Human Low-Density Lipoprotein-Coated Capillaries in

Low-density lipoprotein (LDL) particles were immobilized on the inner wall of a fused-silica capillary and used in a study of the interactions between...
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Anal. Chem. 2005, 77, 3401-3405

Human Low-Density Lipoprotein-Coated Capillaries in Electrochromatography Ruth Kuldvee,† Susanne K. Wiedmer,† Katariina O 2o 1 rni,‡ and Marja-Liisa Riekkola†

Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, FIN-00014 University of Helsinki, Helsinki, Finland, and Wihuri Research Institute, Kalliolinnantie 4, FIN-00140, Helsinki, Finland

Low-density lipoprotein (LDL) particles were immobilized on the inner wall of a fused-silica capillary and used in a study of the interactions between LDL and neutral drugs in electrochromatography. The effect of coating parameters (pH, ionic strength of the coating solution, duration of the coating procedure) on the properties and stability of the coating was examined. The stability of the coating was highest when the pH of the coating solution was under the pI value of the LDL particles. Interactions of unmodified LDL coatings with drugs were compared with those of acetylated LDL coatings. Acetylation of LDL neutralizes the positive charge on the lysine residues of the protein component of LDL particles, and acetylated LDL was used as a reference to examine the effect of the positively charged amino acids in the unmodified coating. Under similar coating conditions, acetylated LDL coating yielded stronger EOF evidently due to the decreased number of positive charges on LDL particles. The interactions of the unmodified and acetylated LDL coatings with steroids aldosterone, testosterone, and progesterone were comparable, which indicates that the density of immobilized LDL particles is not appreciably altered by acetylation. As expected, the strength of the interactions between steroids and the LDL coating increased with hydrophobicity of the drug. Low-density lipoproteins (LDL) are lipid transport particles specialized for cholesterol carriage in the blood.1 Their physiological role is to provide cells with the cholesterol they need for growth of membranes and for synthesis of steroidogenic hormones. In pathological conditions, LDL-derived cholesterol accumulates in the inner layer of the arterial wall, the intima, and may lead to development of atherosclerosis, the major cause of cardiovascular diseases.2 It is reasonable to assume that elevated level of plasma LDL cholesterol is a strong risk factor for coronary heart disease. LDL particles can be used to deliver hydrophobic drugs to their cellular targets, and they already play an important role as * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +358 9 19150253. † University of Helsinki. ‡ Wihuri Research Institute. (1) Brown, M. S.; Kovanen, P. T.; Goldstein, J. L. Science 1981, 212, 628635. (2) Brown, M. S.; Goldstein, J. L Science 1986, 232, 34-47. 10.1021/ac048187q CCC: $30.25 Published on Web 04/12/2005

