Anal. Chem. 2003, 75, 6728-6731
An Assay of Ganglioside Using Fluorescence Image Analysis on a Thin-Layer Chromatography Plate Tomohiro Hayakawa and Mitsuhiro Hirai*
Department of Physics, Gunma University, 4-2 Aramaki, Maebashi 371-8510, Japan
A new and simple fluorometric method for determine quantities of gangliosides ranging from pico- to nanomoles is reported. Spraying hydrochloric acid followed by a heating treatment, sugars (glucose, galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid (sialic acid)), gangliosides (GM1, GD1a, GT1b), and asialoganglioside (asialoGM1) on thin-layer chromatography plates produced fluorescence under 365nm UV light. This fluorescence production of each sample was greatly dependent on the heating temperature. As sialic acid fluoresced readily at lower temperature (∼90 °C), we were able to distinguish sialic acid easily from other sugars tested. To determine gangliosides based on this sialic acid fluorescence, calibration curves for gangliosides were obtained by high-performance thin-layer chromatography and the image-analyzing system equipped with a CCD camera. The observed fluorescence images were analyzed using image-analyzing softwares, and the determined calibration curves for ganglioside-bound sialic acids were reproducible and showed a high linearity in a wide range from 47 pmol to 4.5 nmol. Since the fluorescence from sialic acid is easily measurable on TLC plates and is sensitive over a wide range of sample concentration, the present method is applicable for quantitative determination of gangliosides. Gangliosides are a class of glycosphingolipids composed of a ceramide attached to an oligosaccharide chain containing one or more sialic acid residues. For most gangliosides expressed in vertebrate cells, the oligosaccharide chain consists of aldohexoses (e.g., glucose, galactose, fucose, N-acetylgalactosamine, and Nacetylglucosamine) except for sialic acid, which is ketohexose. Since sialic acid is a characteristic component of gangliosides, determination methods for gangliosides such as the resorcinolHCl-Cu2+ reagent method1 are based on the coloration of sialic acids. The resorcinol-HCl-Cu2+ reagent detection on highperformance thin-layer chromatography (HPTLC) plates has generally been the method of choice for separation, identification, and quantitation of gangliosides. The developing patterns and the Rf values of major gangliosides on HPTLC plates have been well * To whom correspondence should be addressed. Tel: +81-27-220-7554. Fax: +81-27-220-7551. E-mail:
[email protected]. (1) Svennerholm, L. Biochim. Biophys. Acta 1957, 24, 604-611.
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characterized in various solvent systems.2-7 In many previous studies, the visualized bands of gangliosides on HPTLC plates by resorcinol-HCl-Cu2+ reagent were quantitated with a TLC scanning densitometer, measureing the absorbance of the bands at 580 nm where the sialic acid chromophore has maximum absorption.1 The reactions and coloration mechanisms of resorcinol-HCl-Cu2+ reagent are not yet fully understood. On the other hand, even without special reagents, sugars themselves can form colored compounds in strong acids, particularly after heating. The rate of formation of these compounds varies with different sugars, concentration of acid, and temperature.8,9 Although the colors of the formed compounds are stable, they are rather pale and not distinct in comparison with those obtained by the resorcinol and orcinol methods.10 However, under UV light, these compounds produce clear yellow-green fluorescence either in solutions or on TLC plates. It is known that the color/ fluorescence reactions of sugars are based on their abilities to form furfural derivatives, and ketoses such as sialic acids form the derivatives much more rapidly than aldoses. Therefore, specific fluorescence for sialic acids can be obtained by adjusting the heating time and temperature of the reaction, which would be applicable to the quantitative determination of gangliosides on TLC plate. In the previous studies, we have quantitated gangliosides using a resorcinol method with HPTLC.11-14 To search for a more rapid, sensitive, and simple alternative to currently available methods for gangliosides, we studied the characteristics of the fluorescence production of the sugars, gangliosides, and asialoganglioside on HPTLC plates, taking advantage of video image analysis. (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14)
