ANALYTICAL
BIOCHEMISTRY
A Simple
SHINJI Fuculty
90, 662-670 (1978)
and Specific Determination Biological Samples
of Glycine
in
OHMORI. MIKIKO IKEDA, YOKO WATANABE, AND KAZUHIRO HIROTA
of Pharmacrutical
Sciencrs. Okayamu University, Tsushimu-Nrtka-I. Japan
Oknyama
755
Received May 18, 1978 A simple specific assay was developed for the determination of glycine in a solution containing other amino acids. Hippuric acid was obtained after reacting glycine with benzoyl chloride and was extracted with ethyl acetate. It was then reacted with acetic anhydride. p-dimethylaminobenzaldehyde, and pyridine for color development. The amount of glycine (I to 100 pg) in the original solution could be determined by measuring the absorbance (458 nm) of this chromogen. This procedure was applied on an amino acid mixture, urine, serum, blood. and liver homogenate.
Since Fisher (1) isolated glycine from casein hydrolysate as ethyl glycinate with 79% yield, this amino acid has been determined by methods based on gravimetry (2,3), calorimetry (4-9), or enzymatic analysis (10). Some of these methods have been applied for the determination of glycine in biological samples (6) or in protein hydrolysates (5,9). However, they were inadequate for practical use because sensitivity (6) or specificity (5.9) of the chemical reaction was low. Recently, glycine has been widely determined using an amino acid analyzer as a dependable method. However, this procedure is quite time-consuming when glycine concentration should be determined in a solution containing other amino acids. Calorimetric determination of hippuric acid in urine or rat liver homogenate was established in our previous paper (11). This method was based on the color reaction of hippuric acid with acetic anhydride, pyridine, and p-dimethylaminobenzaldehyde (12). In this paper, we have applied this method for the determination of glycine in urine, serum, blood, or rat liver homogenate as well as in the mixture of common amino acids and established a highly sensitive and specific assay of glycine in such samples. CHEMICALS
AND INSTRUMENTS
Peptides were purchased from the Protein Research Foundation (Osaka) and Sigma Chemical Company. All other reagents (analytical grade) were 0003.2697/78/0902-0662$02.00/O CopyrIght G 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
662
DETERMINATION
OF GLYCINE
663
obtained from Wako Pure Chemical Industries Ltd. (Osaka). Urine, serum, and blood were supplied by the people in our laboratory. Copper-ammonia reagent was prepared by mixing 1 M cupric sulfate with 2.8% ammonia solution (1:3, v/v). Amino acid analysis was carried out in a Hitachi KLA-5 amino acid analyzer (column size, 9 x 550 mm) at 55°C using sodium citrate buffer (pH 3.25 and 4.25). Norleucine was used as an internal standard. Visible absorption spectrum was taken in a Hitachi 124 spectrophotometer. The solvent was evaporated under reduced pressure in an evaporator equipped with a mechanical vibrator (Taiyo Scientific Instrument Co. Ltd., Tokyo). Eight samples were treated at the same time. Benzoylation was carried out in the same apparatus. A Vortex mixer (Ikemoto Rikakogyo Co. Ltd., Tokyo) was also used for vigorous mixing of the sample. Thin-layer chromatography was performed on silica gel (Wako-gel B-SFM); Solvent 1, toluene:acetic acid:water (3:2: 1, v/v/v): Solvent 2, n-butanol:acetic acid:water (4: 1:5, v/v/v). EXPERIMENTAL
Glycine in Amino Acid Mixture A sample solution (0.1 ml) was mixed with 1 N NaOH (1.0 ml) and benzoyl chloride (50 ~1) in a 20-ml test tube. After 15 min of vigorous stirring at 4o”C, the mixture was acidified with 0.15 ml of 43% phosphoric acid and extracted with 5 ml of benzene-n-hexane (1: 1, v/v). The organic layer containing benzoic acid was removed thoroughly by aspiration and evaporation. Hippuric acid in the aqueous layer was extracted into 7 ml of ethyl acetate. An aliquot (1.4 ml) of the ethyl acetate layer was transferred to another test tube and was evaporated to dryness under reduced pressure in the evaporator. The concentration of hippuric acid was determined calorimetrically following our previous report (11) except that the concentration of p-dimethylaminobenzaldehyde was modified to avoid hippuric acid-independent color development in the original concentration. The dried residue obtained above was dissolved in 1.0 ml of acetic anhydride and the solution was vigorously mixed with 2.0 ml of 0.4%p-dimethylaminobenzaldehyde solution in pyridine. After incubation at 40°C for 1 h, the absorbance was measured against a blank solution containing acetic anhydride and 0.4% p-dimethylbenzaldehyde in pyridine. Glycine in Urine Prior to benzoylation, endogenous hippuric acid present in urine had to be removed. Urine (0.25 ml) was mixed with water (0.25 ml) and 6 N HCl (50 ~1) in a lo-ml centrifuge tube. Hippuric acid in the aqueous solution
664
OHMORI
ET AL.