© 2005 American Chemical Society

drug delivery vehicles in tumor therapy.3,4 Knowledge of the nature and extent of interactions between drugs and LDL is important, therefore, if we are to understand and properly estimate the therapeutic effects as well as the side effects of drugs. Probing of the interactions between oxidized LDL particles and antioxidant drugs may also assist in the search for ways to protect LDL from oxidative modifications and thereby reduce the risk of atherosclerotic disease. The interactions between LDL and drugs have been studied by several HPLC methods5,6 as well as by high-performance frontal analysis combined with capillary electrophoresis (CE).7-9 The latter method has been used to study the relationship between LDL oxidation state and the drug binding affinity. LDL particles interact strongly with the capillary wall when LDL samples are injected into uncoated fused-silica capillaries.10 In most of studies reported in the literature, successful analysis of LDL has been achieved only after adsorption of the particles had been suppressed through permanent11 or dynamical12,13 coating of the capillary wall. An exception is the work of Liu et al., where a novel sample preparation technique allowed them to separate two LDL particle species without a modification of the capillary wall.14 In this work, we immobilized unmodified and acetylated LDL particles on the fused-silica capillary wall and optimized the parameters for immobilization to obtain a stable LDL coating for electrochromatography experiments. The stability of the coating (3) Urien, S. In Protein Binding and Drug Transport; Tillement, J. P., Lindenlaub, E., Eds.; Schattauer Verlag: Stuttgart, 1986; pp 63-75. (4) Bonneau, S.; Vever-Bizet, C.; Morlie`re, P.; Mazie`re, J.-C.; Brault, D. Biophys. J. 2002, 83, 3470-3481. (5) Eley, J. G.; Halbert, G. W.; Florence, A. T. J. Pharm. Pharmacol. 1989, 41, 858-860. (6) Covac, M. I.; Fito, M.; Lamuela-Raventos, R. M.; Sebastia, N.; De La TorreBoronat, C.; Marrugat, J. Int. J. Clin. Pharmacol. Res. 2000, 20, 49-54. (7) Mohamed, N. A. L.; Kuroda, Y.; Shibukawa, A.; Nakagawa, T.; Gizawy, S. E.; Askal, H. F.; El Kommos, M. E. J. Chromatogr. 2000, 875, 447-453. (8) Kuroda, Y.; Cao, B.; Shibukawa, A.; Nakagawa, T. Electrophoresis 2001, 22, 3401-3407. (9) Kuroda, Y.; Watanabe, Y.; Shibukawa, A.; Nakagawa, T. J. Pharm. Biomed. Anal. 2003, 30, 1869-1877. (10) Ceriotti, L.; Shibata, T.; Folmer, B.; Weiller, B. H.; Roberts, M. A.; de Rooij, N. F.; Verpoorte, E. Electrophoresis 2002, 23, 3615-3622. (11) Schmitz, G.; Mollers, C.; Richter, V. Electrophoresis 1997, 18, 1807-1813. (12) Stocks, J.; Miller, N. E. J. Lipid Res. 1998, 39, 1305-1309. (13) Weiller, B. H.; Ceriotti, L.; Shibata, T.; Rein, D.; Roberts, M. A.; Lichtenberg, J.; German, J. B.; de Rooij, N. F.; Verpoorte, E. Anal. Chem. 2002, 74, 1702-1711. (14) Liu, M.-Y.; McNeal, C. J.; Macfarlane, R. D. Electrophoresis 2004, 25, 29852995.

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was estimated by measuring the electroosmotic flow (EOF) mobilities. Three steroids provided models to study the interactions between immobilized LDL and neutral hydrophobic drugs. EXPERIMENTAL SECTION Materials. Dimethyl sulfoxide (DMSO) was from Oy FFChemicals Ab (Yli Ii, Finland). EDTA disodium salt, HEPES, d-aldosterone, and acetic acid (99.7%, d 1.049 kg/L) were from Sigma Chemical Co. (St. Louis MO). Testosterone and progesterone were from Merck (Darmstadt, Germany). Equipment. Electrophoretic measurements were made with a Hewlett-Packard 3DCE system (Agilent, Waldbronn, Germany) equipped with a diode array detector (detection at 200 and 254 nm) and air thermostating of the capillary. The data acquisition rate was 100 Hz and the response time of the detector 0.1 s. Uncoated fused-silica capillaries were from Composite Metal Services Ltd. (Worcestershire, U.K.). Dimensions were 50-µm i.d. and 375-µm o.d. The length of the capillary to the detector was 30.0 cm with a total length of 38.5 cm. Methods. Preparation of LDL. Human LDL (d ) 1.019-1.050 g/mL) was isolated from the plasma of a healthy volunteer by sequential ultracentrifugation in the presence of 3 mM EDTA.15,16 Briefly, solid KBr was added to plasma to adjust its density to 1.019 g/mL, and the plasma was centrifuged for 24 h at 35 000 rpm in a 50 Ti rotor to float VLDL and IDL. VLDL and IDL were removed and the density of the bottom fractions was adjusted to 1.050 g/mL with solid KBr. After ultracentrifugation for 72 h at 35 000 rpm, LDL was recovered from the top of the centrifuge tubes, recentrifuged (d ) 1.060 g/mL) for 24 h at 35 000 rpm and dialyzed extensively against LDL buffer (1 mM EDTA and 150 mM sodium chloride in water, pH 7.4, adjusted with sodium hydroxide). The amount of LDL is expressed in terms of its protein concentration, which was determined by the method of Lowry et al.17 with bovine serum albumin as standard. Preparation of Acetyl-LDL. Acetyl-LDL (ac-LDL) was obtained by acetylating the lysine residues of apoB-100 with acetic anhydride.18 The protein concentration of ac-LDL was 1.13 mg/mL. Sample and Buffer Preparation. The concentration of background electrolyte (BGE) solution was 8 mM (ionic strength 20 mM), with pH adjusted to the desired value with 1.0 M sodium hydroxide. For calculations of ionic strength of our background electrolyte, we used the pKa value of 7.2 for phosphoric acid. The BGE solution did not contain any LDL. The concentrations of original LDL samples were 4.5-5.5 mg/ mL except when interday RSD of LDL-coated capillaries was measured. Then LDL concentration varied in the range of 4.57.0 mg/mL. The LDL solutions used for coating of the capillary contained (if not stated otherwise) one-fourth of the original LDL (unmodified or modified) sample and three-fourths of distilled water or the running BGE solution. The stock solutions of steroids were prepared in methanol. The steroid sample consisted of 20 µg/mL aldosterone, 20 µg/ mL testosterone, and 50 µg/mL progesterone in BGE solution. (15) Weisgraber, K. H.; Rall, S. C., Jr. J. Biol. Chem. 1987, 262, 11097-11103. (16) Radding, C. M.; Steinberg, D. J. Clin. Invest. 1960, 39, 1560-1569. (17) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275. (18) Basu, S. K.; Goldstein, J. L.; Anderson, G. W.; Brown, M. S. Proc. Nat. Acad. Sci. U.S.A. 1976, 73, 3178-3182.

