CLIN. CHEM.27/4, 543-548 (1981)
QuantitativeDeterminationof ApolipoproteinsC-I and C-Il in HumanPlasma
by Separate Electroimmunoassays
Michael D. Curry,1 Walter J. McConathy,
Jim D. Fesmire, and Petar Alaupovic2
Separate electroimmunoassays are described for measuring human plasma apolipoproteins C-I and C-Il. Purified
apolipoproteins C-I and C-Il were used in preparing
monospecific antisera and as the primary standards. These
assays are sensitive (maximal sensitivity, 20 ng), specific,
rapid, precise (the within- and between-assay coefficients
of variation for both assays were 5 and 8%, respectively),
and accurate (accuracy was based on comparison of
calculated and measured C-I, C-Il, and C-Ill contents of an
ApoC-containing column-eluent fraction) and are applicable to measurement of C-I and C-Il polypeptides in whole
plasma and density classes. However, plasma samples
with triglyceride (triacylglycerol) concentrations >6000
mg/L must be delipidized before analysis for C-Il, as must
those with >12 000 mg/L before analysis for C-I polypeptide. Mean concentrations (and SD) of C-I in plasma of
normolipidemic subjects and hyperlipoproteinemic phenotypes Ila, lIb, IV, and V were 60 (15), 70 (20), 100(20),
100 (20), and 260 (94) mg/L, respectively. The corresponding C-Il values were 40(20), 43 (20), 68(20), 65 (20),
and 210 (70), respectively. C-I and C-Il concentrations in
patients with phenotypes lib, IV, or V significantly (p <
0.001) exceeded those in normal persons or phenotype
ha. The observed correlations (r = 0.92 and r = 0.94)
between triglyceride and C-I and C-Il values suggest that
these two polypeptides, like C-Ill, are excellent plasma
markers for assessing the state of triglyceride metabolism.
AddItIonal Keyphrases:
lipoproteins
.
hyperlipoprotein.
triglyceride
transport and
emia
reference intervals
metabolism
The discovery and isolation of an apolipoprotein
C-phospholipid complex from human plasma VLDL3 provided the
initial evidence for the participation of ApoC in the transport
and metabolism of triglyceride (1-3). It is now generally accepted that ApoC or its C-I, C-Il, and C-Ill polypeptides
play
an important
role in the catabolism
of triglyceride-rich
lipoproteins
(4). Various studies have demonstrated
the specific
effects of C-I, C-lI, and C-Ill on lipoprotein
lipase (EC
Laboratory of Lipid and Lipoprotein Studies, Oklahoma Medical
Research Foundation and Department of Biochemistry
and Molecular
Biology, University of Oklahoma Health Sciences Center, Oklahoma
City, OK 73104.
‘Department
of Pathology, University of Colorado Medical Center,
Denver, CO.
2Address correspondence to P. Alaupovic, Ph.D., Head, Laboratory
of Lipid and Lipoprotein
Studies, Oklahoma Medical Research
Foundation, 825 N.E. 13th St., Oklahoma City, OK 73104.
‘ Nonstandard
abbreviations
used: A-I and A-Il, polypeptides of
apolipoprotein A; ApoC, apolipoprotein C, consisting of C-I, C-Il, and
C-Ill polypeptides; ApoD, apolipoprotein
D; ApoE, apolipoprotein
E; LP-B, lipoprotein B, characterized by apolipoprotein
B; VLDL,
very-low-density lipoproteins; LDL, low-density lipoproteins; HDL,
high-density
phoresis.
lipoproteins;
and
PAGE,
polyacrylamide
Received Nov. 10, 1980; accepted Jan. 9, 1981.
gel electro-
3.1.1.34) activity,
whereas C-Il activates
hydrolysis
of triglyceride, and the C-I and C-Ill polypeptides
and a deficiency
of C-Il inhibit the reaction (5-7).
The role(s) played by C-I and C-Il in normal or deranged
lipid metabolism
cannot
be ascertained
without
specific,
sensitive,
precise, and accurate
assays that are applicable
to
both plasma and isolated lipoproteins.
Unfortunately,
no
method currently available for C-I quantification
satisfies
these criteria, and the only satisfactory method described for
measurement of C-I! is a radioimmunoassay
(8). The purpose
of this study was to develop separate electroimmunoassays
for quantification
of C-I and C-Il and to determine the concentrations
of these
lipoproteinemic
two polypeptides
in normal
and hyper-
plasma.
