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Microdetermination of hyaluronan in human plasma by high-performance liquid chromatography with a graphitized carbon column and postcolumn fluorometric detection
Journal of Chromatography B, 879 (2011) 950–954
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Microdetermination of hyaluronan in human plasma by high-performance liquid chromatography with a graphitized carbon column and postcolumn fluorometric detection Hidenao Toyoda a,b,∗ , Fumie Muraki b , Toshio Imanari b , Akiko Kinoshita-Toyoda a a b
Faculty of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan Graduate School of Pharmaceutical Sciences, Chiba University, 1-33, Yayoi, Inage, Chiba 263-8522, Japan
a r t i c l e
i n f o
Article history: Received 20 August 2010 Accepted 3 March 2011 Available online 10 March 2011 Keywords: Hyaluronan Human blood plasma HPLC Graphitized carbon column Fluorometric detection
a b s t r a c t A chemical method for the determination of hyaluronan (hyaluronic acid, HA) has been developed and applied to the human blood plasma. Human blood plasma HA was converted to the Di-HA by digestion with hyaluronidase SD and determined by a sensitive and selective high-performance liquid chromatography (HPLC). The HPLC includes the separation and detection of Di-HA using a graphitized carbon column and fluorometric reaction with 2-cyanoacetamide in an alkaline eluent. The calibration graph for Di-HA was linear over the range 0.2 ng–1 g. It was revealed that the concentration of HA in normal human blood plasma is very low levels (about 24 ng/ml) in comparison to low-sulfated chondroitin 4-sulfate (about 13 g/ml). © 2011 Elsevier B.V. All rights reserved.
1. Introduction Glycosaminoglycans (GAGs) exist in various tissues as major components of the extracellular matrix, and small amounts of GAGs are reported in blood plasma and urine [1,2]. To elucidate the physiological roles of GAGs, many workers have studied analytical methods for determination of GAGs in plasma, serum and urine using chromatography and electrophoresis [3–5]. Highperformance liquid chromatography (HPLC) is extensively utilized for the microdetermination of unsaturated disaccharides produced enzymatically from GAGs in biological and clinical fluids. The unsaturated disaccharides have been generally separated on chemical-bonded type silica columns (ODS-, amino- and amidebonded type silica column) by several precolumn [6–8] and postcolumn [9–11] derivatization methods or mass spectrometric methods [12–15]. Hyaluronan (hyaluronic acid, HA) is a kind of GAGs composed of alternating 1,3-glucuronic acid and 1,4-N-acetylglucosaminidic bonds and Di-HA is produced by enzymatic digestion (Fig. 1). HA has been postulated as playing important roles in vivo, based on the observations of the elevated HA levels in blood plasma and
∗ Corresponding author at: Faculty of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan. Tel.: +81 77 561 5738; fax: +81 77 561 2659. E-mail address: [email protected] (H. Toyoda). 1570-0232/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2011.03.007
the excessive urinary HA excretion in the case of several diseases related to inflammation and cancer [16–19]. However, it is hard to study the biological functions of HA, because normal HA concentrations in blood plasma and urine is too low to determine precisely [20]. In blood plasma, low-sulfated chondroitin 4-sulfates (LSC) are major GAGs [3,21]. When plasma GAGs are digested enzymatically, large amounts of Di-0S are produced and interfere with the measurement of HA. Furthermore, the chromatographic separation of Di-HA and Di-0S is very difficult, because Di-HA is the C-4 epimer of N-acetylgalactosamine of Di-0S (Fig. 1). In this paper, we established a sensitive and selective method for the determination of HA and demonstrated the application to human plasma samples. 2. Materials and methods 2.1. Reagents and materials Standard unsaturated disaccharides of [2-acetamido-2-deoxy3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose (Di-HA), 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-galactose (Di-0S), 2-acetamido-2-deoxy-3-O-(d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose (Di-4S)], hyaluronidase SD from Streptococcus dysgalactiae (EC 4.2.2), chondroitinase ABC (EC 4.2.2.4) and chondroitinase ACII (EC 4.2.2.5) were obtained from Seikagaku (Tokyo, Japan). A
H. Toyoda et al. / J. Chromatogr. B 879 (2011) 950–954
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Fig. 1. Enzymatic digestion of HA and chondroitin sulfate. Di-HA is the C-4 epimer of N-acetylgalactosamine of Di-0S.
