTERONE
h) IN MEN, WOMEN AND
(/l&z4
CHILDREN
WITH
DISORUERS
Disease
Cast5
Ages
Addison’s disease hdrenal adenoma Adrenal carcinoma Adrenal hyperplasia Adrenal tumour Congenital adrenal hyperplasia
Delayed puberty Eunuchoidism Hirsutlsm Hypopituitary hypogonadism Impotence Precocious puberty Primary testicular hypogonadism St&-Leventhal syndrome Virilizing adrenal tumour Virilizing ovarian tumour
Pathological values In Table IV the values of testosterone various endocrine disorders, are given.
Testosterone
y.
$vzth.
I6.3-j3.5
I””
16.7-20.5
none none
2”.4-34,7 130.2-436.4
100 100
68-j-352.7
32”.3-504.7 220.7-420.’ 16.5-215.6 13.4-250.2 16.X-74.6 3.6-10.6 1.2.5-26.1 3.2-147.” 9.7-30.’ 8.8-18.3 4.6-8.3 6.8-20.6 4.8-70.8 1X8.2 -4’2.3
IO”
273.4-380.7
100 100 IO0 100 100
25 58.1 28.6 5”
Epitestosterone 7.8-20.1 10.3-12.8 8.8-12.5 93.1-446.7 45.8-418.8 198.7-224.3 130.4-218.2 7,5-‘73.2 5.6-162.4 10232.8 1.9-4.1 5.8-12.5 1.3-80.6 4.3-12.3 4 3-X.8
o/o patk.
__-
5” llOtlf2 none TOO IO0 IO0 IO0
57.’ IO0 IO0 ‘“0 75 29 71.4 7.5 IO0
87.1 1””
3.1-3.7 r,7-11.7 2.3-36.7 150.6-326.5
100
17X.3-2j8.4
IO0
IO0 30
and epitestosterone
77.8 42 IO0
in patients with
DISCUSSIOK
The method described here for the determination of urinary conjugated testosteronc and epitestosterone is simple and reproducible. It is suitable for use in normal healthy men, women and particularly in children and in patients with various endocrine disorders. A fluorimetric technique is applied due to its extremely high sensitivity. The fluorescence of testosterone in sulphuric acid has been previously reported13-16. This reaction is relatively specific; also, epitestosterone in sulphuric acid is highly fluorogenie like testosterone. Sulphuric acid-ethanol-induced fluorescence for testosterone determination was first described by Wilsonl’ and, by an adaptation of this reaction, testosterone and epitestosterone were first measured in normal adults and in patientsz. Testosterone is primarily excreted as the glucuronide’*lR, a very small quantity is also excreted as sulphate and as the free steroidI*. It has heen reported that testosterone sulphate was identified and measured from pooled normal male urine in the range of 5-10 pug/q hl9. An appreciable amount of free testosterone could be excreted in urine under certain clinical conditions. Therefore, in the piesent method, free steroids are extracted before an attempt is made to measure the testosterone glucurC&L. Chim. Ada,
33 (1971) 2r5-227
226 onide after enzyme hydrolysis.
PAL Acid hydrolysis
was avoided as this forms the artifacts
of steroid molecules or transformation products 20. It was found from the published work that simultaneous benzene extraction and acid hydrolysis method for determining urinary testosterone in men, women and children, interfered with gas-chromatographic assay of testosterone in some urine specimens, especially from women21. It was concluded that the method should be limited to use with urine specimens from male subjects only. In the other published methods employing acid hydrolysisS2, it was found that this procedure interfered with the gas-chromatographic assays and the required sensitivity needed for application to children’s urine could not be achieved. There was no significant increase of 24-h output of testosterone and epitestosterone when solvolysis procedure was included with the method; similar findings have already been reported. Moreover, this step released various pigments from the urine which were contaminating the extracts. Therefore, solvolysis procedure23 was not used. The fractionation of enzyme-hydrolysed urines on the Sephadex columnl” and paper-chromatographic separation of testosterone from epitestosterone and their further purification in the same way, appeared to achieve a specific determination of these two steroids without any appreciable loss. In certain clinical conditions it was found useful to measure epitestosterone at the same time as testosterone. Little is known at the present time about the origin and significance of epitestosterone excretion. It has been shown thatZ4 epitestosterone is not derived from testosterone and, although its production rate is only 3% of that of testosterone, its excretion rate is about 33o/Oof that of testosterone in the adult male; the excretion of epitestosterone was consistently higher than normal range in all hirsute patients 25. It was also reported, separately, that the rate of formation of epitestosterone from testosterone in blood was 30 times higher in hirsute patients than in normal men and women, irrespective of virilism. The sulphuric acid-ethanol-induced fluorimetric estimation of testosterone and epitestosterone appears to be very sensitive and specific. Various other steroids which could interfere with the fluorescence are easily separated by the chromatographic systems employed. When this method is compared with a gas-chromatographic procedure developed by the author, fair agreement between the results is found. The values for urinary testosterone and epitestosterone in adult women and men, in literature, cover a wide range. The values from adults without any known endocrine disorders, given here, are in good agreement with the values published elsewhere’0,‘4,26-31. In children, the values for pre-pubertal children also agree well with those of other workers. Negligible amounts of these steroids are found in prepubertal children32-37. Increased urinary concentrations of testosterone and epitestosterone have been found in various patients with different adrenal disorders such as adrenal adenoma, adrenal carcinoma, adrenal hyperplasia, congen;tal adrenal hyperplasia and virilizing adrenal tumour. Some of the patients suffering from hirsutism and Stein-Leventhal syndrome also give high values for testosterone and epitestosterone. From these results, therefore, it appears that the method described here provides reliable conditions for the separation and quantitative determination of urinary conjugated testosterone in human adults and in children.
Clin. Chim. Acta, 33 (1971) 215-227
URINARY
TESTOSTERONE
AND
227
EPITESTOSTERONE
ACKNOWLEDGEMEXTS
I wish to thank Professor Dr. E. F. Pfeiffer, Professor Dr. W. Teller and other physicians and surgeons for the interest which they took during the course of this work; Professor W. Klyne, Westfield College, London, for generously providing reference compounds from the Medical Research Council Steroid Reference Section; Professor V. H. T. James, St. Mary’s Hospital Medical School, London, Dr. R. W. H. Edwards, Institute of Child Health, London, British Postgraduate Medical Federation, Dr. G. H. Thomas, University of Birmingham and Dr. M. B. Lipsett, Endocrinology Branch, National Cancer Institute, Bethesda, Maryland, U.S.A. for kindly going through the manuscript and making valuable suggestions. Finally, my thanks go to Mrs. M. R. Pal, for help in preparing
the manuscript.
