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Determination of 2-pyrrolidone-5-carboxylic and α-ketoglutaramic acids in human cerebrospinal fluid by gas chromatography
ANALYTICAL
BIOCHEMISTRY
103,
118-126 (1980)
Determination of 2-Pyrrolidone-5-Carboxylic and a-Ketoglutaramic Acids in Human Cerebrospinal Fluid by Gas Chromatography’ ARTHUR J. L. COOPER,AMIYA K. DHAR, HENN KUTT, AND THOMAS E. DUFFY Departments
of Neurology
and Biochemistry, New York, New
Cornell University York 10021
Medical
College,
Received July 2, 1979 A gas chromatographic method is described for the quantitation of some substituted 2-pyrrolidones of biological interest. These compounds are converted to the trimethylsilylated derivatives with N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA). The derivatized pyrrolidones are well separated by gas chromatography, and flame ionization provides a sensitive technique for their detection. The method was applied to the simultaneous estimation of 2-pyrrolidone-S-carboxylic acid and 5-hydroxy-2-pyrrolidone-5carboxylic acid (the cyclic lactam and predominant form of cr-ketoglutaramic acid) in human cerebrospinal fluid. Both compounds were found to be increased in cerebrospinal fluid of patients with hepatic encephalopathy. It is suggested that the increases are secondary to elevated amino acids in cerebrospinal fluid and plasma.
The concentration of a-ketoglutaramic acid, the cY-keto acid analog of glutamine, is elevated in the cerebrospinal fluid (CSF)* of patients with hepatic encephalopathy (1,2) and appears to be directly related to the degree of neurological impairment (1,2). a-Ketoglutaramic acid probably arises via transamination of glutamine; glutamine transaminase (~-glutamine:2-oxo-acid aminotransferase, EC 2.6.1.15) activity has been detected in human brain (3). a-Ketoglutaramate has previously been assayed by conversion to a-ketoglutarate in a reaction catalyzed by w-amidase (w-amidodicarboxylate amidohydrolase, EC 3.5.1.3), followed by the fluorometric determination
of cY-ketoglutarate (1,2,4). This method has several disadvantages. (i) o-Amidase is not available commercially and must be prepared from rat liver. (ii) cr-Ketoglutaramate exists overwhelmingly in a cyclic (i.e., enzymatitally unreactive) form (5-7) and the apparent K, of 2-3 mM is relatively high; therefore incubation times of up to 2 h are necessary to insure complete conversion of a-ketoglutaramate to a-ketoglutarate. In order to make the determination of a-ketoglutaramic acid in human CSF more amenable to routine clinical analysis, a gas chromatographic (gc) procedure has been developed. The gc procedure also measures 2-pyrrolidone-5carboxylic acid,3 an important metabolite of the y-glutamyl cycle (8). The present findings confirm that a-ketoglutaramic acid is elevated in the CSF of patients with hepatic encephalopathy and further show that 2-pyrrolidone-5-carboxylic acid is also substantially elevated in the CSF from this group of patients. Thus, the simul-
i This work was supported by U. S. Public Health Service Grant AM16739. A.J.L.C. is a recipient of a United States Public Health Service Career Development Award NS00343. T.E.D. is an Established Investigator of the American Heart Association. Part of this work was presented at the 8th Annual Meeting of the American Society for Neurochemistry, Denver, Colo., March 1977. 2 Abbreviations used: CSF, cerebrospinal fluid; gc, gas chromatography; BSTFA, N,O-bis(trimethylsilyl)ttifluoroacetamide; TMS, trimethylsilyl. 0003-2697/80/050118-09$02.00/O Copyri~t All rights
0 1980 by Academic Press. Inc. of reproduction in any form reserved.
3 Synonyms: pyroglutamic acid, 5-oxoproline, 2-pyrrolidone-carboxylic acid. 118
5-0x0-
GAS CHROMATOGRAPHY
taneous estimation of 2-pyrrolidone-S-carboxylic acid and a-ketoglutaramic acid in CSF offers a reliable diagnostic test for neurological dysfunction secondary to liver disease. MATERIALS
N,O-bis(Trimethylsilyl)-trifluoroacetamide (BSTFA) and acetonitrile (silylating grade) were obtained from Pierce Chemical Company, Rockford, Illinois. The barium salts of cr-ketoglutaramic and N-methyl-a-ketoglutaramic acids were prepared essentially as described by Meister (5). Enzymatic and gas chromatographic analyses (below) revealed less than 1% contamination of these compounds with 2-pyrrolidone-5-carboxylic acid. Barium a-keto-[U-14C]glutaramate (3.6 &i/pmol) was prepared as described by Duffy et al. (4). L-2-Pyrrolidone-5carboxylate was obtained from Sigma Chemical Company, St. Louis, Missouri. L-[U-14C]-2-Pyrrolidone5carboxylic acid (250 &i/pmol) was obtained from New England Nuclear, Boston, Massachusetts. Dowex-2 (AG 2-X8, 200400 mesh, chloride form) was purchased from Bio-Rad Laboratories, Rockville Centre, N.Y. L-N-Methyl-2-pyrrolidone-S-carboxylic, DL-pyrrolidone-5-methyl-5-carboxylic, and DL-2-pyrrolidone-4-carboxylic acids and or-5-carboxymethyl-2-pyn-olidone were gifts of Dr. Owen Griffith (Biochemistry Department, Cornell University Medical College). They were prepared from the corresponding amino acids (9) by a modification of the procedure of Wilson and Cannan (10). DL-Hydroxy-2-pyrrolidone, prepared according to the method of de Mayo and Reid (1 l), was a gift of Dr. de Mayo (Department of Chemistry, University of Western Ontario). The aromatic benzaldehydes and 2-pyrrolidone were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. METHODS
Standardization ketoglutaramate
of solutions containing and 2-pyrrolidone-j-car-
OF 2-PYRROLIDONES
119
boxylate. Barium a-ketoglutaramate was initially standardized by conversion to (Yketoglutarate with w-amidase, followed by the fluorometric determination of cY-ketoglutarate with glutamate dehydrogenase (4). A more convenient calorimetric assay was also devised. Aliquots (10 ~1) were heated with 0.05 ml of 0.1% 2,4-dinitrophenylhyrazine in 2 N HCl at 100°C for 30 min; 0.94 ml of 1 N NaOH was then added and absorbance at 430 nm was read against a blank lacking (Yketoglutaramate. The extinction coefficient under these conditions is 10.6 x 103.4 2-Pyrrolidone-5-carboxylic acid was converted to glutamic acid by heating at 100°C in 1 N HClfor 1 h(lO). Analiquot(lOpl)was assayed spectrophotometrically for glutamate according to the method of Lowry and Passonneau (13). Determination of retention times of the TM.3 derivatives of various pyrrolidones. About 10 pg of each compound was dissolved in 100 ~1 of water, added to a ReactiVial (Pierce, Rockford, Ill.), and evaporated to dryness under a stream of dry nitrogen (barium salts were converted to the free acids by passage through small Dowex 50 H+ columns). To the dried residue was added 50 ~1 of BSTFA; the vial was then sealed and heated at 60°C for 5 min, and the supernate was subjected to gc analysis as described below. This procedure was sufficient to completely silylate each of the pyrrolidones that was tested, except ar-ketoglutaramic acid (5-hydroxy-2-pyrrolidone-5-carboxylic acid). Complete silylation of a-ketoglutaramic acid required 1 h of heating; apparently, ad-
4 Since a-ketoglutaramic acid exists mostly in a cyclized form, it does not readily form a hydrazone (5). However, prolonged heating of cY-ketoglutaramic acid in acid yields a-ketoglutaric acid and succinic semialdehyde (12). Paper chromatographic analysis of the heated cr-ketoglutaramic acid-2,4-dinitrophenylhydrazone solution revealed the presence of two hydrazones with R, values identical to those of the dinitrophenylhydrazones of a-ketoglutaric acid and succinic semialdehyde; a third, unidentified spot was CY- probably due to the 2,4-dinitrophenylhydrazone of a-ketoglutaramic acid.
