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Lipids and Lipoproteins |
-cyclodextrin sulfate
1
Department of Central Laboratory, Kumamoto University Hospital, 1-1-1, Honjo, Kumamoto 860, Japan.
2
Faculty of Pharmaceutical Sciences, Kumamoto University,
5–1, Oe-honmachi, Kumamoto 862, Japan.
3
Department of Laboratory Medicine, Kumamoto University,
School of Medicine, 1-1-1, Honjo, Kumamoto 860, Japan.
a Author for correspondence. Fax (81) 96-362-7540;
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-cyclodextrin sulfate.
Of the surfactants tested, POE-POP with a higher molecular mass of the
POP block and a greater hydrophobicity reduced the reactivity of
cholesterol in lipoprotein fractions; the reactivity in descending
order was LDL 
VLDL > chylomicron 
HDL. Gel
filtration chromatographic studies revealed that POE-POP removed lipids
selectively from the LDL fraction and allowed them to participate in
the cholesterol esterase–cholesterol oxidase coupling reaction system.
By contrast, -cyclodextrin sulfate reduced the reactivity of
cholesterol, especially in chylomicrons and VLDL. A combination of
POE-POP with -cyclodextrin sulfate provided the required selectivity
for the determination of LDL-C in serum in the presence of magnesium
ions and a small amount of dextran sulfate without precipitating
lipoprotein aggregates. There was a good correlation between the
results of LDL-C assayed by the proposed method and the
beta-quantification reference method involving 161 sera with
triglyceride concentrations ranging from 0.3 to 22.6 mmol/L. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A wide variety of methods has been used for determining LDL-C in serum, including a precipitation-based method (3), electrophoresis (4), HPLC (5), sequential and density-gradient ultracentrifugation (6), Friedewald formula (7), and immunoseparation (8). Of these, the Friedewald formula is the most commonly used approach in clinical laboratories; however, it is limited to specimens from fasting subjects and specimens with triglyceride concentrations <4.5 mmol/L (9). Although beta-quantification by ultracentrifugation is the currently accepted reference method for measuring LDL-C in serum (10), it requires relatively large volumes of serum, long turnaround time, and is unsuitable for routine laboratory testing. Therefore, there is a great clinical need to develop a convenient and reliable method for measuring LDL-C in serum without resorting to any fractionation procedures.
Interaction of surfactants with lipoproteins has been the focus of many studies (11)(12). In general, for the determination of total cholesterol in serum, excess amounts of surfactants have been used for solubilizing all the lipoprotein cholesterol in a nonspecific manner and allowing it to freely participate in the enzymatic reaction system. By contrast, an approach for the direct measurement of LDL-C in serum has been proposed on the basis of the sequential use of two surfactants that show differential selectivities towards lipoprotein fractions (13).
Recently, we established a direct method for measuring HDL-C in serum,
with the combined use of polyethylene glycol-modified enzymes and
-cyclodextrin sulfate (14)(15). In these
studies -cyclodextrin sulfate, which has a highly concentrated
negative charge and possesses heparin-mimicking activity, was used to
reduce the reactivity of cholesterol in chylomicron and VLDL fractions
in the presence of magnesium ions, without the need for precipitation
of these lipoprotein fractions. Furthermore, our preliminary studies
have shown that a polyoxyethylene–polyoxypropylene block polyether
(POE-POP) reduced the reactivity of cholesterol, especially in HDL,
suggesting that a combination of POE-POP with -cyclodextrin sulfate
could provide the needed selectivity for the determination of LDL-C in
serum. Building upon these studies, this paper deals with the potential
application of POE-POP and -cyclodextrin sulfate in developing a
novel direct assay for LDL-C in serum without prior fractionation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Cyclodextrin was donated by Nihon Shokuhin
Kako Co. The sodium salt of -cyclodextrin sulfate was prepared
according to the nonregioselective method described previously
(16). The average degree of substitution of sulfate groups
in -cyclodextrin sulfate was confirmed to be between 12.0
± 0.5 (mean ± SD of five different lots) by fast-atom bombardment
mass spectrometry and elemental analysis. Dextran sulfate (from
dextran, with an average molecular mass of 500 kDa) and
3-(N-morpholino)propanesulfonic acid (MOPS) were obtained
from Sigma Chemical Co. and Dojin Chemical Co. respectively. Free and
conjugated bilirubin dissolved in 30 g/L bovine serum albumin solution
(ditaurobilirubin) were obtained from International Reagent Co.