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All solutions were stored at + 4 °C. Capillary Coating. Before coating, the fresh capillary was submitted to either sodium hydroxide or hydrochloric acid pretreatment. The sodium hydroxide pretreatment consisted of the following steps: 20-min flush with 1 M NaOH, 10-min flush with 0.1 M NaOH, and 20-min flush with deionized water at a pressure of 940 mbar. The hydrochloric acid pretreatment consisted of the following steps: 20-min flush with 1 M HCl, 10min flush with 0.1 M HCl, and 20-min flush with deionized water at a pressure of 940 mbar. LDL coating was applied to the capillary inner surface as follows: after preconditioning, the capillary was flushed with LDL solution at 50 mbar for 20-40 min and left to stand filled with the LDL solution for 15 min. EOF Measurements and CE Separations. For measurement of the stability of the coatings, 6-12 successive runs with DMSO as EOF marker were performed in freshly coated capillaries. The EOF marker was hydrodynamically injected into the capillary by applying a pressure of 50 mbar for 3 s. Before the first run, the capillary was flushed for 5 min with BGE solution. Between runs, the capillary was flushed for 2 min with BGE solution. The duration of the runs ranged from 10 to 20 min depending on the mobility of the EOF marker. The total time of voltage application to one capillary coating was altogether 60-120 min. Where the EOF mobilities were close to zero, the effective mobilities were determined by the method of Williams and Vigh.19 CE separation conditions were as follows: voltage 20 kV, temperature of the capillary cassette 25 °C, and injection of steroids for 3-5 s at 50 mbar. Before runs and before each injection, the capillary was flushed for 2 min (940 mbar) with the BGE solution. RESULTS AND DISCUSSION LDL particles are spherical with a diameter of ∼22 nm. Apolipoprotein B-100 (apoB-100) is wrapped around the particle, keeping it intact. The interactions between LDL particles and the capillary wall are expected to be both hydrophobic (between the exposed lipids and capillary wall) and electrostatic. ApoB-100 determines the charge of the particle and the contribution of electrostatic interactions. The pI value of apoB-100 is 6.620 in solution and ∼5.521,22 when it is bound to lipids. The total charge of apoB-100 depends on the pH of the environment and is negative at physiological pH (which is also the pH at which we carried out the majority of our experiments). Even though the total charge of the LDL particle is negative, we presume that electrostatic interactions can take place between the negatively charged capillary wall and positively charged domains on apoB-100. In support of this, LDL particles have been found to bind to glycosaminoglycans via ionic interactions between the positively charged amino acids of apoB-100 and the negatively charged glycosaminoglycan chains.23,24 Immobilization of LDL particles on the fused-silica capillary wall offers a new approach to studying the interactions between (19) Williams, B. A.; Vigh, G. Anal. Chem. 1996, 68, 1174-1180. (20) http://au.expasy.org/cgi-bin/pi_tool1?P04114@noft. (21) Ghosh, S.; Basu, M. K.; Schweppe J. S. Anal. Biochem. 1972, 50, 592601. (22) Hurt-Camejo, E.; Camejo, G.; Rosengren, B.; Lopez, F.; Wiklund, O.; Bondjers, G. J. Lipid Res. 1990, 8, 1387-1398. (23) Camejo, G.; Hurt-Camejo, E.; Wiklund, O.; Bondjers, G. Atherosclerosis 1998, 139, 205-222. (24) Iverius, P.-H. J. Biol. Chem. 1972, 247, 2607-2613.

