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Measurement of lipoprotein particle sizes using dynamic light
Sakurai, Toshihiro; Trirongjitmoah, Suchin; Nishibata, Yuka;
Namita, Takeshi; Tsuji, Masahiro; Hui, Shu-Ping; Jin, Shigeki;
Shimizu, Koichi; Chiba, Hitoshi
Citation Annals of Clinical Biochemistry, 47(5): 476-481
Doc URL http://hdl.handle.net/2115/44222
Ann Clin Biochem 2010;47:476-481,
This is the final draft, after peer-review, of a manuscript
published in Annals of Clinical Biochemistry. The definitive
version, detailed above, is available online at
Type article (author version)
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
Measurement of lipoprotein particle sizes using dynamic light scattering
A short title: Measurement of lipoprotein particle sizes using DLS
Toshihiro Sakurai1, Suchin Trirongjitmoah2, Yuka Nishibata1, Takeshi Namita2, Masahiro
Tsuji3, Shu–Ping Hui4, Shigeki Jin1, Koichi Shimizu2 and Hitoshi Chiba1
1Faculty of Health Sciences, Hokkaido University, Sapporo, Japan; 2Graduate School of
Information Science and Technology, Hokkaido University, Sapporo, Japan; 3Division of
Internal Medicine, Health Sciences University of Hokkaido Hospital, Sapporo, Japan;
4Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido,
Corresponding author: Hitoshi Chiba, Faculty of Health Sciences, Hokkaido University, Kita
12, Nishi 5, Kita–ku, Sapporo 060–0812, Japan. Phone: +81–11–706–3698, Email:
Competing interests: None.
Funding: This research was partly supported by a Grant–in Aid for Scientific Research from
the Japan Society for the Promotion of Science and also by the Knowledge Cluster Initiative
(Bio–S Sapporo), the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Ethical approval: The study was approved by the ethics review board at the Faculty of
Health Sciences, Hokkaido University (approval number 09–38).
Guarantor: Hitoshi Chiba.
Contributorship: Toshihiro Sakurai and Hitoshi Chiba researched literature and conceived
the study. Yuka Nishibata, Shu–Ping Hui and Shigeki Jin were involved in lipoprotein
separations and data analysis. Suchin Trirongjitmoah, Takeshi Namita and Koichi Shimizu
were involved in data analysis of DLS. Masahiro Tsuji was involved in gaining ethical
approval, patient recruitment. Toshihiro Sakurai wrote the first draft of the manuscript. All
authors reviewed and edited the manuscript and approved the final version of the manuscript.
Acknowledgements: The DLS analysis was carried out with FDLS–3000 at the OPEN
FACILITY, Hokkaido University Sousei Hall. The Central Research Laboratory, Faculty of
Health Sciences, Hokkaido University, kindly provided with the work space.
Background: A simple method for the measurement of LDL particle sizes is needed in
clinical laboratories because a predominance of small dense LDL (sd LDL) has been
associated with coronary heart disease. We applied dynamic light scattering (DLS) to measure
lipoprotein particle sizes, with special reference to sd LDL.
Methods: Human serum lipoproteins isolated by a combination of ultracentrifugation and gel
chromatography, or by sequential ultracentrifugation, were measured for particle size using
Results: The sizes of polystyrene beads, with diameters of 21 and 28 nm according to the
manufacturer, were determined by DLS as 19.3 ± 1.0 nm (mean ± SD, n = 11) and 25.5 ± 1.0
nm, respectively. The coefficients of variation for the 21–nm and 28–nm beads were 5.1%
and 3.8% (within–run, n = 11), and 2.9% and 6.2% (between–run, n = 3), respectively. The
lipoprotein sizes determined by DLS for lipoprotein fractions isolated by chromatography
were consistent with the elution profile. Whole serum, four isolated lipoprotein fractions
(CM+VLDL+IDL, large LDL, sd LDL, and HDL) and a non–lipoprotein fraction isolated by
sequential ultracentrifugation were determined by DLS to be 13.1 ± 7.5 nm, 37.0 ± 5.2 nm,
21.5 ± 0.8 nm, 20.3 ± 1.1 nm, 8.6 ± 1.5 nm, and 8.8 ± 2.0 nm, respectively.
