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Determination of pyrrolidone carboxylate and γ-glutamyl amino acids by gas chromatography
ANALYTICAL
BIOCHEMISTRY
69, 100-l 13 (1975)
Determination
of Pyrrolidone
Carboxylate
y-Glutamyl
Amino Acids Chromatography
SHERWIN
WILK
AND MARIAN
and
by Gas
ORLOWSKI
Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 Received January 7, 1975: accepted June 4, 1975 Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and y-glutamyl amino acids are described. These intermediates of the y-glutamyl cycle were separated by ion exchange chromatography and converted to their Nacyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-lpropanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several y-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.
Pyrrolidone carboxylate, an intermediate in mammalian metabolism, has been found in the free state in tissues and body fluids (1-3) and in the bound form at the N-terminus of a number of proteins and biologically active peptides (for a review see 4). The formation of free pyrrolidone carboxylate is catalyzed by y-glutamyl cyclotransferase (5) in a reaction that involves the cyclization of the y-glutamyl moiety of y-glutamyl amino acids. y-Glutamyl amino acids are in turn formed either in a synthetic reaction catalyzed by y-glutamylcysteine synthetase (6) or in a transfer reaction catalyzed by y-glutamyl transpeptidase (7). These reactions are involved in the synthesis and degradation of glutathione. It has been proposed that the reabsorption of amino acids in the kidney is mediated by the membrane-bound enzyme y-glutamyl transpeptidase (8,9) and that this process is coupled to the synthesis and degradation of glutathione in reactions that have been later integrated into a cyclic process called the y-glutamyl cycle (10). This proposal and the discovery of patients excreting large amounts of pyrrolidone carboxylate in the urine (11) stimulated interest in the study of pyrrolidone carboxylate and y-glutamyl amino acids as intermediates of the cycle. Pyrrolidone carboxylate has been previously determined by gas chromatography (11,12). These methods quantitated the methyl ester deriva100 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
y-GLUTAMYL
CYCLE
INTERMEDIATES
101
tive using flame ionization detection. The sensitivity of this detector is not sufficient for the quantitation of the low levels of pyrrolidone carboxylate present in normal tissues and body fluids. We describe here the quantitative determination of pyrrolidone carboxylate and y-glutamyl amino acids in tissues and body fluids by gas chromatography utilizing electron capture detection. N-acyl ester derivatives were prepared by reaction with a mixture of 20% pentafluoropropanol in pentafluoropropionic anhydride ( 13). The excellent electron capture response to these derivatives makes possible the determination of nanogram amounts of these intermediates in material of biological origin. MATERIALS
Pentafluoropropionic anhydride was obtained from Pierce Chemical Co., Rockford, Ill. 2,2,3,3,3-pentafluoro-I-propanol was obtained from Peninsula Chemical Research Gainesville, Fla. These reagents were used without further purification. Analytical grade Dowex 50 (AG50W-X4; 100-200 mesh; H+ form) and analytical grade Dowex 1 (AGl-X4; 100-200 mesh; H+ form) were obtained from Bio Rad Laboratories, Rockville Centre, N.Y. Dowex 50 was purified to remove all ultraviolet absorbing material as described (14). The Dowex 1 resin was converted to the hydroxide form by washing with 20 vol of 1 N NaOH followed by water until a neutral pH was obtained. The hydroxide form was converted to the acetate form by washing with 2 vol 2 N acetic acid followed by water until a neutral pH was obtained. Dowex 1 acetate was stored under distilled water. Dowex 1 formate was prepared by washing the hydroxide form of the resin with 2 vol 2 N formic acid. The formate form of the resin was packed into a 2.5 x 60 cm column and washed daily with 1 column vol of 0.25 N formic acid. Portions of this resin were removed from the column for the determination of pyrrolidone carboxylate, and washed with distilled water until the effluent attained a neutral pH. This washing procedure was necessary for removal of substances that interfered in the gas chromatographic determination of pyrrolidone carboxylate. Nanograde solvents were obtained from the Malinckrodt Chemical Works. Pyrrolidone carboxylate and all amino acids were purchased from the Sigma Chemical Co., St. Louis, MO. The DL-piperidone carboxylate was prepared by the cyclization of DL-aaminoadipic acid as described by Dieckman (15). The product was recrystallized from ethanol. Generally labeled [‘“Cl glutamate (sp act 260 mCi/mmole) was obtained from Schwarz-Mann, Orangeburg, NY. [ 14C] pyrrolidone carboxylate was prepared by heating [ ‘,C] glutamate for 6 hr at 145°C in a sealed tube. The product was purified as previously described (16). The y-glutamyl amino acids were obtained as described in previous publications ( 14,16.17).
102
WILK
AND
ORLOWSKI
All coated gas chromatographic supports were obtained from The Applied Science Laboratories, State College, Pa. Chromatography was carried out on 6 ft X 4 mm coiled glass columns. A Packard 7400 series gas chromatograph equipped with two 250 mCi tritium foil electron capture detectors and a Hewlett-Packard model 7620 A gas chromatograph equipped with a Ni63 electron capture detector were employed. Aliquots of cerebrospinal fluid samples, drawn for diagnostic purposes were kindly supplied by Dr. M. Yahr of the Department of Neurology of the Mount Sinai School of Medicine. These samples were immediately frozen after collection. Freshly voided urine was obtained from healthy human volunteers and processed immediately after collection or immediately frozen. The frozen samples were assayed within 24-48 hr after collection. Creatinine was determined by the method of Jaffe (18). METHODS Purijcation of Pyrrolidone Carboxylate Acids for Gas Chromatography
and y-Glutamyl
Amino
Male Swiss albino mice weighing approximately 30 g were used in these studies. The animals were decapitated and the brain, liver, and kid:tey rapidly removed and frozen on dry ice. The tissues were weighed and homogenized in 5 vol of cold 1% picric acid using a Potter-Elvehjem glass homogenizer equipped with a motor-driven Teflon pestle. The homogenate was centrifuged for 15 min at 30008 in a Sorvall refrigerated centrifuge and the protein-free supematant decanted. To aliquots of the protem-free supematant (usually 2 ml) was added 7.15 pg piperidone carboxylate as an internal standard in the determination of pyrrolidone carboxylate. Urine (0.1 ml + 7.15 pg piperidone carboxylate), cerebrospinal fluid (1 .O ml + 7.15 pg piperidone carboxylate), and the protein-free tissue aliquot were each applied to the top of separate Dowex 50 (H+) columns (0.6 X 6 cm). Pyrrolidone carboxylate and piperidone carboxylate which are not adsorbed on Dowex 50 were quantitatively recovered by washing the column with 5 ml H,O. The combined effluents were applied to the top of a Dowex 1 formate column (0.6 x 6 cm). The column was washed with 10 ml water and 10 ml 0.08 M formic acid. Pyrrolidone carboxylate and piperidone carboxylate were eluted with 10 ml 0.25 M formic acid and the eluate evaporated to dryness in a 50 ml round bottom flask using a Buchler flash evaporator. The residue was dissolved in 2.5 ml methanol, quantitatively transferred to a 3-ml ground glass stoppered centrifuge tube, and the methanol removed under a stream of dry nitrogen. The residue was taken for derivatization (see Fig. 1).
