Potent Capillary Isotachophoresis (cITP) for Analyzing a Marker of Coronary Heart Disease Risk and Electronegative Low-Density Lipoprotein (LDL) in Small Dense LDL Fraction

Abstract

apillary isotachophoresis (cITP) is a newly established technique for characterizing plasma lipoprotein subfractions according to their electric charges. It separates plasma lipoproteins into 8 fractions, comprising 3 high-density liporotein (HDL) fractions with fast (f), intermediate (I), and slow (s) electric mobility, a chylomicron/remnants fraction (fast very low-density lipoprotein (VLDL)), a VLDL/intermediate density lipoprotein (IDL) fraction (slow VLDL), 2 LDL fractions with fast and slow electric mobility, and a minor LDL fraction. [1][2][3][4][5] In our previous reports, the fast LDL fractions corresponded to electronegative LDL in which some oxidized LDL, VLDL, and small dense LDL etc, the so-called "bad cholesterol", are concentrated. 4 After the light LDL fraction is precipitated from whole serum using heparin-Mg 2+ , electronegative LDL in the small dense LDL fraction can be measured using cITP. 4 In the present study we used this method before and after treatment with fenofibrate. Methods Quantification of Lipoprotein Subfractions by cITPcITP of serum lipoproteins was performed on a Beckman P/ACE MDQ system (Beckman-Coulter Inc, Tokyo, Japan) according to the method of Bottcher et al 6 with some modifications. All of the reagents used in the cITP analysis were purchased from Aldrich-Sigma (Tokyo, Japan) unless indicated otherwise. Dimethylpolysiloxane modified fused silica capillary (AT™-1) was purchased from Alltech Japan Inc (Tokyo, Japan). A 6 l sample of serum was diluted with 14 l of leading buffer consisting of 10 mmol/L HCl and 18 mmol/L ammediol (2-amino-2-methyl-1,3-propanediol) (pH 8.8), prestained with 10 l 0.1 mg/ml NBD C6-ceramide (Molecular Probes, Inc, OR, USA) for 5 min at room temperature, and mixed with 50 l of a mixture containing leading buffer with 0.35% hydroxypropylmethylcellulose, spacers, and 5-carboxy-fluorescein as an internal marker. The spacers included were the same as those described by Bottcher et al, including N-(2-acetamido)-2-aminoethanesulfonic acid, D-glucuronic acid, 1-octanesulfonic acid sodium salt, 3-(N-tris[hydroxymethyl]methylamino)-2-hydroxypropanesulfonic acid, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, L-serine, L-glutamine, L-methionine, and glycine. The terminating buffer contained 24 mmol/L -alanine and 13 mmol/L ammediol, and was adjusted to pH 10.5 with saturated barium hydroxide solution. The sample was injected for 18 s at 20 pounds into a 30 cm-long capillary (inner diameter 180 m), and separation was performed at a constant 30 mA for 1 min and 10 kV for 7 min. The separated zones were monitored with argon-laser-induced fluorescence detection (excitation, 488 nm; emission, 520 nm). Each peak was identified and the peak area in relative fluorescence units was analyzed using 32 Karat Software version 5.0 (Beckman-Coulter Inc). The capillary was washed with leading buffer and water between runs. Separation of Light and Dense LDL Subfractions by Heparin-Mg 2+ PrecipitationThe light LDL subclass was separated from the dense LDL subclas...

Novel Pharmaceutical and Nutraceutical-Based Approaches for Cardiovascular Diseases Prevention Targeting Atherogenic Small Dense LDL

“…However, as recently reviewed by Rivas-Urbina et al [13], a full spectrum of biological roles of electronegative LDL in various pathological conditions remains to be elucidated. Interestingly, it has been shown that the amount of electronegative LDL subfractions determined by capillary isotachophoresis correlates negatively with LDL particle size, thus suggesting that electronegative LDL is accumulated in the sdLDL fraction [14]. In addition, Zhang et al [15] demonstrated that rosuvastatin treatment significantly reduced the electronegative LDL contained in both large and sdLDL, thereby implying another pleiotropic effect of statins.…”

The role of electrophoresis in disease biomarker discovery

“…Correct identification of patients with urothelial carcinoma from those with other malignant and nonmalignant genitourinary disease ranged from 86% to 100%. cITP has been used to search for a biomarker of coronary heart disease risk and electronegative low-density lipoprotein (LDL) in a small dense LDL fraction [55]. The electronegative LDL fraction decreased after treatment with fenofibrate.…”

Recent progress in capillary ITP

“…The same system was used to quantify charge-based low-density lipoprotein (LDL) subfractions, to examine the relation between insulin resistance and the ITP fast-migrating LDL levels [75], to determine three charge-based high density lipoprotein subfractions, and to investigate the effects of the inhibition of cholesteryl ester transfer protein activity by an inhibitor in Japanese white rabbits [76]. Also the same system was used to measure electronegative LDLs in the small dense LDL fraction [77] and a very similar system served for monitoring the changes in lipoprotein profile at hypertriglyceridemia [78].…”