Direct and Simple Fluorescence Detection Method for Oxidized

Dec 31, 2009 - This method can be readily applied for clinical measurement in patients with dyslipidemia using only 1 μL of 1 mg/mL of lipoprotein an...
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Anal. Chem. 2010, 82, 1128–1132

Technical Notes Direct and Simple Fluorescence Detection Method for Oxidized Lipoproteins Takeshi Ikeda,† Makoto Seo,‡ Ikuo Inoue,§ Shigehiro Katayama,§ Toshiyuki Matsunaga,| Akira Hara,| Tsugikazu Komoda,‡ and Mari Tabuchi*,†,⊥ Department of Chemistry, Graduate School of Science, and Research Information Center for Extremophile, Rikkyo University, 3-34-1, Nishi-Ikebukuro, Toshima-ku, Tokyo, 171-8501, Japan, Department of Biochemistry and Department of Endocrinology and Diabetes, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan, and Laboratory of Biochemistry, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan The quantification of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) is currently one of the most important clinical measurements for characterizing metabolic syndrome. However, recent studies have revealed additional factors that may be more strongly associated with the coronary heart disease than simple measurement of LDL or HDL levels, such as small dense (sd) LDL particles and oxidized LDL or HDL particles. Although several methods using enzyme-antibody detection systems or fluorescent probes have been devised to characterize these factors, such methods are expensive to implement for clinical measurements. Here, we present a straightforward analytical method for direct quantitation of oxidized lipoproteins by fluorescence spectrometry, with excitation in the UV (365 ( 10 nm) or visible (470 ( 10 nm) range and emission detected at 450 ( 30 nm or 535 ( 15 nm. This method can be readily applied for clinical measurement in patients with dyslipidemia using only 1 µL of 1 mg/mL of lipoprotein and without the need for any expensive detection antibodies. Using this new technique, biological samples from patients with dyslipidemia showed higher fluorescence intensities than samples from normal subjects when detecting oxidized LDL and light HDL (d ) 1.063-1.125 g/mL), whereas samples from patients with dyslipidemia showed lower fluorescence intensities than samples from normal subjects when measuring oxidized heavy HDL (d ) 1.125-1.210 g/mL) levels. Lipoproteins are complex, nanometer-sized particles consisting of apolipoproteins, cholesterol, a phospholipid monolayer on the * To whom correspondence should be addressed. E-mail: mtabuchi@ rikkyo.ac.jp. † Department of Chemistry, Graduate School of Science, Rikkyo University. ‡ Department of Biochemistry, Faculty of Medicine, Saitama Medical University. § Department of Endocrinology and Diabetes, Faculty of Medicine, Saitama Medical University. | Gifu Pharmaceutical University. ⊥ Research Information Center for Extremophile, Rikkyo University.

