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Is mevalonic acid a precursor of the corticosteroids?
ARCHIVES
OF
BIOCHEMISTRY
AND
IS Mevalonic
Acid MELVIN
From
BIOPHYSICS
the Department
96, 1-3
(1962)
a Precursor J. BRYSON
of Obstetrics
of the
AND
an.d Gynecology, Salt Lake City, Received
August
&lh);
University Utah
Corticosteroids?’ L. SWEAT of Utah
College
of
Medicine,
22, 1961
Bovine adrenal tissue preparations (whole homogenates or a combination of microsomal and supernstant fractions) readily convert acetate-l-C’” to corticosteroids, 14 of which have been isolated and identified. In parallel experiments with aliquots of thr, same adrenal tissue preparations, mewlonate-2-C” was not found to be incorporated into any of these corticosteroids. INTRODUCTION
Evidence that cholesterol is synthesized biologically from simple organic compounds via mevalonic acid, squalene, and a number of precursor steroids relat,ed to lanosterol has been reported (l-4). Steroid hormone synthesis in the adrenal, ovary, and testis also has hcen thought to follow this basir pathway, although some investigators hare quwtioncd whctlicr cholesterol lit9 dircct,l?in the pathway. In this study, nicvalonic~ avid-2-C’” anti :icct.atc-l-Cl’ wx incubatctl in aliquots of scTwa1 :~drru:~l prqwations. Fourteen steroid products including cortisol lvere isolated and identified from the incut)ations \vitll acctat,e: but. no cviclcncc was found that mevalonic acid serves as a precursor for any of these compounds. The rerent report of Sa\-arc1 et crl. (5) that mevalonic acid is not incorporated in the in zlitro biosynthesis of testis hormones or their prccurs0r.c: / is consistent with the results of tlrcw cxpc~rinients.
A 107: fresh bovine adrenal homogenate was prepared in a Potter-Elvehjem homogenizer with 0.25 M sucrose and 0.05 A1 sotliunll,ot:lssiun1~~~l)~)t:~~si~ln~ phosphate buffer (PI-I ’ This study was supported by grants A1803 and RC-4720 from the Institute of -4rthritis and Metabolic Diseases and the Division of General Medical Sience, thr r. S. Publir Health SerT-ice.
7.35). Ten milliliter samples of the whole homogenate or 20 ml. of the homogenate freed from mitochondria, nuclei, and other large granular material were incubated with ~otlium acetate-l-Cl4 (Y. A. 30.5 pc.jing.) 01 mewlonic acid-2-C14 (obtained from Tracer Lab, S. A. 4.03 pc.jmg.) in the presence of 0.004 111 MgClz , 0.0001 M DPNH, 0.0001 Jl TPX, 0.002 111ATP, 0.01 ~11glucose, and 0.007.5 211 ascorbic xi& at 37°C. for 2 hr., aftor wliicll the lipid products n-cre cstractc(l, pwtitioncyl bttwrn 7OyA ethanol :ml ll~~XI11c’, ant1 c~l~ronl~~togr:tphcd by the forlual~litlc rystems of Zaffnroni (6). The rntlioactivc steroid products were located on tlic ch~~omat.ogr~tln by means of an autonlat,ic strip feed colmter and recorder (7) and idcntificd by inctliod~ described in the litcraturo (8-l I). TM-O groups of triplicate aliquots from each of five scparatc :idrcnal preparations ( nlitocllonclri:l-fry, wwe incubated, respectiwly, with acetate-l-Cl” and mevnlonic :arid-2-C’4. With acetate-l-Cl4 3s subst.r:rt.c. scwral adrenal steroicls were isolated antI itlcntifictl from each of t,he incubations. Rclatiwly large quantities of Reichstein’s sutwtancc S, dc~oxycorticosterone, lip-hytiros~~~roge~t~eron~!~ and 6~-Ilydrol;y~?rogc~stcronc. n‘cw identified in t)hesc prcparntions. Pr0gcstr~01ic. 17a-h~dr0x-~]~rog~~t~ero~~e, androst~cnedionc, corticost)crone. and cortisol n-ew prewnt. in i-in:lller conrcntrations. In tn-0 Gniilar cxpwimcnta employing ~vhole
2
BRTSOS
ASD
homogenates, the results were essentially the same. However, the steroid products in these latter experiments (due to the presence of niit~ocliondria) wcrc quantitatively different, in t,hat corticosterone and cortisol were the major products. When mevalonic acid was employed as a substrate, t,here was no indication of the presence of any of the
SWEAT
above steroids in any of the preparations (Table I). In these latter experiments, 2% significant quantity of radioactivity was not& at t,lie solvent’ front as well as at the origin of t,hc chromatogram (chloroformformamide). However, further chromatography of these areas and study of the compounds by means of oxidation and acetyla-
TSBLE !,
f2OMl’AHISOS
OF
THE
~(‘ORI’OR.~TIOS
OF
I ACETATE-l-C
CORTIC~STEROIDS
BI-
THE
“‘I
ANI)
~~F,V.4I.OS.4TE-2-(!”
