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Isopropanol precipitation methodfor thedetermination of
apolipoproteinB specific activityand plasma concentrations during
metabolic studiesof very low density lipoprotein and low
densitylipoprotein apolipoprotein B
Genshi Egusa, David W. Brady, Scott M. Grundy,' and Barbara
V.Howard'Phoenix Clinical Research Section, National Institute of
Arthritis, Digestive, Diabetes and KidneyDiseases, National
Institutes of Health, Phoenix, AZ
Abstract A method has been described for the measurementof apoB
concentration and specific activity in very low densitylipoprotein
(VLDL) and low density lipoprotein (LDL) duringmetabolic studies.
For measurement of specific activity, apoBwas separated from other
apolipoproteins and lipids by pre-cipitation in, and subsequent
washing with, isopropanol. Fordetermination of apoB concentration ,
equal volumes of lipo-protein and isopropanol were mixed, and the
protein contentof the apoB precipitate was measured by the
difference be-tween total lipoprotein protein and the protein
measured inthe supernatant. Evidence that there was no apoB
solubiliza-tion in isopropanol and that precipitated apoB was
virtuallyfree of soluble apolipoproteins was obtained by
electropho-resis. Lipid contamination of the apoB recipitate was
less han1%. The methods were applicable to VLDL, ntermediatedensity
lipoprotein (IDL), and LDL from normolipemic pa-tients with
proteinconcentrations between 187 pg/ml and1898 pg/ml. The data
obtained using isopropanol were highlycorrelated with those using
tetramethylurea, and recoveriesof apoB were similar. Furthermore,
the isopropanol methodhas been demonstrated to yield consistent
data for apoB spe-cific activities in a study of VLDL, IDL, and LDL
metabo-lism.-Egusa, G., D.W.Brady, S. M.Grundy, and B. V.Howard.
Isopropanol precipitation method for the determi-nation of
apolipoprotein B specific activity and plasma con-centrations dur
ing metabolic studies of very low density li-poprotein and low
density lipoproteinapolipoprotein B. J .Lipid Res. 1983. 24:
1261-1267.Supplementary keywords apoB VLDL IDL LDL
Apolipoprotein B (apoB) is the major structural pro-tein for
both very low density lipoproteins (VLDL) andlow density
lipoproteins (LDL). Because of its high mo-lecular weight and
insolubility it has proven difficult toquantify. An important
advance in measurement ofapoB was the report of Kane et al. (1, 2)
that tetra-methylurea (TMU) selectively precipitates apoB in
the
presence of other lower molecular weight apolipopro-teins.
Concentration can be estimated using the Lowryprocedure either by
difference or after resolubilizationand direct measurement.
Resolubilization has been em-ployed for determination of specific
activities of apoBin metabolic studies (3).
Despite the advantages of T M U , it is expensive anddifficult
to obtain in pure form, and it interferes withcolor development in
the Lowry procedure. RecentlyHolmquist et al. (4, 5 ) reported that
isopropanol cansolubilize C and E apolipoproteins, and some
investi-gators have used isopropanol to isolate
apolipoproteins(6-10). In this report we have described methods
usingisopropanol to determine apoB concentration and spe-cific
activity in VLDL and LDL and compared them tomethods using TMU. The
results showed that isopro-panol provides a convenient method
equivalent to thatusing T M U for determining the oncentration and
spe-cific activity of apoB in metabolic studies.
MATERIALS AND METHODSIsolation of lipoproteins
Five hundred ml of venous blood (from a subjectfasted overnight)
was collected in plasmapheresis bags
Abbreviations:VLDL, very low density lipoprotein; DL,
nter-mediate density lipoprotein;LDL, low density lipoprotein; TM
U,1,1,3,3-tetramethylurea; DS, sodium dodecyl sulfate; PAGE,
poly-acrylamide gel electrophoresis; apoB, apolipoprotein B; DTNB,
5.5'-dithiobis-(2-nitrobenzoic cid).' Center for Human Nutrit ion,
University of Texas Health ScienceCenter, Dallas, T X 75235.Address
eprint equests toDr.BarbaraV.Howard,PhoenixClinical Research
Section, NIADDK, National Institutes of Health,4212 North 16th
Street, Phoenix, AZ 85016.