REFERENCES I B. E. I'.MURPHY,
Recent
Progr.
Hormozolte Res.,
25 (1969)
563.
H. WILSONAXD M. B.LIPsETT,J.C~~~. Invest.,42 (1963) 1753. 3 S. G. KORENMAX, T. E. DAVIS, H. WILSONAXD M. B.LIPSETT, Steroids, 3 (1964) 203. 4 S. LIEBERMAN, D. K. FUKUSHIMA, K. DOBRINER, L. B. HARITOX AXD B. PRAETZ, J. Biol. 2 S. G. KORENMAN,
Chem., I82 (1950) 299. 5 R. ANLII~ER, 0. ROHR AWD M. MARTI, Helu. Chim. Acta, 39 (1956) IIOO. 6 K. SCHUBERT AND E. WEHRBERGER, Naturwissenschaften, 47 (1960) 281. 7 S. G. KORENMAN, H. WILSONAND M. B. LIPSETT,J. Biol.Chem., 239 (1964)
8 9 IO II IL 13 14 I5
16 17 18 I9
20 21 22 21
24 25 26 27
28 29
30 3I 32
22 dd 34
35 36 37
1004. R. V. BROOKS AND G. GIULIANI, Steroids,4 (1964) IOI. J. BLAQCIER, R. I. DORFMAX AND E. FORCHIELLI, A&, Endocrinol., 54(Ig67) 208. AUDREY MOXHAM AND J.D.N. NABARRO,~~~~. Chim.Acta, 22 (Ig68)385. B. B. L. BROOKSBANK AND G. A. D. HASLEWOOD, Biochem. J., 80 (1961) 4X8. R. J, BOSCOTT, Nature, 164(194g) 140. J, H. LINFORD AND 0. B. PAULSON,CUX.J. Med. Sci., 30 (1952) 213. J.W. GOLDZIEHER, J.M.BODENCHUKA~D P.NOLAX, AnaZ.Chem., 26(I9j4) X53. H. KALANT, Biochem. J., 6g (1958) 79. H. GERDES AND W. STAIB, Steroids, 6 (1965) 793. H. WILSON, Anal. Biochem., I (1960) 402. A. RI.CAMACHO AND C. J, MIGEON, 1. Clin.Endocrinol., 23 (1963) 3oI. .4. DESSYPRIS, M. A. DROSDOWSKY, pi. L. MCNIVEN AND R. I. DORFMAN, Pvoc. Sot. Exp. Biol. Med., 121 (1966) 1128. R. I.DORFMAN AND F. UNGAR, in Metabolism of Steroid Hormones, Academic Press, New York, 1965, p. 28. P. VESTERGAARD, E. RAABO AXD S. VEDSQ,CZ~~. Chim. Acta, 14(rg66) 540, A. A. A. ISMAIL AXD R. A. HARKNESS, Biochem. J., 99 (1966) 717. S. BUNSTEIN AND S. LIEBERMAN, I.Biol. Chem., 211 (1958) 331. H. WILSON AND M. B. LIPSETT, J: Clin. Endocrinoi.: 26 (1966) 902. A. F. DE NICOLA, R. I. DORFMAN AND E. FORCHIELLI, Strvoids, 7 (1966) 351. W. FUTTERWEIT,N.L.MCNIVEN,L.NARCUS,C.LANTOS,N.DROSDOWSKYAND R.I.DoRFMAX, Steroids, I (1963) 628. H. IBAYASHI, N. NAKAMURA, S.MURAKA~A, T. I!CHIKAWA, T. TANIOKA AXD K.NAKAO, Steroids.3 (1964) 559. R. V. BROOKS, Steroids, 4 (1964) 117. TM. SPARAGANA, Stevoids, 5 (1965) 773. J. W. MCROBERTS, A.D. OLSOX AND W.L. HERRMANX,CZ~~. Chem., I4 (Ig68)565. A. PETERS AND W.CULLEY,C~~~. Chim. Acta, 25(Ig6g) 199. A. VERMEULEN ABD J. C. M. VERPLANCKE, Steroids, 2 (1963) 453. T.M.RosxER.N.F.CONTE. T.H.BRIGGs.P.Y.CHAO. E.M. SUDMAN AXD P.H.FORSHAM, j. Clin. Endockinol., 25 (1965; 95. W.W. CLEVELAND,N.AHMED, D.H.SAXDBERGAND K.SAVARD, Steroids, 8 (1966) 149 D. KNORR, Acta Endocrinol., 54 (1967) 215. D. GUPTA AND HJLLARY BUTLER, Steroids, 14 (1969) 343. H. J. DEGENHART, H. K. A. VESSEL AND R. WILMINK, Pediat. Rex., 4 (1970) 309. Clin. Chim.
Acta,
33 (1971) 215-227
CHIMICA ACTA
URINARY
EXCRETION
MEN, WOMEN
215
OF TESTOSTERONE
AND CHILDREN,
IN HEALTH
AND EPITESTOSTERONE
IN
AND DISEASE
S. B. PAL
Universitiit Ulm, Abteilung Endokrinologie und Stoffwechsel, Zentrum fiir innere Medizin und Kinderheilkunde, D 79 Ulm (Donau) (D.B.R.) (Received
December
zg, 1970)
SUMMARY
A fluorimetric method for the determination of urinary conjugated testosterone and epitestosterone in human subjects is described. According to this method a Io-zo-ml aliquot of a 24-h urine collection was hydrolysed with #I-glucuronidase and sulphatase at pH 5.2 for 72 h, and the hydrolysed urine was filtered and fractionated on a Sephadex column. After the extraction of the liberated steroids, testosterone was separated from epitestosterone by paper chromatography and they were then further purified in the same way. Testosterone and epitestosterone were eluted from paper chromatograms and, after further purification, were measured fluorimetrically with a sulphuric acid-ethanol-induced reaction. After the addition of testosterone glucuronide and epitestosterone alcohol (0.1 pg/ml of each steroid) to give a concentration of 0.1 ,ug-0.4 ,ug to 20 ml water and to a zo-ml aliquot of normal urine, taken through the entire procedure, the mean recoveries of testosterone and epitestosterone from 20 experiments, in each case were: 70.6% and 73.2% from water and 68.8% and 71% from urine. By the application of this method the average testosterone and epitestosterone excretion in IOO normal women (age 18-61 years) was 4.7 pg/z4 h (range 0.8-13.8) and 2.9 pg/z4 h (range 0.6-12.4) respectively; in IOO normal men (age 20-63 years) 47.6 pug/z4 h (range 13-176.4) and 31.4 pg/z4 h (range 8.2-100.1) respectively; and out of 21 normal children, 9 girls (age 7-12 years) 0.8 pg/z4 h (range 0.2-1.6) and 0.5 rug/z4 h (range 0.2-1.1) respectively; 12 boys (age 6-13 year-s) 1.3 pg/z4 h (range 0.4-3.2) and 0.9 ,ug/z4 h (range 0.3-2.6 ) respectively. Results from patients with various endocrine disorders are presented. This method was compared with a gas-liquid chromatographic procedure and good agreements were found. The method appeared to be a clinically useful one.
