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Squalene and cholestanol in normal rabbit aorta
Atherosclerosis Elsevier Publishing
SQUALENE
Company,
Amsterdam
-
AND CHOLESTANOL
V. STEFANOVICH
AND
401
Printed in The Netherlands
IN NORMAL
RABBIT
AORTA
G. KAJIYAMA
Defiartment of Pathology, University Hospital and Departments of Pathology and Biochemistry, Boston Univevsity Medical School, Houseman Medical Research Center, Boston, Mass. 02118 (U.S.A.) (Received
October
IOth, 1969)
SUMMARY
Squalene
and cholestanol
tified and quantitatively
were isolated
determined.
was 4.5 and 7.7 mg respectively,
Key words:
intima
in aortic
and media
from adventitia, were extracted evaporated
The concentration
rabbit
aortic tissue,
of squalene
in 100 g of dry, delipidized
iden-
and cholestanol
tissue.
Cholestanol - Rabbit aorta - Squalene
We would like to report cholestanol
from normal
tissue
here identification of normal
of 50 rabbits combined
using
rabbit.
(New Zealand
and homogenized
essentially
the procedure
and the residue dissolved
in hexane.
and determination For the isolation albino,
mixed
by a Virtis
of squalene of squalene,
sexes) were separated
“45” homogenizer.
of FOLCH et al.1, the Chromatography
and aortic
solvent
Lipids was
was carried out on a
silicic acid column (100-200 mesh) using 6% benzene in hexane for elution of hydrocarbons. Preliminary examination of hydrocarbon fraction by a gas-liquidchromatography (described below) disclosed eight major components (Fig. 1) with one component having the same retention time as reference squalene (Sigma Chemical Co., St. Louis, MO.) on a QF-1 column (2 min) and on a SE column (10.4 min). The 6% benzene-hexane fraction was evaporated to dryness. The residue was dissolved in 24 ml of benzene and added to a saturated solution of thiourea in methanol (160 ml) and kept at 0” C overnights. The collected crystals of a squalene-thiourea adduct were dissolved in water, squalene extracted with light petroleum and subjected to thinlayer chromatography (TLC) on Silica gel G (washed with chloroform-methanolThis work was aided by N.I.H. Grant No. HE-07327 of the University Hospital.
and by the General Research Support grant
.4 thevosclevosis, 1970, 11:401-403
V. STEFANOVICH,
402
10.4
20.8
G. KAJIYAMA
31.2
Minutes
Fig. 1. Gas-liquid chromatogram (6% SE-30 on Diatoport S) on hydrocarbons isolated from rabbit aorta.
acetic acid (10 : 10 : 1, by vol.) using hexane-benzene development. The position of squalene (RF 0.30) squalene-visualized
by spraying
(98 : 2, v/v) as a system for was obtained using standard
with 3 y0 iodine in chloroform.
Squalene
was eluted
(2 : 1, v/v), solvent evaporated to dryness in 0.5 ml of chloroform. Gas-liquid chromatography (GLC)
from silica gel with chloroform-methanol and the residue dissolved was performed
with a F & M model 402 chromatograph
flame-ionization Diatoport
detector.
Glass columns
S and 6’ x 3/16” containing
3 y0 QF- 1 on Gaschrom
was 225°C for both columns.
Detector
temperature
heater
pressure
was 5 PSI.
was 300°C. Nitrogen In this condition
reference
squalene
retention
equipped
with a hydrogen
were 4’ x 3/16” containing
times of squalene
Q. Column
6% SE-30
was 230°C and temperaturein isolated
from rabbit
on
temperature flash
aorta and of
was on the QF-1 column 2 min, and on the SE-30 column
10.4 min.
In addition, the low resolution mass spectrum of fragmentation pattern of a compound isolated from rabbits aorta was identical with that of the authentic squalene; furthermore, infrared spectra (KBr pellet) of both compounds were essentially identical. The amount of squalene found in the aortae of rabbits, as determined by gas chromatography, was found to be 4.5 mg f 1.4 (mean f standard deviation from four determinations) per 100 g of dry, delipidized tissue. Using the same procedure, the recovery of reference squalene was 90 %. Cholestanol was isolated from rabbit aorta, identified and determined in the following manner: 40 rabbits’ aortas (New Zealand, albino, mixed sexes) were combined and homogenized with Virtis “45” homogenizer in chloroform-methanol (2 : 1, v/v). The homogenized sample was filtered, solvent evaporated and lipids extracted with ether-hexane (1 : 1, v/v). The solution was washed with water, dried (NasSOd) and evaporated to dryness. The residue was dissolved in hexane and chromatographed on a silicic acid (100-200 mesh) column. The sterol fraction was eluted with benzene. The solvent was evaporated and subjected to thin-layer chromatography on Silica gel Atherosclerosis,
1970, 11: 401-403
SQUALENE
AND CHOLESTANOL
G in a system
consisting
IN NORMAL RABBIT
with chloroform. After evaporation obtained. The unsaturated sterols diethylether
solution
however
by iodine vapor).
