UNCORRECTED PROOF 1 2 Partial filling affinity capillary electrophoresis with cationic 3 poly(vinylpyrrolidone)-based copolymer coatings for studies on human 4 lipoprotein–steroid interactions 5 Ai-Jun Wang a , Kati Vainikka a , Joanna Witos a , Lucia D’Ulivo a , Geraldine Cilpa a , Petri T. Kovanen b , 6 Katariina Öörni b , Matti Jauhiainen c , Marja-Liisa Riekkola a, * 7 a Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, FIN-00014 Helsinki, Finland 8 b Wihuri Research Institute, FIN-00140 Helsinki, Finland 9 c National Institute for Health and Welfare, Public Health Genomics Research Unit and Finnish Institute for Molecular Medicine, FIN-00251 Helsinki, Finland 10 12 article info 13 Article history: 14 Received 30 September 2009 15 Received in revised form 18 November 2009 16 Accepted 18 November 2009 17 Available online xxxx 18 Keywords: 19 Affinity capillary electrophoresis 20 Partial filling technique 21 Affinity constant 22 High- and low-density lipoproteins 23 Steroids 24 Copolymers 25 26 abstract 27 Human plasma lipoproteins have strong hydrophobic interactions with steroids and their fatty acyl deriv- 28 atives such as estradiol fatty acyl esters. In this work, affinity capillary electrophoresis with the partial 29 filling technique was applied to study the hydrophobic interactions between lipoproteins, which are 30 nanometer-sized particles, and nonconjugated steroids. The capillaries were first rinsed with one of 31 two novel poly(vinylpyrrolidone) (PVP)-based cationic copolymers that were strongly adsorbed onto 32 the fused-silica surface via electrostatic interactions. This surface treatment greatly suppressed the 33 adsorption of lipoproteins. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles 34 were then employed in the coated capillaries as pseudostationary phase in the partial filling mode. 35 The changes in corrected migration times of steroids increased linearly with the filling time of the lipo- 36 proteins. The affinity constants between the steroids and lipoproteins were calculated, and the most 37 hydrophobic steroid studied, progesterone, had stronger affinity than testosterone or androstenedione 38 toward both LDL and HDL. Affinity between steroids and LDL was stronger than that between steroids 39 and HDL. Interactions between the steroids and lipoproteins were mainly nonspecific with particle lipid 40 components, whereas some were site specific with the apolipoproteins. The developed technique has 41 great potential for determination of the affinity of various compounds toward lipoproteins. 42 Ó 2009 Elsevier Inc. All rights reserved. 43 44 45 Introduction 46 Human plasma lipoproteins, which are nanometer-sized parti- 47 cles, are spherical molecular structures composed of a core of 48 insoluble lipids (triacylglycerols and cholesteryl esters) and a sur- 49 rounding surface layer of more polar lipids (phospholipids and 50 cholesterol) and specific lipid-binding proteins (apolipoproteins) 51 [1]. In general, lipoproteins are divided into five major classes— 52 chylomicrons (CMs), 1 very low-density lipoproteins (VLDLs), inter- 53 mediate-density lipoproteins (IDLs), low-density lipoproteins (LDLs), 54 and high-density lipoproteins (HDLs)—on the basis of their density 55 [2]. Regarding atherosclerosis, perhaps the most investigated lipo- 56 proteins have been LDL and HDL. The physiological role of LDL is 57 to provide cells with the cholesterol they need for growth of mem- 58 branes and synthesis of steroidogenic hormones [3]. In pathological 59 conditions, LDL-derived cholesterol accumulates in macrophages in 60 the inner layer of the arterial wall, the intima, and affects the devel- 61 opment of atherosclerosis and cardiovascular diseases [4]. HDL par- 62 ticles have the ability to facilitate cholesterol efflux from peripheral 63 tissues and macrophage foam cells and to transfer the cholesterol to 64 the liver in a process of reverse cholesterol transport [5–7]. HDL may 65 also afford protection from vascular disease by exerting additional 66 effects that include antioxidant, antiapoptotic, antithrombotic, anti- 67 inflammatory, and vasodilatory functions [8]. HDL has been 68 indicated not only as a marker of risk for the development of prema- 69 ture cardiovascular diseases (CADs) but also as a key mediator of 70 vascular health [8,9]. Plasma lipoprotein particles have special and 71 important functions in the body and a close relationship with the 72 causes of atherosclerosis and coronary artery diseases. Thus, 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.11.028 * Corresponding author. Fax: +358 9 191 50253. E-mail address: marja-liisa.riekkola@helsinki.fi (M.-L. Riekkola). 1 Abbreviations used: CM, chylomicron; VLDL, very low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; CAD, cardiovascular disease; SDS, sodium dodecyl sulfate; PEO, poly (ethyleneoxide); HPMC, hydroxypropylmethylcellulose; PVP, poly(vinylpyrrolidone); VP, vinylpyrrolidone; VI, vinylimidazole; P(VP-co-EDMAEMAES), poly[(1-vinylpyrr- olidone)-co-(2-ethyldimethylammonioethyl methacrylate ethyl sulfate)]; MW, molecular weight; GPC, gel permeation chromatography; P(VP-co-MVIC, poly[(1-vinylpyrrolidone)-co-(3-methyl-1-vinylimidazolium chloride)]; BGE, back- ground electrolyte; DMSO, dimethyl sulfoxide; EOF, electroosmotic flow; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; PBS, phosphate-buf- fered saline; RSD, relative standard deviation; ACE, affinity capillary electrophoresis. Analytical Biochemistry xxx (2009) xxx–xxx Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio YABIO 9740 No. of Pages 9, Model 5G 10 December 2009 ARTICLE IN PRESS Please cite this article in press as: A.-J. Wang et al., Partial filling affinity capillary electrophoresis with cationic poly(vinylpyrrolidone)-based copolymer coatings for studies on human lipoprotein–steroid interactions, Anal. Biochem. (2009), doi:10.1016/j.ab.2009.11.028