Journal of Nrurochwusrrr.. 197U. VoI 30. pp. 291 -293 Pergamon Prew. Printed in Great Britain
SHORT COMMUNICATION
Verification and quantification of GABA in human cerebrospinal fluid (Received 7 April 1977. Accepted 22 June 1977)
1976; ENNAet a/., 19776). tion medium (ENNA& SNYDER, MEASCREMEWof putative neurotransmitters and their For the assay, frozen rat brain synaptic membrane pellets metabolites in human cerebrospinal fluid (CSF) has pro1975) were homogenized in 0.05 M-Trisvided new insights into the neurochemical characteristics (ENNA& SNYDER, of CNS disorders (KLAWANS. 1971; CURZONet 01.. 1972; citrate buffer (pH 7.1 at 4°C) at a concentration of 1 mg protein/ml. Sufficient Triton X-100 was added to the memWOOD et a/., 1977a. 19776). Since y-aminobutyric acid brane suspension to yield a 0.05% (v/v) concentration of (GABA)is felt to be, quantitatively, one of the more important neurotransmitters in the mammalian CNS ( B ~ &M detergent and the suspension was incubated at 37°C for IVERSEN. 1971) and abnormalities in GABAergic transmis- 30 min. then centrifuged at 48.000 g for 10 min. The Triton X-100 supernatant was discarded and the pellet was resuser al.. sion have been implicated in epilepsy (VAN GELDER pended to 0.05 M-Tris-citrate and centrifuged at 48,000 g 1972), Huntington's disease (PERRY ~t a/.. 1973; BIRD & IVERSEN, 1974; ENNAet a/., 1976). cerebrovascular disease for 10 min to wash off the excess detergent. After repeating this washing procedure a second time, the pellet was resus(ACHAR et al., 1976). meningitis (BURVAKOVA et a/., 1975) and psychiatric illness (ROBERTS, 1972; ROBERTS er a/.. pended to 0.05 M-Tris-citrate and centrifuged at 48,000 g concentration of approx I mg protein/ml. One ml portions 1976; LANGER et a/., 1975). Methods to accurately determine the quantity of this amino acid in human CSF may of this membrane suspension were placed into 15 ml Soraid in the diagnosis and treatment of some CNS disease vall centrifuge tubes containing 250pl of untreated CSF. 750 p1 H 2 0 . and 8 m-C3H]GABA (New England Nuclear. states. Recently, several reports have appeared on attempts to Boston. MA, 36.73 Ci/mmol). All samples were analyzed measure CSF GABA using a variety of techniques but, in triplicate. The samples were incubated at 4°C for 5 min since some investigators have been unable to detect and the reaction terminated by centrifugation at 48,000 g measurable quantities of GABA in this fluid, the issue of for 10min. The resultant pellet was rinsed rapidly and CSF GABA remains in doubt (GLAESER& HARE. 1975; superiicially with 15 ml ice-cold H 2 0 . and bound radioacACHAR et a/., 1976: PERRY & HANSEN.1976; GROSSMAN tivity was extracted into 1 ml of Protosol (New England e l a/.. 1976; ENNAet a/.. 1977b; Htilzlxca et a/., 1977). Nuclear, Boston, MA), lOml of toluene phosphor were In the present study, GABA levels were estimated on the added and radioactivity assayed by liquid scintillation same human CSF samples using different analytical tech- spectrometry (Packard Tricarb Model 3385 or 3375 Packniques in an attempt to demonstrate conclusively. and to ard Instrument Co., Downers Grove, IL). Standard curves quantitate accurately, GABA in CSF. for displacement of C3H]-GABA by nonradioactive GABA were determined for each experiment using 250 pl of H 2 0 containing various concentrations of unlabeled GABA in MATERIALS AND METHODS place of the CSF. Using these volumes. this assay can For this study. CSF was obtained from 24 patients hav- readily detect as little as IOpmol of GABA in the incubaing various CNS disorders. One patient had dystonia mus- tion medium (ENNAet a/., 1977b). Other qualitative proculorum deformans and 5 patients had arteriovenous mal- cedures have indicated that the only substance in the CSF capable of displacing C3H]-GABA at these dilutions is formations without seizures. Eighteen patients had intractGABA itself (ENNAet a/., 1977b). able epilepsy and were chronically maintained on antifonThe GABA content in the CSF samples was also detervulsant medication. All patients were given low mono& HARE. amine diets during the 2 weeks prior to CSF sampling. mined using ion exchange-fluorometry (GLAESER 1975). For this procedure, the CSF samples were thawed. Patients were restricted to absolute bedrest and oral intake then deproteinized by