INTRODUCTION
It is generally considered that the quantitative determination of free testosterone (17/Chydroxyandrost-4-en-3-one) in plasma is the best available parameter of androgen secretion. Testosterone is the most powerful naturally occurring androCliPZ.Chim. A&Z. 33
(1971)
215-227
216
PAL
gen-it is secreted largely by the Leydig cells of the testis in men, and small amounts are also produced by the adrenal cortex in both sexes and by the ovary in women. However, Murphy1 reported that of all the steroids tested so far by the competition of various steroids with tritiated testosterone in late pregnancy plasma, dihydrotestosterone was found to be the most potent, being about three times as effective as testosterone itself. Tissues of non-endocrine origin can also convert steroid precursors, especially d4-androstenedione (androst-4-ene-3,r7-dione) and dehydroepiandrosterone (@hydroxyandrost-5-en-r7-one) to testosterone. A number of methods for the determination of free testosterone in plasma have been published in recent years but all these methods are very time consuming and are not always suitable for diagnostic work. The determination of urinary testosterone or urinary production rate&” are very useful in some clinical investigations such as in women with Cushing’s syndrome, SteinLeventhal syndrome, virilizing ovarian neoplasm and idiopathic hirsutism, children with congenital adrenal hyperplasia, virilizing adrenocortical tumours and men with hypogonadism secondary to hypopituitarism or primary hypogonadism, eunuchoidism and impotence. Testosterone was first isolated from urine after parenteral administration of the steroid in large doses*. It was extracted from an adrenal carcinoma5. The isolation and determination of testosterone glucuronide from the mine of normal men and women was first reported by Schubert and WehrbergeP. Epitestosterone (r7a-hydroxyandrost-4-en-j-one) which is the r7-hydroxy epimer of testosterone, was first isolated by Korenman et aZ.7 from the urine of both normal men and women; isolation from normal human urine was also reported by Brooks and Giuliani* in 1964. As our knowledge of testosterone metabolism is becoming increasingly important, it was found necessary to develop a suitable method for the estimation of urinary testosterone and epitestosterone. In certain disease states, such as hirsutism and the Stein-Leventhal syndrome, the determination of epitestosterone level becomes meaningful in spite of the fact that little is known about its biological significance. It has been suggested that the origin of epitestosterone can be the resultant of both peripheral and endocrine gland secretions. Although testosterone glucuronide is not a unique metabolite of testosterone, the measurement of urinary testosterone is more closely related to testosterone production than is the determination of urinary 17ketosteroids under certain clinical conditions. There are various published methods for the estimation of urinary testosterone, testosterone and epitestosterone, separately or combined, but only a few are suitable for performing estimations in children’s urine. Testosterone is primarily excreted in urine as the glucuronide and a very minute amount of testosterone sulphate and free testosterone are also excreted. Therefore, the determination of urinary testosterone glucuronide appears to be a useful method to study androgen metabolism. The aim of the present study was to develop a clinically useful method to determine urinary testosterone and epitestosterone in men, women and particularly in children, and patients with various endocrine disorders. This method is in many ways similar to one which was previously publishedlO. After a preliminary study, good agreement was found when the present method was compared with a gas-liquid chromatographic procedure.
Clin. Chim.
Acta,
33 (1971) 215-227
URINARYTESTOSTERONE
AND
217
EPITESTOSTERONE
MATERIALS
All chemicals
used were of analytical
grade (Hopkin
& Williams
Ltd., U.K. and
E. Merck AG., Darmstadt). Sue &Helix pomatia. I ml contains IOOOOO units of /?-glucuronidase (Fishman), 50000 units sulphatase (Whitehead). (Industrie Biologique Francaise S.A., Quai du Moulin de Cage-Genevilliers, Seine, France). These quantities of enzymes, contained in a r-ml ampoule, were dissolved into 9 ml of 2 pvlsodium acetate-acetic acid buffer of pH 5.2 before use. Ketodase (/?-glucuronidase) 5000 units/ml (Warner Chilcott Laboratories, New Jersey, U.S.A.) was also used on several occasions. All solvents were of general purpose reagent grade (Hopkin & Williams Ltd.) and were further purified before use according to standard methods. Later on, all solvents used were of pro analysi grade (E. Merck AG). Glass-distilled water was used throughout the work. Glass-stoppered test tubes 15 x 2.5 cm approximate capacity 54 ml and 12.5 x I .5 cm approximate capacity 15 ml were used (Quickfit & Quartz Ltd, U.K.). A fro-cm long glass column (1.5 cm internal diam.) with a stopcock and sintered glass filter was packed with Sephadex G-25 fine for gel filtration (Pharmacia, Uppsala, Sweden) to a height of 30 cm, which was previously soaked in distilled water (changed on alternate days) for 14 days. The column had a reservoir of 50-ml capacity at its top and a ro-ml syphon for collecting the fractions. Testosterone, epitestosterone and other Yeference steroids used were obtained from Steroid Reference Collection (Medical Research Council, Chemistry Department, Westfield College, Hampstead, London, U.K.). Some of these steroids were also available from different sources as gifts, which, when needed, were further purified by paper chromatography and repeated crystallisation from methanol, ethanol and acetone. Melting points were determined on a Kofler hot stage and agreed with published figures. Testosterone glucuronide was purchased from Ikapharm Ltd., Ramat-Gan, 31, Jabotinski Road, Israel. [I+-3H]Testoste~one (N)-3,!-D-glucuronide (specific activity 30-50 C/mmole was purchased from New England Nuclear Corporation, Boston, Massachusetts, U.S.A. [r-%]n-Hexadecane was used as an internal standard for liquid scintillation counting which was purchased from Radiochemical Centre, Amersham, U.K. Toluene scintillator was prepared by adding 3.0 g of 2.5-diphenyloxazole (PPO) and 0.3 g of r,4-bis-z,4-methyl-5-phenyloxozolyl benzene (POPOP) both scintillator grade in I litre of purified toluene and was stored in an amber glass bottle at 4O. Toluene of scintillation grade from E. Merck was also used for preparing scintillator solution. Liquid scintillation countings were made in a Packard Tri-Carb Liquid Scintillation Spectrometer (Model 314 EX), automatic dual channel. Packard Radiochromatogram Scanner (Model 7201) was used for measuring and recording radioactivity on paper chromatograms. a&Diphenyloxazole (PPO), r,4-his-2,4-methyl-5-phenyloxozolyl (dimethyl-POPOP) and high quality glass vials (Low-Potassium-I) were all purchased from Packard Instrument Company, Inc., Box 429, La Grange, Ill, U.S.A. All the extracts for paper chromatography were evaporated to dryness throughout this work in a water bath at 40~ under a stream of nitrogen; similarly, the radioczin. Chim. Acta, 33
