Salivary Amino Acids Determination and Their Changes in Vision Stress Experiments

doi:10.4236/jss.2013.16005

Paper Menu >>

Journal Menu >>

Open Journal of Social Sciences

2013. Vol.1, No.6, 23-25

Published Online November 2013 in SciRes (http://www.scirp.org/journal/jss) http://dx.doi.org/10.4236/jss.2013.16005

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: *[email protected]

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

Introduction

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 illness’ mechanisms. 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.

Experimental

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.

W. TANG ET AL.

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

Stress

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.

Conclusion

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

W. TANG ET AL.

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

Gly

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

Thr

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

Ala

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

Orn

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.

Acknowledgements

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).

REFERENCES

Jones, D. P., Carlson, J. L., & Samiec, P. S. (1998). Glutathione mea-

surement in human plasma evaluation of sample collection, storage

and derivatization conditions for analysis of dansyl derivatives by

HPLC. Clinica Chimica Acta, 275, 175-184.

http://dx.doi.org/10.1016/S0009-8981(98)00089-8

Kang, X. J., Xiao, J., Huang, X., & Gu, Z.Z. (2006). Optimization of

dansyl derivatization and chromatographic conditions in the deter-

mination of neuroactive amino acids of biological samples. Clinica

Chimica Acta, 366, 352-356.

Nakamura, Y., Kodama, H., Satoh, T., Adachi, K., Watanabe, S., Yo-

kote, Y. et al. (2010). Diurnal changes in salivary amino acid con-

centrations. Vivo, 24, 837-842.

Spencer, G.S. (1994). Hormone and metabolite changes with stress in

stress susceptible pie train pigs. Endocrine Regulations, 28, 73-78.

Sun, J., Kang, X. J., Ma, Y. Q., Chen, L. Q., Qu, Z. J., Gu, Z. Z., & Lu,

Z. H. (2009). A potential quick determination used for monitor bio-

logical response of human under stress. 2009 the Regiomal Work-

shop of International Society for the Study of Behavioural Develop-

ment (ISSBD), Nanjing, June 2009, 119-121.

Sun, J., Zheng, S. L., Wang, Y., Kang, X. J., Gu, Z. Z., & Lu, Z. H.

(2010). The preliminary investigation of salivary L-histidine under

computer vision stress. The 7th International Conference on Cogni-

tive Science (ICCS2010), Beijing, 17-20 August 2010, 394.

Yan, S. L., Zhao, G., & Liu, Y. L. (2003). High-performance liquid

chromatographic method for the determination of dansyl-amino acid s.

Journal of Capital Medical University, 24, 338-339.