Translation Series No. 910

TRI,NSLATION SERVICES CAN 7,.DA F4iI ICIEi OR S. T. I. NATIONAL FESEARCH COUNCIL

OTTAWA FISHERIES RESEARCH BOARD OF CANADA CANArg Translation Series No. 910

The metabolism of steroid hormones

By Renata Derowska and Bogdan Szukalski

Original title: Metabolizm hormonciw sterY-dowych. From: Post. Biochem., Vol. 12, pp. 309-345. 1966.

Translated by the Translation Bureau (MJK) Foreign Languages Division Department of the Secretary of State of Canada

Fisheries Research Board of Canada Research Laboratory, Halifax, N. S. 1967 ', -DEPARTMENft OF THE SECRETARY OF STATE SECRÈTARIAT D'ÉTAT 9/0 BUREAU FOR TRANSLATIONS BUREAU DES TRADUCTIONS FOREIGN LANGUAGES DIVISION DES LANGUES DIVISION ÉTRANGÈRES

TRANSLATED rnom - TRADUCTION DE IN TO --• -•À Polish English

SUBJECT - SUJET Steroid Chemistry

AUTHOR - AUTEUR Renata D.erowska and Bogdan Szukalski

TITLE IN ENGLISH - TITRE ANGLAIS

The metabblism of steroid hormones

TITLE IN FOREIGN LANGUAGE - TITRE EN LANGUE dTRANGèRE Metabolizm hormonew sterydowych

REFERENCE - ReFgRENCE (NAME OF BOOK OR PUBLICATION - NOM DU LIVRE OU PUBLICATION Postgpy w Biochemii ( Advances in Biochemistry ) Vol.12

PUBLISHER - eDITEUR

CITY - VILLE DATE PAGES Warsaw 1966 309 - 345 60 typed pages

Dr. D.R. Idler REQUEST RECEIVED FRom Fisheries Research Board OUR NUMBER 9332 REQU(S PAR NOTRE DOSSIER NO

DEFARTmENT of Fisheries TRANSLATOR M ,J eicrtrhynSki MINISTÉRE TRADUCTEUR

YOUR NUMBER 769 - 18 - 14 DATE COMPLETED 14 July 1967 VOTRE DOSSIER N° REMPLIE LE

DATE RECEIVED May 15, 1967 REÇU LE (Advàiiices- in BiocflemiS- t;ir) 9:› "A?- if, (/7

Renata Derowsk:and Bogdan Szukalski

The metabolism of steroid hormones The biosynthesis and catabolism of steroid hormones are reviewed. The physiological importance of steroid hormones, as well as their ever increasing clinical application are the reason why for a number of years investigations of their biosynthesis

and catabolism have been conducted. The basic methods used for that purpose are perfusion of a gland or some other organ with metabolites and an analysis of the resulting products, incubation of metabolites with pieces or homogenates of gland tissue (adrenal glands, testes, ovaries, placenta) and the analysis of blood leaving the gland, blood of the peripheral circulatory system, and urine. The application of tracer compounds accel- erated considerably the progress in this field and supplied much new information, permitting to create a fairly complete 'picture of the metabolism of steroid hormones.

› M.Sc.Eng., Senior Instructor, Institute of General Chemistry, Medical Academy, Warsaw M.D.,M.Sc. (Chem) Assistant Professor, Institute of General Chemistry, Medical Academy in Warsaw, and the II Internal Medicine Division, the Bielariski Hospital in Warsaw. List of abbreviations applied: DHA - dehydroepiandrosterone; androsteAtione 4-androstene-3,17-dione; ACTH - adrenocorticotropic hormone , Translator's note: A Polish-English biochemioal dictionary not being available, the following 1 dictionaried and reference books were used to clarify terminological problems 1. Diotionary of Organic Compounds. Eyree & Spottiewoods. London, 1965 • ' 2. Merok Index, 1965. 3. Physiology and Bioohemistry. Bell,Davidson, Scarborough, 1965. 4. Review of Physiological Chemistry. Harold A. Harper, 1965 5. Biochemistry. West Todd Mason, Van Bruggen, 1966.

•••2 • 2 • The division of steroid hormones is based on their physiological activity conditioned by their chemical structure. All the steroid hormones can be derived from three synthetic hydrocarbons: pregnane built of 21 carbon atoms, androstane of 19 atoms and estrane of 18 atoms. In a live system, cholesterol is the intermediary compound in the process of formation of all steroid hormones. Its complete biosynthesis was the subject of exhaustive works by Supniewski (158) and lately by Clayton (29). We shall deal with changes of cholesterol leading to steroid hormones: gestagens, cortice-steroids androgens and estrogens and with the catabolism of those hormones.

I. Routes of biosynthesis of steroid hormones 1 . "2..q.AIP 521 * The first stage of biosynthesis of pregnancy hormones taking place in the corpus luteum, adrenal glands, testes, ovaries and the placenta, is based on a degradation of the side chain of cholesterol. Solomon et al. (156) established that homogenates of the adrenal gland of a cow produce 2O_fl (II) from cholesterol (I). The subsequent stage of cholesterol transformation after 201 hydroxylation is hydroylation in position

22 with formation of 20/1 , 22 a -dihydroxycholesterol (III) (145) which is transformed into pregnenolone(IV) by the action of 20, 22-desmolase, with detachment of the side chai n in thea form of l'ç Ti-anslatoris note: Term not found in English books. Appnrently a collective name for pregnancy hormones.

*• * 3 . 3

isocaproilx aldehyde (143, 144, 163). Of other oxidized cholesterol derivatives, 22-hydroxycholesterol is a better substrate for de- composing enzymes than cholesterol itself, and 22-ketocholesterol does not decompose in those conditions. 20P 41.ydroxy-20-ketocholesterol is not sensitive to the action of desmolase (30), and therefore cannot be considered as an intermediate product of

the transformation of cholesterol into "gestagene. In pregnenolone in position 3, under the influence of dehydrogenase found in the microsomes of the adrenal glands, a reversible transformation occurs of the hydroxyl group Into the ketone group with formation of Z1 5_ pregnene-3,20-diofflV); subsequently under the influence of A5-3-ketoisomerase a shift of the double bond into a conjugatalposition in relation to the newly formed ketone group takes place. The product of those transformations is the pregnancy hormone progesterone (VI), (scheme 1). The cyclical corpus luteum produces in 24 hours about 20 mg of progesterone, whereas in the cortex of the adrenal glands about 250 mg of it is formed during that period (14, 78). Under the influence of dehydrogenases 20- othydroAy-AA-pregnenone-3 and its isomer 20 f3- / can result from progesterone and from pregnenolone 200C-hydroxy--pregnenol- 3 is formed. Talalay et co-workers ( 161, 176) obtained from Pseudomonas testosteroni the isomerase of 21;5-3-ketesteroids i participating

•••4 See:nOte ion page 2. in formation of progesterone, in crystalline form. It shows its _optimum activity between -

Schemat 1. Biosynteza gestagenôw

Scheme 1. Biosynthesis ofn gestagens" 6 pH and 8 pH, is competitively inhibited by a low concentration 31 of 1718 -estradiol, 19-nortestosterone and 17-dihydroequilenin (67). Among animal tissues, it is in the adrenal glands, where this enzyme is present most abundantly (43, 44). The reversibil- ity of the isomeration reaction is pointed out by investigations conducted by Ward and Engel (177), Who reported that extracts of acetone powder from the adrenal glands of sheep cause a transformation of androster;hone into 3/3 -hydroy„y-45-androsten- 17-one.

2. Corticosteroids Among more than 70 steroids isolated from the cortex of adrenal glands (182) only 7 show to a greater or lesser degree the ability of r.emoving the disturbances occurring after removal

See footnote on page Z.