Conclusions: The proposed DLS method can differentiate the sizes of isolated lipoprotein
particles, including large LDL and sd LDL, and might be used in clinical laboratories in
combination with convenient lipoprotein separation.
Previous studies have reported the relation of small, dense LDL particles (sd LDL) with
coronary heart disease.1–5 The predominance of sd LDL among the LDL subclasses has been
reported to indicate a threefold increase in the risk of myocardial infarction.1 The sd LDL (d =
1.044–1.063 kg/L) is small in size compared to the counterpart of LDL (or large LDL, d =
1.019–1.044 kg/L). Several methods have been reported for the evaluation of LDL particle
sizes, such as high–performance liquid chromatography (HPLC),6 gradient gel electrophoresis
(GGE),7 electron microscopy (EM),8 and nuclear magnetic resonance (NMR).9,10 Hirano et al.
reported that sd LDL–cholesterol levels had a significant inverse correlation with the LDL
particle sizes determined by GGE.11 These previous methods, however, are time–consuming,
laborious, and/or difficult to apply to many samples. Although a commercial sd
LDL–cholesterol reagent is currently available,7 the information provided about the particle
sizes of the lipoproteins targeted by this reagent has been thus far insufficient.7
Dynamic light scattering (DLS) is a method that can estimate the mean nanoparticle
size in fluids by measuring the intensity fluctuation of scattered light. DLS is quickly
performed (i.e., a few minutes) and easy to be used in small spaces such as in clinical
laboratories. Previous studies have reported the use of DLS for measuring the sizes of
lipoproteins, such as human LDL and chylomicron (CM) from human lymph.12–14 However,
the feasibility of using DLS to differentiate lipoprotein subclasses has not been investigated.
In the present study, we isolated five lipoprotein fractions, including large LDL and sd LDL,
using two separation methods: gel filtration after ultracentrifugation and sequential
ultracentrifugation. DLS analysis of the isolated lipoproteins was evaluated to determine
whether DLS can differentiate these lipoproteins, in particular, sd LDL from large LDL.
Materials and methods
Blood was drawn from healthy men (n = 4; range = 22–23 years old) for the experiments
using ultracentrifugation and Sepharose CL4B chromatography (Study 1). Blood was also
obtained from healthy volunteers (n = 11; male:female = 7:4; mean age = 27.7 ± 12.1 years,
range = 21–60 years) for the experiments using sequential ultracentrifugation (Study 2). All
subjects had fasted overnight before blood drawing. Sera were obtained by centrifugation
(2000g, 10 min, room temperature) and were stored at 4°C until use. Ultracentrifugation was
conducted immediately after blood drawing, and all experiments were completed within 2
weeks. Clinical data of the studied subjects are shown in Table 1.
Lipids and apolipoproteins
Lipids were measured by automated enzymatic methods using commercial kits (Denka Seiken
Co., Ltd., Tokyo, Japan): T–CHO (S) for total cholesterol (TC), TG–EX for triglyceride (TG),
LDL–EX (N) for LDL–cholesterol, HDL–EX (N) for HDL–cholesterol, and sd LDL SEIKEN
for sd LDL–cholesterol. Apolipoprotein A–I (apo A–I) and B (apo B) were measured by
automated immunoturbidimetry using commercial kits (Apo A–I and Apo B Auto N Daiichi,
Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan).
