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소장정보 검색

국회도서관 홈으로 정보검색 소장정보 검색
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Title Page

Contents

Abstract 7

I. INTRODUCTION 24

1. BH4 and NTs 24

1.1. BH4 24

1.2. Dopamine 29

1.3. Serotonin 31

1.4. Norepinephrine 31

1.5. Epinephrine 32

1.6. Glutamate 32

1.7. GABA 32

2. Radiofrequency radiation on the central nervous system 33

3. Analysis of NTs 34

4. Aims and objective 35

II. Materials and Methods 36

1. Reagents. 36

2. Determination of biopterins, neurotransmitters and ISs from MS/MS 36

2.1. BH4 and IS (AACA) 36

2.2. BH2 and Biopterin 37

2.3. Dopamine and dopamine-D₄(IS) 37

2.4. Serotonin and serotonin-D₄(IS) 37

2.5. Norepinepherine and norepinephrine-D6 (IS)(이미지참조) 44

2.6. Epinepherine and epinepherine-D₃(IS) 44

2.7. Glutamate and glutamate-D5 (IS)(이미지참조) 51

2.8. GABA and GABA-D6 (IS)(이미지참조) 51

3. Preparation of stock solution, calibration standards, and quality control samples. 51

4. Animals experimentation 56

5. RFR experimental design 57

6. RFR exposure System 57

7. Sample preparation. 63

8. Apparatus and chromatographic conditions. 64

9. BH4 & NTs assay method developed by using LC-MS/MS 65

9.1. BH4 & Dopamine 65

9.2. NTs 74

10. Method validation. 74

11. Statistical analysis. 84

III. Results 85

1. Sample preparation and liquid chromatography. 85

2. Mass spectrometry of BH4, BH2 and biopterin 85

3. Assay Optimization 90

4. Sensitivity and Specificity. 94

4.1. BH4 and dopamine in rat brain tissues 94

4.2. BH4 and NTs in the mouse brain tissue of exposed RFR. 94

5. Linearity. 95

6. BH4 stability and the measurement of BH4 and dopamine in brain tissue. 95

7. Analysis of NT in the exposed RFR mouse brains 97

IV. Discussion 117

1. The measurement of BH4 and dopamine in brain tissue. 117

2. Determination of BH4 and NTs in the exposed RFR mouse brains. 119

V. References 121

국문초록 130

List of Tables

Table 1. The analytic parameters of neurotransmitters in DW by LC-MS/MS. 93

Table 2. The calibration of neurotransmitters in DW by LC-MS/MS. 96

Table 3. Concentration of BH4 & dopamine in Various brain tissues (n＝10) 100

Table 4. The level of Tetrahydrobiopterin (BH4) in mouse brain regions after RFR exposure. 101

Table 5. The level of Dopamine (DA) in mouse brain regions after radiofrequency radiation (RFR) exposure 102

Table 6. The level of Serotonin (5-HT) in mouse brain regions after radiofrequency radiation (RFR) exposure 103

Table 7. The level of Norepinephrine (NE) in mouse brain regions after radiofrequency radiation (RFR) exposure 104

Table 8. The level of epinephrine (EP) in mouse brain regions after radiofrequency radiation (RFR) exposure 105

Table 9. The level of Glutamate (Glu) in mouse brain regions after radiofrequency radiation (RFR) exposure. 106

Table 10. The level of GABA in mouse brain regions after radiofrequency radiation (RFR) exposure. 107

List of Figures

Fig. 1. The biopterin molecular frame in its various oxidation states. 26

Fig. 2. De novo synthetic pathway 27

Fig. 3. BH4 and Synthesis of neurotransmitters 28

Fig. 4. Synthesis of the dopamine (DA), norepinephrine (NE) and epinephrine (EP) from tyrosine. 30

Fig. 5. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of BH4. 38

Fig. 6. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of epsilon-acetamidocaproic acid (AACA). 39

Fig. 7. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of BH2. 40

Fig. 8. Precursor ion and MS/MS breakdown curve (A) and product ion (B) Spectra of biopterin. 41

Fig. 9. Precursor ion and MS/MS breakdown curve (A) and product ion (B) Spectra of dopamine (DA). 42

Fig. 10. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of dopamine-D₄(DA-D₄). 43

Fig. 11. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of serotonin (5-HT). 45