Immunoseparation reagent for the determination of LDL-C and latex
agglutination reagent for the determination of lipoprotein(a) [Lp(a)]
were obtained from Sigma Diagnostics and Daiichi Pure Chemicals Co.
respectively. Human sera were obtained from healthy subjects 1 h after lunch, and the four major lipoprotein fractions [chylomicron, hydrated density (d) <0.950 kg/L; VLDL, d 0.950–1.006 kg/L; LDL, d 1.006–1.063 kg/L; and HDL, d 1.063–1.135 kg/L], isolated by ultracentrifugation according to the method of Hatch and Lees (6), were supplied by Health Care Technology Foundation. All other materials and solvents used were of analytical reagent grade.
gel filtration analysis
The separation of lipoprotein fractions was performed by HPLC
(CCPA 8000, Tosoh) with a gel filtration column (TSK gel Lipopropak XL,
Tosoh) according to the method of Kitamura et al. (17).
The individual lipoprotein fractions or serum samples were eluted with
2.6 mmol/L NaCl, pH 7.0, at a flow rate of 1.0 mL/min. The column
effluent was collected at intervals of 30 s with a fraction
collector (FRAC100, Pharmacia Biotech.). The concentrations of
cholesterol and phospholipids in the fraction separated by HPLC were
determined enzymatically (Determiner TC and PL, Kyowa Medex). The
concentration of total proteins in the fractions separated by HPLC was
determined by the micro BCA protein assay reagent kit (Pierce) and
those of apolipoproteins A-I, A-II, B, C-II, C-III, and E by
immunoturbidimetry (Daiichi Pure Chemicals Co.). There was no apparent
contamination with any other proteins such as serum albumin.
analytical procedure
In the final formulation of reagents used for the determination,
reagent 1 contained -cyclodextrin sulfate (0.5 mmol/L), dextran
sulfate (0.5 g/L), MgCl2 (2.0 mmol/L), and EMSE (1.4
mmol/L) in MOPS buffer (50 mmol/L, pH 6.75); reagent 2 consisted of
CHER (1 kU/L), CHOD (3 kU/L), peroxidase (30 kU/L), 4-AA (2.5 mmol/L),
and POE-POP (4 g/L) in MOPS buffer (50 mmol/L, pH 6.75).
All the specimens were stored at 4 °C and analyzed within 2 days after blood sampling. Four microliters of the serum sample was added to 300 µL of reagent 1. The mixture was incubated at 37 °C for 5 min before adding 100 µL of reagent 2 and incubated for an additional 5 min. The chromophore formed in a coupled reaction with peroxidase was measured spectrophotometrically at 600 nm; alternatively, dual wavelength measurements [600 nm (main) and 700 nm (subsidiary)] were used, especially when assaying turbid or hemolyzed samples, to avoid any interference with the colorimetric assay. The concentration of LDL-C was calculated by using a serum-based calibrator (LDL-C, 2.77 mmol/L, Precinorm L; Boehringer Mannheim). The instruments used for the determination were a Hitachi 911 automated analyzer and a UV 160A spectrophotometer (Shimadzu).
The results of LDL-C in serum assayed by beta-quantification method were obtained by Health Care Technology Foundation, performed according to the Lipid Research Clinic's protocol (18). The immunoseparation reagent for the determination of LDL-C was used to remove HDL and VLDL from serum, leaving LDL in the filtrate, and was quantified with a standard cholesterol assay (8). The concentration of triglycerides in serum was determined enzymatically (Determiner TG, Kyowa Medex) and the concentration of HDL-C in serum was determined by a dextran sulfate–phosphotungstate–MgCl2 precipitation method (Daiichi Pure Chemicals Co.) respectively.
data analyses
The contribution of various structural and physicochemical factors
of surfactants to the selectivity towards LDL-C was statistically
examined with multiple regression analysis (19).