Journal of Lipid Research Volume 24, 1983 1261
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containing citrate dextrose as anticoagulant
(FenwalLaboratories, Deerfield, IL). Plasmawas immediatelyseparated
by centrifugation at 600 g for 20 min (1OOC)and preservative (10%
sodium azide, 0.2 M DTNB,0.5% chloramphenicol) was added (10 p1 per
ml plasma).VLDL was isolated by ultracentrifugation at 40,000rpm
for 16 hr (15C) n a Beckman 60 Ti rotor (Beck-man Instruments,
Inc., Palo Alto, CA) andrecentri-fuged through saline (d 1.006
g/ml) in a 40 Ti rotor.IDL was removed after adjusting the density
to 1 O 19g/ml using NaC1-NaBr (d 1.35 g/ml) and centrifugingat
40,000 pm for 20 hr (15OC). LDL was isolated, afteradjusting the
density to 1.060 g/ml, in a type 50 Tirotor at 50,000 rpmor 20 hr
(15OC) and recentrifugedat d 1.070 g/ml at 40,000 rpm for 20 hr
(15OC). Li-poprotein preparations were dialyzed against EDTA-saline
(d 1.006 g/ml, 100 mM EDTA, pH 7.4) and 3 * I -labeled VLDL and
'251-labeled LDL wereprepared ac-cording to he iodine monochloride
method ofMc-Farlane (1 1) as modified by Langer, Strober,
andLevy(12) and dialyzed against 20 changes of EDTA-saline,pH
7.4.Isolation of apoB for determination ofconcentrationor specific
activity
For the measurement of apoB concentration, 1 mlof lipoprotein
sample was combined with 1 ml 100%isopropanol (analytical grade, J.
T. Baker ChemicalCo.,Phillipsburg, NJ) in a 12 X 75 mm conical
heavy-walledplastic centrifuge tube. After vigorous mixing (1
min)the samples were incubated overnight at room temper-ature (or
at 4OC for samples containing high salt con-centrations) and
centrifuged at 1OOOg for 30 min. ApoB
concentrations in VLDL and LDL were determined bysubtracting the
absorbance (660 nm) of the supernatantfrom the absorbance of the
initial lipoprotein prepa-ration. If on occasion the precipitate
was not completelysedimented by centrifugation, he upernatant
waspassed through a 0.22-pm filter.
For the measurement of apoB specific activity, theprecipitation
was performed as above and the super-natant was discarded. Two ml
of 50% isopropanol wasadded to thepellet and thesample was mixed
vigorouslyand centrifuged as above; this washing procedure
wasrepeated one time. After this, 2 ml of 100% sopropanolwas added
oensure complete lipid extraction. Th emixture was agitated
vigorously until the precipitate wascompletely dispersed, incubated
2 hr at room temper-ature, and then centrifuged as described above.
Afterdiscarding the supernatant,aliquots of 1 N NaOH wereadded to
thepellet; for VLDL or IDL, ca. 1 pl of NaOHper p g of initial
protein; for LDL, ca. 2 p1 of NaOH perpg of nitial protein.
Radioactivity of the NaOH sus-pension was determined immediately in
an auto-gammaspectrometer (Packard Instrument Co., Inc.,
DownersGrove, IL). Then the NaOH suspension was incubatedin a water
bath at 37C until the pellet was completelydissolved. This
requiredapproximately 1 day for VLDLand IDL and 2 to 3 days for
LDL. Protein content ofthis solution was then measured. The
proportion of ra-dioactive iodine bound to lipid in the apoB
precipitateswas assessed by extraction with chloroform-methanol
In order to compare the isopropanol methods to pre-vious
methodology using T M U , identical samples ofVLDL and LDL were
processedor apoB concentration
2: 1 (vol/vol) (1 3).
