Metabolism of cortisol in cushing's syndrome

369

P. C. Ghosh and G. V. Pennington Sheffield and Region Endocrine Investigation Centre, The Jessop Hospital for Women, Sheffield, &gland. Received April 8, 1969

AE%STZiCT /

The urinary output of 6p,llp,l70~,21,tetrahy&rox~pregn-$-ene-3, 20-dione (6p-OH-F) and its 20 dihydro metabolites 6S,ll~,l70~,20+21 pentahydroxypregn-&-ene-j-one(@,20x-OH-F) and $3,11$,17~,2OS-21 pentahydroxypregn-4-ene-j-one(63,20p-OX-P)have been deterLined in patients with Gushing's syndrome. Following the administrationof tritiated cortisol, the spacific activities of cortisol (F) and tetrahydrocortisol(TIE')were compared with the specific activity of 6$-OK-F to demonstrate that adrenal secretion of 6p-OH-P occured, in addition to its formation by the peripheral metabolism of circulating2. Contrary to.earlier findings, the production of ~+CX-Y in Cushing's syndrome expressed as a percentage of the cortisol secretion rate (CSR) was founc to be loner than normal. This would appear to indicate a possible derangement of cortisol metabolism in this syndrome. A tentative explanation of this phenomenon is discussed. 3'hencompared with normal excretion values, the excretion rate of both,20 dihyaro metabolites of 6p-W-F expressed as a percentage of the CSR were found to be elevated. A possible explnnation for this phenomenon is given. Ii‘K?RODUCTIOlI The adrenal secretion of 6@-Oh-P was first demonstratedby Touchstone et al (1), while in adrenal hyperactivity the excretion of &-OH-F in abnormal amounts has also been reported (2). et al

(3) reported

Gray

a markedly elevated level of urinary bp-03-Y

in patients with Gushing's syndrome but concurrentlyfound that the conversion of F to 6P-OH-F was not abnormal.

ilheseaorkers

also failed to demonstrate in the urine the presence of any unconjugated corticosteroidsmore polar than 6P-OiY-F. Y'ceFrstu&y demonstrated that even when the Csi2was increased by a factor of

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20 above the normal, the pattern of cortisol netAolism remained essentially unaltered (3). This result has not been substnntinte&in the ::resent investigation. the 6p-0&F

In the six patients with iushing's syndrome studied,

excretion expressed as a percentage of the CSR wes lower

than that found in the normal population. The isolation and identificationof 2Ck-dihydro-6+C::-?F& its 20p-epimerwas first reported by Dixon and Pennington (4) in late pregnancy urine and their excretion pettern throughout normal and abnormal pregnancies has since been studied in this laboretory (5). The excretion of these ccr~poundshas been measured in tAhreeof the six patients under investigationand their outputs ex;:ressedas a percentage of the CSR was found to be higher than normal, a phenomenon quite different from that fountifor ~+os-F.

Systems for Chromatography Paper

PC-I) benzene:ethyl acetate:methanol:v;:-ater (7:3:10:10by vol) PC-II) benzene:t-butanol:water (70:43:66 by vol)

Silica Gel

TLC-I) distilled water TLC-II) Chloroform:methanol(84~16 by vol) TLC-III) diethyl ether:ethanol (9:l by vol) TLC-IV) chloroform:methanol(92:8 by vol) TLC-V) chloroform:methanol(99:l by vol)

Extraction, Purificetion and Chromatop;rs,~hic Estimation. Extraction of urine, and primary purification of the extract together with tinegas-liquid ckomatographic (G.L.C.) deterknation of 6P-0X-F was carried out as previously described (6). Derivstive formation and the re reducibility of tileassay were also reporteii in the same paper (6P.