Ando, S.; Chang, N.-C.; Yu, R. K. Anal. Biochem. 1978, 89, 437-450. Zanetta, J.-P.; Vitiello, F.; Robert, J. J. Chromatogr. 1977, 137, 481-484. Schnaar, R. L.; Needham, L. K. Methods Enzymol. 1994, 230, 371-389. Yu, R. K.; Ariga, T. Methods Enzymol. 2000, 312, 115-134. Mu ¨ thing, J. J. Chromatogr. 1996, 720, 3-25. Smid, F.; Reinisova, J. J. Chromatogr. 1973, 86, 200-204. Rogers, C. J.; Chambers, C. W.; Clarke, N. A. Anal. Chem. 1966, 38, 18511853. Dische, Z. Methods in Carbohydrate Chemistry I; Academic Press: New York, 1962; Section V. Svennerholm, L. J. Neurochemistry 1956, 1, 42-53. Hayakawa, T.; Hirai, M. J. Appl. Crystallogr. 2003, 36, 489-493. Hirai, M.; Iwase, H.; Arai, S.; Takizawa, T.; Hayashi, K. Biophys. J. 1998, 74, 1380-1387. Hirai, M.; Takizawa, T. Biophys. J. 1998, 74, 3010-3014. Hirai, M.; Takizawa, T.; Yabuki, S.; Hirai, T.; Hayashi, K. J. Phys. Chem. 1996, 100, 11675-11680. 10.1021/ac0346095 CCC: $25.00
© 2003 American Chemical Society Published on Web 10/09/2003
MATERIALS AND METHODS Materials. All solvents and chemicals were of analytical grade or better and were used without further purification. Sugars (D(+)-glucose (>98%), D(+)-galactose (>98%), D(+)-fucose (>97%), N-acetyl-D-galactosamine (>90%), N-acetyl-D-glucosamine (>95%), and N-acetylneuraminic acid (>98%)) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and standard gangliosides (GM1 (∼95%), asialoGM1 (>98%), GD1a (>95%), GT1b (∼97%)) were obtained from Sigma Chemical Ltd. (St. Louis, MO). HPTLC plates (Kieselgel 60, 10 × 10 cm) were purchased from Merck (Darmstadt, Germany), and TLC plates (K6 silica gel 60, 5 × 10 cm) were obtained from Whatman International Ltd. (Maidstone, England). Thin-Layer Chromatography. All HPTLC plates used were predeveloped in methanol-chloroform (1:1 v/v) to remove fluorescent contaminants and were preactivated by heating at 110 °C for 30 min prior to use. The samples were dissolved in chloroform-methanol-water (60:35:8 v/v) or 95% ethanol to give a required concentration and were applied at 1.5 cm from the bottom edge of the plate by using a 10-µL microsyringe. The plate was developed in chloroform-methanol-0.2% CaCl2 (aq) (60:35:8 v/v) in a well-saturated TLC chamber. The plates were then thoroughly dried at 40 °C in a drying oven and sprayed with ∼18% hydrochloric acid. They were heated on a hot plate for 12 min (for 10 min under a glass plate covering, and for 2 min without a glass plate) at various temperatures (from 50 to 180 °C) for production of fluorescent compounds. The temperature of the hot plate surface was monitored with a thermocouple. Video Image Analysis of Fluorescence. To eliminate any artifacts in the fluorescence measurements, dusts in the filters of UV transilluminator were removed carefully. The developed HPTLC plates were placed on the UV transilluminator (365 nm, 250 W), and the fluorescence images of the plates were captured by Image Saver AE-6905C, Atto Co. Ltd. (Tokyo, Japan) equipped with a CCD camera (768 × 493 pixels with 8-bit gray scale). All images were taken using a zoom lens (8.5-51 mm, F1.2) with +3 Dpt. closeup lens, UV-blocking filter, and optical cutoff filter (540 nm). To avoid the effects of inhomogeneous UV irradiation to the TLC plate, the fluorescent bands of the samples were measured at the center of the transilluminator. Fluorescence intensities were obtained by measuring the integrated fluorescence intensities covering the area of the band using imageanalyzing computer software: NIH image (http://rsb.info.nih.gov/ nih-image/). To check the software dependence, Densitograph ver.3.01 (Atto Co. Ltd.) was also employed and we obtained the same results. Background corrections were made on all measurements. RESULTS AND DISCUSSION The characteristics of fluorescence production of the sugars, constituent of the oligosaccharide headgroup of gangliosides in vertebrate cells, were investigated. Figure 1 shows the temperature dependence of the fluorescence production of the sugars on a TLC plate. Under UV light in the present conditions, fluorescent derivatives became visible above ∼80 °C for sialic acids, ∼100 °C for other sugars. For sialic acid, fluorescence production reached maximum at 100-110 °C. The other aldose sugars (glucose, galactose, fucose, N-acetylgalactosamine, N-acetylglcosamine) showed their maximum fluorescence around 140-170 °C. These
Figure 1. Temperature dependence of the fluorescence production of the sugars. All samples were dissolved in 95% ethanol and were spotted on a TLC plate, heated at various temperatures (from 50 to 180 °C at 10 °C intervals) after spraying 18% hydrochrolic acid. Fluorescence measurements on TLC plates were as described in Materials and Methods. Symbols: (4) sialic acid; (O) N-acetylglucosamine; (b) N-acetylgalactosamine; (0) glucose; (9) galactose; and (]) fucose. Smoothed lines for respective samples are shown.