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particle surface, and an interior consisting of triglyceride and cholesterol esters.1 Lipoproteins are classified by their densities into three major groups: high-density lipoprotein (HDL), lowdensity lipoprotein (LDL), and very low-density lipoprotein (VLDL).2 Ultracentrifugation is the traditional method used to separate and define the major lipoprotein classes. Each class plays a critical biological role in the transport of cholesterol and triglycerides. Disorders in lipoprotein metabolism facilitate the initiation and progression of atherosclerosis and related diseases.3 One of the most important clinical measurements today is the quantification of LDL and HDL,4,5 where increased LDL levels shows a positive correlation with coronary heart disease and increased HDL levels shows an inverse correlation. However, recent studies have revealed that smaller and more dense LDL particles (sd LDL) found in the blood are more atherogenic and are strongly correlated with the development of coronary heart disease.6,7 Moreover, oxidized LDL has also been revealed to be strongly associated with the accumulation of cholesterol on vascular walls,8 and elevated plasma levels of oxidized LDL have been found in patients with arteriosclerosis.9 Furthermore, while HDL is classified as “good cholesterol” since HDL prevents arteriosclerosis by collecting excess cholesterol,10,11 the oxidized form of HDL has been reported to have reduced (1) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 2nd ed.; Worth Publishers: New York, 1993. (2) Havel, R. J.; Eder, H. A.; Bragdon, J. H. J. Clin. Invest. 1955, 34, 1345– 1353. (3) Steinberg, D. Atherosclerosis 1983, 3, 283–301. (4) Kannel, W. B. J. Atheroscler. Thromb. 2000, 6, 60–66. (5) Buchwald, H.; Boen, J. R.; Nguyen, P. A.; Williams, S. E.; Matts, L. P. Atherosclerosis 2001, 154, 221–227. (6) Austin, M. A.; Breslow, J. L.; Hennekens, G. H.; Buring, J. E.; Willett, W. C.; Krauss, R. M. J. Am. Med. Assoc. 1988, 260, 1917–1921. (7) Austin, M. A.; King, M. C.; Viranizan, K. M.; Krauss, R. M. Circulation 1990, 82, 495–506. (8) Avogaro, P.; Bon, B. G.; Cazzolato, G. Arteriosclerosis 1988, 8, 79–87. (9) Holvoet, P.; Perez, G.; Zhao, Z.; Brouwers, E.; Bernar, H.; Colllen, D. J. Clin. Invest. 1995, 95, 2611–2619. (10) Miller, G. J.; Miller, N. E. Lancet 1975, 1, 16–19. (11) Carew, T. E.; Koschinsky, T.; Hayes, S. B.; Steinberg, D. Lancet 1976, 1, 1315–1317. 10.1021/ac902018a  2010 American Chemical Society Published on Web 12/31/2009

beneficial properties12 and is converted into a cytotoxic particle,13 suggesting that oxidized HDL may also contribute to the genesis of coronary artery disease. As a result, these oxidized lipoprotein particles have become a focus of attention as a new arteriosclerosis risk factor. At the present time, many methods have been devised to measure lipoprotein particle levels and associated oxidation states. Such methods include a density gradient ultracentrifugal procedure for the rapid isolation of the major lipoprotein classes,14 rapid ultracentrifugation,15 polyacrylamide gradient gel electrophoresis,16 an online enzymatic method using high performance liquid chromatography (HPLC),17,18 a lipoprotein profile approach using capillary electrophoresis and mass spectrometry,19 an ultrafast microchip electrophoresis for each of the classes,20 an improved simple precipitation method for the quantification of sd LDL,21 a measurement of lipid peroxidation in oxidized LDL using thiobarbituric acid-reactive substances,22 a specific enzymeantibody assay for malondialdehyde-modified LDL,23 and approaches using fluorescent lipid probes.24 Among these studies, fluorescently labeled lipoproteins have provided important information on both physical and cellular binding properties of lipoproteins. Two main fluorescence detection methods of lipoproteins in use today involve either protein marker dyes or lipophilic dyes.24 The former is composed of covalent fluorochrome-apolipoprotein conjugates, and the latter is composed of noncovalent bonds with lipids. Many protein marker dyes using fluorochromes that conjugate apolipoproteins through functional groups (e.g., amino, sulfhydryl, aldehyde, carboxyl, or hydroxyl groups) are commercially available. One of the more useful lipophilic dyes for lipoprotein analysis is 3,3′-dioctadecylindocarbocyanine iodide (Dil).24 Lipoproteins are readily labeled with Dil. Nonetheless, the use of Dil is somewhat limited, as the labeling process is complicated and the signal-tonoise ratios offered by this dye can be low for some applications. In addition, Dil does not provide a simple quantification method that distinguishes between each type of lipoprotein particle. To solve these problems, Corsetti et al.25 synthesized an improved lipophilic dye, N,N-dipentadecylaminostyrylpyridinium iodide (DASP), which shows increased fluorescence intensity for detecting lipoproteins. In this study, we investigated the use of DASP-labeled lipoprotein for detecting and quantitating lipoproteins. We find that (12) Nagano, Y.; Arai, H.; Kita, T. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6457– 6461. (13) Alomar, Y.; Negre-Salvayre, A.; Levade, T.; Valdiguie, P.; Salvayre, R. Biochem. Biophys. Acta 1992, 1128, 163–173. (14) Chapman, M. J.; Goldstein, S.; Lagrange, D.; Laplaud, P. M. J. Lipid Res. 1981, 22, 339–358. (15) Pietzsch, J.; Subat, S.; Nitzsche, S.; Leonhardt, W.; Schentke, K.-U.; Hanefeld, M. Biochim. Biophys. Acta 1995, 1254, 77–88. (16) Warnick, G. R.; McNamara, J. R.; Boggess, C. N.; Clendenen, F.; Williams, P. T.; Landolt, C. C. Clin. Lab. Med. 2006, 26, 803–846. (17) Hara, I.; Okazaki, M. Methods Enzymol. 1986, 129, 57–78. (18) Usui, S.; Hara, Y.; Hosaki, S.; Okazaki, M. J. Lipid Res. 2002, 43, 805–814. (19) Macfarlane, R. D. Electrophoresis 1997, 18, 1796–1806. (20) Ping., G.; Zhu, B.; Jabasini, M.; Xu, F.; Oka, H.; Sugihara, H.; Baba, Y. Anal. Chem. 2005, 77, 7282–7287. (21) Hirano, T.; Ito, Y.; Yoshino, G. J. Atheroscler. Thromb. 2004, 12, 67–72. (22) Yagi, K. Chem. Phys. Lipids 1987, 45, 337–351. (23) Kotani, K.; Maekawa, M.; Kanno, T.; Toda, N.; Manabe, M. Biochem. Biophhys. Acta 1994, 1215, 121–125. (24) Maier, O.; Oberle, V.; Hoekstra, D. Chem. Phys. Lipids 2002, 116, 3–18. (25) Corsetti, J. P.; Weidner, C. H.; Cianci, J.; Sparks, C. E. Anal. Biochem. 1991, 195, 122–128.