(1971)
*q--227
218
PAL
active extracts were evaporated directly in glass vials. The radioactive samples were counted after using 15 ml of scintillator solutions at an average efficiency of 35% for 3H with a background of 14 counts/min. Sample measurements were corrected for quenching by the internal standardisation method. The background and radioactive samples were counted long enough to collect IOOOO counts. The paper chromatograms were prepared from a sheet of Whatman No. 2 filter paper without any previous washing and r-cm wide lanes were cut out between the strips. Chromatograms were equilibrated for at least 3 h and developed at room temperature, between zo-22', keeping the tanks in a wooden draft-proof box, later on in a thermostatically controlled hot box at 25". The following solvent systems of Bush were used throughout the work: (A) IOOO ml. pet. ether (b.p. 100-120“) 800 ml methanol, 200 ml water (v/v). (B) 667 ml toluene, 333 ml pet. ether (b.p. 1oo-12o~), 600 ml methanol, 400 ml water (v/v). Ultraviolet lamps “Chromatolite” (Hanovia Ltd., Slough, U.K.) and Uvis (Desaga, Heidelberg) were used throughout this work. A rotary-$lm evaporator (Rotavapor) R was supplied by Biichi (Flawil, Switzerland). The fluorescence reagent was prepared by slowly adding 8 vol. concentrated sulphuric acid from a burette to 2 vol. ice-cold 90% ethanol, the mixture being cooled in ice water. This reagent was made freshly every day. Fluorescence measurements were performed in an Aminco-Bowman Spectrophotofluorometer (American Instrument Company Inc., Silver Spring, Md., U.S.A.) at zero sensitivity and meter multiplier set at 0.01 using silica cuvettes of r-cm light path (external dimensions: 1.2 cmzx4.8 cm high, internal volume 4.8 ml) at fluorescent wavelength 530 nm, activating wavelength 480 nm. No filters were incorporated in the light path. The slit system was as follows: No. I, I/S in., No. 2, r/16 in., No. 3, I/S in., No. 4, I/S in., No. 5,1/16 in., No. 6, I/S in., photomultiplier slit, 1/16 in. A IP 21 photomultiplier tube was used. In certain experiments a Moseley Autograph Model I X-Y recorder (F. 0. Moseley Co., Pasadena, Calif., U.S.A.) was coupled to the fluorimeter. A Pye series 104 gas chromatograph Model 64 fitted with dual flame ionization detectors was used (Pye Unicam Instruments, Cambridge, U.K.). All samples were injected on to the column in solution (benzene or acetone) with a Hamilton I-$ syringe fitted with a rr.5-cm long needle. The gas-liquid chromatography columns 3% SE-30 on 100/120 mesh Gas Chrom 2 in 7-ft. long glass columns, 0.4 cm internal diam., 3% QF-I on 100/120 mesh Gas Chrom Q in 9-ft. long glass columns, o.4-cm internal diam. were purchased ready packed from Pye Unicam Instruments. METHODS Collection of urine specimens Urine was collected over a period of 24 h without any preservative and stored at 4’ until processed. When the volume was less than 500 ml (in case of children) it was made up to this volume with distilled water; urine collections of over 500 ml but of less than I litre were made up to I litre, and those of over I litre were made
URINARYTESTOSTERONEAND
219
EPITESTOSTERONE
up to I litre, and those of over I litre were made made up to 2 litres; distilled water was used in each case. Extractio?z of free q-ketosteroids
and free testosterone
A 5o-ml aliquot of urine from a 24 h collection was extracted in a 2oo-ml capacity separating funnel 3 times with equal volume of diethyl ether. This ether extract was rejected. Enzymatic
hydrolysis of the urine
A zo-ml aliquot of urine was taken in duplicate in glass-stoppered tubes, the pH was adjusted to 5.2 with 2 N hydrochloric acid. To each urine sample, approximately 3000 counts/min of [1,2-H3]testosterone (N)-3-D-glucuronide was added. The contents of the tubes were mixed well and Helix pomatia in buffer (I ml containing IOOOO units of ,%glucuronidase and 5000 units of sulphatase) at pH 5.2 was added to each tube. This was incubated at 37” for 24 h. After this incubation period, another r-ml portion of Helix pomatia (of same concentration as above) was added and incubated for a further period of 48 h. of hydrolysed urine on a Sephadex columlz The hydrolysed urine samples were filtered through a fluted Whatman filter paper No. I of 7.5-cm width and the urines were percolated through a Sephadex column and eventually eluted with distilled water and 6x IO-ml fractions of the eluate were collected. The radioactivity was always found in the second and third fractions. They were pooled together.
Fractionation
Extraction
of steroids
The pooled eluates were extracted 3 times with IO ml of ether and the combined ether extract was washed 3 times with 5 ml N sodium hydroxide solution and 3 times with 5 ml of water and then evaporated to dryness in a rotary-film evaporator. For blank, 20 ml of water was taken, 0.2 ,ug and 0.4 pg of testosterone glucuronide in 20 ml of water were treated as standards. These were also taken in duplicate and were processed as mentioned above. Paper chromatography
The extract was quantitatively transferred to a centrifuge tube with methylene dichloride and this was evaporated to dryness. The residue was dissolved in 0.02 ml of methanol and was spotted on a z.5-cm wide Whatman No. 2 filter paper. Similarly, a mixture of reference testosterone and epitestosterone (2 ,ug of each) was spotted as a guide strip (in duplicate), also, a chromatogram was prepared from an extract of a pooled urine. Chromatograms were developed in a Bush A system, solvent being run for 16 h after overnight equilibration and the chromatograms were dried at room temperature. The guide strips containing reference testosterone and epitestosterone, the chromatogram containing extract from pooled urine, were examined under a UV lamp. The positions of testosterone and epitestosterone were located on the chromatograms. Parts of the chromatograms containing steroids were cut out and eluted with methylene dichloride and were evaporated to dryness. Testosterone and epitestosterone were spotted separately on a 2.5-cm wide Whatman No. z filter paper C&?t. Chim. Acta, 33
(1971)
215-227
220
PAL
and were chromatographed in a Bush Bz system under the same conditions as before. Testosterone, epitestosterone and the guide strips were located and eluted as above. The eluates were evaporated to dryness and dissolved into IO ml of 85% methanol in water and partitioned against 2 ml petroleum ether (b.p. 80-100”).The petroleum ether layer was removed by suction and the methanol was evaporated to dryness under reduced pressure in a rotary film evaporator. Fluorimetry The fluorimetric determination was carried out by adding I ml sulphuric acidethanol reagent from a burette, at r-min intervals, to all the tubes containing steroid residues including blanks and standards. The contents were mixed well and the tubes were warmed up at 60” in a water bath for 15 min and cooled immediately in ice water. The samples were diluted with 1.5ml 95% ethanol, and were mixed well. Fluorescence was read after 15 min at I-min intervals. The activation wavelength and fluorecent wavelength
were 480 nm and 530 nm respectively.
