Direct Conversion of 17 6-Eshadiol-3-glucosiduronate and 17 6 -Estradiol-3-sulfate to their 17-Keto Forms by Human

Kidney Homogenatesl

R. HOB KIRK,"^ N. GREEN, M. NILSEN, AND E). A. JENNINGS Division o f Cdinicwd Biochemistry, Department of Biochemistry, and Department o f Medicine,

University o f Western Ontario, London, Ontario N6A 3K7

Received August 13, 1973

Hobkirk, R., Green, R. N., Nilsen, M. & Jennings, B. A. (1974) Direct Conversion of 17@- Estradisl-3-glucosiduronate and 17p-Estradiol-3-sulfate to their 17-Keto Forms by Human Kidney Homogenates. Can. 1. Bioehem. 52, 15-20

Labelled 17p-estradiol-3-glucosiduronate and 17p-estradiol-3-suIfate were both directly dehydrogenated to their respective 17-keto forms on incubation with human kidney hornogenates. NAD increased the conversion to a greater extent than did NADP. The reverse reaction, even in the presence of NADH or NABPH was not found to a measurable extent, presumably because of rapid oxidation of the cofactors. High or low activity towards the conjugates was accompanied by high or low activity, respectively, towards free 17p-estradiol. These dehydrogenase activities were particularly high in the medulla of one kidney so investigated. Considerable sulfatase activity was usually encountered in these homogenates but little 8-glucuronidase activity was demonstrated under the experimental conditions.

Hobkirk, R., Green, R. N., Nilsen, ha. & Jennings, B. A. (1974) Direct Conversion of 178- Estradiol-3-glucosiduronate and 17p-Estradiol-3-sulfate to their 17-Keto Forms by Human Kidney Homogenatea. Can. J . Biocltem. 52, 15-20

Le 178-oestradiol-3-glucoaiduronate et le 17p-oestradiol-3-sulfate rnarquCs sont tous deux dCshydro&nCs directeaent en leur forme 17-cCto respective par incubation avec des homogCnats de reins humains. Le NAD augmente davantage la conversion que le NADP. La rCaction inverse, m$me en prCsence de NABH ou de NADPH n'est pas mesurable, probablement ii cause de l'oxydation rapide des cofacteurs. L'activitC Clev6e ou faible B 1'Cgard des conjuguCs s'accompagne respectivement d'une activitC ClevCe ou faible vis-h-vis le 17p-oestradiol libre. Ges activitCs dCshydrogCnasiques sont particulibrement Clevtes dans Ia zone mCdullaire du rein. Dans nos conditions expkrimentales, I'activitC de la sulfatase est habituellement considCrable, mais 19activitC de la p-glucuronidase est faible. [Traduit par le journal]

Introduction described irz vivo (1 ) and in vitro using a p%a-

The biological interconversion of 1 7P-estra- cental enzyme preparation (2)- Direct conversion

diol-3-glucosiduronate (E23G):3 and estrone-3- of E I ~ S to E23S Clccurs in the fetus (3) and the

glucosiduronate ( E 1 3 ~ ) on the one hand, and interconversion of these two sulfates occurs in

of 17P-estradiol-3-su1fate (E,3S) and estrone- intact and hemolyzed red blood cells (4-61. It

3-sulfate (E, 3s) on the other, without prior became of some interest to investigate additional

removal of the conjugating groups, seems well sites of such enzymic activity in the human and

establisheds with particular respect to fie human the present study is concerned with kidney tissue. subject E,~G/E>G interconversion has been

Materials and Methods

ISupported by Medical Research Council of Canada (grant No. MT-532).

%search Associate of The Medical Research Coun- cil of Canada.