was extracted with 5 ml of ethyl acetate previously saturated with water under vigorous mixing and the mixture was centrifuged at 1OOOgfor 10 min. The ethyl acetate layer was discarded, and the aqueous layer was again extracted with ethyl acetate, followed by centrifuging and discarding. The aqueous layer (0.1 ml) thus obtained was treated as described under Glycine in Amino Acid Mixture. Glycine in Serum Serum (0.3 ml) was diluted with water (0.7 ml) in a lo-ml centrifuge tube and was mixed with 30% metaphosphoric acid (50 ~1) and 5 ml of chloroform-ethanol (2: 1, v/v). After shaking vigorously for 30 s, the mixture was centrifuged at 1OOOgfor 10 min. The aqueous upper layer (0.7 ml) was withdrawn and mixed with 1 N NaOH (1 .O ml) and benzoyl chloride (50 ~1) in a 20-ml test tube. After the benzoylation, the reaction mixture was concentrated to about half the original volume under reduced pressure. The aqueous solution thus obtained was free from chloroform and ethanol, and the content of hippuric acid was determined as described above except that a 4-ml aliquot of the ethyl acetate extract was assayed after evaporation. Glycine in Whole Blood Heparinized blood (0.2 ml) in a lo-ml centrifuge tube was mixed successively with water (0.8 ml), 30% metaphosphoric acid (0.1 ml), and 5 ml of chloroform-ethanol (2: 1, v/v). After shaking vigorously, the mixture was centrifuged as described above. An aliquot (0.8 ml) of the supernatant was applied to acolumn of Dowex 50W x 8 (H-type, 100/200 mesh, 12 x 40 mm). The column was washed with water (15 ml) and amino acids were eluted with 2 N ammonia solution (30 ml). The eluate was evaporated to dryness under reduced pressure and the residue was dissolved in 1 .O ml of 1 N NaOH. To 0.7 ml of this solution water (0.8 ml), the copper-ammonia reagent (0.1 ml), and benzoyl chloride (50 ~1) were added. The mixture was treated as described above and a 4-ml aliquot of the extract was assayed for hippuric acid. Glycine in Liver Homogenate Rat liver (10 g) was homogenized in 90 ml of 1.15% KC1 with a PotterElvejhem homogenizer with a Teflon pestle and the homogenate was centrifuged at 700s for 10 min. The supernatant solution (0.5 ml) was mixed with water (0.5 ml) and 30% metaphosphoric acid (50 ~1) and was centrifuged at 1OOOgfor 10 min. The solution (0.8 ml) was then mixed with the copper-ammonia reagent (0.1 ml), 1 N NaOH (1.0 ml), and benzoyl chloride (50 ~1). This mixture was treated as described above and a 4-ml aliquot of the extract was assayed for hippuric acid.
DETERMINATION
-
Glycine
665
OF GLYCINE
(ug
) in
Cuvett (1 cm light
path
FIG. 1. Standard curve of glycine. Each 0.1 ml of aqueous glycine solution in different concentration was measured by the procedure under Glycine in Amino Acid Mixture.