Table 1. Effect of Coating Time and Reconditioning between Runs on the Mobility of EOF and on the Stability of the LDL Coatinga description of the measurementb

EOF mobility (10-4 cm2/(V s)) n)6

RSD of EOF for six consecutive runs (%)c

20-min coating (A) 20-min coating (B) 40-min coating (A) 40-min coating (B)

1.5 1.9 1.7 2.1

4.83 6.06 2.30 1.10

a The coating solution was LDL solution diluted 1:3 with distilled water. b (A) capillary was flushed 1 min with coating solution between runs, followed by 1-min BGE flush. (B) capillary was flushed only 2 min with BGE between runs. c The change of EOF during measured consecutive runs was monotonic, i.e., getting slightly faster with every run.

LDL and analytes of interest and to clarifying the role of LDL as a carrier of drugs in the mammalian body. A prerequisite for such investigations is the stability of the LDL coating. Stability of the Coating. Effect of Coating Time. We evaluated the stability of the immobilized particles on the capillary wall by measuring the mobility of EOF during successive runs. After preconditioning of the capillary as described in the Experimental Section (sodium hydroxide pretreatment), the capillary was flushed with LDL solution (diluted with distilled water 1:3 (v/v)) for 20 or 40 min at low pressure (50 mbar). After rinsing, the capillary was left to stand filled with the coating solution for a further 15 min. The excess of LDL was then flushed out with BGE solution (phosphate buffer, pH 7.4, ionic strength 20 mM), and the stability of the coating was determined indirectly by measuring the EOF mobilities in the capillary during six consecutive runs (the total time of high voltage applied was 60 min). Two sets of measurements were made to determine whether the coating needed to be refreshed between runs. In the first set, the capillary was flushed between runs for 1 min with coating solution (containing LDL) and 1 min with BGE solution. In the second set, the capillary was flushed for 2 min with BGE solution between runs. Table 1 shows the results of the measurements. As can be seen from Table 1, the prolongation of the coating time from 20 to 40 min does not influence remarkably the EOF mobility in capillary. However, the RSD values for EOF mobilities are decreased with longer LDL flush (Table 1, second column). Evidently, some time is needed for the LDL coating to achieve a stable state. Table 1 demonstrates also that refreshing the capillary with LDL coating solution between runs caused a slight decrease in the EOF mobilities. At shorter coating time (20-min LDL flush), the LDL flush also increased the stability of the coating, whereas at longer coating time the effect was the opposite. The most stable coating was obtained when the capillary was flushed with LDL solution for 40 min with no LDL flush between runs. This result suggests that once the inner surface of the capillary has been covered with LDL particles at optimum density, a further LDL flush disturbs the established equilibrium. Increase of coating time beyond 40 min did not result in any significant change. And longer time was then needed for stabilization. The optimal coating procedure was thus 40-min flush with coating solution followed by 15-min standing time.

