Anal. Chem. 2006, 78, 2934-2938
Characterization of Prolidase Activity Using Capillary Electrophoresis with Tris(2,2′-bipyridyl)ruthenium(II) Electrochemiluminescence Detection and Application To Evaluate Collagen Degradation in Diabetes Mellitus Jipei Yuan,†,‡ Tao Li,† Xue-Bo Yin,† Li Guo,§ Xiuzhen Jiang,§ Wenrui Jin,‡ Xiurong Yang,† and Erkang Wang*,†
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China, and People’s Hospital of Chaoyang District, Changchun, Jilin 130021, China
A new method for prolidase (PLD, EC 3.4.13.9) activity assay was developed based on the determination of proline produced from enzymatic reaction through capillary electrophoresis (CE) with tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)32+] electrochemiluminescence detection (ECL). A detection limit of 12.2 fmol (S/N ) 3) for proline, corresponding to 1.22 × 10-8 units of prolidase catalyzing for 1min was achieved. PLD activity determined by CE-ECL method was in agreement with that obtained from the classical Chinard’s one. CE-ECL showed its powerful resolving ability and selectivity as no sample pretreatment was needed and no interference existed. The clinical utility of this method was successfully demonstrated by its application to assay PLD activity in the serum of diabetic patients in order to evaluate collagen degradation in diabetes mellitus (DM). The results indicated that enhanced collagen degradation occurred in DM.
protein binding analysis,6 and pharmaceutical determinations.7,8 However, most works were used to validate CE-ECL using standard solutions;9 very few papers have published for analytical techniques of the determination of bioactive analytes, especially that at low concentrations existing in body fluids such as plasma or serum established on the basis of CE-ECL. Also, for the analytes at low concentrations in biological fluids, much effort may be required to optimize the sample pretreatment for CE.2,7,10 This tedious and time-consuming pretreatment limits their utility in clinical analysis. We applied the CE-ECL technique to assay prolidase (PLD, EC 3.4.13.9) existing in human plasma, serum, and erythrocytes by exploiting its inherent character of selectivity and sensitivity in clinical samples. PLD is a cytosolic enzyme that catalyzes the hydrolyzation of the iminodipeptides containing C-terminal prolyl or hydroxylprolyl residues to release proline or hydroxyproline via the following reaction:
Capillary electrophoresis (CE), with high efficiency as its most important feature, is suitable for bioseparations as well as pharmaceutical analysis.1,2 Another advantage of CE for bioanalysis is that the running electrolyte used is compatible with bioanalytes, which is superior to the relatively incompatible organic eluents often used in liquid chromatography.2 Combined with electrochemiluminescence (ECL) of tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)32+] detection, CE has been proved as a powerful analytical tool in amino acid detection,3,4 catecholamine detection,5 drug-
glycylproline + H2O y\z glycine + proline
As proline is rich in collagen, PLD plays an important role in the final stage of collagen degradation and the recycling of proline. Increased PLD activity reflects increased intensity of collagen degradation.11 A metabolism disorder of type IV collagen on the basement membrane often causes debilitating microvascular complications in diabetes mellitus (DM).12 Enhanced collagen
* Corresponding author. Tel: +86-431-5262003. Fax: +86-431-5689711. E-mail:
[email protected]. † Changchun Institute of Applied Chemistry. ‡ Shandong University. § People’s Hospital of Chaoyang District. (1) Huck, C. W.; Stecher, G.; Bakry, R.; Bonn, G. K. Electrophoresis 2003, 24, 3977-3997. (2) Perrett, D. Ann. Clin. Biochem. 1999, 36, 133-150. (3) Bobbitt, D. R.; Jackson, W. A.; Hendrickson, H. P. Talanta 1998, 46, 565572. (4) Wang, X.; Bobbitt, D. R. Anal. Chim. Acta 1999, 383, 213-220.
(5) Kang, J.; Yin, X.; Yang, X.; Wang, E. Electrophoresis 2005, 26, 1732-1736. (6) Zhao, X.; You, T.; Liu, J.; Sun, X.; Yan, J.; Yang, X.; Wang, E. Electrophoresis 2004, 25, 3422-3426. (7) Liu, J.; Cao, W.; Qiu, H.; Sun, X.; Yang, X.; Wang, E. Clin. Chem. 2002, 48, 1049-1058. (8) Cao, W.; Liu, J.; Qiu, H.; Yang, X.; Wang, E. Electroanalysis 2002, 14, 15711576. (9) Yin, X.; Wang, E. Anal. Chim. Acta 2005, 533, 113-120. (10) Liu, J.; Yang, X.; Wang, E. Electrophoresis 2003, 24, 3131-3138. (11) Myara, I.; Myara, A.; Mangeot, M.; Fabre, M.; Charpentier, C.; Lemonnier, A. Clin. Chem. 1984, 30, 211-215.