RESULTS Standard Curve and Yield
of Benzoylation
The standard curve of glycine by the present method is shown in Fig. 1. The curve is linear up to at least 100 pg of glycine and the coefficient of variation calculated from each point is 3.3% (mean value). As little as 1.0 pg of glycine can be measured by the present procedure. The yield in the benzoylation step under the present condition was 99.0 t 4.4%. This value was obtained from the standard curve of authentic hippuric acid. Glycine in Amino Acid Mixture
It was of interest to try the present method on a standard solution for the amino acid analyzer. This consisted of 17 common amino acids and ammonia in each concentration of 2.5 PmoYml except the cystine concentration which was 1.25 pmol/ml. The value of 2.48 2 0.02 pmol glycinelml (n = 3) was obtained by the present method.
666
OHMORI
ET AL.
TABLE RECOVERY
Species Urineb
Serumb
Blood’
Liver”’
OF GLYCINE
Glycine added (kdml) 0 200 300 400 600 loo0
ADDED
1 TO BIOLOGICAL
Glycine determined (&ml. mean t SD) 218 407 526 654 780 1218
t k f 2 t 2
SAMPLES’
Recovery %
6 25 24 43 17 51
95 103 109 94 loo
Mean k SD 100.2 c 6.1
0 20 40 70 100
282 502 69+ 98t 1182
2 4 5 8 6
II0 103 100 90
0 50 100 150
26k 74+ 1132 158+
3 I I 3
96 87 88
0 20 40 60
18+ 38+ 55+ 7lk
0 I I 1
100 93 88
100.8 k 8.3
90.3 t 4.9
93.7 t 6.0
,JVarying amounts of glycine were added to each sample, and glycine was measured by each procedure under Experimental. “N = 3. ‘N=4. dN = 5.
Recovery Tests
For the purpose of applying this procedure to biological samples, recovery tests were carried out. Varying amounts of glycine were added to each sample ofurine, serum, whole blood, or liver homogenate, and the total amount of glycine was measured by each procedure given above. The results are shown in Table 1, indicating that it was possible to recover 90.3 2 4.9 to 100.8 + 8.3% of glycine added. Comparison of Results by the Present Method and Those by the Amino Acid Analyzer
The comparison was carried out by measuring glycine in 11 urine samples and 15 serum samples. These results demonstrate that the present
DETERMINATION
-Gtylmg/dL 2
serum) the Analyzer
667
OF GLYCINE
by
o /
0
2
Y=t.O29X +4.900 r -0.991
* c
g loo0
0 0
V
i/
0
100 -
Glycine
200 (mg/L
urine 1 by the
300 Analyzer
FIG. 2. Relationship between concentrations ofglycine in urine samples and serum samples measured by the amino acid analyzer and those by the present method.
method gave essentially analyzer (Fig. 2). Specificity
the same values as those by the amino
acid
of the Color Reaction
Specificity of the color reaction was studied using different amino acids, peptides, and components in urine. The following compounds did not show any color reaction when each of them was assayed as described under Glycine in Amino Acid Mixture: Asp. Thr, Ser, Glu, Pro, Ala, Val, Met, Ile, Leu, Tyr, Phe, Trp, Lys, Arg, Cys, sarcosine, y-Glu-Glu, y-Glu-Ala, y-Glu-His, y-Glu-Glu.NH,, y-Glu-Val, y-Glu-Ser, y-Glu-Lys, a-Glu-GlubNH,, N-acetylhomocarnosine, N-acetylcarnosine, Tyr-Tyr-Tyr, Phe-Phe, Tyr-Tyr, Phe-Phe-Phe, Tyr-Phe, Lys-Phe-Tyr, Pro-Tyr, Pro-Phe, Ser-Phe, Trp-Phe, Phe-Pro, Phe-Leu, Phe-Trp, Trp-Trp, Tyr-Leu, Phe-Tyr, Trp-Tyr, urea, uric acid, and creatine. On the other hand, some peptides showed the positive color reaction (Table 2). It is noteworthy that all of them had glycine residue at their Cterminals. Absorption spectra of the colored compounds before treating with the copper-ammonia reagent are shown in Fig. 3.
668
OHMORI
ET AL.
TABLE RELATIVE
ABSORFTIVITIES
OF PEPTIDES
2
SHOWING
THE
Amino acid and peptides
POSITIVE
Ratio
COLOR
REACTION
of absorptivities at 458 nm (%I
Glycine
loo 71 34 81 47 23 47 37 11
GIY-i4Y GlY-dY%lY Glutathione S-Methylglutathione Leu-gly ‘W-gly Phe-gly y-Glu-gly
: :.:.‘.