-y-GLUTAMYL
CYCLE
INTERMEDIATES
103
The y-glutamyl amino acids were eluted from the Dowex 50 column with 5 ml 3 N NH,OH. The eluate was evaporated to dryness by flash evaporation, the residue was dissolved in 3 ml H,O and applied to the top of a 0.6 X 7.5 cm column Dowex 1 (acetate). The effluent was collected and the column eluted with 1.5 ml 0.1 N acetic acid. The eluate combined with the effluent contains free amino acids as well as y-glutamyllysine. The y-glutamyl derivatives of glycine, cY-aminobutyrate, vaIine, ,&aminoisobutyrate, leucine, and methionine were eluted with 15 ml of 1 N acetic acid. The y-glutamyl derivatives of phenylalanine, tyrosine, and glutamate were eluted with 15 ml 2 N acetic acid. All eluates were separately evaporated in 50 ml round bottom flasks and the residues derivatized directly in the flasks. Figure 1 summarizes the isolation procedure for pyrrolidone carboxylate and y-glutamyl amino acids. Gas Chromatography
of Pyrrolidone
Carboxylate
To the residue in the 3-ml centrifuge tube was added 0.25 ml of a mixture of 20% 2,2,3,3,3-pentafluoro1-propanol in pentafluoropropionic anhydride. The tube was stoppered and heated for 15 min at 75°C in a sand bath. The tube was then cooled and the reagents removed under a stream of dry nitrogen. The reaction was completed by addition of 0.1 ml pentafluoropropionic anhydride and heating for an additional 5 min at 75°C. The excess anhydride was evaporated under nitrogen, the residue dissolved in 1 ml ethyl acetate and an aliquot of this solution diluted 1 : 5 with ethyl acetate for gas chromatographic analysis. Standard mixtures of 0.39-l .29 pg pyrrolidone carboxylate in the presence of 1.43 pg piperidone carboxylate were similarly carried through the entire isolation procedure and derivatized as described above. The derivatives were dissolved in 1 ml ethyl acetate for gas chromatography. Chromatography was performed on columns of 3% OV-17 coated on gas chrom Q 60/80 mesh and 3% DC-200 coated on gas chrom Q 100/200 mesh in a Packard 7400 series gas chromatograph. Both columns were operated isothermally at 125°C. The flow rates of the OV-17 and DC-200 columns were 20 ml/min and 25 ml/min, respectively. The electrometer was 1 X 1O-y A. The inlet port and detector temperatures were both maintained at 190°C. The ratio of peak heights of pyrrolidone carboxylate to piperidone carboxylate in the standards was plotted against the amount of pyrrolidone carboxylate. Quantitation was based on the ratio of the peak heights of pyrrolidone carboxylate to piperidone carboxylate in the unknown sample. Gas chromatography was also performed in a Hewlett-Packard model 7620A gas chromatograph. Three microliters of a I : 5 dilution of the samples was injected on a 6 ft x 4 mm glass column packed with 3% OV- 17. The column temperature was maintained at 120°C. A mixture of
CAABOXYLATE
50 H*
CARBOXYLATE
CARBOXYLATE
FIG. 1. Schematic outline of the fractionation
PIPERIDONE
DISCARD
+
I
formote
fuse ‘4 somplel
IOml 0.25N
Dorex
PYRROLIDONE
\\
Super!atant
IOml O.OBN formote
IOml Hz0
Effl:en’ Doier-I-formote I
5ml Hz0 I
I
l.Oml CSF +7.15~9 PIPERIDONE CARBOXYLATE
PIPERIDONE
0.1 ml urine + 7.15~9
acid
of pyrrolidone
I
-discord
ISOBUTYRATE LEUCINE
GLUTAMATE
TYROSINE
1 2N acetic acid I y-GLUTAMYL DERIVATIVES OF PHENYLALANINE l5ml
carboxylate and y-glutamyl amino acids.
METHIONINE
B-AMINO
WJTYRATE VALINE
a-AMINO
I IN acetic acid I y-GLUTAMYL DERIVATIVES OF GLYCINE 15ml
5 ml 3N NH;OH I Evoporote I Dorex-l-acetate I
I Precipitate
I 15ml 0.1 N acetic acid I y-GLUTAMYLLYSINE
CARBOXYLATE
in 5 ~01s I% pioic
I Ccntrifuqe I
PIPERIDONE
Tissues - homopcnizc
Add 215~9
y-GLUTAMYL
CYCLE
INTERMEDIATES
105
95% argon and 5% methane was used as the carrier gas and the flow rate was 20 ml/min. Separations were also performed on a 4 ft X 4 mm column packed with 1% XE-60 coated on gas chrom Q 60/80 mesh at 160°C and a flow rate of 20 ml/min. Gas Chromatography
of y-Glutamyl
Amino
Acids
The y-glutamyl amino acids were derivatized directly in the ground glass stoppered round bottom flasks. In the first stage of the derivatization procedure 0.5 ml of the alcohol-anhydride mixture was used. In the second step 0.25 ml of the anhydride was used. The stoppered flasks were sealed with parafilm and placed in a sand bath at 75°C as described for pyrrolidone carboxylate. The derivatives were dissolved in 1 ml ethyl acetate and transferred to aluminum foil covered screw capped vials for storage prior to chromatography. y-Glutamyl amino acids were separated on the 3% DC-200 column. The y-glutamyl derivatives of glycine, cY-aminobutyrate, valine, p-aminoisobutyrate, and leucine were chromatographed at 14o”C, the y-glutamyl derivatives of glutamate, methionine, and phenylalanine at 165”C, and the y-glutamyl derivatives of lysine and tyrosine at 185°C. The flow rate in all cases was 50 ml/min. Biological samples containing peaks which interfered with the determination of individual y-glutamyl peptides on the 3% DC-200 column were chromatographed on 3% OV-17. Standards of y-glutamyl amino acids (10 pg) were processed through the procedure. Quantitation was based on comparison of the peak heights of the compounds assayed in the biological sample with the peak heights of the corresponding standards. Processing of y-glutamyl amino acids through the procedure did not lead to the cyclization of the y-glutamyl residue to pyrrolidone carboxylate. RESULTS Pyrrolidone
Carboxylate
Table 1 shows that considerable amounts of pyrrolidone carboxylate are present in mouse kidney, brain, and liver. It is of interest that the concentrations are remarkably similar in all three tissues. Fig. 2 shows chromatograms of pyrrolidone carboxylate in mouse kidney, brain, and liver. Appreciable amounts of the compound are also found in human urine and cerebrospinal fluid. Several studies were undertaken on the recovery of pyrrolidone carboxylate carried through the procedure. [ 14C] pyrrolidone carboxylate (50,000 cpm) was added to the picric acid extracts of tissues and also to the samples of urine and cerebrospinal fluid. An aliquot of the ethyl acetate solution of the final derivative was counted. These experiments
106
WILK
AND
ORLOWSKI
TABLE
1
LEVELS OF PYRROL~DONE CARBOXYLIC ACID IN TISSUES AND BODY FLUIDS Source Mouse Mouse Mouse Human Human
Number of samples
PCA level f. SE
17 15 1.5 43 29
52.2 nmoles/g k 3.1 58.7 nmoleslg 2 2.3 56.4 nmoles/g f 4.6 237 nmolesimg creatinine 2 17 71 nmolesknl f 13
kidney brain liver urine lumbar spinal fluid
showed that the recovery of pyrrolidone carboxylate consistently exceeded 90%. Similar results were obtained when the peak heights of standard samples of pyrrolidone carboxylate carried through the procedure were compared with the peak heights of the same standards directly derivatized. The recovery of piperidone carboxylate also exceeded 90%. Both compounds behaved identically during ion exchange chromatography and during the derivatizatiou procedure. The derivatives of these two compounds gave single sharp symmetrical peaks that were
LIVER
STANDARDS
KIDNEY
PC
PC
PC
c
1 PCA t
#
1
k
-II 5
IO 0 TIME
5
IO 0
5
IO
(min)
FIG. 2. Gas chromatographic analysis of pyrrolidone carboxylate (PCA) in mouse tissues. PC = piperidone carboxylate (internal standard), 3% OV-17, 12o”C, flow rate = 20 ml/min.
