노환중외 - 복강내감작 20 g ovalbumin & 10 mg AIOH3 1 of N/S 0.5 of mg% ovalbuminaioh3gel 0.5 Nebulization with 0.1% ovalbumin solution in saline for 1 minute b

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1 KISEP Rhinology Korean J Otolaryngol 2005;48: 알레르기비염유발동물모델에서미세투석법에의한아미노산의동정 노환중 서정출 이현순 고의경 전경명 Detection of Ammino Acids Using Microdialysis Technique and Changes of Amino Acid Concentrations in Experimentally Induced Nasal Allergy Model Hwan-Jung Roh, MD, Jung-Chul Suh, MD, Hyun-Sun Lee, MD, Eui-Kyung Goh, MD and Kyong-Myong Chon, MD Department of Otolaryngology, College of Medicine, Pusan National University, Busan, Korea ABSTRACT Background and Objectives:The basic principle of microdialysis is to mimic the function of a capillary blood vessel by perfusing physiologic liquid implanted into the target tissue. Amino acids are supposed to have functions for controlling the homeostasis of normal nasal mucosa and a role in the pathogenesis of allergic rhinitis. However, no studies have been conducted about the existence of amino acids in the nasal cavity. This study measures the concentration of 19 amino acids found in the nasal cavity of normal control and experimentally allergy-induced animal model in order to evaluate the difference in the concentration of amino acids between normal and allergic nasal mucosa. Materials and Method:An experimentally induced nasal allergy model was developed by intraperitoneal and intranasal sensitization with ovalbumin in Dunkin-Hartely guinea pigs according to a programmed protocol. A microdialysis probe was designed to be suitable to nasal mucosa using a Cuprophan hollow fiber (200 μm inner diameter, 300 μm outer diameter, 45 kda molecular weight cut-off, Fitral, AN 69-HF). After verification of the probe, microdialysis was performed in the inferior turbinate submucosa of normal control (N=8) and experimental (N=8) groups. The concentration of 19 amino acids was analyzed using high performance liquid chromatography. Statistical analysis was performed using a student t-test. Results:All 19 amino acids were validated at various concentrations in the nasal cavity. Glutamate (p=0.036) and GABA (p<0.001) concentrations were significantly higher in the experimental group than in the control group. Conclusion:The 19 amino acids measured existed in the nasal cavity at various concentrations, and the concentrations of glutamate and GABA were significantly higher in the allergy group than in the control group. The microdialysis technique is a powerful tool not only to measure endogenous substances for target organ chemistry but also to pharmacokinetically evaluate exogenous drug delivery processes in the nasal cavity. (Korean J Otolaryngol 2005;48:476-81) KEY WORDS:Amino acids Microdialysis Rhinitis Allergic

2 노환중외 - 복강내감작 20 g ovalbumin & 10 mg AIOH3 1 of N/S 0.5 of mg% ovalbuminaioh3gel 0.5 Nebulization with 0.1% ovalbumin solution in saline for 1 minute by ultrasonic nebulizer Intranasal challenge of allergen 50 of 1% ovalbumin solution to each nostril 1 st 2 nd 3 rd 4 th 5 th 1 st IN 2 nd IN 3 rd IN 4 th IN Fig. 1. Schedule for development of the experimentally induced nasal allergy model in guinea pigs. intraperitoneal sensitization, IN intranasal sensitization. 5 days 24 hours Outflow Inflow Hollow fiber Epoxy glue Capillary tube Polyethylene tube-10 Inflow 2 mm A B 8.5 mm Outflow Fig. 2. Schematic drawing of microdialysis catheter. The perfusate enters through the right inlet cannula and passes through an inner cannula all the way to the tip of the probe see enlargement of the tip. when it flows upwards between inner cannula and the membrane, where microdialysis takes place. the liquid leaves the probe through the left outlet cannula A. Schematic drawing of microdialysis catheter for the nasal cavity. Catheter was made with Cuprophan hollow fiber 200 m inner diameter, 300 m outer diameter and microdialysis was made through microdialysis membrane 45 kda weight cut-off, 2 mm in length which was inserted in submucosa of the inferior turbinate and the remaining portion was sealed with epoxy glue B. 477

알레르기비염유발동물모델에서아미노산의동정 비강내감작 - 미세투석관의검정 - Sterilization of the probe Verification Recovery rate Microdialysis after implantation into submucosa of inferior turbinate HPLC UV light 70% alcohol Wash

HPLChigh performance liquid chromatography. - - 미세투석법 Fig. 4. Microdialysis of the nasal cavity in guinea pig.