adding one-third volume of 20% was avoided for 18 h prior to lumbar puncture. Lumbar punctures were performed at 9a.m. in standard fashion aqueous salicylic acid (BLOCKet a/., 1966) and 500 p1 porwith the patient in the lateral decubitus position. In all tions of the deproteinized supernatants were analyzed for cases the same CSF fraction was taken from each patient their GABA content. For the analysis, GABA is automatifor analysis. After collection, CSF samples were immedi- cally separated from interfering components by high resoately placed on ice. divided into two portions and stored lution ion-exchange liquid chromatography followed by at - 7 0 C within 30min. The GABA content of each CSF detection in the flow-stream after reaction with o-phthalalsample was determined using both the radioreceptor assay dehyde (GLAFSER & HARE,1975). During the analyses for this study, a fluorocolorimeter (American Instrument Comof ENNAet a/. (19776) and the ion exchange-fluorometric pany, Silver Spring, MD, Model No. 7440) was utilized method of GLAESER& HARE(1975). With the radioreceptor assay, CSF GABA measurement as the detector resulting in a 1O.fold increase in sensitivity & HARE.1975). is based upon the principle that the amount of C3H]GABA over that previously described (GLAESER bound to rat brain synaptic membranes is inversely related Using this fluorocolorimeter, this method has a lower limit to the amount of unlabeled GABA present in the incubaof sensitivity of approximately 5pmol of GABA (Fig. I). 29 1
292
Short communication 10 plcomoles GABA
Blank analysis
50 picomoles GABA
100 picomoles GABA
I
!
0 c 0
I
0
e -
u-
iJJJLJi 80
90
100
80
Time,
100
90
80
100
90
80
90
loo
mln
FIG.1. Current chromatograms of prepared samples containing no, 10, 50 and 100 pmol GABA used for calibration of ion exchange-fluorometric method (GLAESER 8i HARE,1975). Peaks indicate assay sensitivity of approx 5- 10 pmol/ml GABA. RESULTS AND DISCUSSION The CSF GABA content in all 24 patients was 126 f 13 pmol/ml (s.E.M.) using the radioreceptor assay, and 157 17pmol/ml using the ion exchange-fluorometric procedure. The correlation between the GABA con-
centrations determined by these two different methods for the same CSF samples was highly significant ( r = 0.85; P < 0.001)(Fig. 2). In addition, the mean CSF GABA levels for the I8 medicated seizure patients were 109 f 13 pmol/ml with the radioreceptor assay and 142 18 pmol/ml with the fluorometric assay, whereas the
300 -
”
*
z ,0200 2
/
:
linear
represson r = 085
m= 0.91 n = 24
*;
0
-
0
picomoles/m?00 eachonge-fluoromelric method
loo
Ion
300
FIG.2. Comparison graph of GABA determination in CSF samples obtained from 24 patients employing radioreceptor assay (ordinate) and ion exchange-fluorometric method (abscissa). Highly significant agreement (P < 0.001) of these two methods is indicated by correlation coefficient (r) of 0.85 and plot slope (m)of 0.91.
Short communication mean CSF GABA levels for the 6 unmedicated. seizure-free patients were 177 f 30 pmol/ml and 202 & 44 pmol/ml, with the two procedures respectively. The average difference between the individual samples using the two methods of analysis was 35 _+ 6% (s.E.M.)with the greatest differences observed at the extreme limits of sensitivity for the procedures. In general, the values obtained with the radioreceptor assay were lower than those round using ion exchange- Ruorometry. These findings suggest that these two analytical techniques are measuring the same entity in the CSF. Since these two procedures are fundamentally different and since previous quantitative and qualitative studies have indicated that each technique is specific for GABA. it would appear that GABA is present in human CSF in picomole quantities. Further evidence that the values reported here accurately reflect human CSF GABA levels is provided by the finding that there is a highly significant correlation betwcen CSF GABA measured using the radioreceptor assay procedure and gas chromatography mass spectrometry (GC/MS) (ENNAet a/., 1977a). In addition, others have reported finding similar levels of CSF GABA in man using GC/MS (GROSSMAN et a/., 1976; HUIZINCA et al., 1977). Thus i t would appear that the analytical methods described in this communication have the requisite specificity and sensitivity to monitor GABA in human CSF.