Materials and Methods
Plasma donors. Plasma samples used in this study were
obtained from fasting men and women. Donors were classified
as either normolipidemic
or hyperlipoproteinemic
according
to the procedures recommended
by the Lipid Research Clinics
(9). The hyperlipoproteinemic
subjects were subdivided into
various phenotypes on the basis of criteria outlined by the
Lipid Research
Clinics (9).
Isolation
of lipoprotein
density
fractions.
Lipoprotein
density fractions were isolated from fresh plasma by preparative ultracentrifugation
as previously
described
(10); however, the fractions
were not subjected
to repeated
ultracentrifugations,
so as to avoid unnecessary
losses of apolipoproteins. Pooled plasma was the source of the larger volumes used
for the isolation of ApoC polypeptides.
Isolation of C-I and C-Il polypeptides
and preparation
of
antisera.
Purified
C-I and C-Il were isolated
from both
apoVLDL and apoHDL. The ultracentrifugally
isolated
VLDL and HDL were delipidized with chloroform/methanol
as previously described (11). ApoVLDL was solubilized in 0.1
mol/L (NH4)2CO3 (12), and the soluble fraction was lyophilized, solubilized in 2 mol/L acetic acid, and applied to a
Sephadex G-50 column (110 X 2.5 cm) equilibrated
with 2
mol/L acetic acid. Eluted fractions were monitored at 280 nm,
and those containing ApoC were collected, pooled, and lyophilized. The lyophilisate was dissolved in 2 mol/L acetic acid
and rechromatographed
under the same conditions. ApoHDL
was fractionated on Sephadex G-100 (150 X 5 cm) as previously described (11). The ApoC-containing
fraction from
HDL was chromatographed
on the Sephadex G-50 column to
remove the A-Il dimer. The C-I, C-Il, and C-III-l, and C-II1-2
polypeptides were separated by column chromatography
on
DEAE-cellulose at 6 #{176}C,
in a linear gradient from zero to 0.08
mol/L NaCI in phosphate buffer (1 mmol/L, pH 8.0) containing 6 mol of de-ionized urea per liter. The fractions eluted
from the 30 X 1.5 cm column were monitored by basic and
acidic polyacrylamide
gel electrophoresis
(PAGE) (11). The
unretained
fractions,
eluted
with a NaC1-free
gradient
and
having a single fast-moving band on acidic PAGE characteristic
of C-I, were pooled and designated as the C-I fraction. Fractions eluted with the salt gradient and displaying on basic
PAGE a band with the mobility characteristic
of C-Il were
pooled and designated as the C-Il fraction. These C-I and C-Il
CLINICAL CHEMISTRY,
Vol. 27, No. 4, 1981
543
pools reacted only with their respective
antisera
and their
amino acid compositions
were consistent with those previously
reported
(13, 14).
To increase the immunogenicity
of C-I, we coupled this
polypeptide
to the appropriate
albumin
of the recipient
species by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
HC1, as described by Likhite and Sehon (15). C-I was coupled
to the appropriate
albumin in a weight ratio of 1:5. After the
cross-linking
reaction, the reaction mixture was dialyzed exhaustively
against distilled
water, then lyophilized.
Such a
procedure
was not necessary
for the enhancement
of C-Il
immunogenicity.
Antisera
were prepared
by injecting
New Zealand White
rabbits or Karakul sheep intraperitoneally
with 0.5 mg of the
coupled
C-I or C-Il dispersed
in 1.0 mL of 0.1 mol/L
(NH4)2C03
and an equal volume of Freund’s complete adjuvant. The animals were injected at weekly intervals;
a sufficient antibody
titer was usually obtained
after the fourth injection. Blood was sampled weekly, by heart puncture
from
rabbits and by venipuncture
of the jugular vein of sheep.
The antisera
and antigens
were tested for purity and
specificity
by immunoelectrophoresis
(16) and double diffusion (17) with whole serum, purified
apolipoproteins,
and
specific antisera,
respectively.
Isolation
of A-I, A-I!, C-Ill,
ApoD, ApoE, and LP-B, and preparation
of their corresponding
antisera have been described
previously
(18-21).
Chemical analysis and delipidization.
