The Urinary Excretion of Glucosiduronates of Cortisol and Cortisone B. M. LUTTRELL1 AND A. W. STEINBECK
ABSTRACT. Cortisol and cortisone glucosiduronic acids were synthesised in a MC-labelled form and utilised in a double-isotope derivative procedure for the analysis of cortisol glucosiduronate (FG) and cortisone glucosiduronate (EG) in human urine. Normal adults were found to excrete between 16 and 100 /xg/24 h of FG (n = 14) and between 55 and 120 /i.g/24 h of EG (n = 15). Elevated values were observed in subjects with Cushing's syndrome and following ACTH stimulation. Abnormal excretion
T
HE URINARY excretion of cortisol in man occurs by way of numerous conjugates. Two of these, the C-21 glucosiduronates of cortisol and cortisone occur in minor quantities but are of particular interest be-
Received August 19, 1975. 1 Address: Division of Endocrinology and Metabolism, Prince of Wales Hospital, Randwick, N.S.W., 2031 Australia. The following trivial names and abbreviations are used in the text: cortisol glucosiduronate (FG) = 11)3, 17a-dihydroxy-3,20-dioxopregn-4-en-21yl /3,T>glucosiduronate; cortisol glucosiduronic acid = 11/3, 17a-dihydroxy-3,20-dioxopregn-4-en-21yl /3,Dglucosiduronic acid; FGA = methyl (11)3, 17a-dihydroxy-3,20-dioxopregn-4-en-21yl 2',3',4'-tri-Oacetyl-/3,D-glucosid)uronate; cortisol acetate = 11/3, 17a-dihydroxy-3,20-dioxopregn-4-21yl acetate; cortisol acetate oxime = ll/3,17a-dihydroxy-3-oximino-20oxopregn-4-en-21yl acetate; cortisone glucosiduronate (EG) = 17a-hydroxy-3,ll,20-trioxopregn-4-en-21yl /3,D-glucosiduronate; cortisone glucosiduronic acid = 17a-hydroxy-3,ll,20-trioxopregn-4-en-21yl /3,Dglucosiduronic acid; EGA = methyl (17a-hydroxy3,ll,20-trioxopregn-4-en-21yl 2',3',4'-tri-O-acetyl)3,D-glucosid)uronate; cortisone acetate = 17a-hydroxy3,ll,20-trioxopregn-4-en-21yl acetate; cortisone acetate oxime = 17a-hydroxy-ll,20-dioxo-3-oximinopregn-4-en-21yl acetate; cortisol sulfate = ll/3,17adihydroxy-3,20-dioxopregn-4-en-21yl sulfate; cortisone sulfate = 17a - hydroxy - 3,11,20 - trioxopregn - 4en-21yl sulfate; tetrahydrocortisol (THF) = 3a,ll/3, 17a,21-tetrahydroxy-5/3-pregnan-20-one; tetrahydrocortisone (THE) = 3a,17a,21-trihydroxy-5j3-pregnane-ll,20-dione.
was noted in one patient with hepatic cirrhosis and in one case of cholestatic jaundice. The ratio FG/EG was markedly increased after ACTH stimulation and, in the normal group, was positively correlated to a highly significant degree (P < 0.001) with FG excretion. These two observations suggest that EG excretion is less sensitive than FG excretion to variations in cortisol production. (J Clin Endocrinol Metab 42: 567, 1976)
cause the parent steroids have not suffered biochemical degradation. They are considered together in this report as there is interconversion of cortisol and cortisone in peripheral tissues (1). The occurrence of the two conjugates in human urine wasfirstreported by Pasqualini (2,3) and this claim was supported by Brouillet and Mattox (4) who also used a colorimetric method to estimate the aglycones cortisol and cortisone liberated by enzymatic hydrolysis. The two glucosiduronates and their protonated forms, the glucosiduronic acids, were synthesized and characterised by Mattox et al. (5). With the availability of more specific analytical methods, the current study was undertaken to provide stronger evidence for the excretion of these conjugates in human urine and to determine the effects of changes in endocrine and metabolic status on the levels obtained. In order that their contribution to the overall excretion of corticosteroid metabolites might be assessed, the 17hydroxycorticosteroid levels (17-OHCS) were measured by the method of Few (6). Although this measurement has limited specificity, including some steroids not derived from cortisol or cortisone and excluding some which are, it provides a satisfactory estimate of adrenocortical activity for many clinical purposes. 567
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
The Division of Endocrinology and Metabolism, Prince of Wales Hospital and the Department of Medicine, University ofN.S.W., Randwick, N.S.W. 2031 Australia
568
LUTTRELL AND STEINBECK
TABLE 1. Data from the crystallization of radioactive
steroids with authentic unlabelled ones Fraction
FGA
X-l X-2 X-3 X-4 M-l M-2 M-3 X-l X-2 X-3 M-l M-3 X-l X-2 X-3 M-l M-2
EGA
FAO
Solvent
Isotope ratio
Specific activity cpm/mg 6,480 6,431 6,970 6,950 5,458 5,681 6,740
i-/propanol i-/propanol Ethanol/water Ethanol/water
Ethanol Ethanol Acetone/i-octane
629 635 644
578
method of Bush (8), using Whatman no. 1 chromatography paper unless otherwise specified and radioactive zones were eluted with methanol. Eluates were dried down under an air-stream in a water bath maintained below 40 C. Liquid scintillation counting was performed with a Packard Tri Carb Model 3003 spectrometer. For toluene-soluble samples a solution of 4 g 2,5-diphenyloxazole (PPO) and 40 mg 1,4bis-[2-(5-phenyloxazolyl)]benzene (POPOP) per litre of toluene was used as scintillant. The glucosiduronates were dissolved in 0.5 ml of water and 10 ml of Instagel (Packard) was added. An empirical correction factor was applied for the different counting efficiencies of these media.