....... S-methylglutathione IO.30 u moles1 Glycine 10.13 3’ ) --- Glutathione 10.13 v 1 10.13 -.Gly-Gly (0.13 ‘8 I -..-
Gly-Gly-Gly
lo.13
r
j , I I ./
ft50
100
FIG. 3. Absorption spectra absence of the copper-ammonia
500
nm
Wavelength
of colored compounds reagent.
formed
from
glycine
and peptides
in the
DETERMINATION
A
B
C
D
669
OF GLYCINE
E
F
G
H
I
FIG. 4. Thin-layer chromatogram of substances after benzoylation in the presence or absence of the copper-ammonia reagent. Thin-layer chromatography was performed in solvent 1. Spots near the start line were detected with ninhydrin, and other spots were visualized by ultraviolet absorption. Spots surrounded by a dotted line or a broken line were scarcely or slightly visible, respectively. The size of the spot indicates the amount of material found. (A) authentic hippuric acid: (B) after reaction of glycine with benzoyl chloride; (C) after reaction of glycine with benzoyl chloride in the presence of the copper-ammonia reagent: (D) authentic benzamide; (E) after reaction of Gly-Gly with benzoyl chloride; (F) after reaction of Gly-Gly with benzoyl chloride in the presence of the reagent; (G) authentic benzoic acid; (H) glycine; (I) Gly-Gly.
DISCUSSION
A sensitive and specific procedure was established for the determination of glycine. As described above, peptides which had glycine residue at their C-terminals showed the positive color reaction. However, their interference in the present assay seems to be negligible because peptides, except glutathione, appear to be scarcely present in normal urine, serum, blood, and liver homogenate. Glutathione occurs in liver and red blood cells in relatively large quantities; approximately 4 pmol/g of wet rat liver and 41 to 214 ~mol/lOO ml of blood of vertebrates (13). The masking method for these peptides, especially for glutathione, therefore, had to be devised, on determining glycine in whole blood or liver homogenate. We first applied a copper aqueous solution for masking, but failed because cupurous hydroxide formed in the benzoylation step adsorbed glycine as well as the peptides. The copper-ammonia reagent was thus prepared to solubilize the precipitate and the color development due to glutathione, Gly-Gly , Gly-Gly-Gly , Tyr-Gly, and Phe-Gly was suppressed by the use of the reagent to 98.9, 82.8, 82. I, 73.6, and 89.4%, respectively, compared with that in the ab-
670
OHMORI ET AL.
sence of it. On the other hand, the formation of color due to glycine was not influenced by the same reagent. To clarify the mechanism of the masking effect by the reagent, thin-layer chromatography was carried out using Gly-Gly as a model peptide. Figure 4 shows that the benzoylated Gly-Gly is scarcely found in the presence of the copper-ammonia reagent. The samples (A-I) were also developed in another solvent system 2 to check the spots near the start line in Fig. 4, and the spot in Sample C or F (E) was confirmed as glycine with the R, value of 0.29 or as Gly-Gly with that of 0.65, respectively. The reagent thus seems to act as a chelating agent on peptide bonds as expected and prevent peptides from benzoylation. Glycine, on the other hand, must be benzoylated because its amino group is free, even though it may be coordinated with the copper ion. The authors are very interested in the color reaction of peptides having glycine residues at their C-terminals, and work on the qualification or quantification of glycine residues at C-terminals or the mechanism of the color reaction is in progress. ACKNOWLEDGMENT The present paper is dedicated to the 65th birthday of Professor Takaji Koyama of this faculty.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Fisher, E. (1902) Z. Physiol. Chem. 35, 227. Bergmann, M., and Fox, S. W. (1935) J. Biol. Chem. 109, 317. Town, B. W. (1936) Biochem. J. 30, 1833. Klein, G., and Linser, H. (1932) Z. Physiol. Chem. 205, 251. Patton, A. R. (1935) J. Bid. Chem. 108, 267. Alexander, B., Landwehr, G., and Seligman, A. M. (1945) J. Biol. Chem. 160, 51. Jewel], J. P., Morris, M. J., and Sublett, R. L. (1965) Anal. Chem. 37, 1034. Umberger. C. J., and Fiorese, F. F. (1963) Clin. Chem. 9, 79. Suzuki, S., Hachimori, Y., and Yaoeda, U. (1970) An&. Chem. 42, 101. Berger, S. J., Carter, J. A., and Lowry, 0. H. (1975) Anal. Biochem. 65, 232. Ohmori, S., Ikeda, M., Kira, S., and Ogata, M. (1977) Anal. Chem. 49, 1494. Gaffney, G. W., Schreier, K., Diferrante, N., and Altman, K. I. (1954) J. Biol. Chem. 206, 695. 13. Grunert, R. R., and Phillips, P. H. (1951) Arch, Biochem. Biophys. 30, 217.