Calculation The fluorescence of the extracts was examined in terms of fluorescence intensity (F.I.) given by multiplying the galvanometer reading by the meter multiplier setting of the spectrophotofluorimeter. Urinary testosterone and epitestosterone per 24 h is calculated as follows : F.I.
of sample F.I.
testosterone of standard
in standard
vol. of 24-h urine
IO or 20 ml urine
Wavelength On various occasions the activation and fluorescence wavelengths were checked for solutions of authentic testosterone and epitestosterone which were used as reference steroids. In both cases, maximum fluorescence was obtained at 530 nm when the activation wavelength was 480 nm at 15 min in 60”. The activation fluorescence spectra of urinary extracts from normal human individuals and pathological urines (at high and low concentrations) were parallel as observed with authentic testosterone and epitestosterone and extracts from testosterone and epitestosterone added to urines. Fluovimetric
examination
of a number of steroids relative to testosterone
and epitestosterone
in sul~huric acid-ethanol reagent The fluorescence of steroids available during the course of this work, some of which had chromatographic mobilities similar to those of testosterone and epitestosterone, were estimated according to the fluorimetric procedure mentioned in this text. It is well established that the presence of a double bond in a steroid molecule is a necessary requirement for fluorescence but not essential. Various workers have reported the absence of a characteristic structure in a steroid molecule responsible for sulphuric acid-ethanol-induced fluorescence. 0.4 ,ug of each steroid was taken for estimation. The fluorescence of testosterone and epitestosterone was set at = 100% (see Table I). From this it was found that these steroids did not interfere with the fluorescence of testosterone and epitestosterone.
URINARY TABLE
TESTOSTERONE I
FLUORIMETRIC -
EXAMIKATfOR
OF VARIOUS
STEROIDS
Retatwe 0;’ flUOYeSCf&
Relative y. j?ztcwescence of testosterone
Testosterone Epitestosterone Testosterone acetate Androstenedione Cortisol Corticosterone Desoxycorticosterone r7,GHydroxyprogesterone Cortisone Androsterone Etiocholanolone Epiandrosterone Dehydroepiandrosterone Androstanediol Etiocholanediol Pregnanediol Pregnanctriol I r~-~~~drox~androstelledione An amount METHODS.
221
AND EPITESTOSTERONE
=
100%
epitestostevone
100
IO0
TOO
100
65 35 28 20
15
of =
IOOa/~
66
35 29 21 ‘5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
of 0.4 pg of each steroid was estimated by the fluorimetric procedure described in The Auorescence of testosterone and epitestosterone were set at = IOO”/~in each case.
Accuracy. The accuracy of the method was assessed by determination of the recovery of testosterone and epitestosterone, when testosterone glucuronide and epitestosterone alcohol were added to normal urine and to distilled water respectively. An aqueous solution of testosterone glucuronide and epitestosterone alcohol (0.1 ,ug/ml of each steroid) were added to zo-ml aliquot of normal urine and to zo-ml distilled water to give a concentration of 0.1-0.4 pug,taken through the entire procedure; the mean recoveries from 20 experiments in each case, were 70.6% and 73.2o/o from water and 60.8% and 71% from urine. Similarly, from 20 experiments after adding 0.005 ,uC of pH]testosterone glucuronide, the mean recovery of testosterone was 74.6% and 68.3% from water and urine respectively. Precision. This was examined in terms of the degree of agreement of replicate estimations. The volume of a 24-h normal male urine collection was adjusted to 2 litres with distilled water and was divided into 20 equal parts. The concentration of testosterone and epitestosterone were determined on each sample. The mean value of testosterone was 73.4 ,ug/z4 h -& 5.8 (S.D.). The coefficient of variation was 7.9%. The mean value of epitestosterone was 26.3 ,ug/24 h & 2.6 (S.D.). The coefficient of variation was g,go/O.Similarly, from a normal female urine. The mean value of testosterone was 6.2 ,ug/24 h & 0.51 (SD.). The coefficient of variation was 8.2%. The mean value of epitestosterone was 3.4 ,ug/24 h f 0.35 (SD.). The coefficient of variation was ro.3%. S~ec~~c~~~}. The specificity of testosterone was investigated in terms of constant specific activity by the addition of a known amount of iPHItestosterone glucuronide (60000 disint./min in 0.5 pg) to an aliquot (400 ml) from a pooled male urine and was taken all through the procedure. A large Sephadex column (60 cm x 5 cm) was used for the fractionation of the hydrolysed urine sample. After the separation and purification of [3H]testosterone and epitestosterone, they were carried on further as follows. C&B.Chin%Acta, 33 (1971)
215--2’27
PAL
222
+
/z&h measured fluorimctrically I
/ 4 remainder acetylated chromatographed in Bush A system
Specific activity 702 disint. /min /pg
eluted [3H]Testosterone
J remainder saponified with sodium bicarbonate, chromatographed in Bush A system
4 I 150th counted
acetate
4, r Iroth measured fluorimetrically
J I /5oth counted
Specific activity 718 disint./min/pg
eluted [3H]Saponified $ r 15th measured fluorimetrically
testosterone 4, r /2oth counted Specific activity 686 disint./min//~g
During this experiment, pure testosterone was acetylated and saponified in parallel with [3H]testosterone. These derivatives were used as reference steroids for paper chromatography and as standards for the fluorimetric estimations respectively. The specificity of testosterone was also examined in terms of chromatographic mobility of derivative made out of testosterone isolated and pure testosterone at the same time. A portion of testosterone isolated was oxidised with chromic acid in parallel with pure testosterone. These oxidation products and 20 ,ug androstenedione were spotted on 3 separate chromatograms. When these chromatograms were developed in a Bush A system and were treated with Zimmermann reagent, single spots jn identical positions on each chromatogram positive to Zimmermann reaction were shown. Acetates, formates and propionates of testosterone isolated were also prepared at the same time with pure testosterone and the products formed were chromatographed on papers in parallel. When these chromatograms were treated with suitable reagents, single steroid spots in identical positions were shown. The specificity of epitestosterone was examined in various ways. After preparing derivatives such as acetates, formates, propionates and chromic acid oxidation product, and comparing their chromatographic mobilities against the derivatives prepared from pure epitestosterone, a single steroid spot from epitestosterone isolated was found in the identical position of the pure epitestosterone on the chromatogram in each case when developed in parallel. When pure epitestosterone was added to epitestosterone isolated and developed on paper chromatogram, a single steroid spot was found in each case. To dried sample of epitestosterone, 0.3 ml of 0.5% resorcylaldehyde-glacial acetic acid (v/v) were added. After heating for 8 min at 95” a blue colour developed Clin. Chim. Ada,
33
(1971)
215-227
URINARY
TESTOSTERONE
and the absorption testosterone
AND
EPITESTOSTERONE
spectra were determined.