y-GLUTAMYL
CYCLE
INTERMEDIATES
107
1.3 I.1 0.9 PCA pc
-
O.?-
FIG. 3. Standard curve for the quantitation of pyrrolidone carboxylate (PCA). PC = piperidone carboxylate (internal standard). The concentration of PCA is plotted against the ratio of the peak heights of the two compounds.
separated on a number of liquid phases including OV-17, XE-60, and DC-200. This made it possible to use piperidone carboxylate as an internal standard, and thus to avoid the necessity of adding [14C]pyrrolidone carboxylate to the extracts to compensate for any losses during the isolation procedure. When the ratio of peak heights of pyrrolidone carboxylate to piperidone carboxylate obtained from mixtures of these two standards which were carried through the procedure was plotted against the concentration of pyrrolidone carboxylate in the sample, a straight line was obtained (Fig. 3). The overall accuracy of the procedure was determined by analyzing seven samples containing from 0.39 to I .29 pg pyrrolidone carboxylate. The average difference between the estimated and true values was 6.9%. y-Glutamyl Amino Acids Derivatives of the y-glutamyl amino acids chromatographed as single sharp symmetrical peaks on the DC-200 column. By analogy to the structure of the pyrrolidone carboxylate derivative it is believed that the reaction with pentafluoropropanol and pentafluoropropionic anhydride yields N-acyl ester derivatives of the y-glutamyl amino acids. The chromatographic properties of the derivatives suggest that all functional groups have been derivatized. The y-glutamyl amino acids have been fractionated on a Dowex 1 acetate column (Fig. 1). The neutral peptide y-glutamyllysine elutes with 0.1 N acetic acid. The acidic y-glutamyl derivatives of short chain amino acids are tAuted with 1 N acetic
108
WILK
AND ORLOWSKI TABLE
SEPARATION
OF Y-GLUTAMYL GAS-LIQUID
Peptide y-glutamylglycine y-glutamyl cY-aminobutyrate y-glutamylvaline y-glutamyl P-aminoisobutyrate y-glutamylleucine y-glutamylglutamate y-glutamylmethionine y-glutamylphenylalanine y-glutamyllysine y-glutamyltyrosine
2
AMINO
ACID
DERIVATIVES
BY
CHROMATOGRAPHY”
Column temperature (“C)
Retention time (min)
140 140 140 140 140 165 165 165 185 185
6.6 8.0 8.9 11.4 12.7 8.0 10.9 13.8 8.1 8.4
a y-Glutamyl amino acids were derivatized with a mixture of 20% 2,2,3,3,3-pentafluoro-lpropanol in pentafluoropropionic anhydride as described under methods. Derivatives were chromatographed on a 6 ft x 4 mm i.d. coiled glass column packed with 3% DC-200 coated on gas chrom Q 60180 mesh. NZ flow = 50 mlimin. Column temperature as indicated. Inlet and detector temperatures = 190°C.
TIME (min) 4. Separation of 2.5 X lo-l2 moles each of derivatives of the following y-glutamyl amino acids: (1) y-glutamylglycine: (2) y-glutamyl-a-amino butyrate; (3) y-glytamylvaline; (4) y-ghnamyl-cw-aminoisbutyrate; (5) y-glutamylleucine. 3% DC-200, 140°C flow rate = 50 ml/min. FIG.
‘)‘-GLUTAMYL Kjdney
before
CYCLE INTERMEDIATES methiomne
Kidney
1
after methmnine
“-1
-L
5
6-
IO
TIME
109
meth,o”,ne
5
IO
15
(min)
FIG. 5. Formation of y-glutamylmethionine in mouse kidney 30 min after intraperitoneal administration of methionine (5 pmoleslg). 3% OV- 17, 165°C. flow rate = 60 ml/min. The concentration of y-glutamylmethionine is 3.9 nmoles/g.
acid, and the y-glutamyl derivatives of the aromatic amino acids and of glutamate are eluted with 2 N acetic acid. The recoveries throughout the entire procedure have averaged 70%. By combination of ion exchange chromatography and gas-liquid chromatography it is possible to separate all 10 y-glutamyl peptides studied (Table 2). Fig. 4 shows the gas chromatographic separation of five of these peptides on 3% DC-200. The high electron capture response to these derivatives makes it possible to quantitate the low levels of y-glutamyl amino acids formed after amino acid loading (19). Thirty minutes after intraperitoneal administration of methionine to mice (5 pmoleslg) we detected the presence of y-glutamylmethionine in mouse kidney (Fig. 5). Similarly 30 min followKldney
before
phenylalanme
5
IO
Kidney
after
phenylalonine
I
0
I5 TIME
0
5
IO
15
(min)
FIG. 6. Formation of y-glutamylphenylalanine in mouse kidney 30 min after intraperitoneal administration of phenylalanine (5 pmoleslg). 3% OV-17. 165°C. flow rate = 60 ml/min. The concentration of y-glutamylphenylalanine is 8.1 nmoles/g.
110
WILK
AND
ORLOWSKI
ing administration of phenylalanine (5 @moles/g) to mice, y-glutamylphenylalanine in mouse kidney was found (Fig. 6). These results were confirmed on 1% XE-60 and 3% DC-200. DISCUSSION The quantitative determination of pyrrolidone carboxylate and y-glutamyl amino acids is of considerable importance in studies on the synthesis, degradation, and metabolic function of glutathione. The small concentration of pyrrolidone carboxylate in animal tissues and the lack of analytical procedures for the determination of y-glutamyl amino acids prompted the present study on the application of gas chromatography coupled with electron capture detection for the determination of these compounds. Derivatives of pyrrolidone carboxylate and y-glutamyl amino acids were prepared using a mixture of pentafluoropropanol and pentafluoropropionic anhydride. This reagent which reacts with carboxyl, amino, and hydroxyl functional groups was previously applied for the preparation of derivatives of biologically important acids (13). The response of the electron capture detector to these derivatives is very high thus permitting the determination of low concentrations of these intermediates in tissues. Purification of pyrrolidone carboxylate and of y-glutamyl amino acids by ion exchange chromatography eliminates interference from other compounds in the analyzed sample. In earlier attempts to use gas chromatography for the determination of pyrrolidone carboxylate, its methyl ester derivative was prepared either in a reaction with a mixture of dry hydrogen chloride and methanol (12) or in a reaction with diazomethane (1 l), and determined using the flame ionization detector. This derivative was also used for the detection of pyrrolidone carboxylate in the urine of patients with skin burns (20). The method was successfully employed for the detection in urine and plasma of the markedly elevated concentrations of pyrrolidone carboxylate in patients with pyroglutamic aciduria, an inborn error of metabolism in which large amounts (25-40 g daily) are excreted in the urine (11,2 1). This procedure was also applied to the determination of pyrrolidone carboxylate in the human epidermis where its concentration amounts to an average of 1.6% of the tissue weight of the horny layer (22).