120
COOPER ET AL.
dition of two TMS groups is facile, but addition of the third TMS group is sterically hindered. Gas chromatography and mass spectral analysis. Gas chromatography was carried
out using columns (185 x 0.2 cm) of 3% SE30 coated onto Gas-Chrom Q in a HewlettPackard 5830A gas chromatograph. Samples (2-5 ~1) were injected. Helium was the carrier gas; the flow rate was 30 mumin. The temperatures of the injection port, oven, and flame ionization detector were 180, 130 (isothermal), and 280°C respectively. Gas chromatographic-mass spectrometric analysis of the TMS derivative of cy-ketoglutaramic acid was performed essentially as described above for gas chromatographic analysis except that the elution profile was obtained by subjecting the effluent to mass spectrometric analysis every 8.4 s and plotting the ion current against time. Spectra were determined by electron impact and isobutane chemical ionization techniques (14) using a DuPont 21-492 mass spectrometer.5 Preparation of cerebrospinal fluid for gas chromatography. Cerebrospinal fluid (CSF)
was obtained from patients undergoing routine lumbar puncture for diagnostic or therapeutic purposes; the fluids were stored at -80°C. To 0.5 ml of spinal fluid was added 40 nmol of DL-2-pyrrolidone-4-carboxylic acid as internal standard. The spinal fluid was then added directly to the top of a Dowex 2 column (chloride form; 4 x 0.5 cm). The column was eluted with 10 ml of water followed by 3.0 ml of 0.1 N HCl. The fraction eluting between 1.0 and 3.0 ml of HCl was collected in a 12-ml conical centrifuge tube; 2 ~1 of 50 mM barium chloride was added and the mixture was lyophilized to dryness. To the residue was added 25 ~1 of ethanol and the mixture was agitated for 2 min. After centrifugation at 2000g for 10 min the clear 5 The mass spectrographic studies were supported in part by National Institutes of Health Grant RR-862-01 from the Division of Research Facilities and Resources and were carried out at the Rockefeller University.
supernate was removed, transferred to a Reacti-Vial, and evaporated to dryness under a stream of dry nitrogen. The residue was resuspended in.100 ~1 of methanol and again evaporated to dryness. The residue was taken up in 10 ~1 of acetonitrile and 50 ~1 of BSTFA was added. The mixture was then heated at 80°C for 2 h and a 5+1 sample was analyzed by gas chromatography as described above. Standards contained 40 nmol of 2-pyrrolidone-4-carboxylate and varying amounts of 2-pyrrolidone-5-carboxylate and a+ketoglutaramate in 0.5 ml of mock human CSF (15). The standards were carried through the purification procedure. Recoveries of labeled 2-pyrrolidone-5-carboxylate (6000 dpm) and a-ketoglutaramate (3500 dpm) that were added to 0.5 ml of mock CSF and carried through the purification procedure were consistently greater than 95% after the lyophilization step, and greater than 80% after evaporation within the Reacti-Vial. The peak height ratios of a-ketoglutaramic acid/internal standard and 2-pyrrolidone-5carboxylic acid/internal standard were linear over the range of 5-140 nmol for a-ketoglutaramic acid and 5-50 nmol for 2-pyrrolidone-5-carboxylic acid (Fig. 1). I
I
I
I
I
0
FIG. 1. Standard curves for the quantitation of 2-pyrrolidone-Scarboxylic acid (PCA) and cy-ketoglutaramic acid ((Y-KGM). To O&ml samples of mock cerebrospinal fluid were added 40 nmol of2-pyrrolidone4-carboxylic acid as internal standard and varying amounts of PCA and o-KGM. The standard solutions were taken through the purification procedure and the peak heights of the TMS derivatives were compared.
GAS CHROMATOGRAPHY
Potential sources of error. Prolonged exposure of 2-pyrrolidone-5carboxylic acid and a-ketoglutaramic acid to strong acid yields glutamic acid (10) and a mixture of a-ketoglutaric acid and succinic semialdehyde (12), respectively. Nevertheless, analysis of standards (see Ref. (12)), after extraction in ethanol, revealed less than 1% conversion of 2-pyrrolidone-5carboxylate to glutamate and less than 1% conversion of cr-ketoglutaramate to a-ketoglutarate and to succinate semialdehyde. Glutamic acid and glutamine yield, in part, the TMS derivative of 2-pyrrolidone-5-carboxylic acid when heated with BSTFA. However, these amino acids are removed prior to silylation in the first milliliter of the HCl wash of the Dowex 2 column. Although 2-pyrrolidone-5carboxylic acid can theoretically arise via cyclization reactions of TABLE
121
glutamine and glutamic acid on prolonged storage, this does not appear to be a problem in the present work. We have stored 14Clabeled glutamate and 14C-labeled glutamine for several years at -20°C without appreciable formation of 2-pyrrolidone-5-carboxylic acid (the CSF samples were stored at -80°C). RESULTS Separation of various 2-pyrrolidone analogs by gas chromatography. Table 1 lists
the retention times of TMS derivatives of various 2-pyrrolidone analogs on a 3% SE-30 column. A satisfactory separation of all the derivatized 2-pyrrolidones was obtained. In particular, the TMS derivative of 5-hydroxy2-pyrrolidone-5-carboxylic acid is well separated from that of 2-pyrrolidone-5-carboxylic acid. Mass spectra of the TMS derivative of a-ketoglutaramic acid. Mass spectra ob-
1
RETENTION TIMESOFTHE TMS DERIVATIVES 2-PYRROLIDONE ANALOGSON 3% SE-30"
Compound
OF 2-PYRROLIDONES
OF
Retention time (min)
With solvent front 2-Pyrrolidone DL-2-Pyrrolidone-5-methyl-5-car2.1 boxylic acid DL-5-Hydroxy-2-pyrrofidone 3.5 L-N-Methyl-2-pyrrolidone-5-carboxylic acid 6.1 L-2-Pyrrolidone-5-carboxylic acid 6.6 DL-2-Pyrrolidone-%carboxylic acid 8.7 DL-5-Carboxymethyl-2-pyrrolidone 10.8 Dr-N-Methyl-5-hydroxy-2-pyrrolidone-5-carboxylic acidb 11.3 DL-5-Hydroxy-2-pyrrolidone-5carboxylic acidc*d 11.8 (L For conditions, see text. * The cyclic lactam form of N-methyl-cy-ketoglutaramic acid. c The cyclic lactam form of cr-ketoglutaramic acid. d The retention time refers to the tris(TMS) derivative. If silylation is not complete, a peak at 10.6 min is obtained. This peak is ascribed to the bis(TMS) derivative since on prolonged heating in BSTFA this peak disappears with the concomitant appearance of a peak at 11.8 min.
