Free amino acids (glutamic acid, y-amino-butyric add and
glycine) from the central nervous system of albtno mice have been
indentified and determined quantitatively. This method i8 convenient
for determtnation of certain amino acids iln 1small samples
of nervous tissue (50-1000 mg).
The procedure ios simrpLe, economical and rapid. Separation on
the chromatogram is good and reproduciible, due to a specific
saturation of the chromatographic-tank atmosphe·re

N. Filipović

English

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CROATICA CHEMICA ACTA CCACAA 48 (3) 379-383 (1976) CCA-945 YU ISSN 0011-1643 577.17 Note Determination of some Amino Acids in the Central Nervous System by Thin-layer Chromatography N. Filipovic Department of Pharmacology and Toxicology, Medical Faculty, University of Sarajevo, 71000 Sarajevo, Yugoslavia Received February 19, 1976 Free amino acids (glutamic acid, y-amino-butyric add and glycine) from the central nervous system of albtno mice have been indentified and determined quantitatively. This method i8 con-venient for determtnation of certain amino acids iln 1small samples of nervous tissue (50-1000 mg). The procedure ios simrpLe, economical and rapid. Separation on the chromatogram is good and reproduciible, due to a specific saturation of the chromatographic-tank atmosphe·re. Previous 1studies have indicated that y-amino hutyric acid (GABA), glycine and ·tauriine may act as 1inhibiitory neurotram;;mi:tters, whereas gJ!utamic and aspariic .acid may acit as excitatmy neurotransmitters :iin the central nervous system (CNS) of vertebrata1- 4 • There is a number of methods for the separation a:nd determination of free a:mtno acids fa CNS: thin-layer chromatography (TLC)5•6, paper chromato-graphy (PC)7, and gas-liquid chromatography (GLC)8•9• The most precise method .is the .ion-exchange chromatography by means of automatic amino acid analyser (AAA)10- 11• TLC on silica-gel or :on ceUu1ose layers is very conven1ent for analysis of free amino acids in nervous tissue12• A number of TLC .solvent .systems, especially the one-d~mensional ones, have a shortcoming: poor separa,tion of glycine and sedne. The purpose of this work wais .to obtain good separation of .serine aind glycine, as well as other free amino acids :in the mouse brain. EXPERIMENTAL Materials and Methods Male albino mice weighing 17-23 g, were used in all experiments. The absorbance values were measured with a Beckman DU UV/VIS spectro-photometer. Standard samples of amino acids from F. Hoffman-La Roche and Co., Baisel, Switzerland, were used. Ethanol abs., methanol, nilnhydrine and cadmium acetate were obtained from Merck, Darmstadt, Germany. n-Butanol and glacial acetic acid were products of »Laphoma«, Skopje, while ammonia 25°/o was a product of »Azot«, Gorazde, Yugoslavia. Tissue Preparation and Extraction The animals were decapited, the brains were very quickly weighed and! placed in previously refrigerated glass-homogeniser, coIJJtai:ning 85•0/o ethanol (1 : 8 g/v). The 380 N . FILIPOVIC tissue was homogenized for 90 sec at O 0c and centrifuged at 38 000 g and -10 °c for 30 minutes. The supernatant was decanted and stored evernight at -20 °c to precipitate the lipids. The lipdds were separated by centrifugation at -10 °c and 18 000 g for 10 mililutes. The .supernatant (0.3 ml) was used dfrectly for TLC. Thin-layer Chromatography Commercially available plates (20 X 20 cm) with 0.25 mm thick Silica-gel F 25 layer (Merck, Darmstadt) were used throughout the experiments to control the