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Journal of Chromatography, 494 (1989) 289-296 Bzomedzcal Applzcatzons Elsevler Science Publishers B V , Amsterdam - Prmted m The Netherlands

CHROMBIO 4846

Note

Rapid, quantitative method for the isolation and purification of gangliosides by LIPSEP gel chromatography

KRISHNA KANT, A P JOSH1 and K C GUPTA*

CSIR Centre for Bzochemrcals, Guru Tegh Bahadur Marg, Delhz Unzverszty Campus, Deb1 10 007 (Indra)

(First received February 9th, 1989, revised manuscript recewed May 3rd, 1989)

Ganghosldes are complex acldlc lipids that are normal constituents of mam- malian cell membranes, the highest concentrations being found m neuronal cell membranes [ 1 ] They are glycosphmgohplds that contam at least one slahc acid moiety Isolation of gangliosldes by the method of Folch et al [ 21 mvolves the chloroform-methanol extraction of tissues, followed by the partitioning of ganghosldes m the methanol-saline upper phase. To remove salts and other low-molecular-mass, water-soluble substances, the upper fraction (methanol- salme) 1s dlalysed [ 2,3] exhaustively against distilled water, followed by con- centration and lyophdizatlon. This makes the lsolatlon of ganghosldes a time- consummg and multi-step procedure. Moreover, loss of some less polar species of ganghosldes has been reported [ 41 to occur d the methanol-salme fraction IS dlalysed without prior removal of organic solvents [ 51.

By taking advantage of the acidic properties of ganghosides, a different ap- proach has been proposed for their lsolatlon and purification This mvolves the use of anion exchangers [ 6-121, the total lipid extract of the tissues being applied to an amon-exchange column and the ganghosides eluted with solvents contammg sodium acetate The resulting fraction 1s then subJected to mild alkaline hydrolysis and desalted by &alysls or gel filtration [ 131 and further purified by adsorption [ 14-201 chromatography. Recently, reversed-phase chromatographlc methods [ 21-231 have been described for the concentration and lsolatlon of ganghosldes, mvolvmg the use of Sep-Pak C,, cartridges. The

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gel column is washed with water Ganghosldes are quantitatively eluted with DCE-methanol (2 1, v/v) without the loss of any of the ganghosides Non- slahc acid lipids that were bound more tightly with LIPSEP gel required a higher concentration of DCE m the eluent and eluted later than ganghosides. In conclusion, LIPSEP gel was found to be an economical matrix for the large- scale isolation and purification of ganghosides

ACKNOWLEDGEMENTS

We thank Dr S.V Gangal for his interest m thus work and Dr G Yogee- swaran for critical reading of the manuscript We are also grateful to Mr Sum1 Agarwal for phosphorus determinations m the ganghoside samples

REFERENCES

1

2 3 4 5 6 7 8 9

10

L Svennerholm, m 0 Ouchterlony and J Holmgren (Editors), Cholera and Related Dlar- rheas, 43rd Nobel Symposmm, Stockholm, 1978, Karger, Basle, 1980, p 80 J Folch, M Lees and G H Sloane-Stanley, J Blol Chem ,226 (1957) 497 L Svennerholm, Methods Carbohydr Chem ,6 (1972) 464 J N Kanfer and C Splelvogel, J Neurochem ,20 (1973) 1483 R Ghldom, S Sonnmo and G Tettamantl, Llplds, 13 (1978) 820 R W Ledeen, R K Yu and L F Eng, J Neurochem ,21 (1973) 829 T Momol, S Ando and M Nagal, BlochIm Blophys Acta, 441 (1976) 488 S Ando and R K Yu, J Blol Chem ,254 (1979) 12224 M Iwamon and Y Nagal, Blochim Blophys Acta, 528 (1978) 257 P Fredman, 0 Nllsson, J -L Tayot and L Svennerholm, BlochIm Blophys Acta, 618 (1980) 42

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

T Itoh, Y -T Ll, S -C Ll and R K Yu, J Blol Chem ,256 (1981) 165 S K Kundu, S K Chakravarty, S K Roy and A K Roy, J Chromatogr ,170 (1979) 65 K Ueno, S Ando, R K Yu, J Lipid Res ,19 (1978) 863 D E Vance and C C Sweeley, J Lipid Res ,8 (1967) 621 E Svennerholm and L Svennerholm, Blochlm Blophys Acta, 70 (1963) 432 G M Gray, Nature (London), 207 (1965) 505 B Slddlqul and R H McCluer, J Llpld Res ,9 (1968) 366 S Ando, M Isobe and M Nagal, Blochim Blophys Acta, 424 (1976) 98 S Ando and R K Yu Trans Am Sot Neurochem ,8 (1977) 128 C C Irwin and L N Irwm, Anal Blochem, 94 (1979) 335 H Kubo and M Hoshl, J Llpld Res ,26 (1985) 638 M A Wllhams and R H McCluer, J Neurochem ,35 (1980) 266 S K Kundu and A Suzuki, J Chromatogr ,224 (1981) 249 F J Rolsen, H Bartfeld, R Nagele and G Yorke, Science, 214 (1981) 577 W Dlmpfed, W Moller and U Mengs, m M M Rapport and A Gorlo (Editors), Gangho- sides m Neurological and Neuromuscular Function Development and Repalr, Raven Press, New York, 1981, p 119

26 R W Leeden, J Neuroscl Res ,12 (1984) 147 27 L Svennerholm, Blochlm Blophys Acta, 24 (1957) 604 28 C H Fiske and Y Subba Row, J B1o1 Chem ,66 (1925) 375 29 M A K Markwell, S M Hass, L L Bleber and N E Tolbert Anal Blochem ,87 (1978) 206

296

30 L Svennerholm, Acta Chem Stand ,17 (1963) 239 31 K Suzuki, J Neurochem ,12 (1965) 629 32 M C Byrne, M Sbaschmg-Agler, D A Aqumo, J R Sclafam and R W Leeden, Anal Blochem

148 (1985)163 33 L Svennerholm, J Neurochem , 10 (1963) 613