lipoproteins, and in particular oxidized lipoproteins, could be readily analyzed by fluorospectrometry without requiring any covalent labeling steps. MATERIALS AND METHODS Equipment. A NanoDrop ND-1000 spectrophotometer and a NanoDrop ND-3300 fluorospectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE) were utilized for analyzing absorption and fluorescence spectra, respectively. The fluorospectrophotometer was equipped with excitation light sources at UV (365 ± 10 nm) and visible (470 ± 10 nm) wavelengths. Lipoproteins. HDL and LDL, purified from human plasma, were purchased from Sigma (St. Louis, MO). Other lipoproteins purified from blood samples were also used. Blood samples were obtained from 15 healthy normolipidemic volunteers, aged 23 to 65 years (7 males/8 females), and 21 patients with dyslipidemia, aged 45 to 67 years (10 males/11 females). Patient samples were collected on two occasions under different conditions. The first set of samples was taken after a fast of 12 h or longer and the second set of samples was collected after one month of treatment with fenofibrate. All blood samples were collected into tubes containing solution with 0.1% ethylenediaminetetraacetic acid (EDTA) and buffered at pH 7.4. Serum was separated by centrifugation at 3000 rpm for 20 min at 4 °C and was then subjected to lipoprotein fractionation within 24 h of isolation. During this period, the samples were stored at 4 °C. Each type of lipoprotein particle was recovered from the tubes and dialyzed against phosphate-buffered saline (PBS) containing 3 µM EDTA. The lipoproteins were sterilized by filtration and stored at 4 °C in the dark. All lipoprotein particles, including LDL (d ) 1.006-1.063 g/mL), light HDL (d ) 1.063-1.125 g/mL), and heavy HDL (d ) 1.125-1.210 g/mL), were isolated by sequential ultracentrifugal flotation, with slight modifications from a previously described procedure.26 An Optima-MAX with a MLA80 rotor (Beckman Coulter Inc.) was used for these ultracentrifugation procedures. The experimental procedure and its purpose were thoroughly explained to all the subjects and written consent was obtained. The study protocol was approved by the Ethics Committee of Saitama Medical University. DASP Synthesis and Characterization. DASP was synthesized according to the method reported by Corsetti et al.25 The chemical structure of DASP is shown in Figure 1a, and the absorption and fluorescence spectra of DASP in dimethyl sulfoxide (DMSO) are shown in Figure 1b. The DASP absorption spectrum demonstrated a significant peak at 487 nm, and the DASP fluorescence spectrum exhibited a maximum at 625 nm. Lipoprotein Staining. The quantification of LDL or HDL was carried out using the DASP labeling. For the preparation of DASP-LDL or DASP-HDL, 200 µL of LDL or HDL (3.2 mg/ mL) was added to 5 mM DASP in DMSO. This mixture was incubated for 3 h at room temperature in the dark. To separate the stained LDL or HDL from the free dye, the mixture was then sequentially passed through two gel filtration columns (BD Clontech Ab Microarray buffer kit, BD Bioscience, San Jose, CA). The total protein concentration of DASP-LDL or DASP-HDL was determined by a bicinchoninic acid assay (Wako Purechemi(26) Manzato, E.; Zambom, S.; Marin, R.; Baggio, G.; Crepaidi, G. J. Lipid Res. 1986, 27, 1248–1258.

