New Functions of Glucosamine as a Scavenger of the Lipid

Chem. Res. Toxicol. 2007, 20, 947-953

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New Functions of Glucosamine as a Scavenger of the Lipid Peroxidation Product Malondialdehyde Chui Fang, Mijun Peng, Guolin Li, Jinzhou Tian, and Dazhong Yin* Key Laboratory of Protein Chemistry and DeVelopmental Biology of the Ministry of Education, College of Life Sciences, Hunan Normal UniVersity, Changsha 410081, Hunan, China ReceiVed January 22, 2007

Direct reaction between malondialdehyde (MDA) and glucosamine (GlcN) was studied using highperformance liquid chromatography, liquid chromatography/mass spectrometry, spectrophotometry, and spectrofluorometry. The results indicated that GlcN reacted readily with MDA at supraphysiological conditions to form different products, such as a nonfluorescent enamine with an absorption peak at 281 nm (product 1) and a lipofuscin-like fluorescent (Ex. 392 nm/Em. 454 nm) 1,4-dihydropyridine (product 2). GlcN also greatly inhibited the formation of lipofuscin-like fluorescence induced by MDA reacted with bovine serum albumin. The reaction of GlcN with MDA suggested a novel anticarbonylation function of GlcN in pathophysiological situations related to aging-related diseases and provided insight into the reaction mechanism of GlcN in protecting proteins against carbonyl stress. Introduction Lipid peroxidation and nonenzymatic glycosylation (glycation) reactions are two critical biological side-reactions in the energy metabolism which can cause biochemical modifications of tissue proteins and lead to the formation of advanced glycation end-products (AGEs)1 and advanced lipoxidation endproducts (ALEs). These processes have been widely documented to be responsible for the formation of various age pigment-like fluorophores and many acute and chronic diseases, such as osteoarthritis (OA), atherosclerosis, diabetes mellitus, neuronal degenerative diseases, and even physiological aging (1-6). A variety of reactive carbonyls, the common intermediates derived from glycation and lipid peroxidation, were found to react readily with an amino group of proteins with the formation of protein aggregates, resulting in protein structural and functional alterations (7, 8). Such alterations are now known as “carbonyl toxification” or “carbonyl stress” in the related field (6, 8). Malondialdehyde (MDA) and 4-hydroxynonenal (HNE), in the level of 0.3-30 nmol/mL of human plasma at pathophysiological conditions (2), are the two most well-studied intermediates of oxidative stress. These reactive unsaturated carbonyls can target a variety of amine-containing biological components, such as lysine, arginine, and histidine residue of proteins, to cause cross-linkages and aggregates (9). These reactions have been generally regarded as being involved in causing cellular deteriorations as well as in aging-related extracellular alterations, particularly in the formation of ceroid/lipofuscin-related pigments with characteristic fluorescence (9, 10). In chondrocytematrix and collagen extracts, for instance, MDA and HNE adducts were comprehensively observed with fluorometric techniques and immunoblot analysis (2, 9, 11). * To whom correspondence should be addressed. Tel.: +86 731 8872786. Fax: +86 731 8872786. E-mail: [email protected]. 1 Abbreviations: AGEs, advanced glycation end-products; ALEs, advanced lipoxidation end-products; DAD, diode array detector; GlcN, glucosamine; HNE, 4-hydroxynonenal; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; MDA, malondialdehyde; OA, osteoarthritis; PBS, phosphate buffer solution; TIC, total ion current chromatogram; TMP, 1,1,3,3-tetramethoxypropane.