The protein content
of C-I and C-Il standards
and samples used to assess accuracy
of the electroimmunoassay
was estimated
from amino acid
analyses of 24- and 72-h acid hydrolysates
(22). Triglyceride
and total chloesterol
were determined
as described previously
(9). Plasma was delipidized
with n-butanol/isopropyl
ether
(23), n-heptane
(24), or 1,1,3,3-tetramethylurea
(25).
Polyacrylamide
gel elect rophoresis.
Separation
of
1 ,1,3,3-tetramethylurea-soluble
apolipoproteins
by acidic
PAGE (11) and subsequent
densitometric
scanning of the gels
were performed
essentially
as described
by Kane (25). A
standard
curve was constructed
with purified C-I for quantitative analyses.
Elect roimmunoassays
for C-I and C-Il. Assay conditions
were selected
to yield optimal
immunoprecipitation.
Presumably because the physical-chemical
properties
of C-I and
C-Il are similar, the corresponding
assay conditions
for these
two polypeptides
were essentially
identical.
The supporting
medium
for both assays was prepared
by melting
25 g of
agarose (“Indubiose”
HAA45; Accurate Chemical & Scientific
Corp., Westbury,
NY 11590) per liter of electrophoresis
buffer
on a boiling water bath, with continuous
stirring. We added
50 g of Dextran T-l0 (Pharmacia
Fine Chemicals, Piscataway,
NJ 08850) per liter to the agarose solution and dissolved it by
continual
heating. The agaropectin
content of various lots of
commercial
agarose varied. For optimal
immunoprecipitin
lines with some preparations,
4 g of special agar-Noble
(Difco
Laboratory,
Detroit, MI 48232) had to be added to the agarose-dextran
mixture.
Monospecific
antiserum
to either C-I
or C-Il was mixed with 25 mL of the agarose solution when it
had cooled to 55 #{176}C.
Usually, 1.0 mL of antiserum
sufficed.
This mixture was poured into a mold constructed
as previously described
(20). Alternatively,
antiserum
was conserved
by keeping free of antiserum
the areas of the agarose plate that
normally
are in contact
with sponge wicks. With this arrangement,
about the middle two-thirds
of the plate contained
antibodies.
The agarose gels containing antibodies
were stored
in a humid chamber at 4 #{176}C
overnight
to ensure gel stability.
Eighteen
sample wells, 4 mm in diameter,
were punched out,
with center-to-center
distances
of 10mm, and 10 L of standard or sample that had been diluted with electrophoresis
buffer were delivered
to the sample well with a Drummond
microdispenser.
Alternatively,
a plate was used containing
35
544
CLINICAL CHEMISTRY,
Vol. 27, No. 4, 1981
sample wells, 2.5 mm in diameter
with center-to-center
distance of 5 mm, accommodating
5-iL sample volumes. Solutions of 1,1,3,3-tetramethylurea
(Burdick
& Jackson
Laboratories,
Inc., Muskegon,
MI 49442) were also evaluated
as
diluent. A normolipidemic
plasma usually required a four- to
eightfold dilution. The electrophoresis
buffer for both assays
contained,
per liter, 25 mmol of barbital
and 0.2 mol of
tris(hydroxymethyl)methylamine
(Tris). The pH and conductivity of the buffer were 8.5 and 3.0 mQ’,
respectively.
A
field strength
of 5.5 V cm1 was applied to the electroimmunoassay
plate for 5.5 h; the current
developed
with these
conditions
was 80 to 90 mA. Water circulating
through
the
electrophoresis
platform
was maintained
at 15 #{176}C
with a
Lauda K-2/RD
refrigerated
circulator
(Brinkmann
Instruments, Inc., Westbury,
NY 11590). Immunoprecipitates
were
stained
as previously
described
(20). We measured
rocket
height to the nearest 0.2 mm, from the center of a sample well
to the rocket apex, and the width at one-half this height.
Plasma
references
of known C-I and C-Il content
were
prepared
and stored as previously
described
(21). After the
standard
curves were obtained with use of either C-I and C-Il
polypeptides,
subsequent
analyses were carried out with these
plasma references.
Use of references
prepared
at different
times and periodically
restandardized
with purified
polypeptides
allowed a check of their stability.
Statistical
analyses.
The statistical
analyses
were performed by analyses
of variance
and, when more than two
means were involved, by. Duncan’s multiple-range
test (26).
