Protective effects of endomorphins, endogenous opioid peptides in the brain, on human low density lipoprotein oxidation Xin Lin 1 , Li-Ying Xue 1 , Rui Wang 1,2 , Qian-Yu Zhao 1 and Qiang Chen 1 1 Department of Biochemistry and Molecular Biology, School of Life Science, Lanzhou University, China 2 State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, China It is now commonly recognized that the damaging con- sequences of oxidative stress have been implicated in a variety of very different human disorders, including arteriosclerosis and diseases of the nervous system [1]. A large body of evidence indicates that the brain appears to be particularly vulnerable to oxidative dam- age because of its high oxygen consumption, abundant lipid content, and relatively low antioxidant levels [2,3]. Oxidative damage may play important roles in the slowly progressive neuronal death that is character- istic of several different neurodegenerative disorders, such as Alzheimer’s disease and Parkinson disease [4,5]. Reactive oxygen species (ROS) rapidly oxidize cellular lipids, resulting in the formation of numerous lipid peroxidation products in nerve cells. Oxidatively modified lipids are able to react with cellular and sub- cellular membranes, leading to neuronal cell death [6,7]. Low density lipoprotein (LDL) is present in the brain and is exposed to a highly oxygenated and lipid-enriched environment, making it susceptible to free radical-mediated lipid peroxidation that can result in the formation of oxidized LDL (oxLDL) [7–9]. A number of studies have proved that oxLDL Keywords antioxidant; endomorphins; free radical; lipid peroxidation; low-density lipoprotein Correspondence R. Wang, State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China Fax: +86 931 8912561 Tel: +86 931 8912567 E-mail: wangrui@lzu.edu.cn (Received 3 December 2005, accepted 23 January 2006) doi:10.1111/j.1742-4658.2006.05150.x Neurodegenerative disorders are associated with oxidative stress. Low den- sity lipoprotein (LDL) exists in the brain and is especially sensitive to oxi- dative damage. Oxidative modification of LDL has been implicated in the pathogenesis of neurodegenerative diseases. Therefore, protecting LDL from oxidation may be essential in the brain. The antioxidative effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid pep- tides in the brain, on LDL oxidation has been investigated in vitro. The peroxidation was initiated by either copper ions or a water-soluble initiator 2,2¢-azobis(2-amidinopropane hydrochloride) (AAPH). Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes, thiobar- bituric acid reactive substances, and the relative electrophoretic mobility. Low density lipoprotein oxidative modifications were assessed by evaluat- ing apoB carbonylation and fragmentation. Endomorphins markedly and in a concentration-dependent manner inhibited Cu 2+ and AAPH induced the oxidation of LDL, due to the free radical scavenging effects of endo- morphins. In all assay systems, EM1 was more potent than EM2 and l-glutathione, a major intracellular water-soluble antioxidant. We propose that endomorphins provide protection against free radical-induced neuro- degenerative disorders. Abbreviations AAPH, 2,2¢-azobis(2-amidinopropane hydrochloride); BHT, butylated hydroxytoluene; DNPH, 2,4-dinitrophenylhydrazine; EM1, endomorphin 1; EM2, endomorphin 2; GSH, L-glutathione; LDL, low density lipoprotein; oxLDL, oxidized LDL; REM, relative electrophoretic mobility; ROS, reactive oxygen species; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substance. FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS 1275 is cytotoxic to neurones, further suggesting that oxLDL may not only be involved at the vascular level of neurodegenerative diseases but also could be more directly responsible for the degeneration of neurones [10–12]. Elevated levels of ROS and trace metals, such