Sept. 1969

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371

The determination of t‘ne20-cihydro-6P-OH-Fwas also carried The steroids were isolated from TLC system II out by G.L.C. previously described, (6) 0.1 ml pyridine being used in place of ethy-lenedichloride. 1;suitable aliquot was cnromatographedon a hybrid column (0.9;/; XJ-~O+O.~,eterl!iination of the Cortisol Secretion ;iate CSR), 17 Oxosteroid 117 :,A) 11-Xyrdroxy-corticostroids(17 OiiCS)and Plasma Cortisol. The CX was carried out as described by Cope (lo), 17oxosteroids and 17 oxogek,c steroids by the methods of Appleby et al (11) as modified by "he LledicalZesearch Council Committee on Clinical _kdocrinolow (12,13). Plasma Cortisol was estimated by tilemethod of Peterson et al (UC). LiadioactiveiAaterinls 1,2-31I-Cortisol (specific activity: 5 mc/mg) was obtained from the lkdiochemical Centre, hersham and was purified by thin layer chromatograohyin TLC System IV prior to use. Determination of specific activities Tritiated cortisol wit'na radioactivity of approximately 35,000 q:c v;asadministered orally to patients E5th Gushing's syndrome in 40 ml isotonic saline, ~$-o:I-Y,3 and TXF were isolated and extensively purified. KLio_uotsof the oxidation products of 6+l\;:_1_:: and P were qunantitated by GLC ezldseparate aliquots ivere taken for radioactivity assay. Isolated TTHFwas estimated by the blue tetrazolium method (6) and a suitable aliyuot was taken for raaioactivity counts. Iuentification of cortisol metabolites more polar than 60-Oil-F The urinary ethyl acetate extract was purified following initial purification in TLC System I and 2c& and 208 dihydro-6P-OH-F were isolated as a mixture from TLC system II (I>$0.30). Primary identificationprocedures were carried out as previously reported (4). Additional identificationwas carried out as described by Dixon and ?ennin$on ('7)and by comparative programmes of thin-layer, paper and gas-liquid chromatography.

372

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Separation of the 2ooCand 2013enimers of 63-O&.-I". Aliquots of the combined 204ihydro epimers of 6+0i1-E'were chromatographedon boric acid impregnantedplates(3;; boric acid in water) in TLC system II (modified from 8). '.;-ith boric acid present in the chromztographicsystem, the cycloboxatesformed from the sterically favourable 17x,20$-dialsmoved considerablyfaster than the 17g2&-dials, which did not form such complexes. The uovo ( 254 w) absorbing zones correspondingto the marker ::ereeluted and separately purified in TLC ;;ystemIII. bteroids vrithreduced side chains move extremely slowly in this system, thus facilitating a wider separation of the t:Togroups even nhen tl?eirpolarities in all known systems are fairly close. It was also found possible to separate the two epimers by preliminary.chrom:ltography in :X System I and following elution of the single s?ot, subsequent clhromatography in FC system II v;itha four nour over run. 'Ihis revealed two UOV= absorbing zones corresponding to the marker of 20x and 206 dihydro-6$-@&F, the 20x being more polar than 2013epimer. Aliquots of 2ooCdihydro-6$-O&P and its 20@-epimer detained. either from the boric acid plate or from the FC jystem II yferethen oxidised to 17-0~0 steroias (6). The TlSi deri.vatives cere then prepared using both the methods previously described for ~~-oE-;: and the method described for 2O+ihydro 6p-C:-:-~. _XLlaeri.vetives were chromatographedon two columns; a hybrid (O.g;>E-60+1.C;; KGS) and a I??' In both instances the isolated steroids had identical P NGS. retention times with that of 63,11+!iy~iro~-androst-~;_-ene-3,17-!~ione treated the same nay. It can therefore be assumed tl!atboth the compounds tiifferonly in their si&e clain 0rient:;tion. .-.s -Lne ll-hytioxyl Grouping can only be reacted for 'T';:IX-ether cierivstive formation when pyridine is used in @ace of ethylene dichloride, both have a hydroxyl grouping in the ll-position. Further aliquots of the 2ooCand 2013aihy&ro derivative of 6p-OH-F were reacted for TiiSi-etherformntion, again using nyridine and with the side chain intact (9). Considerzble The resultant products were ;,aschromatographed. separation of the 2Bc and 203 epimers ~,s n.c?Aevedon the 1,;:,CS LJeaks column, although tne retention times v;ereion;;and r-Tide were obtained. The hybrid stationery nhzse column was found to be more suitable although r:ithazeinc the sensratiollfactor of the two epimers is reduced. The 200c-dih;rdro-6+C;i-F and its 2013eTic:er,isolated from ?I) both the sources )iee. boric acid plate an> tne PC Gyste;:: had identical retention times which vferesimilar to those obtained from authentic steroids processed in a similnr fashion.