0.5 1 B
Fig. 1. Effect of T M U and isopropanol on protein measurement
by the Lowry method 14). A. Isopropanol:standard bovine serum
albumin (BSA) (0 - 60 pg ) was mixed with isopropanol and water at
the following finalconcentrations:0 0). (A), 0 (W), 15 0). 0 (V),
5% (V).Eachstandard line was linear with r> 0.99. B. T M U : T M
U was added to standard BSA (0- 60 pg) at the following final
concentrations:0(O),0.5% 0). (A), .5 (A), 2% (W), 3% 0 , (V), nd 5
(V). Each standard ine was inear with r> 0.99.
1262 Journal ofLipid Research Volume 24, 1983
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- m o r i o - m w - - n -m m m w r - nm 4 w m m a m DY- -0
e -%g.2 f +I +I +I tl +I +I +I +I tI +I f las described by Kane
et al. (2) and for specific activity 5. P - - - -ccording to the
method described by Le et al. (3).0 0 0 - - m b W n 0 C . Ie * * *
* w m w a w w0 . I m I O u mmmProtein measurementProtein
concentrations in lipoprotein fractions and
2R
2 nc9 2
dNaOH digests were determined by a modified Lowryprocedure that
employed 1% SDS in reagent A (14). In
nm of A l c M 1 % = 6.6 in order to be independent ofwater of
hydration. The influence of TMU and isopro- - z g o p r- Q) m ~ a w
p- a o p- mpanol on color development was assessed by
comparingassays containing from 0.5% to 25% solvent (Fig. 1).With
increasing TMU concentration, absorbance of the g 6 - :- ::
~SS??~~
+I +I +I +I +I +Iblank rose from 0 to 0.177, and the slope of
the stan- 1 2& f . ? E ? ? ?B g Q ? 9c?ard line decreased. In
contrast, isopropanol in con- m m w a w 2 a n m o acentrations u p
to 25% had no effect on color develop-ment. For all subsequent
experiments, Lowry assays 8 4were performed using standards
containing the same .- ? ? E - ? - n - n mz m m m o Z m r - i o -
;
2 2 +I +I +I +I +I +I +I +I +I +I t l< g 2 OTY 'C9d : m m b a
o -are not all equivalent to albumin in the Lowry proce- - w m o 1
1 c 4 0 d c r ; e r ; r - mdure , the chromogenicity factor for
apoB compared to E s - m * m - bfactor of 1.16 was used for the
isopropanol supernatant zElectrophoresis 5
3 * g c $ G g g t l z g g g g g t la
4 *ll assays the reference bovine serum albumin solutionwas
standardized using an extinction coefficient at 280d
m n a a - + l m m * - r - - + 1o x8 5 & ;
.f 4 2 mmg ge - - e- r'u -E +I +I +I +I t l2
w m a - r - o e w - m w- a m * - O . l m I . a * -0.
- - - - m m-qn.amount of TMU, NaOH, or isopropanol as that of
thesamples. Since it has been shown that apolipoproteins c'g a m a
o m P - W W ~ W P -- -a bovine albumin standard was taken to be
1.0, and asolutions (2).
a m m m m ~o m m o m m-beam+I +I +I t l +I +I- -
>uu
'c xz 2 - - - -k.5 f +I +I +I +I +I - m w - o ma - - b o m m a o
m m.- u > .-e e - m - 0 . I m m m w m m* * * * * mmmm.Ic .