as copper and iron, capable of oxidizing LDL are present in neurodegenerative conditions [13]. Therefore, neuronal cells have to maintain an effective antioxidation system in order to protect themselves against ROS overload and subsequent damage. As described in various excellent reviews, antioxidants keep the fine-tuned balance between the physiological production of ROS and their detoxification [14,15]. Recently, it has been observed that the female sex hormone estrogen can inhibit the oxidation of LDL and can attenuate the cytotoxicity of oxLDL on neur- onal cells [14,16,17]. It is believed that melatonin, a hormone mainly secreted by the pineal gland, is an effective free radical scavenger and antioxidant and thus attenuates the effects of free radical-induced neur- onal damage [18,19]. There are studies investigating the antioxidant activity of melatonin in lipid model systems [20] and also in LDL [21,22]. In addition, it has been reported that the brain monoamines and their metabolites can inhibit lipid peroxidation and protect from oxidative damage in the brain [23]. Recent studies have demonstrated that enkephalins (leu-enke- phalin, met-enkephalin) and their derivatives (5-S-cyst- einyldopaenkephalin, 2-S-cysteinyldopaenkephalin and [d-Ala 2 , d-Leu 5 ]enkephalin) have free radicals scaven- ging activities and the capacity to reduce ROS-induced lipid peroxidation [24–26]. Endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides, have been found in much higher amounts in the human brain [27]. The two peptides differ in one amino acid: EM1 (Tyr-Pro-Trp-Phe-NH 2 ) and EM2 (Tyr-Pro-Phe-Phe- NH 2 ). The major effect of the endomorphins is their antinociceptive action [28,29]. Additionally, endomor- phins can cause vasodilatation by stimulating nitric oxide release from the endothelium [30] and bind to l-opioid receptors to activate G-proteins, regulate gastrointestinal transit, respiratory system and mem- ory [28,31–33]. Endomorphins have been investigated to modulate damage related to inflammatory diseases of the brain [34]. More recently, we have found that endomorphins can scavenge radical, and inhibit lipid peroxidation, DNA and protein oxidative damage [35]. It is worth to note that EM1 is more potent than EM2 and l-glutathione (GSH). Therefore, it is worthy to see if the antioxidant activity of endomor- phins is also valid in LDL. The present study investigates the preventive effects of endomorphins against Cu 2+ - and a water-soluble initiator 2,2¢-azobis(2-amidinopropane hydrochloride) (AAPH)-induced human LDL lipid peroxidation in vitro, the characteristics of which have been exten- sively investigated. The effects of endomorphins were compared to those of GSH. It is suggested that endo- morphins and especially EM1 may provide antioxidant defence in neurodegenerative disorders. Results Inhibition of conjugated diene formation by EM1 and EM2 A set of representative kinetic curves of conjugated dienes formation during the peroxidation of LDL is shown in Fig. 1. It is seen from Fig. 1 that in the absence of exogenous antioxidants, conjugated diene formation was still inhibited for about 20 min (Fig. 1, line a), demonstrating the presence of endogenous anti- oxidants in LDL ) for example, a-tocopherol, carote- noids, ubiquinol-10 [36] ) which can trap the initiating and ⁄ or propagating radicals to inhibit peroxidation. After the inhibition period, absorbance at 234 nm increased fast with time upon Cu 2+ -initiation, indica- ting depletion of the endogenous antioxidants and fast peroxidation of LDL. The change in absorbance was inhibited by addition of EM1 (1.25–5 lm), EM2 (5 lm) and GSH (5 lm) in the inhibition period. After Fig. 1. Formation of conjugated dienes during the peroxidation of LDL at pH 7.4 and 37 °C, initiated with Cu 2+ and inhibited by endo- morphins and GSH. [LDL] ¼ 0.2 mg proteinÆmL )1 ;[Cu 2+ ] ¼ 10 lM. a, uninhibited