The determination of pyrrolidone carboxylate as the methyl ester derivative using flame ionization detection is not sufficiently sensitive to quantitate the small amounts of pyrrolidone carboxylate present in extracts of normal tissues and in normal body fluids. The limit of detectability of this method has been reported as 10 pg/ml (or 77 nmoles/ml) (21) which exceeds the average concentrations found in normal tissues. This limitation is eliminated by the method presented here with its much
y-GLUTAMYL
CYCLE
INTERMEDIATES
111
higher sensitivity. Moreover the introduction of piperidone carboxylate as internal standard compensates for losses during the isolation and derivatization procedure as well as for spontaneous changes in detector sensitivity. In an earlier study (l), we documented the occurrence of free Lpyrrolidone carboxylic acid in body fluids and tissues using a similar but more complex gas chromatographic procedure. This method has been simplified by substitution of a single Dowex 1 formate form column for the Dowex I chloride and DEAE sephadex-chloride columns previously employed. This has resulted in an enhanced recovery of pyrrolidone carboxylate. Moreover introduction of piperidone carboxyiate as internal standard has enhanced the reproducibility of the method. The present values of urinary pyrrolidone carboxylate (237 nmoles/mg creatinine are in good agreement with those reported in our earlier publication (190 nmoleslml urine) since the average concentration of creatinine in urine is close to 1 mg/ml. Similarly the pyrrolidone carboxylate levels of this new group of CSF samples are close to those previously reported. Tissue levels of pyrrolidone carboxylate, were determined in the earlier publication for the guinea pig and therefore are not directly comparable to the values given here for mouse tissue levels. Pyrrolidone carboxylate has been determined in mouse tissues by Van der Werf et ul. (2). In their procedure tissue samples were purified by passage through Dowex 2 chloride and Dowex 50 columns. The eluate containing pyrrolidone carboxylate was hydrolyzed to glutamate either enzymatically or by hydrochloric acid. Glutamate was then determined using an amino acid analyzer. Their values were somewhat lower than the values reported here. The differences may be due to the different strains of mice used in the two studies. On the other hand, Wolfersberger et al. (23) determined pyrrolidone carboxylate in guinea pig tissues by passage of a deproteinized tissue extract through a Dowex 50 column followed by acid hydrolysis of the effluent at 100°C. The glutamate formed was determined by the ninhydrin procedure. Their values for guinea pig tissues are 10 to 80-fold higher than those obtained by us by gas chromatography (1). It is obvious that the method of Wolfersberger et al. is not specific for pyrrolidone carboxylate and that any acidic peptide not retained by the column would yield ninhydrin positive material thus yielding spurious values. The availability of a method for the determination of y-glutamyl amino acids should prove to be useful for the study of these compounds with respect to their significance in mammalian metabolism. Thus we have detected small amounts of y-glutamyl derivatives in mouse tissues after administration of the corresponding free amino acids. Although these compounds give a positive ninhydrin reaction and thus can be detected
112
WILK
AND
ORLOWSKI
using the amino acid analyzer, this method would not be sufficiently sensitive to detect some of the derivatives (e.g., y-glutamylphenylalanine and y-glutamylmethionine) that are formed only in very small amounts of several nanomoles per gram. It remains to be established whether the increase in the sensitivity of amino acid detection by the use of reagents such as a fluorescamine (24) could solve this analytical problem. Since the elution pattern of many y-glutamylamino acids in an amino acid analyzer overlap with the elution pattern of many amino acids (23, a preliminary separation of this class of compounds from other amino acids on anion exchange resins (e.g., Dowex 1 acetate) would probably be required. ACKNOWLEDGMENTS This work was supported in part by Grants from the National Institute of Arthritis, Metabolism and Digestive Diseases (AM 17113), and from the Health Research Council of the City of New York. We thank Miss Charlene Michaud for her untiring and skillful assistance.
REFERENCES 1. Wilk, S., and Orlowski, M. (1973) FEBS L&t. 33, 157-160. 2. Van der Werf, P., Stephani, R. A., and Meister, A. (1974) Proc. Nat. Acad. Sci. USA 71, 1026-1029. 3. Meister, A., Bukenberger, M. W.. Strassberger, M. (1963) Biochem. 2. 338, 2 17-229. 4. Orlowski, M., and Meister, A. (1971) in The Enzymes (Boyer, P. D. ed.), Vol. 4, pp. 123-151, Academic Press, New York. 5. Connell, G. E., and Hanes, C. S. (1965) Nature (London) 177, 377-378. 6. Bloch, K. (1949) J. Biol. Chem. 179, 1245-1254. 7. Hanes, C. S., Hird, F. J. R., and Isherwood, F. A. (1950) Nature (London) 166, 288-292. 8. Binkley, F. (1954) in Glutathione: A symposium (Colowick, S. et al., eds.), p. 160, Academic Press, Inc., New York, N. Y. 9. Orlowski, M. (1963) Arch. Immunol. Thu. Exp. 11, I-61. 10. Orlowski, M. and Meister, A. (1970) Proc. Nat. Acad. Sci. USA 67, 1248-1255. 11. Jellum, E., Kluge, T., Borresen, H. C., Stokke, 0.. and Eldjarn, L. (1970) Stand. J. Clin. Lab. Invest. 26, 327-335. 12. Polgar, P., and Meister, A. (1965) Anal. Biochem. 12, 338-343. 13. Watson, E., Wilk, S., and Roboz, J. (1974) Anal. Biochem. 59, 441-451. 14. Orlowski, M., Richman, P. G., and Meister, A. (1969) Biochemistry 8, 1048-1055. 15. Dieckman, W. (1905) Berichte Deut. Chem. Ges. 38, 1654-1661. 16. Orlowski, M., and Meister, A. (197 1) J. Biol. Chem. 246, 7095-7 105. 17. Orlowski, M., and Meister, A. (1971) Biochemistry 10, 372-380. 18. JatIe, M. (method of) (1965) in Hawk’s Physiological Chemistry (Oser, B. L. ed.). pp. 1040-1042, McGraw Hill, New York. 19. Orlowski. M. and Wilk, S. (1975) Eur. J. Biochem. 53, 581-590. 20. Tham, R., Nystrom, L., and Holmstedt, B. (1968) Biochem Pharmacol. 17, 1735-1738. 21. Eldjarn, L., Jellum. E.. and Stokke. 0. (1972) C/in. Chim. Acta 40, 461-476.
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22. Marstein, S., Jellum, E., and Eldjarn, L. (1973) C/in. Chim. Actu 49, 389-395. 23. Wolfersberger, M. G., Tabachnick, J., Finkelstein, B. S., and Levin, M. (1973) J. fnvest. Dermatol.
60, 278-28
1.
24. Udenfriend, S.. Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M. (1972) Science 178, 87 I-872. 25. Zacharius, R. M.. and Talley, E. A. (1962)Anal Chem 34, 1551-1556.