tained at the exact retention time of a TMSa-ketoglutaramic acid standard on 3% SE-30 are reproduced in Fig. 2. Both the chemical ionization and electron impact spectra are consistent with the structure of the TMS derivative of a-ketoglutaramic acid as being tris(TMS)-5-hydroxy-2-pyrrolidone-S-carboxylic acid. Gas chromatographic analyses of wketoglutaramic acid and 2-pyrrolidone-Scarboxylic acid in human cerebrospinal fluid. Il-
lustrated in Fig. 3 are the gas chromatographic elution profiles of (A) a standard containing 2-pyrrolidone-5-carboxylic acid, internal standard, and m-ketoglutaramic acid, (B) a purified CSF sample from a control subject with normal liver function, and (C) a purified CSF sample from a patient with hepatic encephalopathy. Both CSF samples contained appreciable amounts of 2-pyrrolidone5-carboxylic acid although the concentration of this acid was very much greater in the CSF from the patient with hepatic encephalopathy. a-Ketoglutaramic acid, on the other hand, was pronounced in the CSF of the
122
COOPER ET AL.
loo
f
12711 M+-90 IHOSiMe,)
11721
1731 +SilCH31,
80
M+-llIlCO,SiMe,)
11471 ICHd,Si-0-SPlCH&
A
O@OSiMe, I I1561 H
I
13461 M+-15 ICHJ
x 20.c, 2
I..,,.;.. 3
.*, L , ml1 M+-I17 ICOZSIM~XI
I., 13621
11721
13461 M+-151CHSI
20 A Oo
50
I II loo
I 150
200
FIG. 2. Electron impact (A) and isobutane chemical derivative of cr-ketoglutaramic acid. For details, see are depicted as being derived from the silylated lactam, 5-carboxylic acid. Abbreviations used: M+, molecular
250
I 300
I, 350
400
450
ionization (B) mass spectra of the trimethylsilyl text. Note that the structures of the fragments i.e., tris(trimethylsilyl)-S-hydroxy-2-pyrrolidoneion; Me,, trimethyl.
patient with hepatic encephalopathy, but in the CSF. The data agree closely with was barely detectable in the CSF of the those published previously in which a-ketoglutaramic acid was determined by enzymatic control subject. Figure 4 shows the concentrations of (Y- analysis (1,2). The values obtained by the gc method were generally in good agreeketoglutaramic acid and 2-pyrrolidone-5carboxylic acid in the CSF of 11 control ment with the values obtained by enzymatic subjects who had a variety of neurological analysis; only 3 of the 36 values reported disorders but who had normal liver function in Fig. 4 were appreciably different from tests, and of 25 patients with liver disease. the values obtained by enzymatic analysis.6 Patients with liver disease were further 6 We have recently reported on a spot test for the classified as having no neurological dysfuncsemiquantitative estimation of a-ketoglutaramic acid tion or as having moderate-to-severe en- in human CSF (16). We now report an improved test cephalopathy. A good correlation was noted that does not require a chromatography step and can between the degree of encephalopathy and be used to quickly screen for the presence of abnormal the concentration of a-ketoglutaramic acid a-ketoglutaramic acid in CSF: To 10 ~1 of CSF is added
GAS CHROMATOGRAPHY
OF 2-PYRROLIDONES
123
PCA IS ‘-KGM
I
I
I PCA I
k
l KGM
1 5
:! 0
A
15 0
5
IO
Time
(mln)
1
I:
FIG. 3. Gas chromatographic analyses of 2-pyrrolidone-5carboxylic acid (PCA) and a-ketoglutaramic acid (c+KGM) in 0.5 ml of human cerebrospinal fluid. (A) A standard solution containing PCA (18.2 nmol) and a-KGM (82 nmol) in 0.5 ml of mock human cerebrospinal fluid. (B) Cerebrospinal fluid from a control subject with normal liver function. (C) Cerebrospinal fluid from a subject with hepatic encephalopathy. Each sample contained 40 nmol of internal standard (IS). Isolation and silylation were carried out as described in the text.
The concentration of 2-pyrrolidone-5-carboxylic acid in human CSF also correlated closely with the degree of hepatic encephalopathy. With the exception of two false positives (one from a patient with a duodenal ulcer and a history of gastrointestinal bleeding and another from a patient with a meningioma) the diagnostic correlation of 2-pyrrolidone-5carboxylic acid was comparable to that of a-ketoglutaramic acid. DISCUSSION
The present data (Fig. 4) confirm the previous findings (1,2,4) that cr-ketoglutaramic acid is elevated in the CSF of patients with 2 ~1 of 20% sulfosalicylic acid and the precipitate is removed by centrifugation at 5OOOgfor 2 min. The clear supernate is spotted onto Whatman No. 1 filter paper, 2 ~1 at a time. The paper is sprayed with 1% vanillin (4hydroxy-3-methoxybenzaldehyde) in 1 N HCl in 50% ethanol and allowed to air-dry for 1 h. The paper is then heated at 100°C for 1 min and sprayed with 0.5 N NaOH in 50% ethanol. The presence of a-ketoglutaramic acid is indicated by a red ring around the periphery of the applied spot. The limit of detection is 50 pmol. Semiquantitative analysis may be made by comparing the intensity of the red color against known standards in mock human CSF-sulfosalicylic acid.
liver disease and that the concentration correlates with the degree of encephalopathy. However, the gc method, described above, is more convenient than the enzymatic assay used previously (1,2,4) and promises to be more amenable to clinical work. A new finding is that 2-pyrrolidone-5carboxylic acid levels are also increased in the CSF of patients with moderate-to-severe hepatic encephalopathy (Fig. 4). Gas chromatographic techniques for the estimation of 2-pyrrolidone-5carboxylic acid have been published previously (17- 19). Thus, Polgar and Meister have followed the o-glutamic acid cyclotransferase reaction by converting the enzymatically generated 2-pyrrolidone-5-carboxylic acid to the methyl ester and determining the derivative by gas chromatography (17). Jellum and co-workers have also measured 2-pyrrolidone-5-carboxyhc acid as the methyl ester by a gas chromatographic procedure (18) and used this technique to determine the 2-pyrrolidone-5carboxylic acid content of the urine of a patient with pyroglutamic aciduria (18,20). Finally, Wilk and Orlowski developed a gas chromatography procedure for the determination of 2-pyrrolidone-5-carboxylic acid
124
COOPER ET AL. 300
a-KETOGLUTARAMATE
3oo
2-PYRROLIDONE+CARBOXYLATE
.
