CLB-08983; No. of pages: 3; 4C: Clinical Biochemistry xxx (2015) xxx–xxx

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Short Communication

Study of frozen low density lipoprotein particles by using nanotechnology V. Sgouropoulou a, K. Makedou b,⁎, M. Seitanidou c, M. Trachana a, V. Karagiozaki c, S. Logothetidis c a b c

Pediatric Immunology and Rheumatology Referral Center, 1st Department of Pediatrics, Aristotle University of Thessaloniki, Hippokration General Hospital, Thessaloniki, Greece Laboratory of Biological Chemistry, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece Nanotechnology Lab LTFN, School of Physics, Faculty of Sciences, Aristotle University of Thessaloniki, Greece

a r t i c l e

i n f o

Article history: Received 5 February 2015 Received in revised form 17 March 2015 Accepted 18 March 2015 Available online xxxx Keywords: Low density lipoproteins Frozen Size AFM Nanotechnology

a b s t r a c t Objectives: To investigate the impact of freezing in −80 °C on the structure of isolated low density lipoproteins (LDLs), using nanotechnology, such as Atomic Force Microscopy (AFM). Design and methods: Blood EDTA plasma was obtained from healthy subject and used immediately to isolate LDL by sequential ultracentrifugation at 10 °C in 55,000 rpm for 3 h, using a Beckmann XL-90 ultracentrifuge (75Ti rotor), in the presence of KBr in PBS. LDLs were then diluted with PBS until final concentrations of 5 and 15 mg LDL/dl. After initial observation, samples were frozen in −80 °C for two weeks and observed again after thawing. Experiments were performed in triplicate on two smooth and clean substrates of different hydrophobicity, glass (HOPG) and Si (c-Si). Statistical significance was set at 0.05. Results: Macroscopically, LDL particles formed aggregations in a dendroid layout. There were no differences between images taken from both substrates (HOPG and c-Si). Frozen samples presented significantly smaller LDL particles, than fresh ones. In specific, mean diameter of LDL particle in the fresh LDL sample was 19.77 nm, ranging from 13.34 to 28.76 nm. The frozen LDL sample had a mean diameter of 5.2 nm, ranging from 2.0 to 8.0 nm, which was significantly different from the unfrozen. Conclusions: Atomic Force Microscopy showed that freezing of LDL causes alterations in their size. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

Materials and methods

Studies on lipoproteins isolated by ultracentrifugation reveal the need of using them fresh, immediately after fractionation and isolation. This notion has been supported by a few analytical studies investigating the impact of storage and handling of blood plasma on the accuracy of analysis. Several studies showed an increase in lipid concentrations by freezing [1,2], whereas others showed a decrease [3,4], or no significant alterations in lipid content [5]. Moreover, a later study of Zivkovic et al. [6] has shown that a single freeze–thaw cycle resulted in a variability of about 37% in HDL- and LDL-cholesterol. Only two studies have investigated the effects of multiple freeze–thaw cycles [7,8] and showed no difference between one and three freeze–thaw cycles in − 80 °C. To our knowledge, there is no study using Atomic Force Microscopy (AFM), an instrument able to provide nanometer-scale resolution images and to observe possible changes on lipoprotein particles after freezing and thawing. The aim of the present study was to investigate the impact of freezing in −80 °C on the structure of isolated low density lipoproteins (LDL), using nanotechnology, such as AFM.

For the purpose of the study, blood EDTA plasma was obtained from healthy children, aged 5.6–16 years old, and used immediately to isolate LDL by sequential ultracentrifugation at 10 °C in 55,000 rpm for 3 h, using a Beckmann XL-90 ultracentrifuge (75Ti fixed angle rotor), in the presence of KBr in PBS [9]. LDLs were then diluted with PBS until final concentrations of 5 and 15 mg LDL/dl. After initial observation, samples were stored at − 80 °C for two weeks and observed again, after thawing slowly, at room temperature. Experiments were performed on two smooth and clean substrates of different hydrophobicity (glass and c-Si). After incubation into the LDL solutions, the samples were rinsed gently with Milli-Q water, substrates were dried with mild N2 flow and AFM measurements were performed with Solver P47H Pro (NT-MDT). The observation took place in fields of 10 ∗ 10 μm and 5 ∗ 5 μm. The size of 100 particles of LDL was counted in each one of the 10 fields (5 ∗ 5 μm) observed. The experiment was performed in triplicate, with different samples. Statistical significance was set at 0.05.