Correlations and regression equations were calculated on a PC-9801UF
personal computer (NEC), where a stepwise method was used and the
validity of regression was judged by the F-test value
(>2.0). The data were analyzed statistically by one-way analysis of
variance, with Duncan's multiple comparison test, and P
values <0.01 were viewed as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The isolated lipoprotein fractions had the expected
compositions, which are in good agreement with those of other
compositional studies (20). A total of 381 surfactants (24
cationic, 77 anionic, 249 nonionic, 6 amphoteric, and 25 other types)
were surveyed with regard to the selectivity towards LDL-C. Fig. 1
shows the selectivity indices of surfactants (2 g/L) for LDL-C
(SILDL) in the presence of CHER (1 kU/L) and
CHOD (3 kU/L). The SILDL value is defined here
as the product of the relative reactivity of cholesterol in the LDL
fraction (the first term) and the LDL selectivity (the second term)
according to the equation:
 |
 | (1) |
). In particular, a series of POE-POP, as indicated by open circles in
Fig. 1
, were most superior with respect to LDL selectivity.
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POE-POP is an A-B-A-type triblock copolyether consisting of hydrophilic
polyoxyethylene A blocks attached to a hydrophobic central
polyoxypropylene B block. In this study we used 19 POE-POP with an
average molecular mass ranging from 1100 to 6500 Da. The relative
contribution of structural factors of the POE-POP to the selectivity
towards LDL-C was examined by using multiple regression analysis. In an
initial multiple regression equation, the variable criterion was the
SILDL value, and the explanatory variables were
(a) the average molecular mass of POE-POP, (b)
the molecular mass of the POP block in the POE-POP molecule
(MMPOP), and (c) the hydrophobicity
index for POE-POP as indicated by the fractional molecular mass of the
POP block in the POE-POP molecule (FMPOP),
respectively. When a stepwise method was used and the validity of
regression was judged by the F-test value (>2.0), the first
dependent variable was excluded from the equation and consequently the
following equation was obtained:
 |
 | (2) |
 |
. The partial correlation coefficients for the
MMPOP and FMPOP were
0.548 and 0.535, respectively. Because the magnitudes of the two
coefficients are not significantly different, the two determinants may
contribute equally to the selectivity of POE-POP towards LDL-C. On the
basis of the above results, POE-POP with a MMPOP
value of 3850 Da and a FMPOP value of 90%,
giving a SILDL value of 9.24, was selected as
the most preferred reagent for the determination of LDL-C and used in
the following studies.
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Figure 3
shows the relative reactivity of cholesterol in lipoprotein
fractions as a function of POE-POP concentrations in the presence of
CHER (1 kU/L) and CHOD (3 kU/L). The relative reactivity of cholesterol
in the lipoprotein fractions decreased with increasing concentrations
of POE-POP, with the reactivity decreasing in the order: LDL
VLDL > chylomicron HDL.
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effects of poe-pop on gel filtration of lipoprotein fractions
Insight into the mechanism by which POE-POP exhibits its
selectivity towards LDL-C was gained by gel filtration chromatography.
Fig. 4
shows the effect of POE-POP (4 g/L) on the elution pattern for
the LDL fraction, as obtained by detecting cholesterol, phospholipids,
and proteins in the effluent. In the presence of POE-POP, the first
peak at ~30 min contained predominantly apolipoprotein (apo) B, as
determined by immunoturbidimetry, and cholesterol and phospholipids
eluted later in a separate peak (Fig. 4B
). The lipids isolated from the
LDL particles would be solubilized in the mixed micelles with POE-POP,
making them readily amenable to enzymatic reactions with CHER and CHOD.
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By contrast, when POE-POP at the same concentration as in Fig. 4
was
added to the HDL fraction, it reduced the elution volume of the HDL
fraction, with only a small change in the protein/lipid composition
(Fig. 5
). In fact, the protein fraction shown in Fig. 5B
contained apo
A-I and apo A-II at a molar ratio of 5:1, which is consistent with the
expected protein concentrations for the intact HDL fraction. This
suggests that the POE-POP varies the density or surface charge of the
HDL particles and (or) induces the aggregation of HDL to
macroparticles. If such is the case, the modified HDL particles should
be resistant to the enzymatic reactions, eventually leading to reduced
reactivity of cholesterol in that lipoprotein fraction. In the case of
chylomicron and VLDL, the addition of POE-POP to those lipoprotein
fractions caused no remarkable separation of proteins and lipids (data
not shown). The above results clearly indicate that the
POE-POP-mediated selectivity towards LDL-C can be ascribable to
selective solubilization of cholesterol in the LDL fraction, thus
allowing it to participate in the enzymatic reactions.