Results
Standardization of the C-I and C-Il
Electroimmunoassays
Immunochemically
pure C-I and C-Il preparations
of
known protein content as determined
by amino acid analysis,
dissolved in electrophoresis
buffer containing
urea (8 mol/L),
were assayed and the data used to construct
standard
curves.
The construction
of standard
curves for C-I and C-I! was
based on measurement
of rocket areas rather than height,
because the widths at half height became greater with increasing
antigen
concentration.
Rocket
area and protein
content of C-I standards
were linearly related between 12 and
56 mm2 (r = 0.98; y = 12.5x + 8.3). Similarly,
the C-I! standard curve was linear between 15 and 65 mm2 (r = 0.97; y =
31.2x - 1.7). Between these limits, serially diluted plasma or
lipoprotein
fractions
had essentially
the same slopes as C-I
or C-Il standards.
Evaluation of the Electroimmunoassays
C-I and C-Il in intact and delipidized
plasma.
Delipidization of plasma may allow C-I or C-Il previously
associated
with lipoproteins
of larger size to enter the agarose matrix,
induce self association
(27), denature
antibody reaction sites,
or expose antigenic sites masked by lipid. Each of these effects
may influence the accuracy of the electroimmunoassays.
To
ascertain
the effects of delipidization,
we assayed plasma in
the intact lipoprotein
form and after delipidization
with
1,1 ,3,3-tetramethylurea,
a -heptane,
or n -butanol/isopropyl
ether.
Extraction
with heptane
increased
the measured
amount of both C-I and C-Il in hypertriglyceridemic
plasma
which contained
lipoproteins
of S1 >400, but this procedure
was only partially
effective,
because the highest concentrations of C-I and C-Il were observed after delipidization
with
n-butanol/isopropyl
ether. Apparently
both extraction
procedures allowed C-I and C-Il in the larger lipoproteins
to
migrate
into the agarose matrix.
Like heptane,
1,1,3,3-tetramethylurea
was only partially effective, yielding values for
C-I and C-Il intermediate
to those observed
with intact
plasma and plasma delipidized
by n-butanol/isopropyl
ether.
Table 1. ConcentratIons (mg/L) of C-I and C-Il In Intact and Delipidlzed Plasma Samplesa
Apollpoprot.lns
cholesterol
I
Triglyceride
1580
1970
2550
4470
5690
6660
7000
12260
35960
38650
67380
90500
2130
3460
2640
1840
2950
2430
1860
2020
2870
4500
9300
8400
a Intact (I) and deilpidlzed (0).
after delipidlzatlon.
Because
‘
64
126
100
82
93
113
110
110
0
35
59
60
48
54
47
25
32
c-tic-Il
1.8
d
134b
d
77b,c
105b
d
252b
350b
d
d
210b
263b
2.1
1.7
1.7
1.7
1.4
1.6
1.4
1.3
1.2
1.3
310b
d
250b
1.2
d
d
signIficantly ‘eater
ratio,
I
D
no
increase
than intact lipoprotein levels (p <0.001).
c
no
increase
j
80b,c
70bc
ratios calculated by use of C-Iand C-Il values determined
Weit
Llpoproteins partIally mIgrated into agarose matrIx.
the latter
appeared
to be the best delipidization
procedure,
we studied its effect on the quantification
of C-I
and C-I! in several plasma samples with a wide range of triglyceride concentrations.
Our results
(Table 1) indicate
that plasma need not be
delipidized
for accurate quantification
of C-I until triglyceride
concentrations approach 12000 mgfL and, in the case of C-il,
until triglyceride
concentrations
approach 6000 mg/L. On the
basis of data presented
in Table 1, the correlation
(r) of plasma
C-I and C-I! concentrations
with triglyceride
concentrations
was 0.92 and 0.94, respectively. The close relationship between
C-I and C-I! (r = 0.99) was evident up to triglyceride
concentrations
of 6000 mg/L. However,
as plasma triglyceride
concentrations
increased,
the relative increase in C-Il with
respect to C-I was greater. This was observed as a decrease in
the C-I/C-Il weight ratio from about 2:1 to only slightly greater
than 1:1 (Table 1).