Table 2. Influence of Ionic Strength on the Capillary Coating

ionic strength of the coating solution (mM)

EOF mobility (10-4 cm2/ (V s))

EOF change during five consecutive runs (%)a

11 (LDL sample/water)b 43 (LDL sample/water)c 58 (LDL sample/BGE solution)c 170 (LDL sample/LDL buffer)c 433 (additional NaCl was added)

2.38 2.46 2.18 2.48 3.39

4.6 2.9 1.5 5.5 14

a The change of EOF during measured consecutive runs was monotonic. b The v/v ratio of LDL sample and water was 1:15. c The v/v ratio of LDL sample and the diluting solution was 1:3.

Effect of Preconditioning. In our previous projects (studies of different phospholipid-coated capillaries), it was noticed that acid pretreatment gives better results (i.e., the coatings are more stable),25 a possible reason being that acids dissolve more effectively the impurities on capillary surface. Therefore, we studied also the effect of preconditioning on LDL coating. The effect of the capillary pretreatment was tested by conditioning with hydrochloric acid solution in place of sodium hydroxide. The EOF measurements of 12 successive runs demonstrated that with acid pretreatment the LDL coating had slightly better stability of the coating (Supporting Information, Figure S-1). Therefore, hydrochloric acid pretreatment was used in the remaining studies. Effect of Ionic Strength. The ionic strength of the LDL coating solution may affect the results of the coating. To estimate the effect, we prepared five different coating solutions, which all differed in their ionic strength. We made two different dilutions with distilled water, one dilution with BGE, one dilution with LDL buffer, and one with BGE where additional sodium chloride was added. Then we carried out a regular coating procedure. The results of the coatings are shown in Table 2. It can be seen that it does not affect the EOF mobilities markedly if the LDL sample is diluted with water or BGE or LDL buffer. The RSDs of the EOF mobilities are somewhat higher for the solution with the lowest ionic strength (the ratio of LDL and water is 1:15) and when LDL buffer was used as diluting agent. Addition of sodium chloride to the coating solution (last line in Table 2) increased the EOF mobilities and had a significant effect on their RSDs. During five successive runs, the EOF increased monotonically 14%. The results indicate that, at higher ionic strengths, the interactions between silanol groups on the fused-silica wall and LDL particles are suppressed. However, whether the LDL sample was diluted with water or with BGE solution did not seem to have any significant impact on the coating. Therefore, to ensure the desired pH of the coating solution, the LDL sample was diluted 1:3 (v/v) with phosphate buffer (pH 7.4) in further experiments, unless stated otherwise. Lifetime and Repeatability of Coating. Studies were made of the lifetime of LDL-coated capillaries and the effect of reconditioning on the capillary coating performance. For lifetime studies, the EOF mobilities were measured on 2 successive days with 12 runs on each day. The capillaries were left filled with BGE solution (25) Hautala, J. T.; Wiedmer, S. K.; Riekkola, M.-L. Anal. Bioanal. Chem. 2004, 378, 1769-1776.