293 REFERENCES
ACHAR V. S.. WELSH K., CHABl E.. BARTOSH K. & MEYER J. S. (1976) Neurology 26, 777-780. BIRDE. D. & IVERSEN L. L. (1974) Brain 97,457-572. BLOCKW. D., MARKOVS M. & STEELE B. (1966) Proc. SOC. exp. Biol. Med. 122, 1089-1091. BLOOMF. & IVERSENL. L. (1971) Nature, Lond. 229, 629430. BURYAKOVA A. V., CANDM. S. & SYnNsKy I. A. (1975) Arch. Neurol. 32, 28-31. CURZOh. G., GUMPERT J. & SHARP D. (1972) J . Neurol. Neurosurg. Psychiat. 35, 514-519. ENNAS., BIRDE., BENNETT J., BYLLWDD., YAMAMURAH., IVERSEN L. & SNYDER S. (1976) N . Engl. J . Med. 294, 1305-1 309. ENNAS . J. & SNYDER S. H. (1975) Brain Res. 100, 81-97. ENNAS. J. & SNYDERS. H. (1976) J . Neurochem. 26, 22 1-224. ENNAS. J.. STERN L.. WASTEKG. & YAMAMURAH. (1977a) Arch Neurol. in press. ENNAS. J.. WOODJ. H. & SNYDER S. H. (1977b) J . Neurochem. 28, 1121-1124. GLAESER B. S. & HARET. A. (1975) Biochem. Med. 12. 274282. GROSSMAN R., BEYERC., SHANNON G.. KELLYP. & HABER B. (1976) Proc. SOC. Neurosci. 2, 763 (abst.). A. W., MuSKItT F. A. J., M E U L ~ N Acknowledgements-Supported by USPHS grant HUIZIIGAJ. D., TEELKEN J. V. D. & WOLTHERS B. G. (1977) N. Engl. J . Med. MH-29739, grants from Merck Sharp and Dohme, Eli 2%. 692. Lilly. University of Texas General Research Support H. L. (1971) J . Neurol. Sci. 13, 277-279. Funds, PMA Foundation Research Starter Grant, and the KLAWANS W. E. 8~ V A N Huntington Chorea Foundation to S.J.E. and by USPHS LANCERD. H.. BROW G . L., BUNNEY KAMMEN D. P. (1975) N. Engl. J. Med. 293, 201. grant RR-5414 and the Foundation for Research in HerePERRYT. & HANSENS. (1976) J . Neurochem. 27. ditary Disease to T.A.H. 1537-1 538. T., HANSENS. & KLOSTERM. (1973) N. Engl. J . 'Division of Neurosurgery, J. H. WOOD' PERRY Med. 288, 337-342. Unioersity of Pennsyloania School B. S. GLAESER' of Medicine and s.J. ENNA' ROBERTS E. (1972) Neurosci. Res. f r o g . Bull. 10, 468-482. ROBERTS E., CHASET. N. & TOWERD. B. (1976) G A B A 'Department of Pharmacology, T. A. HARE^ in Nervous System Function. Raven Press. New York. Thomas Jefferson Unicersity School of Medicine, VAN GELDER N. M., SHERWIN A. L. & RASM~JSENT. (1972) Philadelphia. P A , U.S. A. ; Brain Res. 40. 385-393. 'Depart men t s of Pharmacolog y . N eurobiology WOODJ. H., GLAESER B. S.. HARET. A.. SODEJ.. BROOKS and Anatomy, B. R. & VAN BrrREN J. M. (1977~)J . Neurosurg. 47. Unioersity of T e x a s Medical School. 582-589. Houston, Texas. U.S.A. WOODJ. H.. ZIECLER M. G.. LAM C. R.. SHOULSDN I.. BROOKS B. R. & V A N BUREN J. M.(19776) Ann. Neurol. 1. 9 4 9 9 .