647
Methanol/benzene Methanol/benzene Ethanol/toluene
1.43 1.32 1.36 1.41 1.32 1.33 1.84 1.96 1.85 1.81 1.90 1.90 0.46 0.46 0.44 0.49 0.45 0.45
Radiochemical syntheses
4-14C-Steroid glucosiduronic acids were prepared by the Schapiro version (9) of the KoenigsM-3 Knorr synthesis (10) from 4-14C-cortisol (54 X-l Methanol/benzene EAO mCi/mmol) and 4-14C-cortisone (55 mCi/mmol). X-2 Methanol/benzene In this 10 fjiCi of the steroid previously purified X-3 Ethanol/toluene M-l by paper chromatography in system 1, was disM-2 solved in 0.2 ml chloroform and 10 mg silver M-3 carbonate, freshly prepared by the method of Methanol/benzene EAO X-l McCloskey and Coleman (11), and 5 mg methyl X-2 Ethanol/benzene 2,3,4 - tri - O - acetyl -1 - bromo - l-deoxy-a,D-gluX-3 Methanol/benzene M-l curonate were added, and, thereafter, the mixM-2 ture was agitated for 18 h in the dark at room M-3 temperature. The initially formed products, methyl (4- 14 C-ll/3,17a-dihydroxy-3,20-dioxoX-n = crystalline product from nth crystallization. pregn-4-en-21yl /3,D-glucosid)uronate (4-14CM-n = mother liquor residue from nth crystallization. FAO = cortisol acetate oxime. FGA) and its 11-oxo analogue (4-14C-EGA) were EAO = cortisone acetate oxime. purified by chromatography on Celite in system Constancy of specific activity is evidence for the identity of 2 as described for the unlabelled compounds radioactive steroids while constancy of isotope ratio demon(FGA and EGA) by Mattox et al. (5) and identified strates the specificity of the assay. by mixing them with FGA and EGA and repeatedly crystallizing the mixture without resMaterials and Methods olution of the labelled and unlabelled steroids Solvents for chromatography were of reagent (Table 1). 14 grade. Benzene, cyclohexane, and isooctane C-FGA and 14C-EGA were hydrolyzed by were purified by being passed through a column shaking for 2 h at room temperature with 0.05M of silica gel while methanol was distilled. Un- sodium hydroxide solution in dioxane/water labelled steroids were supplied by Roussel (1:1) and the 14C-steroid glucosiduronic acids, Laboratories, Ltd., and purified by crystalliza- formed on acidification with 2M acetic acid, were purified by paper chromatography in tion before use. Radiochemicals were obtained from the Radio- system 3 in which they exhibited Rf values of chemical Centre, Amersham. 3H-Acetic anhy- 0.4 in agreement with the report of Mattox et al. 14 dride was diluted to the desired specific activity (12) for the unlabelled conjugates. The Cand made up to a 10% solution in anhydrous glucosiduronic acids were not crystallized with benzene, and the solution was distilled before unlabelled material for proof of identity since use. Its specific activity was determined by the the latter were only obtained in an amorphous form. Nonetheless, proof of their identity rests method of Kliman and Peterson (7). Paper chromatography followed the general on a number of observations. First, the unlabelled
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
Compound
JCE & M • 1976 Vol 42 • No 3
CORTISOL AND CORTISONE GLUCOSIDURONATES
Analytical method Urines were collected for 24 h into a bottle containing 10 ml glacial acetic acid as a preservative and, when collection was complete, were stored frozen until used. Creatinine measurements were used to assess the adequacy of collection. Each analytical run consisted of duplicate samples of each unknown urine collection, mostly 1% of the 24 h volume, duplicate aliquots of standard solutions of synthetic glucosiduronates, and duplicate blanks. 14 C-Steroid glucosiduronic acid solutions of 0.002 fid approximately, were added to each of the unknowns, to standards, and directly to counting vials. The urine samples were diluted with water to 70 ml and passed through 5 g columns of XAD-2 resin (product of Rohm and Haas). Two volumes of water were then passed through the columns and the aqueous eluates were discarded. 40 ml of methanol was used to elute the steroid conjugates from the columns and afterwards removed under reduced pressure at 40 C to leave a dark oily residue. The residues were chromatographed on Whatman no. 3 chromatography paper in system 3 and subsequently rechromatographed in the same system following evidence of over-loading. Enzymatic hydrolysis was carried out with 2500 U Ketodase (Warner-Chilcott) per sample in 3 ml of 0.1M acetate buffer at pH 4.85 for 16 h at 37 C. The liberated corticosteroids were then extracted with 10 ml ethyl acetate and purified by paper chromatography in system 5. The acetylation of unknowns, standards, and blanks was carried out with 1 drop 3H-acetic
anhydride and 2 drops pyridine overnight at 37 C. 0.5 ml ethanol/water mixture (1:4) was added to stop the reaction, and the cortisol and cortisone acetates were extracted into carbon tetrachloride (5 ml). This was twice washed with 0.5 ml water and then evaporated. At this stage, sufficient unlabelled cortisol acetate and cortisone acetate markers were added to permit the location of steroids on chromatograms by inspection under U.V. light. Chromatography was carried out in systems 6 and 7, successively. In system 7, the two steroid acetates were separated and henceforth treated separately. The samples were then reacted with 100 fxg hydroxylamine hydrochloride and 200 /xg sodium acetate in 0.1 ml methanol at 37 C overnight and the reaction mixtures were applied directly to chromatography papers and developed in system 8. The resulting C-3 oximes of cortisol acetate and cortisone acetate (13) were eluted into counting vials, and the 3H/14C ratio was determined. This ratio was used to calculate the levels of glucosiduronates in the original urine collections after allowance was made for the values obtained in the accompanying blanks and for the weight of the 14C-tracer added. Values were considered unreliable if the difference between duplicates exceeded 15% of their mean and if the recovered 14C-level was too low for accurate counting, usually below 5%. The effective working range of this method was determined by the 3H/14C ratio that could be measured with confidence between the values of 0.5 and 20, which is in turn dependent upon the choice of sample volume and the specific activity of the 3 H-acetic anhydride. Under the conditions employed here a lower limit of 8 /u-g/24 h was set for the working range. The lowest values encountered were 10 and 7 /x,g/24 h for FG and EG, respectively, in a subject with adrenocortical supression following prednisone therapy. Values up to 1000 /i.g/24 h were estimated with appropriate choice of sample volume or dilution. Assessment of performance characteristics for analytical method i) Accuracy. Increasing increments of a standard solution of each of the glucosiduronates were added in duplicate to aliquots from a 24 h urine collection. The results are shown in Fig. 1 where the values estimated are plotted against the weight of steroid added, and the relationship is linear, with a slope agreeing well with that predicted (b = 1). The intercept on the Y
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
glucosiduronic acids, prepared by the same method showed I.R. spectra identical with those of authentic samples. Second, the 14C-tracers, when mixed with authentic cortisol and cortisone glucosiduronic acids and chromatographed in system 3, moved with the U.V. opaque zone. This zone, after elution and hydrolysis with Ketodase, yielded a mean of between 90 and 100% of the predicted value for each conjugate in 12 successive estimations by the double-isotope derivative procedure. Finally, samples of the l4 C-glucosiduronic acids were methylated with diazomethane and acetylated with acetic anhydride in pyridine to reform I4C-FGA and 14CEGA, as demonstrated by chromatography in system 4, and confirmed that the alkaline hydrolysis reaction had not affected the glucuronic acid residue.
569
570
JCE & M • 1976 Vul >\2 • No 3
LUTTRELL AND STEINBECK
0.5 EG (Aig) Added
axis is the value of the conjugate concentration in the sample. ii) Water blank. The contribution of the extraction and other steps prior to acetylation to the "blank" was tested by adding two aliquots of each 14C-tracer to distilled water and treating it as an unknown. After subtracting the acetylation blank and the calculated weight of tracer added, values of ±0.02 (jug were obtained which would represent ±2 /ug/24 h in a typical urine sample.
in) Within-assay variation. Ten replicate samples from a 24 h urine collection were analyzed and a within-assay coefficient of variation of 5% was calculated for each of the two steroid conjugates.
v) Specificity. Proof of specificity of a doubleisotope-derivative analysis rests on evidence as to the radiochemical homogeneity of the final derivative. This evidence is best obtained from the constancy of isotope ratio before and after crystallization with an authentic cold carrier (14). The criteria for proof of identity of radioactive steroids are discussed by Brooks et al. (15). Accordingly, groups of replicate samples, after scintillation counting, were pooled and 20 mg of an appropriate cold carrier was added. The solution was chromatographed on 10 g of alumina (Woelm activity II) using 100 ml toluene and then toluene/dichloromethane 1:1 until the 2 scintillator components PPO and POPOP were eluted, which was ascertained by illuminating the column with U.V. light. The steroid derivative was eluted with methanol/benzene 1:1 and this was crystallized 3 times in succession. In each case, the mother liquor was set aside while the crystalline fraction was carried to the next stage. Samples from all crystalline and mother liquor fractions were dissolved in 0.2 ml methanol and 0.5 ml toluene and 10 ml of toluene scintillant. The 3H/14C ratio was determined and Table 1 shows that this was not decreased following repeated crystallization. Indeed, the glucosiduronate level calculated from the isotope ratio of the last crystalline fraction (X-3), in each case falls within the range of the pooled replicates. Chromatography 1. 2. 3. 4. 5. 6. 7.
iv) Between-assay variation. The length of the procedure and the limited number of samples included in each run preclude calculation of this
8.
systems
Benzene, methanol, water, 100:50:25. Benzene, formamide (5). n-Butanol, water (12). Isooctane, toluene, methanol, water, 225: 275:400:100. Cyclohexane, dioxane, water, 100:100:25. Cyclohexane, dioxane, methanol, water, 100: 75:50:25. Cyclohexane, benzene, methanol, water, 100: 40:100:20. Cyclohexane, benzene, methanol, water, 100: 100:100:50.