peroxidation; b, inhibited with 1.25 l M EM1; c, inhib- ited with 2.5 l M EM1; d, inhibited with 5 lM EM1; e, inhibited with 5 l M EM2; f, inhibited with 5 lM GSH. The results are representa- tive of three independent experiments. Protective effects of endomorphins on huLDL oxidation X. Lin et al. 1276 FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS the inhibition period the rate of the absorbance increase became faster, which is close to the original rate of propagation, demonstrating the exhaustion of the antioxidant. The kinetic data deduced from Fig. 1 are listed in Table 1. It is shown in Fig. 1 and Table 1 that the inhibition period t inh was prolonged in a dose- dependent fashion for EM1, resulting an about 1.6-, 2.8- and 4-fold longer interval than that of the control at 1.25, 2.5 and 5 lm drug, respectively. On the basis of t inh , R inh and R p , the antioxidant activity follows the sequence EM1 > GSH > EM2. Inhibition of TBARS formation by EM1 and EM2 Figure 2 shows the inhibition of thiobarbituric acid reactive substance (TBARS) formation by EM1, EM2 and GSH during the Cu 2+ -induced peroxidation of LDL. After 60 min incubation at 37 °C, 18 nmol TBARSÆmg )1 LDL protein were generated in control experiments; TBARS were instead 15.26, 11.16, 3.84 and 2.06 nmolÆmg )1 LDL protein in the presence of 0.625, 1.25, 2.5 and 5 lm EM1. It is clearly seen that EM1 significantly suppressed rate of TBARS forma- tion and increased the inhibition period in a concentra- tion- and time-dependent manner. EM2 and GSH show similar inhibitory activity. Figure 3 indicates the inhibition of TBARS formation by EM1, EM2 and GSH during AAPH-induced peroxidation of LDL. It was found that addition of EM1 inhibited TBARS for- mation in a dose-dependent manner. EM2 and GSH also diminished the rate of TBARS formation and increased the inhibition period. During both Cu 2+ - and AAPH-induced peroxidation of LDL, the activity sequence is EM1>GSH>EM2. Inhibition of apoB carbonyl formation by EM1 and EM2 Oxidative damage to protein results in the formation of protein carbonyl groups [37]. Therefore, we meas- ured the inhibitory effects of EM1, EM2 and GSH on either Cu 2+ - or AAPH-induced LDL oxidation by apoB carbonyl assay. As shown in Fig. 4, Cu 2+ -induced apoB carbonyl formation were inhibited about 55, 41 and 35.7% at 10, 20 and 40 lm EM1, respectively. At the same experimental concentrations, either EM2 or GSH were also effective. During AAPH-induced LDL oxidation, the carbonyl content Table 1. Kinetic parameters for the Cu 2+ -induced peroxidation of LDL and its inhibition by endomorphins and GSH. The reaction con- ditions and the initial concentration of the substrates are the same as described in the legends of Fig. 1 for reactions conducted in LDL. Data are the means of three determinations. Compounds t inh (min) R p (10 )2 min )1 ) R inh (10 )3 min )1 ) None 20.6 ± 1.6 3.8 ± 0.2 2.1 ± 0.1 EM1 (1.25 l M) 31.9 ± 1.8** 2.3 ± 0.2 1.1 ± 0.1 EM1 (2.50 l M) 56.6 ± 4.2** 1.9 ± 0.2 1.1 ± 0.1 EM1 (5.00 l M) 80.6 ± 4.7** 1.5 ± 0.1 0.9 ± 0.1 EM2 (5.00 l M) 22.5 ± 1.4 2.4 ± 0.2 3.6 ± 0.2 GSH (5.00 l M) 54.4 ± 3.8** 2.2 ± 0.2 1.0 ± 0.1 **P<0.01 compared with the control. Fig. 2. Inhibition of TBARS formation during the Cu 2+ -induced per- oxidation of LDL by endomorphins and GSH at 37 °C. [LDL] ¼ 0.2 mg protein ⁄ mL; [Cu 2+ ] ¼ 10 lM. a, uninhibited peroxidation; b, inhibited with 0.625 l M EM1; c, inhibited with 1.25 lM EM1; d, inhibited with 2.5 l M EM1; e, inhibited with 5 lM EM1; f, inhibited with 5 l M EM2; g, inhibited with 5 lM GSH. Values are mean ± SE (n ¼ 3). Fig. 3. Inhibition of TBARS formation during the AAPH-induced per- oxidation of LDL by endomorphins and GSH at 37 °C. [LDL] ¼ 0.5 mg proteinÆmL )1 ; [AAPH] ¼ 20 mM. a, uninhibited peroxidation; b, inhibited with 10 l M EM1; c, inhibited with 20 lM EM1; d, inhib- ited with 40 l M EM1; e, inhibited