Polystyrene latex (PS) beads (21 nm and 28 nm in diameter, Magsphere, Inc., Pasadena, CA)
were used for calibration of DLS. DLS measurement was conducted using a model
FDLS–3000 (Otsuka Electronics Co. Ltd., Hirakata, Japan). A CONTIN algorithm was used
for DLS analysis to obtain the weight–size distribution.15,16 The laser light was irradiated at
100 mW power at 532 nm wavelength. The fluctuation in scattered intensity was measured at
an angle of 90°, and accumulated 100 times over 5 min. The temperature was set at 25°C for
experiments using the PS beads and at 37°C for the lipoprotein fractions. The PS beads were
measured 11 times for the evaluation of within–run variation and 3 times on 3 consecutive
days for the evaluation of between–run variation. Each lipoprotein fraction (1 ml), diluted by
10– to 20–fold in saline was contained in a 178 mm × 5 mmφ glass tube (Optima Inc., Tokyo,
Japan) and was measured by DLS. The average of three measurements was used for statistical
Ultracentrifugation and gel chromatography
Serum was added with a final concentration of 0.7 mmol/l 5, 5’ –dithiobis (2–nitrobenzoic
acid) and 2.7 mmol/l EDTA–2Na (pH 7.4). The sample was adjusted with KBr to d = 1.225
kg/L and then centrifuged in a RPV–50T rotor (Hitachi, Tokyo, Japan) at 40,000 rpm for 18 h
at 15°C using a Hitachi Himac CP60E ultracentrifuge.17 The total lipoprotein fraction (d <
1.225 kg/L) was obtained by aspiration and then applied to a Sepharose CL4B column (1.6 ×
100 cm) in 5 mmol/l Tris–HCl buffer (pH 7.4) containing 0.15 mol/l NaCl, 0.27 mmol/l
EDTA–2 Na, and 3 mmol/l NaN3. The column was eluted at 4°C, and 3–ml fractions were
collected under continuous monitoring of the absorbance at 280 nm. Each fraction was
measured for apo A–I and apo B to confirm the distribution of LDL and HDL particles. Each
tube bracketing the LDL and HDL fractions was subjected to DLS analysis.
The lipoprotein fractions were isolated from serum by sequential ultracentrifugation
according to the method of Hirano et al.7 with some modifications. Briefly,
ultracentrifugation was performed using a near–vertical tube rotor (MLN–80, Beckman
Coulter, Fullerton, CA) on a model Optima MAX (Beckman Coulter). Serum (2 ml) was
adjusted to d = 1.019 kg/L and then centrifuged at 40,000 rpm for 20 h at 15°C. After
isolating the top fraction (2.5 ml) containing d < 1.019 lipoproteins (CM, VLDL, and IDL),
the bottom fraction was adjusted with KBr solution to d = 1.044 kg/L and then centrifuged
again at 50,000 rpm for 18 h at 15°C. After the top fraction (2.5 ml) containing large LDL
was isolated, the bottom fraction was adjusted to d = 1.063 kg/L and centrifuged further at
50,000 rpm for 18 h at 15°C. After the top fraction (2.5 ml) containing sd LDL was isolated,
the bottom fraction was adjusted to d = 1.225 kg/L and centrifuged further at 50,000 rpm for
20 h at 15°C. The top fraction (2.5 ml) containing HDL and the bottom fraction containing
lipoprotein–free serum proteins, designated as the non–Lp fraction, were recovered.
Lipoprotein separation was confirmed by polyacrylamide gel electrophoresis (LipoPhor,
Jokoh Co., Ltd., Tokyo, Japan).
The sizes determined by DLS for the serum samples and the lipoprotein fractions isolated by
the sequential ultracentrifugation were compared by nonparametric Kruskal–Wallis test. The
particle sizes in the large LDL and sd LDL fractions were compared by Wilcoxon
signed–rank test. The association between the serum TG levels and the particle sizes of the
CM+VLDL+IDL fraction was tested by Spearman’s rank correlation coefficient (Rs value).
P < 0.05 was considered to be statistically significant.
All individuals gave written informed consent to participate in this study. The study was
approved by the ethics review board at the Faculty of Health Sciences, Hokkaido University
(approval number 09–38).