isolated
223 The epitestosterone
from the urine pool gave identical
standard
and epi-
spectra with an absorption
maximum of 585 nm and a peak at 560 nrn11p7. A known amount of pure epitestosterone (3 ,ug) and epitestosterone isolated were heated in 98% formic acid at 95” for 30 min, the fluorescence spectra were determined in the Aminco-Bowman spectrophotofluorometer. Pure epitestosterone and the epitestosterone isolated showed an excitation maximum of 400 nm and a fluorescence maximum of 555 nm. Testosterone didnot fluoresce under these conditions12’9. In three women who had undergone bilateral oophorectomy no testosterone or epitestosterone was detected in the urine.
and adrenalectomy,
Sensitivity. Urine extracts containing 0.01 ,ug of testosterone and 0.01 peg of epitestosterone gave readings on the fluorimeter 6 times above the reagent blank and 4.5-5 times above the paper blank. It was found that less than 0.8,ug/z4 h testosterone or epitestosterone was not significantly different from o, when the volume of 24-h urine collection was 2 litres. Practicability. A fully trained person, when well organised, should be able to perform IO analyses in duplicate per week consisting of 54 working days. From the criteria of reliability of the method examined, it is shown that this method is highly reproducible. Determination of testosterone and e$itestosterone by gas-liquid chromatogra@y The gas-chromatographic estimation of testosterone and epitestosterone was performed on 3% QF-I on 80/100 mesh celite glass columns 9 ft. x 0.4 cm at 235” with nitrogen flow rate 80 ml/min, 3% SE-30 on 100/120 mesh Gas Chrom Z glass columns 7 ft. x 0.4 cm at 230’ with nitrogen flow rate 50 ml/min using trimethylsilyl derivative which was prepared by trimethylchlorosilane. Sample of final testosterone and epitestosterone extracts containing 0.2-0.4 ,ug of each steroid as measured by fluorescence gave values in close agreement when analysed by gas chromatography. Moreover, in each instance, only the single testosterone and epitestosterone peaks were seen, suggesting purity of the final fraction. Under these conditions, 0.2 ,ug of T.4BLE II COMPARISON
OF
THE
PRESENT
FLUORIMETRIC
METHOD
WITH
A
GAS-LIQUID
CHROMATOGRAPHIC
PROCEDURE
Urine
Testosterone
Epitestosterone
Fluorimetry (~124 h) I
II.2
i
4.6 3.2 II.4 5.3 7.5 20.0 14.6
9 10
48.7 106.5
2
3 4 ii
All these estimations the
Gas chromatography (0~123 h)
duplicate
Fluorimetry (zwi24 h)
II.4 4.7 3.1
5.3
11.8
4.8 IO.1
5.0 10.3
5.5
7.4 14.9 20.3 49.0 106.2 were
estimations
done did
in duplicate not
Gas chromatography (N/24 h)
vary
by
and more
the
mean
than
2.1
5.5 2.4
1.8
2.0
8.3 2.1
8.4 2.2
10.2
IO.0
23.8
25.2
38.6
24.1
results
are
given.
The
difference
between
50/:. Clin.
Chim. Acta, 33
(1971)
215-227
PAL
224
testosterone gave a peak height of 20 mm with a relative retention time of 0.50 in relation to cholestane and same amount of epitestosterone gave a peak height of 15 mm with a relative retention time of 0.40. Samples of final testosterone and epitestosterone extracts containing o.z-0.4pg of each steroid as measured by fluorimetric method, as described, gave values in close agreement when analysed by gas chromatography, also, in each case, only the single testosterone and epitestosterone peaks were seen, implying purity of the final fractions (see Table II). RESULTS
The average excretion values of testosterone and epitestosterone from IOO normal women, IOO normal men and 21 normal children, 9 girls and 12 boys (Table III) are presented. All the adult subjects and children studied were in a normal healthy state without any known endocrine, kidney or liver disorders. TARIX III URIrhRYEXCRETION AND -I
CHILDREN
OF
OF
TESTOSTERONE
VARYING
AND
~-___I-.-
Numbcv, SPX
-.I.I-
Age
-. 30 w. 30 12'. 30 w.
18-28
jI-61
4’.
JJ.
m.
yl
In.
20-30 y, 31-41 y.
30 m 423.2 y. IO Ii,. 53-63 Y. 100 m. 9 girls 7-12 y. (2 girls post-pubertal) 12 boys 6 y. 6 m.-r3 (3 boys post-pubertal) _“..__..-
.__.
MES,
WOMEN
--
I.1
(1.4-13.8)
2.j
I.3
(0.8-2.1)
0.9
(oh1.2)
2.0
(ox-rz..*)
4.7 (0.8-13.8) 6z.6 (24.591II.j) 59.3 (22.5--176.-t) j7.s (23.X-55.7) 16.4 (13.0-23.7) 47.6 (13.0-176.4) 0.78 (0.2-1.6)
I00 xv. 30
XORMAL
T&OdWOW ~~~t~~t~st~~an~ (means -+ vmages in @w&hews) I~ -. ~ -.i-. 54 (I.&13.2) 3.3 (o.%r2.4) 5.x (I&1P.l) 3.2 (1.0-8.1)
29-39 Y. 40-50 y.
IO w.
(,UUg/24 h) IS
EPITESTOSTEROSE
AGES
4’.
1~3
-_-.