graphitized carbon column of Carbonex (3.5 m, 100 mm × 4.6 mm I.D.) was purchased from Tonen (Tokyo, Japan). An amino-bonded silica column of TSKgel NH2 -60 (5 m, 150 mm × 4.6 mm I.D.) was obtained from Tosoh (Tokyo, Japan). 2-Cyanoacetamide was obtained from Sigma. All other chemicals used were of analytical reagent grade.
sodium chloride solution was added. Then the mixture was heated in a boiling water bath for 5 min. After being cooled in a water bath, the solution was centrifuged at 2300 × g for 15 min. To 1 ml of the supernatant, 100 l of 0.1 M sodium hydroxide solution and 4 ml of cooled ethanol saturated with sodium acetate were added. The mix-
2.2. Apparatus and chromatographic conditions A flow diagram of liquid chromatography for the determination of HA is shown in Fig. 2. The chromatographic equipment included two high-pressure pumps (L-6000), a fluorescence detector (F1050), a chromato-integrator (D-2500) from Hitachi Instruments (Tokyo, Japan), a sample injector with 20 l loop (Model 7125) from Rheodyne (Rohnert Park, CA, USA), a column thermocontroller (Mini-80) from Taitec (Tokyo, Japan) and a dry reaction bath (DB5) from Shimamura Instrument (Tokyo, Japan). Established HPLC conditions were as follows (Fig. 2): A Carbonex column was eluted at 40 ◦ C with 25 mM sodium phosphate–NaOH buffer (pH 11) in 4% acetonitrile at a flow rate of 0.5 ml/min by using a L-6000 pump. To the effluent was added aqueous 0.5% 2-cyanoacetamide solution at the flow rate of 0.5 ml/min by using a L-6000 pump. The mixture passed through a polytetrafluoroethylene (PTFE) reaction coil (10 m × 0.5 mm I.D.) set in a dry reaction bath thermostated at 110 ◦ C and a following PTFE cooling coil (2 m × 0.25 mm I.D.). The effluent was monitored fluorometrically (excitation 335 nm, emission 395 nm). A 10-l portion of sample solution was loaded via a sample injector with a 20 l loop. 2.3. Preparation of human plasma GAGs Blood was collected from the healthy volunteers. Ethylenediamine tetra acetic acid disodium salt was added to the blood as an anticoagulant. The blood was separated into plasma and cells by centrifugation at 1200 × g for 15 min. Plasma GAGs were separated by our modified method reported previously [9]. To 250 l of human plasma, 100 l of 0.05 M Tris–HCl buffer (pH 8.0) containing 1% actinase E was added, and the mixture was incubated at 45 ◦ C for 3 h. To the solution, 1 ml of 0.1 M acetic acid containing 10%
Fig. 2. Flow diagram of the postcolumn HPLC system for determination of HA with a graphitized carbon column. Column, Carbonex (3.5 m, 100 mm × 4.6 mm I.D.) with a precolumn (3.5 m, 10 mm × 4.6 mm I.D.) at 40 ◦ C; eluent, 25 mM sodium phosphate–NaOH buffer (pH 11) in 4% acetonitrile; reagent, 0.5% 2-cyanoacetamide; reaction temperature, 110 ◦ C; detection, Ex. 335 nm, Em. 395 nm. Other conditions as described in the text.
