1H NMR investigation on interaction between ibuprofen and lipoproteins

Chemistry and Physics of Lipids 148 (2007) 105–111

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H NMR investigation on interaction between ibuprofen and lipoproteins Wenxian Lan a,b , Hang Zhu a,b , Zhiming Zhou a,b , Chaohui Ye a , Maili Liu a,∗

a

Wuhan Center for Magnetic Resonance, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, PR China b Graduate School of Chinese Academy of Sciences, Beijing 100049, PR China Received 22 November 2006; received in revised form 27 April 2007; accepted 27 April 2007 Available online 10 May 2007

Abstract A large number of studies indicate that oxidative modification of plasma lipoproteins, especially low-density lipoprotein (LDL), is a critical factor in initiation and progression of atherosclerosis. We have previously found that ibuprofen (IBP), a potential antioxidant drug to inhibit LDL oxidation, interacted with lipoproteins in intact human plasma. In the present study, we compare the binding affinities of IBP to LDL and HDL (high-density lipoprotein) by 1 H NMR spectroscopy. When IBP is added into the HDL and LDL samples, the N+ (CH3 )3 moieties of phosphatidylcholine (PC) and sphingomyelin (SM) in lipoprotein particles experience the chemical shift up-field drift. Intermolecular cross-peaks observed in NOESY spectra imply that there are direct interactions between ibuprofen and lipoproteins at both hydrophobic and hydrophilic (ionic) regions. These interactions are likely to be important in the solubility of ibuprofen into lipoprotein particles. Ibuprofen has higher impact on the PC and SM head group ( N+ (CH3 )3 ) and (CH2 )n group in HDL than that in LDL. This could be explained by either IBP has higher binding affinity to HDL than to LDL, or IBP induces orientation of the phospholipid head group at the surface of the lipoprotein particles. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: NMR; Lipoprotein; Ibuprofen; Antioxidant; Interaction; Affinity

1. Introduction As the key components in regulation of lipid transport in circulation, lipoproteins are generally classified by their size and density as very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and highdensity lipoprotein (HDL) (Brown and Goldstein, 1986).

∗ Corresponding author. Tel.: +86 27 87197305; fax: +86 27 87199291. E-mail address: [email protected] (M. Liu).

They have a spherical structure with a hydrophobic core of nonpolar triglyceride (TG) and cholesteryl esters (CE) surrounded by an amphipathic surface of apolipoproteins, cholesterol and phospholipids, predominantly phosphatidylcholine (PC) and sphingomyelin (SM) (Hevonoja et al., 2000). HDL and LDL are two of the most abundant and important lipoprotein fractions, and are responsible for cholesterol reversible transportation in blood. It is well established that LDL cholesterol (LDL-C) has positive correlation with atherosclerosis (Cullen and Assmann, 1997), while increasing data shows that HDL cholesterol (HDL-C) is in the con-

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trary (Barter et al., 2003). A large number of studies indicate that oxidative modification of LDL plays a key role in initiation and progression of atherosclerosis (Witztum and Steinberg, 1991; Colles et al., 2001). It has been reported that many antioxidants could inhibit oxidation of LDL in several experimental models (Huang et al., 1999; Zapolska-Downar et al., 1999; Naderi et al., 2003; Upston et al., 2003; Gonen et al., 2005; Tavridou et al., 2006). Most studies were carried out in vitro with isolated LDL focusing on antioxidant efficiency to protect LDL (Zapolska-Downar et al., 1999; Naderi et al., 2003). In order to understand mechanism of the protection, it is essential to study antioxidant–lipoprotein interaction at molecular and atomic resolutions. Nuclear magnetic resonance (NMR) spectroscopy can provide information at both molecule and atomic levels under physiological or ‘near-physiological’ conditions. As a unique tool, NMR has been widely used to investigate drug–protein interaction, and to derive information of binding site and affinity, conformation and dynamics in a noninvasive manner. Such information is included in NMR observable parameters, such as chemical shift (Medek et al., 2000; McCoy and Wyss, 2002; Cui et al., 2003; Yang et al., 2004), relaxation time (Liu et al., 1997a; Veglia et al., 1998; Delfini et al., 2000; Bai et al., 2005), diffusion coefficient (Hajduk et al., 1997; Liu et al., 1997a,b; Luo et al., 1999; Yang et al., 2004), and nuclear Overhauser effects (NOE) (Meyer et al., 1997; Chen and Shapiro, 1998). Another significant advantage of NMR is that magnitude of a NMR signal, regardless of its chemical shift, is proportional only to the number of nuclei responsible for the signal. This makes 1 H NMR a sensitive and useful tool for semi-quantitative and quantitative analysis. Ibuprofen (IBP) is a well-known nonsteroidal antiinflammatory drug and could act as antioxidant to inhibit oxidation of LDL in a high dose-dependent manner over the concentration range 0.1–2.0 mM (100 ␮g/mL LDL) (Zapolska-Downar et al., 1999, 2000). In our previous work, we found that IBP caused chemical shift up-field drifts for the protons of N+ (CH3 )3 moieties of phosphatidylcholine and sphingomyelin, olefinic chains from unsaturated lipids in lipoprotein particles in intact human blood plasma (Yang et al., 2004). Diffusion coefficient measurements suggested that the protons that experienced chemical shift changes were from relatively small and rapidly diffusing lipoprotein particles. From that, we primarily presumed IBP might interact with lipoproteins in plasma (Yang et al., 2004). In this article, we provide evidences that electronic and hydrophobic