chromatograiphic effect in UV-light (254 nm). · The solvent system ethanol-NH40H (250/o) in ratio 100: 28 was used for deve-lopment. The solvent was allowed to ascend 15 cm with the walls of chromart;ographic tank lined with two fi1ter-paper pieces to saturate the atmosphere. As a supple-mentary saturation a 10 ml glass vessel containing 25P/o NH40H was placed in the tank. The development of the chromatogram took about Z hours and was performed twice at room temperature. After the first development the chromatogram was dried at 100 °c for 30 minutes. After the! second development with identical solvent system, the minimum period of drying was two hours at 105 °c, for the removal of even traces of ammonia. The chromatogirams were stained with ninhydrin-butanol reagent following the procedure o.f Barirollier13 . The coloured spots of the amiino acids wexe marked and removed quamiti.tatively iby coveri:ng them w:ith a 15'0/~ so1uticm of ·collodion in acetone. The coloured spots were placed into centdfugation tubes and to each 3.5 ml of a 0.50/o Cd(CH3C00)2 solution and 0.10/o ,glacial •acetic acid were added. The test tubes were put in dark place for one hour and sb.aken periodically. They were centrifuged at 9 500 g for 15 min. The supennatant was decanted into glass cel1s' and absorbance measured at 520 nm. Standard samples of amino acids (0.1 µM/0.1 ml) were 1prepared and amino acid recovery was determined by addition of the relevant amino acid (5 µM/0.5 g of tissue) to birain tissue extracted previosly 5-6 times with an excess of 950/o ethanol. Such samples were subjected to the extraction process. Rechromatography by the method of Fahmy ·et aL14 has shown that the sepa-ration of these three amino acids was good. It was shown that using tMs method as small quantity of tLssue as 50 mg was sufficient to perform the experiment. RESULTS AND DISCUSSION TL chromatogram of a mixture of glutamic acid, GABA and glycine is shown in Figure 1. The Rf values of pure standards of a ,serie·s of .amino acids in chromatographic system used, with each value 1given as the mean of five measurements and accompanied by standard error, are. given in Table I. Recovery vaJ.ues for glutamic acid, GABA and glycine were found to be 920/o, 93.50/o and 90.5°/o ,respeotively, and thei;r concentrations as deter1mined iln alhinio mouse brain were compared with results obtained by Agrawal et al.15 (Ta1ble II). The primary purpose of our work was to obtain ·a good separafaon of serine and glycine, and this problem was solved satisfaotorily. In addi,tion to thts problem, it was poss.ible to obtain a good separation ·of many other amino acids fr.om the albi,no mouse brain (Figure 2.). However, even 1f a good resolution o.f these amino acids was obtained, alanine and threon:ine were Jstill so close together on the chroma,tog,ram, that thiis method for their quantitative analysis should be avotded, It should be stressed that the dhect use of the supernatant w;i:thout pre-vious evaporation gives better results for determination of free amino acids. Evaporatirn1 to dryness and dis19olution of the dry .residue was fou:nd to yield 10-150/o, lower Tecovery. THYN-LAYER CHROMATOGRAPHY 381 Figure 1. Thin-layer chromatogra phic separation of 1. Glutamic acid, 2. GABA and 3. Glycine Figure 2. Thin-layer chromatographic separation of amino acids from albino mouse brain 382 N. FILIPOVIC TABLE I Rrvalues of amino acids in chromatographic system used 1. Glutamine 2. Phosphoethanol amine 3. Ethanol amine 4. Arginine 5. Aspart!c