Infrared study of human serum very-low-density and low-density lipoproteins. Implication of esterified lipid CO stretching bands for characterizing lipoproteins

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Abstract

Fourier-transform infrared (FT-IR) spectroscopy was applied to examine human serum very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) in aqueous solution and in solid film for characterizing lipid components. On the basis of the FT-IR second-derivative spectra for standard samples of triglycerides, cholesterol esters and phospholipids, it was found that the band at 1746 cm−1 for VLDL and the band at 1738 cm−1 for LDL were mainly due to the unsaturated triglycerides and unsaturated cholesterol esters, respectively. The implications of ester CO stretching bands are discussed.

Introduction

Serum lipoproteins are complexes of triglyceride (TG), cholesterol ester (CE), free cholesterol (FC) and phospholipid (PL) with proteins known as apolipoproteins (Morrisett et al., 1975). The hydrophobic core of a lipoprotein particle consists of TG and CE, and the surface of the particle contains amphipathic PL, FC and apolipoproteins. Lipoproteins are mainly classified into chylomicrons, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL) on the basis of density (Havel et al., 1955). The density of each lipoprotein class depends on the composition of lipids and apolipoproteins. VLDL is assembled in the hepatocytes as a range of different size TG-rich particles, containing one apolipoprotein B-100 (apo B-100) molecule and is secreted into plasma. LDL is the main CE-rich particle in plasma, containing one molecule of apo B-100 and no other apolipoproteins and is formed by lipolysis of VLDL. HDL is the smallest and most dense lipoprotein and is packed with apolipoproteins which account for 50% of its mass. The plasma levels of TG and CE are clinically important, because they are thought to reflect disorders of lipid metabolism (for example, Dominiczak, 2000).

Fourier-transform infrared (FT-IR) spectroscopy is a useful method for examining lipid-protein interactions in lipoproteins (Arrondo and Goñi, 1998). Application of FT-IR method to lipoproteins has mainly been focused on analyzing the secondary structure of apolipoproteins rather than lipid components (Herzyk et al., 1987, Goormaghtigh et al., 1989, Bañuelos et al., 1995, Tanfani et al., 1997, Yang et al., 1991). The secondary structures of apo B-100 in LDL components (Herzyk et al., 1987, Goormaghtigh et al., 1989, Bañuelos et al., 1995, Tanfani et al., 1997) and apolipoprotein A-I in HDL (Yang et al., 1991) were analyzed by FT-IR method in combination with band enhancement technique. On the other hand, the lipid components have not been discussed enough by using FT-IR method due to the difficulties in characterizing the contributions due to lipids in the complicated spectra of lipoproteins.

In the present study, we have examined the infrared spectra of VLDL and LDL in aqueous solution and in solid film for characterizing lipid components as well as apolipoproteins. We discuss the implications of ester CO stretching bands for these lipoproteins.

Section snippets

Materials and methods

We used VLDL and LDL (Chemicon International) as lipoprotein classes separated by ultracentifugation. Triolein (TO) (Wako), tripalmitin (TP) (Wako), cholesteryl palmitate (CP) (Wako), cholesteryl linoleate (CL) (Sigma), dioleitoylphosphatidylcholine (DOPC) (Wako) and dipalmitoylphosphatidylcholine (DPPC) (Wako) were used for assigning the spectra of lipoproteins.

FT-IR measurements were performed at 25 °C on a Perkin–Elmer Spectrum-2000 Fourier-transform infrared spectrometer equipped with a TGS

Results

Fig. 1 shows FT-IR spectra for VLDL (a) and LDL (b) in the region of 1800–1500 cm−1, where absorptions of H2O have been subtracted. Three bands were observed in each spectra: esterified lipid CO stretching band (1750–1700 cm−1), amide-I band (1700–1600 cm−1), and amide-II band (1600–1500 cm−1). The relative intensity of the ester CO stretching band to the amide-I band was stronger for VLDL than for LDL. To analyze these absorption spectra in more detail, Fig. 1 also shows the

Discussion

The present study has shown that the region of ester CO stretch (1750–1700 cm−1) can be used as a marker for characterizing TG and CE, which are the main components for VLDL and LDL, respectively. As far as the TG and CE are concerned, the results obtained are summarized as follows: (1) The CO stretching bands for unsaturated TG and unsaturated CE exhibit a band at about 1746 and 1738 cm−1, respectively. As for the unsaturated compounds, there is the trend of νCO(TG)>νCO(CE). (2) The CO

Acknowledgements

The authors appreciate Dr H.M. Heise at the University of Dortmund for his valuable advice. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

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