BIOCHEMISTRY
69, 100-l 13 (1975)
Determination
of Pyrrolidone
Carboxylate
y-Glutamyl
Amino Acids Chromatography
SHERWIN
WILK
AND MARIAN
and
by Gas
ORLOWSKI
Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 Received January 7, 1975: accepted June 4, 1975 Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and y-glutamyl amino acids are described. These intermediates of the y-glutamyl cycle were separated by ion exchange chromatography and converted to their Nacyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-lpropanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several y-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.
Pyrrolidone carboxylate, an intermediate in mammalian metabolism, has been found in the free state in tissues and body fluids (1-3) and in the bound form at the N-terminus of a number of proteins and biologically active peptides (for a review see 4). The formation of free pyrrolidone carboxylate is catalyzed by y-glutamyl cyclotransferase (5) in a reaction that involves the cyclization of the y-glutamyl moiety of y-glutamyl amino acids. y-Glutamyl amino acids are in turn formed either in a synthetic reaction catalyzed by y-glutamylcysteine synthetase (6) or in a transfer reaction catalyzed by y-glutamyl transpeptidase (7). These reactions are involved in the synthesis and degradation of glutathione. It has been proposed that the reabsorption of amino acids in the kidney is mediated by the membrane-bound enzyme y-glutamyl transpeptidase (8,9) and that this process is coupled to the synthesis and degradation of glutathione in reactions that have been later integrated into a cyclic process called the y-glutamyl cycle (10). This proposal and the discovery of patients excreting large amounts of pyrrolidone carboxylate in the urine (11) stimulated interest in the study of pyrrolidone carboxylate and y-glutamyl amino acids as intermediates of the cycle. Pyrrolidone carboxylate has been previously determined by gas chromatography (11,12). These methods quantitated the methyl ester deriva100 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
y-GLUTAMYL
CYCLE
INTERMEDIATES
101
tive using flame ionization detection. The sensitivity of this detector is not sufficient for the quantitation of the low levels of pyrrolidone carboxylate present in normal tissues and body fluids. We describe here the quantitative determination of pyrrolidone carboxylate and y-glutamyl amino acids in tissues and body fluids by gas chromatography utilizing electron capture detection. N-acyl ester derivatives were prepared by reaction with a mixture of 20% pentafluoropropanol in pentafluoropropionic anhydride ( 13). The excellent electron capture response to these derivatives makes possible the determination of nanogram amounts of these intermediates in material of biological origin. MATERIALS
Pentafluoropropionic anhydride was obtained from Pierce Chemical Co., Rockford, Ill. 2,2,3,3,3-pentafluoro-I-propanol was obtained from Peninsula Chemical Research Gainesville, Fla. These reagents were used without further purification. Analytical grade Dowex 50 (AG50W-X4; 100-200 mesh; H+ form) and analytical grade Dowex 1 (AGl-X4; 100-200 mesh; H+ form) were obtained from Bio Rad Laboratories, Rockville Centre, N.Y. Dowex 50 was purified to remove all ultraviolet absorbing material as described (14). The Dowex 1 resin was converted to the hydroxide form by washing with 20 vol of 1 N NaOH followed by water until a neutral pH was obtained. The hydroxide form was converted to the acetate form by washing with 2 vol 2 N acetic acid followed by water until a neutral pH was obtained. Dowex 1 acetate was stored under distilled water. Dowex 1 formate was prepared by washing the hydroxide form of the resin with 2 vol 2 N formic acid. The formate form of the resin was packed into a 2.5 x 60 cm column and washed daily with 1 column vol of 0.25 N formic acid. Portions of this resin were removed from the column for the determination of pyrrolidone carboxylate, and washed with distilled water until the effluent attained a neutral pH. This washing procedure was necessary for removal of substances that interfered in the gas chromatographic determination of pyrrolidone carboxylate. Nanograde solvents were obtained from the Malinckrodt Chemical Works. Pyrrolidone carboxylate and all amino acids were purchased from the Sigma Chemical Co., St. Louis, MO. The DL-piperidone carboxylate was prepared by the cyclization of DL-aaminoadipic acid as described by Dieckman (15). The product was recrystallized from ethanol. Generally labeled [‘“Cl glutamate (sp act 260 mCi/mmole) was obtained from Schwarz-Mann, Orangeburg, NY. [ 14C] pyrrolidone carboxylate was prepared by heating [ ‘,C] glutamate for 6 hr at 145°C in a sealed tube. The product was purified as previously described (16). The y-glutamyl amino acids were obtained as described in previous publications ( 14,16.17).
102
WILK
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All coated gas chromatographic supports were obtained from The Applied Science Laboratories, State College, Pa. Chromatography was carried out on 6 ft X 4 mm coiled glass columns. A Packard 7400 series gas chromatograph equipped with two 250 mCi tritium foil electron capture detectors and a Hewlett-Packard model 7620 A gas chromatograph equipped with a Ni63 electron capture detector were employed. Aliquots of cerebrospinal fluid samples, drawn for diagnostic purposes were kindly supplied by Dr. M. Yahr of the Department of Neurology of the Mount Sinai School of Medicine. These samples were immediately frozen after collection. Freshly voided urine was obtained from healthy human volunteers and processed immediately after collection or immediately frozen. The frozen samples were assayed within 24-48 hr after collection. Creatinine was determined by the method of Jaffe (18). METHODS Purijcation of Pyrrolidone Carboxylate Acids for Gas Chromatography
and y-Glutamyl
Amino
Male Swiss albino mice weighing approximately 30 g were used in these studies. The animals were decapitated and the brain, liver, and kid:tey rapidly removed and frozen on dry ice. The tissues were weighed and homogenized in 5 vol of cold 1% picric acid using a Potter-Elvehjem glass homogenizer equipped with a motor-driven Teflon pestle. The homogenate was centrifuged for 15 min at 30008 in a Sorvall refrigerated centrifuge and the protein-free supematant decanted. To aliquots of the protem-free supematant (usually 2 ml) was added 7.15 pg piperidone carboxylate as an internal standard in the determination of pyrrolidone carboxylate. Urine (0.1 ml + 7.15 pg piperidone carboxylate), cerebrospinal fluid (1 .O ml + 7.15 pg piperidone carboxylate), and the protein-free tissue aliquot were each applied to the top of separate Dowex 50 (H+) columns (0.6 X 6 cm). Pyrrolidone carboxylate and piperidone carboxylate which are not adsorbed on Dowex 50 were quantitatively recovered by washing the column with 5 ml H,O. The combined effluents were applied to the top of a Dowex 1 formate column (0.6 x 6 cm). The column was washed with 10 ml water and 10 ml 0.08 M formic acid. Pyrrolidone carboxylate and piperidone carboxylate were eluted with 10 ml 0.25 M formic acid and the eluate evaporated to dryness in a 50 ml round bottom flask using a Buchler flash evaporator. The residue was dissolved in 2.5 ml methanol, quantitatively transferred to a 3-ml ground glass stoppered centrifuge tube, and the methanol removed under a stream of dry nitrogen. The residue was taken for derivatization (see Fig. 1).