" See footnote on page 1. •••5 .5 • of the cortex. They are cortisol(XV), cortisone(XVI), corticosterone(XI) îl-dehydrocorticosterone(XII), cortexolone(IX) and aldosterone(X)II). Their systematic names and synonyms used are compiled in Table 1. Table 1 Sysjpematic names and used synonyms of fundamental corticosteroids

\C cram° n name . Synon,yms in use Syst emati name

Cortisol hydr000rti sol ; 17 -hydro3eoorticouterone ; A, 4 -pregnene -lip , 174 '21 -triol -3,20-di one Kendall '8 substance 'TH; Rei abstain's substance "M"

Cortisone 7-hydroxy -11 -dehydr000rtiooster one ; Kendall 's substance "E"; Reichstein's substance 'IF'? A4 -pregnene -17J,21 -di ol 1 ,21 -tri one Corti oost erone Kendall 'a substance Hs" R ohstein '9 substance "HI' A4 -pregnene-np,21 -diol -3, 20 -dione

11 -dehydrocorti 4 cc st e ro ne Kendall's substance HA" -pre gnarl , —21 -al -3,11 ,20-tri one

Cortexone 11 -desorycorti cost erone ; 'DOC ; 21 -hydroxyp ro gesterone ; et Reichr+ein's bstance "CI" LN2-preg,non -21-o 1 -3 ,20 -di one

Cortex°lone 17d-hydr oxy cot BX o ne ; 17-hydrox,y -11 -desoxyoortio ester one ; ll -des ox,y oorti s ol ; Reichstein's substance "Sit 4 -pregne.ne -17,4 21 -di ol -3, 20-dl one

Aldosterone A.4 -pre gnene -Ile, 21 --dial -3,20-di —18a1

Apart from cortex hormones proper with molecules built of 18 carbOn atoms, etxx compounds with 18 carbon atoms in the mole- also cule have/been isolated from the adrenal glands, pertaining to female sexual hormones as well as compounds of androgenic pro- perties containing 19 carbon atoms (180, 181). ...6 . 6 .

312

312 R. DABROWSKA, B. SZUKALSKI [41

m 4 0 • 0 0 X tn 0 e e o 9.1 0 O 0 OI1 O fl x 0 o u II el 0 ,p o p O X 0 se • CH > O 0 /IF re) (:)C=, ) t._ & 2. ,C c z -2 0 0 0 , o Translator' s note 11 r. Roman :•rimerals to denote The authors use e steroid hormones both 1.-n the text (in m and tt.0 brackets af ter the nar,é;) in diagrams. ctj However, this sys tem i*pears to be some- »ri what confusing, since 1.n several cases the sanie Roman numeral is used to denote compounds differing buth in naine and stereochemical structure. These numerals are: )(II in diagrams 2 and LMV to LX:k= in dinrrams 11 and 12. LMV, IMI/1 and LXVI1T are shown in diagram 12, but not mentioned in the text 0 I ntbt find any c..„-Çplanation in text books. could On the other hand what, is confusing to me may be clear to a specialist in steroid chemistry. Nevertheless I thought it worthwhile to raise • this point.

••• • 7 • The main route of biosynthesis of glucocorticosteroids is based 313 on hydroxylations of progesterone (VI) in the order 17c.(, 21, 11 p . The substitute present in the molecule makes it more difficult to introduce an -OH group into the position preceding it in a given series. The synthesis of cortisol (XV) requiring three hydroxylations runs mainly over progesterone, 17c(-hydroxyprogesterone (VII) and cortexolone (IX). However, corticosterone (XI) is formed as result of 11p-hydroxylation of cortexone (X). Owing to investigations by Sharma and co-workers (151, 152, 153), who obtained relatively stable preparations of lip -hydroxylase it is known that this reaction is competitively inhibited by

testosterone, androstendione and DHA. Both basic glucocorticc»-- steroids i.e. cortisone and corticosterone can be easily oxidized at C-111.5 , forminQcortisone (XVI) and 11-dehydrocorticosterone (XII). Also a direct hydroxylation of progesterone in position c-ne is possible, which is indicated by isolation from the adrenal glands of hog of -hydroxy-progesterone (XIII) (100) and its transformation into corticosterone and cortisol, and also detection in them of 21-desoxycortisol (XIV) (101). Never- theless, the percent participation of direct le-hydroxylation in the transformation of progesterone indicates, that those are

reserve routes for the synthesis of glucocorticosteroids (41). There is also a route for corticosteroid biosynthesis omitting progesterone, namely: from pregnenolone (IV) via 17d-hydroxypregnenolone (VIII), 170(-hydroxyprogesterone (VII) cortexolone. (IX)

See footnote on page 1. •••8 . 8 . to cortisol (2, 179). This is suppOrted by the experiment by 14 Engel (178) who incubated (4- C) progesterone and (7-3H) 17d-hydroxypregnenolone with homogenates of tumor tissue of adrenal glands and established that both substances became transformed into cortisol with progesterone showing 17% efficiency and 174:L-hydroxypregnenolone 63% efficiency. The existence of a biosynthesis route of cortisol from pregnenolone omitting progesterone and corticosterone explains the non uniform intro- 14 duction of the isotope C in corticosterone and cortisol after administering traced cholesterol and progesterone, at observed earlier by Eichhorn and co-workers (42).

Investigations conducted in recent years indicate the existence of still other possibilities of transformations of pregnenolone. Dobriner and Lieberman (37) detected in the urine of patients treated with ACTH 21-hydroxypregnenolone, and Pasqualini and Jayle (109) found 3,21-bisulfate of this compound. After administering 21-monoacetate of 17 1 , 21- dihydroxypregnenolone, part of the administered compound was found in the urine in the form of 3-monosulfate and a part in metabolised form as alle-tetrahydrocortexone (116, 117). Berliner and co-workers (9) proved the transformation of 21- hydroxypregnenolone into cortexolone in homogenates of adrenal glands of oxen. Recently, Pasqualini and co-workers (110) investigated transformations of 21-hydroxypregnenolone and

•••9 • 9 •

17c,i,21-dihydroxypregnenolone traced wi th tritium in pieces of hypèrplastic human adrenal glands. It 1,,las established that

WI% of the administered 21-hydroxypregnenolone became tran.sformed into cortexon.e and 12% into corticosterone, whereas 9.6% of

171 ,21-dihydroxypregnenolone was transformed into cortexolone and 7.5% into cortisol. It follows from this that pregn.enolone, before transformation of the 45 -3-hydroxy-group into the

A4-3-keto-group can undergo hydroxylation not only in positions

17d, and 111 , but also at the carbon 21, and that f 170(,21- dihydroxypregnenolone is an intermediary metabolite in the biosynthesis of cortisol. The location of enzymatic systems participating in the biosynthesis of the hormones of the adrenal gland cortex has been

a paper by Krawczynski (73). The deepest described in detail in layers of the cortex, the "zona retic-ularis" and partially the tr zona fasciculata" contain in the microsomes 17t[ -hydroxylase and produce cortisol; hOwever, only the most remote from the medulla "zona glomerulosa" contains l8-oxygenase and is able to produce aldosterone. Other hydroxylating enzymes, parti- cularly 11p-hydroxylase from the mitochondria and 21-hydroxylase from the microsomes occur in the whole cortex, so that cortico- steron.e is produced in all the layers, in largest quantities however in the "zona fasciculata".

••.10 . 10 . The adrenal glands are also able to produce hydroxylated

steroid compounds in non typical positions like 2,6 2<' ,613 ,7

and 16e/, of which particular attention is attracted by 160i- hydroxypregnenolone. Bongiovanni (16) detected it for the first time in the urine of children showing a deficiency of dehydro-

genase of 3p -hydroxysteroids. This compound WaS also found in the urine of healthy children and of children with a deficiency of 21-hydroxylase. In vitro it is formed from pregnenolone incubated with the adrenal gland cortex of a foetus, or a newborn, or with hyperplastic cortex. (173, 174). 16c/- hydroxypregnenolone was detected in 1964 in the blood of the unbilical cord (40). The presence of this compound in the urine in the first period of life is probably the result of feeble activity of 3 f, -hydrox-ydehydrogenase and high activity of 160(-hydroxylase, typical for the immature adrenal gland cortex (172). The physiological meaning of 16J-hydroxypregnenolone is not clear so far, it is only known that it has no influence on the water and mineral economy (164). In order to find the precursors of aldosterone, adrenal glands were incubated with various steroid compounds participating in the biosynthesis of other hormones of the cortex. Among them, cortexone intensified considerably the production 1 4. of aldosterone. An application of cortexone traced with C in position 21 led to formation of radioactive aldosterone, which

11 . 11 .

contained 48% Of the radioactivity of the substrate. The isolation . by 'Kahnt and co-workers (63) of crystalline 18-hydro.Kycortexone from adrenal glands proves that cortexone is oxidized in them in position 18, and the detection in adrenal gland extracts of the corresponding hydroxylactone (C-18-aC-20) (157) indicates that the adrenal glands are capable of oxidizing primary alcohol to the carboxyl group. Consequently the main route of aldosterone

synthesis runs from cortexone (X) via 18-hydrol_ycortexone (XVIII) and 18-aldocortexone (XVII) (scheme 3). On the basis of results of experiments with isolated adrenal glands of oxen Dorfman and co-workers (132) proposed a conception of another route for aldesterone bios.ynthesis. It starts from progesterone (VI). 315

. I 'OH /CH 3 0, H CH3 HO, C=0 o Progesterone CH2 140_ .4,--- VI 11p hydro,xy- o Cortexone 1.1P X -.., progesterone' ee cn Corticosterone XII 0 .3 XVII XVIII XI XXIII XXIV tT1 ÇH2OH D:r 0 HO CH2 c,-0 er / el* XXI CH OH 0, H / 2 HO 'C' C=

XXII Schemat 3. Biosynteza aldosteronu Diagram 3. Biosynthesis of aldosterone

I c:n41—j • 13 • which undergoes consecutive hydroxylations in positions 18, 21

and ll r , producing 18-hydroxyprogesterone (XX), 18-hydroxycortexone (XVIII) and 18-hydroxycorticosterone (XXI). The latter transforms into aldosterone(XXII) as result of oxidation of the alcohol group in position 18 (1)4). However, these transformations do not cover all the routes of aldosterone biosynthesis. It can also be produced from corticosterone (6, 119) which undergoes hydroxylation to 18- hydroxycorticosterone (XXI), under the influence of 18-hydroxydehydrogenase the latter transforms into aldosterone (XXII).