Sensitivity
and precision
of the elect roimmunoassays
for
C-I and C-Il. The maximal sensitivity of both the C-I and C-I!
assays was approximately
20 ng per applied
sample;
the
smallest amount that we could accurately
quantify was 50 ng
per sample. The precision
of the C-IT and C-I electroimmunoassays was established
by analyzing
10 plasma samples in
duplicate
every other day for two weeks. The within- and
between-assay
coefficients
5 and 8%, respectively.
of variation
Accuracy of electroimmunoassay
and C-Il. To assess the accuracy
for both assays were
for quantification
of C-I
of the C-I electroimmu-
Table 2. C-I as Measured in Seven Samples of
HDL by Electroimmunoassay
and Polyacrylamide
Gel Electrophoresis
Llpoprotelns,
d 1.063-1.2
EI.ctrolmmunoassay
1 kg/I
PAGE
Concn., mg/L
44
52
56
60
14
18
17
34 (SD 17)
a
W.lght
c-Il
c-I
Total
Not significantly dIfferent from
60
53
43
46
8
25
20
39.5 (SD 20)
pos
results (p >0.10).
noassay, we isolated the HDL fraction,
recentrifuged
until
albumin was removed, and analyzed for C-I by both electroimmunoassay
and the PAGE procedure.
Results obtained
by
electroimmunoassay
were very similar to those estimated
by
densitometric
scanning
of the C-I band resolved
by PAGE
(Table 2).
An alternative
method for assessing the accuracy of the C-I
and C-Il electroimmunoassays
was based on fractions isolated
by gel permeation
chromatography
of delipidized
VLDL. The
fractions were composed predominantly
of C-I, C-il, and C-rn
polypeptides;
less than 3% of the total protein was due to A-il,
ApoD, and ApoE. Because the primary sequences of C-I, C-TI,
and C-Ill are known, the molar concentration
of each poly-
peptide in the “ApoC” fraction could be calculated from the
amino acid analyses. The calculated values for C-I, C-I!, and
C-Ill were compared
with results obtained
by electroimmunoassay (Table 3). Briefly, the number of moles of C-Il! was
calculated
on the basis of their unique histidine
content,
as
previously
described
(28). Because C-I contains
no tyrosine
(29) and the tyrosine contributed
by C-Ill is known, the remaining tyrosine was considered
to represent the C-I! content.
Values for moles of C-Il were obtained
by dividing by five,
because one mole of C-I! contains
five residues
of tyrosine
(30). The calculated values for C-il (74 ± 18 nmol) agreed well
with those measured
by electroimmunoassay
(80 ± 5 nmol).
The calculated values for C-TI and C-Ill were subtracted
from
the total protein content as estimated
from the amino acid
analysis. The difference,
accounting
for the C-! content (104
+ 13 nmol), agreed reasonably
well with that estimated
by
electroimmunoassay
(119 ± 51 nmol).
Quantification
of C-I and C-Il in plasma and lipoprotein
density fractions
by elect roimmunoassay.
Results of quan-
Table 3. C-I, C-Il, and C-Ill In ApoC Fraction as
Determined from Amino Acid Composition and by
Electroimmunoassay (n =
Calcd. from
amino
Apoilpoprotein
Eiectrolmmunoasaay
acid
comp. b
nmol (and SD)
119(51)
80 (5)
216(20)
C-I
C-Il
C-Ill
a Samples
represent
delipidlzed VLDL.
b
the ApoC
104(13)
74(18)
195 (7)
fractIon Isolated by gel chromatoaphy
of
See text.
CLINICAL
CHEMISTRY,
Vol. 27, No. 4, 1981
545
Table 4. Concentrations of C-I and C-Il in Plasma with Normal and Above-Normal Cholesterol and
Triglyceride Concentrations
W.Ight
Cholesterol
2170(360)
Normal plasma
(n
=
68)
=
35)
(n
IV
=
32)
(n
=
52)
=
4)
Ila
(n
llb
V
(n
a
980(390)
c-wc-iu
60(15)
40(20)
1.5
70(20)
43 (20)
1.6
1180(300)
2920 (240)
2340(440)
100 (20)
68 (20)
1.5
2280 (360)
2830 (770)
100 (20)
65(20)
1.5
6270 (3070)
58120(25850)
260 (94)
210 (70)
1.2
SignIfIcantlygreater than normal (p <0.00 1).
spectively). On the other hand, all hypertriglyceridemic
patients had C-I and C-IT values significantly greater than normal (p <0.001). Among the hypertriglyceridemic
patients,
those characterized
by phenotype
V had the highest C-I
C-lI values (p <0.001). However, in comparison
with
molipidemic
subjects or patients with phenotypes
Ha, fib,
IV, the C-I/C-!! weight ratio for patients with phenotype
was decreased
(Table 4).