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overnight, at 25 °C. The results showed that the initial mobility of EOF on day two was slightly lower relative to the last run on day one. The EOF mobility changed ∼4% over 4 first runs on the first day but showed very little tendency to increase during the rest of 20 runs. The RSD of EOF mobilities for 20 successive runs over a 2-day period was 1% (here the change was not monotonic). Experiments with model compounds (see below) demonstrated that the retention factors of the studied analytes (i.e., their interactions with LDL coating) stayed unchanged over a 2-day period. We conclude that an LDL coating can be used for at least 2 days without reconditioning. We next studied the effect of reconditioning the LDL-coated capillary (repetition of the coating procedure without removal of the previous LDL coating). EOF measurements were made on an LDL-coated capillary (LDL flush 40 min), and after soaking in BGE solution overnight (at 25 °C), the capillary was reconditioned the next morning. Reconditioning was done on 3 consecutive days, and each day, 10 runs were measured (Supporting Information, Figure S-2). Even though the EOF mobility did not change dramatically over these 4 days, the mobility of EOF (2.3 × 10-4 cm2/(V s) in freshly coated capillary) was increased after the second and third reconditioning by 10-15%. The first reconditioning appeared to improve the capillary performance; that is, the initial mobility of EOF was the same, but the stability was slightly better (RSD of 10 successive runs 1.9 vs 2.6%). This supports the previous findings that the LDL coating is still intact on the second day of use and its stability may even be improved. The interactions of aldosterone, testosterone, and progesterone with fresh capillary coating and reconditioned capillaries were studied. All three model compounds are neutral drugs, with hydrophobicity increasing from aldosterone to progesterone. As shown in Figure 1A, aldosterone is least retained and progesterone is most strongly retained by the LDL coating. The retention factors, k, in Table 3 show the same trend. As demonstrated in Table 3, the retention factors of the model compounds are not significantly influenced by the reconditioning of the coating. Coating-to-Coating Repeatability. To determine the repeatability of LDL coatings, five capillaries were coated by using the same procedure (see Capillary Coating in the Experimental Section). The LDL samples were diluted 1:3 (v/v) with BGE (phosphate buffer, pH 7.4, I 20 mM). The LDL samples were prepared from different plasma samples and varied in concentration (4.5-7.0 mg/ mL). The age of LDL samples varied from 1 to 4 weeks. The RSD of the EOF mobility in five different capillaries was 6%. Every EOF mobility value was an average of four successive runs. Effect of pH. According to the present model of an LDL particle, apoB-100, which represents the protein part of LDL, is wrapped around the lipid core in beltlike fashion. The charge of the LDL particle depends on the charge of the protein, and the total charge of the protein is pH dependent. It can be expected, therefore, that the pH has a significant effect on the properties of the LDL coating in the capillary. The isoelectric point (pI) of the LDL particles is suggested to be ∼5.5. No data are available on the pI of LDL particles immobilized on the fused-silica capillary wall. We chose the pH range 4.5-7.4 to investigate the effect of pH on the LDL coating. This range ensured that the hypothetical pI of the LDL particle was covered, as well as the physiological pH (7.4). In all coating procedures, the LDL sample was diluted in 3404 Analytical Chemistry, Vol. 77, No. 10, May 15, 2005

Figure 1. Separation of steroids in fresh LDL-coated capillaries. Coating solution: (A) unmodified LDL and (B) ac-LDL, both diluted 1:3 (v/v) with BGE solution. Coating conditions: 40-min rinsing (at 50 mbar) and 15 min standing with coating solution. BGE solution: phosphate, I ) 20 mM, pH 7.4. Running conditions: fused-silica capillary with total length of 38.5 cm (30 cm to the detector) and i.d./ o.d. 50/375 µm; capillary temperature 25 °C; injection 5 s at 50 mbar; running voltage 20 kV; detection at 254 nm. Between runs the capillary was flushed for 2 min with BGE solution. Peaks: 1, aldosterone; 2, testosterone; 3, progesterone. Table 3. Effect of Reconditioning of Capillaries on the Interactions between LDL Coating and Steroids As Measured by the Mobility of Steroids and Retention Factor k aldosterone

testosterone

progesterone

coating

RSDa

k

RSDa

k

RSDa

k

fresh I reconditioning II reconditioning III reconditioning

5.67 2.79 2.72 3.25

0.02 0.02 0.02 0.01

6.09 3.01 2.74 3.20

0.12 0.12 0.12 0.12

6.09 3.21 2.91 3.64

1.06 1.06 1.10 1.07

a RSD of the retention time of corresponding steroid (the mean value of six successive runs).

the corresponding BGE solution. Phosphate buffer was used as BGE solution for pH values 6.5 and 7.4, and acetate buffer for pH values 4.5-5.5. The LDL sample was stable throughout the pH range tested, and capillary coatings were of good stability. As seen from Figure S-3 (Supporting Information), the stability of the capillary wall appeared to be the highest at pH value 4.5, where the total charge of the LDL particles is assumed to be positive. Figure 2 shows the dependence of the EOF mobility on the pH. As can be seen, the EOF mobility increases with pH, as expected, and the linear trendline correlates well with the measured data points (correlation coefficient greater than 0.995). The results show that at pH of ∼5.5 the EOF mobility is zero, which suggests that the pI of an LDL particle attached to the fused-silica capillary wall is close to 5.5. The results further indicate that immobilization of an LDL particle to the capillary wall does not have a significant effect on its pI value, and the interactions between LDL particles