,NTO
ADRENAL
Init,i:tl chromatography of the partially purified extract was performed in the hcs:Lllc-fornlamidr system (note recorded in table). Subsequent chromatography of the radioactive products and its now radioactive st,eroid carrier (100~g.) was carried out either in the above syst.em (H), t,he hexane-twnzeneformamide (HB), the t)enzene~forrilalllide (B), or chloroform-formamide (C,I systems depending on the polar rharacterist,ics of the steroid under investigat,ion. The effective system reported above was one in which the carrier positioned it.self away from the origin and short of t,he solvent front of the chromatogram. In some of the procedures the elution solvents were allowed to drop from the paper (time designated in hours “h”) in order to allow the compounds to migrate furbher from the origin. “f,” “a,” and ‘lo” refer t.o the free st,at,e or t,he acet,ylatetl or oxidized derivatives of the product and its steroid carrier, respertively. The steroids were detected on t,he chromatograms by means of ultraviolet scanning (254 mp). Sat,ur:tted ketones were detect,ed t)y the %immermann reaction. AcetJrl:ttion and oxidation prw cedures were carried out as previously reported (II). Substrate Comvxmls
Oxidation
isolated
4-l’rcgnene-l1~~17a,21~t~riol~ 3,20-dionc I’regllane-llp,lia,21~triol3,20-dione I-l’regnene-J7a,21mdiol-3,11 20.t,rione 4.Pregnene~llp,21-diol-3,20dione I-I’regnene-21.01.3,11,20t rionc 4-I’regnene-Bfl , 21 +liol-3,20dione 4.I’regnene-17ff,21 -tliol-3,20dione 4.Androstene-llp~ol-3,li dionc 4~J’regnene.6P-ol-3,20-dionc 4.l’regnrne-I lfl-ol-3,20-dionc 1~Pregnenc-17u-ol-3,2O-dione
21 -hlono:tcet,at,e 21 ~Monoaceta1.e ,-
21 -Mononcet~atr 21 -Monoacetate 21 -Monoacetate 6,21-Diacetate
+ I
21
-
-Monoacetate
NOIlP
6.Monoacetate Xonc None 21 -Monoacetate
4.Androstene-S, I i-dione 4~T’regnene~3,2@dione (Progesterone) ” b4ild
oxidation
(II).
TVone None
product
Il&Hydrosyandro stcnedioneB Ilp~Hydros?:lrltlro stnnedinne,L Adrenostcrone 3,11-Diketoetiocholenic acid 3,ll~Diketoetiocho~ lenic acid 3,6-Diketoet,iocholrnic acid Androstrnc-3,17dionc Adrenost,rrone 6~JietoJJrogest,erone 11.Ketoprogestrrone Androst,ene-3,17dione 3~k-etoetiocholellic acid So reaction Yo reaction
CORTICOSTEROIDS
FROM
tion procedures gave no evidence that the radioactive material was associated with known steroids.