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combination effects of poe-pop with -cyclodextrin
sulfate
The reactivity of cholesterol in the VLDL fraction persisted with
the sole use of POE-POP at concentrations up to 4 g/L, as shown in Fig. 3
. For accomplishing the direct determination of LDL-C, the
reactivities of cholesterol in all the lipoprotein fractions except for
LDL must be completely abolished. For this purpose, we used
-cyclodextrin sulfate in combination with POE-POP. Our previous
studies have shown that -cyclodextrin sulfate, having a highly
concentrated negative charge and possessing heparin-like activity,
reduces the reactivity of cholesterol, especially in chylomicrons and
VLDL in the presence of magnesium ions, without precipitating those
lipoprotein aggregates. Recent studies have demonstrated that
cyclodextrin sulfates exhibit threshold behavior in their
heparin-mimicking activities; the introduction of ~2 sulfate groups
per glucose unit is adequate (21). Therefore, in this
study we prepared -cyclodextrin sulfate with a degree of sulfation
of ~12, which corresponds to an average substitution of ~2 hydroxyl
groups per glucose unit. Fig. 6
shows the relative reactivity of cholesterol in lipoprotein
fractions as a function of magnesium ions in the presence of the
POE-POP (4 g/L), -cyclodextrin sulfate (0.5 mmol/L), dextran sulfate
(0.5 g/L), CHER (1 kU/L), and CHOD (3 kU/L). The relative reactivity of
cholesterol in the lipoprotein fractions decreased with increasing
concentrations of POE-POP, with reactivity decreasing in the order: LDL
VLDL > chylomicron HDL. Finally, the residual
reactivities with fractions other than LDL were completely abolished by
adding magnesium ions at concentrations >2 mmol/L.
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time course of enzymatic reaction
Figure 7
shows changes with time in absorbance at 550 nm during the
reaction of LDL-C, as determined at intervals of 20 s for 10 min,
with the four lipoprotein fractions and the serum samples (diluted five
times serially with 155 mmol/L NaCl solution) from a patient with
hyperlipemia (total cholesterol 11.12 mmol/L, triglycerides 2.82
mmol/L, HDL-C 0.78 mmol/L). Only LDL-C from both the lipoprotein
fractions and the serum sample exhibited reactivity, reaching a plateau
within ~5 min after the addition of reagent 2.
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calibration curve and linearity
When calibration for the LDL-C assay was performed with control
serum Precinorm L (LDL-C, 2.77 mmol/L, Boehringer Mannheim) for lipid
determination, a straight line passing through the origin was obtained,
and the calibration was stable during the 1-month period, with
absorbance readings varying within 1%. The LDL fraction (LDL-C, 15.5
mmol/L) isolated by ultracentrifugation was diluted with 155 mmol/L
NaCl solution, and linearity of the method was investigated. As shown
in Fig. 8
, good linearity was obtained with the serially diluted
fractions of LDL-C solutions up to 15.5 mmol/L. The minimum detectable
concentration was 0.005 mmol/L. When serum samples from patients with
hyperlipemia (triglycerides 2.3–18.1 mmol/L, total cholesterol
5.2–15.5 mmol/L) were serially diluted with 155 mmol/L NaCl solution
and used for assessment of the linearity, good linearity was obtained,
suggesting that the influence of cholesterol derived from VLDL and
chylomicrons in sera of patients with hyperlipemia was negligible or
nonexistent.
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imprecision and recovery tests
Repeatability and reproducibility of the method with three
different serum samples are shown in Table 2
. For run-to-run precision studies the specimens were stored at
-80 °C and the proposed method indeed worked with the frozen sera.
In all three cases, the values obtained were within 4% of the target
values as recommended by the National Cholesterol Education Program
(NCEP) (22). For the recovery test, 100 µL of the LDL
fraction (LDL-C, 5.82 mmol/L) isolated by ultracentrifugation was added
to 400 µL of the pooled serum (LDL-C, 1.34, 2.72, and 3.54 mmol/L)
and the assay was performed with the reagent. The mean ± SD
recovery was 101.1% ± 3.8% with nine specimens, and ranged between
97% and 105%.