ImD
he isopropanol supernatants and protein precipi- 8*C.-J-m0+1 W b
m * * a + l
9 c?tates were subjected to SDS-polyacrylamide gel electro- 2
mPAGE was performed in a discontinuous buffer system(15) with the
addition of 0.1% SDS to the upper cham- cber buffer. Gels were cast
in 13 X 0.5 cm glass tubes. a & gRunning gels were 7.5%
acrylamide, 0.2% N,N'-meth- q Eylene-bisacrylamide, and stacking
gels were 1.25% ac- 3rylamide and 0.3%
N,N'-methylene-bisacrylamide. ;j 9
8 * * v v w * m a a m m ae4*phoresis (PAGE) (Bio-Rad Lab,
Richmond, CA) (15). g 4.-
4-
A ?g o p z g g g g + l g z g g g g t i2 g0 0 aW 9 c ? - 4 9 d :c
? ? c 9 ' 9 m b m a b *
+I +I +I +I +I +I +I +I +I +I +I-ab - - -hole lipoprotein
samples were delipidated using chlo- croform, and all samples were
suspended in 0.01 M phos- k 2% .phate buffer (pH 7.0) containing 1%
SDS and 1% P- g o m m o w a w w a n fg m m - - 0w n * m m m m on *
m z zercaptoethanol; electrophoresis was conducted at 2 4
mamp/gel until the tracking dye (bromophenol blue)Coomassie
Brilliant blue G250, destained in 10% acetic < e -trophotometer
(Gilford Instruments Inc., Walnut Creek, s 2 g z g z 2 c g z z
zmigrated/distance of tracking dye. s . 5 % mW p- ' 9 o - d :
oMetabolic study C k - -
Volunteer subjects admitted to a metabolic ward con-sumed a
weight-maintaining diet (40 at, 15%protein,45% carbohydrate) for a
week. Sterile and pyrogen-free - Inautologous ' I-labeled VLDL (25
&i) was prepared as
3- J zm 3 P ' 9 9 9 c ? o ? E d : ? ? ' ?
AC O o ? - ? r - "-?"a -??m .- - - - -
t g~ 3 +I tl +I +I +I +I +I +I +I +I +Iacid, and scanned at 595
nm using a Gilford 250 spec- 4 ; 3 ? ? ? ? ? c ~ ~ ? W Q ~ Ea m c r
i q m o * w * n a
w * - n m - r . * n * meached the end of the gels. Gels were
stained using
- -A). Relative migration (R f )was calculated as distance: z g
4 2 ; ; & g g E g gc x ~ b a z - m w m m m
el eln+2
Egusa et al. Determination of apoB specific activity
9 -Mm-
2.P5atnCo-3g.mCo
uC
.--.-.-.-5
?si-25k
e,
gz C
E2z z xG o -2 5 s
; ; 35 30 2 25 g >~ O)0 m &p g z4z. 5 Z o , E Gg 4.2 0
.oP .P.hZo s g 5 . z5 - a.5 LZ E m . 5 9='E 8 2 4m b Mk.;8 . z ; ~
~m-oimo% a l l 0 2h a ws c t : mm . 0 s at1o r y - G. 5 E t I I ml
m m gB g ZLg- 0 2 : :u
3 5 g l 5 5 Cm a & $ 3
m k -
3
u mQ J ' Y C
~ O U U JU < & L >W Q u - u u
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1 I I 1 ,A Isopropanolmethod (pg/ml)0 100 200 300 400 500500
0 )aE 450P\
E.= 400x>0m.p 350c0alP
.-
300
2000-z 600-0 )aQ 1200-05g 800-3b 400-
VLDL ) 3500t.E
A nA E 000
.-LvPg 2000
LDL
I0 400 800 1200600000Isopropanol method (pg/ml)
LDL
0n X A 8 9 9
described above (except that preservative was omittedfrom the
plasma) and injected at time 0. At the indicatedtimes, 4 ml of
plasma was obtained and VLDL, IDL,and LDL were isolated by
ultracentrifugation atd1.006, 1.O19, and1.063 g/ml, respectively. '
I-labeledapoB specific activity of VLDL, IDL, and LDL was
de-termined as described.
RESULTS AND DISCUSSIONConcentrations and specific activities of
'I-labeled
VLDL-apoB and '251-labeledLDL-apoB determinedusing isopropanol
and T M U were compared (Table 1,Fig. 2). Estimatesof apoB
concentration by the twomethods were highly correlated (Fig. 2A).