Figure 2. Fluorescence production of sugars and gangliosides on HPTLC plates at 90 (a) and 160 °C (b) heating. In (a) and (b), lane 1, sialic acid; 2, GM1; 3, GD1a; 4, GT1b; 5, asialoGM1; 6, N-acetylglucosamine; 7, N-acetylgalactosamine; 8, glucose; 9, galactose; and 10, fucose. A total of 10 nmol of aldose sugars (lanes 6-10) and 5 nmol of sialic acid and gangliosides (lanes 1-5) dissolved in chloroform-methanol-water (60:35:8 v/v) were spotted in 0.6-cm streaks on HPTLC plates. The conditions of chromatography and heating treatment of the plates were as described in Materials and Methods. The exposure time for image acquisition was 2 s.
results suggest that we are able to determine ganglioside quantities by measuring the fluorescence of ganglioside-bound sialic acid below ∼100 °C. Panels a and b of Figure 2 show the images of the fluorescence production of each sample on an HPTLC plate heated at 90 and 160 °C, respectively. In (a), at 90 °C, only the samples containing sialic acid (lanes 1-4) showed fluorescence and the fluorescent intensities of gangliosides were dependent on the number of sialic acids they contain. In (b), at the higher temperature of 160 °C, Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
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Figure 3. Visualized bands of sugars and gangliosides using resorcinol-HCl-Cu2+ reagent on HPTLC plate. The identification of lanes (1-10) is as in Figure 2. The conditions of chromatography were as described in Materials and Methods. The heating temperature and time was 90 °C and 10 min, respectively.
browning (charring) of the bands was seen for sialic acid and gangliosides (lanes 1-4), while aldose sugars (lanes 6-10) and asialoGM1 (lane 5) produced a clear fluorescence at this heating temperature. Due to such fluorescence productions of aldoses, minor ganglioside components in lanes 2-4 became visible at 160 °C. Browning was seen in the bands of aldose sugars above the temperature of their maximum fluorescence (data not shown). The observed bands in Figure 2a were confirmed by the resorcinol-HCl-Cu2+ reagent. Figure 3 shows the visualized bands of sugars and gangliosides by the resorcinol reagent on an HPTLC plate. The samples containing sialic acid were visible as blue-violet bands (lanes 1-4), whereas the other bands (lanes 5-10) gave a faint yellowish or pinkish color. This blue-violet coloration by this reagent is a characteristic of sialic acid,1 and these blue-violet bands in Figure 3 correspond to the fluorescent bands in Figure 2 (a) (lanes 1-4). The density of the blue-violet color of the bands is dependent on the number of sialic acids. These results also indicate that only fluorescence from sialic acids was visible under the present coloration condition, that is, 90 °C heating temperature and ∼18% hydrochloric acid concentration.15,16 A relationship between concentration versus fluorescence intensity of gangliosides (GM1 and GT1b) was assessed, ranging in amounts on the HPTLC plates from 12 pmol to 6 nmol. To obtain maximum sensitivity for the ganglioside-bound sialic acid detection, the heating treatment was done at 100 °C for these measurements. Figure 4a shows the image of fluorescence production of gangliosides heated at 100 °C. Respective samples of 2 nmol developed on HPTLC plates were clearly fluoresced. In Figure 4b, a linear relationship was observed in the range from 47 pmol to 4.5 nmol for lipid-bound sialic acids. The lines in Figure 4b represent the results of a linear regression for GM1 and GT1b. For both cases, the values of the linear correlation coefficient are better than 0.997. The slope of the calibration curve of GM1 tends to be slightly greater than that of GT1b. This would be partly due to the contribution from aldose sugars of ganglioside. The same tendency was reported in other studies.21,22 (15) Roe, J. H. J. Biol. Chem. 1934, 107, 15-22. (16) Kulka, R. G. Biochem. J. 1956, 63, 542-548. (17) Hirabayashi, Y.; Hyogo, A.; Nakao, T.; Tsuchiya, K.; Suzuki, Y.; Matsumoto, M.; Kon, K.; Ando, S. J. Biol. Chem. 1990, 265, 8144-8151. (18) Hirabayashi, Y.; Nakao, T.; Matsumoto, M. J. Chromatogr. 1988, 445, 377384. (19) Svennerholm, L.; Fredman, P. Biochim. Biophys. Acta 1980, 617, 97-109.
