ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978
951
Double-Enzymatic Assay for Determination of Glutamine and Glutamic Acid in Cerebrospinal Fluid and Plasma I a n F. Pye,' Clive Stonier, and Edward H. F. McGale" Medical Research Unit, North Staffordshire Hospital Centre, Hartshill, Stoke-on-Trent, Staffordshire ST4 7NY, England
Two different analytical procedures for quantitative determination of glutamine concentration in cerebrospinal fluid and plasma have been compared. A double-enzymatic procedure using Lglutamlne aminohydrolase (glutamine glutamic acid) and L-g1utamate:NAD oxidoreductase (glutamic acid 2oxoglutamate) was found to give accurate and reproducible measurement of glutamine concentration. The between-batch relative standard deviation of the method was 2.1 % (for CSF) and 1.9% (for plasma). Recovery of glutamine added to CSF and plasma prior to deprotelnization and assay was 95.5% and 96.9 % , respectively. I n contrast, unsatisfactory results were obtained using an automatic amino acid analyzer to quantitate this amino acid.
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During a study of t h e interrelationship of the free amino acid composition of cerebrospinal fluid (CSF) and plasma, we found that accurate determination of glutamine concentration was not possible using data obtained from a dual-column automatic amino acid analyzer (1). Previously, we had reported similar problems using a single-column analyzer ( 2 ) . T h e problem of accurate determination of glutamine concentration has been reported by other workers (3-5) and may explain t h e wide variation in values for CSF glutamine concentration reported in t h e literature (6-10). In the present study, we have evaluated a double-enzymatic method for glutamine determination and compared the results with those obtained by automatic amino acid analysis. We found accurate and reproducible results using the enzymatic procedure, whereas t h e automated method had inherent limitations which produce serious errors in glutamine measurement. T h e results obtained by both methods are compared in t h e present study.
Table I. Summary of Analytical Procedure for Determination of Glutamine and Glutamic Acid Concentration b y t h e Double Enzymatic Assay Glutamine 4 glutamic acid 0.2 m L deproteinized plasma, CSF or standard 0.2 m L glutaminase in acetate buffer (pH 4.90) Incubate at 37 "C for 4 5 min 0.2 m L incubate 2.0 m L hydrazine-glycine buffer 0.1 m L ADP solution 0.1 m L NAD solution 0.02 m L glutamate dehydrogenase solution Incubate at 20 "C for 4 5 min Measure fluorescence ( F , )
Glutamic acid 0.2 mL deproteinized plasma, CSF or standard 2.0 m L hydrazine-glycine buffer 0.1 m L ADP solution 0.1 m L NAD solution 0.02 m L glutamate dehydrogenase solution Incubate a t 20 "C for 45 min Measure fluorescence ( F , )
Enzyme blank. Hydrazine-glycine buffer, ADP, NAD and glutamate dehydrogenase. Mix as above, incubate (20 "C for 4 5 min) and measure fluorescence ( F 3 ) . Sample blank. Deproteinized sample (0.2 mL), hydrazine-glycine buffer, ADP and NAD; mix as above, incubate ( 2 0 "C for 4 5 min) and measure fluorescence ( F 4 ) . Reagents. (a) Sodium acetate (0.5 M) adjusted to pH 4.90 with 1.0 M acetic acid and containing 1 unit mL-' of glutaminase
EXPERIMENTAL Methods. S p e c i m e n Deproteinization. Freshly collected venous plasma and CSF specimens were deproteinized ( 2 1 ) by addition of sulfosalicylic acid (0.03 g mL-'), centrifuged (24 000 g, 20 min, 4 "C) and the supernatant used for amino acid analysis immediately or stored at -20 "C prior to analysis. For the double-enzymatic assay, a separate aliquot of the same plasma or CSF specimen (0.5-1.0 mL) was mixed with an equal volume of 1.0 M perchloric acid (standarized by titration with 1.0 M sodium hydroxide), and centrifuged as above. The supernatant was neutralized with 0.9 M potassium hydroxide and stored a t -20 "C prior to analysis. Amino Acid Analysis. This was carried out using a six-sample discrete flow dual-column amino acid analyzer (Model JLC 6AH: Jeol Ltd., 1418 Nakagami, Tokyo, Japan). Details of the analytical procedure have been reported elsewhere ( 1 1 ) . Deproteinized plasma specimens were diluted fivefold prior to analysis; deproteinized CSF specimens were analyzed undiluted. Enzymatic Assay for Glutamine and Glutamic Acid. This was performed by a fluorimetric procedure developed from the methods of Nahorski (12) and C. Record (personal communication).
