ANA1

YTICAL

64, 38Y-‘t()?

BIOC‘HtMISTRY

(lc)75)

A Dansylation

Microassay

Amino H.

MICHAEL

JOSEPH’

A method is described for glutamine, glutamate, asparate. is an extension [:‘H]dansyl chloride

of

a method t 15) with

standards to allow correction An improved chromatographic timates

of the contents

compared with using autoanalytical

those

Acids

the

the for

of these obtained and other

in Brain

AND

determination and taurine based addition

on

JOHN

HAI.LIDAY’

of y-amino in small samples the formation of “C-labeled

incomplete separation amino

for Some

acids

butyric acid. glycine. of brain tissue. This of amino

derivatives with acids as internal

and variable of dansyl-taurine

degrees

in rat cortical

tissue

on an autoanaly\er. methods.

and

of dansylation. employed.

is

with

are

those

reported

in the

Esand

literature

A neurotransmitter role has been proposed for a number of amino acids found in large amounts in the free amino acid pool of the CNS. Krnjevic ( 1) has summarised the evidence suggesting that GABA” and glutamic acid, its biochemical precursor, are inhibitory and excitatory neurotransmitters respectively; metabolically these amino acids are related to glutamine and aspartic acid which also occur in large amounts in the CNS. A neurotransmitter role has also been proposed for aspartate and glycine (2). There is now an extensive literature on amino acid transmitters, which have been the subject of a number of recent reviews (3-8). More recently, taurine, which occurs in the brain in amounts second only to glutamate. has also been proposed as a transmitter candidate (9). Analysis of brain amino acids on an autoanalyser can be performed on 100 mg of tissue (10). A method involving the thin layer chromatography of dinitrophenyl derivatives of amino acids has been described ( 11) and can be used with 10 mg of brain tissue. More sensitive tech’ Present

address:

Harrow. Middlesex. L Present address: W.C.2. :( Abbreviations amino

butyric

acid:

Division

of

Pharmacology used: S.A..

Dansyl. specific

Psychiatry. Department.

Clinical

Research King’s

5-Dimethylaminonaphthalene-\ulphonyl: activity.

College.

Centre. The

Watford Strand.

Road. London.

GABA.

y-

390

JOSEPH

AND

HALLIDAY

niques using fluorescence autoanalysis ( 12). gas-liquid chromatography (13). and glc-mass spectrometry (14) have also been described. The dansylation” technique ( 1.5) was originally described as a highly sensitive method for end-group analysis of proteins or peptides ( 16). and it has been modified to provide a highly sensitive method for the determination of free amino acids in tissues or in protein hydrolysates (17-22). Quantification of the dansyl amino acid derivatives has depended on fluorimetric assay (I 8,19) or the use of [:‘H] or [“Cldansyl chloride (20-22). The yield of the dansylation reaction for each amino acid must also be estimated. and these authors have used parallel experiments with standard amino acids. Many factors influence the yield: side reactions occur, and sufficient reproducibility of reaction conditions is difficult to achieve with the small volumes used in a microassay. In the procedure to be described, these difficulties are overcome by the use of [“~H]dansyl c hl ori d e with ‘-‘C-labeled amino acids as internal standards to monitor the yields of the respective dansyl derivatives. No sophisticated apparatus is required other than a two channel liquid scintillation counter. A brief account of the estimation of glutamate and GABA by a similar double isotope method applied to superfusate of cortical slices has recently appeared (23). Also, during the preparation of this paper a similar method and its application to the determination of amino acids in a tryptic peptide has been described (24). METHODS

Radiochemicals were obtained from the Radiochemical Centre. Amersham. Other chemicals. Analar grade where available, were obtained from British Drug Houses (BDH). Poole. Drummond “Microcap” disposable micropipettes (Shandon Scientific. London) and Oxford Microsamplers (Boehringer, London) were used to dispense small volumes. Micropolyamide thin layer chromatography (tic) plates (F 1700. Schleicher and Schull) were obtained from Anderman. London.