acid 6. Lysine 7. Glutamic aeid 8. GABA 9. Serine 10. Glycine 11. Alanine 12. Threonine 13. Tyrostne 14. Taurine 15. Valine 16. Urea 17. Leucine 18. Iso-Leucine 19. Phenylalan.ine TABLE II Rf± S.E.M. 0 0 3 ± 0.1 10 ± 0.2 17 ± 0.2 21 ± 0.2 28 ± 0.3 38 ± 0.2 42 ± 0.1 45 ± 0.2 52 ± 0.3 56 ± 0.1 61 ± 0.2 62 ± 0.2 70 ± 0.3 70 ± 0.2 75 ± 0.1 75 ± 0.2 77 ± 0.2 Amino acid level in albino mouse brain Amino acid Recovery Our results Agrawal et al.'s O/o µM/g results µM/g Glutamic acid 92.0 9.36 ± 1.09 11.50 GABA 93.5 2.04 ± 0.13 2.52 Glycine 90.5 1.96 ± 0.15 1.61 Our ~results concerni:ng ithe examined levels o.f the 1glutamic acid, GABA and rg,lydne whe:n compared with results of Agrawal et al.15 (Table II.), demon-strate that our values for glutamic acid and GABA are lower, while the value for ;glyc.iine is higher. This fact may be due to a number of different factors, of which the use of different sorts of exper~mental animals need not be the least. It should :be aLso stressed that a 1pe:rmanent saturation wi·th NH3 gives a better resolutiOl!l of amino acids on the chromatogram, compared with filter--paper saturation. A satisfactory separation of aspartic acid, glutamic acid, GABA, serine, glycine and taur.i:ne by means of the method described in tMs work! might be useful in small laibo;ratories without expensive equipment. REFERENCES 1. D. R. Curtis and J. M. Crawford, Ann. Rev. Pharmacol., 9 (1969) 209. 2. J. K., J o h n s on, Brain Res., 37 (1972) 1. 3. K. Kr n j e vi c, Physiol. Rev., 54 (1974) 418. THYN-LAYER CHROMATOGRAPHY 383 4. K. Kr n j e vi c, Brit. Med. Bull., 21 (1965) 10. 5. G. Pat a k i, Diinnschichtchromatographie in der Aminosiiure- und Peptid--Chemie, Walter de Gruyter Co., Berlin, 1966. 6. E. St ah l, Diinnschicht-Chromatographie, Springer-Verlag, Berlin-Heidelberg--New York, 1967, p. 696. 7. J . Mourek, H . C. Agrawal, J.M. Davis, and W. A. Himwich, Brain Res., 19 (1970) 229. 8. H . P. B u r ch f e 1 d and E. E . S to r rs, Biochemical Applications of Gas--Chromatography, Academic Press, New York, 1962, p. 127. 9. R. F. Mc G reg or, Clin. Chim. Acte, 48 (1973) 65 . 10. T. Gerri t sen, M. L. R eh berg, and H. A. W a ism an, Anal. Biochem., 11 (1965) 460. 11. J . V. Benson, M. J . Gordon, and J . A. Patterson, Anal. Biochem., 18 (1967) 228. 12. H. Loth ha 11 er, A. Re t t , and H. Z imp rich, Wiener Klin. Wschr., 83 (1971) 863. 13. J . B arr o 1 i er, N aturwissenschaften, 48 (1961) 404. 14. A. R. Fahmy, A. Niede r wieser G. Pataki, and M. Brenner, Helv . Chim. Acta, 44 (1961) 2022. 15. H. C. Agrawal, .r. M. Davi 1s, and W. A. Himwich, J. Neurochem., 15 (1968) 917. SAZETAK Odredivanje nekih aminokiselina u centralnom nervnom sistemu tankoslojnom hromatografijom Nadan Filipovic Slobodne aminokiseline (glutaminska kiselina, y- aminomaslaona kiselina i glicin) iz centralnog nervnog sistema albino-mifa identif<ikovane su i kvanititativno odredivane. Ta je metoda pogodna za odredivanje tih aminokiselina u malim uzor-cima nervnog 1tkiva (50-1000 mg). Postupaik je jednostavan, ekonomican i brz, a osim toga prufa mogucnost analize i nekih drugih aminok1selina iz centralnog nervnog sistema. Razdvajanje na hromatogramu je dobro i reproducibilno zbog drugacijeg zasicenja atmos.fere hroma-tografskog tanka. INSTITUT ZA FARMAKOLOGIJU I TOKSIKOLOGIJU MEDICINSKOG FAKULTETA UNIVERZITET U SARAJEVU 71000 SARAJEVO Prispj elo 19. velja<!e 1976. 

Determination of some Amino Acids in the Central Nervous System by Thin-layer Chromatography

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