The particle sizes of PS beads determined by DLS were 19.3 ± 1.0 nm (n = 11) for the 21–nm
beads and 25.5 ± 1.0 nm (n = 11) for the 28–nm beads. The coefficients of within–run
variation for the 21–nm and 28–nm beads were 5.1% and 3.8%, respectively, and those of
between–run variation (n = 3) were 2.9% (19.2 ± 0.5 nm) and 6.2% (26.0 ± 1.6 nm),
respectively. The mean sizes estimated in DLS measurements were smaller than those
provided by the manufacturer by approximately 10%. The experimental values were used for
calibration in the following experiments.
Lipoproteins isolated by ultracentrifugation and gel chromatography (Study 1)
The DLS measurements for each lipoprotein fraction isolated from the total lipoproteins (d <
1.225 kg/L) by Sepharose CL4B chromatography are presented in Figure 1. Similar results
were obtained for all subjects examined. The distribution of DLS measurements was
consistent with the elution profile of lipoprotein classes. Elution fraction #44, corresponding
to the peak of apo B, or the major apolipoprotein of LDL, showed a DLS measurement of
21.9 ± 0.5 nm (mean ± SD). Elution fraction #53, corresponding to the peak of apo A–I, or the
major apolipoprotein of HDL, showed a DLS measurement of 8.6 ± 0.6 nm. Between the
LDL and HDL peaks, the average particle size decreased gradually along with the elution
fraction numbers, indicating that LDL and HDL particles co–eluted in these fractions. The
large standard deviations in this sizes region were due to the less number of particles than
other elution numbers.
Lipoproteins isolated by sequential ultracentrifugation (Study 2)
The serum and lipoprotein fractions isolated by sequential ultracentrifugation, namely, the
CM+VLDL+IDL fraction (d < 1.019 kg/L), the large LDL fraction (d = 1.019–1.044 kg/L),
the sd LDL fraction (d = 1.044–1.063 kg/L), the HDL fraction (d = 1.063–1.225 kg/L), and
the non–Lp fraction (d > 1.225 kg/L), were well separated, as demonstrated by
polyacrylamide gel electrophoresis (Figure 2). The average particle sizes determined by DLS
for the 11 subjects were 13.1 ± 7.5 nm for serum, 37.0 ± 5.2 nm for CM+VLDL+IDL, 21.5 ±
0.8 nm for large LDL, 20.3 ± 1.1 nm for sd LDL, 8.6 ± 1.5 nm for HDL, and 8.8 ± 2.0 nm for
non–Lp (Figure 3), with significant differences among the fractions (Kruskal–Wallis test, P =
0.00000051). The particle sizes for the sd LDL fraction were significantly less than those for
the large LDL fraction (P = 0.016). Similar particle sizes were obtained for the HDL and the
non–Lp fractions. The particle sizes for the CM+VLDL+IDL fraction were correlated with
serum TG levels (Rs = 0.93, P = 0.003, Figure 4).
The estimation of particle sizes is possible by measuring the light–scattering intensity of the
particles with the random movements, or Brownian motion. This method is called dynamic
light scattering (DLS), and has been applied to nanoparticles such as proteins, DNA,
liposomes, and detergent micelles. In our experimental system, particles ranging from 0.5 to
5000 nm in diameter are measurable. In general, DLS systems are small in size and easy to be
set up on the benchtop, unlike other systems for measuring lipoprotein particle sizes, such as
EM and NMR. Additionally, DLS is quick (i.e., analysis within a few minutes) and applicable
to the particles in fluid, which is also advantageous for clinical use. One problem with DLS is
that the larger particles have a greater effect on the total scattered intensity.12,18 When a
mixture of differently sized particles is measured, the average size estimated by the DLS
measurement is susceptible to the change in large particles. Therefore, it is recommended to
perform the DLS measurement with some separation technique to eliminate unnecessary large
particles in biological specimens.
Our present experiments using the PS beads verified the reproducibility of DLS
measurements. The discrepancy in the sizes of beads between our DLS measurements and the
values provided by the manufacturer might be due to different methodological bases.