with 40 lM EM2; f, inhibited with 40 l M GSH. Values are mean ± SE (n ¼ 3). X. Lin et al. Protective effects of endomorphins on huLDL oxidation FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS 1277 of apoB was reduced by the addition of EM1, EM2 and GSH in a concentration-dependent manner (Fig. 5). EM1 is more potent than EM2 and GSH, similar to that observed in the LDL oxidation prod- ucts assay mentioned above. Inhibition of apoB fragmentation by EM1 and EM2 Figures 6 and 7 show gel electrophoresis of apoB trea- ted with EM1, EM2 and GSH in the presence of 10 lm Cu 2+ or 20 mm AAPH at 37 °C for 24 h, respectively. The spot of apoB was observed in native LDL (Fig. 6, lane 1), but a degradation pattern of apoB was observed when LDL was incubated with Cu 2+ (Fig. 6, lane 2). Compared to the Cu 2+ -treated band in lane 2, a discernible increase in the intensity of bands was noted with the addition of 10, 20 and 40 lm EM1 (Fig. 6A, lane 3–5). Treatment of apoB with 40 lm EM1, EM2 and GSH in the presence of 10 lm Cu 2+ decreased the extent of apoB fragmenta- tion (Fig. 6B). As shown in Fig. 7A, when LDL was Fig. 4. Protection of apoB carbonyl formation during the Cu 2+ -induced peroxidation of LDL by endomorphins and GSH. LDL (0.5 mg proteinÆmL )1 ) in NaCl ⁄ P i was incubated with 10 lM Cu 2+ and ⁄ or compounds at 37 °C. After incubation for 6 h, carbonyl content was measured as described in Experimental procedures. Values are mean ± SE (n ¼ 3). *P<0.05 compared with GSH. Fig. 5. Protection of apoB carbonyl formation during the AAPH- induced peroxidation of LDL by endomorphins and GSH. LDL (0.5 mg proteinÆmL )1 ) in NaCl ⁄ P i was incubated with 20 mM AAPH and ⁄ or compounds at 37 °C. After incubation for 6 h, carbonyl content was measured as described in Experimental procedures. Values are the mean ± SE (n ¼ 3). *P<0.05 compared with GSH. A 12345 B Fig. 6. SDS ⁄ PAGE of apoB fragmentation induced by Cu 2+ and inhibited by endomorphins and GSH. LDL (1.5 mg proteinÆmL )1 ) was incubated with 10 l M Cu 2+ and ⁄ or compounds in NaCl ⁄ P i (pH 7.4) for 24 h at 37 °C. (A) apoB fragmentation was inhibited by EM1 in the presence of 10 l M Cu 2+ . Lane 1, native LDL; lane 2, 0 l M; lane 3, 10 lM EM1; lane 4, 20 lM EM1; lane 5, 40 lM EM1. (B) apoB fragmentation was inhibited by compounds in the pres- ence of 10 l M Cu 2+ . Lane 1, native LDL; lane 2, 0 lM; lane 3, 40 l M EM1; lane 4, 40 lM EM2; lane 5, 40 lM GSH. A 12345 B Fig. 7. SDS ⁄ PAGE of apoB fragmentation induced by AAPH and inhibited by endomorphins and GSH. LDL (1.5 mg proteinÆmL )1 ) was incubated with 20 m M AAPH and ⁄ or compounds in NaCl ⁄ P i (pH 7.4) for 24 h at 37 °C. (A) apoB fragmentation was inhibited by EM1 in the presence of 20 m M AAPH. Lane 1, native LDL; lane 2, 0 l M; lane 3, 50 lM EM1; lane 4, 100 lM EM1; lane 5, 200 lM EM1. (B) apoB fragmentation was inhibited by compounds in the presence of 20 m M AAPH. Lane 1, native LDL; lane 2, 0 lM; lane 3, 200 l M EM1; lane 4, 200 lM EM2; lane 5, 200 lM GSH. Protective effects of endomorphins on huLDL oxidation X. Lin et al. 1278 FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS incubated with EM1 in the presence of 20 mm AAPH at 37 °C for 24 h, a concentration-dependent increase in the intensity of apoB bands was observed. More- over, we investigated the protective effects of 200 lm EM1, EM2 and GSH against 20 mm AAPH-induced apoB fragmentation (Fig. 7B). On the basis of the intensity of bands, the protective activity follows the sequence of EM1 > GSH > EM2. Inhibition of REM by EM1 and EM2 Using the same sample solutions as used for protein carbonyl analysis, we performed relative electropho- retic mobility (REM) studies. Figure 8A indicates the protective effects of EM1, EM2 and GSH against Cu 2+ -induced LDL oxidation. In the presence of 40 or 20 lm EM1, 10 lm Cu 2+ caused only a 24.5 or 49.4% increase in REM, respectively, but