All patients studied exhibited the clinical manifeststlons of Gushing's syndrome, the diagnosis being conf'imed by the determina-

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STEROIDS

Sept. 1969

tion of a CO&_{., the vrinery output of 17 KS and 17 OHCS and multiple &eterioin:Ltions of ~ksrria cortisol. 'YzbleI.

These values are listed in

The excretion of unconjuzated polar corticosteroids

(6;;_~i;i_i?; 69,2&41~&~ and its 209 epimer) were also found to be sienificsntly elevated (Table II) when compared with the normal ren;e found in four endocrinologicallynormal subjects. (Table III). In one experiment the adrenal secretion of 6+0H-F has been confirmed.

Tritiated cortisol ::'a~ administered to patient (B.B.)

2nd 6,3-&!-F,3'and THF isoleted.

The specific activities of

6,3-0;,7(515.5 ~_'C/'"~) and F (832.5 GLC annul t&t

of TIP (1,139

rq:c/mg)mere determined by

qJc/mg) by the blue tetrazolium reaction.

ii'nis &ata demonstrates that a considerabledilution of 6p-bH-F has taken place and confirms earlier reports that in addition to the peripheral metabolism from cortisol, ~~-oI+-Fis also directly secreted by the adrenal gland (l;ll;) The discrepancy found between the specific ectivities of cortisol and TEF is puzzling, although it substantiatesthe recent 7:ork. of Fukushima et 21 (17).

These .Trorkers /,

me:.isured tne srJccificnctivities of TX7 and THE in a series of patients by at 1e:st three different methods including double isotope aerivstive analysis, and found that the ratio of the specific activity of the tv
and F were measured by G.L.C., previously shown to

measure these compounds only (6).

In all instances an internal

standasd was used for quantitation. DISCUSSION The main purpose of the current investigationwas to

374

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STEROIDS

investigate the in vivo metabolic patterns of cortisol in Gushing's syndrome, the output of polar unconjueated corticosteroidn being considerably elevated. Gray et al (3) while surveying the excretion pattern of cortisol metabolites in a patient v:ithCushing's syndrome found that despite a C.&ii. twenty times higher t'nannormal, cortisol metabolism remained essentially similar to the normal pattern. These workers concluded thst despite a high excretion of all tiovrncortisol metabolites, the proportion of the mets_bolites, when Compared with the C.S.9. remains normal in Gushing's synurome. This observationwas not substantiatedby us when an analysis of polar cortisol metabolites Ins

achieved by GLC deterdnations.

The 6p-OK-i(" output in Gushing's syndrome expressed as a percentage of the total cortisol produced was found to be substantiallylower than the normal, (Table II) although the absolute x:ount excreted remained extremely high.

It is possible that in Gushing's synorome

either the peri_nheralmetaboiism of F to 6p-Oil-Por the adrenal secretion of 6p-OH-P is grossly disturbed.

As a considerable

adrenal secretion of 6+0X-F was found to occur (determinedby the specific activities of F and 6p-OH-F) it is sugGested that the defect is mainly in the peripheral metabolism of F to 6~-OH-F. It is of interest to note that the excretion of the 20-aihydro6p-OH-F corticosteroidsexpressed as percentage of the C.S.H. was higher than the normal (Table II) indicating a metabolic cierangement of cortisol in this disease.

Dixon and Pennington (7) have

recently found that about 47:; of the circulating 2094ihydrocortisol is utilized for the synthesis of 20+dihydro-6p-OH-B in Gushing's syndrome.

If prior reduction of cortisol at the C-20 occurs prior

Sept. 1969

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STEROIDS

to C-6 hydro.xylation, it can be assumed that in Gushing's syndrome proportionatelymore 20-dihydro cortisol than 6p-OH-F is utilized for the formation of ZO-tihydro-6$-O&F.

The metabolism of 20-dihydro-

cortisol in normal subjects has so far not been studied.

It is

possible that in vivo metabolic transfer of 204ihydrocortisol to 20-dihydro-6$-01-;-F is less in Gushing's syndrome than in normal subjects. This would add considerableemphasis to the results found in the f v-tion. present investi,,

The authors gratefully acknowledge the technical assitance of the laboratory staff of the Sheffield and Region Endocrine InvestigationCentre. :ieaxe also grateful for a supply of authentic 6~-OX-F which was received from Dr. S. Bernstein, Lederle Laboratories, U.S.A. Additional quantities of both 6+0:1-F snd 204ihydro metabolites of 6~-OH-F were synthesised in this laboratory by Dr. V:r, R. Dixon. This work was supported by a grant from The tiedical. Research Council of Great BritAn.