Table 5 shows the percent distributions
and absolute
and
norand
V
con-
centrations of C-! and C-!! in the major lipoprotein density
fractions of normal plasma. All density fractions contained
C-I and C-lI polypeptides.
The major portions of both C-I and
C-IT were present in HDL, with a molar ratio of 2.1. Interestingly, lipoproteins
with d <1.019 kgfL and 1.019-1.063 kg/L
contained
C-I and C-IT in proportions
different
from each
other in plasma and HDL. Specifically, lipoproteins
of d
<1.019 kg/L had a low C-I/C-TI molar ratio (0.6), demonstrating their relatively greater concentration
of C-!!. Conversely, lipoproteins of d 1.019-1.063
kg/L had a higher CI/C-I! molar ratio (3.0) than is true of plasma or HDL.
Discussion
Because C-! lacks tyrosine residues (29) and is difficult to
radiolabel, electroimmunoassay
is a particularly
attractive
method for quantification of this apolipoprotein.
A significant
amount of both C-! and C-IT is present on lipoprotein particles
of large
diameters
in patients
with
moderate
to severe
hy-
Table 5. DIstribution of C-I and C-il among
Lipoprotein Density Fractions from Normal
Plasma (n = 10)
Reiatlve density
fraction, kg/I
Molar a ratio,
c-i
concn.,
______________________
mg/I
c-iic-u
c-il
(SD and
% of total)
(plasma)
d <1.019
1.019-1.063
1.063-
1.2
1
d>1.21
a
c-ti
3050(260)
titative determination
of C-! and C-IT in normolipidemic
and
hyperlipoproteinemic
plasma
are presented
in Table 4.
Plasma
from normolipidemic
and hypercholesterolemic
(phenotype
lIa) subjects had comparable C-I (60 and 70 mg/L,
respectively) and C-I! concentrations
(40 and 43 mg/L, re-
-
ratio,
c-i
Triglycedde
Concn., mg/L (and SD)
65 (15)
7 (1.5) (11)
9(1) (15)
40 (7) (65)
6 (2) (9)
0.6
(58)
2.1
25 (9)
2.0
3.0
b
<5 mg/L.
_______________________________________________
546 CLiNICALCHEMISTRY,
concentrations
>12 000 mg/L. The greatest increases in apparent C-! and C-TI concentrations
after delipidization
were
recorded in plasma samples from severely hypertriglyceridemic patients of phenotype V. The larger particles probably
fail to enter the agarose gel; however, the increased values for
C-! and C-I! after delipidization
may be due in part to normally unexposed
antigenic sites. If true, this explanation
may
pertain especially
to C-!!, because in some instances
hypertriglyceridemic plasma samples, which apparently entered the
agarose
gel without difficulty, had greater C-TI measured after
delipidization;
however, the values for C-I were not affected.
There are no reports in the literature
on the plasma C-I
concentrations
for us to compare with our results. However,
our results showing that C-I accounts
in normolipidemic
subjects for 3% of the apolipoprotein
content of lipoproteins
with d <1.019 kg/L is in agreement
with results obtained
by
PAGE (31). In addition,
the C-I values in HDL as measured
in this study by PAGE and by electroimmunoassay
were very
similar,
if not identical.
Reported
results on plasma C-IT
concentrations
as determined
by radioimmunoassay
are in
excellent
agreement
with those estimated
by electroimmunoassay for both normolipidemic
and hyperlipoproteinemic
plasma samples as well as lipoprotein
fractions
(32,33). The
C-TI content of lipoproteins
with d <1.019 kg/L from normolipidemic
subjects
was 8% of the total apolipoprotein
content,
a value similar to reported
results based on C-I!
measurement
by PAGE or isoelectric focusing procedures
(31,
34). Based on our comparison
of data obtained
by PAGE,
amino acid analyses, and reports from the literature, we be-
lieve electroimmunoassay
to be an accurate method for
of both C-I and C-Il in delipidized
and intact
plasma, VLDL, and HDL. However, to ensure accuracy,
plasma samples with triglyceride concentrations greater than
approximately 6000 mg/L must be delipidized before analysis
for C-I!, as must those with triglycerides exceeding 12 000
quantification
mg/L
43 (15)
14 (4) (33)
4 (3) (9)
Relative molecular masses: C-I. 6631; C-lI. 8837(29, 30).
amounts
was an increase in the concentration
of plasma samples with triglyceride
concentrations
exceeding 6000 mg/L. A similar increase in
apparent C-I was observed in plasma samples with triglyceride
pertriglyceridemia.