The efficiency of the latter transformation is mall and in a healthy human only 10% of 18-hydroxycorticosterone is subjected to it. The fact that 18-hydroxycorticosterone occurs mainly in cyclical form, containing a hemi-a_cetal bond between C-18 and C-20, is probably the factor which renders the transformation more difficult. (146). A much minor role is played by the route of aldosterone biosynthesis from progesterone (VI) via 18-hydroxyprogesterone (XX), 18-aldoprogesterone (XIX) and 21-deso.m_yaldosterone (XXIV). The intermediate compound in this synthesis21-desoxyaldosterone, can form nlso from progesterone via lq-hydroxyprogesterone

(XII) and lip ,18-dihydroxyprogesterone (XXIII). All the changes leading to formation of aldosterone occur in the "zona glomerulosa". An exception is the reaction of

• • • 111. • 14 • 18-hydroxycortexone synthesis, which occurs also in the "zona fasciculata" and "zona reticulata" (85, 154). The formation of corticosteroid in the adrenal gland cortex is strongly influenced by ACTH. The mechanism of this action has not yet been sufficiently explained; however it is known that ACTH acts here indirectly by its influence on the synthesis of NADPH, which is the co-enzyme of all the reactions of steroid hydroxylation. ACTH activates the phosphorylases, which increases the metabolism of glycogen in the adrenal glands and causes an increase of the ouantity of 6-phosphoglucose, Oxidation of 6-phosphoglucose is accompanied by a reduction of NADP to NADPH (84). Besides, ACTH acts indirectly on the phosphorylases, stimulating the sylithesis of 3 1 5tAE1 (55) (Scheme 4). The production of glucocorticosteroids depends Rlso to a great extent on environment factors. Stresses are accompanied by an increase of the corticosteroids in the adrenal glands and a temporary reduction of ACTH in the pituitary gland (131). Experimental removal of the pituitary gland causes an atrophy of the adrenal gland cortex, however, leaving intact the "zona glomerulosa", in which aldosterone is formed. A similar atrophy is observed after prolonged administration of corticosteroids (13). Consequently, the production of ACTH depends on the level of corticosteroids circulating in blood; an increase of their quantity inhibits the production of ACTH, a decrease increases

••• 15

lb 15 e the release of ACTH. However it is not known, whether this negative reverse conjugation acts directly or indirectly on the pituitary gland by an increase of the production of its sub- > eminence regulator of CRF (corticotrophin releasing factor) (St) .

Adrenal glycogen ACTH Glikogen nocInerezowy Fosforylaza phosphory 3'5'Amp ACTH 1 fbsfoglikoza 1 phosphoglycose

Kwas + ....--, NADPH'-'-',---:-.---, 6 fasfoglikoza +NADP--).- likorewg. .,---r-e 6 phosphoglyoose pho spmg.i.uco.../a.e //1 \-....., a.old \ \\ / /// 1 \ \\ \ \\ • / 1 1 \ \ // i, \■ \Cholesterol • 1 \\\ \ I I 1 `---,...* i 1 \ I \. . Pregnenolone I 1 ‘ \ ..... ----1,...+ / I 1 / I t \ \ este ron e / / 1 I I\ \ \ Pme \ e...j...--- -----,,....ie / 1 C or t exe;ie, \Kortekson, ,* 17cc OH progestepon e // • \„ . *4 Corticost er o nortykosteron Korteksoly i ortoxolone

Aldosteront Kortyzol Cortisol Schemat 4. Postulowa.ny mechanizm dzialania ACTH na biosyntezq kortykosteryd6w

Scheme L. Postulated mechanism of ACTH action on the biosynthesis of corticosteroids

As far as aldosterone is concerned, it is presently assumed that it is also stimulated by ACTH; however the quantity of endo- genous or exogenous corticotrophin required to produce this effect is much larger than the quantity causing the maximum secretion of glucocorticosteroids.

Translator's note: translated literally. Could not find the correct 161 medical term. 16 • The ability to stimulate corticogenesis is shown also by glütaminic acid and gIutathione and also biogenic amines like adrenalin,, serotonin, and histamine. Recently, Makoff and co-workers (86) have stated that an addition of mall quantities of - serum or plasma from a rat to the homogenate of rat adrenal glands (0.05 ml per 30 mg of adrenal gland cortex) increases distinctly the production of cortexone and corticosterone from progesterone. At the sanie time the ll9 -hydroxylation of progesterone became almost completely inhibited. Electrophoretic investigations have shown, that the factor contained in the plasma, stimulating corticogenesis in the adrenal glands is connected with the p)-globulin fraction. The activity dis- ci appeared after heating the plasma to 56 C for half an hour. Since the above mentioned compounds did not show in this system any stimulating influence on the cortex of the adrenal glands it appears that some new, hitherto unknown, stimulant of corticogenesis is involved.

3. Androgens • The human body produces six substances with androgenic action: testosterone (XXIX), dehydroepiandrosterone (XXV), androsteddione (XXVI), 11r, -hydroxyandrostèridione (XXVII), adrenosterone (XXVIII) and lle-hydro_x_ytestosterone (XXX).

See footnote on page 1. • • • 17 CO

Pregnenolone. Progesterone

• iv

17a-hydrox progesterone VII

17a-hydroxy- 0 pregnenolona VIII HO XXVI XXV 4,1, XXVII XXVIII

OH rn

HO, ,OH

O. XXX Schemat 5. ,Biosynteza androgen6w

Diagram 5. Biosynthesis of 1-4 androgens ■-• co ■-• • 18

As can be seen from scheme 5, which represents the transformatfons leading to the production of those substances from pregnenolone, the precursor of testosterone i.e. androstendionJ (XXVI) can be formed by two routes: via progesterone (VI) and 1701-hydroxypregnenolone (64, 102) and over dehydroepiandrosterone (XXV) (51). The first of them is predominant in gonades, the second in adrenal glands. On the other hand, transformation of androstendione into testosterone occurs only in gonads, since in the vein blood leaving the adrenal glands testosterone has not been detected (139, 183). After hydroxylation of pregnenolone and progesterone in position 17j, a degradation of the side chain occurs, catalysed by 17,20-desmolases. Subsequently, via intermediate compounds androgenic substances are formed like *. DHA (XXV) and androstexidione (XXVI) and also acetic acid from the side chain of the steroid. Oxygen and NADPH as coenzyme take part in this reaction. The transformation of pregnenolone into prOgesterone joining both routes of androgen biosynthesis, occurs under the influence of 3p -hydroxydehydrogenase and then isomerase. Possible is also a transformation of 170(,-hydroxypregnenolone (VIII) into 17o( -hydroxyprogesterone (VII) (scheme 5) Recently, a third route was postulated for the synthesis of testosterone from progesterone without its hydroxylation in pos ition 17 0,(. This possibility is supported by the fact that r 14 after incubation of [7-3H] progesterone and L7- CJ 170C-hydroxyprogesterone with homogenates of an ovarian cyst the ratio

See note on page I. •• • 19 . 19 .

3H//140 in testosterone is higher than in the simultaneously formed androstendione (65) Dorfman is of the opinion, that this way of formation of testosterone is of particular importance after reaching sexual maturity.

Reports concerning the possibility of direct synthesis of DHA from cholesterol (23, 38) in a reaction similar to formation of pregnenolone still require confirmation. However a direct transformation of cholesterol sulfate into DHA sulfate has been proved, when adrenal glands were perfused with a 32 doubly traced cholesterol sulfate (4-C and S) (170).