BIOCHEMISTRY
A Simple
SHINJI Fuculty
90, 662-670 (1978)
and Specific Determination Biological Samples
of Glycine
in
OHMORI. MIKIKO IKEDA, YOKO WATANABE, AND KAZUHIRO HIROTA
of Pharmacrutical
Sciencrs. Okayamu University, Tsushimu-Nrtka-I. Japan
Oknyama
755
Received May 18, 1978 A simple specific assay was developed for the determination of glycine in a solution containing other amino acids. Hippuric acid was obtained after reacting glycine with benzoyl chloride and was extracted with ethyl acetate. It was then reacted with acetic anhydride. p-dimethylaminobenzaldehyde, and pyridine for color development. The amount of glycine (I to 100 pg) in the original solution could be determined by measuring the absorbance (458 nm) of this chromogen. This procedure was applied on an amino acid mixture, urine, serum, blood. and liver homogenate.
Since Fisher (1) isolated glycine from casein hydrolysate as ethyl glycinate with 79% yield, this amino acid has been determined by methods based on gravimetry (2,3), calorimetry (4-9), or enzymatic analysis (10). Some of these methods have been applied for the determination of glycine in biological samples (6) or in protein hydrolysates (5,9). However, they were inadequate for practical use because sensitivity (6) or specificity (5.9) of the chemical reaction was low. Recently, glycine has been widely determined using an amino acid analyzer as a dependable method. However, this procedure is quite time-consuming when glycine concentration should be determined in a solution containing other amino acids. Calorimetric determination of hippuric acid in urine or rat liver homogenate was established in our previous paper (11). This method was based on the color reaction of hippuric acid with acetic anhydride, pyridine, and p-dimethylaminobenzaldehyde (12). In this paper, we have applied this method for the determination of glycine in urine, serum, blood, or rat liver homogenate as well as in the mixture of common amino acids and established a highly sensitive and specific assay of glycine in such samples. CHEMICALS
AND INSTRUMENTS
Peptides were purchased from the Protein Research Foundation (Osaka) and Sigma Chemical Company. All other reagents (analytical grade) were 0003.2697/78/0902-0662$02.00/O CopyrIght G 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
662
DETERMINATION
OF GLYCINE
663
obtained from Wako Pure Chemical Industries Ltd. (Osaka). Urine, serum, and blood were supplied by the people in our laboratory. Copper-ammonia reagent was prepared by mixing 1 M cupric sulfate with 2.8% ammonia solution (1:3, v/v). Amino acid analysis was carried out in a Hitachi KLA-5 amino acid analyzer (column size, 9 x 550 mm) at 55°C using sodium citrate buffer (pH 3.25 and 4.25). Norleucine was used as an internal standard. Visible absorption spectrum was taken in a Hitachi 124 spectrophotometer. The solvent was evaporated under reduced pressure in an evaporator equipped with a mechanical vibrator (Taiyo Scientific Instrument Co. Ltd., Tokyo). Eight samples were treated at the same time. Benzoylation was carried out in the same apparatus. A Vortex mixer (Ikemoto Rikakogyo Co. Ltd., Tokyo) was also used for vigorous mixing of the sample. Thin-layer chromatography was performed on silica gel (Wako-gel B-SFM); Solvent 1, toluene:acetic acid:water (3:2: 1, v/v/v): Solvent 2, n-butanol:acetic acid:water (4: 1:5, v/v/v). EXPERIMENTAL
