Open Journal of Social Sciences
2013. Vol.1, No.6, 23-25
Published Online November 2013 in SciRes (
Open Access 23
Salivary Amino Acids Determination and Their Changes in
Vision Stress Experiments
Wei Tang1, Xuesong Li1, Xiaoxiao Wu1, Yu Wang1, Xuejun Kang1,2*
1Key Laboratory of Child Development and Learning Science of Ministry of Education,
Research Center for Learning Science, Southeast University, Nanjing, China
2Suzhou Key Laboratory of Enviroment and Biosafety, Southeast University, Suzhou, China
Email: *
Received October 2013
Amino acid neurotransmitters represent a major class of compounds that are involved in neuronal com-
munication at CNS synapses, which can provide the basis for a variety of disease diagnosis and treatments
and the study of the mechanism of mental illness. An analytical method for the determination of several
amino acids in saliva was established with reversed-phase high performance liquid chromatography (RP-
HPLC) with UV detector. About ten kinds of amino acids were detected in saliva. Nine subjects have par-
ticipated in the stress experiments which have undergone a 50-min three-dimensional cartoon watching.
The result of the experiment has proved that four kinds of salivary amino acids respond to the vision
stress experiment obviously.
Keywords: Amino Acids; RP-HPLC; Stress Experiments
Stress research in human health and diseases has occupied a
prominent position. According to the literature survey, about 75
to 90% of the diseases have a connection with the activation of
stress mechanisms. Many factors cause stress, such as mechan-
ical trauma, undercooling, overheating, food poisoning, psy-
chological fear, depression, heavily mental tension and so on.
Stress has a variety of assessment methods. Many indicators of
stress response can really and effectively show human being’s
physiological stress response system, such as cortisol, catecho-
lamines and so on. Among them the amino acid neurotransmit-
ters are important stress markers. Glutamate, aspartate, γ-ami-
nobutyric acid, glycine and taurine and many kinds of amino
acids are important neurotransmitters in brain. Glutamate and
aspartate are considered to be excitatory amino acids; while γ-
aminobutyric acid, glycine and taurine are considered to be in-
hibitory amino acids. They are important materials which can
regulate the body's physiological activities. Thus separating and
determining the content of biological tissue’s amino acid neu-
rotransmitters can provide a basis for the diagnosis and the
treatment of many diseases and mental illnessmechanisms. As
a result, the determination and study of stress amino acids are
really important (Xuejun Kang, Jing Xiao, Xiao Huang, &
Zhongze Gu, 2006).
Amino acids are regarded as important nutrients which can
directly involve brain protein synthesis and metabolism, (Spen-
cer GS, 1994) play an important regulatory role on the brain
function and psychological behavior. The researches of amino
acids and nervous system’s function can be traced back to the
forties and fifties last century. In recent years, neurotransmitters
and their receptors, neurophysiological and biochemical phar-
macology research also have many new developments (Yan SL,
Zhao G, & Liu YL, 2003). Studies have shown that some ami-
no acids are involved in learning and memorizing and some
other complex nervous activi tie s (Jones DP, Carlson JL, &
Samiec PS, 1998).
Kodama and co-workers found that concentrations of gly-
cine, alanine, threonine and histidine in the saliva are slightly
elevated, whereas those of glutamic acid and lysine are reduced
during the examination. The enhanced ratio of glycine/glutamic
acid may reflect the change from the excited state to the de-
pressed state (Nakamura Y, Kodama H, Satoh T, Adachi K,
Watanabe S, Yokote Y et al., 2010). Our group’s researches
imply that L-histidine in saliva may be another index in corre-
lation with human stress (Jing Sun, Xuejun Kang, Yuqin Ma,
Liqin Chen, Zijian Qu, Zhongze Gu, & Zuhong Lu, 2009) (Jing
Sun, Shenglan Zheng, Yu Wang, Xuejun Kang, Zhongze Gu, &
Zuhong Lu, 2010).
The aim of the present study was to use HPLC to determine
amino acids, and to evaluate the response of some salivary
amino acids on the learning process of people submitted com-
puter vision stress.
Chemicals and Materials
Methanol (chromatographic purity) was from Chengdu Xin-
du area of the industrial development zone. (Chengdu, China).
Acetonitrile was from Shanghai Ludu Chemical Reagent Co.,
Ltd. (Shanghai, China). Triethyl ammonium was from Sino-
pharm Chemical Reagent Co., Ltd. Tetrabutyl ammonium hy-
droxide was from Shanghai Kefeng Industry Co., Ltd. (Shang-
hai, China). Dansyl chloride and amino acid standards were
from Sigma (St. Louis, MO). Acetone was from Shanghai
Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Po-
tassium bicarbonate, potassium hydroxide and glacial acetic
acid were from Nanjing Chemical Reagent Co., Ltd. (Jiangsu
Province, China). Triply distilled water was used for all studies.
*Corresponding author.
Open Access
Instrument and Chromatographic Conditions
The instruments used are as follows: LC-20AD HPLC; SPD-
10AD UV detector (Shimadzu Corporation); Shimadzu chro-
matography workstation; Shimadzu C18 column (4.6 × 150 mm,
5 μm); GL-20B high speed refrigerated centrifuge (Shanghai
Anting Scientific Instrument Factory); GL-88B vortex mixer
(Jiangsu Haimen Kirin Medical Instrument factory); PHS-2C
pH meter; METTLERAT electronic analytical balance; BF-
2500 positive and negative pressure oil-free vacuum pump
(Shanghai Rate Nako Trading Co., Ltd.); electric constant tem-
perature water bath.
