BIOCHEMICAL

MEDICINE

Measurement

12,

of

274-282

GABA

(1975)

in

Human

Cerebrospinal

B. S. GLAESER AND T. A. Department of Pharmacology, Thomas Jefferson University, Received

Jefferson Philadelphia, October

Fluid

HARE

Medical College of Pennsylvania 19107

21, 1974

INTRODUCTION Gamma-aminobutyric acid (GABA) has been implicated as a possible inhibitory neurotransmitter and as such, may play a role in the pathogenesis of certain neurological disorders. For example, deficient GABA levels have been demonstrated in specific areas of the brains of patients with Huntington’s chorea, after death, when compared to normal brains (1). Currently there is a great deal of interest in exploiting this observation through drug treatment designed to produce elevation of brain GABA levels (2-7). These attempts are being carried out under the assumption that, like dopamine in Parkinson’s disease, the reduced GABA levels are to some extent responsible for the disorder and that correction of this deficiency may benefit the patients. Unfortunately, since brain samples are not available from the patients being treated, there is no way to monitor the influence of these treatments except through judgment of clinical response. The observed deficiency of brain GABA could likely be more readily exploited for diagnostic, treatment, and predictive purposes, if the deficiency were reflected, and could be detected in cerebrospinal fluid (CSF). Unfortunately, to our knowledge, there has been no indication in the literature that GABA is a natural constituent of CSF. Even though GABA is present at relatively high levels in brain (1,8-lo), attempts to detect it in CSF have not been successful (11-13) and have demonstrated that earlier reports (14,15) of GABA in CSF were not correct, presumably because of inadequate resolving power of the paper chromatographic procedures which were utilized. Only after administration of GABA to man or animals have levels been elevated enough to be detected in plasma or spinal fluid (7,16,17). GABA has been measured by a variety of chromatographic methods (9-15,18,19). A sensitive procedure for thin layer chromatography of the dansyl derivative of GABA has been described (20). GABA can also be measured to very low levels by enzymatic procedures utilizing GABA 274 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

GABA

IN HUMAN

CSF

275

transaminase and succinic aldehyde dehydrogenase (21). The existing methods, however, have been inadequate for measuring human CSF-GABA in clinical situations because they are either too complicated for routine use, do not provide sufficient resolution or are not sufficiently sensitive. We report here a method for GABA analysis with which GABA in human spinal fluid can be readily measured. The procedure utilizes ionexchange column chromatography and fluorescence detection based on the reaction of ortho-phthalaldehyde (OP) with primary amines as recently described by Roth (22,23). The method is rapid enough (2 hr) for clinical application, has resolution like that of usual physiological fluid amino acid analysis and is sensitive in the picomole range. MATERIALS

AND

METHODS

Buffers and Reagents

A two-buffer elution procedure was utilized. These buffers were made from stock concentrates which were 3 M citric acid (Fisher Scientific Corp.), 12 N LiCl (Lithium Corp. of America, Bessemer City, N.C.) and 3 N LiOH (Lithium Corp. of America). During the first attempts at these analyses a substantial amount of interfering background fluorescence was observed. In view of Hamilton and Myoda’s study of interfering contaminants during high sensitivity analyses (24), a buffer purification procedure was devised. This system consisted of a suction flask and cylindrical filtering funnel (4 cm diameter) which contained a 2.5 cm bed of dowex 50 ion-exchange resin (200-400 mesh, 4% crosslink, Sigma Chem. Co., St. Louis, MO.). The buffers were prepared in five liter batches as shown in Table 1. They TABLE 1 COMPOSITION OF ELUTION BUFFERS 3M Citric acid

12N LiCl

Thiodiglycol”

Octanoic acid b

Final volume’

First buffer

175 ml

130 ml

25 ml

0.5 ml

5L

Second buffer

185 ml

230 ml

25 ml

0.5 ml

5L

n Pierce Chemical Co., Rockford, 111. * Eastman Organic Chem., Rochester, N.Y. c Water used for buffer preparation was deionized to >500,000 ohm specific resistance (Continental Water Conditioning Corp., El Paso, Texas, Model #200). Buffers were prepared as indicated in the above table, then filtered through Dowex 50 resin and taken to appropriate pH (first buffer 4.55, second buffer 5.50) with 3N LiOH as described in the text.