Fig. 12. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of serotonin-D₄(5-HT-D₄). 46

Fig. 13. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of norepinephrine (NE). 47

Fig. 14. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of norepinephrine-D6 (NE-D6).(이미지참조) 48

Fig. 15. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of epinephrine (EP). 49

Fig. 16. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of epinephrine-D₃(EP-D₃). 50

Fig. 17. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of glutamate (Glu). 52

Fig. 18. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of glutamate-D5 (Glu-D5).(이미지참조) 53

Fig. 19. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of GABA. 54

Fig. 20. Precursor ion and MS/MS breakdown curve (A) and product ion (B) spectra of GABA-D6.(이미지참조) 55

Fig. 21. Schematics of radiofrequency radiation (RFR) exposure design 58

Fig. 22. Mouse body weight variation with time in the radiofrequency radiation (RFR) exposed system. 59

Fig. 23. Dissection of mouse brain regions. 60

Fig. 24. The liquid chromatographic (HPLC) system and quadrupole tandem mass spectrometer coupled with electrospray ionization (ESI-MS/MS) system. 61

Fig. 25. Radiofrequency radiation exposure instrument. Wave Exposer V20 is used for radiation exposure of mice to 835 ㎒ at the SAR value 4.0 W/㎏. 62

Fig. 26. BH4 LC-MS/MS chromatogram in matrix DW using column Capcell PAK MGII (2.0 × 50 ㎜,3 um) & mobile phase (A: 15% MeOH, B: 20% MeOH, C: 30% MeOH, D: 50% MeOH). 66

Fig. 27. BH4 LC-MS/MS Chromatogram in brain matrix using column Capcell PAK MGII (2.0 × 50 ㎜, 3 um) & mobile phase MeOH : DW (0.1% Formic acid) (30 : 70, v/v).... 67

Fig. 28. BH4 LC-MS/MS Chromatogram in brain matrix using column Gemini-NX 5u C18 110A (2.0 × 150 ㎜, 5um) & mobile phase MeOH : DW(0.1% formic acid) (20 : 80, v/v) (A: Double Blank, B: 1000 ng/g Spiked in brain matrix). 68

Fig. 29. BH4 LC-MS/MS chromatogram in brain matrix using column Gemini-NX 5u C18 110A (2.0 × 150 ㎜, 5um) & mobile phase gradient (ACN : 5 -〉 50%, 5min) (A: Standard Stock, B: 200 ng/g Spiked in brain matrix, C: Sample (FC-1W-1C)) 69

Fig. 30. BH4 & IS LC-MS/MS chromatogram in brain matrix using column Luna 3u HILIC 200A (2.0 × 150 ㎜; 3um) & mobile phase ACN : DW (75 : 25,v/v3 0.1% formic acid).... 70

Fig. 31. BH4 & IS LC-MS/MS chromatogram in brain matrix using new column Luna 3u HILIC 200A (2.0 × 150 ㎜; 3um) & mobile phase ACN : DW (75 : 25, v/v, 0.1% formic acid).... 71

Fig. 32. BH4 & IS LC-MS/MS chromatogram in DW using Polar-Diol (2.0 × 150 ㎜; 3um) & mobile phase ACN : DW (A: AACA (IS) in ACN 75%, B: BH4 in ACN 75%, C: AACA (IS) in ACN 85%, D: BH4 in ACN 85%,E: AACA (IS) in ACN 90%,F:... 72

Fig. 33. BH4 LC-MS/MS chromatogram in brain matrix using Polar-Diol(2.0 × 150㎜; 3 um) & mobile phase ACN : DW (75:25, v/v, 0.1% formic acid)(A:AACA (IS) in double blank, B: BH4 in double blank). 73

Fig. 34. BH4 LC-MS/MS chromatogram in brain matrix using Polar-Imidazole (2.0 × 150 ㎜; 3 um) & mobile phase ACN : DW (75:25, v/v, 10 mM ammonium formate) (A: AACA (IS) in double blank, B: BH4 in double blank, C: AACA(IS) in standard 100... 75

Fig. 35. Characteristic chromatograms of rat brain homogenate as blank using Polar-Imidazole (2.0 × 150 ㎜; 3 um) & mobile phase ACN : DW (75:25, v/v, 10 mM ammonium formate) 76