On t,he basis of Bloch’s (1) schemefor the formation of t,he theoretical &carbon isoprenoid intermediate, the maximum incorporation of radioactive carbons from carboxyl-labeled acetat’e is 2 per unit. The maximum number of labeled carbon atoms in an isoprenoid unit from mevalonic acid2-C14 is 1. As the specific activity of the nic~valonic acid and sodium acetate emt)loyotl in the present cxpcriments was 0.60 anal2.60 p.c.,‘pmole,rcspert,ively, the specific activity of an isoprenoid unit xynt8hesized from the acetate is 8.3 times that, of an isoprenoid unit syntlicsizcd from meralonic acitl. The experiments in this study were sc% up in a maimc’r which would allow ready clttcction of tbc quant,itg of steroids exlwctccl to 1~ formed from this quantit~y of mcvalonatc~ witli this magnitude of radioactivity. Further, if cxogcnously adtled rlwvalonic~ acitl wcrc :L precursor of the wrticostcroitls, 01w would cxpcct appreciable c~oirlpcnsationfor this ion-cr actjiritJy on tlic I):wis that nwvalonic acicl has bwn reported to be a mow &icicnt precursor for cholestcrol synthesis than ncct~wtc.Pol>jak I2‘1has rclwrtctl that ~vhcreas only a fcIv inillimiwoniole:: of acetate are incorporated into qualcne anti ~l~olceterol, 0.5-l .O qroles of mcvalonic acid is converted by the same liver prtparat~ion. Parallel cxperiment,s in this study with liver demonstrated rapid incorporation of mevalonic acid into cholcstcrol.
MEVBLONIC
ACID?
:3
Three general possibilities exist as explanations of the results of these experiments : (aj An enzymic or physical barrier to exogenously added mevalonic acid is present in the adrenal. (13) Competitive inhibition bctwecn the D and L forms of the Incvalonic acid is more effective in the adrenal than the liver. (cl The adrenal synthesizes steroid hormones through a route otlrcr than via mcvalonic acid.
1.
2. 3. 1. 5. 6. 7. s. 9.
10.
11.
OF
BIOCHEMISTRY
AND
IS Mevalonic
Acid MELVIN
From
BIOPHYSICS
the Department
96, 1-3
(1962)
a Precursor J. BRYSON
of Obstetrics
of the
AND
an.d Gynecology, Salt Lake City, Received
August
&lh);
University Utah
Corticosteroids?’ L. SWEAT of Utah
College
of
Medicine,
22, 1961
Bovine adrenal tissue preparations (whole homogenates or a combination of microsomal and supernstant fractions) readily convert acetate-l-C’” to corticosteroids, 14 of which have been isolated and identified. In parallel experiments with aliquots of thr, same adrenal tissue preparations, mewlonate-2-C” was not found to be incorporated into any of these corticosteroids. INTRODUCTION
Evidence that cholesterol is synthesized biologically from simple organic compounds via mevalonic acid, squalene, and a number of precursor steroids relat,ed to lanosterol has been reported (l-4). Steroid hormone synthesis in the adrenal, ovary, and testis also has hcen thought to follow this basir pathway, although some investigators hare quwtioncd whctlicr cholesterol lit9 dircct,l?in the pathway. In this study, nicvalonic~ avid-2-C’” anti :icct.atc-l-Cl’ wx incubatctl in aliquots of scTwa1 :~drru:~l prqwations. Fourteen steroid products including cortisol lvere isolated and identified from the incut)ations \vitll acctat,e: but. no cviclcncc was found that mevalonic acid serves as a precursor for any of these compounds. The rerent report of Sa\-arc1 et crl. (5) that mevalonic acid is not incorporated in the in zlitro biosynthesis of testis hormones or their prccurs0r.c: / is consistent with the results of tlrcw cxpc~rinients.
A 107: fresh bovine adrenal homogenate was prepared in a Potter-Elvehjem homogenizer with 0.25 M sucrose and 0.05 A1 sotliunll,ot:lssiun1~~~l)~)t:~~si~ln~ phosphate buffer (PI-I ’ This study was supported by grants A1803 and RC-4720 from the Institute of -4rthritis and Metabolic Diseases and the Division of General Medical Sience, thr r. S. Publir Health SerT-ice.
