Use of capillary electrophoresis to evaluate protective effects of methylglyoxal scavengers on the activity of creatine kinase

Jinyu Ma1, 2

Xiaofang Peng1

Ka-Wing Cheng1

Feng Chen1

Dajin Yang2

Bo Chen2*Mingfu Wang1

1School of Biological Sciences,The University of Hong Kong,Hong Kong, P. R. China

2Key Laboratory of ChemicalBiology and Traditional ChineseMedicine Research (Ministry ofEducation), Hunan NormalUniversity, Changsha, Hunan,P. R. China

Original Paper

Use of capillary electrophoresis to evaluateprotective effects of methylglyoxal scavengers onthe activity of creatine kinase

Methylglyoxal (MGO) is a highly reactive a-oxoaldehyde formed endogenously innumerous enzymatic and nonenzymatic reactions. The reactions between MGO andvarious amino residues in proteins not only result in inactivation of enzymes, butalso lead to the formation of different detrimental advanced glycation endproducts(AGEs). Recently, it was reported that creatine kinase (CK, EC 2.7.3.2) activity couldbe reduced or even lost under incubation with MGO in vitro. In this study, an effi-cient CE analytical method was developed for the evaluation of CK activity. Based onthis CE method, the inhibitory effect of MGO on CK activity was confirmed. SeveralMGO scavengers such as aminoguanidine (AG) and some thiols showed obvious pro-tective effects on CK activity against MGO. Furthermore, tiopronin (TP), a hepatopro-tective drug, was found for the first time to counteract MGO-induced inhibition ofCK activity in CK reaction. Meanwhile, TP also retained adenosine diphosphate(ADP) generation level in plasma treated with MGO, which implies that this drugmay have potential protective effect on other enzymes which are associated withadenine nucleotide metabolism. Besides, the established CE approach can be uti-lized as a model for screening effective MGO scavengers by monitoring CK-catalyzedconversion between adenosine triphosphate and ADP.

Keywords: Capillary electrophoresis / Creatine kinase / Methylglyoxal / Thiols / Tiopronin /

Received: April 25, 2008; revised: May 30, 2008; accepted: June 6, 2008

DOI 10.1002/jssc.200800241

1 Introduction

Methylglyoxal (MGO), a physiological metabolite, is ahighly reactive a-oxoaldehyde formed endogenously innumerous enzymatic and nonenzymatic reactions (Mail-lard reaction) [1, 2]. Many studies have proven modifica-tions of biological macromolecules, mostly proteins,with this reactive carbonyl compound. MGO can reactwith various amino residues of proteins to form differentdetrimental advanced glycation endproducts (AGEs) [3,4]. It has been reported that reactions between MGO andarginine residues lead to the formation of imidazolones,such as Nd-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-orni-

thine (MG-H1), 2-amino-5-(2-amino-5-hydro-5-methyl-4-imidazolon-1-yl)pentanoic acid (MG-H2), and argpyrimi-dine [5–7], while reactions between MGO and lysinecould yield MGO-derived lysine –lysine crosslinks, Ne-(1-carboxyethyl)lysine (CEL) and MGO-derived lysine dimer1,3-di(Ne-lysino)-4-methyl-imidazolium (MOLD) [8, 9].Hence, the activities of modified proteins could be influ-enced to some extent. The modification of proteins byMGO is a signal for their degradation and the accumula-tion of AGEs in vivo has been implicated in the develop-ment of diabetic complications [10, 11], atherosclerosis[12], Alzheimer's disease [13, 14], aging, and oxidativestress [14, 15].