SHORT COMMUNICATION
Verification and quantification of GABA in human cerebrospinal fluid (Received 7 April 1977. Accepted 22 June 1977)
1976; ENNAet a/., 19776). tion medium (ENNA& SNYDER, MEASCREMEWof putative neurotransmitters and their For the assay, frozen rat brain synaptic membrane pellets metabolites in human cerebrospinal fluid (CSF) has pro1975) were homogenized in 0.05 M-Trisvided new insights into the neurochemical characteristics (ENNA& SNYDER, of CNS disorders (KLAWANS. 1971; CURZONet 01.. 1972; citrate buffer (pH 7.1 at 4°C) at a concentration of 1 mg protein/ml. Sufficient Triton X-100 was added to the memWOOD et a/., 1977a. 19776). Since y-aminobutyric acid brane suspension to yield a 0.05% (v/v) concentration of (GABA)is felt to be, quantitatively, one of the more important neurotransmitters in the mammalian CNS ( B ~ &M detergent and the suspension was incubated at 37°C for IVERSEN. 1971) and abnormalities in GABAergic transmis- 30 min. then centrifuged at 48.000 g for 10 min. The Triton X-100 supernatant was discarded and the pellet was resuser al.. sion have been implicated in epilepsy (VAN GELDER pended to 0.05 M-Tris-citrate and centrifuged at 48,000 g 1972), Huntington's disease (PERRY ~t a/.. 1973; BIRD & IVERSEN, 1974; ENNAet a/., 1976). cerebrovascular disease for 10 min to wash off the excess detergent. After repeating this washing procedure a second time, the pellet was resus(ACHAR et al., 1976). meningitis (BURVAKOVA et a/., 1975) and psychiatric illness (ROBERTS, 1972; ROBERTS er a/.. pended to 0.05 M-Tris-citrate and centrifuged at 48,000 g concentration of approx I mg protein/ml. One ml portions 1976; LANGER et a/., 1975). Methods to accurately determine the quantity of this amino acid in human CSF may of this membrane suspension were placed into 15 ml Soraid in the diagnosis and treatment of some CNS disease vall centrifuge tubes containing 250pl of untreated CSF. 750 p1 H 2 0 . and 8 m-C3H]GABA (New England Nuclear. states. Recently, several reports have appeared on attempts to Boston. MA, 36.73 Ci/mmol). All samples were analyzed measure CSF GABA using a variety of techniques but, in triplicate. The samples were incubated at 4°C for 5 min since some investigators have been unable to detect and the reaction terminated by centrifugation at 48,000 g measurable quantities of GABA in this fluid, the issue of for 10min. The resultant pellet was rinsed rapidly and CSF GABA remains in doubt (GLAESER& HARE. 1975; superiicially with 15 ml ice-cold H 2 0 . and bound radioacACHAR et a/., 1976: PERRY & HANSEN.1976; GROSSMAN tivity was extracted into 1 ml of Protosol (New England e l a/.. 1976; ENNAet a/.. 1977b; Htilzlxca et a/., 1977). Nuclear, Boston, MA), lOml of toluene phosphor were In the present study, GABA levels were estimated on the added and radioactivity assayed by liquid scintillation same human CSF samples using different analytical tech- spectrometry (Packard Tricarb Model 3385 or 3375 Packniques in an attempt to demonstrate conclusively. and to ard Instrument Co., Downers Grove, IL). Standard curves quantitate accurately, GABA in CSF. for displacement of C3H]-GABA by nonradioactive GABA were determined for each experiment using 250 pl of H 2 0 containing various concentrations of unlabeled GABA in MATERIALS AND METHODS place of the CSF. Using these volumes. this assay can For this study. CSF was obtained from 24 patients hav- readily detect as little as IOpmol of GABA in the incubaing various CNS disorders. One patient had dystonia mus- tion medium (ENNAet a/., 1977b). Other qualitative proculorum deformans and 5 patients had arteriovenous mal- cedures have indicated that the only substance in the CSF capable of displacing C3H]-GABA at these dilutions is formations without seizures. Eighteen patients had intractGABA itself (ENNAet a/., 1977b). able epilepsy and were chronically maintained on antifonThe GABA content in the CSF samples was also detervulsant medication. All patients were given low mono& HARE. amine diets during the 2 weeks prior to CSF sampling. mined using ion exchange-fluorometry (GLAESER 1975). For this procedure, the CSF samples were thawed. Patients were restricted to absolute bedrest and oral intake then deproteinized by adding one-third volume of 20% was avoided for 18 