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interfering substances
Addition of conjugated bilirubin at final concentrations up to
0.68 mmol/L (0.14, 0.27, 0.41, 0.55, and 0.68 mmol/L) to the pooled
serum produced a slightly negative error of up to 10% in the assay
result. No adverse effect was observed when free bilirubin (up to 0.68
mmol/L), ascorbic acid (up to 2.84 mmol/L), hemoglobin (up to 5 g/L),
Intralipos (up to 10 g/L, The Green Cross Co.), EDTA-2Na (up to 0.27
mmol/L), or citrate (up to 0.34 mmol/L) was included during the
measurement of LDL-C with this assay. However, when citrate at a final
concentration of 12.9 mmol/L was used as an anticoagulant for whole
blood, it produced a slightly negative error of 7.8% in the assay
result.
comparison with other methods
The proposed method was compared with ultracentrifugation
(beta-quantification method of the Lipid Research Clinic's Program)
with serum samples from healthy volunteers (n = 86), patients with
hyperlipemia (triglycerides <4.5 mmol/L, n = 41), and from
patients with hyperlipemia (triglycerides 
4.5 mmol/L, n = 34).
For serum samples from these three populations, there was a high degree
of correlation between the results of the proposed method and those of
the beta-quantification method, with r = 0.989 or
better. As shown in Fig. 9
, the mean percentage bias of standard deviation for LDL-C
determined by the proposed method in comparison with the
beta-quantification method was 0.73% ± 5.78% (0.03 ± 0.16
mmol/L) with serum samples from healthy volunteers, showing 1.01%
± 5.27% (0.05 ± 0.18 mmol/L), and those from patients with
hyperlipemia, showing no significant difference (P <0.5).
There was no increase in the percentage bias, even when the
concentrations of triglycerides and HDL-C were high. Correlations
between the proposed method and other methods (Friedewald formula,
immunoseparation, and HPLC method) are summarized in Table 3
. In the serum samples from healthy volunteers, the results
obtained by the proposed method were well correlated with other methods
(r = 0.98–0.99), but in those from patients with
hyperlipemia (triglycerides 4.5 mmol/L), the correlation coefficient
increased in the order: Friedewald formula 
immunoseparation
< HPLC < beta-quantification method.
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The LDL fraction used in this study is heterogeneous, and contains remnant particles of IDL (d 1.006–1.019 kg/L) and Lp(a) (d 1.050–1.080 kg/L). The proposed method for measuring LDL-C would actually include the contribution of IDL and Lp(a), since there was a good correlation between the results of LDL-C assayed by the proposed method and the beta-quantification method. This estimate is supported by the fact that 77.6% ± 3.3% of total cholesterol (0.39 ± 0.13 mmol/L) in the IDL fraction isolated by ultracentrifugation responded to the proposed method. On the other hand, no direct evidence is available to clarify whether the proposed method would include particles of Lp(a), because of the difficulty in isolating the Lp(a) from the LDL and HDL fractions by ultracentrifugation. Nevertheless, there was no increase in the percentage bias for LDL-C determined by the proposed method in comparison with the beta-quantification method as the concentration of Lp(a) increased. This indicates that the proposed method would also include Lp(a).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We established a direct method for determining the concentration of
LDL-C in serum in a convenient format with the combined use of POE-POP
and -cyclodextrin sulfate. Only a very small sample volume (4 µL)
is needed for this method, without the need for isolation of LDL. LDL-C
can be determined in a short time (10 min) by this homogeneous method,
which can be easily automated.
The strategy used for direct measurement of LDL-C in serum is based on
the cooperative actions of POE-POP as a quencher for HDL-C and
-cyclodextrin sulfate as a quencher for chylomicrons and VLDL-C.
This is distinctly different from the approach used for the homogeneous
assay for the direct measurement of LDL-C in serum described previously
(13), in which one surfactant selectively hydrolyzes
chylomicrons, VLDL, and HDL in a non-color-forming reaction, and then
another surfactant hydrolyzes only LDL for color development.