in the presence of 40 lm EM2 or GSH, 10 lm Cu 2+ caused a 95.4 or 66.2% increase in REM, respectively. Thus, EM1 is at least twice or three times as effective as GSH or EM2 in protecting against Cu 2+ -induced apoB modification in LDL. As shown in Fig. 8B, exposure of LDL to 20 mm AAPH in the presence of an increasing concentration of EM1 (50–200 lm) resulted in a dose-dependent decrease in REM. However, EM2 and GSH were not able to prevent AAPH-induced apoB modification in LDL. Discussion It is well established that oxidative stress is not simply a by-product of degenerative processes or the end product of nerve cell death but may directly initiate neurodegeneration. Although oxLDL has been studied primarily for its role in the development of atheroscler- osis, recent studies have identified that oxidative modi- fication of LDL is capable of eliciting cytotoxicity, differentiation, and inflammation in neuronal cells [10– 12,38]. Suppression of oxidative modification of LDL by antioxidants may be effective in preventing and treating neurodegenerative diseases [8,16]. In the pre- sent study, two different initiating assays were used. One is copper which is one of the major potential sources of free radical production in the brain [39]. Cu 2+ -induced LDL peroxidation is generally believed to involve reductive activation of Cu 2+ as the first stage. The reductive activation may be accomplished by a net transfer of one electron to produce Cu + which is a strong pro-oxidant and can rapidly generate the ultimate initiating radicals by a Fenton-type reac- tion with peroxides or by forming an electron transfer complex with molecular oxygen. Another is AAPH, a water-soluble initiator, which decomposes at physiolo- gical temperature producing alkyl radicals (R • ) fol- lowed by fast reaction with oxygen to give alkyl peroxyl radicals (ROO • ) to initiate LDL peroxidation (LOO • ). In the presence of an antioxidant molecule, AH, either the initiating peroxyl radical and ⁄ or the propagating lipid peroxyl radical can be trapped and a new antioxidant radical, A • , produced. If the A • is a stabilized radical (e.g. a-tocopheroxyl radical or ascor- bate radical) which can promote the rate-limiting hydrogen abstraction reactions and undergo fast ter- mination reactions, the peroxidation would be inhib- ited [40]. The formation of lipid peroxidation products is a phenomenon common in most types of neurone damage associated with oxidative stress [7,10]. Cu 2+ - induced LDL peroxidation is generally monitored by UV spectroscopy since the primary peroxidation Fig. 8. Inhibition of the increase in REM of LDL during the Cu 2+ -induced (A) or AAPH-initiated (B) peroxidation of LDL by endo- morphins and GSH. LDL (0.5 mg proteinÆmL )1 ) in NaCl ⁄ P i was incu- bated with 10 l M Cu 2+ or 20 mM AAPH and ⁄ or compounds at 37 °C. After incubation for 6 h, REM of LDL was measured as described in Experimental procedures. Values are the mean ± SE (n ¼ 6). *P < 0.05 and **P < 0.01 compared with GSH, respectively. X. Lin et al. Protective effects of endomorphins on huLDL oxidation FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS 1279 products of polyunsaturated fatty acids in LDL are hydroperoxides possessing a conjugated diene structure which shows characteristic UV absorption at 234 nm [41]. The rate of the chain propagation, R p , the inhibited rate of propagation by antioxidants, R inh , and the inhi- bition period, t inh , can be easily obtained from spectro- photometric data. In the present work, we can find from Fig. 1 and Table 1 that on the basis of t inh , R inh and R p , the antioxidant activity follows the sequence EM1 > GSH > EM2 in conjugated diene formation. Moreover, TBARS formation is also used to detect lipid peroxidation products in LDL oxidation. It is clearly seen by comparison of Fig. 1 with Figs 2 and 3 that the antioxidant activity follows the