Slightly lowervalues were obtained by TMU when the protein massof
VLDLwas low ( ~ 6 0 0g/ml); somewhat lower valuesalso were noted
for LDL-apoB measured by the TMUprocedure. This could be because
the TMU procedure
I I 10 20000 60000 1000 1200 0 400 800 1200 1600 2000B Initial
protein mass p g / m l ) Initial protein mass pg/ml)Fig. 2. A .
Relationships between apoB concentra tion of V L D L and L D L
determined using isopropanol andT M U . . Comparison of apoB
specific activity in V L D L and L D L measured using isopropanol
(A) and T M U0) ver a wide range of initial protein
concentrations
requires greater dilution of the TMU supernatant toeliminate the
effect of T M U on the color developmentof the Lowry procedure
(2).
Specific activities of poB in VLDL and LDL by thetwo methods
were virtually identical using the two sol-vents (Table 1).
Furthermore, when using isopropanol,specific activities of VLDL
apoB and LDL apoB wereconstant over a wide range of apoB
concentrations (187to 1898 pg/ml) (Fig. 2B). With isopropanol, the
recov-ery of apoB after solubilization of the precipitates
av-eraged 76.8% forVLDL and 86.0% or LDL. Recoveryof apoB in VLDL
appeared to ncrease in both methodswith increasing initial protein
mass. It is, thus, likely thatthe loss represents loss of
precipitated apoB during theextensive washing procedure.
Solubilization of he apoBprecipitate in LDL after isopropanol
treatment usuallyrequired 2-3 days incubation with 1 N NaOH. For
thisreason, the radioactivity measurements were made di-rectly
after mixture with NaOH; this was done becauseof the rapid decay of
isotopes generally employed in
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VLDL LDL
E
1.0
Fig. 3. Densitometric scan of S DS-polyacrylamide gel
electrophoresis of isopropanol supernatants and washedprecipitates:
VLDL, left panel; LDL, right panel. Top of gel is at the left; the
abscissa is the relative m igrationcompared to the tracking dye
Rj). . Intact VLDL and L DL preparations. B. ApoB precipitates
obtained usingisopropanol. C. Supernatants a fter isopropanol
precipitation of apoB.
metabolic studies. Th e apoB precipitated by isopropa-nol was
more readily soluble in both NaOH and elec-trophoresis buffer than
precipitates obtained with TMU.This appears to be a definite
advantage of isopropanol.
Specificity of he isopropanol precipitation procedurewas
confirmed by comparing SDS-PAGE of sopropanolprecipitates and
supernatantswith those of whole VLDL(Fig. 3, left) and LDL (Fig. 3,
right). Electrophoresis ofisopropanol supernatants on SDS-PAGE
revealed no
presence of apoB when either LDL or VLDL were pro-cessed (Fig.
3C). Inaddition,no obviousCoomassieblue-stainable bands (which
would ndicate the presenceof large aggregates) were noted in the
stacking gel.Thus, apoB precipitation was complete. In the
sampleshown on the gel, the apoE peak was small,
indicatingsomepossible oss or degradation during
processing.Electrophoresis after resolubilization of the
isopropanolprecipitate disclosed no apoE and very little
contami-
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TABLE 2. Proportion of radioactivity bound to lipid in patients
with high triglyceride. However, the apoBin apoB precipitates
precipitationusing sopropanolhas not been verifiedIsopropanol
Method T M U Method using lipoproteins from patients with a variety
of hy-
VLDL apoB 1.02 * 0.11 0 . 4 6 * 0.07%LDL apoB 0 . 7 2 f 0.07
0.12* 0 . 0 4 %~
VLDL apoB and LDL apo B precipitates obtained as described
inMethods for specific activity measurement were extracted with
chlo-roform-methanol 2:l for 30 min. Values are mean SEM (N = 6 )of
lipid radioactivity/total radioactivity.
nation (4 ) by apoC (Fig. 3B). The TM U precipitatescontained
similar proportionsf apoC (data nothown).
