Isolation and identification of etiocholan-3α-ol-17-one and etiocholane-3α, 11β-diol-17-one from human blood plasma

ARCHIVES

OF

BIOCHEMISTRY

Isolation

AND

and

BIOPHYSICS

150-153

92,

Identification

(1961)

of Etiocholan-3cy-ol-l7-one

Etiocholane-3a!,llp-diol-17-one Human GEORG From

W. OERTEL

the Department

of Rioloyical

from

Blood

Plasma’

21~1) KRISTEX

Chemistry, I-niversity Salt Lake City, 7,‘tah Received

July

and

R. EIK-r\jES of 7ltah,

t’ollege

of Medicine,

14, 1960

Two hitherto unidentified 17.ketosteroids were isolated from 600 ml. of normal human male plasma. By such rriteria as infrared and sulfuric acid spectra and mot&t> of the free, the ucctylat,ed. and the oxidized compounds in several systems of paper chromatography, these 17-ketosteroids were identified as etiochol:tn-3ol-ol-17.one and etiocholane~3~-ll~-diol-l7-one. INTRODUCTION

t,he aqueous phase.2 AJ~ sliyuot of t,he combined extrnrts of solvolyzate and hydrolyzate was assayed for total 17.KS by the Zimmermnnn reacCon (5). The remaining material was separated into a ketonir :rnti a non-ketonic fraction by Girard-T reagent (6). The ketonic fraction was chromatographed on paper in propylene glycolmethylc~clohesnne (7), using testosterone ag reference compound. After drying, n small sectiorp of t,he paper strip was stained with alkaline mdinitrohenzene (8). Four distinct Zimmermann-positive spots were detected, corresponding to thy location of l>HlL4 (frnct,ion A), androsterone (frartion B), etiocholan-3ru-olI7-one (etiochol’ anolone; fraction C), and etiocholanc-3a, lip, I diol-17.one (llp~hydros~-rt.ic,cholanolone; fracl tion D). The appropriate spots were cut, out and eluted with methanol (M&H), and the dried eluntes were rechromatogral)~led separately in forJn:LJnit~e-hex~ne-t)enZ~Jle (9) and ethylene gl?col-Jneth~lc~cl(~hexnric, running rorresponding standards simultaneousl?’ 011 separate st,rips. After final paper chromatography, nliquots of the four different fractions obtained were wsnyed for 17. KS, while the remaining material was purified on aluminum oxide. The dry residue of each fraction was applied to :I column (height. 2.5 rm.; diameter 0.5 qm.) of aluminlun oxide (“Woelm”, almost neutral, activity 1) prepared with benzene, using three times 1 ml. benzene for complete transfer of the LlJLkJlowJl material to the CO~LIJ~JJ. The cdLiJIJJJ, was washed with 10 ml. benzene before 17.KS were’ eluted with 15 ml. of 55; MeOH in benzene. These fractions bvere collected and evaporated to dryness1

Of the 17-ket’oxt’eroids (IT-KS) present in systemic human blood plasma, only s-androsten-3/3-ol-17-one (dehydroepiandrost’erone, DHEAJ and androstan-3cu-ol-li-oue (androsterone) have been isolated and properly identified (1, 2). These steroids represent t,he major fraction of plasma li-KS. In recent experiments involving the est,imation of DHFX, androst)erone, and total 17-KS in plasma,? it was observed that t,he concentration of total 17-KS alwa.ys exceeded the sum of DHE;\ and androsterone, indira,ting the presence of additional 17-KS. In this communicat,ion we wish t,o report the isolation and identification of hithert,o unidentified plasma IT-KS. RIETHC)

1)s

Six hundred milliliters pooled plasma from normal male subjects was estracted t,hree times with 900 ml. ethyl acctnte-ether (1:I v/v). The aqueous phase was deproteinized and purified as described 11~ Kernel (3). For cleavage of 17.KS conjugates the purified extract was subjected to solvolysis (4), followed t>?- hot, acid hydrolysis of 1 This work ~vas partly supported from the U. S. Public Health Service,

by grants Bethesda,

Maryland.

* Hudson, 1960.

B. and

Oertel,

(:.