4. Estroans The group of the estrogen hormones is composed of three steroid compounds described long time ago: estrone (XL) and estriol (XXXVIII), as well as 1713 -estradiol (XLI) showing the highest biological activity. Its hydroxyl group in position 17 is easily oxidized to a ketone group under the influence of 17 -dehydrogenase. Estrone formed at this occasion is 10-12 times less active than estradiol. The transformation of estradiol into estrone is a reversible process regulating the quantity of 17p-estradiol circulating in the body (46 ). The main site of synthesis of 17/3 -estradiol and estrone in non-pregnant women are the ovaries. Estriol detected in urine is to a great extent a product of metabolism of 17 i5 -estradiol and estrone. In pregnant women it is produced

...20 • 20 . by the placenta, which synthesises also other estrogens (83). Estrone was obtained for the first time from biological material (urine of a pregnant woman) as early as in 1929, and its complete synthesis was achieved in 1948. In spite of that, the biosynthesis of estrogen attained a conclusive work-out only during the recent years, although not all its details have been clarified in full so far. Dorfman proposed the first hypothesis on this subject in 1956. It assumed the participation in the biosynthesis of equilin and equilenin, i. e. steroids with an unsaturated ring B e as intermediate metabolites, which would be transformed into estrone,as result of saturation of the double bonds present in this ring.

The results of other authors (84), however, contradicted this hypothesis and pointed to the participation of androgenic compounds in the biosynthesis of estrogens. Thus Savard and co-workers (142), after introducing 17[-dihydroequilenin 14 traced with C into the blood of a gestating mare, isolated from the urine radioactive equilenin, whereas the products of reduction of the ring B i. e. equilin and estrone were not radioactive. Heard end. co-workers (56) fed traced testosterone to a mare and detected in the urine radioactive estrone; Besh and co-workers (11) noted a distinct increase in secretion of estrogens in human urine after administering androgenic compounds. A further confirmation of the suggestion concerning

...21 . 21 . the role of androgens as metabolic intermediaries in thc; bi o_ synthesis of estrogen hormones consisted in establishing the presence of DEA, androstendione and testosterone in pices of a normal ovary (65, 150, 187).

The transition of androgens into estrogens is based on the removal of the methyl group at C-10 and aromatization of the ring A with simultaneous reduction of the carbonyl group at C-3. The detection by Meyer (90) in homogenates of adrenal glands of oxen of the 19-hydroxylase capable of transforming androstendione And DHA into their 19-hydroxyl derivatives made it possible to forward some suggestions on the theme of the mechanism(ofeliminatioh of the methyl group. The same author proved (91), that 19-hydroxyandrostendione, incubated with homogenates of adrenal glands, the placenta or pieces of ovaries, transforms easier into estrone than its non-hydrwLylated homologue in position 19, although the yield of this reaction was very small (2-6%). The experiments of Meyer were con- tinued by Ryan (133, 134) using placenta tissue for his arperi- ments. It transpired that the enzymes required for transformation of 19-hydroxyandrostendione are present in the micro- samal fraction of the placenta cells and that they act in the presence of NADPH and atmospheric oxygen. Owing to having learned these facts, Ryan was able to increase the percentage of transformation of 19-hydroxyandrostendione into estrone to 60-80%. Thus the 19-hydroxylation is the first step towards * See footnote on page 1. . . .22 • 22 elimination of the methyl group and 19-hydroxyandrostendione is an important intermediary metabolite on the way to the

aromatization of the ring A. It can undergo the following

, d ri CO g o • HORMONY STERYDOWE 321. [13] • E 0 • .-3 •-■ 32.1. U)0 000 L. 0,1

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(1.) Biosy

Biosy 6.

am cu . r c.) Co 1 Diag

cioe at

. . . 2 3 . 23 . a) Dehydrogenation of the side carbinol to the aldehyde group .with'formation of 19-aldoandrostenedione (XXXV).

h) dehydrogenation of the bond between 01 and 0 2 which leads to 19-hydroxyandrostadienedione (XXXVI) and subsequently as the result of oxidation of the primary alcohol group to 19-aldo- androstadienedione (XXXVII). c) separation of the whole side group C-19 in the form of formaldehyde with formation of 19-norandrostenedione (XXXIV) (97,

157). Those three metabolites are transformed into estrone as a result of dehydrogenation or separation of formaldehyde (20). Also a transformation of androstenedione into 19-hydroxyandrostadienedione (X1XVI) over androstadienedione (XXXII') (scheme 6) is possible. It was found that during the incubation of the placenta 19-nor-combinations undergo a change into estrogens with much more difficulty than steroids having the side croup C-19 (134). Table 2 representing the degree of transformation of various

androgenic compounds into estrogens shows that 19-norLderivatives undergo this transformation in not more than 5-10%, whereas 19-hydroxyandrostenedione changes in as much as 80%.

...24 • 24 Table 2 Transformation of androgenic conmounds into estrogens

T- ablica 2 Przerniana zwiszkOw androgennych w estrogeny

% przemia- trape for- Nazwa androgenu mation into ny w estron estrone

A1,4-androstadiene-3,/7-dion 35 A4-androstenediont 60 Dehydroepiandrosteront 40 5m-Al-androsteridione 10 19-hydroky-A4-androsterldione 80 19 nor-M-an drostenedione. 5 19 nor-5x Al-androsterfdionz- 10

At the same time, it follows from investigations by Dorfman and co-workers, that 19-e0doandrostenedione is transformed into estrone by the microsomes of the placenta much faster than 19-hydroxyandrostenedione. Because the transformation of 19-norandrostenedione into estrone is very slow, it seems that the following sequence of transformations is of fundamental importance for estrone synthesis: androstenedione 19-hydroxyandrostenedione (XXXII)---19-aldoandrostenedione (XXXV) estrone (XL). The side route of the biosynthesis runs via DHA (over A5- androstene-3 13 , 16D1, rip -triol (XXXI))and probably over These two substances namely were trans- hydroytestosterone. formed with a considerable efficiency into estriol (XXXVIII) and into 160(-hydroxyestrone by the placenta microsomes and NADPH (135). Results obtained by Wettsteinandco-workers (182)

6..25 . 25 . are proof that 16-hydroxysteroids play an important role in the 323 •metabolism of steroids; they reported that 16-d-hydroxytestosterone is, besides estriol, the predominant steroid produced by the placenta.

Reversible transformations of estrone into 17p (XLI) and in some species of animals also into 17d-estradiol (XXXIX) catalyzed by specific 17-dehydrogenases occur not only in vivo in tissues secreting hormones but also in vitro in the presence

of liver, or kidney tissue and blood (171).

II Transformations of steroid hormones in the body system The dialysing and hydrophobic hormone molecules produced by the glands enter the blood circulation system, where they become soluble in water and lose their ability of dialysis. These changes can be explained by formation of compounds of P 10. steroids with proteins: transcortine albumins and p-globulins. Recently the composition of the carbohydrate component of trans-

cortine became known (35). This o11 -globulin contains after reduction to dry mass: 5.4% of hexoses, 4.7% hexosoamine, 3.2% of sialic acid and 0.8% of fucose. The presence in the molecule of a steroid of the ketone group in position 3, a double bond between the carbons 4 and 5 and a hydroxyl group in positions 11 are necessary for the formation of compounds of a steroid with a protein (16). The period of a steroid remaining in the blood is short. They are subjected in the liver to various structural transformations,

Translator's note: ...26 Repeated as in original. Could not located this term in English text books . • 26 which are mostly irreversible and the forming catabolites show a low biological activity or lack it altogether. Finally these catabolites are combined with glucuronic acid and in man are evacuated mainly with urine. Among the transformations which the steroid hormones undergo in the organism, reduction processes are the most essential. They occur primarily in ring A, where hydrogen joins the third, fourth, and fifth carbon atom. Because of saturation of the ethylene bond two different dihydro-derivatives are formed, depending on the space orientation assumed by the hydrogen atom at C-5. This is connected with the peculiarities of the hydro- genases catalyzing the reaction:

-hydrogenase isolated from the microsome fraction of the liver and .A4-5 f3 -hydrogenase obtained from the supernatant (49). After saturation of the double bond a reduction of the ketone group occurs at C-3, which becomes assymetric. The space orientation of the hydroxyl-group formed can be 3d* (trans) or

3 3 (cis). Thus from each steroid undergoing those two reduction stages four isomers can be formed: 3D./.5 1 , 3f3 , 3015[?, , and 3f(350 These catabolites are usually called tetrahydro-derivatives and denoted with the letter ni preCeding the name of the hormone. In steroids of the type C21 also the ketone group in position 20 can be reduced. In such case hexahydro-derivatives in two possible isomeric forms 20>7 and 20/ are formed. It should be underlined that the ketone group in position 20 can . 27 • undergo a reduction process before saturation of the double bond

" and the reduction of the ketone group in C-3. The dihydro-derivates formed, maintain in such a case the double bond between the carbons 4 and 5 and the ketone group in position 3. In the molecules of steroids of the pregnane

series a reduction of the primary alcohol group may occur at C-21, as the result of which 21-desoxy-metabolites are formed playing an important role in transformations of aldosterone. Apart from reduction, steroids can also be oxidized. The

presence of the hydroxyl group at C-17, adjacent to the 0(-ketol side chain makes possible a separation of this chain catalyzed by 17„20-desmolase occurring in the microsomal fraction; 17- ketosteroids, compounds of the 019 type, are formed in such a case. About 3-5% of the compounds discussed are transformed in this way. The routes of estrogen catabolism lead mainly over oxidation reactions. Catabolites hydroxylated in positions 2,6,7,15,16 show a very weak biological activity or none at all. Steroids enter urine almost exclusively in the form of combinations with glucuronic and sulfuric acid. Thus, 95% of corticosteroids and their catabolites appear in the urine in the form of glucoside-uronates, 4% in the form of sulfuric acid esters and only 1% in free form (34). In the case of 17-ketosteroids, glucoside uronates amount to 65%, sulfuric acid esters to 33%, and free ketosteroids appear in urine in trace quantities (57).