200
Controls
Liver dieeose “0 encephobpothy
Hepotlc encepholopothy
Controls
Liver diseose
Hepotlt encepholopothy
encep;:topothy
FIG. 4. cY-Ketoglutaramic acid and 2-pyrrolidone-S-carboxylic acid in human CSF. The two high values of 2-pvrrolidone-S-carboxvlic acid were from the CSF of a patient with a duodenal ulcer (150 pM) and from a-patient with a meningioma (125 PM).
as the N-acyl ester formed in a reaction with pentafluoropropanol and pentafluoropropionic anhydride (19); the technique was used to determine the 2-pyrrolidone-S-carboxylic acid content of various mouse organs and of human urine and CSF. The present technique for the gc determination of 2-pyrrolidone-5-carboxylic acid in human CSF is simpler and more sensitive than the previously published procedures (19,20). In the present procedure, 0.5-ml samples of CSF are used, but this amount is only necessary for the estimation of (Yketoglutaramic acid and the 2-pyrrolidoneS-carboxylic acid peak is often off-scale (Fig. 3). The present technique is adequate to measure 1.5 nmol of 2-pyrrolidone-5carboxylic acid in 100 ~1 of human CSF and has been used to analyze as little as 0.5 nmol in 25-~1 samples by reducing the volume of the silylating mixture from 60 to 20 ~1. Wilk and Orlowski (19) report a concentration of 2-pyrrolidone-5-carboxylic acid in human lumbar spinal fluid of 71 +- 13 (SE) PM (n = 29). However, no details were given regarding the medical status of the patients from whom CSF samples were obtained. In the present series, the “normal” concentration of 2-pyrrolidone-5-carboxylic
acid in the CSF was lo-35 PM (Fig. 4) (includes control subjects and patients with liver disease who did not exhibit neurological impairment). In patients with hepatic encephalopathy, the range was 42- 145 PM. The origin of 2-pyrrolidone-5-carboxylate in CSF is unknown. The concentration of 2-pyrrolidone-5-carboxylate in “normal” CSF, reported here, is comparable to that reported previously for normal plasma (21); therefore, it is conceivable that CSF 2-pyrrolidone-5-carboxylate arises from plasma by equilibration across the choroid plexus. Alternatively, pyrrolidone-5-carboxylate in CSF may arise from enzymatic reactions of the y-glutamyl cycle within the brain. Thus, Orlowski er al. have shown that the human brain contains appreciable y-glutamylcyclotransferase activity (22). Tate et al. have shown that four key enzymes (including y-glutamylcyclotransferase) of the y-glutamyl cycle are well represented in various regions of the rat brain but are especially rich in choroid plexus (23). These authors drew an analogy with the kidney and speculated that the y-glutamyl cycle may play a significant role in the transport of amino acids between blood and CSF (23). If this hypothesis were correct, 2-pyrrolidone-5-carboxylate would
GAS CHROMATOGRAPHY
be expected to accumulate in CSF during amino acid transport since the 2-pyrrolidone5carboxylate amidohydrolase (5oxoprolinase) reaction appears to be a rate-limiting step in the operation of the y-glutamyl cycle (cf. Ref. (24)). Indeed, Orlowski and Wilk have shown that 30 min after a single intraperitoneal injection of a mixture of amino acids into mice the concentration of 2-pyrrolidone-5carboxylate is significantly raised in the brain (24). Therefore, the increase in 2-pyrrolidone-5-carboxylate in the CSF of patients with hepatic encephalopathy (Fig. 4) may be due to an increase in brain yglutamylcyclotransferase activity secondary to the elevated CSF and plasma concentrations of amino acids that are typically observed in such patients (e.g., Refs. (25-27)). Amino acid stimulation of the y-glutamyl
cycle may even account for elevated %-pyrrolidone-5-carboxylate in the CSF of the patient with a duodenal ulcer (Fig. 4) since chronic gastrointestinal bleeding is known to raise plasma concentrations of amino acids (28). The increased concentration of ol-ketoglutaramic acid in the CSF of patients with hepatic encephalopathy may also be related to increased amino acid concentrations in plasma and CSF. Thus, we have previously proposed that an increase in certain brain amino acids, notably phenylalanine and methionine, would lead to an increase in their corresponding cr-keto acids via transamination with a-ketoglutaric acid (3). Increased cy-keto acid levels would, in turn, stimulate glutamine transaminase activity, the effect being to increase the concerted reaction:
cY-ketoglutaric acid + L-amino acid E L-glutamic a-keto acid + L-glutamine a-ketoglutaric
acid + L-glutamine
+ L-glutamic
1. Vergara, F., Plum, F., and Duffy, T. E. (1974) Science 183, 81-83. 2. Duffy, T. E., Vergara, F., and Plum, F. (1974) Publ.
Assoc.
Res.
New.
Men!.
Dis.
53,
39-52. 3. Cooper, A. J. L., and Gross, M. (1977) J. Neuruthem. 28, 771-778. 4. Duffy, T. E., Cooper, A. J. L., and Meister, A. (1974) J. Biol. Chem. 249, 7603-7606. 5. Meister, A. (1952) J. Biol. Chem. 200, 571-589. 6. Otani, T. T., and Meister, A. (1957)J. Biol. Chem. 224, 137- 148. 7. Hersh, L. B. (1972) Biochemisrry 10, 2884-2891. 8. Orlowski, M., and Meister, A. (1971) Proc. Nat. Acad. Sri. USA 67, 1248-1255. 9. Griffith, 0. W., and Meister, A. (1977) Proc. Nar. Acad.
Sci.
USA
74, 3330-3334.
acid + a-ketoglutaramic
acid acid
to Wilson, H., and Cannan, R. K. (1937) J.
Biol.
119, 309-331. de Mayo, P., and Reid, S. T. (1962) Chem. Ind., 1576- 1577. Duffy, T. E., Cooper, A. J. L., and Vergara, F. (1976) Bioorg. Chem. 5, 351-366. Lowry, 0. H., and Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, pp. l&t185, Academic Press, New York. Munson, M. S. B., and Field, F. H. (1966)J. Amer. Chem. Sot. 88, 2621-2630. Merlis, J. K. (1940) Amer. J. Physiol. 131, 67-72. Cooper, A. J. L. (1978) Anal. Biochem. 90, 444446. Polgar, P., and Meister, A. (1965) Anal. Biochem. 12, 338-343. Jellum, E., Kluge, T., Borresen, H. C., Stokke, O., and Eldjarn, L. (1970) Sand. J. Clin. Lab. Chem.
11.
12 . 13.
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acid + a-keto acid
+ L-amino acid + cu-ketoglutaramic
If this hypothesis is correct, then despite the fact that cu-ketoglutaric acid is a poor substrate for brain glutamine transaminase (3), net transamination between cr-ketoglutaric acid and glutamine will be promoted by elevated concentrations of certain amino acids.
125
OF 2-PYRROLIDONES
14. 15. 16. 17. 18.
Invest.
26, 327-335.
19. Wilk, S., and Orlowski, M. (1975) Anal. Biochem. 69, 100-113. 20. Eldjarn, L., Jellum, E., and Stoke, D. (1972) Ctin. Chim. Acta 40, 461-476. 21. Palekar, A. G., Tate, S. S., Sullivan, J. F., and Meister, A. (1975)Biochem. Med. 14, 339-345.
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COOPER ET AL.
22. Orlowski, M., Richman, P. G., and Meister, A. (1%9) Biochemistry 8, 1048-1055. 23. Tate, S. S., Ross, L. L., and Meister, A. (1973) Proc. Nat. Acad. Sci. USA 70, 221 l-2214. 24. Orlowski, M., and Wilk, S. (1975)&r. J. Biochem. 53, 581-590. 25. Record, C. O., Buxton, B., Chase, R. A., Curzon, G., Murray-Lyon, I. M., and Williams, R. (1976) Eur.
J. Clin.
Invest.
6, 387-394.
26. Fischer, J. E., Yoshimura, N., Aguirre, A., James, J. H., Cummings, M. G., Abel, R. M., and Deindoctfer, F. (1974)Amer. J. Surg. 127,40-47. 27. Van Sande, M., Mardens, Y., Adriaenssens, K., and Lowenthal, A. (1970) J. Neurochem. 17, 125- 135. 28. Ansley, J. D., Isaacs, J. D., Rikkers, L. F., Kutner, M. H., Nordlinger, B. M., and Rudman, D. (1978) Gastroenterology 75, 570-579.