h prior to lumbar puncture. Lumbar punctures were performed at 9a.m. in standard fashion aqueous salicylic acid (BLOCKet a/., 1966) and 500 p1 porwith the patient in the lateral decubitus position. In all tions of the deproteinized supernatants were analyzed for cases the same CSF fraction was taken from each patient their GABA content. For the analysis, GABA is automatifor analysis. After collection, CSF samples were immedi- cally separated from interfering components by high resoately placed on ice. divided into two portions and stored lution ion-exchange liquid chromatography followed by at - 7 0 C within 30min. The GABA content of each CSF detection in the flow-stream after reaction with o-phthalalsample was determined using both the radioreceptor assay dehyde (GLAFSER & HARE,1975). During the analyses for this study, a fluorocolorimeter (American Instrument Comof ENNAet a/. (19776) and the ion exchange-fluorometric pany, Silver Spring, MD, Model No. 7440) was utilized method of GLAESER& HARE(1975). With the radioreceptor assay, CSF GABA measurement as the detector resulting in a 1O.fold increase in sensitivity & HARE.1975). is based upon the principle that the amount of C3H]GABA over that previously described (GLAESER bound to rat brain synaptic membranes is inversely related Using this fluorocolorimeter, this method has a lower limit to the amount of unlabeled GABA present in the incubaof sensitivity of approximately 5pmol of GABA (Fig. I). 29 1
292
Short communication 10 plcomoles GABA
Blank analysis
50 picomoles GABA
100 picomoles GABA
I
!
0 c 0
I
0
e -
u-
iJJJLJi 80
90
100
80
Time,
100
90
80
100
90
80
90
loo
mln
FIG.1. Current chromatograms of prepared samples containing no, 10, 50 and 100 pmol GABA used for calibration of ion exchange-fluorometric method (GLAESER 8i HARE,1975). Peaks indicate assay sensitivity of approx 5- 10 pmol/ml GABA. RESULTS AND DISCUSSION The CSF GABA content in all 24 patients was 126 f 13 pmol/ml (s.E.M.) using the radioreceptor assay, and 157 17pmol/ml using the ion exchange-fluorometric procedure. The correlation between the GABA con-
centrations determined by these two different methods for the same CSF samples was highly significant ( r = 0.85; P < 0.001)(Fig. 2). In addition, the mean CSF GABA levels for the I8 medicated seizure patients were 109 f 13 pmol/ml with the radioreceptor assay and 142 18 pmol/ml with the fluorometric assay, whereas the
300 -
”
*
z ,0200 2
/
:
linear
represson r = 085
m= 0.91 n = 24
*;
0
-
0
picomoles/m?00 eachonge-fluoromelric method
loo
Ion
300
FIG.2. Comparison graph of GABA determination in CSF samples obtained from 24 patients employing radioreceptor assay (ordinate) and ion exchange-fluorometric method (abscissa). Highly significant agreement (P < 0.001) of these two methods is indicated by correlation coefficient (r) of 0.85 and plot slope (m)of 0.91.
Short communication mean CSF GABA levels for the 6 unmedicated. seizure-free patients were 177 f 30 pmol/ml and 202 & 44 pmol/ml, with the two procedures respectively. The average difference between the individual samples using the two methods of analysis was 35 _+ 6% (s.E.M.)with the greatest differences observed at the extreme limits of sensitivity for the procedures. In general, the values obtained with the radioreceptor assay were lower than those round using ion exchange- Ruorometry. These findings suggest that these two analytical techniques are measuring the same entity in the CSF. Since these two procedures are fundamentally different and since previous quantitative and qualitative studies have indicated that each technique is specific for GABA. it would appear that GABA is present in human CSF in picomole quantities. Further evidence that the values reported here accurately reflect human CSF GABA levels is provided by the finding that there is a highly significant correlation betwcen CSF GABA measured using the radioreceptor assay procedure and gas chromatography mass spectrometry (GC/MS) (ENNAet a/., 1977a). In addition, others have reported finding similar levels of CSF GABA in man using GC/MS (GROSSMAN et a/., 1976; HUIZINCA et al., 1977). Thus i t would appear that the analytical methods described in this communication have the requisite specificity and sensitivity to monitor GABA in human CSF.