The brain sample (up to 10 mg) was rapidly frozen in liquid nitrogen and subsequently homogenised by hand in a conical Eppendorf reaction tube (vol 1.5 ml) in 10 vol 0.48 M cold perchloric acid. The homogenate was centrifuged at 10.000~ for 20 min at 4°C; 10 PI of the supernatant was transferred to a reaction tube (2 x 17 mm i. d.; made from Pyrex glass tubing). Blanks (IO ~1 0.48 M perchloric acid) and standard amino acid mixture (see Results) were also carried through the method rou-

MICROASSAY

FOR

AMINO

ACIDS

391

amino acids continely. Two microliters of a mixture of “C-labeled taining glutamic acid (30.8 nCi: S.A. 785 nCi/nmole). aspartic acid (30.8 nCi: S.A. 134 nCi/nmole), glutamine (15.4 nCi; S.A. 48 nCi/nmole), glycine ( IS.4 nCi/: S.A. 114 nCi/nmole). and GABA (1.4 nCi: S.A. --“8 nCi/nmole) was added and carefully mixed. Two microliters of 1 M potassium carbonate was then added and mixed. the tubes cooled to 4°C. and the precipitated potassium perchlorate centrifuged down. [“Hldansyl chloride of the required specific activity was prepared by mixing [:‘H]dansyl chloride (5.1 mCi/pmole: 250 $I’i/ml) in benzene with a 2 mg/ml (7.4 mM) solution of dansyl chloride in acetone in the ratio 4: 1 (v/v). The final specific activity of the dansyl chloride was verified by measuring the radioactivity and the optical density at 369 nm of a suitable dilution. Ten microliters of the diluted [“Hldansyl chloride solution was placed in a reaction tube and evaporated to dryness under a stream of nitrogen at 35°C. The residue was taken up in 2 ~1 acetone (the specific activity was not affected by drying and redissolving). and ? ~1 of the above tissue extract containing the “C-labeled amino acid internal standards and the potassium carbonate was added. The contents of the tube were mixed and incubated at room temperature for 30 min in the dark. Scaled up control experiments indicated that the pH during the incubation was maintained between 9 and 9.5. The contents of the reaction tube were then evaporated to dryness under a stream of nitrogen at 37°C and the residue taken up in 5 ~1 of a mixture of acetone and acetic acid (3: 2 v/v). After centrifugation a known volume (approx 0.3 ~1) of the supernatant was applied under a stereomicroscope near to one corner of a micropolyamide tic plate (3.5 X 3.5 cm) with a fine calibrated microcapillary using a stream of cold air. Chromatography was carried out in small covered tanks (Quickfit weighing bottles. base diam 4.5 cm) essentially as previously described (20. 15, 26). The plates wet-e developed in the first dimension in formic acid (1.5% v/v). dried carefully. and developed in the second dimension in benzene-acetic acid (9: 1 v/v). An extended development (with an uncovered tank) or a second development in the second dimension in benzene-acetic acid was necessary to adequately resolve dansyl-glutamate and dansyl-aspartate. For the estimation of taurine. the single development in the second dimension was in “880” ammonia (30% v/v: about 10% ammonia w/w). The dansyl derivatives on the developed chromatograms were visualised under ultraviolet light (Fig. 1). and were identified by comparison with a previouxly prepared map of Ih dansylated amino acid standards. Where the amount of dansylated amino acid was too small to be detected under the ultraviolet light (e.g.. dansyl-GABA in blank dcterminations) appropriate standard dansylated amino acids were applied at the

392

JOSEPH

AND

HALLIDAY

(a)

lb)

id)

3t

2 tt

-

1

1

(e) F~ti. I. Developed thin layer- chromatograms of dansylated standard amino acids: (a). (h): and of dansylated extract from rat brain: (c), td). Plates (b). Cd) sectioned vertically after running in first dimension. Solvent: I. Formic acid, l.5?4 (v/v): 2. benzene-acetic acid,

9: I (v/v):

3. “880”

ammonia,

30%

(v/v).