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H. Toyoda et al. / J. Chromatogr. B 879 (2011) 950–954
ture was left to stand overnight at 4 ◦ C and centrifuged at 2300 × g for 15 min at 4 ◦ C. The precipitate was washed twice with 1 ml of ethanol, and then dried under the vacuum. The resulting precipitate was dissolved in 50 l of water. 2.4. Enzymatic digestion For the analysis of LSC in human blood plasma, 10 l of 0.2 M Tris–acetate buffer (pH 8.0) and 20 l of an aqueous solution containing chondroitinase ABC (50 mIU) and chondroitinase ACII (50 mIU) were added to a 5-l portion of the sample solution and incubated at 37 ◦ C for 3 h. A 10-l portion of the mixture was loaded onto the HPLC. For the analysis of HA in human blood plasma, 10 l of 0.2 M sodium phosphate buffer (pH 6.2) and 10 l of an aqueous solution containing hyaluronidase SD (10 mIU) were added to a 20-l portion of sample. The mixture was incubated at 37 ◦ C for 16 h, and a 10-l portion of the mixture was loaded onto the HPLC. 3. Results and discussion 3.1. Analysis of GAGs in human blood plasma by using an amino-bonded silica column LSC is the major class of GAGs in blood plasma [3,21]. Human plasma GAGs were digested with chondroitinase ABC and chondroitinase ACII, then the unsaturated disaccharides from LSC were analyzed by HPLC conditions described previously [9]. Typical chromatogram and the HPLC conditions are shown in Fig. 3. Table 1 summarizes the compositions of disaccharide units from human plasma samples. 3.2. Digestion of blood plasma GAGs with hyaluronidase SD LSC includes a number of non-sulfated units in repeating disaccharide units of chondroitin 4-sulfate (Fig. 4) and is present in a form of a proteoglycan named bikunin [22,23]. By using the LSC, bikunin is held together with heavy chains to form inter-␣-trypsin inhibitor family in blood plasma and some biological fluids [21,24]. Although hyaluronidase SD is the enzyme that yields Di-HA from HA, the enzyme also digests non-sulfated moieties in chondroitin sulfates to form Di-0S simultaneously (Fig. 4). The resulting DiHA and Di-0S were analyzed by HPLC with a graphitized carbon column and fluorometric postcolumn derivatization. 3.3. Chromatographic separation and detection of the unsaturated disaccharides from HA It is great significant to overcome the interference with large amounts of Di-0S from LSC for the analysis of trace amounts of HA in human plasma. We have reported HPLC conditions for the separation of Di-0S and Di-HA with an amino-bonded type silica
Fig. 3. Analysis of LSC in human blood plasma as their unsaturated disaccharides after chondroitinase ABC and ACII digestion by using an amino-bonded column. Sample size: 10 l. Peaks: 1 = Di-0S; 2 = Di-HA; 3 = Di-4S. HPLC conditions were as follows: a TSKgel NH2 column (5 m, 150 mm × 4.6 mm I.D.) with a precolumn (5 m, 10 mm × 4.6 mm I.D.) was eluted at 40 ◦ C with 10 mM ammonium-formate buffer (pH 5.0) containing 10 mM sodium sulfate in 4% acetonitrile at a flow rate of 0.5 ml/min. To the effluent were added 0.1 M NaOH and 1% 2-cyanoacetamide solution at the flow rate of 0.2 ml/min by using a double-plunger pump. The mixture passed through a PTFE reaction coil (10 m × 0.5 mm I.D.) set in a dry reaction bath thermostated at 110 ◦ C and a following PTFE cooling coil (2 m × 0.25 mm I.D.). The effluent was monitored fluorometrically (excitation 346 nm, emission 410 nm).
column [25,26]. The satisfactory separation was achieved with an eluent containing borate buffer and the method was applied to the measurement of human urinary HA. However, the separation was insufficient to determine the concentration of HA in human plasma. Graphitized carbon columns were introduced into HPLC methods [27,28] and utilized for the separation of carbohydrates [29]. We have found that a graphitized carbon column is very effective in the separation of each unsaturated disaccharide from GAGs [30]. In this study, HPLC was employed with a graphitized carbon column and fluorometric detection using 2-cyanoacetamide for the excellent chromatographic separation and high sensitivity. The optimized conditions and flow diagram are summarized in Fig. 2 and a typical chromatogram is shown in Fig. 5. Calibration graphs for Di-0S and Di-HA were linear in the range 0.2 ng–1 g. The relative standard deviations at 1 ng were less than 2% (n = 5) for Di-0S and Di-HA. The graphitized carbon column is so stable under extreme conditions of pH (0–14), salt concentration and temperature that the column lifetime was improved significantly
Table 1 Determination of unsaturated disaccharides produced enzymatically from human plasma GAGs. Case no.
Sex
Age
1 2 3 4 5 6 Mean ± SD
M M M M F F
22 23 29 38 23 25
M: male, F: female.