was extracted
(95 : 5, v/v). The solvent
RF values
of isolated
Louis, MO.) were identical glass column
without
and reference
and reference
time of cholestanol
cholestanol
was 1.27%
(Sigman
chromatography
Q, as previously cholestanol
to cholesterol
Infrared spectra (KBr pellet) of cholestanol reference cholestanol were identical. The content gas chromatography
acidification,
with hexane-
and residue chromatographed (98 : 2, v/v) as a system for develop-
(RF 0.40). Gas-liquid
cholestanol
retention
were eluted
was evaporated
with 3 y0 QF-1 on a Gaschrom
times of isolated relative
Sterols
of solvent, a total amount of 30 mg of sterols was were removed by the ROSENFELD’S procedures.
on a silica gel G plate using chloroform-acetone ment.
403 acetic acid (225 : 70 : 6, by
of hexane-diethylether-glacial
vol.). RF value for sterols was 0.25 (visualized
The aqueous
AORTA
of the total
Chemical
Co., St.
was performed
described.
were identical
on a
The retention (7.2 min). The
was 1.096. isolated from rabbit aorta and of of cholestanol, as determined by a
sterols
and 7.7 mg & 0.8 (mean
standard deviation from four determinations) per 100 g of dry, delipidized Using the same procedure, the recovery of reference cholestanol was 95 %.
f
tissue.
REFERENCES J., M. LEES AND G. H. SLOANE-STANLEY, A simple method for the isolation and purification of total lipids from animal tissues, 1. biol. Chem.. 1957. 266: 497. 3 GOODMAN, DEW. S., AND G. POPJAK, St;dies on the biosynthesis of cholesterol, Part 12 (Synthesis of ally1 pyrophosphates from mevalonate and their conversion into squalene with liver enzymes), J. Lipid Res., 1960, 1: 286. 3 ROSENFELD, R. S., Analysis of sterol extracts for cholestanol, And. Biochem., 1965, 12: 483. 1 FOLCH,
Atherosclevosis,
1970, 11: 401-403
SQUALENE
Company,
Amsterdam
-
AND CHOLESTANOL
V. STEFANOVICH
AND
401
Printed in The Netherlands
IN NORMAL
RABBIT
AORTA
G. KAJIYAMA
Defiartment of Pathology, University Hospital and Departments of Pathology and Biochemistry, Boston Univevsity Medical School, Houseman Medical Research Center, Boston, Mass. 02118 (U.S.A.) (Received
October
IOth, 1969)
SUMMARY
Squalene
and cholestanol
tified and quantitatively
were isolated
determined.
was 4.5 and 7.7 mg respectively,
Key words:
intima
in aortic
and media
from adventitia, were extracted evaporated
The concentration
rabbit
aortic tissue,
of squalene
in 100 g of dry, delipidized
iden-
and cholestanol
tissue.
Cholestanol - Rabbit aorta - Squalene
We would like to report cholestanol
from normal
tissue
here identification of normal
of 50 rabbits combined
using
rabbit.
(New Zealand
and homogenized
essentially
the procedure
and the residue dissolved
in hexane.
and determination For the isolation albino,
mixed
by a Virtis
of squalene of squalene,
sexes) were separated
“45” homogenizer.
of FOLCH et al.1, the Chromatography
and aortic
solvent
Lipids was
was carried out on a
silicic acid column (100-200 mesh) using 6% benzene in hexane for elution of hydrocarbons. Preliminary examination of hydrocarbon fraction by a gas-liquidchromatography (described below) disclosed eight major components (Fig. 1) with one component having the same retention time as reference squalene (Sigma Chemical Co., St. Louis, MO.) on a QF-1 column (2 min) and on a SE column (10.4 min). The 6% benzene-hexane fraction was evaporated to dryness. The residue was dissolved in 24 ml of benzene and added to a saturated solution of thiourea in methanol (160 ml) and kept at 0” C overnights. The collected crystals of a squalene-thiourea adduct were dissolved in water, squalene extracted with light petroleum and subjected to thinlayer chromatography (TLC) on Silica gel G (washed with chloroform-methanolThis work was aided by N.I.H. Grant No. HE-07327 of the University Hospital.
and by the General Research Support grant
.4 thevosclevosis, 1970, 11:401-403
V. STEFANOVICH,
402
10.4
20.8
G. KAJIYAMA
31.2
Minutes
Fig. 1. Gas-liquid chromatogram (6% SE-30 on Diatoport S) on hydrocarbons isolated from rabbit aorta.
acetic acid (10 : 10 : 1, by vol.) using hexane-benzene development. The position of squalene (RF 0.30) squalene-visualized
by spraying
(98 : 2, v/v) as a system for was obtained using standard
with 3 y0 iodine in chloroform.