...2 8 • 28 • Combinations of steroids with sulfuric and glucuronic acids 'occur in the liver (1) 8). Sulfokinase transfers the remainder of sulfuric acid from 5 1 -phosphosulfate of adenosin-3 1 -phosphoric acid to the hydroxyl group of the steroid (89, 104). Glucoside uronates are formed as the result of a reaction between steroids and tridiné_apipsphate - glucoronic acid. The enzyme catalyzing this reaction occurs in the liver microsomes (58). Owing to investigations by Jayle et cons. (106,107,108,111, 112,113,114,115,121) the relationship between the structure of the steroid and the type of conjugation and the placing of the remaining glucuronic and sulfuric acids was clarified. Com- pounds having the arrangement ,A4-3-keto- and a hydroxyl-group in position 17 are evacuated in 75% as glucoside uronates and only in 25% as esters of sulfuric acid. On the other hand, corticosteroids lacking the hydroxyl group in position 17 are evacuated mainly in the form of sulfates. Tetrahydrogen-derivatives transfer into the urine mainly in the form of àlucoside uronates, and the location of the attachment of the glucoronic acid is conditioned by the structure of the steroid: the presence in position 11 of a group containing oxygen favors formation of 3-glucoside uronates; its lack favors formation of 21-glucoside uronates. 3f-hydroxy-17-ketosteroids, to which DHA and epiandrosterone belong, are in first place esterified with sulfuric acid; however, also glucoside.uronate of DHA has

...29 e 29 e been detected (155). On the other hand, 3d -hydroxy-steroids conjugate mainly with glucuronic acid. Esterification by sulfuric acid is not always the last stage of metabolism of steroid hormones. It was found, that precursors of SOM8 hormones also occur in the form of combinations with sulfuric acid (24, 25, 130) and the adrenal gland cortex produces not only free DHA but also its sulfate (8, 175). Initially, DHA sulfate was detected only in pathological adrenal glands (7), later however it was found that it is produced also by healthy adrenal glands (160). Also there are data indicating that steroid sulfates are biologically active (140).

1. Corticosteroids The story of endogenic steroids and also of those introduced for therapeutic purposes was investiated in recent years mostly 14 by means of tracer compounds with the radioactive isotope 0 in position 4 (82, 92, 93, 124). The investigations were concerned mainly with two natural compounds: cortisol and corticosterone. It was found that 20 minutes after an intravenous injection of cortisol, combinations of the steroid with glucoronic acid appear in the blood. After two hours the Quantities of free and combined hormone become equal. During this period about 14%, after 48 hours about 90% of the introduced dose of the hormone transfer into urine. About 4-5% are evacuated with the faeces (62). A measure of the steroids disappearing from the system is the so called biological half-life, i.e. the period

. • .30 30 . after which the concentration of the steroid introduced into the blood falls to a half of the initial value. The biological half-life for cortisol is 80-90 minutes (77). The transformations to which cortisol and cortisone are subjected in the human body are presented in scheme 7. The main metabolites of cortisol (XV) and cortisone (XVI) evacuated with urine are the tetrahydro-derivatives: TH-cortisol (XLV), TH-cortisone (LVI) (95), allo-TH-cortisol (XLVI), allo-TH-cortisone (LVII), with a pronounced predominance of .5-derivatives in relation to 5d:-..derivatives (5rS/5,>(= 9) . are formed over the stage of dihydro- Tetrahydro-derivatives derivatives: DU-cortisols (XLIII) and (XLIV) and DH cortisones (LIV and LV) in which the double bond in ring A became hydrogenated. The product of further reduction of tetrahydro-derivatives are cortols (XLIX, L, LI, LII) and cortolones (LX, LXI, LXII, LXIII) in which also the ketone group in the side chain is reduced. Among the cortols and cortolones 5/5 -isomers are predominant. On the other hand, among the products of cortisol reduction -isomers were never detected in man. The 3f3 stereo- specifity of tetrahydro-derivatives is influenced probably by the hydroxyl-group in position 11, because so far an isomer of 3o(-5,0(-TH-cortisone has not been detected, whereas 3C5(_ TH-cortisol is an important metabolite (112).

31 . 31 . 326-7

HU-

LXII

tDpfi wit 20)3-a1locortolone LXIII CH2OH LV

LIU HO-

Schemat 7. Katabolizm kortyzolu i kortyzonu Scheme 7. Catabolism of cortisol and corti_sone

... 32 • 32 • The following derivatives are also known: 2007-D1i-cortibol (XLII) and 20p(-DH-cortisone (LIII), as well as their isomers 20 forming as the result of a reduction of the ketone group in the side chain (82, 124). The tetrahydro-derivatives of cortisol and cortisone undergo partial transformation consisting in separation of the side chain at the carbon 17 and leading to formation of inert 17- ketosteroids (the so called fraction of 11-oxo-17-ketosteroids.) In this way from TH- cortisol,11/-hydroxyetiocholanolone ()LVII)

and 11/1 -hydroxyandrosterone (XLVIII) and from Th- cortisone 11-ketoetiocholanolone (LVIII) and 11-ketoandrosterone (LIX) are formed. A feature peculiar to this fraction of 17-ketosteroids is the presence of oxygen in position 11 in their molecules. Besides, this fraction belongs mainly to the etiocholane series (hydrogen at C-5 in ois-position and thus differs from 17- ketosteroids formed from 11/S-hydrox.yandrostenedione and adrenosterone belonging to the androstane series (hydrogen at C-5 in position trans). Interesting conclusions with regard to the catabolism of cortisol can be drawn from the findings by Gray and co-workers (52) who established, that within 48 hours after administering traced cortisol, 90% of the radioactivity passed into the urine, although the fraction of the Silber-Porter chromogenes contained only 55% of radioactivity. The level of 17-ketosteroids in urine did not change in those conditions, which proves that the

•••33 • 33 • transformation into 17-ketosteroids does not play any major role in the removal of excess cortisol from the system. Con- sequently, non-typical metabolites are probably formed, which evade the control by the experimenting scientist during the generally applied extraction of urine with chloroform (59, 61, 98). Such metabolite can be e. g. 13-hydroxycortisol, since Katz and co-workers (66) observed a considerable increase of its quantity in the urine of patients with the Cushing's syndrome and after administering ACTH. Similarly in newborns, whose liver has not yet developed fully the capability of con- jugating steroids with glucuronic acid, the evacuation of 6r -hydro.x.ycortisol with urine is high (66). According to the opinion of Lipman and co-workers (99) these results are proof of the existence of a separate metabolic route, which in normal conditions plays an insignificant role in comparison with the changes leading to TH-cortisol and its glucoside. uronate, however, in condition of pathological excess production of the hormone becomes an additional means of evacu- ating the excess of cortisol. The same authors think that

6 --hydroxycortisol is rather a product of peripheral metabolism of endogenic cortisol end not the result of transformations taking place in the gland itself.

.••34- • 34 • Somewhat diff-z-:arent is the picture concerning the third basic hormone of tble adrenal gland cortex i.e. corticosterone. In first place, afer intravenous administering, about 75% of the dosis passes 11_- -nto urine. The biological half-life of corticosterone is 70-110 minutes. It can undergo a transformation into a dehydrogenat-,ed derivative, 11-dehydrocorticosterone, similarly as cortisol into cortisone. In the course of the system transformatfons in the molecules of corticosterone and 329 11-dehydrocorticosterone, the ring A is subjected to reduction and dihydro-derivatives and subsequently tetrahydro-derivatives are formed (schame 8).