T h e following trivial names and abbreviations are used: 17B-estradiol-3-glucosiduronate (E,3@) = 178- hydroxyestra- 1,3,5(10)-trien-3-yI-~-~~glucopyranosiduro- nate; estrone-3-glucosiduronate (E13@) = 17-oxoestra- B ,3,5(10)- trien - 3 - yl-8- D - glucopyranosiduronate; 178 - estradiol-3-sulfate (E23S) = 178-hydroxyestra-1,3,5(10)- trien-3-yhsulfate; estrone-3-sulfate (EP3S) = 17-oxoestra- 1,3,5(10)-trien-3-yl-sulfate.

Biochemicals and Clzeanicals E1-6,7-3H-3S9 specific activity (S.A.) = 48 Ci/mmo19

was purchased from New England Nuclear (Canada) (NEN), Dorval, Que. It was purified by published methods (7) and part was reduced to Ez-6,7-"W-3S with NaBHI followed by purification ( 8 ) . El-4-14C-3S (S.A. = 45 Ci/mol) was prepared chemically (9) and puri- fied as above. Ea-6,7-%-3G-"C and &-6,7-3H-3G-14C. i.e. doubly labelled glucosiduronate with the glucuronyl groups generally labelled with "C, were prepared and purified exactly as previously published ( 1 ) . E?-4-"C (S.A. = 52 Ci/mol) was purchased from NEN.

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16 CAN. 1. BIOCHEM. V8L. 52, 1934

Unlabelled steroids were purchased from Schwarz- Mann, Orangeburg, N.Y., and unlabelled E136 (Na' salt) from Sigma Chem. Co., St. Louis, Mo. NAD, NADH, NABP, and NADPH were obtained from Sigma; saccharo- 1,4-lactone from Calbiochem, kos Angeles, Calif.; and pglucuronidase (Ketodase) from Warner-Chilcott, Morris Plains, N,J. Mylase P (con- taining phenolsulfatase) was a product of Schwarz- Mann.

DEAE-Sephadex (A-25 1 was obtained from Phar- rnacia (Canada) ktd., Montreal, Que., and Amberlite XAD-2 resin from Rohm & Haas, Philadelphia, Pa. AII other chemicals, reagents, solvents, etc., were of acceptable grade and were purified, where necessary (71, before use.

Tissue Prepstration and dncubatisn Human kidney tissue, removed for a variety of

clinical reasons (see Table I ) , was transported in cold saline (usually within 30 min) to the laboratory. The capsule was removed and the tissue cut into small cubes. In most instances a mixture of cortex and medulla was obtained, the former predominating. In one instance cortex and medulla were dissected out separately. One-gram tissue portions (wet weight after blotting on filter paper) were homogenized by hand in a glass homogenizer with 10 ml0.1 M phosphate buffer, pH 7.4, containing 0.04 M nicotinarnide. These steps were performed at 4 OC. In some instances homog- enates were stored at - 15 "C for up to 6 days without obvious loss of enzyme activity.

Methanolic solutions of substrates were measured into Erlenmeyer flasks and dried under Nn. Amounts employed were as follows (all x 10Vd.p.m.); 3H-En3S -- 1.76-6.23; 3H-E13S = 1.8; 'H/"C-E23G = 0.51- 0.82(3H), 0.12-0.19(14C) ; 8H/14C-E13G = 1.04('H), 0.24(l4C); "C-Ea -- 1.44. NAD, NADW, NADP, or NADBH (each 1.4 pmol) was added, where required, in 0.2 rnl buffer. Five milliliters of homogenate were added to each flask and incubation was carried out at 3 V C , for times of 1-2 h, in air, in a Dubnoff shaking incubator. Blank experiments contained buffer or bailed tissue hoxmsgenate. Incubation was terminated by adding 25 rnl cold methanol and the flasks, after being mixed, were placed at - 15 "C at least overnight.