Damages and alterations of cartilage collagen are common pathologies of cartilage aging in OA (12-16). Progressive reduction of tensile properties of collagen during aging has been extensively documented in literature (13). In aging-related OA, the degeneration of collagen was found to be “originated around chondrocytes and extended into the cartilage” (14). In recent studies, the accumulation of AGEs in cartilage, implying one of the important molecular mechanisms in the development of OA, provided profound biochemical explanation of cellular changes in cartilage during aging (17-19). Glucosamine (GlcN) is an amino monosaccharide (2-amino2-deoxy-D-glucose), which is an essential component of mucopolysaccharides and chitin. Such amino-containing saccharides have been identified in mucous secretions, connective tissues, tendons, ligaments, cartilage, and skin (20). Because of its high concentration in joint tissues and beneficial effects, it is widely applied in clinics to relieve arthritic complaints and OA symptoms (20-22). Although glucosamine is widely used as an over the counter remedy for osteoarthritis, its efficacy and reaction mechanisms remain a subject to debate. At present, GlcN has been suggested as an antioxidant in the OA treatment due to its “free radical scavenging” capability (23). Since such saccharide does not have a conjugated ring structure to scavenge free radicals, we hypothesize that its antioxidative stress function may be based on a carbonyl-amino reaction (anticarbonyl stress) potential as an underestimated mechanism in the field. In this paper, we demonstrate that GlcN reacted readily with MDA under supraphysiological conditions which may thus protect biomaterials against deterioration of toxic carbonyls. Similarly, MDA modification on BSA was also significantly inhibited by GlcN at different concentrations.

Materials and Methods Materials. GlcN‚HCl (purity > 99.0%) was purchased from Sangon (Shanghai, China). 1,1,3,3-tetramethoxypropane (TMP) was obtained from Fluka Chemie AG (Buchs, Switzerland). The GlcN stock solution (50 mM) was prepared by dissolving 1.077 g of GlcN into 100 mL of 0.2 M phosphate buffer solution (PBS) (pH 7.4). A fresh MDA stock solution (10 mM) was prepared by hydrolyzing

10.1021/tx700059b CCC: $37.00 © 2007 American Chemical Society Published on Web 05/05/2007

948 Chem. Res. Toxicol., Vol. 20, No. 6, 2007 1,1,3,3-tetramethoxypropane (TMP) according to a method described by Kikugawa et al. (10). Thus, 0.085 mL (0.5 mmol) of TMP was mixed with 2 mL of 1.0 M HCl and shaken at 40 °C for about 2 min. After the TMP was fully hydrolyzed, the pH was adjusted to 7.4 with 6.0 M NaOH, and the stock solution was finally made up to 50 mL with 0.2 M PBS (pH 7.4). The stock solution was checked by measuring absorbance at 266 nm using MDA ) 31 500. Other chemicals used were all of analytic grade from BioRad (Shanghai, China). Reactions of GlcN with MDA at Different Concentrations and pHs. For investigating reaction kinetics, GlcN (5.0 mM) was incubated with different concentrations of MDA in 0.2 M PBS at 37 °C (pH 7.4). On the opposite, MDA at a fixed concentration (5.0 mM) was incubated with GlcN at different concentrations in PBS at 37 °C. Reaction products were diluted to 1/6000 with the same buffer before spectrofluorometric measurements. To study pH effect, GlcN (5.0 mM) was incubated with MDA (5.0 mM) in 0.2 M citrate buffer pH 5.0, 0.2 M phosphate buffer pH 6.0, pH 7.0, pH 8.0, and 0.2 M carbonate buffer pH 9.1, respectively. The reaction products were diluted to 1/3000 in corresponding buffer before spectrofluorometric measurements. HPLC Analysis of the Reaction Mixture of GlcN and MDA. To obtain principal GlcN+MDA reaction products, GlcN (5.0 mM) was incubated with MDA (5.0 mM) in 0.2 M PBS at 37 °C for 24 h, and the reaction products were analyzed by HPLC. HPLC analysis was performed on a reverse-phase C18 analytical column (250 mm × 4.6 mm, 10 µm) on a Waters Alliance 2690 HPLC system with a model 996 photodiode array detector. The mobile phase consisted of solution A (0.1% trifluoroacetic acid in water) and solution B (acetonitrile, 100%). The flow rate was 0.5 mL/ min, and the elution gradient was as follows: 0-5 min, 100% A; 5-25 min, 20% B. Fluorescence Analysis of the Reaction Mixture of GlcN and MDA. Fluorescence spectra of the reaction mixture were characterized by utilizing a Perkin-Elmer LS 50 B spectrofluorimeter (Norwalk, U.S.A.). LC/MS Analysis of the Reaction Products of GlcN and MDA. To assay their molecular weight of principal GlcN+MDA reaction products, GlcN (5.0 mM) was incubated with MDA (5.0 mM) in 0.2 M PBS at 37 °C for 24 h, and the reaction products were analyzed by LC/MS. LC/MS experiments were carried on an LC/ MS-2010 quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) source (LC/MS-ESI). The positive electrospray ionization mode was applied for detection. Separation was done by a Thermo Hypersil-Keystone Hypurity C18 (150 mm × 2.1 mm, 5 µm) analytical column in the following conditions: solution A was 20 mM ammonium acetate (pH 5.0) and solution B was 100% methanol, the flow rate was 0.2 mL/min, and the elution proportion was 95% A and 5% B all the time. The wavelength of the SPD-M10Avp diode array detector (DAD) was set in the range of 200-300 nm. The ESI/MS source was set as follows: the temperatures were maintained at 250 °C, 250 °C, and 200 °C for the probe, CDL, and block, respectively. The voltages were set at 4.5 kV, -30 V, 25 V, 150 V, and 1.5 kV for the probe, CDL, Q-array 1,2,3 bias, Q-array radio frequency, and detector, respectively. The flow rate of nebulizer gas was 1.5 L/min. The ions used for selected ion monitoring were chosen by scanning in the positive ion mode from m/z ) 50-600. Effect of GlcN on the MDA Modified Proteins. BSA (10 mg/ mL) was incubated with MDA (1.0 mM) in the absence or presence of different concentrations of GlcN (GlcN and MDA were added simultaneously to the reaction system) in PBS. The reaction was conducted at 37 °C for 24 h. Modified protein was precipitated by adding isometric 20% trichloroacetic acid (TCA) and centrifuged (3000g) for 3 min. In order to fully remove GlcN+MDA flurescent products, the pellet was washed for 3 times and precipitated with 20% TCA and collected after centrifugation. Finally, the precipitated protein was redissolved in 0.2 M PBS (pH 7.4), and the concentration was adjusted to 10 mg/mL. The sample was diluted 30-fold before the fluorescence was read at an excitation wavelength of 395 nm and an emission wavelength of 460 nm.