' 20a-DH-kbrty- 20/3-DH-koriy- 20 P-Dli-oorti oo st erone kosteron • kosteron oast 20e1-DH-oorti DH-kortykosteron TH-kortykosteran DH-oorti o oat erone TH-oortioosterone Kortukosteron c Q r-ti oosteroné allo-DH-kortykastero- n allo-TH-kortykosteron Allo -DII-oortioosterone allo -T1-1-oorti oost er one

1' DH-11-dehydro- TH-11-dehydro- korleosteiron ,kantesieraft • 11-dehiaro- oortrbosterone oor.i co sterone kafqicrelva: oortl TEt ?rows allo-DH-11-dehydro- allo-TH-11-clehydro- *eatykosiem 31coatyketeeRbt Gorti oo st ero no oortioosterone 2013-DH-77-dehyciro. - jcoteetetec2it ort.' ra&uronet Schemat 8:211etabolizm kortykosteronu i 11-dehydrokortykosteronu

Scheme 8. The metabolism .of corticosterone and 11-dahydrocorticosteron

The main metioolite of corticosterone is allo-TH-corticosterone, with theratio of the isomer quantity 5/3/5,c in the urine being same-e5.ut lower than 1. Similarly like in the case

...35 35 of cortisone, allo-TE-11-dehydrocorticosterone has not been identified (112).

It dhould be emphasized, that corticosterone and 11-dehydrocorticosterone do not form 17-ketosteroids, because compounds lacking the hydroxyl group in position 17 are not submitted to oxidation with a loss of the side chain.

Human adrenal glands produce also cortexolone although in very mnall quantities. The basic route of its transformation in the body system consists in reduction leading to formation of tetrahydro-derivatives. Besides, a reduction of the side chain with formation of pregnane-3 D(,17cC - diol, 20-one and pregnane-3,17c, 20G<-triol can take place. With regard to the presence of a hydroJ,y1 group in position 17,it cari also undergo oxidation with a separation of the side chain and formation of 17-ketosteroids„ derivatives of etiocholanolone, which do not differ at all from the 17-ketosteroids forming from androgenic compounds. It should only be added, that in such a case etiocholanolon is formed in predominant quantities (scheme 9).

Transformations of cortexone occur similarly and hydrogenation of the ring A leads to TH-cortexone. By partial reduction of the side chain progesterone may be formed (76). Simultaneous reduction of the ring A and the side chain leads to pregnanediols. From more than thirty metabolites of glucocorticosteroids

„.36 36 so far detected in urine, a part has been determined quantita- tively (105) (Table 3). It is of interest that in individual species of animals the routes of evacuation of steroids differ from those observed in humans. In rat, e.g. the administered cortisol is evacuated mainly through the intestine (185), because 3 hours after sub- cutaneous introduction of the hormone, 83% of the dosis was detected in the bile. Since, however, only about 66 % of the steroid was evacuated with the faeces, a secondary resorption of the steroid from the intestine into the blood must be taking place.

• Pregnate3a,17c420a-triol Pregnan-3a,17a-dio1-20-on. N KoPteksolon DH-korteksolone---■ TH-korteksolont. 2,-rtexo one oortexolone oorteprolone -Andrusterone Etiocholanolone

. Schemat 9. Katabolizm korteksolonu

Scheme 9. Catabolism of cortexolone

...37 • 37 .

Table 3 Evacuation of glucocorticosteroids and their metabolites : in urine in mg per 24 hrs (cited according to(105)with author's permission)

Tablica 3 Wydalanie glukokortykosteryd6w i ich metabolit6w w moczu w mg na dobg (cyt. wg 105, za zezwoleniem autora)

Mçiczyini Kobiety Me rl if c m A Et Kortyzol Corti sol 0,47 0,27 (0,13-1,21) (0 —0,88) TH-kortyzol T H -o ort i s o 2,19 1,19 (0,81-4,24) (0,78-2,95) Allo-TH-kortyzol 0,92 0,57 cortisol (0,39-1,58) (0,33-1,20)

Kortyzon cortisone 0,33 0,22 (0,11-0,70) (0,09-0,68) 3,06 1,76 T H-leg raj° „ (1,14-5,70) (0,78-5,03)

Kortykosteron 0,29 0,09 cortioosterone (0 —0,97) (0 —0,24) 0,54 0,34 TH-kocCrlecitoeiVâ ro ne (0,10-0,99) - (0,13-0,60) Allo-TH-kco;s-tylcosto r 0,63 0,34 001' i o er o n e . se (0,19-1,50) (0,10-0,56)

11-debydrokooltytliogtom r 0 0,26 0,07 ° (0 —0,83) (0 —0,19) TH41-{lehydrokortykoste- ron Millie 0,49 0,30 (0,09-1,15) (0,06-0,67) .

Korteksolon C 'et e x°1 ° e 0,22 0,07 (0 —0,74) (0 —11,27) TH-korteksolon 0,02 lee 110119 _c_ort crol one '

In the guinea pig 65% of the steroid administered passes into the bile and a considerable part of it is also àbsorbed into the blood and 50Dsequently evacuated with urine.

... 38 b.D

CH2OH OH CH2OH OH 0 —C—H CH2OH drO 0 —C—H .1101

x LXIV XXI I o o

x N tJ OH OH Fe CH2°H CH2OH OH çH2oH o O —c--Hc=o / 0 —C—H d=o Isi

HO HO HO' H LXVI DI LXVIII LXIX

Schemat 10. Kataboliz,m aldosteronu

Diagram 10• Catabolism of aldosterone

CAD

itg • 39 •

Aldosterone is evacuated with urine mainly in the form of 32)2_ C:18 glucoside uronate (22, 120). The main metabolite of aldosterone is tetrahydroaldosterone 5/3 / 30C (LXVIII), formed as the result of saturation of the double bond at C-4 and a reduction of the ketone group to an alcohol group at C-3 (166, 167). Other isomeric tetrahydro-aldosterones were detected in urine in smaller quantities: 507,3ci(LXVI) and 5D(,30,C (LXVII)* also the dihydro-derivatives 5c/-dihydroaldosterone (LXIV),

5p - dihydroaldosterone (LXV). These compounds have often a hemi-acetal bond between the carbons in positions 11 and 18 (68, 70, 72). Not long time ago a report was made on isolation from human urine of 3d,18,21-tr1hydroxy-5 16-pregnane-11,20- dione (LXIX) and also of other interesting catabolites of aldosterone, in which the cyclical hemiacetal 11-18 is trans-

formed into a double ring acetal 11 —.18--2O (69, 1)4.7) (Scheme 10). In the plasma, aldosterone appears mainly in free state and in combinations with proteins (32, )4.8). Its concentration amounts to 0.008-0.03 /2-g/100 ml. On the other hand, tetrahydroaldosterone, the concentration of which in the plasma is five times as high as that of aldosterone, occurs mainly as glucoside uronate. (118). The biological half-life of aldosterone is 18-25 minutes (165). The quantity of aldosterone evacuated in the urine of a healthy human varies within the limits of 40-180 /u-g per 24 hrs.(122).

•••40 • 40 • It increases considerably in some diseases, e.g. in the so called Cdnnis syndrome (primary aldosteronism) and in liver cirrhosis

(secondary aldosteronism) etc. In the urine of healthy humans also 18-hydroxycorticosterone was detected, occurring in quantities twice as large as those of aldosterone (168). The main metabolite of 18-hydroxycorticosterone is also its tetrahydro-derivative.

Recently, Ulick and co-workers (169) detected a new syndrome, for which a very low level of aldosterone in urinp,but a level of 18-hydroxycorticosterone much higher than normal are characteristic. This is probably a metabolic defect consisting in lack of the enzyme catalysing the transformation of 18-hydroxycorticosterone into aldosterone.