Chondroitinase digestion (g/ml)
Hyaluronidase digestion (g/ml)
Di-OS
Di-4S
Total
Di-HA
8.0 7.9 7.3 10.0 9.8 6.8 8.3 ± 1.3
5.7 3.8 2.6 6.2 5.9 4.8 4.8 ± 1.4
13.7 11.7 9.9 16.2 15.7 11.6 13.1 ± 2.5
0.017 0.012 0.033 0.025 0.028 0.030 0.024 ± 0.008
H. Toyoda et al. / J. Chromatogr. B 879 (2011) 950–954
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Fig. 4. Structure and enzymatic digestion of LSC.
against an amino-bonded silica column, which is normally used for the separation of unsaturated disaccharides.
3.4. Analysis of HA in human blood plasma by using a graphitized carbon column Our sensitive HPLC method was used for the determination of the unsaturated disaccharides produced from HA in human blood plasma by hyaluronidase SD digestions (Fig. 6). We could determine trace amounts of HA by the excellent chromatographic separation of Di-0S and Di-HA with a graphitized carbon column and sensitive fluorometric post-column detection (Fig. 6). The Di-HA was only about 1/400th of Di-0S. Table 1 shows the results obtained from the analyses of 6 samples (4 male and 2 female).
Fig. 6. Separation of Di-0S and Di-HA produced from human plasma GAGs with hyaluronidase SD digestion on a graphitized carbon column. Other conditions as in Fig. 5.
4. Conclusion We set out to develop methodology that would facilitate the measurement of HA from small amounts of human blood plasma. This method is unique in that it employs the digestion with hyaluronidase SD, the separation with a graphitized carbon column and the fluorometric post-column detection with 2-cyanoacetamide. As described above, this highly sensitive and selective HPLC was first applied to the determination of HA in small amounts of blood plasma samples as precise chemical method. This method is very effective for the determination of trace amounts of HA in various biological fluids. References Fig. 5. Typical chromatogram of standard unsaturated disaccharides using a graphitized carbon column. Sample size: 10 l (1 ng of each sugar). Peaks: 1 = Di-0S; 2 = Di-HA. Other conditions as in Fig. 2.
[1] A. Calatroni, P.V. Donnelly, N. Di Ferrante, J. Clin. Invest. 48 (1969) 332. [2] D.P. Varadi, J.A. Cifonelli, A. Dorfman, Biochim. Biophys. Acta 141 (1967) 103. [3] R. Hata, S. Ohkawa, Y. Nagai, Biochim. Biophys. Acta 543 (1978) 156.
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H. Toyoda et al. / J. Chromatogr. B 879 (2011) 950–954 G.J. Lee, H. Tieckelmann, J. Chromatogr. 222 (1981) 23. S. Honda, T. Ueno, K. Kakehi, J. Chromatogr. 608 (1992) 289. A. Kinoshita, K. Sugahara, Anal. Biochem. 269 (1999) 367. K.M. Whitham, J.L. Hadley, H.G. Morris, S.M. Andrew, I.A. Nieduszynski, G.M. Brown, Glycobiology 9 (1999) 285. M. Ambrosius, K. Kleesiek, C. Gotting, J. Chromatogr. A 1201 (2008) 54. H. Toyoda, Anal. Sci. 4 (1988) 381. H. Toyoda, T. Nagashima, R. Hirata, T. Toida, T. Imanari, J. Chromatogr. B: Biomed. Sci. Appl. 704 (1997) 19. H. Toyoda, A. Kinoshita-Toyoda, S.B. Selleck, J. Biol. Chem. 275 (2000) 2269. T. Oguma, H. Toyoda, T. Toida, T. Imanari, J. Chromatogr. B: Biomed. Sci. Appl. 754 (2001) 153. R. Lawrence, S.K. Olson, R.E. Steele, L. Wang, R. Warrior, R.D. Cummings, J.D. Esko, J. Biol. Chem. 283 (2008) 33674. Z. Zhang, J. Xie, H. Liu, J. Liu, R.J. Linhardt, Anal. Chem. 81 (2009) 4349. N. Volpi, Anal. Biochem. 397 (2010) 12. C.C. Passerotti, A. Bonfim, J.R. Martins, M.F. Dall’Oglio, L.O. Sampaio, A. Mendes, V. Ortiz, M. Srougi, C.P. Dietrich, H.B. Nader, Eur. Urol. 49 (2006) 71. H. Levesque, B. Delpech, X. Le Loet, P. Deshayes, Br. J. Rheumatol. 27 (1988) 445.