same sequence in spite of the activity being monitored by conjugated diene formation or by TBARS production, and in spite of the peroxidation being initiated by Cu 2+ or by peroxyl radicals (generated by AAPH). Increased tissue protein carbonyls have been detected in numerous human diseases, such as rheumatoid arth- ritis, ischaemia–reperfusion injury to heart muscle and Alzheimer’s disease [42]. Protein carbonyl formation is a biomarker of protein oxidation and has some advan- tages over lipid peroxidation products because the for- mation of protein-bound carbonyl groups seems to be a common phenomenon of protein oxidation, and because of the relatively early formation and relative stability of oxidized proteins [42]. As shown in Figs 4 and 5, EM1 is more active than EM2 and GSH. In addition, the present study also showed that endomor- phins inhibited not only the lipid peroxidation of LDL but also the fragmentation of apoB of LDL and increase in REM in a concentration-dependent manner. The results presented in this paper provide evidence that endomorphins ) endogenous opioid peptides in the brain ) are very efficient in protecting LDL against Cu 2+ - and AAPH-induced oxidative modifica- tion. The inhibitory activity of EM1 is much more effective than that of tested EM2 and GSH, a major intracellular water-soluble antioxidant. It is obvious that these micromolar in vitro concentrations are signi- ficantly higher than endomorphins levels normally detected in the body. Nevertheless, the following should be taken into account. Firstly, to reach a pro- nounced and well-detectable oxidative damage in vitro, one has to use rather high concentrations of oxidants in vitro and, consequently, high concentrations of endomorphins are necessary. Secondly, recent studies have demonstrated that endogenous opioid peptides are released from cells during inflammation and stress, and reach high levels at theses sites. Finally, drugs for effective prevention of damage or treatment may reach pharmacological levels rather than physiological concentrations. Moreover, the general biological activ- ities of endomorphins act through l-opioid receptors, whereas the antioxidant activity of endomorphins is not dependent on opioid receptors. Very probably the neuroprotective effects of endomorphins result from a combination of the different modes of action. In the case of Cu 2+ -induced LDL oxida- tion ) where lipid peroxyl radicals are generated indi- rectly from a series of redox reactions ) EM1 had no apparent copper-binding effect, as judged by both spectral study and lack of quenching of the intrinsic drug absorption by copper (data not shown). Our results indicate that EM1 can trap the lipid peroxyl radicals (LOO • ) derived indirectly from copper on the surface of LDL particles and behave well as chain breaking antioxidant against Cu 2+ -induced LDL per- oxidation. Furthermore, Cu 2+ -induced LDL peroxida- tion is considered to be more relevant to the in vivo situation than the AAPH-induced peroxidation, since the former most likely involves a site-specific attack of the apoB, whereas the latter produces a more-or-less random attack of free radicals in LDL. Also, oxida- tively modified of LDL by Cu 2+ exhibits biological properties very similar, if not identical, to those of cell-oxidized LDL. In addition, a growing body of data supports a significant role for redox active metals, Cu, as key modulators of the pathogenic pathways that underlie neurodegenerative disorders and oxLDL- mediated neuronal damage in vivo, would depend on the availability of Cu [39,43]. Recently, we have reported that endomorphins can directly scavenge galvinoxyl radicals and AAPH- derived alkyl peroxyl radicals [35]. The difference of EM1 and EM2 is primarily Trp and Phe at position 3. Trp does not possess phenolic hydrogens and only has an indole ring, similar to melatonin ) a hormone mainly