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Figure 1. (a) Chemical structure of DASP; R ) (CH2)14-CH3. (b) The absorption spectrum (dashed line) of DASP dissolved in DMSO and the corresponding fluorescence spectrum (solid line) with excitation at 365 nm.

cal, Japan). Ultrapure water (Sigma, St. Louis, MO) was used for dilution of LDL or HDL. Lipoprotein Oxidations. The oxidative modifications of lipoproteins in vitro were achieved by various methods. A common oxidative reagent, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH; Wako Purechemical Industries Ltd. Japan), was used as a reagent-based oxidative method. Two other lipoprotein oxidation approaches, an air-oxidative method and a UV-oxidative method, were performed by incubation for a maximum of 50 h at room temperature under dark conditions. The UV-oxidative method was performed by irradiation of the samples with ultraviolet light (Spectroline ENF-280, Spectronics Corporation, Westbury, NY). The oxidation studies were carried out without DASP labeling. RESULTS AND DISCUSSION Direct Fluorescence Detection Method for Lipoproteins without Covalent Modification. Figure 2Aa,Aa’ shows the excitation spectra of LDL or HDL with and without DASP labeling. The DASP-labeled LDL spectrum had a significant absorption at 470 nm. On the other hand, unlabeled LDL showed the absorption at 280 nm. The DASP-labeled HDL excitation spectrum showed the absorptions at 240, 280, and 480 nm, and unlabeled HDL showed the absorptions at 240 and 280 nm. Given these excitation spectra, we selected two excitation wavelengths (UV (365 nm ± 10 nm) and visible (470 ± 10 nm) ranges) for subsequent experiments. Figure 2Ba,Ba’ shows the emission spectra of DASPlabeled LDL at various concentrations (0.09-0.69 mg/mL) when excited at 365 or 470 nm. Similarly, Figure 2Bb,Bb’ shows the emission spectra of unlabeled LDL at various concentrations (0.64-4.26 mg/mL) when excited at these wavelengths. As shown in Figure 2Ba,Ba’,Bb,Bb’, an increase in LDL concentration produces corresponding fluorescence intensity increases. Figure 2Bc,Bc’ shows the correlation between fluorescence intensity and concentrations of DASP-labeled or unlabeled LDL, monitored at 600 nm. The maximum fluorescence intensity ratios for DASPlabeled LDL fluorescence/unlabeled LDL fluorescence were 28 for 365 nm excitation (Figure 2Bc) and 206 for 470 nm excitation (Figure 2Bc’). Although the fluorescence intensity of unlabeled LDL was much less than that of DASP-labeled LDL, fluorescent emissions 1130