2934 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006
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10.1021/ac051594x CCC: $33.50
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© 2006 American Chemical Society Published on Web 03/28/2006
synthesis and deposition in DM were found by many groups,12-14 while conflicting results were obtained concerning collagen degradation.14-16 Hypertriglyceridemia (HTG) is one of common features of diabetes17 and another risk factor causing microangiopathies.18,19 As there is no accordant conclusion about collagen degradation in DM, and PLD activity can reflect the intensity of collagen degradation, we assayed serum PLD activity to assess collagen degradation in DM with/without HTG in this paper. So far, the method of PLD assay widely used is spectroscopic colorimetry with proline detected by Chinard’s method.11 However, the Chinard’s reagent can react not only with proline but also with some other amino acids and sugars existing in plasma,20 which makes the results ambiguous. Although matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,20 highperformance liquid chromatography,21 and isotachophoresis22 have been used in PLD activity assay, the complicated pretreatment and determination procedures limit their clinical application. So, the development of a fast, easy-to-operate PLD activity assay is critically important in clinical application. In this paper, with a combination of CE and ECL, a simple and sensitive method for prolidase activity assay was developed by detecting proline produced from prolidase-catalyzing glycylproline hydrolyzation. It is distinct from previous simple applications of CE-ECL to standard samples, as the analyte is a bioactive substance existing in clinic samples. The pretreatment and determination procedures were also simplified compared to other PLD assay techniques and to the traditional CE-ECL methods. The new method was applied to detect PLD activity in diabetes to investigate whether there was a difference in collagen degradation between the diabetic patients and normal subjects. EXPERIMENTAL SECTION Chemicals. Tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate was obtained from Aldrich Chemical (Milwaukee, WI). Glycylproline was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). The physiological buffer saline consisted of 8.500 g/L NaCl, 0.4370 g/L NaH2PO4‚2H2O, and 2.579 g/L Na2HPO4‚ 12H2O. Ninhydrin, 2.5 g, dissolved in a mixture of glacial acetic acid (60 mL) and 6 mol/L orthophosphoric acid (40 mL) was used as the Chinard reagent. All the solutions were prepared with water purified by a Milli-Q system (Millipore, Bedford, MA) and stored at 4 °C. All 45 serum samples coming from outpatients at People’s Hospital of Chaoyang District (Changchun, China) were divided into five groups according to American Diabetes Association (12) Roy, S.; Maiello, M.; Lorenzi, M. J. Clin. Invest. 1994, 93, 438-442. (13) Kotajima, N.; Kimura, T.; Kanda, T.; Obata, K.; Kuwabara, A.; Fukumura, Y.; Kobayashi, I. J. Diabetes Complications 2000, 14, 13-17. (14) Trevisan, R.; Yip, J.; Sarika, L.; Li, L. K.; Viberti, G. J. Am. Soc. Nephrol. 1997, 8, 1133-1139. (15) Sumida, Y.; Ura, H.; Yano, Y.; Misaki, M.; Shima, T. Horm. Res. 1997, 48, 23-28. (16) Erbagci, A. B.; Araz, M.; Erbagci, A.; Tarakcioglu, M.; Namiduru, E. S. Clin. Biochem. 2002, 35, 263-268. (17) Jenkins, A. J.; Rowley, K. G.; Lyons, T. J.; Best, J. D.; Hill, M. A.; Klein, R. L. Curr. Pharm. Des. 2004, 10, 3395-3418. (18) Brassard, P.; Robinson, E. Arctic Med. Res. 1995, 54, 116-124. (19) Ebeling, P.; Koivisto, V. A. Acta Diabetol. 1997, 34, 33-38. (20) Kurien, B. T.; Patel, N. C.; Porter, A. C.; Kurono, S.; Matsumoto, H.; Wang, H.; Scofield, R. H. Anal. Biochem. 2004, 331, 224-229. (21) Harada, M.; Fukasawa, K. M.; Hiraoka, B. Y.; Fukasawa, K.; Mogi, M. J. Chromatogr. 1990, 530, 116-121. (22) Mikasa, H.; Arata, J.; Kodama, H. J. Chromatogr. 1984, 310, 401-406.