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
FIG. 1. Vertical bars show the duplicate estimates of the weight of cortisol glucosiduronate (FG) and of cortisone glucosiduronate (EG) found in a series of aliquots of a normal urine collection to which increasing amounts of the synthetic glucosiduronates had been added. The predicted line of slope, b = 1 is also shown.
parameter from replicates of a urine collection. An analysis of the estimations for standards in 12 successive assays indicates the extent of variation likely. For instance, the mean estimate was 91% of the weight added for cortisol glucosiduronate with a coefficient of variation of 12%; for cortisone glucosiduronate the corresponding figures were 94% and 11%.
571
CORTISOL AND CORTISONE GLUCOSIDURONATES Subjects
siduronate (FG), cortisone glucosiduronate (EG), and 17-hydroxycorticosteroids (17-OHCS) in normal and other subjects Subject
Status
FG
EG
/xg/24h
/xg/24 h
FG/ EG
17-OHCS mg/24 h
1.0 1.1 0.6 0.5 0.9
8.2 9.5
H.T. B.L. M.R. R.V. R.F. H.B. P.G. H.L. J.E. R.S. D.F. M.H. D.A. D.K.
N N N N N N N N N N N N
94 100 69 55
50 16 51 53 66 73
95 94 120 110 56 83 88 60 78 55 82 77
N
77
68
N
A.Y.
N basal ACTH inf basal ACTH inf C.S. basal ACTH inf C.S. C.S. C.S. C.S. C.S. H.C. H.C. H.C. H.C. C.J.
33 32 49 320 16 130 92 630 900 110 350 170 140 58 68
79 70 79 170 47 110 140 190 430 130 340 110 350 110
68 33
37 75
0.6 0.3 0.7 1.0 0.8 0.9 1.1 0.4 0.5 0.6 1.9 0.3 1.2 0.7 4.8 2.1 0.9 1.0 1.5 0.4 0.5 1.8 1.8 0.4
100
160
0.6
J.C. J.C. S.M. S.M. A.R. A.R. W.B. H.H. F.D. A.H. J.B. W.L. A.O. A.O. A.L. J.S.
51
38
11.8 5.2 8.4 4.1
14.4 4.2 8.4
10.3 8.4
13.0 13.0 49.0 8.3 22.0 12.3 37.0 58.0 25.6 13.9 28.8 4.0 3.5 4.0 6.1
10.0
N = 1formal.
c.s. = Cushing's syndrome.
H.C. = Hepatic cirrhosis. C.J.= = Cholestatic jaundice. ACTH inf=ACTF[ infusion.
creasing with higher FG excretion and the correlation coefficient was +0.84 (Fig. 2). Results This trend is also evident after ACTH stimuThe values for FG and EG glucosiduro- lation and higher than normal values for the nates were based on the molecular weights FG/EG ratio were found in 2 patients with of their anions, 538 and 536, respectively. Cushing's syndrome but not in others. HowThe results are shown in Table 2. ever, in Cushing's syndrome the excretion The group of 14 normal subjects showed of at least one glucosiduronate was beyond a mean urinary FG excretion of 59 /u,g/24 h the normal range and in 4 subjects the exwith individual values ranging from 16- cretions of both glucosiduronates were ele100 /ug/24 h. The corresponding mean for vated. EG was 81 fig/24 h and the range was 55After ACTH stimulation, the FG excretion 120 /xg/24 h for 15 normal subjects. increased at least 6-fold but that of EG was FG excretion had a greater variability less, the highest increase attained being 2.3 than EG excretion, the ratio FG/EG in- times the basal value.
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
The normal subjects, of whom there were 7 men and 8 women between 19 and 58 years of age, were free from known endocrine or metabolic abnormalities and the women were not using oral contraceptives. Those subjects studied before and during ACTH infusion were females between 15 and 34 years of age and under evaluation for possible Cushing's syndrome and in one (A.R.) the diagnosis was later established. After a basal 24 h urine collection, 250 (xg tetracosactrin was given as a constant intravenous infusion over an 8 h period and urine collections continued over several 24 h periods. The collection for the day of infusion was analysed. The subjects with Cushing's syndrome, 2 male and 4 female between 34 and 52 years of age, were patients in whom the diagnosis was established by acceptable methods. Two (A.R and A.H.) underwent adrenalectomy with remission of symptoms and, on histological examination, adrenocortical hyperplasia was found. The patient H.H. had recurrent Cushing's syndrome from functioning remnants after adrenalectomy and persistent over-secretion of ACTH. The condition in W.B. was an ectopic syndrome from bronchial carcinoma. The 3 patients with hepatic cirrhosis, in each case due to alcohol, were all men between 34 and 67 years of age and were hospitalized with gross symptoms of the disorder. A.O. and W.L. each had pronounced gynecomastia and testicular atrophy; A.L., a patient with a 10 year history of the disease, also had massive ascites. The patient J.S., a male 54 years of age, had cholestatic jaundice from ethylestrenol (Orabolin).
TABLE 2. 24 h urinary excretion of cortisol gluco-
LUTTRELL AND STEINBECK
572
JCE & M • 1976 Vol 42 • No 3
1.0
O.5
1). Also, the isotope ratios obtained during 50
100
FG (Aig/24hr)
FIG. 2. Correlation of the 24 h excretion of cortisol glucosiduronate (FG) and the cortisol glucosiduronate/ cortisone glucosiduronate (FG/EG) ratio in normal adults. The correlation coefficient is r = + 0.84. The regression line is y = bx + a where b = 0.0096 and a = 0.18.
The patients with hepatic cirrhosis also showed variable results; 2 (W.L. and A.L.) had normal excretions and the other a depressed E.G. excretion on two successive occasions (38 and 37 /u.g/24 hr). The patient J.S. with cholestatic jaundice had an elevated EG excretion, and his FG was at the limit of normal, but the 17-OHCS value was normal. Both conjugates represent a small but variable proportion of the steroids measure as 17-OHCS, up to 1% in each case for the normal group (based on the weight of the aglycone only), and when abnormal values are included, levels of up to 2% occur. Discussion Previous workers, when studying cortisol and cortisone glucosiduronates, were restricted to examining the products of enzymatic hydrolysis, while in this study, it was possible to purify the conjugates by paper
analysis required that cortisol or cortisone conjugates within urine samples have diluted the 14C-tracers initially added. They are presumed to be the C-21 glucosiduronates of these steroids since they have a mobility in system 3 identical with authentic samples and are hydrolyzed by Ketodase which has considerable specificity for the ^-conjugates of glucuronic acid. Also in another study, one of us (B.L.) has shown that Ketodase will not hydrolyze the C-21 sulfates of cortisol and cortisone. The conjugates were extracted from urine by a small scale version of the XAD-2 column technique described by Bradlow (16) and the extract, which also contained sulfates and unconjugated steroids, was purified by paper chromatography in system 3. 14C-Labelled cortisol, cortisone, cortisol sulfate, and cortisone sulfate were added to a number of samples to demonstrate their separation from the glucosiduronates in the system. After enzymatic hydrolysis of the glucosiduronates, the procedure differed little from other published methods for the double-isotope derivative analysis of corticosteroids (17-19). The isotope ratio finally obtained was tested by the pooling of replicates after counting and diluting the radioactive products with authentic steroids and crystallizing the mixture (Table 1).