dnvestigats'sn sf Products Methanol extracts were filtered through Whatman 40

paper and the precipitates washed with cold methanol. Filtrates were adjusted to 10% (v/v) with H20 and extracted once with hexane. The aqueous methanol phases were evaporated to dryness, taken up in HaO, and chromatographed on a $0 crn A-25 column in 04 .4 M NaC% (glucosiduronates; Ref. 8) or in 0-0.8 M NaCl (sulfates; Ref. 8). Before chromatography, where a TI-labelled sulfate was the substrate, laC-K3S was added as internal standard. Separated En3S, En3S, E13G, and E23G were recovered via Amberlite XAD-2 resin (10). Ek3S was subjected to incubation with Mylase P (3) and the free El crystallized with carrier before and after acetylation (7)) . Es3G was crystallized either directly with carrier E13G to constant S.A. and isotope ratio and/or after Ketodase hydrolysis (500 umits/ml for 24 h at 37 "C) with carrier En before and after acetylation (7). Free (unconjugated) radio-

activity from A-25 columns ($) was extracted with benzene and subjected to thin-layer chromatography, after mixing with 100 pg each of unlabelled EX and Er, om silica gel in cyclohexane - ethyl acetate ( f : 1 ; Ref. 11 ). Separated zones, visualized under ultraviolet light, were scraped off, eluted, and counted. On occasion these were crystallized with carrier El and En.

All radioactive counting was performed on a Unilux IIA Nuclear Chicago spectrometer set for 'H (45% efficiency) and 'T ((60% efficiency) and using a xylene- based scintillation fluid, Aquasol (NEN) , with the addition of methanol or M20 as the situation required.

Results The radioactivity incubated in each experi-

ment could be accounted for to the extent of 85-180% after the experimental procedure. No interconversion of conjugates, and little or no hydrolysis (none for glucosiduronates, < 2% for sulfates), occurred in the absence of tissue homogenates or with boiled tissue. All glmcosid- uronate peaks were completely hydrolyzed by Ketodase and this hydrolysis was inhibited by saccharolactone. On occasion, radioactive peaks were eluted from A-25 columns before unconju- gated compounds. These were minor in amount in almost every case and have not thus far been identified.

Fig. I shows the chromatographic pattern ob- tained after incubation sf doubly labelled E23G with whole kidney homogenate (tissue No. 3, Table 1 ) without added cofactors. Table 2 shows crystallization data for the E13G formed in that same experiment. Table 3 contains data on the interconversion of E23G and E13G in a number of homogenates from different kidneys. Conver- sion of E23G to E13G without change of isotope ratio occurred in all homogenates and appeared to be somewhat better stimulated by NAD than by NADP where such comparison was made. This was also apparent for E2 + El conversion, the free steroid having been released by limited hydrolysis of glucosiduronate during incubation. In the one experiment where cortex and medulla were separated, conversion of E23G to E13G without added cofactors was greater (some 2.5- fold as judged by E13G production) in the cor- tex. In the presence of exogenous NAB, how- ever, the medullary activity was increased more than 10-fold whereas that for the cortex showed a 2.5-fold rise. In no instance could a conversion of E13G to E23G of > 1 % be detected, even in the presence of exogenops NADH or NABPH (see further below).

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HOBKIRK EF AL.: 170-ESTRADIQL CONVERSIONS

FRACTION NO. ( EACH 5ml 1 FIG. 1 . Chromatographic pattern (60 cm A-25 column) of the conjugate fraction formed by incubating

E236 (isotope ratio = 4.3) with a whole homogenate of kidney No. 3 (see Table 1 ) without added cofactors. (0) 14C.

TABLE 1 . Kidney tissues used in incubations

Tissue No. Age (years) Sex Diagnosis

F Normal* F Normal * F Normal* F Eary glomerulsnephritis M Late glomerulonephritis F Early hydronephrosis

*Surgical specimen used was uninvolved in any disease process and was remote from, and unaffected by, the reason for nephrectomy.