Glycine in Amino Acid Mixture A sample solution (0.1 ml) was mixed with 1 N NaOH (1.0 ml) and benzoyl chloride (50 ~1) in a 20-ml test tube. After 15 min of vigorous stirring at 4o”C, the mixture was acidified with 0.15 ml of 43% phosphoric acid and extracted with 5 ml of benzene-n-hexane (1: 1, v/v). The organic layer containing benzoic acid was removed thoroughly by aspiration and evaporation. Hippuric acid in the aqueous layer was extracted into 7 ml of ethyl acetate. An aliquot (1.4 ml) of the ethyl acetate layer was transferred to another test tube and was evaporated to dryness under reduced pressure in the evaporator. The concentration of hippuric acid was determined calorimetrically following our previous report (11) except that the concentration of p-dimethylaminobenzaldehyde was modified to avoid hippuric acid-independent color development in the original concentration. The dried residue obtained above was dissolved in 1.0 ml of acetic anhydride and the solution was vigorously mixed with 2.0 ml of 0.4%p-dimethylaminobenzaldehyde solution in pyridine. After incubation at 40°C for 1 h, the absorbance was measured against a blank solution containing acetic anhydride and 0.4% p-dimethylbenzaldehyde in pyridine. Glycine in Urine Prior to benzoylation, endogenous hippuric acid present in urine had to be removed. Urine (0.25 ml) was mixed with water (0.25 ml) and 6 N HCl (50 ~1) in a lo-ml centrifuge tube. Hippuric acid in the aqueous solution
664
OHMORI
ET AL.
was extracted with 5 ml of ethyl acetate previously saturated with water under vigorous mixing and the mixture was centrifuged at 1OOOgfor 10 min. The ethyl acetate layer was discarded, and the aqueous layer was again extracted with ethyl acetate, followed by centrifuging and discarding. The aqueous layer (0.1 ml) thus obtained was treated as described under Glycine in Amino Acid Mixture. Glycine in Serum Serum (0.3 ml) was diluted with water (0.7 ml) in a lo-ml centrifuge tube and was mixed with 30% metaphosphoric acid (50 ~1) and 5 ml of chloroform-ethanol (2: 1, v/v). After shaking vigorously for 30 s, the mixture was centrifuged at 1OOOgfor 10 min. The aqueous upper layer (0.7 ml) was withdrawn and mixed with 1 N NaOH (1 .O ml) and benzoyl chloride (50 ~1) in a 20-ml test tube. After the benzoylation, the reaction mixture was concentrated to about half the original volume under reduced pressure. The aqueous solution thus obtained was free from chloroform and ethanol, and the content of hippuric acid was determined as described above except that a 4-ml aliquot of the ethyl acetate extract was assayed after evaporation. Glycine in Whole Blood Heparinized blood (0.2 ml) in a lo-ml centrifuge tube was mixed successively with water (0.8 ml), 30% metaphosphoric acid (0.1 ml), and 5 ml of chloroform-ethanol (2: 1, v/v). After shaking vigorously, the mixture was centrifuged as described above. An aliquot (0.8 ml) of the supernatant was applied to acolumn of Dowex 50W x 8 (H-type, 100/200 mesh, 12 x 40 mm). The column was washed with water (15 ml) and amino acids were eluted with 2 N ammonia solution (30 ml). The eluate was evaporated to dryness under reduced pressure and the residue was dissolved in 1 .O ml of 1 N NaOH. To 0.7 ml of this solution water (0.8 ml), the copper-ammonia reagent (0.1 ml), and benzoyl chloride (50 ~1) were added. The mixture was treated as described above and a 4-ml aliquot of the extract was assayed for hippuric acid. Glycine in Liver Homogenate Rat liver (10 g) was homogenized in 90 ml of 1.15% KC1 with a PotterElvejhem homogenizer with a Teflon pestle and the homogenate was centrifuged at 700s for 10 min. The supernatant solution (0.5 ml) was mixed with water (0.5 ml) and 30% metaphosphoric acid (50 ~1) and was centrifuged at 1OOOgfor 10 min. The solution (0.8 ml) was then mixed with the copper-ammonia reagent (0.1 ml), 1 N NaOH (1.0 ml), and benzoyl chloride (50 ~1). This mixture was treated as described above and a 4-ml aliquot of the extract was assayed for hippuric acid.