2. Androgens

Sandberg and Slaunwhite (138), using the isotope method, investigated the course of testosterone in the human body. Among other things they established that 14% of testosterone administered to humans passes into the bile, whereas only 6% is detected in the faeces. This discrepancy can be explained by a secondary resorption of a part of the steroid from the bowels into the blood system. 90% of the radioactivity administered is evacuated with the urine, with more than a half of it in the form of compounds with glucuronic and sulfuric

acids. The kidney clearance of glucoside urotanes is much higher.than that of sulfates, which explains the presence 1, . 41

1251 HORMONY STERYDOWE 333 333

e ron te tos

u tes teron of tos

tes

lism izrn l

e ><> bo "1:1é) bo ta ta Ca Ka

11. • H

$-!txt) ai

. . . 42 . 42 •

of androgens in the blood mainly in the form of sfflfuric acid esters, whereas in urine their glucosid e ilvonates are predominant. Scheme 11 presents the transformations of testosterone, which forms an oxidizing-reducing system with androstenedione. The routes of transformations are apparently the same for both compounds and, like for the majority of other steroid hormones consists in gradual hydrogenation of the ring A, and for androstenedione also in a reduction of the ketone group in position 17. In the first phase of the reduction, two isomeric saturated diones are formed: androstane-3,17-dione (LXX) and etiodholane-3,17-dione (LXXI) differing by the space con- figuration of the hydrogen at C-5. The reduction of the ketone group at 0-3 leads to formation of four isomers, of which androsterone (LXXII) and epiandrosterone (LXXIII) originate from androstariàione and etiocholanolone (LXXIV) and epietiocholanolone (LXXV) from etiocholaAione. In those metabolites also the ketone group at C-17 may undergo reduction which creates the theoretical possibility of formation of four isomeric androstane-3,17-diols (LXXVI), (LXXVII), (LXXVIII), (LXXIX) and four etiocholane-3,17-diols; of these, howeverl two only were detected in urine: the isomer 30C„5c417(5 (LXXVII) and the isomer 30(,5 f171b (LXXXI).

•••43 • 43 Table 4 Evacuation of 17 -ketosteroids with human urine in mg per 24. hrs (according to(105)with permission of the author)

' Tabiica 4 Wydalanie 17-ketosteryd6w w moczu ludzi w mg na dobç (wg 105) za zezwoleniem autora

MçZczyini Kobiety tg pn Women Dehydroepiandrosterone. 1,72 0,98 (0,43-3,84) (0,37-3,51) Androsterone. 2,78 2,23 (1,68-5,54) . (0,82-6,52) Etiocholanolone. 3,77 3,10 (2,29-8,20) (1,55-6,57) //r3-hydroXyandrosterone. . 0,65 0,65 (0 —1,54) (0,39-1,09) 1/-ketoandrosterona 0,34 0,68 ' (0 —0,74) (0 —2,07) 14-hydroXyetiocho1ano- 1one. 1,10 0,74 (0 —2,99) (0,34-1,37) 11-ketoetiocholanolone 0,89 1,00 (0,23-2,58) (0,28-1,83) .

Of metabolites of testosterone, androsterone and etiocholanolone are formed in the largest quantities; they represent nearly one half of the 17-ketosteroids in urine. On the other hand their 3p-isomers : epiandrosterone and epietiocholanolone are found in urine in insignificant quantities. Besides testosterone and androstenedione, which are formed in the Leydig cells in the testes, some androgens are formed in the cortex of the adrenal glands. They are: adrenosterone, -hydroxyandrostenedione, androstenedione, and DHA (99). DHA is partially eliminated in urine in unchanged form, or it partially undergoes transformations, the products of which are:

• • • Lill • 44 • androsterone, etiocholanolone, androstenedione and alsoit\ 4_ androstene-3i3 -diol produced in very small quantities. Table 4 shows the evacuation in the course of 24. hours of the individual 17-kebosteroids, which are, as known, products of transformations both of testosterone gad of hormones of the adrenal glands. Testosterone shows the strongest adrogenic action. DEA action is about 15 times weaker and the main metabolite of testosterone, androsterone is about six times weaker (125). Other testosterone catabolites: etiocholanolone, epiandrosterone, androstenedione and etiocholaAione R-re even less active or do not Show any activi4 at all (10). 3. Estro,Rens The main routes of metabolic inactivation of estrogens run over oxidation reactions. Estrone and 17 -estradiol undergo mutual transformations in vivo and therefore it was difficult to determine, which of them is the precursor of the more oxidized metabolites. It is known on the basis of experiments by Fidhman and co-workers (45), who investigated the products of transformation of intravenously administered [6,7- III 17)-( -estradiol and ji6-14-cl estrone, and also from works by other authors (18, 75, 96), that estrone is the main, if not the only substrate of hydroxylation. In animals, estrone can undergo transformation into 171- estradiol (XXXIX); in humans, however, this compound does not

• • • 145 . 45 . appear. As the result of hydroxylation of estrone in position 16 tuo isomers are formed: 16C(-hydroxyestrone (LXXVIII) and 16P -hydrox,yestrone (LXXIX). Ihese compounds undergo further a direct reduction to estriol (XXXVIII), 16-epiestriol (LXXXII), 17-epiestriol (LXXXIII) and 16,17-epiestriol (LXXXIV) (87, 88 , 103), or over intermediary compounds: 16cketoestrone (LXXX) and 16-keto-17p-estradio1 (LXXXI) (94, 136). Another equivalent route of estrone transformations, running similarly as transformations of catecholamines, leads over methoxylation (4, 5, 57). Axelrod (5) noted that during incubation of natural and synthetic estrogens with pieces of livers of rats and rabbits a hydroxylation of those compounds takes place in position C-2. Fishman (47) presented proof of existence of this process in vivo in humans; he isolated W-4C-12-hydroxyestrone from human urine after intravenous injection of [16-14C-117[3 -estradiol. Of interest is also the occurrence both in animals and plants of enzymatic activity catalyzing the methoxylation of 2-hydroxyestrone (Lxxvi ) to 2-methoxyestrone (LXXVII) and of 2-hydroxy-1713-estradiol (LXXIV) to 2-methoxy-l7 I?) -estradiol (LXXV). Axelrod and Goldzieher (4) also established methoxylation of those compounds, as well as of estriol in different human tissues.

••414-6 4., CO Crà

?J b 9 w w o

? PJ W N

› t-4 (n

HO UÇXX1

Schemat 12. Katabolizm estrogen6w ., Diagram 12. Catabolism of estrogenb I Cr)e'ci'l 47 Estrone can be also transformed into lactone (LXXXVII) which is sfour times less active. This transformation was damon- strated in vitro under the influence of H202 (33). Recently the possibility is pointed out of oxidizing estrone also in other positions like C-6, C-7, C-11, C-18 (19, 21, 26, 80,

81). During investigations of the metabolism of estrogens in the human foetus a new, so far unknown metabolite was detected, 15-c1 -hydroxyestrone showing not more than 0.001 of the activity of estradiol (149) and 155/-hydroxy-178 -estradiol (186). The biological importance of 15-hydroxylation has not been clarified; it is assumed that it may protect the foetus from the intense activity of 1718 -estradiol and in life after the foetus stage it may be one of important metabolic transformations of 17p- estradiol. (71).

Roberts and Szego (129, 159) proved that estrogens circulate in the blood mainly in the form of complexes with p-globulins of the plasma and also as glucoside uronates. An essential role in formation and elimination of those compounds is played by the functions of the liver (3). Estrogens administered orally pass into the blood, where from part of them is evacuated with urine (table 5).

• • •11-8

• 48 • Table 5 Evacuation of estrone, rip -estradiol and estriol with urine in ,tg per 24 hrs (according to(36lwith permission of the authors)

• • Tablica 5 Wydalanie estronu, 1713-estradiolu i estriolu w moczu w Fig na dobg (wg 36) za zezwoleniem autordw Estrone.. 17fl-cstradiol Estriol

Kobiety z normalnym cyk- Women with a normal eyole lem a) faza pomenstrualna 2,8 0,7 4,2 a) post -enstrual phase - (0,2— 7,2) ( — ) (1,9— 11,4) b) faza owulacyjna 20,1 7,9 30,9 b) ovulation phase 14,8-27,4) (2,8-21,8) (8,1-119,0) c) faza lutealna 11,3 5,0 21,1 o) luteal phase (5,1-25,0) (2,1-10,6) (5,0— 89,0)

Kobiety w okresie klimak- Women in olimaoterium period terium 1,4 0,3 4,1

MçiczyLni- . 5,4 1,5 3,5 Men (3,0— 8,2) (0 — 6,3) (0,8— 11,0)

• Part of the estrogens passes from the blood to the bile, with the latter into the intestine, where it undergoes a secondary resorption into the blood circulatory system. A certain part of those estrogens leaves this intestine-liver circuit and is eliminated with the faeces. In sheep, the elimination of estrogens with urine does not exceed 2% of the 171-estradiol administered, whereas the evacuation with the faeces amounts to 35-45% (184). As follows from recent reports (1), also tissues of some male glands are able to metabolize estrogens. In normal and pathological tissues of the prostate gland estrone undergoes reduction to 17- /3 (0.8-4.3%), 16-hydroxylation to estriol (2%), oxidation and methoJvlation to 2-hydroxyestrone

( 0.9 -4.9%) and 2-methoxyestrone (1.l-14.9%). •••1-1-9 • 49 • 4. Gestagens' * In contrast with other steroid horluones, progesterone appears in the plasma mainly in free form (90%). Its transformations occur in first place in the •liver and are similar to transformations of corticosteroids, i.e. a reduction of the ring A and subseouently of the ketone group in the side chain take place. An attachment of hydrogen to the carbon atoms in positions 3, 5 and 20 results in a theoretical possibility of formation of 8 isomeric pregnanediols, three of which have been isolated from the urine of healthy persons (513, 301. , 20o1 ; 5 01,3 c(,20£(; 5d,3/2),20d) and two from the urine of persons with tumors on the adrenal glands (5 P,3p,20f3; 5, 3(b,20e). The remaining three isomers could not be detected in biological material so far.