276

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were not adjusted to their final pH so that they were still acidic (pH < 2) because at that pH, contaminating primary amines would be expected to remain on the dowex-50 purification filter. The filter was regenerated with 500 ml 0.3 N LiOH followed by 500 ml deionized water followed by 500 ml of one of the above buffers. After this washing procedure was complete, the rest of the 5 liter batch of buffer was pulled through the filter. The purified buffer (now about 4500 ml) was then adjusted to final pH (4.55 for first buffer, 5.50 for second buffer) with 3 N LiOH (approximately 275 ml for first buffer, 410 ml for second buffer). The resulting buffers (first buffer, pH 4.55, 0.1 M citrate, 0.47 N Li; and second buffer, pH 5.50,O.l M citrate, 0.76 N Li) were placed in reservoirs which were connected to the pump through solenoid valves. The air entering the reservoirs passed through traps containing solid citric acid to protect against accumulation of ammonia from the air. The OP reagent was prepared as follows. One mole of boric acid in about 800 ml H,O was titrated to pH 9.6 with LiOH and the resulting solution taken to 1 liter. To this 1 N borate buffer, 0.4 g orthophthalaldehyde (Sigma Chemical Co.) and 10 ml of ethanol were added to make the reagent stock solution. This stock solution was stable to storage at room temperature. To prepare the OP reagent from the stock solution, 0.15 ml mercaptoethanol (Sigma Chemical Co.) was added per liter and the resulting reagent placed under 3 psi air pressure in a reservoir which was connected to the reagent pump. Under these conditions the reagent was stable for at least 10 days. Apparatus

A flow diagram of the analytical apparatus is shown in Fig. 1. The apparatus was basically derived from a Phoenix Precision Instrument Co. amino acid analyzer model 6800 (Philadelphia, Pa.). Samples were applied using a Mark Instrument Co. Automatic Sample Applicator, model 310-8 (Villanova, Pa.) which had eight 0.6 cm id. x 6 cm glass precolumns each containing a 1 cm bed of cation-exchange resin. The elutrient from the sample applicator flowed through capillary telIon tubing to a 70°C water jacketed 0.9 cm id. x 66 cm column having adjustable plunger fittings at each end (Mark Instrument Co.) and packed with Spherix cation-exchange resin Type XX 987-60-o (Phoenix Precision Instrument Co.). Elutrient from the column flowed through a manifold at which point the OP reagent was introduced. The elutrientreagent mixture passed through a 5 minute delay coil of capillary teflon tubing before entering a Farrand model A fluorometer (Farrand Optical Co., New York) containing a 4 mm i.d. x 7 mm o.d. x 2 cm long flowthru cell made from clear fused quartz T 21 Suprasil tubing (Amersil Inc., Hillside, New Jersey). The ends of the flow cell contained rubber

GABA

CSF

IN HUMAN

277 ow 3ps1

rrogent

rl

recorder

1

+

1 fhmromefer $ waste

FIG. 1. Flow diagram of analytical apparatus.

stoppers through which holes had been drilled to accommodate the teflon tubing. The fluorometer light source was stabilized with a 500 watt constant voltage transformer (harmonic type CVS, Sola Electric Co., Elk Grove Village, Illinois). The fluorometer contained a 7-60 optical filter in the excitation position and a 3-72 plus a 4-96 optical filter in the emission position since the fluorescence to be measured was optimum at 340 nm excitation and 455 nm emission. The signal from the fluorometer was recorded using the amino acid analyzer recorder (O-l mV range) for which additional zero suppression was provided using a 1.5 V battery and a potentiometer. The areas under the peaks on the recording were quantified by multiplying the height of the peak above baseline times the width of the peak at halfheight. One of the buffers or the regeneration solution (0.3 N LiOH) was delivered to the buffer pump depending on the position of solenoid operated valves. The output of the pump was directed to one of the sample precolumns by the S-port valve of the automatic sample applicator. The operation of the buffer pump, reagent pump, solenoid valves and

278

GLAESER

AND

recorder was automatically controlled croswitches in the sample applicator.