Abbreviations: LDL, low density lipoproteins; HDL, high density lipoproteins; AFM, Atomic Force Microscopy. ⁎ Corresponding author at: Aristotelous 45, 55236 Panorama, Thessaloniki, Greece. E-mail address: [email protected] (K. Makedou).

Results AFM observation showed that LDL particles formed aggregations in a dendroid layout (Fig. 1). The images of the samples of 5 mg LDL/dl were

http://dx.doi.org/10.1016/j.clinbiochem.2015.03.010 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Please cite this article as: Sgouropoulou V, et al, Study of frozen low density lipoprotein particles by using nanotechnology, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.03.010

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V. Sgouropoulou et al. / Clinical Biochemistry xxx (2015) xxx–xxx

Fig. 1. Dendroid aggregations of LDL particles on substrate of Si, in two different fields of 10 ∗ 10 μm (a) and 5 ∗ 5 μm (b).

more clear, with fewer LDL particles and allowed to measure the size of LDL in a much better way than those of the concentration of 15 mg LDL/dl, in which LDL aggregated in dense masses and prevented us from evaluating the size of them. There were no differences between images taken from both substrates (glass and c-Si). Frozen samples presented with significantly smaller LDL particles (Fig. 2), than the fresh ones (Fig. 1b). In the magnification of 5 ∗ 5 μm

LDL particles after thawing were not distinct. Therefore, we proceeded to a higher magnification (1.45 ∗ 1.45 μm) in which the particle could be measured. We noticed that although the magnification in Fig. 2 was higher, LDL particles were even smaller than in Fig. 1b. In specific, mean diameter of LDL particles in the fresh samples was 19.77 nm, ranging from about 13.34 to 28.76 nm, whereas the frozen and thawed LDL samples presented a mean diameter of 5.2 nm, ranging from 2.0 to 8.0 nm.

Fig. 2. Images of aggregations of LDL particles after one freeze–thaw cycle in a field of 1.45 ∗ 1.45 μm.

Please cite this article as: Sgouropoulou V, et al, Study of frozen low density lipoprotein particles by using nanotechnology, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.03.010

V. Sgouropoulou et al. / Clinical Biochemistry xxx (2015) xxx–xxx

Conclusions The findings of our study indicate that the size of LDL particles is affected by freezing isolated lipoproteins, after sequential ultracentrifugation, in −80 °C. The particles become smaller and this might influence their electrophoretic or chromatographic mobility and, subsequently, the results of an LDL study with electrophoresis or chromatography, respectively. Nevertheless, this is only an observation that needs further analytical investigation. It is not clear whether these smaller particles are intact LDL particles with decreased lipid content, as previous studies have demonstrated [3,4] or fragments of bigger LDL particles that have been produced by freezing, as Wakamatu et al. indicated [10]. Moreover, it would be interesting to investigate whether shock freezing (b− 130 °C) has different impact of the images of LDL, in contrast to slow freezing. In conclusion, the present study shows, for the first time, by using nanotechnology, that freezing seems to have an impact on plasma LDL structure.

Conflict of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

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Please cite this article as: Sgouropoulou V, et al, Study of frozen low density lipoprotein particles by using nanotechnology, Clin Biochem (2015), http://dx.doi.org/10.1016/j.clinbiochem.2015.03.010

Study of frozen low density lipoprotein particles by using nanotechnology.

To investigate the impact of freezing in -80°C on the structure of isolated low density lipoproteins (LDLs), using nanotechnology, such as Atomic Forc...
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