(0.4-3.2)
45.9
..~
(0.X-11.6)
(I6.2-93.2)
38.8 (20.2%1OO.I) 40.5 ('2.2-30.5) 11.-j (XL-17.6) 31.4 (8.c-Ioo.x) O.j0
(0.2-1.1)
0.90 (0.3-2.6) -
I.“._- -
From I00 normal women (age r8-61 years) the mean values of testosterone and epitestosterone were 4.7 pug/z4 h (range 0.8-13.8) and 2.9 pug/z4 h (range 0.6-12.4) respectively. These women were divided into 4 age groups at IO-yearly intervals, and 30 subjects from one group, aged between 29 and 39 years, gave the highest values of testosterone and epitestosterone, mean 5.8 pg/z4 11(range 1.0-12.1) mean 3.2 ,~q/‘z4 h (range r.o-S.1) respectively. From IOO normal men (age 20-63 years) the mean values of testosterone and epitestosterone were 47.6 ,,xgl24 h (range 13-176.4) and 31.4 ,~g/24 h (range 8.2-100.1) respectively. As before, from 4 age groups, 30 men aged between 42 and 52 years, showed a marked decline in testosterone and epitestosterone excretion, mean 57.8 ,ug/24 h (range 23.x--55,7) and mean 40.5 yg/z4 h (range 12.2-30.5) respectively. From 9 girls (age 7-12 years) (z girls were post-pubertal) the mean values of testosterone and epitestosterone were 0.7s pgj24 h (range 0.2-1.6) and 0.50 pug/24 h (range 0.2-x. I) respectively. Cli,n. Chim. Ada,
33
(1971)215~-227
URINARY
TESTOSTEROKE
AND
225
EPITESTOSTEKONE
From 12 boys (age 6 years 6 months-13 years) (3 boys were post-pubertal) mean values of testosterone and epitestosterone were 1.3 rug/24 h (range 0.4-3.2) 0.90 rug/24 h (range 0.3-2.6) respectively. TABLE
the and
IV
URINARY
EXCRETION
EKDOCRINE
OF TESTOSTERONE
AND
EPITESTOSTERONE
h) IN MEN, WOMEN AND
(/l&z4
CHILDREN
WITH
DISORUERS
Disease
Cast5
Ages
Addison’s disease hdrenal adenoma Adrenal carcinoma Adrenal hyperplasia Adrenal tumour Congenital adrenal hyperplasia
Delayed puberty Eunuchoidism Hirsutlsm Hypopituitary hypogonadism Impotence Precocious puberty Primary testicular hypogonadism St&-Leventhal syndrome Virilizing adrenal tumour Virilizing ovarian tumour
Pathological values In Table IV the values of testosterone various endocrine disorders, are given.
Testosterone
y.
$vzth.
I6.3-j3.5
I””
16.7-20.5
none none
2”.4-34,7 130.2-436.4
100 100
68-j-352.7
32”.3-504.7 220.7-420.’ 16.5-215.6 13.4-250.2 16.X-74.6 3.6-10.6 1.2.5-26.1 3.2-147.” 9.7-30.’ 8.8-18.3 4.6-8.3 6.8-20.6 4.8-70.8 1X8.2 -4’2.3
IO”
273.4-380.7
100 100 IO0 100 100
25 58.1 28.6 5”
Epitestosterone 7.8-20.1 10.3-12.8 8.8-12.5 93.1-446.7 45.8-418.8 198.7-224.3 130.4-218.2 7,5-‘73.2 5.6-162.4 10232.8 1.9-4.1 5.8-12.5 1.3-80.6 4.3-12.3 4 3-X.8
o/o patk.
__-
5” llOtlf2 none TOO IO0 IO0 IO0
57.’ IO0 IO0 ‘“0 75 29 71.4 7.5 IO0
87.1 1””
3.1-3.7 r,7-11.7 2.3-36.7 150.6-326.5
100
17X.3-2j8.4
IO0
IO0 30
and epitestosterone
77.8 42 IO0
in patients with
DISCUSSIOK
The method described here for the determination of urinary conjugated testosteronc and epitestosterone is simple and reproducible. It is suitable for use in normal healthy men, women and particularly in children and in patients with various endocrine disorders. A fluorimetric technique is applied due to its extremely high sensitivity. The fluorescence of testosterone in sulphuric acid has been previously reported13-16. This reaction is relatively specific; also, epitestosterone in sulphuric acid is highly fluorogenie like testosterone. Sulphuric acid-ethanol-induced fluorescence for testosterone determination was first described by Wilsonl’ and, by an adaptation of this reaction, testosterone and epitestosterone were first measured in normal adults and in patientsz. Testosterone is primarily excreted as the glucuronide’*lR, a very small quantity is also excreted as sulphate and as the free steroidI*. It has heen reported that testosterone sulphate was identified and measured from pooled normal male urine in the range of 5-10 pug/q hl9. An appreciable amount of free testosterone could be excreted in urine under certain clinical conditions. Therefore, in the piesent method, free steroids are extracted before an attempt is made to measure the testosterone glucurC&L. Chim. Ada,
33 (1971) 2r5-227
226 onide after enzyme hydrolysis.
PAL Acid hydrolysis
was avoided as this forms the artifacts
of steroid molecules or transformation products 20. It was found from the published work that simultaneous benzene extraction and acid hydrolysis method for determining urinary testosterone in men, women and children, interfered with gas-chromatographic assay of testosterone in some urine specimens, especially from women21. It was concluded that the method should be limited to use with urine specimens from male subjects only. In the other published methods employing acid hydrolysisS2, it was found that this procedure interfered with the gas-chromatographic assays and the required sensitivity needed for application to children’s urine could not be achieved. There was no significant increase of 24-h output of testosterone and epitestosterone when solvolysis procedure was included with the method; similar findings have already been reported. Moreover, this step released various pigments from the urine which were contaminating the extracts. Therefore, solvolysis procedure23 was not used. The fractionation of enzyme-hydrolysed urines on the Sephadex columnl” and paper-chromatographic separation of testosterone from epitestosterone and their further purification in the same way, appeared to achieve a specific determination of these two steroids without any appreciable loss. In certain clinical conditions it was found useful to measure epitestosterone at the same time as testosterone. Little is known at the present time about the origin and significance of epitestosterone excretion. It has been shown thatZ4 epitestosterone is not derived from testosterone and, although its production rate is only 3% of that of testosterone, its excretion rate is about 33o/Oof that of testosterone in the adult male; the excretion of epitestosterone was consistently higher than normal range in all hirsute patients 25. It was also reported, separately, that the rate of formation of epitestosterone from testosterone in blood was 30 times higher in hirsute patients than in normal men and women, irrespective of virilism. The sulphuric acid-ethanol-induced fluorimetric estimation of testosterone and epitestosterone appears to be very sensitive and specific. Various other steroids which could interfere with the fluorescence are easily separated by the chromatographic systems employed. When this method is compared with a gas-chromatographic procedure developed by the author, fair agreement between the results is found. The values for urinary testosterone and epitestosterone in adult women and men, in literature, cover a wide range. The values from adults without any known endocrine disorders, given here, are in good agreement with the values published elsewhere’0,‘4,26-31. In children, the values for pre-pubertal children also agree well with those of other workers. Negligible amounts of these steroids are found in prepubertal children32-37. Increased urinary concentrations of testosterone and epitestosterone have been found in various patients with different adrenal disorders such as adrenal adenoma, adrenal carcinoma, adrenal hyperplasia, congen;tal adrenal hyperplasia and virilizing adrenal tumour. Some of the patients suffering from hirsutism and Stein-Leventhal syndrome also give high values for testosterone and epitestosterone. From these results, therefore, it appears that the method described here provides reliable conditions for the separation and quantitative determination of urinary conjugated testosterone in human adults and in children.