Fig. 2 shows the chromatographic pattern ob- tained after incubating "-E23S and IT-E2, simultaneously, with whole kidney homogenate (tissue No. 3, Table 1 ) in the presence of added NAD. The E13S formed contained :%I3 but was devoid of proving that no sulfurylation of E2 (or El from substrate E2) had occurred. Thus direct E23S -, E13S conversion was indicated. Table 4 contains data for E23S and E13S con- version in a number of experiments. Dehydroge- nation of the former was noted in each case and stimulation by NAD appeared greater than by NADP, at least as judged by the amount of un- changed E23S at the end of incubation. A con- siderable degree of hydrolysis, however, tended

to complicate the picture so that good estimates of dehydrogenation were difficult to obtain. Stimulation of conversion of E, to El by cofac- tors appeared to parallel that of E23S to E13S by the same homogenate. Reduced cofactors acted in a similar manner to their oxidized forms, pre- sumably due to efficient oxidation of the former to the latter by the tissue homogenates. This also probably explains the virtuai absence of E13S -, E23S conversion, even with added NADH or NADPH (see also above for E13G -, E23G con- version). In the single instance where cortex and medulla were incubated separately, considerable dehydrogenase activity was apparent in both. The addition of NAD caused almost complete

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CAN. 9. BIQCHEM. VOL. 52. 1974

TABLE 2. Identification of labelled E13G formed from ::M /i4C-E23C (isotope ratio - 4.3) il~cilbated with kidney Jmomogea~ate witt~out added cofactors (tissue No. 3, Table 1)

S.A. (d.p.rn. /mg)

Stage of purification 3~ 14C w ,/14g:

72 500 d.p.rn. 3H + 16 320 d.p.m. 14C from co lum~~ + 20.2 mg carrier E,3G

Crystallization from methanol-H20 XLSI* M LI XkSfl MLII

XLSll + MLIl incubated with Ketodase +Els crystaBlization from methanol

XLSlII XLSlV

XkSlV acetylated + El-3-acetate CrystalIization from methanol-H20

XLSV

*XLS = crystals, ML = n~other liquor.. tCaiculrated from S.A. of conjugate crystnllizatio~?. $Calculated from §.A. of XESIV.

TABLE 3. Metabolism of "H /IT-E23G and E13G by kidney homogenates

Metabolites (% total identified) Incubatiort time

Tissue No.* dh) Substrate Cofactors E l E, E13GP E23G

1 Cortex I Cortex 1 Cortex 1 Medulla 1 Medulla 2 2 2 3 3 3 3 3 3 4 4 4

0 NAD NADP

0 NAD

0 NAD

0 0

NAB NADP

0 NABH NADPM

0 NAD NADP

*See Table 8 ; hornogenate is that s f whole kidney except where indicated. ?In every instance isotope ratio equals that of Es36 incubated. $ A single value refcrs to €1 + €2: individual vafues for El and Ez are indicated in some experiments.

conversion of E24S in the medulla (and of E2 to E l ) but one must note the large degree sf hy- drolysis sf the substrate (Table 4, tissue No. 1, medulla).

Examination of Table 4 suggests higher sul- fate hydrolysis in the presence than in the ab- sence of cofactors, in some sf the incubations. The reason for such a finding remains entirely

obscure. It is also of interest to note that the same homogenates as used in the present study showed no hydrolytic activity towards testsster- one-1 7-sulfate and in only a single instance was any activity (6 96 hydrolysis) seen with dehydrs- isoandrosterone-4-sulf ate as substrate. These ex- periments are not detailed in the present study in view of their negative nature.

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HOBKIRK EB AL.: 178-ESTRADIOL CONVERSIONS

FIG. 2. Chromatographic pattern (60 cm A-25 column) s f the products formed by incubating W-E23S + 14C-Et with a whole homogenate of kidney No. 3 (see Table I ) in the prersence of added NAD. ( X ) W, (9) I4C.

