Vol.2, No.7, 722-730 (2010) Health doi:10.4236/health.2010.27110 Influence of poloxamer 407 on fractional and subfractional composition of serum lipoproteins of mice Tatyana A. Korolenko1*, Fedor V. Tuzikov2,3, Thomas P. Johnston4, Natalia A. Tuzikova2,3, Elena E. Filjushina1, Viktoriya M. Loginova1, Natalia G. Savchenko1 1Institute of Physiology, Siberian Branch of Russian Academy of Medical Sciences, Novosibirsk, Russia; *Corresponding Author: [email protected] 2Boreskov Institute of Catalysis, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia 3Novosibirsk State University, Novosibirsk, Russia 4University of Missouri-Kansas City, Kansas City, USA Received 4 March 2010; revised 22 March 2010; accepted 25 March 2010. ABSTRACT Serum Lipoprotein Fractions and Subfractions Using a novel small-angle X-ray scattering (SAXS) method for determination of fractional and sub- 1. INTRODUCTION fractional composition of lipoproteins (LPs), a significant elevation of total cholesterol-lipop- Changes of different classes of circulating lipoproteins roteins (C-LP) and, especially, total triglyceride- are the important indicies of lipid metabolism in physi- lipoproteins (TG-LP), was shown in this work. ology and pathology; lipoproteins have been shown to Among the LP fractions, poloxamer 407 was also play a regulatory role in vivo [1,2]. The main lipo- shown to significantly increase proatherogenic protein classes consist of pro-atherogenic low-density li- total C-LDL, TG-LDL and, especially, their pre- poproteins (LDL), very-low-density lipoproteins (VLDL), cursors C-VLDL and TG-VLDL, while only ex- and anti-atherogenic high density lipoproteins (HDL), hibiting a moderate increase in the antiathero- and are widely used as common lipid biomarkers in genic C-HDL and TG-HDL fractions. With regard atherosclerosis [1]. However, the biological role of sub- to the VLDL subfractions, significant elevations classes of lipoproteins is still under investigation. The were observed in both subfractions studied; regulatory role of lipoproteins has been shown to relate namely, C-VLDL1-2 and C-VLDL3-5. Similar chang- to many intracellular processes, primarily to plasma es were noted in the TG-VLDL1-2 and TG- VLDL3-5 membrane permeability and fluidity as the result of subfractions. The C-IDL and TG-IDL subfrac- changes in the concentration of plasma membrane cho- tions were increased significantly (20- to 30- lesterol, with subsequent modifications of receptors and fold), while the C-LDL1-3 subfraction was mod- transmembrane proteins (transmitters). In this process, erately (3- to 5-fold) increased at 48 hrs and at the role of total HDL and HDL subfractions have espe- day 4. In the moderately elevated (2- to 4-fold) cially high importance in connection with their unique anti-atherogenic HDL fraction, the C-HDL2 sub- capacity to accept cholesterol from the plasma mem- fraction was increased more significantly (4- brane and transport them to other classes of lipoproteins. fold) compared to the C-HDL3 subfraction; how- Hyperlipidemia is one of the main risk factors in the ever, both C-HDL subfractions returned to base- development of cardiovascular and cerebrovascular dis- line by day 4. The elevation in the TG-HDL2 eases common in contemporary society. In addition to subfraction was observed only at 24 hrs. Mouse the elevation of plasma LDL-cholesterol and VLDL- models of hyperlipidemia and atherosclerosis cholesterol, hypertriglyceridemia is suggested to be an are useful to evaluate the role of “individual” additional independent risk factor for atherosclerosis and LPs, as well as their fractions and subfractions, coronary heart disease [3]. According to recent data, in hyperlipidemia and the genesis of athero- atherosclerosis is not only a metabolic lipid disease, but sclerosis. has also been considered to result from inflammation (chronic inflammatory disease). Statins have been re- Keywords: Poloxamer P-407; Dyslipidemia; ported to not only lower LDL by inhibiting the activity Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/ T. A. Korolenko et al. / HEALTH 2 (2010) 722-730 723 of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lesterolgenesis and down-regulating low-density lipo- reductase, but also by reducing inflammation to endo- protein receptor expression [6]. The mechanism of ele- thelial cells. vation of different classes and, especially subfractions of For the prevention of atherosclerosis, it is important to lipoproteins with pro- and anti-atherogenic effects, is study the mechanisms of the early effects of hyperlipi- still not known. demia on different cell types involved in the pathogene- Small-angle X-ray scattering (SAXS) is a small-angle sis of atherosclerosis. Therefore, it is necessary to know