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
chromatography. Furthermore, as 14Ctracers were added before the extraction procedure, a correction could be made for procedural losses to individual samples, whereas previous methods only allowed a group correction for losses following hydrolysis. The conjugates investigated have not been unequivocally characterized, but the study provides further evidence to support the structures proposed by Brouillet and Mattox (4). In this regard, the steroids present are confirmed to be cortisol and cortisone by the crystallization of their doublelabelled oxime derivatives to a constant isotope ratio with authentic cold carrier (Table
CORTISOL AND CORTISONE GLUCOSIDURONATES
view when discussing the urinary metabolites isolated after a pulse injection of labelled cortisol in human subjects. Cortisone is known to be a minor secretion product of the adrenal gland, but cortisone found in peripheral plasma is largely the oxidation product of cortisol (22). In the Cushing's syndrome group, all subjects showed an elevation of one parameter above the normal range although 2 of them had normal 17-OHCS values. Thus, determinations of FG and EG excretion appear to hold some potential for the detection of Cushing's syndrome; however, the group was small and further studies are indicated. A limitation would be expected when studying subjects with impaired hepatic or renal function, since these organs and the small intestine are considered to be the major sites of glucuronic acid conjugation (23), and abnormal values might thereby occur. Of the 3 patients with severe alcoholic hepatic cirrhosis, W.L. and A.L. had normal results but A.O. on 2 successive occasions had a markedly depressed EG excretion of 38 and 37 /xg/24 h. In this regard Srivastava et al. (1) noted a tendency towards lower than normal values of plasma cortisone in liver and kidney disease. An abnormal result was also recorded for subject J.S. who had cholestatic jaundice induced by ethylestrenol. His EG excretion was elevated and the FG value was at the top of the normal range, but the 17-OHCS was normal. Acknowledgment The authors wish to express their gratitude to Dr. Vemon R. Mattox, Professor of Biochemistry of the Mayo Clinic, Minnesota, for helpful advice and for authentic samples of steroid conjugates, which he generously provided.
References 1. Srivastava, L. S., E. E. Werk, Jr., and K. Thrasher, L. J. Sholiton, R. Kozera, W. Nolten, and H. C. Knowles, Jr., Plasma cortisone concentration as measured by radioimmunoassay, J Clin Endocrinol Metab 36: 937, 1973. 2. Pasqualini, J. R., Corticosteroides urinaires:
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
The mean values for urinary excretion of FG and EG for the normal group were 59 and 81 fxg/24 h, respectively, while Brouillet and Mattox (4) in a group of 7 normal subjects obtained values of 37 and 94 /-ig/24 h after adjusting their values to include the weight of the glucuronic acid residue. This suggests good agreement as there is considerable overlap in individual values, the samples are small and the analytical methods employed differ considerably. The FG/EG ratio is markedly increased after ACTH stimulation, suggesting that cortisol excretion via the EG pathway varies less with cortisol production than the excretion via the FG pathway, and, if an assumption is made that FG excretion varies directly with cortisol production in normal subjects, additional support is found for this contention in the highly significant (P < 0.001) correlation observed between the FG/EG ratio and FG excretion for this group. This interpretation is compatible with the observation of Srivastava et al. (1) that plasma levels of cortisone were not obviously affected by an ACTH injection or by acute stress, although 2 patients with Cushing's Syndrome had elevated values of cortisone relative to cortisol. In the present series 2 patients had EG values elevated relative to FG (J.B. and A.R.) suggesting that the relationship observed between FG/EG and FG may not hold with prolonged ACTH stimulation and hypercortisolemia. While the FG/EG relationship has not previously been reported, the ratio of tetrahydrocortisol/tetrahydrocortisone (THF/ THE) has been extensively investigated. Zumoff e£ al. (20) recently summarised the data and indicate that the THF/THE ratio is increased in chronic lymphatic leukemia, hypothyroidism, administration of cortisol or following ACTH stimulation, and as a non-specific consequence of illness. Also, it is independent of age and sex in normal subjects. It appears likely that the observed changes in the THF/THE and the FG/EG ratios reflect changes in the availability of cortisol. Gallagher et al. (21) adopt a similar
573
574
3.
5.
6. 7. 8. 9. 10. 11. 12.
13. 14.
Metabolites tetrahydrogenes du cortisol et de la corticosterone, C R Acad Sci [D ] (Pans) 250: 1929, 1960. Pasqualini, J. R., Mode de conjugaison et fractionnement des Metabolites de corticosteroides urinaires apres administration d'ACTH, Ada Endocrinol [Suppl](Kbh) 51: 1071, 1960. Brouillet, J. C , and V. R. Mattox, Evidence for the presence of glucuronides of cortisone and cortisol in human urine, J Clin Endocrinol Metab 26: 453, 1966. Mattox, V. R., J. E. Goodrich, and W. D. Vrieze, Synthesis of C-21 glucosiduronates of cortisone and related corticosteroids, Biochemistry 8: 1188, 1969. Few, J. D., A method for the analysis of urinary 17-hydroxycorticosteroids, J Endocrinol 22: 31, 1961. Kliman, B., and R. E. Peterson, Double Isotope derivative assay of aldosterone in biological extracts, J Biol Chem 235: 1639, 1960. Bush, I. E., The chromatography of steroids, Permagon Press, Oxford, London, New York, and Paris, 1961. Shapiro, E., Synthesis of steroid glucuronides, Biochem J 33: 385, 1939. Koenigs, N., and E. Knorr, Ber Deut Chem Ges 34: 957, 1901. McCloskey, C. M., and G. H. Coleman, /3-DGlucose-2,3,4,6-tetraacetate, Organic Syntheses Coll. vol. Ill, Wiley, New York, 1955, p. 434. Mattox, V. R., J. E. Goodrich, and W. D. Vrieze, Chromatographic mobilities and partition coefficients of synthetic corticosteroid glucosiduronates, Steroids 18: 147, 1971. Poos, G. I., and L. H. Sarett, U.S. 3074979, C A 58: P14072e, 1963 (Abstract). Axelrod, L. R., C. Mattijssen, J. W. Goldzieher,
15.
16. 17.
18.
19.
20.
21.
22.
23.
JCE & M • 1976 Vol 42 • No 3
and J. E. Pulliam, Definitive identification of Microquantities of radioactive steroids by recrystallization to constant specific activity, Ada Endocrinol [Suppl](Kbh) 99: 166. Brooks, C. J. W., R. V. Brooks, K. Fotherby, J. Grant, A. Klopper, and W. Klyne, The identification of steroids J Endocrinol 47: 265, 1970. Bradlow, H. L., Extraction of steroid conjugates with a neutral resin, Steroids 11: 265, 1968. Stachenko, J., C. Laplante, and C. J. P. Giroud, Double isotope derivative assay of aldosterone, corticosterone and cortisol, Can J Biochem 42: 1275, 1964. Coghlan, J. P., M. Wintour, and B. A. Scoggins, The measurement of corticosteroids in adrenal vein blood of sheep, Aust J Exp Biol Med Sci 44: 639, 1966. Fraser, R., and V. H. T. James, Double isotope assay of aldosterone, corticosterone, and cortisol in human peripheral plasma,/ Endocrinol 40: 59, 1968. Zumoff, B., H. L. Bradlow, D. K. Fukashima, and L. Hellman, Increase in the tetrahydrocortisol/ tetrahydrocortisone ratio from cortisol-4-l4C: A nonspecific consequence of illness,/ Clin Endocrinol Metab 39: 1120, 1974. Gallagher, T. F., D. K. Fukashima, and L. Hellman, Clarification of discrepancies of cortisol secretion rate, 7 Clin Endocrinol Metab 31: 625, 1970. Dazord, A., J. Saez, and J. Bertrand, Metabolic clearance rates and interconversion of cortisol and cortisone, / Clin Endocrinol Metab 35: 24, 1972. Meittinen, T. A., and E. Leskinen, Glucuronic acid pathway, In Fishman, W. H. (ed.), Metabolic Conjugation and Metabolic Hydrolysis, vol. I, Academic Press, New York and London, 1970, p. 169.
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
4.