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Figure 4. In (a), an image of fluorescence production of gangliosides. 2 nmol of respective samples dissolved in 95% ethanol, developed on HPTLC plates. Lanes 1 and 2, standard GM1; lanes 3 and 4, gangliosides from bovine brain extracted by the methods described elsewhere (17-20); and lanes 5 and 6, standard GT1b. In (b), calibration curves of GM1 (b) and GT1b (O) analyzed by HPTLC and image analysis. Ganglioside standards were prepared by making 2-fold serial dilutions of the stock solutions of gangliosides in 95% ethanol. Respective samples were spotted on HPTLC plates and subjected to chromatography. Detector response values are the mean ( standard deviation of triplicate samples. The inset in the Figure 4b shows an enlargement of the region of the plot from 0 to 0.8 nmol. For (a) and (b), heating treatments were done at 100 °C and fluorescence measurements were as described in Materials and Methods.
It is generally accepted that classical slit-scanning densitometries give more accurate and sensitive results than do video image densitometry.23 However, the level of sensitivity obtained in this study demonstrates the practicality of this method in determining a wide range of detectable concentrations compared with that of other determination results for gangliosides on TLC. Namely, (20) Momoi, T.; Ando, S.; Nagai, Y. Biochim. Biophys. Acta 1976, 441, 488497. (21) Mullin, B. R.; Poore, C. M. B.; Rupp, B. H. J. Chromatogr. 1984, 305, 512513. (22) Wiesner, D. A.; Sweeley, C. C. Anal. Chim. Acta 1995, 311, 57-62. (23) Petrovic, M.; K-Macan, M.; Ivankovic, D.; Matecic, S. J. AOAC Int. 2000, 83, 1457-1462.
Ando et al. reported that they observed a linear relationship between the detector response and the sialic acid content in the range of 90 pmol-10 nmol by using scanning densitometry and resorcinol-HCl reagent.2 Watanabe and Mizuta showed a linearity from 5 to 100 pmol for various glycosphingolipids by labeling the samples with 5-hydroxy-1-tetralone reagent.24 Wiesner and Sweely were able to obtain linear responses from 30 pmol to 5 nmol ganglioside-bound sialic acid by using resorcinol-HCl reagent and image densitometry.22 Mullin et al. reported that they observed a linearity from 1 to 50 pmol for ganglioside-bound sialic acid with resorcinol-HCl reagent and scanning densitometry.21 In the above case, they heated the TLC plates at high temperature (140 °C) to attain the highly sensitive detection; therefore, the contribution of aldose sugars of gangliosides would not be negligible, as mentioned in Figure 1. The fluorescence intensities from sialic acid on HPTLC plates were rather stable for over one week after the coloration treatments. However, the background intensities of the HPTLC plate were increased with the passage of time, which would be due to the adsorption of various chemicals in the air on the surface of the HPTLC plates. Therefore, the determination measurements after the heating treatment were done at a defined time interval for all measurements. The present method of fluorescence assay for sialic acids was intended to determine gangliosides that have sialic acids as the only ketose. Although it is not possible to distinguish between sialic acids and other ketose sugars using this method, to our knowledge, sialic acids are the only ketoses extracted in the lipid fraction of vertebrate cells or tissues by general methods for ganglioside isolation.19,25,26 Therefore, we are able to identify (24) Watanabe, K.; Mizuta, M. J. Lipid Res. 1995, 36, 1848-1855. (25) Suzuki, K. Life Sci. 1964, 3, 1227-1233. (26) Ledeen, R. W.; Yu, R. K.; Eng, L. F. J. Neurochem. 1973, 21, 829-839.
gangliosides by their temperature-dependent fluorescence production and by their Rf values on HPTLC plates. CONCLUSION In the present study, spraying ∼18% hydrochloric acid followed by heating treatment, we could obtain clear fluorescence from sialic acid on HPTLC plates. The fluorescence production of ganglioside-bound sialic acid was highly linear from 47 pmol to 4.5 nmol on HPTLC plates (Figure 4b). Since aldose sugars produce fluorescence at higher temperature (140-170 °C) (Figure 1), it is noted that the methods described here would also be useful for the analysis of glycolipids other than gangliosides by adjusting the heating temperature. The present method combines techniques already available, that is, instruments employed in this studystransilluminator, a video image analyzing system, and image analyzing computer softwaresare those commonly used in typical biochemical laboratories for the detection of electrophoresed bands of DNA or protein. This method gives a simple, low-cost, and reproducible quantitative determination of glycolipids on TLC plate without special reagents or time-consuming fluorescent labeling. ACKNOWLEDGMENT We are thankful to Mr. Satoru Abe for his technical assistance. We also thank Professor Dr. Naohisa Kochibe of Gunma University for helpful discussions.
Received for review June 5, 2003. Accepted September 12, 2003. AC0346095
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