interactions are likely to be important in the solubility of ibuprofen into lipoprotein particles. We also show that ibuprofen has higher binding affinity to HDL than to LDL. 2. Materials and methods 2.1. Materials Ibuprofen sodium salt was purchased from Sigma (Poole, Dorset, UK). Na2 EDTA, KBr, NaCl were from Shanghai Chemical Reagent Corporation (China). All chemicals were used without further purification. For convenience, the numbering systems and molecular structure of IBP are shown in Scheme 1. 2.2. Isolation of HDL and LDL Vein blood was taken from a fasted female subject. The sample was moved into a tube containing 0.1% EDTA and centrifuged at 3000 rpm for 20 min to obtain plasma. HDL and LDL were isolated by one-step density gradient ultracentrifugation (Chung et al., 1980). After adjusting density to 1.30 g/mL by adding solid KBr, the plasma sample was distributed into polycarbonate centrifuge tubes. The density gradient solution (1.006 g/mL) was layered over the density-adjusted plasma. The tubes were ultracentrifuged with a Beckman Ti 80 rotor at 50,000 rpm for 350 min at 4 ◦ C. Lipoproteins were dialyzed extensively at 4 ◦ C against phosphate buffer, pH 7.0, containing, e.g., 137 mM NaCl, 2.7 mM KC1, 10 ␮M Na2 EDTA, 0.01 M Na-phosphate, resulting in fractions of HDL and LDL. The lipoprotein samples were stored at 4 ◦ C until required for NMR analysis. The PAGE-SDS analysis of isolated HDL and LDL fractions indicated absence of significant contamination with plasma proteins and other lipoprotein fractions. Protein concentrations were measure using modification of the Lowry procedure (Lowry et al., 1951) with bovine serum albumin as a standard. The protein concentrations of HDL and LDL samples are 0.39 and 0.31 g/L, respectively.

Scheme 1. The structure and numbering systems of ibuprofen.

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2.3. NMR sample preparation

3. Result and discussion

Three 0.40 mL lipoprotein samples were taken from the storages of HDL and LDL, respectively. In each of the samples, 0.10 mL deuterium phosphate buffer (pD 7.0, 0.1 M Na2 -phosphate) was added for NMR magnetic field frequency lock, and certain amount of IBP was introduced to make final IBP concentrations of 0.0, 10.0, and 20.0 mM, respectively. TSP was added as an internal chemical shift standard (final TSP concentration 0.025%). All samples stored in refrigerator (4 ◦ C) before use and all experiments were completed in a week after lipoprotein isolated. The concentration ratio of IBP to LDL is comparable to the reported value (ZapolskaDownar et al., 2000).

3.1. Binding affinity difference of ibuprofen to HDL and LDL

2.4. NMR measurements All NMR experiments were carried out on a Varian INOVA500 spectrometer operating at 1 H frequency of 500.12 MHz. The samples were equilibrated in the probe for above 30 min at 37 ◦ C before NMR experiments. One dimension (1D) 1 H NMR spectra were obtained conventionally with water suppression using Watergate W5 pulse sequence with double-echo (Liu et al., 1998) with 256 scans, 32k data points and a pulse repetition time of 5 s. These time domain data were multiplied by an exponential function equivalent to a 0.3 Hz line-broadening factor and were zero-filled by a factor of two prior to Fourier transformation (FT). NOESY spectra were acquired by standard water suppressed NOESY sequence, with mixing time of 200 ms and 2048 (F2) × 256 (F1) complex data points with 32 scans. The time domain data were weighted using a sinebell (π/2–π) window function and zero-filled to 1024 (F1) before FT. The bipolar gradient longitudinal eddycurrent delay (LED-BPP) sequence (Wu et al., 1995) was used to measure the self-diffusion coefficients using a diffusion time (Δ) of 100 ms, an eddy current recovery time (Te ) of 50 ms and a relaxation delay (RD) of 2 s with the gradient pulse duration (δ) of 1.0 ms and strength (G) increased linearly from 10 to 55 G/m in 16 steps. The areas of the well-resolved peaks from the phospholipid head group ( N+ (CH3 )3 ) in presence of 20 mM IBP were used for diffusion coefficient calculation by the two-parameter exponential fitting according to equation A(b) = A(0) exp(−bD), where b = (2γδG)2 (Δ − δ/3), γ is the 1 H gyromagnetic ratio, A(b) and A(0) are the NMR signal areas in the presence and absence of the gradient pulses, and D is the diffusion coefficient. The estimated error on D is approximately 5% from the fitting process.