(L-glutamine aminohydrolase, E.C. 3.5.1.2., from Sigma Chemical Co., Ltd., London); (b) Hydrazine-glycine buffer: Hydrazine sulfate (52 g L-') and glycine (38 g L-') adjusted to pH 8.60 with 5.0 M sodium hydroxide; (c) ADP, 2.0 mmol L-'; (d) NAD, 10.0 mmol L-I, prepared fresh on the day of use; (e) glutamate dehydrogenase, 450 units mL-' (L-g1utamate:NAD oxidoreductase, E.C. 1.4.1.2.,from Boehringer Co., Ltd., London). The analytical procedure is summarized in Table I. The reduction of NAD was measured using a Hitachi Model 203 Fluorescence Spectrophotometer (Perkin-Elmer, Ltd., Beaconsfield, Kent, England). The excitation wavelength used was 365 nm and the analyzer wavelength was 460 nm. Standard solutions of glutamine (containing 500 and 1000 pmol L-') and a control solution consisting of an aliquot of a pooled deproteinized CSF or plasma specimen were analyzed with each hatch of CSF and plasma specimens. The instrument zero was set using a blank solution consisting of hydrazine-glycine buffer (2.0 mL), ADP (0.1 mL), NAD (0.1 mL); the instrument 1007~was set using a quinine sulfate solution. Calculation. For glutamine concentration in sample, standard or control, corrected fluorescence (70r ) = F1 - F2/2. For glutamic acid concentration, corrected fluorescence = ( F , - F3 - F4)/2.The concentration of glutamine or glutamic acid in the deproteinized sample was calculated by direct proportion to the standard glutamine corrected fluorescence. Under the conditions of assay, there is a stochiometric conversion of glutamine to glutamic acid following incubation with glutaminase, so that either amino acid may be used as a standard for the method.
Present address, Department of Neurology, Leicester Royal Infirmary, Leicester, LE 5WW, England.
RESULTS AND DISCUSSION C o m p a r i s o n of A m i n o Acid A n a l y z e r a n d E n z y m a t i c R e s u l t s f o r g l u t a m i n e Concentration. Figure 1 compares
0003-2700/78/0350-0951$01 .OO/O
0 1978 American Chemical Society
952
ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978
Table 11. Recovery of Glutamine Added t o Plasma a n d CSF Prior t o Deproteinization and Assay Using the Double Enzymatic Methoda
Specimen Plasma Plasma Plasma I
.
.
230
r30
, 6CC
I
. 830
.
I
ZC3
w m t w CM
. UCI
. iC?
Figure 1. Comparison of CSF (a) and plasma (b) glutamine concentrations measured by amino acid analysis (X) and enzymatic procedure (Y). The ideal (- - - ) and actual (-) linear regression lines are indicated. For CSF: Correlation coefficient = 0.57, Slope (X/Y) = 0.65, Intercept (on Y axis) = 267 pmol L-', No. of observations = 21. For plasma: Correlation coefficient = 0.42, Slope (X/Y) = 0.24, Intercept (on Y axis) = 502 pmol L-', No. of observations = 28
620 825 1025
CSF
0
CSF CSF CSF
200 400 600
266 461 626 860
Recovery, '/c
... 93.8 98.1 98.8 Mean = 96.9 S.D. = 3.9
...
97.5 90.0 99.0 Mean = 95.5 S.D. = 4.8 a All recovery values are the mean of two determina tions.
c L L ? 3 c s : s v : r
C O R R E C T E , P C A