Figure 1. Relationship between ECL intensity and concentration of Mn2+. Substrate, 1.0 × 10-3 mol/L glycylproline; 3.0 × 10-2 mol/L phosphate buffer, pH 7.6; incubation, 37 °C for 30 min; 5.0 × 10-3 mol/L Ru(bpy)32+; detection potential, 1300 mV(vs Ag/AgCl).
criteria of diabetes mellitus proposed in 199723 and National Cholesterol Education Program’s (NCEP) Adult Treatment Panel III criteria of hypertriglyceridemia.24 The five groups were normal fasting glucose (NFG) without and with HTG, DM groups without and with HTG, and impaired fasting glucose group. Extraction of Plasma and Erythrocytes. About 2 mL of anticoagulant human blood was collected in a 5-mL centrifuge tube and centrifuged at 1500 rpm for 10 min. The supernatant was then collected in a 1.5-mL microcentrifuge tube, stored at -20 °C. The erythrocytes were washed with physiological buffer saline until the supernatant was achromatous and clear. After the number of erythrocytes was counted using a hemocytometer, the erythrocytes were also stored at -20 °C. CE-ECL Detection System. ECL detection was carried out with a MPI-A capillary electrophoresis ECL detector (Xi’an Remax Electronics Co. Ltd., Xi’an, China cooperating with the Changchun Institute of Applied Chemistry, Changchun, China), using a threeelectrode system consisting of a 500-µm-diameter platinum wire as working electrode, Ag/AgCl (KCl saturated) as reference electrode, and a 1-mm-diameter platinum wire as counter electrode. The detection potential was set at 1300 mV. The detection cell was filled with 5.0 × 10-3 mol/L Ru(bpy)32+ in 3.0 × 10-2 mol/L phosphate buffer (pH 7.5). The potential of the photomutiplier tube was set at 750 V. An uncoated fused-silica capillary (Yongnian Optical Conductive Fiber Plant, Yongnian, China) of 25-µm i.d, 360-µm o.d, and 41 cm in length was used and rinsed with 0.1 mol/L NaOH, water and running buffer each for 10 min before use. The sample was electrokinetically injected at 10 kV for 10 s, and the separation voltage was 18 kV. Enzyme-Catalyzed Reaction. Plasma, serum, or erythrocyte lysate was diluted with 5-fold phosphate buffer. A 20-µL sample of this diluted solution was mixed with MnCl2 and glycylproline, and then the mixture was diluted to 200 µL with phosphate buffer. The mixture was incubated at 37 °C for 30 min. After a 80 °C waterbath for 5 min to terminate the reaction, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was (23) Hilton, D. J.; O’Rourke, P. K.; Welborn, T. A.; Reid, C. M. Med. J. Aust. 2002, 176, 104-107. (24) NCEP. JAMA-J. Am. Med. Assoc. 2001, 285, 2486-2497.
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Figure 2. Relationship between ECL intensity and CGP. (1) CGP from 1.0 × 10-4 to 0.1 mol/L; (2) CGP from 1.0 × 10-4 to 5.0 × 10-3 mol/L. Cofactor, 1.0 × 10-3 mol/L Mn2+; 3.0 × 10-2 mol/L phosphate buffer, pH 7.6; incubation, 37 °C for 30 min; 5.0 × 10-3 mol/L Ru(bpy)32+; detection potential, 1300 mV (vs Ag/AgCl).
Figure 4. Relationship between ECL intensity and incubation time (1) Within 60 min; (2) within 30 min. Substrate, 1.0 × 10-2 mol/L glycylproline; cofactor, 1.0 × 10-3 mol/L Mn2+; 3.0 × 10-2 mol/L phosphate buffer, pH 7.6; incubation, 37 °C; 5.0 × 10-3 mol/L Ru(bpy)32+; detection potential, 1300 mV (vs Ag/AgCl).
Figure 3. Relationship between ECL intensity and pH. Substrate, 1.0 × 10-2 mol/L glycylproline; cofactor, 1.0 × 10-3 mol/L Mn2+; incubation, 37 °C for 30 min; 5.0 × 10-3 mol/L Ru(bpy)32+; detection potential, 1300 mV (vs Ag/AgCl).
Figure 5. Electropherograms of (1) 1.0 × 10-4 mol/L proline, (2) plasma after incubation, (3) serum after incubation, (4) plasma blank, and (5) serum blank.The plasma and serum samples were without preincubation. Substrate, 1.0 × 10-2 mol/L glycylproline; cofactor, 1.0 × 10-3 mol/L Mn2+; 3.0 × 10-2 mol/L phosphate buffer, pH 7.6; incubation, 37 °C for 30 min; 5.0 × 10-3 mol/L Ru(bpy)32+; detection potential, 1300 mV (vs Ag/AgCl).
electrokinetically injected at 10 kV for 10 s and separated at 18 kV for CE-ECL analysis. For the erythrocyte lysate, the supernatant should be diluted to 10-fold before CE analysis. The unit of PLD activity used in this paper was unit per liter, with the definition of 1 U/L hydrolyzing 1.0 µmol of glycylproline per min per liter of plasma or serum at 37 °C. Statistical Analysis. Linear regression was used to assess the dependence of PLD activity on the independent variable of serum glucose. The results were submitted to statistical analysis using Newman-Keuls test. Two-tailed p values