There
of C-TI after delipidization
Vol. 27, No. 4. 1981
I)
Not quantIfIed;
for C-I.
In plasma
of normolipidemic
subjects,
the C-I and C-Il
are mainly confined to HDL. Concentrations
of
C-I, C-!!, and C-Ill in plasma are increased significantly
in
polypeptides
phenotypes
lIb, IV, and V. This increase in ApoC peptides
occurs in VLDL or LDL, or both; except for patients with
phenotype
V, the relative contributions
of ApoC polypeptides
in HDL remains within the normal range (28, and unpublished results). Because values for C-I and C-Il in plasma were
normal
in hypercholesterolemic
(phenotype
ITa) patients,
we
conclude that the abnormally elevated ApoC is most probably
ascribable to defective triglyceride metabolism. A previous
report showed that plasma samples from patients
of phenotype lIb and IV were characterized
by a disproportionate
increase in C-Ill concentration,
and results from this study indicated a variability
in the relative proportions
of C-I and C-I!
in major lipoprotein
density
classes (35, 36). Our results
confirm the qualitative
changes observed
for C-!! in VLDL
by others (37, 38) and establish
the normal quantitative
relationships
for C-I/C-!! in VLDL (0.6), LDL (3.0), and HDL
(2.1). Not surprisingly,
both C-I and C-TI, like C-Ill, are useful
for assessing the efficiency of triglyceride catabolism (28).
Furthermore,
in hypertriglyceridemia,
the relative amounts
of C-I, C-Il, and C-Ill are useful for detecting and identifying
specific abnormal
lipoprotein
species. The low C-I/C-Il
ratio
observed for phenotype
V plasma probably reflects the greater
proportion of lipoproteins of d <1.019 kg/L. The characteristically normal ratio of C-I/C-Il
in phenotypes
lIb and IV
patients
may result from the presence of abnormal
triglyceride-rich lipoprotein
species in LDL, in addition
to VLDL.
Evidence is convincing (33) that lipoproteins
with the greatest
proportion
of C-Il are preferred substrates
for lipoprotein
lipase or promote the highest activation (33), or both. Possibly,
the regulation of triglyceride catabolism in vivo depends in
part
on the proportion
of ApoC peptides. The absence of C-!!
has been documented
in one family as a probable
cause of
hypertriglyceridemia
(7). Interestingly,
results from ongoing
studies in our laboratory
revealed that in lipoproteins
of d
<1.019
kgfL from familial hypertriglyceridemic
patients, C-I!
was increased
in absolute terms but not to the same proportions as C-I or C-Ill; the C-I/C-I! ratio was 0.8 (unpublished).
This probably accounts for the observation
by Kashyap et al.
(33) that hypertriacylglyceridemic
subjects had less lipoprotein lipase activator per unit of VLDL ApoC-Il. Perhaps some
hypertriglyceridemic
states result from a relative deficiency
of C-I!, due to either a regulatory
defect or merely a limited
capacity to synthesize
C-I! at a rate equivalent
to that of tri-
glyceride.
In summary, this study indicates that electroimmunoassay
is an accurate technique
for quantification
of apolipoproteins
C-! and C-!!. It may be applied to intact lipoproteins
if samples with particles of S1 >400 are delipidized before analysis.
Results suggest that, like C-Ill, both C-I and C-IT are markers
for assessing the efficiency of triglyceride catabolism. More
importantly,
data on C-I, C-TI, and C-Ill together may be
useful for differentiating
fying specific abnormal
hypertriglyceridemias
lipoprotein
species.
and identi-
This work was supported in part by USPHS Grant HL-23181 and
by the resources of the Oklahoma Medical Research Foundation. We
thank Mr. K. Miller and Mr. T. Gross for valuable technical assistance
and Mrs. M. Farmer for secretarial assistance.
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