MICROASSAY

FOR

AMINO

393

ACIDS

origin before the sample and cochromatographed to locate the spots. The spots on the chromatogram corresponding to the amino acids to be estimated were ringed with pencil, cut out. and placed in a scintillation vial. One milliliter of 10% hyamine hydroxide in methanol was added to elute the dansyl derivative followed by IO ml of toluene scintillant (4.15 g PPO (2,5-diphenyl oxazole) and 0.1 I1 g dimethyl POPOP ( I .4-di(4-methyl-5-phenyloxazol-2-yl)-benzene) per litre of toluene). :‘Hand ‘Y-activities were estimated using preset channels on a Nuclear Chicago Mark 11 scintillation counter. counting efficiencies being determined by the external standard channels ratio method. The ratio (R) of “H- to “C-activities in each dansyl amino acid will depend only on the specific activity of the dansyl chloride and on the specific activity of the amino acid reacting with it. The latter will be determined by the extent of the dilution of the added (exogenous) amount of “C-labeled amino acid by the (endogenous) amount of the amino acid present in the tissue extract. Thus for a monodansylated derivative R =

specific activity of dansyl chloride (SAD(.) specific activity of amino acid reacting with it

_ S&x . (AAm A A :/:

+ AA, 0.99 for each amino acid in each experiment. Table I also gives the mean slopes and intercepts from four separate assays. As discussed in Methods the slopes and intercepts can also be predicted from the specific activity of the [:‘H]dansyl chloride, the specific activity of the “C-labeled amino acids. and the amount of “C-labeled amino acid added. It can be seen in Table I that the observed mean slopes (co]. 3) agree reasonably with the predicted values (co]. 6). Omission of either ‘Y-labeled amino acids or

398

JOSEPH

DILUTION

OF BRAIN

AND

HALLIDAY

TABLE 2 E~FRACT. RECOVERY. AND APPROXIMAI-F. OF DANSYLATION REACTION

YIELD

[“Hldansyl chloride from a blank determination results in a lack of detectable activity at the spots corresponding to the dansyl derivatives of the amino acids under study. located with standard unlabeled dansyl amino acids. Thus the 3H/‘4C ratios obtained by carrying a blank through the method should represent only the amount of amino acid added with the “C-labeled amino acids. (This amount as a proportion of that found in brain extract is shown in Table 2 (col. I) since it is important that the amount of internal standard should be small relative to the amount of endogenous material.) However, the observed blank values (Table 1, col. 4) (which agree with the intercepts of the calibration curves (col. 3), since the correlation coefficient is so high), are somewhat higher than these predicted values (col. 5).” This observation could be accounted for by an inaccuracy in the quoted specific activities of the ‘“Clabeled amino acids, although this seems unlikely. (The specific activity of the dansyl chloride is verified by the agreement of observed and predicted slopes.) These considerations, however, do not invalidate the determination of amino acids by reference to the standard curves. Results are presented as ~moles/lOO mg protein and pmoles/g wet wt assuming 100% recovery of amino acids in the perchloric extract. Dilution of acid extract from brain results in a proportionate fall of the “H/‘-C ratio, and addition of standard amino acids to brain results in an additive increase. The results of a typical experiment are given in Table 2 (~01s. 2-6); also the recovery of the dansyl-amino acids (~01s. 7.8) es3 As a proportion amount to: GABA

of the amino acid levels found in rat brain. these unexplained blanks X’S, glycine IS%, glutamine 3%. glutamate 1%. aspartate 45%.