Previously, poloxamers including POE-POP have been successfully
utilized as surface modifiers for improving the stability of latex
particles (23) and as vehicles for transdermal drug
delivery (24). In this study, POE-POP showed limited
specificity towards LDL-C, solubilizing it into mixed micelles and thus
allowing it to participate in the enzymatic reaction. Some surfactants
can be used to isolate apo B and lipids from the LDL fraction
(25)(26), but show no selectivity towards
LDL-C under the present conditions. Although the mechanism that confers
such LDL selectivity to POE-POP is not clear, it is possible that the
surfactant may be able to recognize differences in hydrated density,
net charge, or size of the various lipoprotein fractions. As shown in
Fig. 3
, the higher the molecular mass of the POP block and
hydrophobicity, the greater the selectivity towards LDL-C. Apo A-I and
apo A-II in the HDL particle are known to be water soluble, whereas apo
B in the LDL fraction has an extremely large molecular mass of 540 kDa
and is highly hydrophobic in nature (27). Apo B and its
aggregates isolated from the LDL particle by POE-POP may be solubilized
in the complex form with the surfactant and have an apparent molecular
mass of 2000–3000 kDa, as judged by the elution volume in the gel
filtration. In such a complex, the peripheral hydrophilic POE blocks
should point freely in solution, whereas the central hydrophobic POP
block anchors the surfactant to the hydrophobic surface of the
apolipoprotein. On the other hand, the inability of POE-POP to remove
apo A-I and apo A-II from the HDL particle may be explained in part by
the hydrophilicity of those apolipoproteins and the physical size of
the POE-POP micelles, which may not allow them to penetrate the
interior of the HDL particle and thus may not solubilize the lipids.
The heparin-like activity of -cyclodextrin sulfate was utilized to
reduce the reactivity of cholesterol in chylomicrons and VLDL in a
manner similar to that reported previously
(14)(15). -Cyclodextrin sulfate has an
average molecular mass of 2194 Da, which is ~100 times smaller than
those of the polyanions used in precipitation-based methods.
Furthermore, the charge density of -cyclodextrin sulfate is higher
than those of the polyanions because of spatial constraints imposed on
the sulfates by the hexasaccharide ring structure. In the presence of
magnesium ions, -cyclodextrin sulfate may form water-soluble and
submicron-sized complexes with those lipoproteins with a low
protein/lipid ratio, which are resistant to the enzymatic reactions. In
fact, there was no noticeable visible precipitation during the
measurement of LDL-C with this assay. By contrast, when dextran sulfate
was included instead of -cyclodextrin sulfate, it increased the
turbidity of the reaction mixture to such an extent that it interfered
with the determination of LDL-C in serum.
The proposed method for measuring LDL-C would include the contribution of IDL and Lp(a), as the Friedewald equation or beta-quantification method does. Because all of the particles of this wide-density LDL population are atherogenic, the proposed method might be a more sensitive indicator of risk for premature coronary artery diseases than a method that has the limited specificity for the narrow-density LDL population.
In conclusion, the homogeneous assay described here is simple and reliable for measuring LDL-C in serum without prior fractionation and should prove to be quite useful in the routine clinical laboratory.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-cycrodextrin. Clin Chem 1995;41:717-723.
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M. Esteban-Salan, A. Guimon-Bardesi, J. M. de la Viuda-Unzueta, M. N. Azcarate-Ania, P. Pascual-Usandizaga, and E. Amoroto-Del-Rio Analytical and Clinical Evaluation of Two Homogeneous Assays for LDL-Cholesterol in Hyperlipidemic Patients Clin. Chem., August 1, 2000; 46(8): 1121 - 1131. [Abstract] [Full Text] [PDF]
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M. Nauck, M. S. Graziani, D. Bruton, C. Cobbaert, T. G. Cole, F. Lefevre, W. Riesen, P. S. Bachorik, and N. Rifai Analytical and Clinical Performance of a Detergent-based Homogeneous LDL-Cholesterol Assay: A Multicenter Evaluation Clin. Chem., April 1, 2000; 46(4): 506 - 514. [Abstract] [Full Text] [PDF]
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,
SILDL value for POE-POP; vertical bar
represents the mean of the SILDL values in each
row.
, LDL;
, VLDL; 
, chylomicron.
,
155 mmol/L NaCl solution; (B) 