-y-GLUTAMYL
CYCLE
INTERMEDIATES
103
The y-glutamyl amino acids were eluted from the Dowex 50 column with 5 ml 3 N NH,OH. The eluate was evaporated to dryness by flash evaporation, the residue was dissolved in 3 ml H,O and applied to the top of a 0.6 X 7.5 cm column Dowex 1 (acetate). The effluent was collected and the column eluted with 1.5 ml 0.1 N acetic acid. The eluate combined with the effluent contains free amino acids as well as y-glutamyllysine. The y-glutamyl derivatives of glycine, cY-aminobutyrate, vaIine, ,&aminoisobutyrate, leucine, and methionine were eluted with 15 ml of 1 N acetic acid. The y-glutamyl derivatives of phenylalanine, tyrosine, and glutamate were eluted with 15 ml 2 N acetic acid. All eluates were separately evaporated in 50 ml round bottom flasks and the residues derivatized directly in the flasks. Figure 1 summarizes the isolation procedure for pyrrolidone carboxylate and y-glutamyl amino acids. Gas Chromatography
of Pyrrolidone
Carboxylate
To the residue in the 3-ml centrifuge tube was added 0.25 ml of a mixture of 20% 2,2,3,3,3-pentafluoro1-propanol in pentafluoropropionic anhydride. The tube was stoppered and heated for 15 min at 75°C in a sand bath. The tube was then cooled and the reagents removed under a stream of dry nitrogen. The reaction was completed by addition of 0.1 ml pentafluoropropionic anhydride and heating for an additional 5 min at 75°C. The excess anhydride was evaporated under nitrogen, the residue dissolved in 1 ml ethyl acetate and an aliquot of this solution diluted 1 : 5 with ethyl acetate for gas chromatographic analysis. Standard mixtures of 0.39-l .29 pg pyrrolidone carboxylate in the presence of 1.43 pg piperidone carboxylate were similarly carried through the entire isolation procedure and derivatized as described above. The derivatives were dissolved in 1 ml ethyl acetate for gas chromatography. Chromatography was performed on columns of 3% OV-17 coated on gas chrom Q 60/80 mesh and 3% DC-200 coated on gas chrom Q 100/200 mesh in a Packard 7400 series gas chromatograph. Both columns were operated isothermally at 125°C. The flow rates of the OV-17 and DC-200 columns were 20 ml/min and 25 ml/min, respectively. The electrometer was 1 X 1O-y A. The inlet port and detector temperatures were both maintained at 190°C. The ratio of peak heights of pyrrolidone carboxylate to piperidone carboxylate in the standards was plotted against the amount of pyrrolidone carboxylate. Quantitation was based on the ratio of the peak heights of pyrrolidone carboxylate to piperidone carboxylate in the unknown sample. Gas chromatography was also performed in a Hewlett-Packard model 7620A gas chromatograph. Three microliters of a I : 5 dilution of the samples was injected on a 6 ft x 4 mm glass column packed with 3% OV- 17. The column temperature was maintained at 120°C. A mixture of
CAABOXYLATE
50 H*
CARBOXYLATE
CARBOXYLATE
FIG. 1. Schematic outline of the fractionation
PIPERIDONE
DISCARD
+
I
formote
fuse ‘4 somplel
IOml 0.25N
Dorex
PYRROLIDONE
\\
Super!atant
IOml O.OBN formote
IOml Hz0
Effl:en’ Doier-I-formote I
5ml Hz0 I
I
l.Oml CSF +7.15~9 PIPERIDONE CARBOXYLATE
PIPERIDONE
0.1 ml urine + 7.15~9
acid
of pyrrolidone
I
-discord
ISOBUTYRATE LEUCINE
GLUTAMATE
TYROSINE
1 2N acetic acid I y-GLUTAMYL DERIVATIVES OF PHENYLALANINE l5ml
carboxylate and y-glutamyl amino acids.
METHIONINE
B-AMINO
WJTYRATE VALINE
a-AMINO
I IN acetic acid I y-GLUTAMYL DERIVATIVES OF GLYCINE 15ml
5 ml 3N NH;OH I Evoporote I Dorex-l-acetate I
I Precipitate
I 15ml 0.1 N acetic acid I y-GLUTAMYLLYSINE
CARBOXYLATE
in 5 ~01s I% pioic
I Ccntrifuqe I
PIPERIDONE
Tissues - homopcnizc
Add 215~9
y-GLUTAMYL
CYCLE
INTERMEDIATES
105
95% argon and 5% methane was used as the carrier gas and the flow rate was 20 ml/min. Separations were also performed on a 4 ft X 4 mm column packed with 1% XE-60 coated on gas chrom Q 60/80 mesh at 160°C and a flow rate of 20 ml/min. Gas Chromatography
of y-Glutamyl
Amino
Acids
The y-glutamyl amino acids were derivatized directly in the ground glass stoppered round bottom flasks. In the first stage of the derivatization procedure 0.5 ml of the alcohol-anhydride mixture was used. In the second step 0.25 ml of the anhydride was used. The stoppered flasks were sealed with parafilm and placed in a sand bath at 75°C as described for pyrrolidone carboxylate. The derivatives were dissolved in 1 ml ethyl acetate and transferred to aluminum foil covered screw capped vials for storage prior to chromatography. y-Glutamyl amino acids were separated on the 3% DC-200 column. The y-glutamyl derivatives of glycine, cY-aminobutyrate, valine, p-aminoisobutyrate, and leucine were chromatographed at 14o”C, the y-glutamyl derivatives of glutamate, methionine, and phenylalanine at 165”C, and the y-glutamyl derivatives of lysine and tyrosine at 185°C. The flow rate in all cases was 50 ml/min. Biological samples containing peaks which interfered with the determination of individual y-glutamyl peptides on the 3% DC-200 column were chromatographed on 3% OV-17. Standards of y-glutamyl amino acids (10 pg) were processed through the procedure. Quantitation was based on comparison of the peak heights of the compounds assayed in the biological sample with the peak heights of the corresponding standards. Processing of y-glutamyl amino acids through the procedure did not lead to the cyclization of the y-glutamyl residue to pyrrolidone carboxylate. RESULTS Pyrrolidone
Carboxylate
Table 1 shows that considerable amounts of pyrrolidone carboxylate are present in mouse kidney, brain, and liver. It is of interest that the concentrations are remarkably similar in all three tissues. Fig. 2 shows chromatograms of pyrrolidone carboxylate in mouse kidney, brain, and liver. Appreciable amounts of the compound are also found in human urine and cerebrospinal fluid. Several studies were undertaken on the recovery of pyrrolidone carboxylate carried through the procedure. [ 14C] pyrrolidone carboxylate (50,000 cpm) was added to the picric acid extracts of tissues and also to the samples of urine and cerebrospinal fluid. An aliquot of the ethyl acetate solution of the final derivative was counted. These experiments
106
WILK
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ORLOWSKI
TABLE
1
LEVELS OF PYRROL~DONE CARBOXYLIC ACID IN TISSUES AND BODY FLUIDS Source Mouse Mouse Mouse Human Human
Number of samples
PCA level f. SE
17 15 1.5 43 29
52.2 nmoles/g k 3.1 58.7 nmoleslg 2 2.3 56.4 nmoles/g f 4.6 237 nmolesimg creatinine 2 17 71 nmolesknl f 13
kidney brain liver urine lumbar spinal fluid
showed that the recovery of pyrrolidone carboxylate consistently exceeded 90%. Similar results were obtained when the peak heights of standard samples of pyrrolidone carboxylate carried through the procedure were compared with the peak heights of the same standards directly derivatized. The recovery of piperidone carboxylate also exceeded 90%. Both compounds behaved identically during ion exchange chromatography and during the derivatizatiou procedure. The derivatives of these two compounds gave single sharp symmetrical peaks that were
LIVER
STANDARDS
KIDNEY
PC
PC
PC
c
1 PCA t
#
1
k
-II 5
IO 0 TIME
5
IO 0
5
IO
(min)
FIG. 2. Gas chromatographic analysis of pyrrolidone carboxylate (PCA) in mouse tissues. PC = piperidone carboxylate (internal standard), 3% OV-17, 12o”C, flow rate = 20 ml/min.