[18] A. Engström-Laurent, N. Feltelius, R. Hällgren, A. Wasteson, Ann. Rheum. Dis. 44 (1985) 614. [19] B. Delpech, P. Bertrand, C. Maingonnat, Anal. Biochem. 149 (1985) 555. [20] T.C. Laurent, J.R. Fraser, FASEB J. 6 (1992) 2397. [21] T. Imanari, A. Shinbo, H. Ochiai, T. Ikei, I. Koshiishi, H. Toyoda, J. Pharmacobiodyn. 15 (1992) 231. [22] W. Gebhard, K. Hochstrasser, H. Fritz, J.J. Enghild, S.V. Pizzo, G. Salvesen, Biol. Chem. Hoppe Seyler 371 (1990) 13. [23] H. Toyoda, S. Kobayashi, S. Sakamoto, T. Toida, T. Imanari, Biol. Pharm. Bull. 16 (1993) 945. [24] L. Zhuo, V.C. Hascall, K. Kimata, J. Biol. Chem. 279 (2004) 38079. [25] H. Toyoda, K. Motoki, M. Tanikawa, K. Shinomiya, H. Akiyama, T. Imanari, J. Chromatogr. 565 (1991) 141. [26] H. Akiyama, H. Toyoda, S. Yamanashi, Y. Sagehashi, T. Toida, T. Imanari, Biomed. Chromatogr. 5 (1991) 189. [27] O. Chiantore, I. Novak, D. Berek, Anal. Chem. 60 (1988) 638. [28] B.J. Bassler, R. Kaliszan, R.A. Hartwick, J. Chromatogr. 461 (1989) 139. [29] K. Koizumi, J. Chromatogr. A 720 (1996) 119. [30] A. Mada, H. Toyoda, T. Imanari, Anal. Sci. 8 (1992) 793.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Microdetermination of hyaluronan in human plasma by high-performance liquid chromatography with a graphitized carbon column and postcolumn fluorometric detection Hidenao Toyoda a,b,∗ , Fumie Muraki b , Toshio Imanari b , Akiko Kinoshita-Toyoda a a b
Faculty of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan Graduate School of Pharmaceutical Sciences, Chiba University, 1-33, Yayoi, Inage, Chiba 263-8522, Japan
a r t i c l e
i n f o
Article history: Received 20 August 2010 Accepted 3 March 2011 Available online 10 March 2011 Keywords: Hyaluronan Human blood plasma HPLC Graphitized carbon column Fluorometric detection
a b s t r a c t A chemical method for the determination of hyaluronan (hyaluronic acid, HA) has been developed and applied to the human blood plasma. Human blood plasma HA was converted to the Di-HA by digestion with hyaluronidase SD and determined by a sensitive and selective high-performance liquid chromatography (HPLC). The HPLC includes the separation and detection of Di-HA using a graphitized carbon column and fluorometric reaction with 2-cyanoacetamide in an alkaline eluent. The calibration graph for Di-HA was linear over the range 0.2 ng–1 g. It was revealed that the concentration of HA in normal human blood plasma is very low levels (about 24 ng/ml) in comparison to low-sulfated chondroitin 4-sulfate (about 13 g/ml). © 2011 Elsevier B.V. All rights reserved.