Amountsof ipid-bound adioiodine ound in theapoB precipitate were
quantified (Table 2). In all caseslipids accounted for less than 1%
of total radioactivity.In addit ion there was no detectable lipid
mass after iso-propanolextractions. To determinewhether
triglyc-eride was co-precipitated with apoB when
isopropanoltechniques were employed on VLDL from
patientsithhypertriglyceridemia, a patientwith VLDL-triglycerideof
2229 mg/dl was studied. ['HITriolein in a small vol-ume of ethanol
was added to the VLDL preparation(2470 pgof protein/ml) and the
apoBwas precipitatedusing isopropanol as described above. Less than
1% of'H-radioactivity was associated with the apoB pellet af-ter
the usual washing procedure. Efficient delipidationrequired
dispersing the pellet completely (using a glassrod if necessary)
and agitating frequently dur ing incu-bation. This suggests that
efficient removal of triglyc-eride can be accomplishedusing these
procedures,even
~ C F VLDL
...JL
I I I I 1 I I IHour0 10 20 30 40 50 60 70
Fig.4. ApoB specific activity of VLD L, IDL , and DL after
injectionof ' 'I-labeled VLDL . Data were obtained from a
normolipemic vol-unteer, and the curves were generated for VLDL (A
- - - A), IDL0 - - - O), and LDL (V - - V) y a multicompartmental
model asdescribed by Phair et al. (16).
perlipemias or disorders of apoprotein composition.The time
courses of specific activities of '3'I-labeled
apoB in VLDL, IDL, and LDL n a representative met-abolic study
using isopropanol are shown in Fig. 4. Al-though confirmation f
apoB pool size requires radioim-munoassay or other direct
quantitative techniques, theisopropanol method yields specific
activity and concen-trationdata suitable for metabolicstudies. The
highionic strengths in the LDL and IDL fractions made ef-ficient
precipitation of apoB more difficult; this couldbe overcome by
lowering the incubation temperatureto 4C. No difference was found
in contamination ofapoB with other apolipoproteins when
precipitates ob-tained at 25C or 4Cwere examined using DS-PAGE(data
not shown).
The above results indicate hat isopropanol is equallyas
effective as TMU for precipitatingapoBand ormeasurements of its
concentration and specific activity.Compared to TMU, isopropanol is
cheaper and moreeasily maintained in pure form; this assures more
con-sistent results in long term studies. Furthermore, iso-propanol
has two other distinct advantages over TMU:it does not interfere
with the protein measurement bythe Lowry procedure, and the apoB
precipitatedy iso-propanol is solubilized in NaOH or
electrophoresisbuffer more easi1y.lWe wish to express our
appreciation to Dr. G loria Vega forsupplying hyperlipidemic
plasma, to Dr. Julian Marsh for dis-cussions of apoB techniques,
and to Dr. Barbara Vasquez forher advice o n electrophoresis
procedures. We also thank Ms.Verna Kuwanhoyioma for help in
preparation of the manu-script.Manuscript received 26 November 1982
and in revisedform 6 May 1983.
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foridentification of subunit species of apoproteins in
serumlipoproteins. Anal. Biochem. 53: 350-364.2. Kane, J. P. T.
Sata, R. L. Hamilton, and R. . Havel.197 5. A poprotein composition
of very low density lipo-proteins of human serum.]. Clin. Invest. 5
6 1622-1634.3 . Le, N . A . ,J. S. Melish, B. C . Roach, H . N.
Ginsberg, andW. V. Brown. 1 97 8. Direct measurement of apoproteinB
spec ific activity in '251-labeled ipoproteins.]. Lipid Res.19: 5 7
8 -5 8 4 .4. Holmquist, L. , and K Carlson. 19 77 . Selective
extractionof human serum very low density apolipoproteins
withorganic solvents. Bwchim. Biophys. Acta. 493: 4 0 0 -4 0 9 .5.
Holmquist, L., K . Carlson, and L. A. Carlson. 1978 . Com-parison
between the use of isopropanol and tetramethy-Iura for the
solubilization and quantitation of human
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serum very low density apolipoproteins.An al. Biochem. 88:6.
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Egusa et al. Determination of apoB specific activity 1267