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Figure 1. Principal GlcN+MDA reaction products measured by HPLC analysis. GlcN (5.0 mM) was incubated with MDA (5.0 mM) in 0.2 mM PBS (pH 7.4) at 37 °C for 24 h. Following HPLC separation, the absorption of reaction mixture was measured at 243 nm (A), 281 nm (B), and 388 nm (C), respectively.

Results Principal GlcN+MDA Reaction Products Assayed Using HPLC Technique. After GlcN (5.0 mM) was incubated with MDA (5.0 mM) in 0.2 M PBS, pH 7.4, at 37 °C for 24 h, the reaction mixture was eluted at acidic pH through HPLC. As shown in Figure 1, MDA was estimated at 243 nm with the retention time at 5.264 min (Figure 1A). The first new product (product 1) which was estimated at 281 nm was observed with the retention time at 3.425 min (Figure 1B). The second new product (product 2) which was estimated at 388 nm was observed with the retention time at 12.672 min (Figure 1C). Product 1 did not show any fluorescence, while product 2 showed a stable lipofuscin-like blue (Ex. 392 nm/Em. 454 nm) fluorescence (Figure 2). The standard absorbance spectrum of MDA showed a peak at 266 nm in a neutral condition, while in an acidic condition, its absorption peak was at 243 nm (Figure 3). The GlcN had no absorption in the range of 220-400 nm except it had a strong light quenching effect near 200 nm (Figure 3). The UVabsorption spectrum showed that the maximum of product 1 was at 281 nm whereas product 2 showed three peeks at 234, 263, and 388 nm, respectively (Figure 3). Effect of Different MDA/GlcN Concentrations on Fluorescence Intensity. The fluorescence yields of 5.0 mM GlcN

Reaction of Glucosamine with Malondialdehyde

Figure 2. Fluorescence spectra of product 2 obtained by incubation of GlcN and MDA for 24 h.

Figure 3. UV-absorption spectrum of GlcN+MDA products. The UVabsorption spectra of MDA and GlcN+MDA products were obtained from HPLC with a model 996 photodiode array detector.