y-GLUTAMYL
CYCLE
INTERMEDIATES
107
1.3 I.1 0.9 PCA pc
-
O.?-
FIG. 3. Standard curve for the quantitation of pyrrolidone carboxylate (PCA). PC = piperidone carboxylate (internal standard). The concentration of PCA is plotted against the ratio of the peak heights of the two compounds.
separated on a number of liquid phases including OV-17, XE-60, and DC-200. This made it possible to use piperidone carboxylate as an internal standard, and thus to avoid the necessity of adding [14C]pyrrolidone carboxylate to the extracts to compensate for any losses during the isolation procedure. When the ratio of peak heights of pyrrolidone carboxylate to piperidone carboxylate obtained from mixtures of these two standards which were carried through the procedure was plotted against the concentration of pyrrolidone carboxylate in the sample, a straight line was obtained (Fig. 3). The overall accuracy of the procedure was determined by analyzing seven samples containing from 0.39 to I .29 pg pyrrolidone carboxylate. The average difference between the estimated and true values was 6.9%. y-Glutamyl Amino Acids Derivatives of the y-glutamyl amino acids chromatographed as single sharp symmetrical peaks on the DC-200 column. By analogy to the structure of the pyrrolidone carboxylate derivative it is believed that the reaction with pentafluoropropanol and pentafluoropropionic anhydride yields N-acyl ester derivatives of the y-glutamyl amino acids. The chromatographic properties of the derivatives suggest that all functional groups have been derivatized. The y-glutamyl amino acids have been fractionated on a Dowex 1 acetate column (Fig. 1). The neutral peptide y-glutamyllysine elutes with 0.1 N acetic acid. The acidic y-glutamyl derivatives of short chain amino acids are tAuted with 1 N acetic
108
WILK
AND ORLOWSKI TABLE
SEPARATION
OF Y-GLUTAMYL GAS-LIQUID
Peptide y-glutamylglycine y-glutamyl cY-aminobutyrate y-glutamylvaline y-glutamyl P-aminoisobutyrate y-glutamylleucine y-glutamylglutamate y-glutamylmethionine y-glutamylphenylalanine y-glutamyllysine y-glutamyltyrosine
2
AMINO
ACID
DERIVATIVES
BY
CHROMATOGRAPHY”
Column temperature (“C)
Retention time (min)
140 140 140 140 140 165 165 165 185 185
6.6 8.0 8.9 11.4 12.7 8.0 10.9 13.8 8.1 8.4
a y-Glutamyl amino acids were derivatized with a mixture of 20% 2,2,3,3,3-pentafluoro-lpropanol in pentafluoropropionic anhydride as described under methods. Derivatives were chromatographed on a 6 ft x 4 mm i.d. coiled glass column packed with 3% DC-200 coated on gas chrom Q 60180 mesh. NZ flow = 50 mlimin. Column temperature as indicated. Inlet and detector temperatures = 190°C.
TIME (min) 4. Separation of 2.5 X lo-l2 moles each of derivatives of the following y-glutamyl amino acids: (1) y-glutamylglycine: (2) y-glutamyl-a-amino butyrate; (3) y-glytamylvaline; (4) y-ghnamyl-cw-aminoisbutyrate; (5) y-glutamylleucine. 3% DC-200, 140°C flow rate = 50 ml/min. FIG.
‘)‘-GLUTAMYL Kjdney
before
CYCLE INTERMEDIATES methiomne
Kidney
1
after methmnine
“-1
-L
5
6-
IO
TIME
109
meth,o”,ne
5
IO
15
(min)
FIG. 5. Formation of y-glutamylmethionine in mouse kidney 30 min after intraperitoneal administration of methionine (5 pmoleslg). 3% OV- 17, 165°C. flow rate = 60 ml/min. The concentration of y-glutamylmethionine is 3.9 nmoles/g.
acid, and the y-glutamyl derivatives of the aromatic amino acids and of glutamate are eluted with 2 N acetic acid. The recoveries throughout the entire procedure have averaged 70%. By combination of ion exchange chromatography and gas-liquid chromatography it is possible to separate all 10 y-glutamyl peptides studied (Table 2). Fig. 4 shows the gas chromatographic separation of five of these peptides on 3% DC-200. The high electron capture response to these derivatives makes it possible to quantitate the low levels of y-glutamyl amino acids formed after amino acid loading (19). Thirty minutes after intraperitoneal administration of methionine to mice (5 pmoleslg) we detected the presence of y-glutamylmethionine in mouse kidney (Fig. 5). Similarly 30 min followKldney
before
phenylalanme
5
IO
Kidney
after
phenylalonine
I
0
I5 TIME
0
5
IO
15
(min)
FIG. 6. Formation of y-glutamylphenylalanine in mouse kidney 30 min after intraperitoneal administration of phenylalanine (5 pmoleslg). 3% OV-17. 165°C. flow rate = 60 ml/min. The concentration of y-glutamylphenylalanine is 8.1 nmoles/g.
110
WILK
AND
ORLOWSKI
ing administration of phenylalanine (5 @moles/g) to mice, y-glutamylphenylalanine in mouse kidney was found (Fig. 6). These results were confirmed on 1% XE-60 and 3% DC-200. DISCUSSION The quantitative determination of pyrrolidone carboxylate and y-glutamyl amino acids is of considerable importance in studies on the synthesis, degradation, and metabolic function of glutathione. The small concentration of pyrrolidone carboxylate in animal tissues and the lack of analytical procedures for the determination of y-glutamyl amino acids prompted the present study on the application of gas chromatography coupled with electron capture detection for the determination of these compounds. Derivatives of pyrrolidone carboxylate and y-glutamyl amino acids were prepared using a mixture of pentafluoropropanol and pentafluoropropionic anhydride. This reagent which reacts with carboxyl, amino, and hydroxyl functional groups was previously applied for the preparation of derivatives of biologically important acids (13). The response of the electron capture detector to these derivatives is very high thus permitting the determination of low concentrations of these intermediates in tissues. Purification of pyrrolidone carboxylate and of y-glutamyl amino acids by ion exchange chromatography eliminates interference from other compounds in the analyzed sample. In earlier attempts to use gas chromatography for the determination of pyrrolidone carboxylate, its methyl ester derivative was prepared either in a reaction with a mixture of dry hydrogen chloride and methanol (12) or in a reaction with diazomethane (1 l), and determined using the flame ionization detector. This derivative was also used for the detection of pyrrolidone carboxylate in the urine of patients with skin burns (20). The method was successfully employed for the detection in urine and plasma of the markedly elevated concentrations of pyrrolidone carboxylate in patients with pyroglutamic aciduria, an inborn error of metabolism in which large amounts (25-40 g daily) are excreted in the urine (11,2 1). This procedure was also applied to the determination of pyrrolidone carboxylate in the human epidermis where its concentration amounts to an average of 1.6% of the tissue weight of the horny layer (22).