HARE

by adjustable

cam-operated

mi-

Procedure

CSF specimens were obtained by lumbar puncture, and kept chilled less than two hours before storage at -20°C. After an average of two days they were thawed, deproteinized and then stored at -20°C for periods ranging from one month to one year. Deproteinization was accomplished by adding l/3 volume of a 20% aqueous solution of sulfosalicylic acid as described by Block et al. (25). GABA standards were routinely also subjected to the deproteinization procedure which did not alter the amount of GABA detected. Aliquots of standards or of the deproteinized samples were applied to the sample applicator precolumns and pushed into the resin with air pressure (20 psi). The buffer pump operated at the rate of 60 ml/hr producing approximately 300 psi pressure. The reagent pump operated at the rate of 67 ml/hr producing approximately 100 psi pressure. Fifty-five min after the start of an analysis, the buffer changed from first buffer to second buffer, and the reagent pump and recorder came on. LiOH (0.3 N) was pumped into the column during the interval between 80 min and 90 min followed by first buffer so that after 120 min the column was ready for the next sample. The reagent pump and recorder turned off at 105 min. Amino

acid analyses

Standard amino acid analyses for physiological fluids were carried out by a method routinely used in our laboratory. This amino acid analysis procedure utilized three lithium citrate buffers and the same ion exchange column described for the new GABA procedure. During the amino acid analyses, a first buffer (pH 2.95, 0.1 M citrate, 0.3 N Li) was pumped (60 ml/hr) for 210 min followed by a second buffer (pH 4.2, 0.1 M citrate, 0.3 N Li) for 85 min followed by a third buffer (pH 6.0, 0.1 M citrate, 0.8 N Li). Ninety-five min after the beginning of the analysis, the column temperature was switched from 40°C to 70°C. GABA was eluted from the column after 345 min, i.e., 12-16 min after the third buffer breakthrough. As the GABA was eluted, it was detected using the fluorescent procedure described above for the new GABA method. RESULTS

Figure 2a ence during when GABA Figure 2b

AND

DISCUSSION

presents the elution of a blank sample showing no interferthe time, 4-8 minutes after second buffer breakthrough, would normally be eluted from the column. shows the elution of a GABA standard. GABA was eluted

GABA

IN HUMAN

; I

100 picomoles

CSF

GABA 1 I

279 humon I

CSF

t

GABA h

;;,A 070 time

r-

90

in minutes

FIG. 2. Typical analysis of 200 picomoles of GABA, brospinal fluid, and a blank by the method described.

1.O ml deproteinized

human cere-

95 min after the beginning of the analysis which was about 5 min after the second buffer front. Figure 2c shows a typical analysis of 1.0 ml deproteinized human CSF which indicated the presence of 530 picomoles of GABA. Figure 3 presents a standard curve for the GABA analysis which was linear over the range tested (SO-800 picomoles). Recovery of GABA added to CSF was 8% in duplicate experiments as shown in Table 2.

1T :

64 x x0 8 E -0

?!2

L/i ,z’ 0

# 400 picomoles

800 GABA

FIG. 3. Standard curve for GABA measurement. Relative Peak Area refers to area under the GABA peak determined by multiplying the measured height above baseline times the measured width at half-height.

280

GLAESER

AND

TABLE RECOVERY

mldeproteinized CSF (1)

(2)

OF INTERNAL

2 GABA

Picomoles GABA added

0.50 0.25

none 100

HARE

STANDARD

Picomoles GABA detected” 143 k 6 (SD) 159 !I 20

% Recovery 89%

a Data from duplicate experiments.

To compare the adequacy of resolution of the new method with an established procedure (8,11), a complete amino acid analysis was carried out in duplicate on 1.0 ml aliquots of deproteinized human CSF. These analyses showed an estimated GABA level of 400 picomoles/ml of CSF for this sample. Triplicate analysis of the same CSF sample by the new procedure described in this paper indicated a GABA content of 394 + 39 (SD) picomoles/ml. Since the complete amino acid analysis is usually considered adequate for resolving GABA from interfering materials in brain extract (8,l l), the agreement between these two methods of analysis indicates that the new procedure provides essentially the same adequacy of resolution with CSF as does the complete amino acid analysis. As a test of the new method, GABA was measured in 1.0 ml aliquots of deproteinized CSF specimens from 25 patients with a variety of neurological disorders. The results of these analyses indicated the mean GABA level to be 270 + 260 (SD) picomoles/ml of CSF. The wide range for these analyses is not surprising since all of the patients had neurological disorders and brain GABA levels are known to be altered in certain neurological disorders (1). It seems probable that changes in the brain levels may have been reflected in the spinal fluid. The capability for GABA measurement in CSF may be useful in a number of ways. This measurement may function as a diagnostic test for certain disorders; and, it may also be useful in studying the pharmacology of drugs designed to influence brain GABA levels, or in genetic studies of certain neurological disorders. In view of the evidence that GABA may be an inhibitory neurotransmitter in the CNS, and the possibility that altered brain levels of GABA may be reflected in the CSF, determination of GABA levels in CSF of patients with neurological disorders may provide valuable information about the chemical abnormalities of some of these disorders. For example, preliminary indication from our study of patients with Huntington’s chorea has indicated deficient CSF-GABA relative to control patients (26). These studies can now be expanded to include a variety of other neurological conditions. Measurement of components of CSF has provided relatively little information to date because only a few of the components are present at