Clin. Chim. Acta, 33 (1971) 215-227
URINARY
TESTOSTERONE
AND
227
EPITESTOSTERONE
ACKNOWLEDGEMEXTS
I wish to thank Professor Dr. E. F. Pfeiffer, Professor Dr. W. Teller and other physicians and surgeons for the interest which they took during the course of this work; Professor W. Klyne, Westfield College, London, for generously providing reference compounds from the Medical Research Council Steroid Reference Section; Professor V. H. T. James, St. Mary’s Hospital Medical School, London, Dr. R. W. H. Edwards, Institute of Child Health, London, British Postgraduate Medical Federation, Dr. G. H. Thomas, University of Birmingham and Dr. M. B. Lipsett, Endocrinology Branch, National Cancer Institute, Bethesda, Maryland, U.S.A. for kindly going through the manuscript and making valuable suggestions. Finally, my thanks go to Mrs. M. R. Pal, for help in preparing
the manuscript.
REFERENCES I B. E. I'.MURPHY,
Recent
Progr.
Hormozolte Res.,
25 (1969)
563.
H. WILSONAXD M. B.LIPsETT,J.C~~~. Invest.,42 (1963) 1753. 3 S. G. KORENMAX, T. E. DAVIS, H. WILSONAXD M. B.LIPSETT, Steroids, 3 (1964) 203. 4 S. LIEBERMAN, D. K. FUKUSHIMA, K. DOBRINER, L. B. HARITOX AXD B. PRAETZ, J. Biol. 2 S. G. KORENMAN,
Chem., I82 (1950) 299. 5 R. ANLII~ER, 0. ROHR AWD M. MARTI, Helu. Chim. Acta, 39 (1956) IIOO. 6 K. SCHUBERT AND E. WEHRBERGER, Naturwissenschaften, 47 (1960) 281. 7 S. G. KORENMAN, H. WILSONAND M. B. LIPSETT,J. Biol.Chem., 239 (1964)
8 9 IO II IL 13 14 I5
16 17 18 I9
20 21 22 21
24 25 26 27
28 29
30 3I 32
22 dd 34
35 36 37
1004. R. V. BROOKS AND G. GIULIANI, Steroids,4 (1964) IOI. J. BLAQCIER, R. I. DORFMAX AND E. FORCHIELLI, A&, Endocrinol., 54(Ig67) 208. AUDREY MOXHAM AND J.D.N. NABARRO,~~~~. Chim.Acta, 22 (Ig68)385. B. B. L. BROOKSBANK AND G. A. D. HASLEWOOD, Biochem. J., 80 (1961) 4X8. R. J, BOSCOTT, Nature, 164(194g) 140. J, H. LINFORD AND 0. B. PAULSON,CUX.J. Med. Sci., 30 (1952) 213. J.W. GOLDZIEHER, J.M.BODENCHUKA~D P.NOLAX, AnaZ.Chem., 26(I9j4) X53. H. KALANT, Biochem. J., 6g (1958) 79. H. GERDES AND W. STAIB, Steroids, 6 (1965) 793. H. WILSON, Anal. Biochem., I (1960) 402. A. RI.CAMACHO AND C. J, MIGEON, 1. Clin.Endocrinol., 23 (1963) 3oI. .4. DESSYPRIS, M. A. DROSDOWSKY, pi. L. MCNIVEN AND R. I. DORFMAN, Pvoc. Sot. Exp. Biol. Med., 121 (1966) 1128. R. I.DORFMAN AND F. UNGAR, in Metabolism of Steroid Hormones, Academic Press, New York, 1965, p. 28. P. VESTERGAARD, E. RAABO AXD S. VEDSQ,CZ~~. Chim. Acta, 14(rg66) 540, A. A. A. ISMAIL AXD R. A. HARKNESS, Biochem. J., 99 (1966) 717. S. BUNSTEIN AND S. LIEBERMAN, I.Biol. Chem., 211 (1958) 331. H. WILSON AND M. B. LIPSETT, J: Clin. Endocrinoi.: 26 (1966) 902. A. F. DE NICOLA, R. I. DORFMAN AND E. FORCHIELLI, Strvoids, 7 (1966) 351. W. FUTTERWEIT,N.L.MCNIVEN,L.NARCUS,C.LANTOS,N.DROSDOWSKYAND R.I.DoRFMAX, Steroids, I (1963) 628. H. IBAYASHI, N. NAKAMURA, S.MURAKA~A, T. I!CHIKAWA, T. TANIOKA AXD K.NAKAO, Steroids.3 (1964) 559. R. V. BROOKS, Steroids, 4 (1964) 117. TM. SPARAGANA, Stevoids, 5 (1965) 773. J. W. MCROBERTS, A.D. OLSOX AND W.L. HERRMANX,CZ~~. Chem., I4 (Ig68)565. A. PETERS AND W.CULLEY,C~~~. Chim. Acta, 25(Ig6g) 199. A. VERMEULEN ABD J. C. M. VERPLANCKE, Steroids, 2 (1963) 453. T.M.RosxER.N.F.CONTE. T.H.BRIGGs.P.Y.CHAO. E.M. SUDMAN AXD P.H.FORSHAM, j. Clin. Endockinol., 25 (1965; 95. W.W. CLEVELAND,N.AHMED, D.H.SAXDBERGAND K.SAVARD, Steroids, 8 (1966) 149 D. KNORR, Acta Endocrinol., 54 (1967) 215. D. GUPTA AND HJLLARY BUTLER, Steroids, 14 (1969) 343. H. J. DEGENHART, H. K. A. VESSEL AND R. WILMINK, Pediat. Rex., 4 (1970) 309. Clin. Chim.
Acta,
33 (1971) 215-227