Figure 4. Fluorescence produced from reaction of GlcN with different concentrations of MDA. MDA (at indicated concentration) and GlcN (5.0 mM) were incubated in 0.2 M PBS (pH 7.4) at 37 °C for 48 h. The relation between fluorescence yield and MDA concentration (48 h) is shown in the inset. Results are means of triplicates ( SD.

reacting with MDA in different concentrations (0, 1.25, 2.5, 5.0, 10.0, 20.0 mM) are shown in Figure 4. During 48 h incubation (no significant increase afterward), the fluorescence intensity increased in direct proportion to the MDA concentration. A discernible sharp increase was observed when MDA concentration was higher than 5.0 mM indicating a favorable

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Figure 5. Fluorescence produced from reaction of MDA with different concentrations of GlcN. MDA (5.0 mM) and GlcN (at indicated concentration) were incubated in 0.2 M PBS (pH 7.4) at 37 °C for 48 h. The relation between fluorescence yield and GlcN concentration (48 h) is shown in the inset. Results are means of triplicates ( SD.

Figure 6. Effect of incubation pH on GlcN+MDA fluorescence formation. GlcN and MDA were incubated at 37 °C in 0.2 M corresponding buffer for 48 h. Results are means of triplicates ( SD.

formation of the fluorescent polymer, product 2, requiring excessive amount of MDA (Figure 4). The fluorescence yields of 5.0 mM MDA reacting with GlcN in different concentrations (0, 1.25, 2.5, 5.0, 10.0, 20.0 mM) are shown in Figure 5. The fluorescence intensity increased in direct proportion to the GlcN concentration only up to the equimolar (5.0 mM:5.0 mM) reaction system. An excessive amount of GlcN, the 10.0 mM and 20.0 mM GlcN versus 5.0 mM MDA, was shown to inhibit the formation of fluorescent product 2 at 48 h, suggesting that the formation of nonfluorescent product 1 was the preferred reaction in the model system (Figure 5). Effect of pH on Fluorescence Intensity of GlcN+MDA Reaction. The effect of incubation pH on the fluorescence intensity (Ex. 392 nm/Em. 454 nm) of products of the GlcN (5.0 mM) and MDA (5.0 mM) interaction is given in Figure 6. The fluorescence intensity of product in neutral conditions (pH 6.0, 7.0) was remarkably higher than that in either the acidic conditions (pH 5.0) or basic conditions (pH 8.0, 9.1). Whereas the highest fluorescence intensity of the reaction mixture after 48 h incubation was under the condition of pH 7.0, the reaction mixture of GlcN and MDA at pH 9.1 hardly showed any fluorescence increase (Figure 6). Identification of Reaction Products by LC/MS. LC/MS was employed to identify the reaction products. While the reaction

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Figure 7. LC/MS analysis of reaction mixture after incubating for 24 h. (A) The DAD chromatogram. (B) The total ion chromatogram. (C) m/z 234, 256, and 489 selective ion chromatogram (product 1). (D) m/z 314 selective ion chromatogram (product 2). (E) The mass spectra corresponding to the total ion chromatogram at retention time 2.360 min. (F) The mass spectra corresponding to the total ion chromatogram at retention time 4.490 min.

mixture was incubated for about 24 h, a total ion current chromatogram (TIC) (Figure 7B) in comparison with a DAD chromatogram showed that the peaks of product 1 were at 2.360 min. Although product 2 was not discernible in the TIC (Figure 7B), probably because it was obscured by the noises of product 1, it was separated by HPLC and identified in DAD with a retention time at 4.490 min (Figure 7A). The selective ion chromatographs of product 1 and product 2 were demonstrated in parts C and D of Figure 7, respectively. The mass spectra corresponding to the retention time of product 1 showed three peaks: m/z 234 [MP1 + H]+, m/z 256 [MP1 + Na]+ and m/z 489 [2MP1 + Na]+, respectively (Figure 7E). On the other hand, the mass spectra corresponding to the retention time of product 2 showed only a main peak at m/z 314 [MP2 + H]+ (Figure 7F). The possible molecular structures of products 1 and 2 are illustrated in Figure 8 according to information obtained above and the knowledge in the related field. The formation mechanisms of product 2 were similar to the mechanisms of the other fluorescent dihydropyridine derivatives which were clearly elaborated by Kikugawa (24) and confirmatively reviewed by Esterbauer and co-workers (2). The detailed process was therefore omitted from this report. Effect of GlcN on the MDA Modified Proteins. As shown in Figure 9, when BSA (10 mg/mL) was incubated with 1.0 mM MDA at 37 °C for 24 h, the lipofuscin-like fluorescence