1. Introduction Glycosaminoglycans (GAGs) exist in various tissues as major components of the extracellular matrix, and small amounts of GAGs are reported in blood plasma and urine [1,2]. To elucidate the physiological roles of GAGs, many workers have studied analytical methods for determination of GAGs in plasma, serum and urine using chromatography and electrophoresis [3–5]. Highperformance liquid chromatography (HPLC) is extensively utilized for the microdetermination of unsaturated disaccharides produced enzymatically from GAGs in biological and clinical fluids. The unsaturated disaccharides have been generally separated on chemical-bonded type silica columns (ODS-, amino- and amidebonded type silica column) by several precolumn [6–8] and postcolumn [9–11] derivatization methods or mass spectrometric methods [12–15]. Hyaluronan (hyaluronic acid, HA) is a kind of GAGs composed of alternating 1,3-glucuronic acid and 1,4-N-acetylglucosaminidic bonds and Di-HA is produced by enzymatic digestion (Fig. 1). HA has been postulated as playing important roles in vivo, based on the observations of the elevated HA levels in blood plasma and
∗ Corresponding author at: Faculty of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan. Tel.: +81 77 561 5738; fax: +81 77 561 2659. E-mail address: [email protected] (H. Toyoda). 1570-0232/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2011.03.007
the excessive urinary HA excretion in the case of several diseases related to inflammation and cancer [16–19]. However, it is hard to study the biological functions of HA, because normal HA concentrations in blood plasma and urine is too low to determine precisely [20]. In blood plasma, low-sulfated chondroitin 4-sulfates (LSC) are major GAGs [3,21]. When plasma GAGs are digested enzymatically, large amounts of Di-0S are produced and interfere with the measurement of HA. Furthermore, the chromatographic separation of Di-HA and Di-0S is very difficult, because Di-HA is the C-4 epimer of N-acetylgalactosamine of Di-0S (Fig. 1). In this paper, we established a sensitive and selective method for the determination of HA and demonstrated the application to human plasma samples. 2. Materials and methods 2.1. Reagents and materials Standard unsaturated disaccharides of [2-acetamido-2-deoxy3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose (Di-HA), 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-galactose (Di-0S), 2-acetamido-2-deoxy-3-O-(d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose (Di-4S)], hyaluronidase SD from Streptococcus dysgalactiae (EC 4.2.2), chondroitinase ABC (EC 4.2.2.4) and chondroitinase ACII (EC 4.2.2.5) were obtained from Seikagaku (Tokyo, Japan). A
H. Toyoda et al. / J. Chromatogr. B 879 (2011) 950–954
951
Fig. 1. Enzymatic digestion of HA and chondroitin sulfate. Di-HA is the C-4 epimer of N-acetylgalactosamine of Di-0S.
graphitized carbon column of Carbonex (3.5 m, 100 mm × 4.6 mm I.D.) was purchased from Tonen (Tokyo, Japan). An amino-bonded silica column of TSKgel NH2 -60 (5 m, 150 mm × 4.6 mm I.D.) was obtained from Tosoh (Tokyo, Japan). 2-Cyanoacetamide was obtained from Sigma. All other chemicals used were of analytical reagent grade.
sodium chloride solution was added. Then the mixture was heated in a boiling water bath for 5 min. After being cooled in a water bath, the solution was centrifuged at 2300 × g for 15 min. To 1 ml of the supernatant, 100 l of 0.1 M sodium hydroxide solution and 4 ml of cooled ethanol saturated with sodium acetate were added. The mix-
2.2. Apparatus and chromatographic conditions A flow diagram of liquid chromatography for the determination of HA is shown in Fig. 2. The chromatographic equipment included two high-pressure pumps (L-6000), a fluorescence detector (F1050), a chromato-integrator (D-2500) from Hitachi Instruments (Tokyo, Japan), a sample injector with 20 l loop (Model 7125) from Rheodyne (Rohnert Park, CA, USA), a column thermocontroller (Mini-80) from Taitec (Tokyo, Japan) and a dry reaction bath (DB5) from Shimamura Instrument (Tokyo, Japan). Established HPLC conditions were as follows (Fig. 2): A Carbonex column was eluted at 40 ◦ C with 25 mM sodium phosphate–NaOH buffer (pH 11) in 4% acetonitrile at a flow rate of 0.5 ml/min by using a L-6000 pump. To the effluent was added aqueous 0.5% 2-cyanoacetamide solution at the flow rate of 0.5 ml/min by using a L-6000 pump. The mixture passed through a polytetrafluoroethylene (PTFE) reaction coil (10 m × 0.5 mm I.D.) set in a dry reaction bath thermostated at 110 ◦ C and a following PTFE cooling