BIOCHEMISTRY
103,
118-126 (1980)
Determination of 2-Pyrrolidone-5-Carboxylic and a-Ketoglutaramic Acids in Human Cerebrospinal Fluid by Gas Chromatography’ ARTHUR J. L. COOPER,AMIYA K. DHAR, HENN KUTT, AND THOMAS E. DUFFY Departments
of Neurology
and Biochemistry, New York, New
Cornell University York 10021
Medical
College,
Received July 2, 1979 A gas chromatographic method is described for the quantitation of some substituted 2-pyrrolidones of biological interest. These compounds are converted to the trimethylsilylated derivatives with N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA). The derivatized pyrrolidones are well separated by gas chromatography, and flame ionization provides a sensitive technique for their detection. The method was applied to the simultaneous estimation of 2-pyrrolidone-S-carboxylic acid and 5-hydroxy-2-pyrrolidone-5carboxylic acid (the cyclic lactam and predominant form of cr-ketoglutaramic acid) in human cerebrospinal fluid. Both compounds were found to be increased in cerebrospinal fluid of patients with hepatic encephalopathy. It is suggested that the increases are secondary to elevated amino acids in cerebrospinal fluid and plasma.
The concentration of a-ketoglutaramic acid, the cY-keto acid analog of glutamine, is elevated in the cerebrospinal fluid (CSF)* of patients with hepatic encephalopathy (1,2) and appears to be directly related to the degree of neurological impairment (1,2). a-Ketoglutaramic acid probably arises via transamination of glutamine; glutamine transaminase (~-glutamine:2-oxo-acid aminotransferase, EC 2.6.1.15) activity has been detected in human brain (3). a-Ketoglutaramate has previously been assayed by conversion to a-ketoglutarate in a reaction catalyzed by w-amidase (w-amidodicarboxylate amidohydrolase, EC 3.5.1.3), followed by the fluorometric determination
of cY-ketoglutarate (1,2,4). This method has several disadvantages. (i) o-Amidase is not available commercially and must be prepared from rat liver. (ii) cr-Ketoglutaramate exists overwhelmingly in a cyclic (i.e., enzymatitally unreactive) form (5-7) and the apparent K, of 2-3 mM is relatively high; therefore incubation times of up to 2 h are necessary to insure complete conversion of a-ketoglutaramate to a-ketoglutarate. In order to make the determination of a-ketoglutaramic acid in human CSF more amenable to routine clinical analysis, a gas chromatographic (gc) procedure has been developed. The gc procedure also measures 2-pyrrolidone-5carboxylic acid,3 an important metabolite of the y-glutamyl cycle (8). The present findings confirm that a-ketoglutaramic acid is elevated in the CSF of patients with hepatic encephalopathy and further show that 2-pyrrolidone-5-carboxylic acid is also substantially elevated in the CSF from this group of patients. Thus, the simul-
i This work was supported by U. S. Public Health Service Grant AM16739. A.J.L.C. is a recipient of a United States Public Health Service Career Development Award NS00343. T.E.D. is an Established Investigator of the American Heart Association. Part of this work was presented at the 8th Annual Meeting of the American Society for Neurochemistry, Denver, Colo., March 1977. 2 Abbreviations used: CSF, cerebrospinal fluid; gc, gas chromatography; BSTFA, N,O-bis(trimethylsilyl)ttifluoroacetamide; TMS, trimethylsilyl. 0003-2697/80/050118-09$02.00/O Copyri~t All rights
0 1980 by Academic Press. Inc. of reproduction in any form reserved.
3 Synonyms: pyroglutamic acid, 5-oxoproline, 2-pyrrolidone-carboxylic acid. 118
5-0x0-
GAS CHROMATOGRAPHY
taneous estimation of 2-pyrrolidone-S-carboxylic acid and a-ketoglutaramic acid in CSF offers a reliable diagnostic test for neurological dysfunction secondary to liver disease. MATERIALS
N,O-bis(Trimethylsilyl)-trifluoroacetamide (BSTFA) and acetonitrile (silylating grade) were obtained from Pierce Chemical Company, Rockford, Illinois. The barium salts of cr-ketoglutaramic and N-methyl-a-ketoglutaramic acids were prepared essentially as described by Meister (5). Enzymatic and gas chromatographic analyses (below) revealed less than 1% contamination of these compounds with 2-pyrrolidone-5-carboxylic acid. Barium a-keto-[U-14C]glutaramate (3.6 &i/pmol) was prepared as described by Duffy et al. (4). L-2-Pyrrolidone-5carboxylate was obtained from Sigma Chemical Company, St. Louis, Missouri. L-[U-14C]-2-Pyrrolidone5carboxylic acid (250 &i/pmol) was obtained from New England Nuclear, Boston, Massachusetts. Dowex-2 (AG 2-X8, 200400 mesh, chloride form) was purchased from Bio-Rad Laboratories, Rockville Centre, N.Y. L-N-Methyl-2-pyrrolidone-S-carboxylic, DL-pyrrolidone-5-methyl-5-carboxylic, and DL-2-pyrrolidone-4-carboxylic acids and or-5-carboxymethyl-2-pyn-olidone were gifts of Dr. Owen Griffith (Biochemistry Department, Cornell University Medical College). They were prepared from the corresponding amino acids (9) by a modification of the procedure of Wilson and Cannan (10). DL-Hydroxy-2-pyrrolidone, prepared according to the method of de Mayo and Reid (1 l), was a gift of Dr. de Mayo (Department of Chemistry, University of Western Ontario). The aromatic benzaldehydes and 2-pyrrolidone were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. METHODS
Standardization ketoglutaramate
of solutions containing and 2-pyrrolidone-j-car-
OF 2-PYRROLIDONES
119
boxylate. Barium a-ketoglutaramate was initially standardized by conversion to (Yketoglutarate with w-amidase, followed by the fluorometric determination of cY-ketoglutarate with glutamate dehydrogenase (4). A more convenient calorimetric assay was also devised. Aliquots (10 ~1) were heated with 0.05 ml of 0.1% 2,4-dinitrophenylhyrazine in 2 N HCl at 100°C for 30 min; 0.94 ml of 1 N NaOH was then added and absorbance at 430 nm was read against a blank lacking (Yketoglutaramate. The extinction coefficient under these conditions is 10.6 x 103.4 2-Pyrrolidone-5-carboxylic acid was converted to glutamic acid by heating at 100°C in 1 N HClfor 1 h(lO). Analiquot(lOpl)was assayed spectrophotometrically for glutamate according to the method of Lowry and Passonneau (13). Determination of retention times of the TM.3 derivatives of various pyrrolidones. About 10 pg of each compound was dissolved in 100 ~1 of water, added to a ReactiVial (Pierce, Rockford, Ill.), and evaporated to dryness under a stream of dry nitrogen (barium salts were converted to the free acids by passage through small Dowex 50 H+ columns). To the dried residue was added 50 ~1 of BSTFA; the vial was then sealed and heated at 60°C for 5 min, and the supernate was subjected to gc analysis as described below. This procedure was sufficient to completely silylate each of the pyrrolidones that was tested, except ar-ketoglutaramic acid (5-hydroxy-2-pyrrolidone-5-carboxylic acid). Complete silylation of a-ketoglutaramic acid required 1 h of heating; apparently, ad-
4 Since a-ketoglutaramic acid exists mostly in a cyclized form, it does not readily form a hydrazone (5). However, prolonged heating of cY-ketoglutaramic acid in acid yields a-ketoglutaric acid and succinic semialdehyde (12). Paper chromatographic analysis of the heated cr-ketoglutaramic acid-2,4-dinitrophenylhydrazone solution revealed the presence of two hydrazones with R, values identical to those of the dinitrophenylhydrazones of a-ketoglutaric acid and succinic semialdehyde; a third, unidentified spot was CY- probably due to the 2,4-dinitrophenylhydrazone of a-ketoglutaramic acid.