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Fig. 1 shows 1 H NMR spectra of HDL (A1–A3) and LDL (B1–B3) in absence (A1, B1) and presence of 10.0 mM (A2, B2) and 20.0 mM (A3, B3) ibuprofen. Spectral region contained resonance of the N+ (CH3 )3 moieties of phosphocholine head group and (CH2 )n (L2) are enlarged and plotted at the right column to emphasize ibuprofen induced chemical shift changes. The resonances of the N+ (CH3 )3 head group are deconvoluted into two peaks using Lorentzian lineshapes (Origin7.0, OriginLab Cooperation). The lineshape, chemical shifts (δ3.25, δ3.24) and area ratio (1.5:1) of the two peaks in absence of IBP for LDL are in good agreement with that of the native LDL (Murphy et al., 1997). The two peaks are, accordingly, assigned to phosphatidylcholine (PC, δ3.25) and sphingomyelin (SM, δ3.24) of the LDL. For HDL, the two peaks appear at δ3.22 and δ3.21 with area ratio of about 5.5:1 in absence of IBP. Considering HDL contained about 80% PC and 13% SM (Fournier et al., 1997; Khrenov et al., 2002), we assign the peak at δ3.22 for PC and δ3.21 for SM. The fitting results are presented in the expanded plots (Fig. 1) using dotted lines for PC (red), SM (blue) and the difference (green) between experimental and simulated profile of the band. When 10 and 20 mM IBP are added into the HDL and LDL samples, chemical shifts of the N+ (CH3 )3 head groups of both PC and SM are shifted toward the higher field (Fig. 1, expansions). The chemical shift changes are −2.5 and −5.5 Hz for SM of HDL and LDL, −21.5 and −16.5 Hz for PC of HDL and LDL, respectively, at 10 mM IBP. The values are increased to −8.0 and −8.5 Hz for SM of HDL and LDL, −34.0 and −27.5 Hz for PC of HDL and LDL, respectively, at 20 mM IBP. These results indicated that IBP has larger effects on the N+ (CH3 )3 head groups of PC than that of SM, and on HDL than LDL. The latter is further supported by observable chemical shift up-field changes of the (CH2 )n group after addition of 10 and 20 mM IBP (Fig. 1, expansions). One potential explanation for this finding is that the PC enriched nanoenvironments (that are low in cholesterol) would much easily allow associations with the ibuprofen molecules than the SM enriched nanoenvironments (that are high in cholesterol) (Hevonoja et al., 2000). Diffusion coefficients measured from the N+ (CH3 )3 head groups (PC and SM) are identical for both HDL (6.1 and 6.1 × 10−7 cm2 s−1 ) and LDL (2.5 and 2.5 × 10−7 cm2 s−1 ) at 37 ◦ C and in presence of

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Fig. 1. 1 H NMR spectra of HDL (A1–A3) and LDL (B1–B3) in absence (A1, B1) and presence of 10 mM (A2, B2) and 20 mM (A3, B3) ibuprofen (IBP). Regions contained resonances of N+ (CH3 )3 and (CH2 )n are enlarged and plotted at the right side for each of the spectra. Resonances of the N+ (CH3 )3 groups are deconvoluted into two Lorentzian lines (dotted line) and assigned to phosphatidylcholine (PC, in red) and sphingomyelin (SM, in blue), respectively. The fitting differences are given at the bottom (green line) of the corresponding plot. Assignments of the major peaks are labeled: I2, I3, I4/7, I5/6, I8, I9 and I10 for ibuprofen. For lipoproteins: PC and SM for N+ (CH3 )3 of phosphatidylcholine (PC) and sphingomyelin (SM); L1: CH3 ; L2: (CH2 )n ; L3: CH2 CH2 COO; L4: (CH2 )n CH2 CH ; L5: CH2 CH2 COOC ; L6: CH CHCH2 CH CH ; L7: HC CH CH2 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