Creatine kinase (CK, EC 2.7.3.2), also known as creatinephosphokinase (CPK), is an enzyme widely found in mus-cular tissues throughout the body and also in the brain.CK isoenzymes are involved in cellular energy metabo-lism by catalyzing the reversible transfer of a high energyphosphate group from adenosine triphosphate (ATP) tocreatine, producing phosphocreatine and adenosinediphosphate (ADP) [16, 17]. In clinical examinations, ele-

Correspondence: Dr. Mingfu Wang, School of Biological Scien-ces, The University of Hong Kong, Pokfulam Road, Hong Kong,P. R. ChinaE-mail: [email protected]: +852-22990340

Abbreviations: ADP, adenosine diphosphate; AG, aminoguani-dine; AGE, advanced glycation endproducts; ATP, adenosine tri-phosphate; CK, creatine kinase; Cys, cysteine; GSH, reduced glu-tathione; MGO, methylglyoxal; RS, reaction solution; TP, tiopro-nin; Vc, vitamin C

* Additional Corresponding Author: Professor Bo Chen, E-mail:[email protected].

i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

2846 J. Ma et al. J. Sep. Sci. 2008, 31, 2846 – 2851

J. Sep. Sci. 2008, 31, 2846 –2851 Electrodriven Separation 2847

vation of CK levels is an important indicator of damageto muscle and measurement of CK in blood has beenused as a diagnostic test for screening suspected musclediseases [18, 19]. Recently, it was reported that the activ-ity of CK was reduced or even lost when incubated withdifferent concentrations of MGO under physiologicalconditions in vitro [20, 21]. Hence, it is reasonable to useaminoguanidine (AG), a typical scavenger for reactivecarbonyl compounds [22], to save the active cysteine (Cys)residues in CK by reacting with MGO. Besides, as thiolgroup of Cys is the main target of glycation due to itsstrong nucleophilic property, compounds with thiolgroups are supposed to assume the role of protectiveagents to protect CK activity from MGO.

Conventional methods adopted for measuring CKactivity are colorimetric assays and CK activity wasmainly determined by monitoring the proton genera-tion during the reaction of ATP and creatine catalyzed byCK with the indicator thymol blue at 597 nm [20, 23]. Inaddition, in clinical laboratories, CK level proportionateto its activity was generally tested based upon enzymecoupled reactions, in which the ATP (or ADP) amount inthe system can be reflected by measuring the absorptionof produced NADPH (or NADH) at 340 nm [24, 25]. How-ever, the measurement of production of proton andNADPH (or NADH) is apparently less reliable and conven-ient when compared with the direct measurement of theamount of ADP and ATP. Though the separation of ATPand ADP can also be achieved by ion-pair high perform-ance chromatography, its use is limited to simple sys-tems without much interference from the matrix. Forthis consideration, an efficient CE analytical method forthe evaluation of CK activity was developed in this study.CE is a powerful analytical tool with rapid separationand requiring only a small sample load, especially forionic species [26, 27], and has demonstrated its strongcapacity to resolve nucleotides (such as ATP and ADP) invarious biological samples (cells, blood, and plasma)[28–31]. Moreover, CE offers an ideal approach to moni-tor enzymatic reactions, such as enzymatic conversionsof ATP to ADP catalyzed by hexokinase and MgATPase,respectively [32–36]. Based on this established CEmethod, the inhibitory effect of MGO on CK activity wasconfirmed. Meanwhile, effects of AG and several thiolcompounds including Cys, reduced glutathione (GSH),and tiopronin (TP) in protecting against MGO-mediatedattenuation of CK activity were investigated. Consideringantioxidants may also be able to interrupt glycation ofproteins with MGO, the protective effect of vitamin C (Vc)on CK was also studied. All tested compounds showedobvious protective effects on CK activity while Vc pre-sented relatively weaker effects on the maintenance ofCK activity. In addition, the study of CK activity influ-enced by MGO was also tested in plasma. CK activities inboth TP-free and TP-containing plasma after incubation

with MGO were examined. Our results showed that MGOcould decrease CK activity in plasma and TP is competentfor alleviating the MGO-induced reduction of activities ofsome enzymes engaging in adenine nucleotide metabo-lism.