120
COOPER ET AL.
dition of two TMS groups is facile, but addition of the third TMS group is sterically hindered. Gas chromatography and mass spectral analysis. Gas chromatography was carried
out using columns (185 x 0.2 cm) of 3% SE30 coated onto Gas-Chrom Q in a HewlettPackard 5830A gas chromatograph. Samples (2-5 ~1) were injected. Helium was the carrier gas; the flow rate was 30 mumin. The temperatures of the injection port, oven, and flame ionization detector were 180, 130 (isothermal), and 280°C respectively. Gas chromatographic-mass spectrometric analysis of the TMS derivative of cy-ketoglutaramic acid was performed essentially as described above for gas chromatographic analysis except that the elution profile was obtained by subjecting the effluent to mass spectrometric analysis every 8.4 s and plotting the ion current against time. Spectra were determined by electron impact and isobutane chemical ionization techniques (14) using a DuPont 21-492 mass spectrometer.5 Preparation of cerebrospinal fluid for gas chromatography. Cerebrospinal fluid (CSF)
was obtained from patients undergoing routine lumbar puncture for diagnostic or therapeutic purposes; the fluids were stored at -80°C. To 0.5 ml of spinal fluid was added 40 nmol of DL-2-pyrrolidone-4-carboxylic acid as internal standard. The spinal fluid was then added directly to the top of a Dowex 2 column (chloride form; 4 x 0.5 cm). The column was eluted with 10 ml of water followed by 3.0 ml of 0.1 N HCl. The fraction eluting between 1.0 and 3.0 ml of HCl was collected in a 12-ml conical centrifuge tube; 2 ~1 of 50 mM barium chloride was added and the mixture was lyophilized to dryness. To the residue was added 25 ~1 of ethanol and the mixture was agitated for 2 min. After centrifugation at 2000g for 10 min the clear 5 The mass spectrographic studies were supported in part by National Institutes of Health Grant RR-862-01 from the Division of Research Facilities and Resources and were carried out at the Rockefeller University.
supernate was removed, transferred to a Reacti-Vial, and evaporated to dryness under a stream of dry nitrogen. The residue was resuspended in.100 ~1 of methanol and again evaporated to dryness. The residue was taken up in 10 ~1 of acetonitrile and 50 ~1 of BSTFA was added. The mixture was then heated at 80°C for 2 h and a 5+1 sample was analyzed by gas chromatography as described above. Standards contained 40 nmol of 2-pyrrolidone-4-carboxylate and varying amounts of 2-pyrrolidone-5-carboxylate and a+ketoglutaramate in 0.5 ml of mock human CSF (15). The standards were carried through the purification procedure. Recoveries of labeled 2-pyrrolidone-5-carboxylate (6000 dpm) and a-ketoglutaramate (3500 dpm) that were added to 0.5 ml of mock CSF and carried through the purification procedure were consistently greater than 95% after the lyophilization step, and greater than 80% after evaporation within the Reacti-Vial. The peak height ratios of a-ketoglutaramic acid/internal standard and 2-pyrrolidone-5carboxylic acid/internal standard were linear over the range of 5-140 nmol for a-ketoglutaramic acid and 5-50 nmol for 2-pyrrolidone-5-carboxylic acid (Fig. 1). I
I
I
I
I
0
FIG. 1. Standard curves for the quantitation of 2-pyrrolidone-Scarboxylic acid (PCA) and cy-ketoglutaramic acid ((Y-KGM). To O&ml samples of mock cerebrospinal fluid were added 40 nmol of2-pyrrolidone4-carboxylic acid as internal standard and varying amounts of PCA and o-KGM. The standard solutions were taken through the purification procedure and the peak heights of the TMS derivatives were compared.
GAS CHROMATOGRAPHY
Potential sources of error. Prolonged exposure of 2-pyrrolidone-5carboxylic acid and a-ketoglutaramic acid to strong acid yields glutamic acid (10) and a mixture of a-ketoglutaric acid and succinic semialdehyde (12), respectively. Nevertheless, analysis of standards (see Ref. (12)), after extraction in ethanol, revealed less than 1% conversion of 2-pyrrolidone-5carboxylate to glutamate and less than 1% conversion of cr-ketoglutaramate to a-ketoglutarate and to succinate semialdehyde. Glutamic acid and glutamine yield, in part, the TMS derivative of 2-pyrrolidone-5-carboxylic acid when heated with BSTFA. However, these amino acids are removed prior to silylation in the first milliliter of the HCl wash of the Dowex 2 column. Although 2-pyrrolidone-5carboxylic acid can theoretically arise via cyclization reactions of TABLE
121
glutamine and glutamic acid on prolonged storage, this does not appear to be a problem in the present work. We have stored 14Clabeled glutamate and 14C-labeled glutamine for several years at -20°C without appreciable formation of 2-pyrrolidone-5-carboxylic acid (the CSF samples were stored at -80°C). RESULTS Separation of various 2-pyrrolidone analogs by gas chromatography. Table 1 lists
the retention times of TMS derivatives of various 2-pyrrolidone analogs on a 3% SE-30 column. A satisfactory separation of all the derivatized 2-pyrrolidones was obtained. In particular, the TMS derivative of 5-hydroxy2-pyrrolidone-5-carboxylic acid is well separated from that of 2-pyrrolidone-5-carboxylic acid. Mass spectra of the TMS derivative of a-ketoglutaramic acid. Mass spectra ob-
1
RETENTION TIMESOFTHE TMS DERIVATIVES 2-PYRROLIDONE ANALOGSON 3% SE-30"
Compound
OF 2-PYRROLIDONES
OF
Retention time (min)
With solvent front 2-Pyrrolidone DL-2-Pyrrolidone-5-methyl-5-car2.1 boxylic acid DL-5-Hydroxy-2-pyrrofidone 3.5 L-N-Methyl-2-pyrrolidone-5-carboxylic acid 6.1 L-2-Pyrrolidone-5-carboxylic acid 6.6 DL-2-Pyrrolidone-%carboxylic acid 8.7 DL-5-Carboxymethyl-2-pyrrolidone 10.8 Dr-N-Methyl-5-hydroxy-2-pyrrolidone-5-carboxylic acidb 11.3 DL-5-Hydroxy-2-pyrrolidone-5carboxylic acidc*d 11.8 (L For conditions, see text. * The cyclic lactam form of N-methyl-cy-ketoglutaramic acid. c The cyclic lactam form of cr-ketoglutaramic acid. d The retention time refers to the tris(TMS) derivative. If silylation is not complete, a peak at 10.6 min is obtained. This peak is ascribed to the bis(TMS) derivative since on prolonged heating in BSTFA this peak disappears with the concomitant appearance of a peak at 11.8 min.