secreted by the pineal gland ) which has been reported to be able to protect from oxidative damage in the central nervous system [18]. However, free amino acids such as Trp and Phe cannot react with galvinoxyl radicals (data not shown). Therefore, it is suggested that both the indole ring on the Trp as well as the side chain on the indole nucleus are essential for the antioxidant activity of EM1. The most active hydrogen of EM1 might be H-10 on the Trp, which is an allylic hydrogen. It is well known that allylic hydro- gens are very active and easily abstracted by free radi- cals. In addition, conjugation with -NH on the indole shall further weaken the C–H-10 bond. The mechanis- tic details are worthy of further study. Lipid peroxidation is increased in neurophathologi- cal conditions such as Alzheimer’s and Parkinson disease [10]. Recent studies have reported that oxLDL Protective effects of endomorphins on huLDL oxidation X. Lin et al. 1280 FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS is cytotoxic to neurones and application of antioxi- dants may attenuate neurone death. Thus, inhibition of LDL peroxidation by antioxidants becomes an attractive therapeutic strategy to prevent and treat neurodegenerative diseases. This has led to a great deal of research devoted to the prevention of lipid peroxi- dation in LDL by antioxidants [8,17]. However, the great pharmacological disadvantages of many antioxi- dants are their very limited passage through the blood–brain barrier [14]. Therefore, the existence of antioxidants in the brain protective systems or with much better blood–brain barrier permeation may be essential. The recently demonstrated powerful antioxid- ant activities of certain amines and imines may be the starting point for developing neuroprotective anti- oxidants [44]. Our data demonstrate that endomor- phins may inhibit the formation of oxLDL and thus minimize subsequent oxLDL-induced toxicity. We pro- pose that the neuroprotective activity of endomorphins may provide new insights into therapeutics of neuro- degenerative diseases and a new understanding for oxidative stress in the brain. Experimental procedures Chemicals EM1 and EM2 were synthesized in our laboratory [45]. The purity of the compounds was determined by HPLC (> 95%) and their structures were verified by MS and amino acid analysis. Agarose, 2,4-dinitrophenylhydrazine (DNPH), thiobarbituric acid (TBA) and GSH were from Sigma (St Louis, MO). AAPH and butylated hydroxytolu- ene (BHT) were from Aldrich (Milwaukee, WI). Sudan Black B, Acrylamide and bis-acrylamide were from BBI (Markham, CA). All other chemicals were of the highest quality available. LDL isolation Blood collected into the anticoagulant EDTA (final concen- tration 3 mm) was taken from healthy volunteers. LDL (1.019–1.063 gÆmL )1 ) was isolated from the plasma by a discontinuous density gradient centrifugation procedure as described elsewhere [41] at 140 000 g for 6 h using a HITA- CHI 55P-72 ultracentrifuge in KBr solution at 4 °C in the presence of EDTA (100 lm). The isolated LDL fraction was then dialysed with phosphate buffer (NaCl ⁄ P i pH 7.4) containing 100 lm EDTA to prevent oxidation during dialysis. EDTA was removed by dialysis with NaCl ⁄ P i prior to the oxidation experiments. The concentration of protein was determined by the method of Lowry et al. [46]. LDL was stored in the dark at 4 °C and used within 1 week. Determination of conjugated dienes The ability of endomorphins to inhibit Cu 2+ -mediated LDL oxidation was evaluated in quartz cuvettes through continuous spectrophotometric monitoring of absorbance increase at 234 nm, reflecting conjugated diene formation during peroxidative processes [41]. LDL (0.2 mg pro- teinÆmL -1 ) was incubated at 37 °C using a Shimadazu (Kyoto, Japan) model UV-260 spectrophotometer. Oxida- tion was initiated by 10 lm CuSO 4 . EM1, EM2 and GSH were added to