Progesteronè. •

Pregnandlone Allopegnondione.

Pregnan-3a-o1,20-on Pregnan-3/3-o1,20-on A ollopœgnan-3a- 1,20-on A1lopregnan-3,6-01,20-a7

/\ I\ I\ I\ Pregnarn3a,20a-dio1 Pregnanr313,20a-diol A11opregnanc3c420a-dio1 Allopregnanc3,0 20a-ctiol

Pregnaneea,2013-dio1 Pregnanh313,2013-diol A11opregnare3a,2013-diol A1lopregnote-3/3,2013-diol Schemat 13. Katabolizm progesteronu

Scheme 13. Catabolism of progesterone

See note on page 2

...50 50 After administering traced progesterone to pregnant women, the radioactivity is divided equally between urine and faeces. In this respect progesterone behaves in a different way than other steroid hormones, whose metabolites are eliminated mainly with urine.

A generally accepted index of a quantitative estimate of progesterone formed in the system is the level of pregnanediol in the urine, although this compound is also a catabolite of cortexone. However, the pregnanediol originating from cortexone transformations or adrenal gland progesterone amounts to not more than 1% of the quantity contained in the urine of a woman in the period of a full cycle of ovulation. The participation of the adrenal gland cortex can increase considerably in pathological conditions, e.g. 100-200 times in hyperactivity caused by the presence of adrenal gland tumors.

III. Chan.es of metabolism of steroid hormones as result of enzymatic blocks of steroid 'genesis Owing to the development of clinical biochemistry it became possible in the course of the recent years to detect various anomalies of the biosynthesis of steroid hormones, being the cause of serious disease syndromes. In this way was clan- 339 fied the essence of the natural adrenal-sexual complex, the classification of which only a few years ago had a purely clinical character and was based on different degrees of virilisation changes observed in patients.

•• • 51 At present time, the classification of the adrenal-sexual complex is referred to biochemical bases and discerns three main forms conditioned by the lack of specific enzymes participating in the biosynthesis of hormones of the adrenal gland cortex: 21-hydroxylase, 11(3 -hydroxylase and dehydrogenase of 3 -hydroxysteroids (162). Characteristic clinical aspects correspond to those genetically conditioned enzymatic blocks.

A partial block of 21-hydroxylase is the cause of the so called simple virilism - the most common form of the adrenal- sexual syndrome. A complete block of this enzyme causes the appearance of clinical syndromes defined as virilism with loss of salt. A block of 11 -hydroxylase produces a kind of virilism with excessive arterial pressure, although cases were also reported, when inhibition of lip -hydroxylation was the cause of virilism without excess pressure (15, 50). The last of the so far detected natural enzymopathies of steroid genesis is the block of the dehydrogenase of 3p -hydroxysteroids. A complete failure of the activity of this enzyme causes death of the patient in spite of application of substitute hol.monal medi- cation. These blocks are a cause of serious biochemical changes both in the composition of intermediary metabolites and the final products of metabolism eliminated with urine. In a partial block of 21-hydroxylase, excessive quantities of four precursors of cortisol accumulate in the system: 17(1 -hydroxyprogesterone/ 11 17o( -dihydroxyprogesterone and in a gmaller

ç. .11052 . 52 . degree progesteron.e and 11 -hydrox-yprogesterone ( scheme 2).

Among more than 20 catabolites of those compounds isolated from the urine of patients, the following appear in larger quantities: pregnane-3t1,17c!,20oC-triol and pregnane-3K,170C-dio1-20-one originating from 17c( -hydroxyprogesterone; pregnane-3 e< 200( -di ol resulting from progesterone and pregnane-30(,20oC-dio1-11-one which is formed from 110(-hydroxyprogesterone (186).

As follows from scheme 5, an increase of the quantity of

17-hydroxyprogesterone and 17 -hydroxypregnenolone must cause an increase of the production of adrenal gland androgens i.e. androstenedione, 11 p -hydro.xy androstenedione and DH_A., which leads to evacuation with the urine of both, the compoun.ds named and the products of their further catabolism: androsterone, etiocholanolone, 11p -hydroxy, androsterone and 11-ketoetiocholanolone (17, 60). With a complete block of 2l-hydroxylase, apart from the enumerated changes in the composition of the urine metabolites, a very low level or even absence of tetrahydrocortisol is observed, caused by inhibition of cortisol production in the adrenal glands. In a deficit of 11(3. -hydroxylase described for the first time by Eberlein and Bongiovanni (39) considerable quantities of nortexone and cortexolone accumulate in the system (scheme 2), and the urine of the patients is rich in tetrahydro-derivatives 340

...53 . 53 . of those compounds (53). Accumulation of progesterone and lrci-hydroxyprogesterone takes also place, although to a considerably smaller degree than in the block of 2l-hydro.xylase. On the other hand, 11-hydro.v-derivatives, as could be expected, do not appear at all in the urine. Similarly the androgen of adrenal origin, 11t3 -hydroxyandrostenedione does not form at all, whereas androstenedione and DHA are produced in large quantities. Fractions •of 11-oxo-17-ketosteroids are absent in the urine.

Chromatographi e investigations of compounds of the corticosterone series also produced a proof of a block of 11p-hydroxylation: detection in the urine of tetrahydrocortexone, which normally does not appear there. The evacuation of aldosterone has not be examined so far in patients with this type of enzymatic block; however, the presence in the molecule of a hydroxyl group in position 11 permits to assume that its production decreases.

Attempts were made to explain the mechanism of the arterial excess pressure in a block of 11p-hydroxylase. Original suggestions that the high pressure is caused by cortexolone were not confirmed, since even prolonged administering of this compound to patients did not cause any increase of the excess pressure (12). At present time it is assumed, that the substance responsible for the excess pressure is cortexone, whose

.54 • 54 hypertension action is known both from experiments with animals and from clinical observations. However, this hypothesis does not explain all the facts observed. It is difficult e.g. to

explain the observations by Bongiovanni (15) and Chaptal and co-workers (27), that sometimes patients showing biochemical changes typical for the block of 11/5 -hydrolase and eliminatQ

ing considerable quantities of cortexone with the urine, do not have any excess pressure. However, it is possible that the excess pressure appears only after transgression of a certain limit of cortexone in the blood, which may occur or not occur, depending on the degree of blockage, or an unequal individual sensitivi4 to the action of cortexone. It is also possible that the appearance of excessive pressure is conditioned by the relative quantity of cortexone in respect to cortisol. Small quantities of cortisol produced by the adrenal glands in the case of a not complete block compensate perhaps the pressure increasing action of the irregular metabolite. In a block of dehydrogenase of 38-hydroxysteroids, which catalyses one of the first stages of corticogenesis i.e. the transformation of pregnenolone into progesterone, a complete inhibition of the biosynthesis of adrenocortical hormones takes place, as well as an accumulation of large quantities of pregnenolone. Since, however hydroxylating enzymes are active,

a whole series of pregnenolone derivatives is formed, hydroxylated

...55 . 55 in positions 11(3, 17c1 and 21, and to a emaller degree in positions 16, 20 and 6 (9). As far as androgens are concerned, hyperproduction of DHA which dominates also in the urine is observed.

Genetic investigations by Childs and co-workers (28) and Prader and co-workers (126) lead to the conclusion, that the enzymatic blocks discussed are connected with an autosomal recessive gene, which causes clinical symptoms only in homo- zygotic individuals. A different gene is responsible for each of the three fundamental enzymopathies. On the other hand, it seems that the simple form of virilism and its form with loss of salt constitute a characteristic transferred by the same gene and differ only by the degree of blockage of 21-hydroxylation. Readers interested on a wider scale in the problems discussed in this paper will find ample data in the recently published monograph "Metabolism of Steroid Hormones" by R.J. Dorfman and F. Ungar, Academic Press, New York 1965 (716 pages.).

• ..56 • 56 LITERATURE

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