\\-.,

unpublished, 150

ISOLATION

OF

TABLE ~\IORILITY

OF “l)LAHMh

ETIOCHOLANOLOKE"

I

AND

OF PAI'ER

ITS

RT" 2.25 Kf 0.27

Propylene glyrol-met hylcyclohexane Formamitle~hexane-hcllzene Formamide-hexane Ethylene glycol-methylcyclohexane mobility

IS

DIFFERENT

SE.STEMS

'

Acetylated

~

Unknown

compound

Oxidized

compound

system Ullk~OW

iz Relative

DERIVATIVES

CHROMATOGRhPHY Free com~mnd

Solvent

151

17-KETOSTEROIDS

compared

i R, to that,

0.19

Authentic

2.23 0.26

.4uthentic 1 Unknown ~~~ ___

~ R, 0.70 ~ Rf 0.88 R, 0.83

~ 0.81 0.89 0.83

8uthentic

R,0.20

0.21

0.48

of t&osterone.

under nitrogen. The dry material then was dissolved in 5 ml. MeOH and divided into different sliquots. One aliquot of fraction C was acet,yl:rted with acetic anhydride-1-C” (10) and the ncetylated material chrom:ltogrnphetl in propylene glycolmethylcyclohrsnne and form:lmide-hesane. 9 second aliquot was oxidized with chromic acid, and the oxidation protlurt was suhmit,ted to paper chromatography in formamide-hes:llie. The t,hirtl aliquot was dissolved in 0.5 ml. cont. sulfuric acid and its absorption spectrum registered in a Beckman l)I< 2 recording spectrophotometer. The last aliquot of fraction C was suhject)cd to infrared analysis (11). Similar experiments were performed with nliquot,s of 1 he more polar fraction I). For paper chromatography of t,he Cl,‘-acetylatcd material the solvent systems propylene glgrol-methylcyclohc~xanc and formamide-hex~~ne-beIlzenc \vere used, while the oxidized compound were chromntographed in formaniidc-henzerle. The sulfuric acid sprct~rum of free and oxidized material \vas compared to that of authentic ll~~hydroxy:111d rtiocholnne-3,lI etioc,hol:lllolone ,17-triol, respect,ivcly. For final identification an aliquot of the fret wmpound was eul~mitted to infrared analysis;.

In an ext,ra.ct of 600 ml. human plasma, 1162.8 pg. 17-KS were found after solvolysis and hot acid hydrolysis. Of this amount, 1056.6 pg. was released by solvolysis, tht rest by hot, acid hydrolysis. Besides DHEA and androsterollc, two addit ional IT-KS were detected by spot, tests, wit,11 R, values (R, values) corresponding to those of etiocholauolont and 11/Chydroxyetiocholanolone. Upon acet#ylat’ion or oxidat,ion of aliquots from fraction C, the react,ion products exhibited mobilitirs similar to those of aut,hen-

0.1 -

zoo

I 300

I 400

I 500

w

FIG. 1. Sulfuric et.iocholan-3cu-ol-17.one lated from plasma.

arid

spectra of (.l) authentic and (U) rompound iso-

I;Ic. 2. Infrared spectra of (A) authentic etiocholan~3~-ol-l’i-one and CR) compound isolated from plasma. Both the unknown and the known compound were subjected to chromatogrnph,v on aluminum oxide hefore infrared analysis. The potassium bromide microtechnique as tlescrihed t)y hlason ww used (II).

tic etiocholanolone acet’ate and et’iocholane3, Ii-dione, respect’ively (Table I). The sulfuric acid spectrum of fraction C corresponded t’o that, of authent,ic etiocholanolone (Fig. 1). Likewise, the infrared spectrum of fraction C wa,s found to be in adequate agrcement w&h the infrared spect,rum of authentic

152

OEHTEL

.4NI)

EIK-NES

TABLE 0~'

MOBILITY

Free Solvent

compound

Acetylated

ITS

I>ERIvATIVE~

compound

(

Oxidized

IN

compound

system Unknowl

Propylene glycol-toluene Formamide-benzene Ethylene glycol-methylcyclohexane Formamide-hexane-benzene 0 Relative

II

"PLASMA 11~.HY~ROXYETI~~'H~L~~OLO.UE!' AND DIFFERENT SYSTEMS OF PAPER CHROMATOGRAFHT

mobility

RTC 0.30 Rf 0.29 RT 0.18

compared

to that

Authentic,

0.32 0.31 0.17

.4uthenti(

r\uthentic

Kj 0.87

0.84

Rf 0.73

0.72

Rf 0.72

0.71

of testost,erone.