coil (2 m × 0.25 mm I.D.). The effluent was monitored fluorometrically (excitation 335 nm, emission 395 nm). A 10-l portion of sample solution was loaded via a sample injector with a 20 l loop. 2.3. Preparation of human plasma GAGs Blood was collected from the healthy volunteers. Ethylenediamine tetra acetic acid disodium salt was added to the blood as an anticoagulant. The blood was separated into plasma and cells by centrifugation at 1200 × g for 15 min. Plasma GAGs were separated by our modified method reported previously [9]. To 250 l of human plasma, 100 l of 0.05 M Tris–HCl buffer (pH 8.0) containing 1% actinase E was added, and the mixture was incubated at 45 ◦ C for 3 h. To the solution, 1 ml of 0.1 M acetic acid containing 10%
Fig. 2. Flow diagram of the postcolumn HPLC system for determination of HA with a graphitized carbon column. Column, Carbonex (3.5 m, 100 mm × 4.6 mm I.D.) with a precolumn (3.5 m, 10 mm × 4.6 mm I.D.) at 40 ◦ C; eluent, 25 mM sodium phosphate–NaOH buffer (pH 11) in 4% acetonitrile; reagent, 0.5% 2-cyanoacetamide; reaction temperature, 110 ◦ C; detection, Ex. 335 nm, Em. 395 nm. Other conditions as described in the text.
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H. Toyoda et al. / J. Chromatogr. B 879 (2011) 950–954
ture was left to stand overnight at 4 ◦ C and centrifuged at 2300 × g for 15 min at 4 ◦ C. The precipitate was washed twice with 1 ml of ethanol, and then dried under the vacuum. The resulting precipitate was dissolved in 50 l of water. 2.4. Enzymatic digestion For the analysis of LSC in human blood plasma, 10 l of 0.2 M Tris–acetate buffer (pH 8.0) and 20 l of an aqueous solution containing chondroitinase ABC (50 mIU) and chondroitinase ACII (50 mIU) were added to a 5-l portion of the sample solution and incubated at 37 ◦ C for 3 h. A 10-l portion of the mixture was loaded onto the HPLC. For the analysis of HA in human blood plasma, 10 l of 0.2 M sodium phosphate buffer (pH 6.2) and 10 l of an aqueous solution containing hyaluronidase SD (10 mIU) were added to a 20-l portion of sample. The mixture was incubated at 37 ◦ C for 16 h, and a 10-l portion of the mixture was loaded onto the HPLC. 3. Results and discussion 3.1. Analysis of GAGs in human blood plasma by using an amino-bonded silica column LSC is the major class of GAGs in blood plasma [3,21]. Human plasma GAGs were digested with chondroitinase ABC and chondroitinase ACII, then the unsaturated disaccharides from LSC were analyzed by HPLC conditions described previously [9]. Typical chromatogram and the HPLC conditions are shown in Fig. 3. Table 1 summarizes the compositions of disaccharide units from human plasma samples. 3.2. Digestion of blood plasma GAGs with hyaluronidase SD LSC includes a number of non-sulfated units in repeating disaccharide units of chondroitin 4-sulfate (Fig. 4) and is present in a form of a proteoglycan named bikunin [22,23]. By using the LSC, bikunin is held together with heavy chains to form inter-␣-trypsin inhibitor family in blood plasma and some biological fluids [21,24]. Although hyaluronidase SD is the enzyme that yields Di-HA from HA, the enzyme also digests non-sulfated moieties in chondroitin sulfates to form Di-0S simultaneously (Fig. 4). The resulting DiHA and Di-0S were analyzed by HPLC with a graphitized carbon column and fluorometric postcolumn derivatization. 3.3. Chromatographic separation and detection of the unsaturated disaccharides from HA It is great significant to overcome the interference with large amounts of Di-0S from LSC for the analysis of trace amounts of HA in human plasma. We have reported HPLC conditions for the separation of Di-0S and Di-HA with an amino-bonded type silica
Fig. 3. Analysis of LSC in human blood plasma as their unsaturated disaccharides after chondroitinase ABC and ACII digestion by using an amino-bonded column. Sample size: 10 l. Peaks: 1 = Di-0S; 2 = Di-HA; 3 = Di-4S. HPLC conditions were as follows: a TSKgel NH2 column (5 m, 150 mm × 4.6 mm I.D.) with a precolumn (5 m, 10 mm × 4.6 mm I.D.) was eluted at 40 ◦ C with 10 mM ammonium-formate buffer (pH 5.0) containing 10 mM sodium sulfate in 4% acetonitrile at a flow rate of 0.5 ml/min. To the effluent were added 0.1 M NaOH and 1% 2-cyanoacetamide solution at the flow rate of 0.2 ml/min by using a double-plunger pump. The mixture passed through a PTFE reaction coil (10 m × 0.5 mm I.D.) set in a dry reaction bath thermostated at 110 ◦ C and a following PTFE cooling coil (2 m × 0.25 mm I.D.). The effluent was monitored fluorometrically (excitation 346 nm, emission 410 nm).
