천식 쥐의 혈장과 폐 조직에서 액체크로마토그래피–텐덤질량분석법을 이용한 폴리아민의 프로파일 분석
Polyamine Profiling Analysis by Liquid Chromatography–Tandem Mass Spectrometry in Plasma and Lung Tissues of Mice with Asthma
- 주제(키워드) Polyamine , Liquid chromatography–tandem mass spectrometry , Asthma mouse , Star pattern recognition
- 발행기관 아주대학교
- 지도교수 이광, 백만정
- 발행년도 2017
- 학위수여년월 2017. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000024572
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Endogenous aliphatic and acetylated polyamines (PAs) are known as essential components to regulate about metabolic functions. Imbalance of polyamine metabolism may be bringing about the several pathological processes. In this study, a new sample preparation method and multiple reaction monitoring (MRM) mode for simultaneous measurement of four aliphatic and five acetylated PAs in plasma and lung tissue samples was developed by liquid chromatography–tandem mass spectrometry (LC−MS/MS). Characteristic ions in MRM mode with electrospray ionization (ESI) permitted the accurate and selective analysis of PAs. In the optimal conditions, this method showed good linearity (r ≥ 0.9970) for the range of 0.5–10 ng with limit of detection (LOD) of 0.003–0.8 pmol and limit of quantification (LOQ) of 0.01–2.6 pmol. The repeatabilities of intra and inter days as a percentage of relative standard deviation (% RSD) varied from 0.4–15.4 (% RSD). And accuracy as a percentage of relative error (% RE) was from -17.3–16.2 (% RE). When applied to the analysis of PAs in plasma and lung tissue samples from control and asthma mice, nine PAs were positively identified and determined by LC−MS/MS. The percentage composition of putrescine and spermidine in plasma as major PAs ranged from 20.33 ± 4.51–22.13 ± 2.37 and 67.85 ± 2.84–69.59 ± 5.98, respectively. While, percentage composition of spermidine and spermine as major PAs in lung tissue ranged from 27.61 ± 1.71–28.74 ± 5.91 and 67.60 ± 5.99–69.25 ± 2.08, respectively. In the asthma group compared to control group, level of N1-acetylspermine (p<0.03) was significantly reduced in plasma, while level of putrescine (p<0.02) was significantly increased in lung tissue. The percentage composition of each PA from asthma group was normalized to the corresponding mean value of control group, which were used for creating star pattern graph. The distorted star graphic pattern of the asthma group was different from nonagonal shape of the control group. Therefore, the present PA profiling method by LC−MS/MS will be useful for the biochemical monitoring of various disease state including asthma.
more목차
Ⅰ. INTRODUCTION . 1
Ⅱ. EXPERIMENTAL 4
1. Chemicals and reagents . 4
2. Preparation of standard solutions . 4
3. Sample preparation of plasma and lung tissue with asthma 4
4. Liquid chromatographytandem mass spectrometry . 8
5. Method validation for analysis of PAs . 10
6. Animal model 11
(1) Animals and ethics statement . 11
(2) Sensitization and provocation of airway inflammation with OVA 11
7. Sample preparation of PA profiling analysis from mice plasma 13
8. Sample preparation of PA profiling analysis from mice lung tissue 14
9. Star pattern recognition analysis 15
Ⅲ. RESULTS 16
1. Method development for PA profiling analysis 16
(1) Optimization of Multiple reactions monitoring (MRM) mode 16
(2) Optimization of chromatographic and mass spectral condition . 16
(3) Optimization of matrix effects. 17
2. Method validation of profiling analysis of PAs 24
3. PA profiling analysis in mice plasma with asthma . 27
4. PA profiling analysis in mice lung tissue with asthma 30
5. Star pattern recognition analysis of PAs in mice plasma with asthma . 33
6. Star pattern recognition analysis of PAs in mice lung tissue with asthma . 36
Ⅳ. CONCLUSION . 39
REFERENCES . 40