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Figure 2. (A) Excitation spectra of LDL (a) or HDL (a’) with (solid line) and without (dashed line) DASP labeling. (B) The emission spectra of DASP-labeled LDL at each concentration (0.09-0.69 mg/mL), with excitation at 365 nm (a) or 470 nm (a’) and unlabeled LDL at each concentration (0.64-4.26 mg/mL) excited at 365 nm (b) or 470 nm (b’). Numerical values in the figure show lipoprotein concentration. Plots of fluorescence intensity ratios vs concentration for labeled LDL and unlabeled LDL excited at 365 nm (c) or 470 nm (c’) and emission detected at 600 nm. Plots of fluorescence intensity ratios vs concentration for labeled LDL excited at 365 nm (d) or 470 nm (d’) and emission detected at 420, 450, 480, and 600 nm for (d) or 520, 550, 580, and 600 nm for (d’). (C) Plots of fluorescent intensity ratios vs concentration (ng/mL total protein) for labeled HDL and unlabeled HDL excited at 365 nm (a) or 470 nm (a’) and emission detected at 600 nm. Plots of fluorescence intensity ratios vs concentration (mg/mL total protein) for labeled LDL excited at 365 nm (b) or 470 nm (b’) and emission detected at 420, 450, 480, and 600 nm for (b) or 520, 550, 580, and 600 nm for (b’).

Figure 3. Fluorescent properties of LDL (left) or HDL (right) under various in vitro oxidation conditions. Sample fluorescent intensity for LDL (a) or HDL (a’) where air-oxidation occurs over long time periods; for LDL (b) or HDL (b’) when oxidized in air under dark conditions at room temperature; for LDL (c) and HDL (c’) when oxidized with ultraviolet under dark conditions at room temperature; and for LDL (d) or HDL (d’) when oxidized with AAPH (0, 0.4, 4.0, and 40 mM). All samples were excited at 365 nm and monitored at 450 nm. All results are represented in units of relative fluorescent intensity per milligram of lipoprotein.

from unlabeled LDL were evident when exciting at 365 nm and detecting at 450 ± 30 nm or exciting at 470 nm and detecting at 535 ± 15 nm (Figures 2Bd and 2Bd’). Similarly, although the fluorescence intensity of unlabeled HDL was much less than that of DASP-labeled HDL monitored at 600 nm (Figure 2Ca,Ca’), unlabeled HDL showed measurable fluorescence when excited at 365 or 470 nm and detecting emission at 450 ± 30 or 535 ± 15 nm (Figures 2Cb,Cb’). All of the data showed a close correlation between concentration and intensity at these wavelengths and concentrations. These results indicate that LDL or HDL can be directly analyzed with only 1 µL of at least 1 mg/mL of lipoprotein and without any troublesome conjugation procedures. Oxidized Lipoprotein Properties during in Vitro Oxidation. We further investigated the application of this direct fluorescence detection method to quantitating oxidized lipoproteins. Figure 3 shows the fluorescence intensity of LDL with

different in vitro oxidation levels. All were carried out without DASP labeling. Although a freshly isolated LDL sample showed low intensities, an older LDL sample, for which 1 year had passed after the sample had been isolated, showed a 137-fold higher fluorescence intensity than the freshly collected LDL, when excited at 365 nm and detected at 450 nm (Figure 3a). Similarly, an old HDL sample showed 96-fold higher intensity than a freshly collected HDL sample (Figure 3a’). We believe that this increase in fluorescence intensity is derived from in vitro oxidation that takes place over the passage of time. To confirm the influence of in vitro oxidation, we further investigated the oxidation properties of lipoproteins for several oxidation conditions. Figure 3b shows the relative fluorescence intensity of LDL, which was oxidized by incubation in air under dark conditions at room temperature, and Figure 3b’ shows the Analytical Chemistry, Vol. 82, No. 3, February 1, 2010

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Figure 4. Differences in LDL, light HDL, and heavy HDL fluorescence among normal subjects (white), patients with dislipidemia (black), and patients treated for one month with fenofibrate (gray). All samples were excited at 365 nm, and detection occurred at 450 nm. All results are represented in units of relative fluorescent intensity per milligram of lipoprotein. (* P < 0.05 and **P < 0.01).