Figure 2. Influence of pH on the mobility of EOF in LDL-coated capillaries. The BGE solution was phosphate for measurements at pH 7.4 and 6.5 and acetate for measurements at pH 5.5-4.5. Ionic strength of the BGE solution was 20 mM. DMSO was used as EOF marker. EOF measurements were carried out at 200 nm. Coating and running conditions as in Figure 1.

Figure 3. Comparison of EOF mobility and stability of the coating in LDL and ac-LDL-coated capillaries at pH values 7.4 and 5.0. Coating and running conditions as in Figure 1.

and capillary wall are to a large extent governed by hydrophobic forces. At pH below 5.5, the EOF was reversed (Figure 2). Effect of Modification. To determine whether the LDL interactions with capillary wall are hydrophobic or electrostatic, we tested ac-LDL as an example of an LDL modification for the capillary coating. The ac-LDL has less positive charge than unmodified LDL. Thus, if the binding to the capillary is mediated mainly by the positively charged domains of protein (apoB-100), the modified lipoproteins should bind less effectively. Figure 3 shows the EOF mobilities of the unmodified LDL coating and the ac-LDL coating at pH values of 7.4, which is relevant as physiological pH, and 5.0, at which the unmodified LDL is below its pI value. Comparison of the EOF values at pH 7.4 shows that the EOF mobilities are faster in the ac-LDL-coated capillary. The lower positive charge of the ac-LDL particles than unmodified LDL particles explains the difference. The coating stability is better for the ac-LDL-coated capillaries: the RSD of the EOF mobility over 12 successive runs

(including the first runs) was ∼3% for the unmodified LDL capillary and just 0.2% for the ac-LDL-coated capillary. When the coating and running pH were lowered to 5.0, where the unmodified LDL coating exhibits EOF close to zero (or slightly reversed EOF), the EOF mobility in the ac-LDL-coated capillary was still positive (movement toward the cathode) and the difference in the pH-related EOF mobility was smaller than for the unmodified LDLcoated capillaries. The cathodic EOF at pH 5.0 in the ac-LDLcoated capillary is in keeping with expectation, as the pI of the ac-LDL is clearly lower than that of unmodified LDL. As demonstrated in Figure 1A,B, the separation of steroids in unmodified and ac-LDL-coated capillaries follows exactly the same pattern. The shorter analysis time in the ac-LDL capillaries is caused by the greater EOF mobility. The retention factors of the three steroids exhibit the same trend in LDL and ac-LDL-coated capillaries at pH 7.4, being only slightly higher for the unmodified LDL capillary (Supporting Information, Table S-1). The experiments demonstrate that fused-silica capillaries can be coated with unmodified or modified LDL particles and that both coatings are stable enough to allow study of the influence of different factors on the behavior of the particles, as well as on the interactions between LDL particles and analytes of interest. CONCLUSION A method has been developed for the analysis of LDL-steroid interactions by capillary electrochromatography. The results demonstrate that both modified and unmodified LDL particles can be successfully bound to the fused-silica capillary wall. Reconditioning of the coating by flushing with coating solution between runs is not required. The coating is stable for at least 2 days and is insensitive to minor changes in experimental setup (10-fold changes in ionic strength, dilution of the coating solution, or coating time). The coating is stable over pH range 4.5-7.4. Capillary electrochromatography with LDL-coated capillaries gives a promise for a new approach to LDL-drug interaction studies, making it possible to perform studies under widely different conditions. ACKNOWLEDGMENT Financial support was provided by the Academy of Finland under Grants 210194 and 20914 (M.-L.R.), 206296 (M.-L.R. and R.K.), 78785 (S.K.W.). and 80631 (K.O ¨ .). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review December 9, 2004. Accepted March 17, 2005. AC048187Q

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