increased by 25-fold as compared with the BSA control. The addition of different concentration of GlcN (0.5, 1.0, 2.0, 4.0, and 8.0 mM) inhibited the formation of lipofuscin-like fluorescence by 23%, 30%, 48%, 69%, and 76% respectively. Our results suggested that GlcN might inhibit AGEs/ALEs formation by reacting directly with MDA, thus preventing the formation of MDA adducts and cross-linkages to proteins.

Discussion Although aging is regarded as “by far the most important risk factor for the development of OA” (25), by which mechanism that aging is involved in the development of this degenerative disease remains uncertain. Repetitive loading induced fatigue-damages of the cartilage network were recognized as one of the mechanisms involved in the pathologies of OA (26). Recently, the age-associated accumulation of AGEs was reported to augment tissue stiffness and degenerate the physicochemical properties of collagens in cartilage (18, 27, 28). On the other hand, lipid peroxidation of chondrocytes appeared to be involved in both physiological and pathological processes in cartilage. MDA and HNE, the major unsaturated carbonyl products of lipid peroxidation, have been found largely responsible for cytopathological effects observed during oxidative stress of lipid peroxidation (2, 29). Reaction products of MDA and HNE with amino group of proteins are well-

Reaction of Glucosamine with Malondialdehyde

Figure 8. Proposed structures of GlcN+MDA reaction products. Dotted lines indicate bonding positions during product formation.

Figure 9. Protective effect of GlcN on MDA modification of BSA. BSA (10 mg/mL) was incubated alone or with MDA (1.0 mM) in the absence or presence of different concentrations of GlcN (0.5, 1.0, 2.0, 4.0, 8.0 mM) in 0.2 M PBS, pH 7.4, at 37 °C for 24 h. After protein was precipitated by trichloroacetic acid, and the concentration was adjusted to 0.33 mg/mL, the lipofuscin-like fluorescence was measured at Ex. 395 nm/Em. 460 nm. Results are means of triplicates ( SD.

documented (30), and studies of carbonyl toxicity, therefore, provide clues to a profound nature of oxidative stress. Our results demonstrated for the first time that GlcN reacted with MDA in supraphysiological conditions and formed carbonylated products. Isolated by HPLC and assayed by different techniques, two products were identified in this reaction: a nonfluorescent compound, product 1, and a fluorescent compound, product 2. According to the ion mass measured by LC/ MS-ESI, the products 1 and 2 were proposed to be an enamine derivative and a 1,4-dihydropyridine adduct, respectively (Figure 8). The proposed structure of products suggested that the molecular proportion of GlcN:MDA was 1:1 in product 1, and 1:3 in product 2. As high concentrations of MDA may form acetaldehyde under certain conditions (31, 32), two molecules of MDA and one molecule of acetaldehyde were proposed to