Software for FDLS–3000 gives the size distribution of scatters against the size interpreted
from the weight of the particles. It is referred to as the weight–size distribution. The size
distribution against the size interpreted from the scattered light intensity is referred to as the
intensity–size distribution. The weight–size distribution is much more sensitive for small
particles compared to the intensity–size distribution. The latter has been reported to give
larger mean sizes than the former.15,16,19
The measurement of lipoprotein particle sizes has been conducted using GGE in many
reports and by DLS in few reports. In a previous study using DLS, the LDL fraction isolated
by ultracentrifugation was determined to have diameters of 22.9 ± 1.0 nm.20 The particle sizes
of LDL isolated by density gradient ultracentrifugation were reported as 23.1 ± 0.1 nm by
DLS and 26.1 ± 0.1 nm by GGE, with a strong correlation between methods (r = 0.78).12
GGE is known to give larger values than DLS, possibly because LDL can change its shape by
interactions with the gel matrix and electric field.8,12 The size of HDL isolated by a
precipitation technique using polyethylene glycol was reported to be as large as 8.8 nm
(mean) by DLS.21 Other previous studies have reported LDL sizes of 21.1 ± 0.9 nm by NMR
and 20–27 nm by EM, and HDL sizes of 9.1 ± 0.5 nm by NMR and 6–12 nm by EM.22,23
Thus, the current results of 21.5 ± 0.8 nm for large LDL and 8.6 ± 1.5 nm for HDL are
consistent with previous reports.
In addition to DLS and GGE, HPLC has been used for lipoprotein size estimation.
Mean LDL and HDL particle sizes of 25.3 nm and 11.3 nm, respectively, have been
reported.24 In another study using HPLC, the LDL isolated by ultracentrifugation was
measured as 25.5 ± 0.9 nm, with good correlation with the measurement by GGE in the same
study (r = 0.88).6 In terms of reproducibility, DLS (<6.2% in this study) is worse than HPLC
(<1%)6 and GGE (1%–3.5%),25–27 but seems acceptable for clinical use.
In the present study, we found no difference in the particle size between HDL and
non–Lp (Figure 3). The non–Lp fraction contained many kinds of serum proteins, dominated
by albumin and immunoglobulins. Most of these proteins are smaller than HDL, but some
proteins, such as IgM, are as large as lipoproteins. Such large molecules should have raised
the DLS measures for the non–Lp fraction.
Our CM+VLDL+IDL fraction actually contained only VLDL and IDL because the
serum was sampled from normal young subjects after overnight fasting. The positive
correlation between the serum TG levels and the DLS measurements for this fraction (Figure
4) indicates that the elevation of serum TG levels is associated with the increase in particle
sizes of VLDL and IDL. Hence, the DLS measurement for TG–rich lipoproteins is useful to
detect the delay in TG–rich lipoprotein metabolism.
In a previous study using GGE, the LDL sizes were compared between subjects with
preponderance of large buoyant LDL (called pattern A by the authors) and the rest (pattern
B);28 the LDL diameters were 26.8 ± 0.3 nm and 25.1 ± 0.4 nm for patterns A and B,
respectively. In our study, the diameters for the large LDL and the sd LDL fractions were
21.5 ± 0.8 nm and 20.3 ± 1.1 nm, respectively. As described above, this discrepancy can be
explained by methodological differences. In a previous study using cryo–electron microscopy,
the LDL particle sizes for the patterns A and B were 20.1 ± 1.7 nm and 17.9 ± 1.5 nm,
respectively, which are similar to our results.8 To the best of our knowledge, this is the first
report of DLS measurements of isolated sd LDL.
In conclusion, DLS analysis is feasible for the measurement of particle sizes of isolated
lipoprotein fractions. When DLS is coupled with a convenient isolation technique, the
accurate and precise measurement of lipoprotein particle size could be possible in clinical
laboratories. Further study on the clinical application of DLS is ongoing in our laboratory.
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