7.35). Ten milliliter samples of the whole homogenate or 20 ml. of the homogenate freed from mitochondria, nuclei, and other large granular material were incubated with ~otlium acetate-l-Cl4 (Y. A. 30.5 pc.jing.) 01 mewlonic acid-2-C14 (obtained from Tracer Lab, S. A. 4.03 pc.jmg.) in the presence of 0.004 111 MgClz , 0.0001 M DPNH, 0.0001 Jl TPX, 0.002 111ATP, 0.01 ~11glucose, and 0.007.5 211 ascorbic xi& at 37°C. for 2 hr., aftor wliicll the lipid products n-cre cstractc(l, pwtitioncyl bttwrn 7OyA ethanol :ml ll~~XI11c’, ant1 c~l~ronl~~togr:tphcd by the forlual~litlc rystems of Zaffnroni (6). The rntlioactivc steroid products were located on tlic ch~~omat.ogr~tln by means of an autonlat,ic strip feed colmter and recorder (7) and idcntificd by inctliod~ described in the litcraturo (8-l I). TM-O groups of triplicate aliquots from each of five scparatc :idrcnal preparations ( nlitocllonclri:l-fry, wwe incubated, respectiwly, with acetate-l-Cl” and mevnlonic :arid-2-C’4. With acetate-l-Cl4 3s subst.r:rt.c. scwral adrenal steroicls were isolated antI itlcntifictl from each of t,he incubations. Rclatiwly large quantities of Reichstein’s sutwtancc S, dc~oxycorticosterone, lip-hytiros~~~roge~t~eron~!~ and 6~-Ilydrol;y~?rogc~stcronc. n‘cw identified in t)hesc prcparntions. Pr0gcstr~01ic. 17a-h~dr0x-~]~rog~~t~ero~~e, androst~cnedionc, corticost)crone. and cortisol n-ew prewnt. in i-in:lller conrcntrations. In tn-0 Gniilar cxpwimcnta employing ~vhole
2
BRTSOS
ASD
homogenates, the results were essentially the same. However, the steroid products in these latter experiments (due to the presence of niit~ocliondria) wcrc quantitatively different, in t,hat corticosterone and cortisol were the major products. When mevalonic acid was employed as a substrate, t,here was no indication of the presence of any of the
SWEAT
above steroids in any of the preparations (Table I). In these latter experiments, 2% significant quantity of radioactivity was not& at t,lie solvent’ front as well as at the origin of t,hc chromatogram (chloroformformamide). However, further chromatography of these areas and study of the compounds by means of oxidation and acetyla-
TSBLE !,
f2OMl’AHISOS
OF
THE
~(‘ORI’OR.~TIOS
OF
I ACETATE-l-C
CORTIC~STEROIDS
BI-
THE
“‘I
ANI)
~~F,V.4I.OS.4TE-2-(!”