Fig. 36. Characteristic chromatograms of rat brain spiked with BH4 (10 ng/g), Dopamine (10 ng/g) and IS using Polar-Imidazole (2.0 × 150 ㎜; i.d., 3 um) & mobile phase ACN : DW (75:25, v/v, 10mM ammonium formate) 77

Fig. 37. Characteristic chromatograms of rat brain spiked with BH4 (200 ng/g), dopamine (200 ng/g) and IS using Polar-Imidazole (2.0 × 150 ㎜; 3 um) & mobile phase ACN : DW (75:25,v/v, 10mM ammonium formate) 78

Fig, 38. Characteristic chromatograms of rat brain tissue (corpus striatum) using Polar-Imidazole (2.0 × 150 ㎜; i.d., 3 um) & mobile phase ACN : DW (75:25, v/v, 10 mM ammonium formate) 79

Fig. 39. The LC-MS/MS chromatograms of BH4(A), AACA(B), dopamine(C) and dopamine-D4 (D) using Polar-Imidazole (2.0 × 150 ㎜; i.d., 3um) & mobile phase ACN : DW (75:25, v/v, 10 mM ammonium formate).(이미지참조) 80

Fig. 40. The LC-MS/MS chromatograms of GABA(A), GABA-D6 (B),glutamate (C), glutamate-D5(D), dopamine (E), dopamine-D₄(F), norepinepherine (G), norepinepherine-D6 (H) using Polar-Imidazole (2.0 × 150 ㎜; i.d., 3 um) & mobile...(이미지참조) 81

Fig. 41. The LC-MS/MS chromatograms of serotonin (A), serotonin-D₄ (B), epinepherine (C), epinepherine-D₃(D), AACA (E), BH4 (F) using Polar-Imidazole (2.0 × 150 ㎜; i.d., 3 um) & mobile phase ACN : DW (80:20,v/v, 10 mM ammonium... 82

Fig. 42. The LC-MS/MS Chromatograms of NTs (A: serotonin (5-HT), B: serotonin-D₄ (5-HT-D₄), C: norepinephrine (NE), D: norepinephrine-D6 (NE-D6), E: epinephrine (EP), F: epinephrine-D3 (EP-D₃), G: glutamate (Glu), H: glutamate-D5 (Glu-D5), I: GABA, J:...(이미지참조) 83

Fig. 43. Product ion mass spectra used in multiple reactions monitoring for BH4 (precursor ion m/z 242.1) 86

Fig. 44. Product ion mass spectra used in multiple reactions monitoring for dopamine (DA) (precursor ion m/z 154.1) 87

Fig. 45. Product ion mass spectra used in multiple reactions monitoring for AACA (precursor ion m/z 174.1) 88

Fig. 46. Product ion mass spectra used in multiple reactions monitoring for BH2 (precursor ion m/z 240.0) 89

Fig. 47. Product ion mass spectra used in multiple reactions monitoring for biopterin (precursor ion m/z 238.0) 91

Fig. 48. Characteristic chromatograms from mixture of biopterin (A), BH2 (B), BH4 (C). 92

Fig. 49. Relative stability of BH4 with time (n＝4). Graph showing relative stability of BH4 at different conditions (temperature,DTE, DTE+HCI) with time. Data points were normalized by the initial concentrations of BH4 (time 0).... 99

Fig. 50. The level of BH4 in mice midbrain (MD) with time after radiofrequency radiation (RFR) exposures (n＝6). 108

Fig. 51. The level of BH4 in mouse striatum (ST) with time after radiofrequency radiation (RFR) exposures(n＝6). 109

Fig. 52. The level of BH4 in mouse pituitary gland (PT) with time after radiofrequency radiation (RFR) exposures (n＝6). 110

Fig. 53. The level of DA in mouse striatum (ST) with time after radiofirequency radiation (RFR) exposures (n＝6). 111

Fig. 54. The level of norepinephrine (NE) in mouse brain stem (BS) with time after radiofrequency radiation (RFR) exposures (n＝6). 112

Fig. 55. The level of norepinephrine (NE) in mice pituitary glands (PT) with time after radiofrequency radiation (RFR) exposures (n＝6). 113

Fig. 56. The level of glutamate (Glu) in mice pituitary gland (PT) with time after radiofrequency radiation (RFR) exposures (n＝6). 114