2 Experimental

2.1 Materials and instruments

ATP, ADP, CK (from rabbit muscle), AG, MGO (40% aque-ous solution), Cys, GSH, and Vc were purchased fromSigma–Aldrich (St. Louis, MO, USA). Creatine, H3BO3, andNa2B4O7 N 10H2O were purchased from Hengxin Chemi-cal Reagent (Shanghai, China). NaOH, HClO4, H3PO4, andNaH2PO4 were purchased from Sinopharm ChemicalReagent (Shanghai, China). Tetrabutylammonium bro-mide and magnesium acetate were obtained from Ker-mel Chemical Reagent (Tianjin, China). TP was pur-chased from National Institute for the Control of Phar-maceutical and Biological Products (Beijing, China). TP-free and TP-containing plasma were provided by XiangyaHospital of Central South University (Hunan, China).HPLC-grade ACN was purchased from Tedia (Fairfield,OH, USA). Water for carrier buffer used in CE analysiswas prepared by Milli-Q purification system from Milli-pore (Bedford, MA, USA). CE measurements were carriedout with a Beckman Coulter P/ACE MDQ CE System (Full-erton, CA, USA). Migration times and peak areas wereanalyzed with the 32 KaratTM software. The capillarieswere 60.2 cm in length (50.1 cm to the detector) and hadan od of 375 lm and an id of 75 lm.

2.2 Evaluation of CK activity by CE analysis undertwo pH conditions

All solutions were prepared in 5 mM borate buffer (pH 9and 7.4, respectively). The borate buffer solution (pH 9.0and 7.4) was prepared by titration using the H3BO3

(5 mM) and Na2B4O7 N 10H2O (1.25 mM) solution, and pHof the solution was monitored with a pH meter. The reac-tion solution (RS) was composed of 24 mM creatine,4 mM ATP, and 5 mM magnesium acetate. CK reactionwas initiated by adding 10 lL of 11.5 lM CK solution to100 lL of RS. Borate buffer (pH 9 or 7.4) was added tomake up the volume to 1 mL. The CE running buffer wascomposed of A (100 mM NaH2PO4, 5 mM tetrabutylam-monium bromide, adjusted to pH 3.5 with H3PO4) and B(ACN) in the ratio 90:10 v/v, was freshly prepared and fil-tered through a 0.4 lm filter. CE was run in the reversepolarity mode (anode on the detector side) with UV detec-tion at 257 nm. The capillary columns used were coatedwith c-aminopropyltrimethoxysilane to minimize theEOF of the solvent. Samples were injected for 5 s (38.2 Pa)and a voltage of 26 kV at low pressure (19.1 Pa) was


2848 J. Ma et al. J. Sep. Sci. 2008, 31, 2846 – 2851

applied across the electrodes for 8 min. The capillary col-umn was conditioned at the beginning of each day byrinsing with the running buffer for 5 min at high pres-sure (717.0 Pa). At the end of the day, the column wasrinsed with deionized water at 956.0 Pa for 5 min andthen stored overnight. Based on the CE method men-tioned above, the generated ADP in CK reaction wasdetermined after incubation for 9 min at 378C. The CKactivity can be reflected by the amount of ADP convertedfrom ATP.

2.3 Protection of CK activity from MGO

2.3.1 Inhibitory effect of MGO on CK activity

10 lL of 11.5 lM CK was diluted with 1 mL borate buffer(pH 7.4) with or without 10 lL of 50 mM MGO. Then sam-ples were mixed well and incubated at 378C for 0.5, 1.0,1.5, 2.0, 3.0, and 4.0 h, respectively. Subsequently, sam-ples were added with 100 lL of RS and ready for CE anal-ysis after incubation for 9 min.

2.3.2 Protective effects of chemical agents on CKactivity influenced by MGO

All solutions were prepared in 5 mM borate buffer(pH 7.4). Borate buffer (1 mL, pH 7.4) was added with mix-ture of 10 lL of 100 mM compounds (AG, Cys, GSH, TP,and Vc) or 10 lL borate buffer (pH 7.4), 10 lL of 11.5 lMCK, and 10 lL of 50 mM MGO, and then were mixedtogether and incubated at 378C for 2 h. Subsequently,samples were added with 100 lL of RS. Besides, samplesolution containing only CK was prepared as control.Using the same CE method described in Section 2.2, CKactivities in different samples were measured. Moreover,direct effects of selected compounds on CK activity werealso investigated as described above in MGO-free reactionsystems.