The determination of pyrrolidone carboxylate as the methyl ester derivative using flame ionization detection is not sufficiently sensitive to quantitate the small amounts of pyrrolidone carboxylate present in extracts of normal tissues and in normal body fluids. The limit of detectability of this method has been reported as 10 pg/ml (or 77 nmoles/ml) (21) which exceeds the average concentrations found in normal tissues. This limitation is eliminated by the method presented here with its much
y-GLUTAMYL
CYCLE
INTERMEDIATES
111
higher sensitivity. Moreover the introduction of piperidone carboxylate as internal standard compensates for losses during the isolation and derivatization procedure as well as for spontaneous changes in detector sensitivity. In an earlier study (l), we documented the occurrence of free Lpyrrolidone carboxylic acid in body fluids and tissues using a similar but more complex gas chromatographic procedure. This method has been simplified by substitution of a single Dowex 1 formate form column for the Dowex I chloride and DEAE sephadex-chloride columns previously employed. This has resulted in an enhanced recovery of pyrrolidone carboxylate. Moreover introduction of piperidone carboxyiate as internal standard has enhanced the reproducibility of the method. The present values of urinary pyrrolidone carboxylate (237 nmoles/mg creatinine are in good agreement with those reported in our earlier publication (190 nmoleslml urine) since the average concentration of creatinine in urine is close to 1 mg/ml. Similarly the pyrrolidone carboxylate levels of this new group of CSF samples are close to those previously reported. Tissue levels of pyrrolidone carboxylate, were determined in the earlier publication for the guinea pig and therefore are not directly comparable to the values given here for mouse tissue levels. Pyrrolidone carboxylate has been determined in mouse tissues by Van der Werf et ul. (2). In their procedure tissue samples were purified by passage through Dowex 2 chloride and Dowex 50 columns. The eluate containing pyrrolidone carboxylate was hydrolyzed to glutamate either enzymatically or by hydrochloric acid. Glutamate was then determined using an amino acid analyzer. Their values were somewhat lower than the values reported here. The differences may be due to the different strains of mice used in the two studies. On the other hand, Wolfersberger et al. (23) determined pyrrolidone carboxylate in guinea pig tissues by passage of a deproteinized tissue extract through a Dowex 50 column followed by acid hydrolysis of the effluent at 100°C. The glutamate formed was determined by the ninhydrin procedure. Their values for guinea pig tissues are 10 to 80-fold higher than those obtained by us by gas chromatography (1). It is obvious that the method of Wolfersberger et al. is not specific for pyrrolidone carboxylate and that any acidic peptide not retained by the column would yield ninhydrin positive material thus yielding spurious values. The availability of a method for the determination of y-glutamyl amino acids should prove to be useful for the study of these compounds with respect to their significance in mammalian metabolism. Thus we have detected small amounts of y-glutamyl derivatives in mouse tissues after administration of the corresponding free amino acids. Although these compounds give a positive ninhydrin reaction and thus can be detected
112
WILK
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using the amino acid analyzer, this method would not be sufficiently sensitive to detect some of the derivatives (e.g., y-glutamylphenylalanine and y-glutamylmethionine) that are formed only in very small amounts of several nanomoles per gram. It remains to be established whether the increase in the sensitivity of amino acid detection by the use of reagents such as a fluorescamine (24) could solve this analytical problem. Since the elution pattern of many y-glutamylamino acids in an amino acid analyzer overlap with the elution pattern of many amino acids (23, a preliminary separation of this class of compounds from other amino acids on anion exchange resins (e.g., Dowex 1 acetate) would probably be required. ACKNOWLEDGMENTS This work was supported in part by Grants from the National Institute of Arthritis, Metabolism and Digestive Diseases (AM 17113), and from the Health Research Council of the City of New York. We thank Miss Charlene Michaud for her untiring and skillful assistance.
REFERENCES 1. Wilk, S., and Orlowski, M. (1973) FEBS L&t. 33, 157-160. 2. Van der Werf, P., Stephani, R. A., and Meister, A. (1974) Proc. Nat. Acad. Sci. USA 71, 1026-1029. 3. Meister, A., Bukenberger, M. W.. Strassberger, M. (1963) Biochem. 2. 338, 2 17-229. 4. Orlowski, M., and Meister, A. (1971) in The Enzymes (Boyer, P. D. ed.), Vol. 4, pp. 123-151, Academic Press, New York. 5. Connell, G. E., and Hanes, C. S. (1965) Nature (London) 177, 377-378. 6. Bloch, K. (1949) J. Biol. Chem. 179, 1245-1254. 7. Hanes, C. S., Hird, F. J. R., and Isherwood, F. A. (1950) Nature (London) 166, 288-292. 8. Binkley, F. (1954) in Glutathione: A symposium (Colowick, S. et al., eds.), p. 160, Academic Press, Inc., New York, N. Y. 9. Orlowski, M. (1963) Arch. Immunol. Thu. Exp. 11, I-61. 10. Orlowski, M. and Meister, A. (1970) Proc. Nat. Acad. Sci. USA 67, 1248-1255. 11. Jellum, E., Kluge, T., Borresen, H. C., Stokke, 0.. and Eldjarn, L. (1970) Stand. J. Clin. Lab. Invest. 26, 327-335. 12. Polgar, P., and Meister, A. (1965) Anal. Biochem. 12, 338-343. 13. Watson, E., Wilk, S., and Roboz, J. (1974) Anal. Biochem. 59, 441-451. 14. Orlowski, M., Richman, P. G., and Meister, A. (1969) Biochemistry 8, 1048-1055. 15. Dieckman, W. (1905) Berichte Deut. Chem. Ges. 38, 1654-1661. 16. Orlowski, M., and Meister, A. (197 1) J. Biol. Chem. 246, 7095-7 105. 17. Orlowski, M., and Meister, A. (1971) Biochemistry 10, 372-380. 18. JatIe, M. (method of) (1965) in Hawk’s Physiological Chemistry (Oser, B. L. ed.). pp. 1040-1042, McGraw Hill, New York. 19. Orlowski. M. and Wilk, S. (1975) Eur. J. Biochem. 53, 581-590. 20. Tham, R., Nystrom, L., and Holmstedt, B. (1968) Biochem Pharmacol. 17, 1735-1738. 21. Eldjarn, L., Jellum. E.. and Stokke. 0. (1972) C/in. Chim. Acta 40, 461-476.
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22. Marstein, S., Jellum, E., and Eldjarn, L. (1973) C/in. Chim. Actu 49, 389-395. 23. Wolfersberger, M. G., Tabachnick, J., Finkelstein, B. S., and Levin, M. (1973) J. fnvest. Dermatol.
60, 278-28
1.
24. Udenfriend, S.. Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M. (1972) Science 178, 87 I-872. 25. Zacharius, R. M.. and Talley, E. A. (1962)Anal Chem 34, 1551-1556.