3 알레르기비염유발동물모델에서아미노산의동정 비강내감작 - 미세투석관의검정 - Sterilization of the probe Verification Recovery rate Microdialysis after implantation into submucosa of inferior turbinate HPLC UV light 70% alcohol Wash with sterile normal saline Bacterial culture 10% glucose solution 2 /min24% 19 amino acids 60 min 30 min4 Storage at 70 Fig. 3. Diagram of microdialysis procedures for the nasal cavity. HPLChigh performance liquid chromatography. - - 미세투석법 Fig. 4. Microdialysis of the nasal cavity in guinea pig. AMicrodialysis catheter was connected to microinfusion pump and the perfusate was collected in Eppendrof tube put in the ice filled container, and tracheostomy was done during microdialysis. BMicrodialysis catheter was inserted in submucosa of the inferior turbinate under microscope. A B 478 Korean J Otolaryngol 2005;48:476-81

4 노환중외 Table 1. Mean concentrations of 19 amino acids between control and experimentally induced nasal allergy groups pmol/ Amino acids Control n32 Allergy n32 p-value Alanine Aspartate GABA* Glutamate* Leucine Lysine Methionine Phenylalanine Proline Taurine Tyrosine Arginine Cysteine Glycine Histidine Isoleucine Serine Threonine Valine n32 means 4 times measurement in 8 guinea pigs. p

5 알레르기비염유발동물모델에서아미노산의동정 Korean J Otolaryngol 2005;48:476-81

6 노환중외 REFERENCES 1) Durham SR. Mechanisms and treatment of allergic rhinitis. In: Alan GK editor. Scott-Brown s Otolaryngology. 6th ed. Oxford: Butterworth- Heinemann;1997. p.4/6/1-4/6/16. 2) Hofford JM, Milakofsky L, Pell S, Fish JE, Peters SP, Pollice M, et al. Level of amino acids and related compounds in bronchoalveolar lavage fluids of asthmatic patients. Am J Respir Crit Care Med 1997; 155: ) Dicpinigaitis PV, Spungen AM, Bauman WA, Absgarten A, Almenoff PL. Inhibition of bronchial hyperresponsiveness by the GABA-agonist baclofen. Chest 1994;106: ) Aas P, Tanso R, Fonnum F. Stimulation of peripheral cholinergic nerves by glutamate indicates a new peripheral glutamate receptor. Eur J Pharmacol 1989;164: ) Ungerstedt U. Microdialysis-principles and applications for studies in animals and man. J Int Med 1991;230: ) Krogstad AL, Roupe G. Microdialysis of histamine in the skin of patients with macrocytosis. Exp Dermatol 2001;10: ) Kawaguchi S, Majima Y, Sakakura Y. Nasal mast cells in experimentally induced allergic rhinitis in guinea-pigs. Clin Exper Allergy 1994; 24: ) Lawrence AJ, Jarrott B. L-glutamate as a neurotransmitter at baroreceptor afferents: Evidence from in vivo microdialysis. Neuroscience 1994;58: ) Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329: ) Aas P, Fonnum F. Amino acids as modulators of cholinergic nerves in airways. Agents Actions Suppl 1990;31: ) Huxtable RJ, Sebring LA. Sulfur amino acids, Biochemical and clinical aspects. In: Progress in clinical and biological research. Proceedings of an international symposium and the 5th annual meeting of the Japanese Research; New York: Alan R Liss;1983. p ) Bligh J. Amino acids as central synaptic transmitters or modulators in mammalian thermoregulation. Fed Proc 1981;40: ) Ong J, Kerr DI. GABA-receptors in peripheral tissues. Life Science 1990;46: ) Tohda Y, Ohkawa K, Kubo H, Muraki M, Fukuoka M, Nakajima S. Role of GABA receptors in the bronchial response: Studies in sensitized guinea-pigs. Clin Exp Allergy 1998;28: ) Dicpinigaitis PV. Effect of the GABA-agonist baclofen on bronchial responsiveness in asthmatics. Pulm Pharmacol Ther 1999;12: ) Yang WH, Drouin MA, Herbert M, Mao Y, Karsh J. The monosodium glutamate symptom complex: Assessment in a double-blind, placebocontrolled, randomized study. J Allergy Clin Immunol 1997;99: ) McCulloch PF, Paterson IA, West NH. An intact glutamatergic trigeminal pathway is essential for the cardiac response to simulated diving. Am J Physiol 1995;269: ) Bertrand B, Dab R, Daele J, Cauwenberg P, Broeck R. Clinical efficacy of N-acetyl-aspartyl-glutamic acid nasal spray in children suffering from pollinosis: A double-blind multicentric study. Rhinology 1995;33: ) Miadonna A, Milazzo N, Salmaso C, Cottini M, Lorini M, Tedeschi A. N-acetyl-aspartyl-glutamic acid inhibits cellular recruitment and mediator release during the late allergen-induced nasal reaction. Eur J Clin Pharmacol 1998;54:

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