tained at the exact retention time of a TMSa-ketoglutaramic acid standard on 3% SE-30 are reproduced in Fig. 2. Both the chemical ionization and electron impact spectra are consistent with the structure of the TMS derivative of a-ketoglutaramic acid as being tris(TMS)-5-hydroxy-2-pyrrolidone-S-carboxylic acid. Gas chromatographic analyses of wketoglutaramic acid and 2-pyrrolidone-Scarboxylic acid in human cerebrospinal fluid. Il-
lustrated in Fig. 3 are the gas chromatographic elution profiles of (A) a standard containing 2-pyrrolidone-5-carboxylic acid, internal standard, and m-ketoglutaramic acid, (B) a purified CSF sample from a control subject with normal liver function, and (C) a purified CSF sample from a patient with hepatic encephalopathy. Both CSF samples contained appreciable amounts of 2-pyrrolidone5-carboxylic acid although the concentration of this acid was very much greater in the CSF from the patient with hepatic encephalopathy. a-Ketoglutaramic acid, on the other hand, was pronounced in the CSF of the
122
COOPER ET AL.
loo
f
12711 M+-90 IHOSiMe,)
11721
1731 +SilCH31,
80
M+-llIlCO,SiMe,)
11471 ICHd,Si-0-SPlCH&
A
O@OSiMe, I I1561 H
I
13461 M+-15 ICHJ
x 20.c, 2
I..,,.;.. 3
.*, L , ml1 M+-I17 ICOZSIM~XI
I., 13621
11721
13461 M+-151CHSI
20 A Oo
50
I II loo
I 150
200
FIG. 2. Electron impact (A) and isobutane chemical derivative of cr-ketoglutaramic acid. For details, see are depicted as being derived from the silylated lactam, 5-carboxylic acid. Abbreviations used: M+, molecular
250
I 300
I, 350
400
450
ionization (B) mass spectra of the trimethylsilyl text. Note that the structures of the fragments i.e., tris(trimethylsilyl)-S-hydroxy-2-pyrrolidoneion; Me,, trimethyl.
patient with hepatic encephalopathy, but in the CSF. The data agree closely with was barely detectable in the CSF of the those published previously in which a-ketoglutaramic acid was determined by enzymatic control subject. Figure 4 shows the concentrations of (Y- analysis (1,2). The values obtained by the gc method were generally in good agreeketoglutaramic acid and 2-pyrrolidone-5carboxylic acid in the CSF of 11 control ment with the values obtained by enzymatic subjects who had a variety of neurological analysis; only 3 of the 36 values reported disorders but who had normal liver function in Fig. 4 were appreciably different from tests, and of 25 patients with liver disease. the values obtained by enzymatic analysis.6 Patients with liver disease were further 6 We have recently reported on a spot test for the classified as having no neurological dysfuncsemiquantitative estimation of a-ketoglutaramic acid tion or as having moderate-to-severe en- in human CSF (16). We now report an improved test cephalopathy. A good correlation was noted that does not require a chromatography step and can between the degree of encephalopathy and be used to quickly screen for the presence of abnormal the concentration of a-ketoglutaramic acid a-ketoglutaramic acid in CSF: To 10 ~1 of CSF is added
GAS CHROMATOGRAPHY
OF 2-PYRROLIDONES
123
PCA IS ‘-KGM
I
I
I PCA I
k
l KGM
1 5
:! 0
A
15 0
5
IO
Time
(mln)
1
I:
FIG. 3. Gas chromatographic analyses of 2-pyrrolidone-5carboxylic acid (PCA) and a-ketoglutaramic acid (c+KGM) in 0.5 ml of human cerebrospinal fluid. (A) A standard solution containing PCA (18.2 nmol) and a-KGM (82 nmol) in 0.5 ml of mock human cerebrospinal fluid. (B) Cerebrospinal fluid from a control subject with normal liver function. (C) Cerebrospinal fluid from a subject with hepatic encephalopathy. Each sample contained 40 nmol of internal standard (IS). Isolation and silylation were carried out as described in the text.
The concentration of 2-pyrrolidone-5-carboxylic acid in human CSF also correlated closely with the degree of hepatic encephalopathy. With the exception of two false positives (one from a patient with a duodenal ulcer and a history of gastrointestinal bleeding and another from a patient with a meningioma) the diagnostic correlation of 2-pyrrolidone-5carboxylic acid was comparable to that of a-ketoglutaramic acid. DISCUSSION
The present data (Fig. 4) confirm the previous findings (1,2,4) that cr-ketoglutaramic acid is elevated in the CSF of patients with 2 ~1 of 20% sulfosalicylic acid and the precipitate is removed by centrifugation at 5OOOgfor 2 min. The clear supernate is spotted onto Whatman No. 1 filter paper, 2 ~1 at a time. The paper is sprayed with 1% vanillin (4hydroxy-3-methoxybenzaldehyde) in 1 N HCl in 50% ethanol and allowed to air-dry for 1 h. The paper is then heated at 100°C for 1 min and sprayed with 0.5 N NaOH in 50% ethanol. The presence of a-ketoglutaramic acid is indicated by a red ring around the periphery of the applied spot. The limit of detection is 50 pmol. Semiquantitative analysis may be made by comparing the intensity of the red color against known standards in mock human CSF-sulfosalicylic acid.
liver disease and that the concentration correlates with the degree of encephalopathy. However, the gc method, described above, is more convenient than the enzymatic assay used previously (1,2,4) and promises to be more amenable to clinical work. A new finding is that 2-pyrrolidone-5carboxylic acid levels are also increased in the CSF of patients with moderate-to-severe hepatic encephalopathy (Fig. 4). Gas chromatographic techniques for the estimation of 2-pyrrolidone-5carboxylic acid have been published previously (17- 19). Thus, Polgar and Meister have followed the o-glutamic acid cyclotransferase reaction by converting the enzymatically generated 2-pyrrolidone-5-carboxylic acid to the methyl ester and determining the derivative by gas chromatography (17). Jellum and co-workers have also measured 2-pyrrolidone-5-carboxyhc acid as the methyl ester by a gas chromatographic procedure (18) and used this technique to determine the 2-pyrrolidone-5carboxylic acid content of the urine of a patient with pyroglutamic aciduria (18,20). Finally, Wilk and Orlowski developed a gas chromatography procedure for the determination of 2-pyrrolidone-5-carboxylic acid
124
COOPER ET AL. 300
a-KETOGLUTARAMATE
3oo
2-PYRROLIDONE+CARBOXYLATE
.