LUTTRELL AND STEINBECK
ABSTRACT. Cortisol and cortisone glucosiduronic acids were synthesised in a MC-labelled form and utilised in a double-isotope derivative procedure for the analysis of cortisol glucosiduronate (FG) and cortisone glucosiduronate (EG) in human urine. Normal adults were found to excrete between 16 and 100 /xg/24 h of FG (n = 14) and between 55 and 120 /i.g/24 h of EG (n = 15). Elevated values were observed in subjects with Cushing's syndrome and following ACTH stimulation. Abnormal excretion
T
HE URINARY excretion of cortisol in man occurs by way of numerous conjugates. Two of these, the C-21 glucosiduronates of cortisol and cortisone occur in minor quantities but are of particular interest be-
Received August 19, 1975. 1 Address: Division of Endocrinology and Metabolism, Prince of Wales Hospital, Randwick, N.S.W., 2031 Australia. The following trivial names and abbreviations are used in the text: cortisol glucosiduronate (FG) = 11)3, 17a-dihydroxy-3,20-dioxopregn-4-en-21yl /3,T>glucosiduronate; cortisol glucosiduronic acid = 11/3, 17a-dihydroxy-3,20-dioxopregn-4-en-21yl /3,Dglucosiduronic acid; FGA = methyl (11)3, 17a-dihydroxy-3,20-dioxopregn-4-en-21yl 2',3',4'-tri-Oacetyl-/3,D-glucosid)uronate; cortisol acetate = 11/3, 17a-dihydroxy-3,20-dioxopregn-4-21yl acetate; cortisol acetate oxime = ll/3,17a-dihydroxy-3-oximino-20oxopregn-4-en-21yl acetate; cortisone glucosiduronate (EG) = 17a-hydroxy-3,ll,20-trioxopregn-4-en-21yl /3,D-glucosiduronate; cortisone glucosiduronic acid = 17a-hydroxy-3,ll,20-trioxopregn-4-en-21yl /3,Dglucosiduronic acid; EGA = methyl (17a-hydroxy3,ll,20-trioxopregn-4-en-21yl 2',3',4'-tri-O-acetyl)3,D-glucosid)uronate; cortisone acetate = 17a-hydroxy3,ll,20-trioxopregn-4-en-21yl acetate; cortisone acetate oxime = 17a-hydroxy-ll,20-dioxo-3-oximinopregn-4-en-21yl acetate; cortisol sulfate = ll/3,17adihydroxy-3,20-dioxopregn-4-en-21yl sulfate; cortisone sulfate = 17a - hydroxy - 3,11,20 - trioxopregn - 4en-21yl sulfate; tetrahydrocortisol (THF) = 3a,ll/3, 17a,21-tetrahydroxy-5/3-pregnan-20-one; tetrahydrocortisone (THE) = 3a,17a,21-trihydroxy-5j3-pregnane-ll,20-dione.
was noted in one patient with hepatic cirrhosis and in one case of cholestatic jaundice. The ratio FG/EG was markedly increased after ACTH stimulation and, in the normal group, was positively correlated to a highly significant degree (P < 0.001) with FG excretion. These two observations suggest that EG excretion is less sensitive than FG excretion to variations in cortisol production. (J Clin Endocrinol Metab 42: 567, 1976)
cause the parent steroids have not suffered biochemical degradation. They are considered together in this report as there is interconversion of cortisol and cortisone in peripheral tissues (1). The occurrence of the two conjugates in human urine wasfirstreported by Pasqualini (2,3) and this claim was supported by Brouillet and Mattox (4) who also used a colorimetric method to estimate the aglycones cortisol and cortisone liberated by enzymatic hydrolysis. The two glucosiduronates and their protonated forms, the glucosiduronic acids, were synthesized and characterised by Mattox et al. (5). With the availability of more specific analytical methods, the current study was undertaken to provide stronger evidence for the excretion of these conjugates in human urine and to determine the effects of changes in endocrine and metabolic status on the levels obtained. In order that their contribution to the overall excretion of corticosteroid metabolites might be assessed, the 17hydroxycorticosteroid levels (17-OHCS) were measured by the method of Few (6). Although this measurement has limited specificity, including some steroids not derived from cortisol or cortisone and excluding some which are, it provides a satisfactory estimate of adrenocortical activity for many clinical purposes. 567
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
The Division of Endocrinology and Metabolism, Prince of Wales Hospital and the Department of Medicine, University ofN.S.W., Randwick, N.S.W. 2031 Australia
568
LUTTRELL AND STEINBECK
TABLE 1. Data from the crystallization of radioactive
steroids with authentic unlabelled ones Fraction
FGA
X-l X-2 X-3 X-4 M-l M-2 M-3 X-l X-2 X-3 M-l M-3 X-l X-2 X-3 M-l M-2
EGA
FAO
Solvent
Isotope ratio
Specific activity cpm/mg 6,480 6,431 6,970 6,950 5,458 5,681 6,740
i-/propanol i-/propanol Ethanol/water Ethanol/water
Ethanol Ethanol Acetone/i-octane
629 635 644
578
method of Bush (8), using Whatman no. 1 chromatography paper unless otherwise specified and radioactive zones were eluted with methanol. Eluates were dried down under an air-stream in a water bath maintained below 40 C. Liquid scintillation counting was performed with a Packard Tri Carb Model 3003 spectrometer. For toluene-soluble samples a solution of 4 g 2,5-diphenyloxazole (PPO) and 40 mg 1,4bis-[2-(5-phenyloxazolyl)]benzene (POPOP) per litre of toluene was used as scintillant. The glucosiduronates were dissolved in 0.5 ml of water and 10 ml of Instagel (Packard) was added. An empirical correction factor was applied for the different counting efficiencies of these media.