MICROASSAY

MlNO

r /

ACIDS

400

JOSEPH

COMPARISON

AND

TABLE METHOD

OF DANSYL ~molesig

wet

HALLIDAY

3 WITH

AUTOANALVSER

wt

RESULTS”

~moles/lOO

mg protein

~___~. Autoanalyset GABA Glycine

2.24 0.85

Glutamine Glutamate Aspartate

the

” Parallel text.

analysis

t i

Dansyl

chloride

.____

Autoanalyset

Dansyl

chloride

0.39 0.07

2.61 0.90

-t 0.27 f 0.17

1.49 0.57

k 0.30 t 0.08

1.74 0.60

t 0.16 iz 0.13

6. IO 2 0.57 II.38 2 0.84 7.24 + 0.35

7.09 10.86 2.34

i 0.64 i 0.59 -t 0.45

4.07 7.58 1.69

-c 0.50 t 0.79 5 0.27

4.71 7.20 I.55

iz 0.47 t 0.35 t 0.32

of perchloric

extracts

of corticer

from

rat brain

(II = 8) as described

in

timated from the 14C-activities observed on chromatograms from standards and brain samples. The mean levels of the six amino acids estimated in cortex from male Piebald Viral Glaxo (PVG) rats (200-250 g) are compared with results obtained in other studies in Table 3. It can be seen that results in the present study are generally comparable with, although somewhat higher than, those obtained in previous studies. Some previous studies have underestimated the content of glutamine due to partial destruction during column chromatography at 60°C. The higher levels of taurine found in these animals is confirmed by a recent report (3 1). employing the (Iphthalaldehyde reaction, of rat cortex taurine levels of 8.9 & 0.8 pmoles/g wet wt. The marked variation in taurine levels with age in the rat will contribute to the wide range of levels quoted in the literature. The results of parallel analysis of cortices from male PVC rats (200-250 g) by the dansylation technique and by an autoanalytical technique (28) are shown in Table 4. Results are given in terms of pmoles/g wet wt and pmoles/ 100 mg protein. These results confirm that the present technique gives values consistent with autoanalyser results, but on a small fraction of the tissue. Our studies have shown that in rat brain, amino acid levels in frontal cortex are higher than those in parietal cortex (Emson and Joseph, in preparation), and this together with differences in the animals (age, body weight, strain, and diet) could account for the differences observed in Table 3 between the present and previous studies. CONCLUSION

As described the method is suitable for amounts of tissue down to 1 mg. There is scope for further reduction in quantity since only about 1% of the initial amino acid extract is finally applied to the tic plate. The method has been applied to the most abundant amino acids in rat brain,

MICROASSAY

FOR

AMINO

40 I

ACIDS

however, the same principles should be applicable to the determination of other amino acids or other substances reacting with dansyl chloride. The use of ‘“C-labeled amino acid internal standards allows the amino acids to be estimated accurately in spite of variable yields due to side reactions and the formation of secondary dansylated products (e.g., from glutamate-see above. and GABA - Ref. 17). The problem of having to quantify the amount of material in a volatile solvent applied to the tic plate is also avoided. Taurine, being a p-amino acid, gives a more reliable yield on dansylation ( 17). and this study has shown that it can be quantitatively estimated when separated chromatographically from dansyl hydroxide. ACKNOWLEDGMENTS Financial support r.1. H.) is gratefully and the

Dr. S. R. automated

from the Epilepsy Research acknowledged, as are helpful

Snodgrass. amino acid

Thanks analyses

are also on Dr.

Fund (M. discussions

H. J.) and the McRobert Trust with Dr. T. B. B. Crawford

due to Dr. J. S. de Bellcroche who A. J. Thomas’ equipment at Imperial

performed College.

London.

REFERENCES I. Krnjevic, K. ( 1970) Ntrtrrrc (f.

A dansylation microassay for some amino acids in brain.

ANA1 YTICAL 64, 38Y-‘t()? BIOC‘HtMISTRY (lc)75) A Dansylation Microassay Amino H. MICHAEL JOSEPH’ A method is described for glutamine, gluta...
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