inhibit the peroxidation. The absorbance at 234 nm was measured every 5 min against appropriate ref- erence cuvettes for the duration of the experiment. Every experiment was repeated three times and the results were reproducible within 10% experimental deviation. Determination of TBARS The formation of TBARS was used to monitor lipid per- oxidation [47]. LDL (0.2 or 0.5 mg proteinÆmL )1 ) was incubated at 37 °C in NaCl ⁄ P i , pH 7.4. The peroxidation was initiated by either 10 lm Cu 2+ or 20 mm AAPH and inhibited by EM1, EM2 and GSH. The reaction mixture was gently shaken at 37 °C and aliquots of the reaction mixture were taken out at specific intervals to which a tricholoroacetic acid ⁄ TBA ⁄ HCl stock solution (15% w ⁄ v trichloroacetic acid; 0.375% w ⁄ v TBA; 0.25 m HCl) was added, together with 0.02% w ⁄ v BHT. This amount of BHT completely prevents the formation of any nonspecific TBARS, as well as preventing decomposi- tion of AAPH during the subsequent boiling. The solu- tion was heated in a boiling water bath for 15 min. After cooling, the precipitate was removed by centrifugation. TBARS in the supernatant was determined at 532 nm. Results were calculated as nmol TBARSÆmg LDL pro- tein )1 , using a molar extinction coefficient of 156 000 m )1 Æcm )1 [47]. Determination of apoB carbonylation ApoB carbonyls were measured spectrophotometrically with the use of the carbonyl specific reagent DNPH as pre- viously reported [48]. Briefly, LDL (0.5 mg proteinÆmL )1 ) was incubated with either 10 lm Cu 2+ or 20 mm AAPH, with or without different concentration of EM1, EM2 and GSH. After incubation for 6 h at 37 °C, 0.5 mL 10 mm DNPH in 2 N HCl was added to 1 mL of the incubation mixture and incubated at room temperature for 1 h. Fol- lowing addition of 0.5 mL 20% tricholoroacetic acid, the samples were incubated on ice for 10 min and centrifuged at 4000 g for 10 min. Protein pellets were washed three times with 3 mL ethanol ⁄ ethyl acetate (1 : 1, v ⁄ v) and dis- solved in 6 m guanidine (pH 2.3). The peak absorbance at 370 nm was used to quantify protein carbonyls. X. Lin et al. Protective effects of endomorphins on huLDL oxidation FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS 1281 Determination of apoB fragmentation The measurement apoB fragmentation was performed by vertical electrophoresis as described previously [49] using a 7.5% SDS ⁄ PAGE at a constant current of 20 mA for 90 min. Oxidation of LDL (1.5 mg proteinÆmL )1 )in NaCl ⁄ Pi was initiated by either 10 lm Cu 2+ or 20 mm AAPH and inhibited by EM1, EM2 and GSH. After incubation for 24 h at 37 °C, 1 mm EDTA or 0.02% (w ⁄ v) BHT was added to the reaction mixture to prevent further oxidation, respectively. Then, the samples were mixed with an equal volume of 2 · SDS ⁄ PAGE sample buffer (100 mm Tris ⁄ HCl pH 6.8, 4% SDS, 20% glycerol, 10% b-mercaptoethanol, 0.01% Bromophenol blue), heated at 100 °C for 5 min and loaded onto a 7.5% acrylamide SDS-containing gel. The gels were stained with 0.05% Coomassie Brilliant Blue R-250 and photo- graphed. Determination of REM The negative surface charge of apoB was determined by agarose gel electrophoresis as described previously [49]. LDL (0.5 mg proteinÆmL )1 ) was incubated with either 10 lm Cu 2+ or 20 mm AAPH, with or without different concentration of EM1, EM2 and GSH. After incubation for 6 h at 37 °C, the samples were examined by electro- phoresis at 100 V for 30 min in 50 mm barbital buffer (pH 8.6) on 0.5% agarose gels and stained with Sudan Black B. The REM was defined as the ratio of migrating distance of oxidized LDL to that of native LDL. Statistical analysis Results are expressed as mean ± SE. For most experi- ments, mean values were compared using Student’s t-test to evaluate statistical differences. In the figures, symbols * and ** indicate P < 0.05 and P < 0.01, respectively. 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