The mobile phase was 35:1.5:80 methanol/acetonitr ile/water
(V/V/V) containing 1.3% triethylammonia and 1.1% tetrabutyl
ammonium hydroxide. The mobile phase was adjusted to PH
2.53 with concentrated sulfuric acid, and then was filtrated with
a 0.45 μm microporous organic membrane. The UV detection
wavelength was 221 nm . The flow rate of the mobile phase was
1.5 ml/min. The injection volume was 20 µl. The column tem-
perature was 35˚C.
Experimental Procedure
Accurately weighed thirteen kinds of amino acids, glycine,
glutamate, glutamine, Aspartate, taurine, arginine, hydroxypro-
line, threonine, ornithine, lysine, serine, alanine, γ-aminobutyric
acid and mixed with triply distilled water to prepare 1. 0 mg/ml
standard solution. These solutions were diluted daily to work-
ing concentrations with triply distilled water. The dansyl chlo-
ride solution was prepared just before derivatization by dis-
solving 200 mg of Dns in 10 ml of acetone.
Subjects were 9 graduates undergoing a 50-min three-di-
mensional cartoon watching. Saliva sampl es were spit in a cen-
trifuge tube after rinsing the mouth with water. Participants in
this experiment provided three salivary samples, 20-min before
the vision stress experiment, at the time of the process end and
20-min post-process.
50 μl of the standard solution or the test saliva sample was
added to 50 μl of buffer solution of 2 mol/l KOH-KHCO3 (PH
= 9.8) and 20 mg/ml of dansyl chloride (acetone) solution
(50μl). After capping the tube and mixing vigorously for 5 s,
the mixture was reacted in the dark at 60˚C in a water bath for
60 min. Then, an aliquot (20 µl) of acetic acid was added into
the tube to stop the reaction. The mixture was centrifuged at
10,000 × g for 5 min. The 20 µl supernatant of the reaction
mixture was injected into the HPLC system.
Results and Discussion
Method Validation
Good linearity was observed over the concentration range of
0.5 ~ 100 µg/ml, and the correlation coefficient of 13 kinds of
amino acids is from 0.9934 to 0.9999. The LOD is 0.1 - 0.5
µg/ml. The precision of the method was evaluated as RSD 0.3%
- 8.8% (n = 6). The content of each amino acid in saliva is in
the range of 0.3 - 5.0 µg/ml. The chromatograms of blank, the
amino acid standard mixture, and the samples are shown as A,
B, C in Figures 1 and 2.
The Response of Salivary Amino Acids to Vision
Saliva was collected before and after the stress experiment,
Figure 1.
Typical HPLC separations of target amino acids; (A) A blank (deriva-
tization solution with no analyte); (B) 13 amino acid standards; (C)
Human saliva sample.
Figure 2.
Chromatograms of salivary a mino acids of one subject. (a) Blank; (b) 5
μg/ml amino acid standard solution; (c) Sample at 20-min before the
vision stress experiment; (d) Sample at the end of vision stress experi-
ment; (e) Sample at 20-min after the vision stress experiment.
respectively, and amino acid concentrations were measured by
using HPLC. The effect of stress on the amino acid concentra-
tion was significantly observed only for glycine, threonine,
alanine, ornithine. At the end of the vision stress experiment,
those four amino acids were increased, whereas they were re-
duced at 20-min after the stress. The results were shown in
Table 1 and Figure 3.
The present study demonstrated that thirteen kinds of amino
acids can be detected in the human saliva by the method we
have developed. Salivary glycine, threonine, alanine, ornithine
Open Access
Table 1.
The content of fou r kinds of amino acids in saliva of subjects (μg/ml).
Subject 1 2 3 4 5 6 7 8 9
Before 2.84 0.79 3.35 7.36 11.04 7.32 3.72 3.00 4.54
End 2.95 3.78 10.36 7.78 22.20 8.42 10.47 9.19 8.96
20’post 2.91 1.42 6.58 4.11 13.43 8.32 9.36 8.49 3.11
Before 1.68 1.44 5.50 8.49 4.05 3.74 1.77 5.66 6.97
End 1.85 4.48 5.74 19.53 9.66 5.04 3.94 8.20 7.43
20’post 1.48 3.00 2.91 9.09 3.33 2.87 3.41 4.52 6.86
Before 1.16 0.46 1.86 2.27 4.45 3.19 1.63 1.79 2.83
End 0.95 1.78 3.15 5.57 6.36 5.12 1.77 4.47 2.86
20’post 1.12 0.88 1.64 2.60 5.62 3.91 1.49 3.26 2.77
Before 1.95 0.97 1.66 5.66 17.50 5.29 6.07 5.76 19.40
End 2.90 5.33 6.69 18.54 26.37 5.65 10.88 28.24 25.50
20’post 2.38 2.05 4.64 5.46 8.69 3.80 1.60 25.60 21.20
Figure 3.
The responsing curve of four kinds amino ac-
ids during stress experiment.
levels appear to increase during the vision stress experiment
and to reduce after that. The results showed that salivary gly-
cine, threonine, alanine, ornithine may potentially be another
markers responding to psychological stress, though more com-
prehensive experiments are needed to support this conclusion.
This work was supported by the National Basic Research
Program (No. 2012CB933302), the National Natural Science
Foundation of China (No. 81172720, No. 21307086), and Col-
leges and universities in Jiangsu Province plans to graduate
research and innovation projects (CXLX12_0121).
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