corresponding data for HDL. Figure 3c,c’ shows the fluorescent intensity of LDL and HDL samples, respectively, that were irradiated with UV in the absence of oxygen while incubating at room temperature, and Figure 3d,d’ shows the fluorescent intensity of LDL or HDL oxidized with AAPH. Although a slight difference in oxidation profiles was detected between LDL and HDL samples, higher concentrations of AAPH produced higher fluorescent intensity for both LDL and HDL. These data indicate that fluorescence intensities of LDL or HDL samples are reflective of lipoprotein oxidation levels. Formation of lipid peroxidation in biological tissues leading to the generation of blue fluorescence (excitation maxima of 340-380 nm and emission maxima of 420-490 nm) has been previously reported.27 However, the present study represents the first time that a relationship between fluorescence and oxidized lipoprotein has been demonstrated. Application of the Method to Aid Diagnosis of Dyslipidemia. The goal of this study is to apply this method to aid clinical diagnoses, especially for dyslipidemia. We, therefore, investigated the fluorescence properties of lipoproteins in clinical samples from patients having dyslipidemia. As shown in Figure 4, the fluorescence of LDL samples from patients with dyslipidemia showed higher intensities than that from LDL samples obtained from normal subjects. The light HDL samples showed a pattern similar to that seen with the LDL samples. An inverse correlation was demonstrated for heavy HDL, as the fluorescence levels of heavy HDL samples from patients with dyslipidemia were lower than those of heavy HDL samples from normal subjects. Our LDL data shown in Figure 4 indicates that the in vivo LDL oxidation levels are high for patients with coronary artery disease, which agrees with previous reports.8,9 The levels of oxidized HDL from patients with coronary artery disease have been reported to

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be higher than those from normal subjects.28 Furthermore, HDL from patients with coronary artery disease has been reported to have a higher susceptibility to lipid peroxidative modification than LDL.29 Our data shows that light HDL oxidative levels found in patients with dyslipidemia are higher than those from normal subjects. However, we also recognized an inverse correlation between patient dyslipidemia and heavy HDL oxidation levels, which suggests that oxidative mechanisms may be different between light HDL and heavy HDL. To further explore this hypothesis, we investigated the in vivo oxidative levels of samples from patients that were given fenofibrate treatment. The patients treated with fenofibrate for 1 month showed decreased levels of LDL and light HDL, while there were increased levels of heavy HDL. This finding suggests that the function of heavy HDL may be restored by fenofibrate treatment. In addition, the oxidation levels of heavy HDL from normal subjects may conceivably be higher than those from untreated patients with dyslipidemia. The levels of in vivo and in vitro oxidized LDL or HDL have been described in many recent reports. However, to our knowledge, the present study is the first time that a difference in oxidative properties between light HDL and heavy HDL has been proposed. Finally, we have demonstrated that oxidized lipoproteins can be rapidly and directly analyzed by fluorescence spectrometry, without requiring any covalent labeling. The quantification of LDL or HDL can be carried out using the DASP labeling, but this label was not required for the oxidation studies. The measurement proceeds with excitation occurring in the UV (365 ± 10 nm) or visible (470 ± 10 nm) range and emission maxima detected at 450 ± 30 nm or 535 ± 15 nm and can be completed within several seconds of measuring time. This method can be readily employed with only 1 µL of at least 1 mg/mL of lipoprotein and without using any expensive antibodies. Additional clinical investigations will further explore the effectiveness of this new method in studying oxidative mechanisms of heavy HDL. ACKNOWLEDGMENT This work was supported by the Frontier Project “Adaptation and Evolution of Extremophile” from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Rikkyo University Special Fund for Research (SFR). We greatly appreciate the assistance in the experiments of Ms. Noriko Fukushima at Saitama Medical University.

Received for review December 7, 2009.

September

8,

2009.

Accepted

AC902018A (27) Fletcher, B. L.; Dillard, C. J.; Tappel, A. L. Anal. Biochem. 1973, 52, 1–9. (28) Nakajima, T.; Matsunaga, T. Ann. Clin. Biochem. 2004, 41, 309–315. (29) Hurtado, I.; Fiol, C.; Gracia, V.; Caldu, P. Atherosclerosis 1996, 125, 39– 46.