DETERMINATION
-
Glycine
665
OF GLYCINE
(ug
) in
Cuvett (1 cm light
path
FIG. 1. Standard curve of glycine. Each 0.1 ml of aqueous glycine solution in different concentration was measured by the procedure under Glycine in Amino Acid Mixture.
RESULTS Standard Curve and Yield
of Benzoylation
The standard curve of glycine by the present method is shown in Fig. 1. The curve is linear up to at least 100 pg of glycine and the coefficient of variation calculated from each point is 3.3% (mean value). As little as 1.0 pg of glycine can be measured by the present procedure. The yield in the benzoylation step under the present condition was 99.0 t 4.4%. This value was obtained from the standard curve of authentic hippuric acid. Glycine in Amino Acid Mixture
It was of interest to try the present method on a standard solution for the amino acid analyzer. This consisted of 17 common amino acids and ammonia in each concentration of 2.5 PmoYml except the cystine concentration which was 1.25 pmol/ml. The value of 2.48 2 0.02 pmol glycinelml (n = 3) was obtained by the present method.
666
OHMORI
ET AL.
TABLE RECOVERY
Species Urineb
Serumb
Blood’
Liver”’
OF GLYCINE
Glycine added (kdml) 0 200 300 400 600 loo0
ADDED
1 TO BIOLOGICAL
Glycine determined (&ml. mean t SD) 218 407 526 654 780 1218
t k f 2 t 2
SAMPLES’
Recovery %
6 25 24 43 17 51
95 103 109 94 loo
Mean k SD 100.2 c 6.1
0 20 40 70 100
282 502 69+ 98t 1182
2 4 5 8 6
II0 103 100 90
0 50 100 150
26k 74+ 1132 158+
3 I I 3
96 87 88
0 20 40 60
18+ 38+ 55+ 7lk
0 I I 1
100 93 88
100.8 k 8.3
90.3 t 4.9
93.7 t 6.0
,JVarying amounts of glycine were added to each sample, and glycine was measured by each procedure under Experimental. “N = 3. ‘N=4. dN = 5.
Recovery Tests
For the purpose of applying this procedure to biological samples, recovery tests were carried out. Varying amounts of glycine were added to each sample ofurine, serum, whole blood, or liver homogenate, and the total amount of glycine was measured by each procedure given above. The results are shown in Table 1, indicating that it was possible to recover 90.3 2 4.9 to 100.8 + 8.3% of glycine added. Comparison of Results by the Present Method and Those by the Amino Acid Analyzer
The comparison was carried out by measuring glycine in 11 urine samples and 15 serum samples. These results demonstrate that the present
DETERMINATION
-Gtylmg/dL 2
serum) the Analyzer
667
OF GLYCINE
by
o /
0
2
Y=t.O29X +4.900 r -0.991
* c
g loo0
0 0
V
i/
0
100 -
Glycine
200 (mg/L
urine 1 by the
300 Analyzer
FIG. 2. Relationship between concentrations ofglycine in urine samples and serum samples measured by the amino acid analyzer and those by the present method.
method gave essentially analyzer (Fig. 2). Specificity
the same values as those by the amino
acid
of the Color Reaction
Specificity of the color reaction was studied using different amino acids, peptides, and components in urine. The following compounds did not show any color reaction when each of them was assayed as described under Glycine in Amino Acid Mixture: Asp. Thr, Ser, Glu, Pro, Ala, Val, Met, Ile, Leu, Tyr, Phe, Trp, Lys, Arg, Cys, sarcosine, y-Glu-Glu, y-Glu-Ala, y-Glu-His, y-Glu-Glu.NH,, y-Glu-Val, y-Glu-Ser, y-Glu-Lys, a-Glu-GlubNH,, N-acetylhomocarnosine, N-acetylcarnosine, Tyr-Tyr-Tyr, Phe-Phe, Tyr-Tyr, Phe-Phe-Phe, Tyr-Phe, Lys-Phe-Tyr, Pro-Tyr, Pro-Phe, Ser-Phe, Trp-Phe, Phe-Pro, Phe-Leu, Phe-Trp, Trp-Trp, Tyr-Leu, Phe-Tyr, Trp-Tyr, urea, uric acid, and creatine. On the other hand, some peptides showed the positive color reaction (Table 2). It is noteworthy that all of them had glycine residue at their Cterminals. Absorption spectra of the colored compounds before treating with the copper-ammonia reagent are shown in Fig. 3.