200
Controls
Liver dieeose “0 encephobpothy
Hepotlc encepholopothy
Controls
Liver diseose
Hepotlt encepholopothy
encep;:topothy
FIG. 4. cY-Ketoglutaramic acid and 2-pyrrolidone-S-carboxylic acid in human CSF. The two high values of 2-pvrrolidone-S-carboxvlic acid were from the CSF of a patient with a duodenal ulcer (150 pM) and from a-patient with a meningioma (125 PM).
as the N-acyl ester formed in a reaction with pentafluoropropanol and pentafluoropropionic anhydride (19); the technique was used to determine the 2-pyrrolidone-S-carboxylic acid content of various mouse organs and of human urine and CSF. The present technique for the gc determination of 2-pyrrolidone-5-carboxylic acid in human CSF is simpler and more sensitive than the previously published procedures (19,20). In the present procedure, 0.5-ml samples of CSF are used, but this amount is only necessary for the estimation of (Yketoglutaramic acid and the 2-pyrrolidoneS-carboxylic acid peak is often off-scale (Fig. 3). The present technique is adequate to measure 1.5 nmol of 2-pyrrolidone-5carboxylic acid in 100 ~1 of human CSF and has been used to analyze as little as 0.5 nmol in 25-~1 samples by reducing the volume of the silylating mixture from 60 to 20 ~1. Wilk and Orlowski (19) report a concentration of 2-pyrrolidone-5-carboxylic acid in human lumbar spinal fluid of 71 +- 13 (SE) PM (n = 29). However, no details were given regarding the medical status of the patients from whom CSF samples were obtained. In the present series, the “normal” concentration of 2-pyrrolidone-5-carboxylic
acid in the CSF was lo-35 PM (Fig. 4) (includes control subjects and patients with liver disease who did not exhibit neurological impairment). In patients with hepatic encephalopathy, the range was 42- 145 PM. The origin of 2-pyrrolidone-5-carboxylate in CSF is unknown. The concentration of 2-pyrrolidone-5-carboxylate in “normal” CSF, reported here, is comparable to that reported previously for normal plasma (21); therefore, it is conceivable that CSF 2-pyrrolidone-5-carboxylate arises from plasma by equilibration across the choroid plexus. Alternatively, pyrrolidone-5-carboxylate in CSF may arise from enzymatic reactions of the y-glutamyl cycle within the brain. Thus, Orlowski er al. have shown that the human brain contains appreciable y-glutamylcyclotransferase activity (22). Tate et al. have shown that four key enzymes (including y-glutamylcyclotransferase) of the y-glutamyl cycle are well represented in various regions of the rat brain but are especially rich in choroid plexus (23). These authors drew an analogy with the kidney and speculated that the y-glutamyl cycle may play a significant role in the transport of amino acids between blood and CSF (23). If this hypothesis were correct, 2-pyrrolidone-5-carboxylate would
GAS CHROMATOGRAPHY
be expected to accumulate in CSF during amino acid transport since the 2-pyrrolidone5carboxylate amidohydrolase (5oxoprolinase) reaction appears to be a rate-limiting step in the operation of the y-glutamyl cycle (cf. Ref. (24)). Indeed, Orlowski and Wilk have shown that 30 min after a single intraperitoneal injection of a mixture of amino acids into mice the concentration of 2-pyrrolidone-5carboxylate is significantly raised in the brain (24). Therefore, the increase in 2-pyrrolidone-5-carboxylate in the CSF of patients with hepatic encephalopathy (Fig. 4) may be due to an increase in brain yglutamylcyclotransferase activity secondary to the elevated CSF and plasma concentrations of amino acids that are typically observed in such patients (e.g., Refs. (25-27)). Amino acid stimulation of the y-glutamyl
cycle may even account for elevated %-pyrrolidone-5-carboxylate in the CSF of the patient with a duodenal ulcer (Fig. 4) since chronic gastrointestinal bleeding is known to raise plasma concentrations of amino acids (28). The increased concentration of ol-ketoglutaramic acid in the CSF of patients with hepatic encephalopathy may also be related to increased amino acid concentrations in plasma and CSF. Thus, we have previously proposed that an increase in certain brain amino acids, notably phenylalanine and methionine, would lead to an increase in their corresponding cr-keto acids via transamination with a-ketoglutaric acid (3). Increased cy-keto acid levels would, in turn, stimulate glutamine transaminase activity, the effect being to increase the concerted reaction:
cY-ketoglutaric acid + L-amino acid E L-glutamic a-keto acid + L-glutamine a-ketoglutaric
acid + L-glutamine
+ L-glutamic
1. Vergara, F., Plum, F., and Duffy, T. E. (1974) Science 183, 81-83. 2. Duffy, T. E., Vergara, F., and Plum, F. (1974) Publ.
Assoc.
Res.
New.
Men!.
Dis.
53,
39-52. 3. Cooper, A. J. L., and Gross, M. (1977) J. Neuruthem. 28, 771-778. 4. Duffy, T. E., Cooper, A. J. L., and Meister, A. (1974) J. Biol. Chem. 249, 7603-7606. 5. Meister, A. (1952) J. Biol. Chem. 200, 571-589. 6. Otani, T. T., and Meister, A. (1957)J. Biol. Chem. 224, 137- 148. 7. Hersh, L. B. (1972) Biochemisrry 10, 2884-2891. 8. Orlowski, M., and Meister, A. (1971) Proc. Nat. Acad. Sri. USA 67, 1248-1255. 9. Griffith, 0. W., and Meister, A. (1977) Proc. Nar. Acad.
Sci.
USA
74, 3330-3334.
acid + a-ketoglutaramic
acid acid
to Wilson, H., and Cannan, R. K. (1937) J.
Biol.
119, 309-331. de Mayo, P., and Reid, S. T. (1962) Chem. Ind., 1576- 1577. Duffy, T. E., Cooper, A. J. L., and Vergara, F. (1976) Bioorg. Chem. 5, 351-366. Lowry, 0. H., and Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, pp. l&t185, Academic Press, New York. Munson, M. S. B., and Field, F. H. (1966)J. Amer. Chem. Sot. 88, 2621-2630. Merlis, J. K. (1940) Amer. J. Physiol. 131, 67-72. Cooper, A. J. L. (1978) Anal. Biochem. 90, 444446. Polgar, P., and Meister, A. (1965) Anal. Biochem. 12, 338-343. Jellum, E., Kluge, T., Borresen, H. C., Stokke, O., and Eldjarn, L. (1970) Sand. J. Clin. Lab. Chem.
11.
12 . 13.
REFERENCES
Res.
acid + a-keto acid
+ L-amino acid + cu-ketoglutaramic
If this hypothesis is correct, then despite the fact that cu-ketoglutaric acid is a poor substrate for brain glutamine transaminase (3), net transamination between cr-ketoglutaric acid and glutamine will be promoted by elevated concentrations of certain amino acids.
125
OF 2-PYRROLIDONES
14. 15. 16. 17. 18.
Invest.
26, 327-335.
19. Wilk, S., and Orlowski, M. (1975) Anal. Biochem. 69, 100-113. 20. Eldjarn, L., Jellum, E., and Stoke, D. (1972) Ctin. Chim. Acta 40, 461-476. 21. Palekar, A. G., Tate, S. S., Sullivan, J. F., and Meister, A. (1975)Biochem. Med. 14, 339-345.
126
COOPER ET AL.
22. Orlowski, M., Richman, P. G., and Meister, A. (1%9) Biochemistry 8, 1048-1055. 23. Tate, S. S., Ross, L. L., and Meister, A. (1973) Proc. Nat. Acad. Sci. USA 70, 221 l-2214. 24. Orlowski, M., and Wilk, S. (1975)&r. J. Biochem. 53, 581-590. 25. Record, C. O., Buxton, B., Chase, R. A., Curzon, G., Murray-Lyon, I. M., and Williams, R. (1976) Eur.
J. Clin.
Invest.
6, 387-394.
26. Fischer, J. E., Yoshimura, N., Aguirre, A., James, J. H., Cummings, M. G., Abel, R. M., and Deindoctfer, F. (1974)Amer. J. Surg. 127,40-47. 27. Van Sande, M., Mardens, Y., Adriaenssens, K., and Lowenthal, A. (1970) J. Neurochem. 17, 125- 135. 28. Ansley, J. D., Isaacs, J. D., Rikkers, L. F., Kutner, M. H., Nordlinger, B. M., and Rudman, D. (1978) Gastroenterology 75, 570-579.