Clinical Chemistry 46: 1202-1203, 2000;
(Clinical Chemistry. 2000;46:1202-1203.)
© 2000 American Association for Clinical Chemistry, Inc.
LDL Particle Size by Gradient-Gel Electrophoresis Cannot Be Estimated by LDL-Cholesterol/Apolipoprotein B Ratios
Daisuke Furuya1,
Atsuhito Yagihashi1,
Syunichi Nasu1,
Teruo Endoh1,
Tohru Nakamura1,
Reiko Kaneko1,
Chinatsu Kamagata1,
Daisuke Kobayashi1 and
Naoki Watanabea,1
1
Department of Clinical Laboratory Medicine, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-Ku, Sapporo 060-8543, Japan
a author for correspondence: fax 81-11-622-7502, e-mail watanabn{at}sapmed.ac.jp
LDL particles have been shown to be heterogeneous in
size, density, and composition. Heterogeneity of LDL particles with
respect to size has been demonstrated by analytic ultracentrifugation
(1), density-gradient ultracentrifugation, gradient-gel
electrophoresis (GGE), high-performance gel-filtration
chromatography, dynamic light scattering, and electron
microscopy. Among these methods, GGE is the most reliable and widely
used, but it has the drawback of being labor-intensive. Recently, the
ratio of the LDL-cholesterol (LDL-chol) concentration to the
LDL-apolipoprotein B (LDL-apo B) concentration has been used as an
alternative index of hyperapobetalipoproteinemia and small dense LDL
(2)(3)(4)(5). We therefore studied the relationship between LDL
particle size as measured by GGE and as estimated by the
LDL-chol/LDL-apo B ratio.
LDL particle size was quantified by the above two methods in samples
from healthy controls (n = 49) and from hyperlipemic
subjects (n = 81). The hyperlipemic samples in this study were
defined according to the criteria of the Japan Atherosclerosis Society
[total cholesterol 
2200 mg/L; triglycerides (TGs) 1500
mg/L] (6). GGE was carried out using a 2.4–15.2%
Multilipo nondenaturing gradient gel (Daiichi Pure Chemical).
Serum samples (5 µL) were electrophoresed on the gel at 25 mA for
2 h. Colloidal gold with standard particle sizes (19.9 and 25.9 nm
as confirmed by electron microscopy) was used as a marker for LDL
particle size. The colloidal gold particle sizes were also estimated by
GGE from a linear calibration curve of the diameter vs the migration
distance, with L-lactate dehydrogenase (8.4 nm),
catalase (10.4 nm), ferritin (12.2 nm), and thyroglobulin (17.0 nm) as
calibrators of known size (7). Bands were analyzed by a
personal density scanning imager (PDSI; Molecular Dynamics).
Concentrations of LDL-chol and LDL-apo B were measured by a Hitachi
7170 automated analyzer (Hitachi), using Choletest® LDL, and Apo B
reagent kits (Daiichi Pure Chemical). These analyses were based on
homogeneous enzymatic assay (8) and turbidimetric
immunoassay (9) methods, respectively.
The LDL-chol and LDL-apo B concentrations as well as the LDL particle
sizes as measured by GGE and LDL-chol/LDL-apo B ratios are summarized
as the mean ± SD in Table 1
. A significant difference in LDL particle size was demonstrated
between hyperlipemic and control samples by both GGE and the
LDL-chol/LDL-apo B ratio. When LDL particle sizes measured by GGE were
plotted against those determined in the same blood samples as
LDL-chol/LDL-apo B ratios (Fig. 1
), LDL-chol/LDL-apo B ratios showed a weak negative correlation
with LDL particles sizes measured by GGE in control samples (Fig. 1A
)
and a weak positive correlation in hyperlipemic samples (Fig. 1B
). In control samples, LDL-chol/LDL-apo B ratios were
1.14–1.39, and LDL particle size measured by GGE was 21.38–25.41 nm.
Among hyperlipemic samples, despite reports that the LDL-chol/LDL-apo B
ratio reflects the size of small dense LDL, some samples contained
normal-sized LDL particles as determined by GGE but showed low
LDL-chol/LDL-apo B ratios. This result reflects the fact that
measurement of LDL-chol by the homogeneous enzyme assay is affected by
high concentrations of TGs, which leads to underestimation of the
LDL-chol concentration (8)(10). LDL size
estimated by the LDL-chol/LDL-apo B ratio showed more negative
correlation with TG concentrations than that estimated by GGE
(r = -0.68 vs -0.27). TG concentrations in these two
samples were particularly high (8260 and 11 430 mg/L; Fig. 1B
, 
).
In contrast, some samples shown to contain small LDL particles by GGE
showed high LDL-chol/LDL-apo B ratios. The LDL-chol, LDL-apo B, and TG
concentrations (Fig. 1B
, 
), respectively, were 1550, 1360, and 1810
mg/L in one sample and 1660, 1320, and 1890 mg/L in the other. When the
LDL-chol/LDL-apo B ratio is used to evaluate LDL particle size, the
size in these samples will be misreported as normal.
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Figure 1. Comparison of LDL particle size between GGE and
LDL-chol/LDL-apo B in control subjects (A) and
subjects with hyperlipemia (B).
, misleadingly high LDL-chol/LDL-apo B ratios; ,
spuriously low LDL-chol/LDL-apo B ratios (reflecting
hypertriglyceridemia).
|
|
The presence of small, dense LDL increases the risk of atherosclerotic
cardiovascular disease beyond that associated with normal LDL
(11). Given the importance of LDL particle size
determination, it should be performed by GGE rather than rapidly
estimated by the LDL-chol/LDL-apo B ratio.
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