TABLE 4. Metabolism of Er3S and E,3S by kidney I~ornsgenates

Metabslities (% total identified) Incubation time

Tissue No.* (h) Substrate Cofactors El ES E13Sf E23S

1 Cortex I Cortex 1 Cortex 1 Cortex 1 Cortex 1 MeduIla 1 Medulla 3 A

2 2 3 4 4 4 5 5 6 6

0 NAD NABP NADH NADPH

(3

NAB 0

NAD 0

NAD 0

NAD NADP

8 NAB

0 NABH

'See Table I ; homogenate is that of whole kidney except where indicated. t161 each case 3H-El3S plus added 14C-E13S was hydrolyzed with Mylase P and crystallized with carrier EI without change in isotope ratio. !A single value refers to Er + E?; individual values for El and E! are indicated for most experiments.

Discwsisn is evidence for the direct conversion of Ea3S to The data obtained establish the presence of an E13S and of E, to El, besides a high sulfatase

active dehydrogenation of E236 to form E13G activity towards phenolic steroid sulfates. The in human renal homogenates. In addition, there degree sf involvement of these various enzymes

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20 CAN. J . BIOCHEM. VOL. 52, 1953

in physiological circumstances is not yet under- stood although a direct interconversion of E23G and E13C3 has been demonstrated in the human i~ V ~ V B (1 ). It is not possible to conclude, on the basis of the present results, whether dehydroge- nation of E23G, E23S, and E2 is catalyzed by one single enzyme, or by different enzymes. It should, however, be noted that where low activ- ity towards E23G was seen, poor dehydrogena- tion of E23S was also apparent, even with added cofactors (see tissue No. 2, Tables 3 and 4). Conversely, high activity towards both conju- gates and towards EP appeared to accompany each other in some hsmsgenates (see tissue No. I , medulla, Tables 3 and 49. It is difficult to con- ceive, at this time, of a specific purpose for these enzymes in the overall economy of the cell, and even in the metabolism of the estrogenic hor- m o n e ( ~ ) in the human. The present study does, however, add a further possible tissue site for steroid conjugate conversion. It will be necessary to study the intracellular location and specificity of these kidney enzymes as well as their possible presence in other tissues.

It also may be relevant to note that Hellman et a!. (12) have shown the presence in human kidney of a 11P hydroxysteroid dehydrogenase acting upon cortisol as substrate. It has been sug- gested by these workers that this enzyme could

represent a cellular capture mechanism for cortisol.

We are grateful to Brs. 9. Wyatt and L. M. Mc- Aninch (Department of Urology) and to Drs. M. Smout and A. C. Wallace (Department of Pathology) for obtaining the kidney tissue employed in the above study.

1. Hobkirk, R. & Nilsen, M. (1978) Steroids 15, 649- 667

2. Roy, A. & Slaunwhite, W. W. (1969) Steroids 64, 327-332

3. Emerman, S., Dancis, J., Levitz, M., Wiqvht, N. & Diczfalusy, E. (1965) J . Clin. Endocrinsl. 25, 639- 648

4. Jaeobsohn, G. M. & Hochberg, R. B. (1968) J. Biol. Chem. 243,2985-2994

5. Mulder, E., Earners-Stahlhofen, G. J. M. & Van Der Mealen, H. J. (1972) Biochim. Biophys. Acta 260,290-297

6- Mulder, E., Lamers-Stahlhofen, G. J. M. & Van Der Molen, H. J. (1972) Biochem. I . 127, 649- 659

7. Hobkirk, R., Nilsen, M. & Blahey, P. R. (1969) J. Clin. Endscrinol. 29,328-337

8. Hobkirk, W., Musey, P. & Nilsen, M. (8969) Steroids 14, 19 8-206

9. Fieser, L. F. (1948) J. Anz. Chent. Soc. 70, 3232- 3237

18. Bradlow, H. L. ( 1968) Steroids 11,265-272 1 1 . Eisboa, B. P. & Diczfalusy, E. (1962) Acta Endoc-

rdrrok. (Kbh.) 40,60-8 1 12. Hellman, L., Nakada, F., Zurnoff, B., Fukushirna,

D., Bradlow, H. L. & Galllagher, T. 1;. (1971 ) J. Clln. Endocrine!. 33,52-62

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