scattering technique where the elastic scattering of the types of lipoproteins involved in hyperlipidemia at X-rays (wavelength 0.1 to 0.2 nm) by a sample, which the early stages of atherosclerosis. It is well known that has inhomogeneities in the nanometer range, is recorded during the process of atherosclerosis, foam cells initially at very low angles. This angular range contains informa- trap the oxidized LDL molecules via scavenger receptors. tion about the shape and size of macromolecules, char- The oxidized LDL molecule is digested and transformed, acteristic distances of partially ordered materials, pore and is then presented to T lymphocytes, initiating the sizes, and other data. SAXS is capable of delivering classic immunological reaction, and subsequently stimu- structural information of macromolecules between 5 and lates the inflammatory response. Formation of lipid-laden 25 nm, of repeat distances in partially ordered systems of cells (mainly smooth muscle cells and macrophages) is up to 150 nm. SAXS is used in the characterization of followed by increased secretion of pro-inflammatory various materials. In the case of biologic macromole- cytokines (like IL-6) and other factors (several types of cules, such as proteins, the advantage of SAXS, over matrix metalloproteases), which promote inflammation. crystallography, is that a crystalline sample is not needed. Statin treatment has a pleiotropic protective effect, not The materials can be solid or liquid and they can contain only by inhibiting HMG-CoA reductase, but also by ex- solid, liquid, or gaseous domains (so-called particles) of erting its anti-inflammatory action. the same or another material in any combination. SAXS The changes of serum lipoprotein levels responsible is accurate, non-destructive, and usually requires only a for pro- and anti-atherogenic action have been studied minimum of sample preparation. earlier [2]. However, recently, with help of new methods The aim of the present investigation was to evaluate for characterizing different lipoprotein fractions and the lipoprotein-cholesterol and lipoprotein-triglyceride subfractions, some new data have been obtained on their fractions and subfractions in hyperlipidemia induced by role in the pathogenesis of atherosclerosis [4]. For this a single dose of poloxamer 407 to mice. Our overarching reason, the investigation of experimental models of hy- goal was to quantify the changes in the serum lipopro- perlipidemia is useful. tein levels after poloxamer 407 treatment by utilizing a Poloxamer 407 is a block copolymer comprised of novel technique; specifically, small-angle X-ray scatter- polyoxyethylene and polyoxypropylene units, which is ing (SAXS). known for its biocompatibility and potential to deliver different medications for a variety of disease states [5,6]. 2. MATERIALS AND METHODS Following acute administration, poloxamer 407 was shown to induce significant hyperlipidemia, a model 2.1. Materials which has been used for testing several hypolipidemic drugs (statins, fibrates, and nicotinic acid) [7-11]. With Male CBA/C57BL mice (breeding station of the Institute chronic administration of poloxamer 407 (4 months) to of Cytology and Genetics, Russian Academy of Sciences, mice, fibrofatty aortic lesions are developed, which are Novosibirsk, Russia) having a body mass of 20-25 g similar in size and number to those observed in classic were used. Poloxamer P-407 (Pluronic F-127, Sigma) diet-induced mouse models of atherogenesis [12,13]. was administered to mice, as a single, i.p. injection, in a Hyperlipidemia in acute poloxamer 407-induced ad- dose of 1000 mg/kg. The animals were decapitated at 3, ministration to rodents was characterized by a dramatic 24, 48 hrs, and then at 4, 5, 7, 12, 14 days after a single elevation of both plasma cholesterol and triglycerides, dose of poloxamer 407. All experiments followed the which is usually not observed in patients with athero- official “Rules for the work involving experimental sclerosis. In the poloxamer 407 mouse model of athero- animals” and “Ethical Committee Recommendations of sclerosis, the mechanism of cholesterol and TG elevation working with laboratory animals”. is associated with inhibition of cholesterol 7-hydro- Serum was obtained after centrifugation of blood sam- xylase and lipoprotein lipase, respectively [13], and not ples at 3000 × g for 15 min at 4C (Eppendorf Centri- dependent on PPARα [14]. It was also shown that a sin- fuge 5415R, Germany) and stored at –70C until analy- gle injection of poloxamer 407 administration to mice sis of total cholesterol (C), triglycerides (TG) of lipo- caused hypercholesterolemia by inducing transient cho- proteins (LP): C-LP, TG-LP, their fractions, and subfrac- Copyright