20 mM IBP. The values are similar to that in absence of IBP at the same temperature, HDL (5.7 × 10−7 cm2 s−1 ) and LDL (2.6 × 10−7 cm2 s−1 ). This indicates that the two components (PC and SM) are from a same species, HDL or LDL, respectively. HDL and LDL consists of 50% and 20% protein, 24% and 16% PC, 3.6% and 4.8% SM, respectively (Fournier et al., 1997; Murphy et al., 1997; Cushley and Okon, 2002; Khrenov et al., 2002). It has been reported that there are an apoB-100 (500 kDa) and approximately 3000 lipid molecules, including 700 phospholipid, in an LDL particle, and two apoA-I (28 kDa) molecules and approximately 200 lipid molecules in discoidal HDL (Hevonoja et al., 2000). Although HDL and LDL have similar structure, the compositional variation results in different physical and chemical properties for the two lipoprotein fractions (Ala-korpela et al., 1995). From the

1 H NMR spectra of the HDL and LDL at 10 mM IBP, we

get ratios of the peak areas of the IBP phenyl group (four protons) and N+ (CH3 )3 head groups (nine protons), I47:PC:SM, as 4.00:1.32:0.25 (HDL) and 4.00:1.03:0.57 (LDL, after deconvolution). Since only about 80% of the N+ (CH3 )3 head group of PC in LDL is NMR observable (Yeagle et al., 1977, 1978; Murphy et al., 1997), the concentrations of PC and SM can be estimated, from the NMR peak areas by using IBP as internal reference, as 1.5 and 0.3 mM for the HDL samples, 1.2 and 0.6 mM for the LDL samples, respectively. The molar ratios of IBP to PC are 10:1.5 and 20:1.5 for the HDL samples, 10:1.2 and 20:1.2 for the LDL samples, while the molar ratio of IBP to SM is 10:0.3 and 20:0.3 for the HDL samples, 10:0.6 and 20:0.6 for the LDL samples. Since IBP to PC ratios are close for the HDL and LDL samples, but N+ (CH3 )3 head group of PC

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and (CH2 )n group in HDL experience larger chemical shift up-field drift than that in LDL after addition of IBP, this may imply that IBP has stronger interaction with HDL than that with LDL. Because there is one set of chemical shift for each of the IBP peaks with sharp line shapes and there is no obvious diffusion coefficient change for the lipoproteins in presence of IBP, it is unlikely that there were many IBP inserted into the lipoprotein particles. Instead, the interaction may be limited at surface of the lipoprotein, and the bound and free forms of ibuprofen are in fast exchange, at least at NMR time scale. Considering the N+ (CH3 )3 and (CH2 )n groups are ionic and hydrophobic, respectively, while ibuprofen consists of a propionic acid (charged) and a isobutylphenyl (hydrophobic) groups, it is likely that both ionic and hydrophobic interactions are involved. To provide the direct evidence, we carried out the NOESY experiments. 3.2. Direct evidence of interaction between IBP and lipoproteins Nuclear Overhauser effect (NOE) is caused by dipole-dipole interaction or cross-relaxation and can provide unique spatial relationship between two nuclei ˚ in distance. Intensity of cross-peak of less than 5 A in NOE correlation spectrum (NOESY) is proportional to inverse six order of internuclear distance (Jeener et al., 1979; Macura and Ernst, 1980). The approach is now adopted to characterize intermolecular interaction between ibuprofen and the lipoproteins. Fig. 2 shows parts of NOESY spectra of HDL (A and B) and LDL (C and D) in absence (A and C) and presence (B and D) of 10 mM ibuprofen, respectively. For the NOESY experiments, a mixing time of 200 ms was chosen so that intermolecular NOE cross-peaks become observable. Because of spin diffusion, intramolecular 1 H–1 H NOE cross-peaks are all observable between (CH2 )n (L2) and each of the acyl groups of the lipoproteins, CH3 (L1), (CH2 )n CH2 CH (L4), CH2 CH2 COO (L5), CHCH2 CH (L6), HC CHCH2 (L7), and N+ (CH3 )3 (Fig. 2). In the presence of 10 mM IBP, NOE cross-peaks from H3 (I3) to H2 (I2), H4/7 (I4/7) and H5/6 (I5/6) of ibuprofen are visible (Fig. 2B and D). Most importantly, intermolecular NOE cross-peaks are observed between I3 and N+ (CH3 )3 , phenyl protons (I4/7, I5/6) and L2 for both HDL and LDL sample. In addition, a crosspeak between I8 and L2 is also visible in Fig. 2B (HDL). The intermolecular NOE cross-peaks are indicated by arrows in the figure. A reasonable description for the I3 N+ (CH3 )3 cross-peak may be formation of electrovalent bond between N+ (CH3 )3 of the phospholipids in

Fig. 2. Regions of 2D NOESY spectra of HDL (A and B) and LDL (C and D) in absence (A and C) and presence (B and D) of 10.0 mM ibuprofen to highlight the cross-peaks of intermolecular interaction between ibuprofen and the lipoproteins. Cross-peaks between the lipoproteins and ibuprofen are indicated by arrows.