293 REFERENCES
ACHAR V. S.. WELSH K., CHABl E.. BARTOSH K. & MEYER J. S. (1976) Neurology 26, 777-780. BIRDE. D. & IVERSEN L. L. (1974) Brain 97,457-572. BLOCKW. D., MARKOVS M. & STEELE B. (1966) Proc. SOC. exp. Biol. Med. 122, 1089-1091. BLOOMF. & IVERSENL. L. (1971) Nature, Lond. 229, 629430. BURYAKOVA A. V., CANDM. S. & SYnNsKy I. A. (1975) Arch. Neurol. 32, 28-31. CURZOh. G., GUMPERT J. & SHARP D. (1972) J . Neurol. Neurosurg. Psychiat. 35, 514-519. ENNAS., BIRDE., BENNETT J., BYLLWDD., YAMAMURAH., IVERSEN L. & SNYDER S. (1976) N . Engl. J . Med. 294, 1305-1 309. ENNAS . J. & SNYDER S. H. (1975) Brain Res. 100, 81-97. ENNAS. J. & SNYDERS. H. (1976) J . Neurochem. 26, 22 1-224. ENNAS. J.. STERN L.. WASTEKG. & YAMAMURAH. (1977a) Arch Neurol. in press. ENNAS. J.. WOODJ. H. & SNYDER S. H. (1977b) J . Neurochem. 28, 1121-1124. GLAESER B. S. & HARET. A. (1975) Biochem. Med. 12. 274282. GROSSMAN R., BEYERC., SHANNON G.. KELLYP. & HABER B. (1976) Proc. SOC. Neurosci. 2, 763 (abst.). A. W., MuSKItT F. A. J., M E U L ~ N Acknowledgements-Supported by USPHS grant HUIZIIGAJ. D., TEELKEN J. V. D. & WOLTHERS B. G. (1977) N. Engl. J . Med. MH-29739, grants from Merck Sharp and Dohme, Eli 2%. 692. Lilly. University of Texas General Research Support H. L. (1971) J . Neurol. Sci. 13, 277-279. Funds, PMA Foundation Research Starter Grant, and the KLAWANS W. E. 8~ V A N Huntington Chorea Foundation to S.J.E. and by USPHS LANCERD. H.. BROW G . L., BUNNEY KAMMEN D. P. (1975) N. Engl. J. Med. 293, 201. grant RR-5414 and the Foundation for Research in HerePERRYT. & HANSENS. (1976) J . Neurochem. 27. ditary Disease to T.A.H. 1537-1 538. T., HANSENS. & KLOSTERM. (1973) N. Engl. J . 'Division of Neurosurgery, J. H. WOOD' PERRY Med. 288, 337-342. Unioersity of Pennsyloania School B. S. GLAESER' of Medicine and s.J. ENNA' ROBERTS E. (1972) Neurosci. Res. f r o g . Bull. 10, 468-482. ROBERTS E., CHASET. N. & TOWERD. B. (1976) G A B A 'Department of Pharmacology, T. A. HARE^ in Nervous System Function. Raven Press. New York. Thomas Jefferson Unicersity School of Medicine, VAN GELDER N. M., SHERWIN A. L. & RASM~JSENT. (1972) Philadelphia. P A , U.S. A. ; Brain Res. 40. 385-393. 'Depart men t s of Pharmacolog y . N eurobiology WOODJ. H., GLAESER B. S.. HARET. A.. SODEJ.. BROOKS and Anatomy, B. R. & VAN BrrREN J. M. (1977~)J . Neurosurg. 47. Unioersity of T e x a s Medical School. 582-589. Houston, Texas. U.S.A. WOODJ. H.. ZIECLER M. G.. LAM C. R.. SHOULSDN I.. BROOKS B. R. & V A N BUREN J. M.(19776) Ann. Neurol. 1. 9 4 9 9 .