647
Methanol/benzene Methanol/benzene Ethanol/toluene
1.43 1.32 1.36 1.41 1.32 1.33 1.84 1.96 1.85 1.81 1.90 1.90 0.46 0.46 0.44 0.49 0.45 0.45
Radiochemical syntheses
4-14C-Steroid glucosiduronic acids were prepared by the Schapiro version (9) of the KoenigsM-3 Knorr synthesis (10) from 4-14C-cortisol (54 X-l Methanol/benzene EAO mCi/mmol) and 4-14C-cortisone (55 mCi/mmol). X-2 Methanol/benzene In this 10 fjiCi of the steroid previously purified X-3 Ethanol/toluene M-l by paper chromatography in system 1, was disM-2 solved in 0.2 ml chloroform and 10 mg silver M-3 carbonate, freshly prepared by the method of Methanol/benzene EAO X-l McCloskey and Coleman (11), and 5 mg methyl X-2 Ethanol/benzene 2,3,4 - tri - O - acetyl -1 - bromo - l-deoxy-a,D-gluX-3 Methanol/benzene M-l curonate were added, and, thereafter, the mixM-2 ture was agitated for 18 h in the dark at room M-3 temperature. The initially formed products, methyl (4- 14 C-ll/3,17a-dihydroxy-3,20-dioxoX-n = crystalline product from nth crystallization. pregn-4-en-21yl /3,D-glucosid)uronate (4-14CM-n = mother liquor residue from nth crystallization. FAO = cortisol acetate oxime. FGA) and its 11-oxo analogue (4-14C-EGA) were EAO = cortisone acetate oxime. purified by chromatography on Celite in system Constancy of specific activity is evidence for the identity of 2 as described for the unlabelled compounds radioactive steroids while constancy of isotope ratio demon(FGA and EGA) by Mattox et al. (5) and identified strates the specificity of the assay. by mixing them with FGA and EGA and repeatedly crystallizing the mixture without resMaterials and Methods olution of the labelled and unlabelled steroids Solvents for chromatography were of reagent (Table 1). 14 grade. Benzene, cyclohexane, and isooctane C-FGA and 14C-EGA were hydrolyzed by were purified by being passed through a column shaking for 2 h at room temperature with 0.05M of silica gel while methanol was distilled. Un- sodium hydroxide solution in dioxane/water labelled steroids were supplied by Roussel (1:1) and the 14C-steroid glucosiduronic acids, Laboratories, Ltd., and purified by crystalliza- formed on acidification with 2M acetic acid, were purified by paper chromatography in tion before use. Radiochemicals were obtained from the Radio- system 3 in which they exhibited Rf values of chemical Centre, Amersham. 3H-Acetic anhy- 0.4 in agreement with the report of Mattox et al. 14 dride was diluted to the desired specific activity (12) for the unlabelled conjugates. The Cand made up to a 10% solution in anhydrous glucosiduronic acids were not crystallized with benzene, and the solution was distilled before unlabelled material for proof of identity since use. Its specific activity was determined by the the latter were only obtained in an amorphous form. Nonetheless, proof of their identity rests method of Kliman and Peterson (7). Paper chromatography followed the general on a number of observations. First, the unlabelled
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
Compound
JCE & M • 1976 Vol 42 • No 3
CORTISOL AND CORTISONE GLUCOSIDURONATES
Analytical method Urines were collected for 24 h into a bottle containing 10 ml glacial acetic acid as a preservative and, when collection was complete, were stored frozen until used. Creatinine measurements were used to assess the adequacy of collection. Each analytical run consisted of duplicate samples of each unknown urine collection, mostly 1% of the 24 h volume, duplicate aliquots of standard solutions of synthetic glucosiduronates, and duplicate blanks. 14 C-Steroid glucosiduronic acid solutions of 0.002 fid approximately, were added to each of the unknowns, to standards, and directly to counting vials. The urine samples were diluted with water to 70 ml and passed through 5 g columns of XAD-2 resin (product of Rohm and Haas). Two volumes of water were then passed through the columns and the aqueous eluates were discarded. 40 ml of methanol was used to elute the steroid conjugates from the columns and afterwards removed under reduced pressure at 40 C to leave a dark oily residue. The residues were chromatographed on Whatman no. 3 chromatography paper in system 3 and subsequently rechromatographed in the same system following evidence of over-loading. Enzymatic hydrolysis was carried out with 2500 U Ketodase (Warner-Chilcott) per sample in 3 ml of 0.1M acetate buffer at pH 4.85 for 16 h at 37 C. The liberated corticosteroids were then extracted with 10 ml ethyl acetate and purified by paper chromatography in system 5. The acetylation of unknowns, standards, and blanks was carried out with 1 drop 3H-acetic
anhydride and 2 drops pyridine overnight at 37 C. 0.5 ml ethanol/water mixture (1:4) was added to stop the reaction, and the cortisol and cortisone acetates were extracted into carbon tetrachloride (5 ml). This was twice washed with 0.5 ml water and then evaporated. At this stage, sufficient unlabelled cortisol acetate and cortisone acetate markers were added to permit the location of steroids on chromatograms by inspection under U.V. light. Chromatography was carried out in systems 6 and 7, successively. In system 7, the two steroid acetates were separated and henceforth treated separately. The samples were then reacted with 100 fxg hydroxylamine hydrochloride and 200 /xg sodium acetate in 0.1 ml methanol at 37 C overnight and the reaction mixtures were applied directly to chromatography papers and developed in system 8. The resulting C-3 oximes of cortisol acetate and cortisone acetate (13) were eluted into counting vials, and the 3H/14C ratio was determined. This ratio was used to calculate the levels of glucosiduronates in the original urine collections after allowance was made for the values obtained in the accompanying blanks and for the weight of the 14C-tracer added. Values were considered unreliable if the difference between duplicates exceeded 15% of their mean and if the recovered 14C-level was too low for accurate counting, usually below 5%. The effective working range of this method was determined by the 3H/14C ratio that could be measured with confidence between the values of 0.5 and 20, which is in turn dependent upon the choice of sample volume and the specific activity of the 3 H-acetic anhydride. Under the conditions employed here a lower limit of 8 /u-g/24 h was set for the working range. The lowest values encountered were 10 and 7 /x,g/24 h for FG and EG, respectively, in a subject with adrenocortical supression following prednisone therapy. Values up to 1000 /i.g/24 h were estimated with appropriate choice of sample volume or dilution. Assessment of performance characteristics for analytical method i) Accuracy. Increasing increments of a standard solution of each of the glucosiduronates were added in duplicate to aliquots from a 24 h urine collection. The results are shown in Fig. 1 where the values estimated are plotted against the weight of steroid added, and the relationship is linear, with a slope agreeing well with that predicted (b = 1). The intercept on the Y
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
glucosiduronic acids, prepared by the same method showed I.R. spectra identical with those of authentic samples. Second, the 14C-tracers, when mixed with authentic cortisol and cortisone glucosiduronic acids and chromatographed in system 3, moved with the U.V. opaque zone. This zone, after elution and hydrolysis with Ketodase, yielded a mean of between 90 and 100% of the predicted value for each conjugate in 12 successive estimations by the double-isotope derivative procedure. Finally, samples of the l4 C-glucosiduronic acids were methylated with diazomethane and acetylated with acetic anhydride in pyridine to reform I4C-FGA and 14CEGA, as demonstrated by chromatography in system 4, and confirmed that the alkaline hydrolysis reaction had not affected the glucuronic acid residue.
569
570
JCE & M • 1976 Vul >\2 • No 3
LUTTRELL AND STEINBECK
0.5 EG (Aig) Added
axis is the value of the conjugate concentration in the sample. ii) Water blank. The contribution of the extraction and other steps prior to acetylation to the "blank" was tested by adding two aliquots of each 14C-tracer to distilled water and treating it as an unknown. After subtracting the acetylation blank and the calculated weight of tracer added, values of ±0.02 (jug were obtained which would represent ±2 /ug/24 h in a typical urine sample.
in) Within-assay variation. Ten replicate samples from a 24 h urine collection were analyzed and a within-assay coefficient of variation of 5% was calculated for each of the two steroid conjugates.
v) Specificity. Proof of specificity of a doubleisotope-derivative analysis rests on evidence as to the radiochemical homogeneity of the final derivative. This evidence is best obtained from the constancy of isotope ratio before and after crystallization with an authentic cold carrier (14). The criteria for proof of identity of radioactive steroids are discussed by Brooks et al. (15). Accordingly, groups of replicate samples, after scintillation counting, were pooled and 20 mg of an appropriate cold carrier was added. The solution was chromatographed on 10 g of alumina (Woelm activity II) using 100 ml toluene and then toluene/dichloromethane 1:1 until the 2 scintillator components PPO and POPOP were eluted, which was ascertained by illuminating the column with U.V. light. The steroid derivative was eluted with methanol/benzene 1:1 and this was crystallized 3 times in succession. In each case, the mother liquor was set aside while the crystalline fraction was carried to the next stage. Samples from all crystalline and mother liquor fractions were dissolved in 0.2 ml methanol and 0.5 ml toluene and 10 ml of toluene scintillant. The 3H/14C ratio was determined and Table 1 shows that this was not decreased following repeated crystallization. Indeed, the glucosiduronate level calculated from the isotope ratio of the last crystalline fraction (X-3), in each case falls within the range of the pooled replicates. Chromatography 1. 2. 3. 4. 5. 6. 7.
iv) Between-assay variation. The length of the procedure and the limited number of samples included in each run preclude calculation of this
8.
systems
Benzene, methanol, water, 100:50:25. Benzene, formamide (5). n-Butanol, water (12). Isooctane, toluene, methanol, water, 225: 275:400:100. Cyclohexane, dioxane, water, 100:100:25. Cyclohexane, dioxane, methanol, water, 100: 75:50:25. Cyclohexane, benzene, methanol, water, 100: 40:100:20. Cyclohexane, benzene, methanol, water, 100: 100:100:50.