1.

Touchstone, J-C., Kasparow, ILOand Rosenthal, O., FED. PRO& 18, 340 (1959)

2.

Touchstone, J.C. and Blakemore, W.S., J. CLIN. ENDOCR. 21, 263, (1961)

3*

Gray, CO HO, Greenaway, J.b;.,Holness, ND& J. RxDocR. 3, lyy (1962)

4.

Dixon, 5. R. and Pennington, G. V.,

59

Ghosh, P.C. and Pennington, GV.,

Unpublished.

6.

Ghosh, P-C. and Pennington, G.W.,

STEROIDS, l&(2): 247,

7.

Dixon, !%R. and Pennin#on, G.::'.,STEROIDS,2,

8.

Schneider, JOJO and Lewbart, bl., TElXAHEmON. 20, 243,

Ye

Rosenfeld, B.S.,

10.

STEROIDS. 4, I47

and Shaw DA,

JO Endoor. &

281 (1966)

423, (1968)

(1964)

Cope, C.L. and Black, E.G., BRIT. MED. J. 3

(1969)

1020 (19@)

(1964.)

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STEROIDS

11.

Apl;leby,J-I., Gibson, G., Norymberslci,J.K..and Stubbs, ?P&, BIOWEBi. J. 60, 453, (1955).

12.

Xedicnl Research Council Committee on Clinical Endocrinolog. L_~NX!L' &- UJ5 (1963)

13.

Medical Research Council Committee on Clinical dndocrinology. LANXT & 1% (1969)

14.

Feterson, 9.5, Karrer, A. and Guerra, S-L., AN1LiT~ 05% 2, U-4-3(1957)

15.

Burstein, S-, kimocll, 5 L., Illaiber, 3, CLIII.YIuLOCR.2, 491 (1967)

16.

&iley, d., 5. ENDOCR. 28, 31,

17.

IXkushima, DeK., Bradlow, ii.L-,Hellman, L. and Gallagher, T.P., 2. CLIN. ~xDoca. 28, 1618 (1968)

i.L.

ad,

Gut,

..r.,

(1964).

STEROIDS

Sept. 1969

The

systematic

names of

steroids

used

377

in the text by trivial names

or abbreviation are: dione. E'= Cortisol = llp,l,7~,21-trihy~roqpregn-4_ene-3,20 21-tetrahydroxypregn-4-ene-)-oxxe. 20‘~(oc)-di~ydro_Cortisol = 21~,17o~,2U~(oc), fiednisolone = llj3,17~,21-trihydroqpregn-1,4-&one-520 dione. T?iF= tetrahyb.rocortisol = 3cc,Ll$, 17~~21-tetrahydroxy-J@-pregnan-20-one. THX = tetrahydrocortisone= jg170~,21-trihydsoxy-5p-pregnan-ll, 200dione.

6$-O&F

= 6$-bydroxycortisol

6~,2@+}-W-F ir_-ene-3-one.

= 4% 11~3,I?=, 21_tetrahydro~ree~ne-~a~e.

= 2Ox(j3)-dihydro-6~-OH-.F

= 4% lJ.@t 17% X4@),

2bpentabydroxypreg

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c. s.2. 6p43_~

a3

556-O 383.8 630.8 454.4

67

38

55

82

E.F.

B.bb

J.R.

J.V. 0.55

1.14

1.00

0.

1.40

L123.3

76

0.92

46 of C.&R.

B.B.

hours)

663.2

(pd24

72

(mg/U, hours)

G.B.

Patient

136.0

51*5

117.0

6p, 2bO;-I-F &p/24. hours)

0=u,

0.13

0*17

3 of C.S.&

210.6

0.38

0.25

97.2

c.6.a.

;; of

0.26

SIOUX-S)

176.0

(f~g/24

6~,20i3-0+~

6~,2bLPI-F and 6$,20+0!1-E' in Gushing's Syndrome The urinary output of 6$-011--11', and the determination of the rate of excretion as a percentage of the total cortisol secreted

380

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