668
OHMORI
ET AL.
TABLE RELATIVE
ABSORFTIVITIES
OF PEPTIDES
2
SHOWING
THE
Amino acid and peptides
POSITIVE
Ratio
COLOR
REACTION
of absorptivities at 458 nm (%I
Glycine
loo 71 34 81 47 23 47 37 11
GIY-i4Y GlY-dY%lY Glutathione S-Methylglutathione Leu-gly ‘W-gly Phe-gly y-Glu-gly
: :.:.‘.
....... S-methylglutathione IO.30 u moles1 Glycine 10.13 3’ ) --- Glutathione 10.13 v 1 10.13 -.Gly-Gly (0.13 ‘8 I -..-
Gly-Gly-Gly
lo.13
r
j , I I ./
ft50
100
FIG. 3. Absorption spectra absence of the copper-ammonia
500
nm
Wavelength
of colored compounds reagent.
formed
from
glycine
and peptides
in the
DETERMINATION
A
B
C
D
669
OF GLYCINE
E
F
G
H
I
FIG. 4. Thin-layer chromatogram of substances after benzoylation in the presence or absence of the copper-ammonia reagent. Thin-layer chromatography was performed in solvent 1. Spots near the start line were detected with ninhydrin, and other spots were visualized by ultraviolet absorption. Spots surrounded by a dotted line or a broken line were scarcely or slightly visible, respectively. The size of the spot indicates the amount of material found. (A) authentic hippuric acid: (B) after reaction of glycine with benzoyl chloride; (C) after reaction of glycine with benzoyl chloride in the presence of the copper-ammonia reagent: (D) authentic benzamide; (E) after reaction of Gly-Gly with benzoyl chloride; (F) after reaction of Gly-Gly with benzoyl chloride in the presence of the reagent; (G) authentic benzoic acid; (H) glycine; (I) Gly-Gly.
DISCUSSION
A sensitive and specific procedure was established for the determination of glycine. As described above, peptides which had glycine residue at their C-terminals showed the positive color reaction. However, their interference in the present assay seems to be negligible because peptides, except glutathione, appear to be scarcely present in normal urine, serum, blood, and liver homogenate. Glutathione occurs in liver and red blood cells in relatively large quantities; approximately 4 pmol/g of wet rat liver and 41 to 214 ~mol/lOO ml of blood of vertebrates (13). The masking method for these peptides, especially for glutathione, therefore, had to be devised, on determining glycine in whole blood or liver homogenate. We first applied a copper aqueous solution for masking, but failed because cupurous hydroxide formed in the benzoylation step adsorbed glycine as well as the peptides. The copper-ammonia reagent was thus prepared to solubilize the precipitate and the color development due to glutathione, Gly-Gly , Gly-Gly-Gly , Tyr-Gly, and Phe-Gly was suppressed by the use of the reagent to 98.9, 82.8, 82. I, 73.6, and 89.4%, respectively, compared with that in the ab-
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OHMORI ET AL.
sence of it. On the other hand, the formation of color due to glycine was not influenced by the same reagent. To clarify the mechanism of the masking effect by the reagent, thin-layer chromatography was carried out using Gly-Gly as a model peptide. Figure 4 shows that the benzoylated Gly-Gly is scarcely found in the presence of the copper-ammonia reagent. The samples (A-I) were also developed in another solvent system 2 to check the spots near the start line in Fig. 4, and the spot in Sample C or F (E) was confirmed as glycine with the R, value of 0.29 or as Gly-Gly with that of 0.65, respectively. The reagent thus seems to act as a chelating agent on peptide bonds as expected and prevent peptides from benzoylation. Glycine, on the other hand, must be benzoylated because its amino group is free, even though it may be coordinated with the copper ion. The authors are very interested in the color reaction of peptides having glycine residues at their C-terminals, and work on the qualification or quantification of glycine residues at C-terminals or the mechanism of the color reaction is in progress. ACKNOWLEDGMENT The present paper is dedicated to the 65th birthday of Professor Takaji Koyama of this faculty.
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