column [25,26]. The satisfactory separation was achieved with an eluent containing borate buffer and the method was applied to the measurement of human urinary HA. However, the separation was insufficient to determine the concentration of HA in human plasma. Graphitized carbon columns were introduced into HPLC methods [27,28] and utilized for the separation of carbohydrates [29]. We have found that a graphitized carbon column is very effective in the separation of each unsaturated disaccharide from GAGs [30]. In this study, HPLC was employed with a graphitized carbon column and fluorometric detection using 2-cyanoacetamide for the excellent chromatographic separation and high sensitivity. The optimized conditions and flow diagram are summarized in Fig. 2 and a typical chromatogram is shown in Fig. 5. Calibration graphs for Di-0S and Di-HA were linear in the range 0.2 ng–1 g. The relative standard deviations at 1 ng were less than 2% (n = 5) for Di-0S and Di-HA. The graphitized carbon column is so stable under extreme conditions of pH (0–14), salt concentration and temperature that the column lifetime was improved significantly
Table 1 Determination of unsaturated disaccharides produced enzymatically from human plasma GAGs. Case no.
Sex
Age
1 2 3 4 5 6 Mean ± SD
M M M M F F
22 23 29 38 23 25
M: male, F: female.
Chondroitinase digestion (g/ml)
Hyaluronidase digestion (g/ml)
Di-OS
Di-4S
Total
Di-HA
8.0 7.9 7.3 10.0 9.8 6.8 8.3 ± 1.3
5.7 3.8 2.6 6.2 5.9 4.8 4.8 ± 1.4
13.7 11.7 9.9 16.2 15.7 11.6 13.1 ± 2.5
0.017 0.012 0.033 0.025 0.028 0.030 0.024 ± 0.008
H. Toyoda et al. / J. Chromatogr. B 879 (2011) 950–954
953
Fig. 4. Structure and enzymatic digestion of LSC.
against an amino-bonded silica column, which is normally used for the separation of unsaturated disaccharides.
3.4. Analysis of HA in human blood plasma by using a graphitized carbon column Our sensitive HPLC method was used for the determination of the unsaturated disaccharides produced from HA in human blood plasma by hyaluronidase SD digestions (Fig. 6). We could determine trace amounts of HA by the excellent chromatographic separation of Di-0S and Di-HA with a graphitized carbon column and sensitive fluorometric post-column detection (Fig. 6). The Di-HA was only about 1/400th of Di-0S. Table 1 shows the results obtained from the analyses of 6 samples (4 male and 2 female).
Fig. 6. Separation of Di-0S and Di-HA produced from human plasma GAGs with hyaluronidase SD digestion on a graphitized carbon column. Other conditions as in Fig. 5.
4. Conclusion We set out to develop methodology that would facilitate the measurement of HA from small amounts of human blood plasma. This method is unique in that it employs the digestion with hyaluronidase SD, the separation with a graphitized carbon column and the fluorometric post-column detection with 2-cyanoacetamide. As described above, this highly sensitive and selective HPLC was first applied to the determination of HA in small amounts of blood plasma samples as precise chemical method. This method is very effective for the determination of trace amounts of HA in various biological fluids. References Fig. 5. Typical chromatogram of standard unsaturated disaccharides using a graphitized carbon column. Sample size: 10 l (1 ng of each sugar). Peaks: 1 = Di-0S; 2 = Di-HA. Other conditions as in Fig. 2.
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