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
FIG. 1. Vertical bars show the duplicate estimates of the weight of cortisol glucosiduronate (FG) and of cortisone glucosiduronate (EG) found in a series of aliquots of a normal urine collection to which increasing amounts of the synthetic glucosiduronates had been added. The predicted line of slope, b = 1 is also shown.
parameter from replicates of a urine collection. An analysis of the estimations for standards in 12 successive assays indicates the extent of variation likely. For instance, the mean estimate was 91% of the weight added for cortisol glucosiduronate with a coefficient of variation of 12%; for cortisone glucosiduronate the corresponding figures were 94% and 11%.
571
CORTISOL AND CORTISONE GLUCOSIDURONATES Subjects
siduronate (FG), cortisone glucosiduronate (EG), and 17-hydroxycorticosteroids (17-OHCS) in normal and other subjects Subject
Status
FG
EG
/xg/24h
/xg/24 h
FG/ EG
17-OHCS mg/24 h
1.0 1.1 0.6 0.5 0.9
8.2 9.5
H.T. B.L. M.R. R.V. R.F. H.B. P.G. H.L. J.E. R.S. D.F. M.H. D.A. D.K.
N N N N N N N N N N N N
94 100 69 55
50 16 51 53 66 73
95 94 120 110 56 83 88 60 78 55 82 77
N
77
68
N
A.Y.
N basal ACTH inf basal ACTH inf C.S. basal ACTH inf C.S. C.S. C.S. C.S. C.S. H.C. H.C. H.C. H.C. C.J.
33 32 49 320 16 130 92 630 900 110 350 170 140 58 68
79 70 79 170 47 110 140 190 430 130 340 110 350 110
68 33
37 75
0.6 0.3 0.7 1.0 0.8 0.9 1.1 0.4 0.5 0.6 1.9 0.3 1.2 0.7 4.8 2.1 0.9 1.0 1.5 0.4 0.5 1.8 1.8 0.4
100
160
0.6
J.C. J.C. S.M. S.M. A.R. A.R. W.B. H.H. F.D. A.H. J.B. W.L. A.O. A.O. A.L. J.S.
51
38
11.8 5.2 8.4 4.1
14.4 4.2 8.4
10.3 8.4
13.0 13.0 49.0 8.3 22.0 12.3 37.0 58.0 25.6 13.9 28.8 4.0 3.5 4.0 6.1
10.0
N = 1formal.
c.s. = Cushing's syndrome.
H.C. = Hepatic cirrhosis. C.J.= = Cholestatic jaundice. ACTH inf=ACTF[ infusion.
creasing with higher FG excretion and the correlation coefficient was +0.84 (Fig. 2). Results This trend is also evident after ACTH stimuThe values for FG and EG glucosiduro- lation and higher than normal values for the nates were based on the molecular weights FG/EG ratio were found in 2 patients with of their anions, 538 and 536, respectively. Cushing's syndrome but not in others. HowThe results are shown in Table 2. ever, in Cushing's syndrome the excretion The group of 14 normal subjects showed of at least one glucosiduronate was beyond a mean urinary FG excretion of 59 /u,g/24 h the normal range and in 4 subjects the exwith individual values ranging from 16- cretions of both glucosiduronates were ele100 /ug/24 h. The corresponding mean for vated. EG was 81 fig/24 h and the range was 55After ACTH stimulation, the FG excretion 120 /xg/24 h for 15 normal subjects. increased at least 6-fold but that of EG was FG excretion had a greater variability less, the highest increase attained being 2.3 than EG excretion, the ratio FG/EG in- times the basal value.
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
The normal subjects, of whom there were 7 men and 8 women between 19 and 58 years of age, were free from known endocrine or metabolic abnormalities and the women were not using oral contraceptives. Those subjects studied before and during ACTH infusion were females between 15 and 34 years of age and under evaluation for possible Cushing's syndrome and in one (A.R.) the diagnosis was later established. After a basal 24 h urine collection, 250 (xg tetracosactrin was given as a constant intravenous infusion over an 8 h period and urine collections continued over several 24 h periods. The collection for the day of infusion was analysed. The subjects with Cushing's syndrome, 2 male and 4 female between 34 and 52 years of age, were patients in whom the diagnosis was established by acceptable methods. Two (A.R and A.H.) underwent adrenalectomy with remission of symptoms and, on histological examination, adrenocortical hyperplasia was found. The patient H.H. had recurrent Cushing's syndrome from functioning remnants after adrenalectomy and persistent over-secretion of ACTH. The condition in W.B. was an ectopic syndrome from bronchial carcinoma. The 3 patients with hepatic cirrhosis, in each case due to alcohol, were all men between 34 and 67 years of age and were hospitalized with gross symptoms of the disorder. A.O. and W.L. each had pronounced gynecomastia and testicular atrophy; A.L., a patient with a 10 year history of the disease, also had massive ascites. The patient J.S., a male 54 years of age, had cholestatic jaundice from ethylestrenol (Orabolin).
TABLE 2. 24 h urinary excretion of cortisol gluco-
LUTTRELL AND STEINBECK
572
JCE & M • 1976 Vol 42 • No 3
1.0
O.5
1). Also, the isotope ratios obtained during 50
100
FG (Aig/24hr)
FIG. 2. Correlation of the 24 h excretion of cortisol glucosiduronate (FG) and the cortisol glucosiduronate/ cortisone glucosiduronate (FG/EG) ratio in normal adults. The correlation coefficient is r = + 0.84. The regression line is y = bx + a where b = 0.0096 and a = 0.18.
The patients with hepatic cirrhosis also showed variable results; 2 (W.L. and A.L.) had normal excretions and the other a depressed E.G. excretion on two successive occasions (38 and 37 /u.g/24 hr). The patient J.S. with cholestatic jaundice had an elevated EG excretion, and his FG was at the limit of normal, but the 17-OHCS value was normal. Both conjugates represent a small but variable proportion of the steroids measure as 17-OHCS, up to 1% in each case for the normal group (based on the weight of the aglycone only), and when abnormal values are included, levels of up to 2% occur. Discussion Previous workers, when studying cortisol and cortisone glucosiduronates, were restricted to examining the products of enzymatic hydrolysis, while in this study, it was possible to purify the conjugates by paper
analysis required that cortisol or cortisone conjugates within urine samples have diluted the 14C-tracers initially added. They are presumed to be the C-21 glucosiduronates of these steroids since they have a mobility in system 3 identical with authentic samples and are hydrolyzed by Ketodase which has considerable specificity for the ^-conjugates of glucuronic acid. Also in another study, one of us (B.L.) has shown that Ketodase will not hydrolyze the C-21 sulfates of cortisol and cortisone. The conjugates were extracted from urine by a small scale version of the XAD-2 column technique described by Bradlow (16) and the extract, which also contained sulfates and unconjugated steroids, was purified by paper chromatography in system 3. 14C-Labelled cortisol, cortisone, cortisol sulfate, and cortisone sulfate were added to a number of samples to demonstrate their separation from the glucosiduronates in the system. After enzymatic hydrolysis of the glucosiduronates, the procedure differed little from other published methods for the double-isotope derivative analysis of corticosteroids (17-19). The isotope ratio finally obtained was tested by the pooling of replicates after counting and diluting the radioactive products with authentic steroids and crystallizing the mixture (Table 1).