HDL/LDL and the carboxyl of ibuprofen. Therefore, the CH3 (I3), which is next to the carboxyl, is expected to be close to the N+ (CH3 )3 head group in space. For a similar reason, the NOE peaks between (CH2 )n (L2) of the phospholipids and aromatic protons (I4/7, I5/6) of ibuprofen may be the direct evidence of the hydrophobic interaction between ibuprofen and HDL or LDL particles. It is noticed that NOE peak intensity/area of L2-(I4/7, I5/6) for the HDL–IBP system (Fig. 2B) is larger than that for the LDL–IBP system (Fig. 2D), which coincides with the larger chemical shift change of the L2 peak in HDL than in LDL. This may be understood as that affinity of the hydrophobic interaction between HDL and ibuprofen is higher than that between LDL and ibuprofen. However, an alternative explanation for the IBP induced PC and SM head group ( N+ (CH3 )3 ) chemical shift change may due to the orientation of the phospholipid head group at the surface of the lipoprotein particles. 4. Conclusion In this paper, we characterized interaction between lipoproteins (HDL and LDL) and ibuprofen (IBP), a potential antioxidant to inhibit oxidation of lipopro-

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teins, using NMR based chemical shifts and NOE measurements. It is observed that chemical shifts of the N+ (CH3 )3 moieties of phosphatidylcholine (PC) and sphingomyelin (SM) are drifted toward high field when ibuprofen is added into HDL and LDL samples. In addition, (CH2 )n group experiences a similar chemical shift up-field changes. The ibuprofen induced chemical shift changes of the PC is significant larger than that of SM for both lipoproteins. While up-field drifts of N+ (CH3 )3 head group in HDL is larger that in LDL. The intermolecular NOE cross-peaks indicate the direct interactions between proton pairs of L2 and (I4/7, I5/6), I3 and N+ (CH3 )3 , I2 and L2, I8 and L2 (only for HDL). The results revealed that both hydrophobic and ionic interactions are likely to be important in the solubility of ibuprofen into lipoprotein particles. Ibuprofen has higher impact on the PC and SM head group ( N+ (CH3 )3 ) and (CH2 )n group in HDL than that in LDL. This could be explained by either IBP had higher binding affinity to HDL than to LDL, or IBP induced orientation of the phospholipid head group at the surface of the lipoprotein particles. Acknowledgements This work is supported by the grants from the Natural Science Foundation (#20635040, #20475061, #20610104), and 973 Project of China (2002CB713806). References Ala-Korpela, M., Oja, J., Lounila, J., Jokisaari, J., Savolainen, M.J., Kesaniemi, Y.A., 1995. Structural-changes of lipoprotein lipids by 1 H NMR. Chem. Phys. Lett. 242, 95–100. Bai, G.Y., Cui, Y.F., Yang, Y.H., Ye, C.H., Liu, M.L., 2005. A competitive low-affinity binding model for determining the mutual and specific sites of two ligands on protein. J. Pharm. Biomed. Anal. 38, 588–593. Barter, P., Kastelein, J., Nunn, A., Hobbs, R., 2003. High density lipoproteins (HDLs) and atherosclerosis; the unanswered questions. Atherosclerosis 168, 195–211. Brown, M.S., Goldstein, J.L., 1986. A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47. Chen, A., Shapiro, M.J., 1998. NOE pumping: a novel NMR technique for identification of compounds with binding affinity to macromolecules. J. Am. Chem. Soc. 120, 10258–10259. Chung, B.H., Wilkinson, T., Geer, J.C., Segrest, J.P., 1980. Preparative and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor. J. Lipid Res. 21, 284–291. Colles, S.M., Maxson, J.M., Carlson, S.G., Chisolm, G.M., 2001. Oxidized LDL-induced injury and apoptosis in atherosclerosis— potential roles for oxysterols. Trends Cardiovasc. Med. 11, 131–138. Cui, Y.F., Wen, J., Sze, K.H., Man, D., Lin, D.H., Liu, M.L., Zhu, G., 2003. Interaction between calcium-free calmodulin and IQ motif of

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