0.4

etate aud etiocholane - 3,11,17 - t’rione, respectively. The sulfuric acid spectra of Fraction D and its oxidation product, are shou-n in Pigs. 3 and 4. From these spectra as well as from the infrared spectrum seenin Fig. 5 the presence of 1lb-hydroxyetiocholanolone in fra,ction D is clearly indicated.

r

1>1scuss10?; 200

400

300

500

w

FIG. 3. Sulfuric acid spectra of (A) authentic etiocholane-3or, lip-diol-17.one and (B) compound isolated from plasma. 0.5 0.4

I\

Y z 0.3 2 : \\\ w .--,J z 0.2

Ii‘ ‘, ,*’ \\ ‘-._ ---a.

0.1Y-i

IA

200

300

mp

Two hitherto unidentified 17-KS were isolated from 600 ml. pooled plasma of normal male subjeck As judged by mobilit,y of t’he free, the acetylated, and the oxidized compound in fraction C using different systems of paper chromatography, by sulfuric acid and infrared absorpt,inn spectra this IT-KS fract’ion consists of etiocholanolone. The other component (fraction D), which is considerably more polar, apparemly represents lip - hydroxyetiocholanolone. The mobility of the free, the acetylated, or oxi-

_. - - - -- ___ _g

400

FIG. 4. Sulfuric acid spectra of (A) and (R) etiocholanc-3,11,17-trione “plasma etiocholane-3~,ll~~diol-l7-one”.

500 authentic oxidized

etiocholanolone. However, t,here was evidence of small amounts of cont~aminat~ing material (Fig. 2). Fract,ion D containing t,he more polar material showed chromat,ographic properties of authentic Ilo-hydroxyetiocholanolone (Table II). Following acetylation or oxidation of aliquots from fraction D, the mobility of the reaction prodwAs compared favorably with t’hat of 1lp-hydroxyetiorholanolone ac-

Fro. 5. Infrared specta of (A) authentic etiocholane-3ol, llp-diol-17.one and (H) compound isolated from plasma. Both the unknown and the known compounds were subjected to chromatography on aluminum oxide before infrared an alysis. The potassium bromide microt,echniyne as _ described by Mason was used (11).

diwd material in several systems of paper chromat’ography, sulfuric acid and infrared spectra permit this conclusion. I-sing the extraction and purificsation met hods described previously, the following concent,rations of 17-KS were found in 100 ml. blood plasma obtained from one normal, male subject: 90 ~g. DHEA, 60 pg. androsterone, 31 pg. etiocholanolone, and 14 pg. 11 (I-hydroxyetiorholanolone. ,411 quantitations were made by the Zimmermann reaction. From this preliminary study, plasma etiocholanolone maj comprise an important fraction of total pl:wma 17-W in man. The authors wish to express their appreciation to I)r. IAX) T. Samuels for advire and ~ncouragcmcnt, and to I)r. Chnrles Nugent, 1)ep:trtment of bIedipinc, University of lit ah, for obtaining

2. MIGEON, C. J., J. Liiol. (‘hem. 218, Ml (l%(i). 3. KORSEL, I>.. .J. Lab. Clir~. ,Ved. 54, (iS9 (1!)59) ; (Cf. 11. MO). 4. BCTSTEIN. S., AND LIERERXAS, S., J. Rio/. Chem. 233, 331 11958); (Cf. 11. 333). 5. ~~~~ERI\IAXN. \v., Tyitamine II. Horwonc 5, 121 (19Ni. W. H., Ef~do6. PINC’GS, c;., ASD PEARLMAS, crindogy 29,413 (1941). 7. Sava~u, Ii., J. [. L.? Proc. Sot. Esptl. Rio2. Jletl. 94, 120 (19X). l’ittsburgh Conference 11. hlaso~, IV. B., “The on A4nslytic:ll Chemistry and Applied Spcctrow~p~,” March 1958; Icf. Perkin