2.4 Protective effect of tiopronin against MGO inplasma

2.4.1 Sample preparation

Plasma samples with and without TP were obtained fromvolunteers. The concentration of TP in TP-containingplasma from volunteers after administration of this drugwas determined to be around 5 ppm [37]. Plasma sample(1 mL, both TP-free and TP-containing plasma) was addedwith 20 lL of 50 mM MGO (or 20 lL of 5 mM borate buf-fer, pH 7.4) and incubated at 378C for 4 h. Then, sampleswere added with 100 lL of RS. After incubation foranother 30 min at 378C, samples were added with 0.1 mL25% HClO4 to precipitate proteins and vortex mixed for1 min, and then centrifuged for 10 min at 10000 rpm. Asmall quantity of NaOH was added to 0.8 mL of the super-natants for neutralization. After further centrifugation

at 10000 rpm for 10 min, the supernatant of the sampleswas taken for CE analysis.

2.4.2 Evaluation of protective effect of TP

Prior to actual analysis, the capillary was conditioned byrinsing with running buffer for 5 min under high pres-sure (717.0 Pa). Then, samples were injected for 5 s(38.2 Pa) and a voltage of 26 kV at low pressure (19.1 Pa)was applied across the electrodes for 8 min. At last, thecapillary was rinsed with deionized water at 956.0 Pa for5 min. The amount of ADP determined in each samplewas referred to the reflection of activities of enzymesinvolved in the ADP conversion from ATP in plasmaincluding CK.

3 Results and discussion

According to our study, changes in the activity of CK inconverting ATP to ADP can be much more convenientlymeasured with CE instrument compared to traditionalcolorimetric methods. Another demerit of these colori-metric assays is that analysis is usually carried out underalkaline condition (pH 9) [20, 38], which is far more alka-line than that of human physiological systems. In orderto be closer to the physiological condition, the currentstudy mainly employed pH 7.4 reaction medium. Never-theless, our analysis began with comparison of the activ-ity of CK under two pH conditions (pH 7.4 and 9.0) so asto get an insight into the pH-dependent behavior of thisenzyme. As shown in Fig. 1, the current analytic methodachieved good separation between ATP and ADP, thusfacilitating accurate quantitative evaluation. It wasfound that pH 7.4-reaction medium was more favorablefor the conversion of ATP to ADP when compared withpH 9-medium in terms of sensitivity of detection. This isprobably due to the fact that CK-catalyzed conversion ofATP to ADP is accompanied by proton generation. In analkaline environment (i.e., pH 9), protons generated aredepleted. This forces the reaction equilibrium to shift inthe direction toward ADP generation, thus replenishingthe system with protons.

Figure 2 shows the time-dependent inhibition of CKactivity by MGO as expressed by the reduction in ADPgeneration (CK activity) in the system added with MGOrelative to the control. It was reported that MGO prob-ably lead to inactivation of CK via glycation of its activeCys residues [20]. For this consideration, compoundswith MGO trapping abilities are supposed to be potentialprotective agents for CK activity. Some known MGO scav-engers (AG, Cys, GSH) along with Vc and TP (a potentialMGO scavenging agent) were selected in our study toexamine their protective effects. All the compoundstested did not exhibit observable effects on the activity ofCK when added alone. However, in systems where MGO



was added following treatment with the above pur-ported MGO scavengers, protective effects against MGO-induced inactivation of CK were observed (Fig. 3). It isclear that guanidine group of AG and sulfhydryl group ofthiol compounds contributed respectively to their pro-tective effect by nucleophilic addition to MGO. Interest-ingly, Vc, though less effective than the MGO scavengers,could also retain CK activity to some extent. Protectiveeffect of Vc may be due to its strong antioxidant activity.