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react with one molecule of GlcN to form dihydropyridine adducts as reported similarly in the literature (33, 34). Our data showed that the reaction mechanism of MDA with GlcN was also similar to that of MDA reacted with other amine compounds, such as with melatonin (35), histamine (36), pyridoxamine (37), dopamine, and serotonin (38). The effect of GlcN and MDA concentrations on GlcN+MDA reactions showed that when reacting with 5.0 mM MDA, excessive GlcN failed to enhance the fluorescence yield due to preferred formation of nonfluorescent product 1 (Figure 5). In contrast, when reacting with 5.0 mM GlcN, high MDA concentration enhanced the fluorescence intensity progressively, which was most possibly owing to the crosslinking and polymerization potential of MDA tending to form the fluorescent product 2, the 1,4-dihydropyridine (Figure 4). The produced fluorophore showed the typical ceroid/lipofuscin-like fluorescence (Ex. 310-395/Em. 395-460), which implicated that GlcN could react, thus reduce toxic carbonyl compounds under certain conditions (9). The pH effects on fluorescence intensity have often been observed in relevant studies (9). In neutral and alkaline conditions the predominant form of MDA is the enolate anion, whereas at acidic pH, MDA mainly exists in the tautomeric undissociated enol form as β-hydroxy acrolein, which may hinder the reactivity of this molecule (2). In this study, nonetheless, the highest fluorescence intensity of the reaction products was found at pH 7.0; while in basic condition, the reaction mixture of GlcN and MDA hardly showed any fluorescence. MDA was frequently reported to react with amino compounds forming fluorescent products such as dihydro-pyridinium derivatives (30, 39-43). In our experimental conditions, MDA (1.0 mM) modified BSA showed lipofuscin-like fluorescence at 460 nm when excited at 395 nm, which was in accordance with the fluorescence of dihydropyridine. Our results demonstrated that GlcN inhibited the development of fluorescence of MDA+BSA adducts which implied a preventive effect on the formation of AGEs/ALEs. Although the MDA concentration (1.0 mM) added to BSA was higher than the average physiological level, it is reasonable to consider that biological tissues or cells may accumulate chemicals on their membrane which may raise the MDA concentration to such a “higher” level in microenvironment. The inhibitive effect of GlcN against MDA+BSA was nevertheless verified in a model system in vitro. These observations implied a general reactivity of amino compounds toward toxic carbonyls, especially unsaturated carbonyls. Such MDA-resulted carbonyl toxification has also been confirmed in a few cellular model systems in our laboratory (44). The degenerative effect of MDA on growth and proliferation of human bone marrow mesenchymal cells was inhibited by GlcN and related amino compounds. Similarly, the increased viscosity and rigidity of erythrocytes induced by MDA were also found reduced significantly by a number of amino compounds (nonpublished data). On the basis of the general biochemical mechanisms of age pigment formation, we proposed a carbonyl toxification theory of aging (9, 45), which was recently developed to be an essential biochemical mechanisms of aging (6). Although the role of carbonyl-scavenging in their health benefits is speculative and needs to be experimentally verified, a variety of anticarbonylative amine containing compounds have been found to be very effective in preventing aging-related degenerative diseases as well as stress-associated situations (the so-called subhealth situations). These include L-dopamine in treating dementia (46,

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47), statins in remedying cardiovascular diseases (48, 49), aminoguanidine in defending glycation-related diabetes mellitus (50), procaine (the key element of the antiaging drug, GH3), and carnosine (51-53) in preventing a broad spectrum of degenerative diseases. Our laboratory has been also among the first to report a series of anticarbonyl stress studies, such as the reactions of melatonin and serotonin with MDA (35), histamine with MDA (36), and pyridoxamine with MDA (37). The aminocarbonyl reaction seemed to be a crucial biochemical process following oxidative and glycation stresses. Therefore, it is reasonable to believe the anticarbonylation and even decarbonylation strategies may lead to a profound understanding and development in the field of preventing subhealth and stresses in pathophysiology. We will with this paper stress for critical attention to achieve more anticarbonylation remedies to battle against aging-related degenerative diseases. Though amino compounds are found to be more and more important in the practice of anticarbonyl stress, the toxicity of carbonyl stress can be better prohibited by the thiol compounds, particularly by glutathione. More detailed information about glutathione in decarbonylation, however, is beyond the scope of this paper. In summary, our study demonstrated that GlcN reacted with MDA to form different conjugated complexes, which could either be fluorescent or nonfluorescent. As one of the aminecontaining anticarbonylation chemicals in pharmacology, GlcN nevertheless prohibited the MDA+BSA induced fluorescence formation. These data suggested a novel scavenging function of GlcN to avoid crosslinking reactions associated with the formation of AGEs and ALEs. Beyond its proposed free radical quenching potential, the prohibitive effect of GlcN on carbonyl stress may be one of the molecular mechanisms of the GlcN treatment in defending degenerative diseases, such as OA. Acknowledgment. We are grateful for the technical support of Dr. Zhou at the Proteomic Laboratory of Hunan Normal University. We also thank Dr. Chen for assistance with LC/ MS analyses. This study was supported by the Distinguished Professor Position Fund of Hunan Normal University and the National Key Basic Research ProgrammesThe Syndrome Defined by TCM, Grant 2003CB517104. This information is available free of charge via the Internet at http://pubs.acs.org.

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