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
chromatography. Furthermore, as 14Ctracers were added before the extraction procedure, a correction could be made for procedural losses to individual samples, whereas previous methods only allowed a group correction for losses following hydrolysis. The conjugates investigated have not been unequivocally characterized, but the study provides further evidence to support the structures proposed by Brouillet and Mattox (4). In this regard, the steroids present are confirmed to be cortisol and cortisone by the crystallization of their doublelabelled oxime derivatives to a constant isotope ratio with authentic cold carrier (Table
CORTISOL AND CORTISONE GLUCOSIDURONATES
view when discussing the urinary metabolites isolated after a pulse injection of labelled cortisol in human subjects. Cortisone is known to be a minor secretion product of the adrenal gland, but cortisone found in peripheral plasma is largely the oxidation product of cortisol (22). In the Cushing's syndrome group, all subjects showed an elevation of one parameter above the normal range although 2 of them had normal 17-OHCS values. Thus, determinations of FG and EG excretion appear to hold some potential for the detection of Cushing's syndrome; however, the group was small and further studies are indicated. A limitation would be expected when studying subjects with impaired hepatic or renal function, since these organs and the small intestine are considered to be the major sites of glucuronic acid conjugation (23), and abnormal values might thereby occur. Of the 3 patients with severe alcoholic hepatic cirrhosis, W.L. and A.L. had normal results but A.O. on 2 successive occasions had a markedly depressed EG excretion of 38 and 37 /xg/24 h. In this regard Srivastava et al. (1) noted a tendency towards lower than normal values of plasma cortisone in liver and kidney disease. An abnormal result was also recorded for subject J.S. who had cholestatic jaundice induced by ethylestrenol. His EG excretion was elevated and the FG value was at the top of the normal range, but the 17-OHCS was normal. Acknowledgment The authors wish to express their gratitude to Dr. Vemon R. Mattox, Professor of Biochemistry of the Mayo Clinic, Minnesota, for helpful advice and for authentic samples of steroid conjugates, which he generously provided.
References 1. Srivastava, L. S., E. E. Werk, Jr., and K. Thrasher, L. J. Sholiton, R. Kozera, W. Nolten, and H. C. Knowles, Jr., Plasma cortisone concentration as measured by radioimmunoassay, J Clin Endocrinol Metab 36: 937, 1973. 2. Pasqualini, J. R., Corticosteroides urinaires:
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
The mean values for urinary excretion of FG and EG for the normal group were 59 and 81 fxg/24 h, respectively, while Brouillet and Mattox (4) in a group of 7 normal subjects obtained values of 37 and 94 /-ig/24 h after adjusting their values to include the weight of the glucuronic acid residue. This suggests good agreement as there is considerable overlap in individual values, the samples are small and the analytical methods employed differ considerably. The FG/EG ratio is markedly increased after ACTH stimulation, suggesting that cortisol excretion via the EG pathway varies less with cortisol production than the excretion via the FG pathway, and, if an assumption is made that FG excretion varies directly with cortisol production in normal subjects, additional support is found for this contention in the highly significant (P < 0.001) correlation observed between the FG/EG ratio and FG excretion for this group. This interpretation is compatible with the observation of Srivastava et al. (1) that plasma levels of cortisone were not obviously affected by an ACTH injection or by acute stress, although 2 patients with Cushing's Syndrome had elevated values of cortisone relative to cortisol. In the present series 2 patients had EG values elevated relative to FG (J.B. and A.R.) suggesting that the relationship observed between FG/EG and FG may not hold with prolonged ACTH stimulation and hypercortisolemia. While the FG/EG relationship has not previously been reported, the ratio of tetrahydrocortisol/tetrahydrocortisone (THF/ THE) has been extensively investigated. Zumoff e£ al. (20) recently summarised the data and indicate that the THF/THE ratio is increased in chronic lymphatic leukemia, hypothyroidism, administration of cortisol or following ACTH stimulation, and as a non-specific consequence of illness. Also, it is independent of age and sex in normal subjects. It appears likely that the observed changes in the THF/THE and the FG/EG ratios reflect changes in the availability of cortisol. Gallagher et al. (21) adopt a similar
573
574
3.
5.
6. 7. 8. 9. 10. 11. 12.
13. 14.
Metabolites tetrahydrogenes du cortisol et de la corticosterone, C R Acad Sci [D ] (Pans) 250: 1929, 1960. Pasqualini, J. R., Mode de conjugaison et fractionnement des Metabolites de corticosteroides urinaires apres administration d'ACTH, Ada Endocrinol [Suppl](Kbh) 51: 1071, 1960. Brouillet, J. C , and V. R. Mattox, Evidence for the presence of glucuronides of cortisone and cortisol in human urine, J Clin Endocrinol Metab 26: 453, 1966. Mattox, V. R., J. E. Goodrich, and W. D. Vrieze, Synthesis of C-21 glucosiduronates of cortisone and related corticosteroids, Biochemistry 8: 1188, 1969. Few, J. D., A method for the analysis of urinary 17-hydroxycorticosteroids, J Endocrinol 22: 31, 1961. Kliman, B., and R. E. Peterson, Double Isotope derivative assay of aldosterone in biological extracts, J Biol Chem 235: 1639, 1960. Bush, I. E., The chromatography of steroids, Permagon Press, Oxford, London, New York, and Paris, 1961. Shapiro, E., Synthesis of steroid glucuronides, Biochem J 33: 385, 1939. Koenigs, N., and E. Knorr, Ber Deut Chem Ges 34: 957, 1901. McCloskey, C. M., and G. H. Coleman, /3-DGlucose-2,3,4,6-tetraacetate, Organic Syntheses Coll. vol. Ill, Wiley, New York, 1955, p. 434. Mattox, V. R., J. E. Goodrich, and W. D. Vrieze, Chromatographic mobilities and partition coefficients of synthetic corticosteroid glucosiduronates, Steroids 18: 147, 1971. Poos, G. I., and L. H. Sarett, U.S. 3074979, C A 58: P14072e, 1963 (Abstract). Axelrod, L. R., C. Mattijssen, J. W. Goldzieher,
15.
16. 17.
18.
19.
20.
21.
22.
23.
JCE & M • 1976 Vol 42 • No 3
and J. E. Pulliam, Definitive identification of Microquantities of radioactive steroids by recrystallization to constant specific activity, Ada Endocrinol [Suppl](Kbh) 99: 166. Brooks, C. J. W., R. V. Brooks, K. Fotherby, J. Grant, A. Klopper, and W. Klyne, The identification of steroids J Endocrinol 47: 265, 1970. Bradlow, H. L., Extraction of steroid conjugates with a neutral resin, Steroids 11: 265, 1968. Stachenko, J., C. Laplante, and C. J. P. Giroud, Double isotope derivative assay of aldosterone, corticosterone and cortisol, Can J Biochem 42: 1275, 1964. Coghlan, J. P., M. Wintour, and B. A. Scoggins, The measurement of corticosteroids in adrenal vein blood of sheep, Aust J Exp Biol Med Sci 44: 639, 1966. Fraser, R., and V. H. T. James, Double isotope assay of aldosterone, corticosterone, and cortisol in human peripheral plasma,/ Endocrinol 40: 59, 1968. Zumoff, B., H. L. Bradlow, D. K. Fukashima, and L. Hellman, Increase in the tetrahydrocortisol/ tetrahydrocortisone ratio from cortisol-4-l4C: A nonspecific consequence of illness,/ Clin Endocrinol Metab 39: 1120, 1974. Gallagher, T. F., D. K. Fukashima, and L. Hellman, Clarification of discrepancies of cortisol secretion rate, 7 Clin Endocrinol Metab 31: 625, 1970. Dazord, A., J. Saez, and J. Bertrand, Metabolic clearance rates and interconversion of cortisol and cortisone, / Clin Endocrinol Metab 35: 24, 1972. Meittinen, T. A., and E. Leskinen, Glucuronic acid pathway, In Fishman, W. H. (ed.), Metabolic Conjugation and Metabolic Hydrolysis, vol. I, Academic Press, New York and London, 1970, p. 169.
Downloaded from https://academic.oup.com/jcem/article-abstract/42/3/567/2684570 by Nagoya University user on 13 January 2019
4.
LUTTRELL AND STEINBECK