Fig. 57. The level of glutamate (Glu) in mice Cerebellum (CB) with time after radiofrequency radiation (RFR) exposures (n＝6). 115

Fig. 58. The level of GABA in mice cerebellum (CB) with time after radiofrequency radiation (RFR) exposures (n＝6). 116

초록보기

 테트라히드로비옵테린 (BH4) 는 중추신경계 내에서 도파민(DA), 세로토닌 (5-HT) 및 노르에피네프린 (NE)을 생합성 하는 과정에서 꼭 필요한 보조인자이며, 산화질소 신타아제에 의해 산화질소를 생산하는 보조인자이다. BH4 대사체의 변화는 파킨슨씨병, 페닐케톤뇨증과 신장부전증과 같은 다양한 질병과 밀접한 관련이 있는 것으로 알려져 있다. 심부전, 고혈압, 심장비대, 아테롬성 동맥 경화증과 당뇨병들은 심장혈관계에서 BH4 농도와 관련이 있다고 알려져 오고 있다. 그러므로, 다양한 질환과 연계된 진단평가에 적용가능한 믿을 수 있는 방법으로 BH4의 농도를 측정하는 것이 절실히 필요하며 BH4 의 농도를 조절하는 것이 다양한 질환에 대한 잠재적 치료법을 알아내는 것에 쓰여 질 수 있다.

단순하고 빠른 액체크로마토그래피 텐덤 질량분석기(LC-MS/MS)를 이용한 방법으로 쥐 및 생쥐 내에서 입실론-아세타미도카프로산 (AACA) 과 동위 원소로 치환된 내부 표준물질를 이용하여 BH4, DA, 5-HT, NE, 에피네프린 (EP), 글루타민산염 (Glu) 과 감마아미노뷰티르산 (GABA) 의 측정방법을 개발하였다. 아세토니이트릴을 사용하여, 시료내의 단백질을 침전시키고 상등액은 Sepax Polar-Imidazole (2.1 mm x 100 mm, 내경, 3 ㎛) 컬럼으로 이동상은 10mM 암모니늄 포름산염이 함유된 아세토니이트릴/물 (75:25, 부피비)로 제조하여 유속 300 uL/min 으로 BH4 및 DA을 분리하였다. 5-HT, NE, EP, Glu 및 GABA를 정량하기 위하여 Luna 3u C18 (3.0 mm x 150 mm, 내경, 3um) 컬럼을 사용하여 개미산이 1% 함유된 아세토니이트릴/물 (20:80) 혼합액을 350 uL/min 유속으로 흘려주었다. 전체 크로마토그래픽 운영 시간은 5.5분이 소요되었다. 시료분석에서 방법은 입증되었다. 검정선의 직선구간은 생쥐 뇌 조직 에서 10-2000 ng/g 농도에서 BH4 (r2 = 0.995), 10-5000 ng/g 농도에서 DA (r2 = 0.997), 20-10000 ng/g 농도에서 5-HT (r2 =0.994), NE (r2 =0.993), EP (r2 =0.993) 그리고 0.2 - 200 ug/g 농도에서 Glu (r2=0.996), GABA (r2=0.999) 였다.

생쥐 뇌에 고주파 방사(RFR)를 피폭 시킨 후, 선조체에서 BH4 와 DA는 의미심장하게 감소하였지만, 5-HT는 변화가 없었다. 중뇌에서 BH4는 의미있게 감소하였다. 흥미롭게도, 뇌하수체에서 BH4, NE 와 Glu 는 처음에는 의미있게 감소한 후 1주후 부터는 증가 하였다. 소뇌에서 Glu 와 GABA는 초기에 증가하였으나 1주 후 부터는 감소하는 경향을 보였다.

이상과 같이, 쥐와 생쥐 뇌에서 LC-MS/MS 결과는 BH4, DA, 5-HT, NE, EP, Glu 와 GABA 의 정량분석에 대하여 유용한 수단으로 수립되고 얻어졌다. 제시된 연구는 중추신경계에서 잠재적으로 병태생리학적 역할을 하는 바이오마커로서의 BH4 가능성과 생쥐 뇌에서 고주파 방사를 피폭하여 BH4 와 신경전달물질 DA, 5-HT, NE, EP, Glu 및 GABA와의 관계를 나타낸다.

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