Squalene
was eluted
(2 : 1, v/v), solvent evaporated to dryness in 0.5 ml of chloroform. Gas-liquid chromatography (GLC)
from silica gel with chloroform-methanol and the residue dissolved was performed
with a F & M model 402 chromatograph
flame-ionization Diatoport
detector.
Glass columns
S and 6’ x 3/16” containing
3 y0 QF- 1 on Gaschrom
was 225°C for both columns.
Detector
temperature
heater
pressure
was 5 PSI.
was 300°C. Nitrogen In this condition
reference
squalene
retention
equipped
with a hydrogen
were 4’ x 3/16” containing
times of squalene
Q. Column
6% SE-30
was 230°C and temperaturein isolated
from rabbit
on
temperature flash
aorta and of
was on the QF-1 column 2 min, and on the SE-30 column
10.4 min.
In addition, the low resolution mass spectrum of fragmentation pattern of a compound isolated from rabbits aorta was identical with that of the authentic squalene; furthermore, infrared spectra (KBr pellet) of both compounds were essentially identical. The amount of squalene found in the aortae of rabbits, as determined by gas chromatography, was found to be 4.5 mg f 1.4 (mean f standard deviation from four determinations) per 100 g of dry, delipidized tissue. Using the same procedure, the recovery of reference squalene was 90 %. Cholestanol was isolated from rabbit aorta, identified and determined in the following manner: 40 rabbits’ aortas (New Zealand, albino, mixed sexes) were combined and homogenized with Virtis “45” homogenizer in chloroform-methanol (2 : 1, v/v). The homogenized sample was filtered, solvent evaporated and lipids extracted with ether-hexane (1 : 1, v/v). The solution was washed with water, dried (NasSOd) and evaporated to dryness. The residue was dissolved in hexane and chromatographed on a silicic acid (100-200 mesh) column. The sterol fraction was eluted with benzene. The solvent was evaporated and subjected to thin-layer chromatography on Silica gel Atherosclerosis,
1970, 11: 401-403
SQUALENE
AND CHOLESTANOL
G in a system
consisting
IN NORMAL RABBIT
with chloroform. After evaporation obtained. The unsaturated sterols diethylether
solution
however
by iodine vapor).
was extracted
(95 : 5, v/v). The solvent
RF values
of isolated
Louis, MO.) were identical glass column
without
and reference
and reference
time of cholestanol
cholestanol
was 1.27%
(Sigman
chromatography
Q, as previously cholestanol
to cholesterol
Infrared spectra (KBr pellet) of cholestanol reference cholestanol were identical. The content gas chromatography
acidification,
with hexane-
and residue chromatographed (98 : 2, v/v) as a system for develop-
(RF 0.40). Gas-liquid
cholestanol
retention
were eluted
was evaporated
with 3 y0 QF-1 on a Gaschrom
times of isolated relative
Sterols
of solvent, a total amount of 30 mg of sterols was were removed by the ROSENFELD’S procedures.
on a silica gel G plate using chloroform-acetone ment.
403 acetic acid (225 : 70 : 6, by
of hexane-diethylether-glacial
vol.). RF value for sterols was 0.25 (visualized
The aqueous
AORTA
of the total
Chemical
Co., St.
was performed
described.
were identical
on a
The retention (7.2 min). The
was 1.096. isolated from rabbit aorta and of of cholestanol, as determined by a
sterols
and 7.7 mg & 0.8 (mean
standard deviation from four determinations) per 100 g of dry, delipidized Using the same procedure, the recovery of reference cholestanol was 95 %.
f
tissue.
REFERENCES J., M. LEES AND G. H. SLOANE-STANLEY, A simple method for the isolation and purification of total lipids from animal tissues, 1. biol. Chem.. 1957. 266: 497. 3 GOODMAN, DEW. S., AND G. POPJAK, St;dies on the biosynthesis of cholesterol, Part 12 (Synthesis of ally1 pyrophosphates from mevalonate and their conversion into squalene with liver enzymes), J. Lipid Res., 1960, 1: 286. 3 ROSENFELD, R. S., Analysis of sterol extracts for cholestanol, And. Biochem., 1965, 12: 483. 1 FOLCH,
Atherosclevosis,
1970, 11: 401-403