GABA

IN HUMAN

CSF

281

levels which can readily be detected. Since fluorogenic reagents are now available for detecting primary amines with greatly increased sensitivity, it is reasonable to expect that similar methods can be developed for measuring many of the other components of CSF which have not yet been adequately studied. Simultaneous measurement of various neurologically active substances should be more useful than simply measuring GABA, in view of the often stated possibility that the ratio of these agents is more critical than is the actual level of any one of them. These types of analyses offer much potential for increasing our knowledge of CNS chemistry, neurological abnormalities and the actions of drugs on the CNS. SUMMARY An automatic method is described utilizing ion-exchange column chromatography and fluorescence detection after reaction with orthophthalaldehyde to determine levels of GABA in human CSF. The standard curve was linear over the range tested (5O-WO picomoles). The method is rapid enough (2 hrs) for clinical application and provides resolution comparable to that of usual amino acid analyses of physiological fluids. The level of GABA was found to be 270 + 260 (SD) picomoleslml in CSF from 25 patients with a variety of neurological disorders. ACKNOWLEDGMENT We wish to thank Mr. Richard F. Cerruti, Jr. for excellent technical assistance during these studies and to acknowledge support, in part by PHS General Research Support Grant RR-5414.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Perry, T. L., Hansen, S., and Kloster, M. New Engl. J. Med. 288, 337 (1973). Mattson, B., and Persson, S. A. Lnncet ii, 684 (1973). Barbeau, A. Lancer ii, 1499 (1973). Fisher, R., Norris, J. W., and Gilka, L. Lancer i, 506 (1974). Perry, T. L., Hansen, S., and Urquhart, N. Lancet i, 995 (1974). Anden, N. E., Dalen, P., and Johansson, B. Lancer ii, 93 (1973). Perry, T. L., and Hansen, S. J. Neurochem. 21, 1167 (1973). Perry, T. L., Berry, K., Hansen, S., Diamond, S., and Mok, C. J. Neurochem. 18,513 (1971). Shaw, R. K., and Heine, J. D. J. Neurochem. 12, 151 (1%5). Perry, T. L., and Jones, R. T. J. C&z. Invest. 40, 1363 (1961). Perry, T. L., Stedman, D., and Hansen, S. J. Chromatog. 38,460 (1968). Dickinson, J. C., and Hamilton, P. B. J. Neurochem. 13, 1179 (1%6). van Sande, M., Mardens, Y., Adriaenssens, K., and Lowenthal, A. J. Neurochem. 17, 125 (1970). Logothetis, J. Neurology 5, 767 (1955). Logothetis, J. Neurology 8, 299 (1958). van Gelder, N. M., and Elliott, K. A. C. J. Neurochem. 3, 139 (1958).

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17. Tower, D. B., in “Inhibition in the Nervous System and Gamma-Aminobutyric Acid” (Roberts, E., Ed.), p. 562. Pergamon Press, New York, l%O. 18. Wagner, F. W., and Liliedahl, R. L. J. Chromatog. 71, 567 (1972). 19. Levin. E., Lovell, S. J., and Elliott, K. A. C. /. Neurochem. 7, 147 (1961). 20. Auhart, J., Sibiga, S.. Sanders, H.. and Khairallah, E. A. Anal. Biochem. 53, 132 (1973). 21. Otsuka, M., Obata, K., Miyata. Y., and Tanaka, Y. J. Neurochem. 18, 287 (1971). 22. Roth, M. Anal. Chem. 43, 880 (1971). 23. Roth, M., and Hampai, A. J. Chromatog. 83, 353 (1973). 24. Hamilton, P. B., and Myoda, T. T. Clin. Chem. 20, 687 (1974). 25. Block, W. D., Markovs, M. E., and Steele, B. F. Proc. Sot. Exptl. Biol. Med. 122, 1089, (1966). 26. Glaeser, B. S., Vogel, W. H.. Olewieler, D. B., and Hare. T. A. Biochem. Med., (in press).

Measurement of GABA in human cerebrospinal fluid.

BIOCHEMICAL MEDICINE Measurement 12, of 274-282 GABA (1975) in Human Cerebrospinal B. S. GLAESER AND T. A. Department of Pharmacology, Thom...
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