TP (N-(2-mercaptopropionyl)-glycine), which possessesa free thiol group, has been successfully applied to thetherapy of hepatitis [39], cataract [40], and rheumatoidarthritis [41]. As TP demonstrated strong protective effect

on CK activity, its protective effect was further investi-gated in plasma. Such reaction medium provided a sys-tem better simulating the physiological environment. Inthis way, we were able to derive parameters regardingthe protective effect of TP on most enzymes involved inthe conversion between biologically active ATP and ADPincluding hexokinase [32], NTPDase-1 [42] and ATPase[43]. Since no ATP or ADP was determined in the blankplasma (Fig. 4A), ATP was added to plasma samples to pro-vide substrate for the above-mentioned enzymes. Figure4B demonstrates the presence of enzyme activityrequired for the conversion of ATP to ADP in the plasmasamples employed. Interestingly, the zig-zag peak in Fig.4 at migration time 2.5 min has been enlarged whencompared with that in Fig. 1. One of the factors whichmay cause such change in peak shape is the differentcomplexity of the samples. In this regard, the plasmasample was expected to be much more complex than thebuffer solution. Similar to the phenomenon observed inpH 7.4 buffer, generation of ADP in plasma samples wasreduced by nearly 40% after incubation with MGO. Thisalso suggests the potential detrimental consequences ofMGO when it is present or accumulates to high concen-trations in human tissues. Our further investigationfound that intervention with TP almost abrogated suchMGO-induced suppression of ATP–ADP-associatedenzymes. At concentrations as low as 5 ppt, TP was ableto significantly (l80% of the control) retain the enzymeactivity in the plasma samples (Fig. 5). This was supportedby the observation that much higher amount of ADP wasformed in TP-protected than in TP-free samples treatedwith MGO. This suggests that TP left in plasma after oraladmission may be able to counteract MGO's inhibitoryactivity on certain enzymes associated with adeninenucleotide metabolism. On the other hand, in theabsence of MGO, addition of TP alone to plasma sampleshad no effect on ADP generation (Fig. 5). Since MGO is animportant precursor of detrimental AGEs which werereported to be relative to the pathogenesis of diabeticcomplications, atherosclerosis, Alzheimer's disease, etc.,our findings provide useful information for the develop-ment of new clinical applications of drug TP other thantherapy of chronic hepatitis. Besides, some other MGOtrapping agents including AG, Cys, and GSH, or even


Figure 1. Electrophoretic separa-tion of ATP and ADP in CK reaction(257 nm, pH 7.4). The inset showsthe corresponding electric currentof the CE process.

Figure 2. Inhibitory effect of MGO on CK activity with differ-ent incubation times (0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 h,respectively). ADP generation amount-based CK activity insample without addition of MGO was used as control.Results were expressed as means l SD for n = 2, p a 0.01.

Figure 3. Protective effects of several compounds on CKactivity influenced by MGO. ADP generation amount-basedCK activity in sample with only CK was set as control.Results were means l SD for n = 3, p a 0.01.

2850 J. Ma et al. J. Sep. Sci. 2008, 31, 2846 – 2851

antioxidants like Vc were also found to be potential pro-tective agents in glycation-induced health disorders.

4 Concluding remarks

Our results showed that MGO scavengers with guanidineor thiol functional groups have protective effects on CKactivity reduced by MGO in CK reaction. Furthermore,the drug TP, mainly known as a hepato-protective agent,could retain activities of some enzymes effective inadenine nucleotide metabolism in plasma present withMGO. On the other hand, the established CE analysismethod provides a rapid and simple way to evaluate CKactivity by determining ADP generation amount. This CEapproach can be widely applied to complicated biologi-cal samples. In addition, this approach can be utilized asa model for screening effective MGO scavengers via CKreaction which may hopefully aid the development ofnutraceuticals or even potential drugs from naturalplants for MGO-mediated disorders or ailments.

The authors declared no conflict of interest.

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Use of capillary electrophoresis to evaluate protective effects of methylglyoxal scavengers on the activity of creatine kinase