,NTO
ADRENAL
Init,i:tl chromatography of the partially purified extract was performed in the hcs:Lllc-fornlamidr system (note recorded in table). Subsequent chromatography of the radioactive products and its now radioactive st,eroid carrier (100~g.) was carried out either in the above syst.em (H), t,he hexane-twnzeneformamide (HB), the t)enzene~forrilalllide (B), or chloroform-formamide (C,I systems depending on the polar rharacterist,ics of the steroid under investigat,ion. The effective system reported above was one in which the carrier positioned it.self away from the origin and short of t,he solvent front of the chromatogram. In some of the procedures the elution solvents were allowed to drop from the paper (time designated in hours “h”) in order to allow the compounds to migrate furbher from the origin. “f,” “a,” and ‘lo” refer t.o the free st,at,e or t,he acet,ylatetl or oxidized derivatives of the product and its steroid carrier, respertively. The steroids were detected on t,he chromatograms by means of ultraviolet scanning (254 mp). Sat,ur:tted ketones were detect,ed t)y the %immermann reaction. AcetJrl:ttion and oxidation prw cedures were carried out as previously reported (II). Substrate Comvxmls
Oxidation
isolated
4-l’rcgnene-l1~~17a,21~t~riol~ 3,20-dionc I’regllane-llp,lia,21~triol3,20-dione I-l’regnene-J7a,21mdiol-3,11 20.t,rione 4.Pregnene~llp,21-diol-3,20dione I-I’regnene-21.01.3,11,20t rionc 4-I’regnene-Bfl , 21 +liol-3,20dione 4.I’regnene-17ff,21 -tliol-3,20dione 4.Androstene-llp~ol-3,li dionc 4~J’regnene.6P-ol-3,20-dionc 4.l’regnrne-I lfl-ol-3,20-dionc 1~Pregnenc-17u-ol-3,2O-dione
21 -hlono:tcet,at,e 21 ~Monoaceta1.e ,-
21 -Mononcet~atr 21 -Monoacetate 21 -Monoacetate 6,21-Diacetate
+ I
21
-
-Monoacetate
NOIlP
6.Monoacetate Xonc None 21 -Monoacetate
4.Androstene-S, I i-dione 4~T’regnene~3,2@dione (Progesterone) ” b4ild
oxidation
(II).
TVone None
product
Il&Hydrosyandro stcnedioneB Ilp~Hydros?:lrltlro stnnedinne,L Adrenostcrone 3,11-Diketoetiocholenic acid 3,ll~Diketoetiocho~ lenic acid 3,6-Diketoet,iocholrnic acid Androstrnc-3,17dionc Adrenost,rrone 6~JietoJJrogest,erone 11.Ketoprogestrrone Androst,ene-3,17dione 3~k-etoetiocholellic acid So reaction Yo reaction
CORTICOSTEROIDS
FROM
tion procedures gave no evidence that the radioactive material was associated with known steroids.
On t,he basis of Bloch’s (1) schemefor the formation of t,he theoretical &carbon isoprenoid intermediate, the maximum incorporation of radioactive carbons from carboxyl-labeled acetat’e is 2 per unit. The maximum number of labeled carbon atoms in an isoprenoid unit from mevalonic acid2-C14 is 1. As the specific activity of the nic~valonic acid and sodium acetate emt)loyotl in the present cxpcriments was 0.60 anal2.60 p.c.,‘pmole,rcspert,ively, the specific activity of an isoprenoid unit xynt8hesized from the acetate is 8.3 times that, of an isoprenoid unit syntlicsizcd from meralonic acitl. The experiments in this study were sc% up in a maimc’r which would allow ready clttcction of tbc quant,itg of steroids exlwctccl to 1~ formed from this quantit~y of mcvalonatc~ witli this magnitude of radioactivity. Further, if cxogcnously adtled rlwvalonic~ acitl wcrc :L precursor of the wrticostcroitls, 01w would cxpcct appreciable c~oirlpcnsationfor this ion-cr actjiritJy on tlic I):wis that nwvalonic acicl has bwn reported to be a mow &icicnt precursor for cholestcrol synthesis than ncct~wtc.Pol>jak I2‘1has rclwrtctl that ~vhcreas only a fcIv inillimiwoniole:: of acetate are incorporated into qualcne anti ~l~olceterol, 0.5-l .O qroles of mcvalonic acid is converted by the same liver prtparat~ion. Parallel cxperiment,s in this study with liver demonstrated rapid incorporation of mevalonic acid into cholcstcrol.
MEVBLONIC
ACID?
:3
Three general possibilities exist as explanations of the results of these experiments : (aj An enzymic or physical barrier to exogenously added mevalonic acid is present in the adrenal. (13) Competitive inhibition bctwecn the D and L forms of the Incvalonic acid is more effective in the adrenal than the liver. (cl The adrenal synthesizes steroid hormones through a route otlrcr than via mcvalonic acid.
1.
2. 3. 1. 5. 6. 7. s. 9.
10.
11.