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2 Fig. 1. Typical pulse sequence diagram for 2D-correlation spectroscopy (COSY). This consists of preparation, evolution, mixing, and detection periods

3 Fig. 3. The general amino acid backbone structure. The C exists between the amino group (NH3) and carboxyl (COOH) group. The H, H, and H represent protons attached to C, C, and C, respectively. The character R represents the carbon chain, which can be continuous. Fig. 2. General spectrum of 2D-homonuclear MR spectroscopy. This consists of diagonal and cross peaks. A cross peak (ABx) can be made if there is any relationship between proton (A) and proton (B). The characters d and x represent diagonal and cross peaks, respectively. The 1 and 2 axes represent data obtained during t1 (evolution period) and t2 (detection period), respectively. 2D Fourier transform (FT) yields the 2D spectrum with two frequency axes. Fig. 4. The typical 2D-COSY spectrum of the amino acid backbone in Figure 3. This shows diagonal and cross peaks for 3-bond coupling connectivity

4 Fig. 5. Chemical structures of the major human brain metabolites. Proton number is assigned with respect to carbon

5 Table 1. Phantom Ingredients of the Various Metabolites in the Brain Mimicking Solution Compound Abbreviation CAS no. Concentration Potassium phosphate monobasic KH 2 PO 4 [ ] 50.0 mm Soduim hydroxide NaOH [ ] 56.0 mm Sodium azide [ ] 0.01 % L-Glutamic acid Glu [ ] 12.5 mm L-Glutamine Gln [ ] 12.5 mm Creatine hydrate Cr [ ] 10.0 mm Choline chloride Cho [ ] 3.0 mm N-Acetyl-L-aspartatic acid NAA [ ] 12.5 mm Myo-inositol Ins [ ] 7.5 mm DL-Lactic acid Lac [ ] 5.0 mm Magnevist Gd-DPTA 1 ml/l a b Fig. 6. In vitro 2D MRS spectra for human brain metabolites including from 0 ppm to 12 ppm for full scale data. (a) COSY spectrum (b) NOESY spectrum. Cross peaks were observed at 7.9 ppm as well as from 1.0 ppm to 4.5 ppm (arrow)

6 Table 2. Chemical shift ( ) a and COSY/NOESY Connectivities of the Typical Brain Metabolites by Proton MRS. b Compound (ppm) Resonance COSY Connectivity NOESY Connectivity NAA 2.02 CH 3 CH 2 (3,H )+CH 2 (3,H ) CH 3 + NH 2.50 CH2(3,H ) 2.70 CH2 (3,H ) 4.39 CH(2) 8.00 NH c Cr 3.03 CH 3 CH 3 +CH CH 2 Cho 3.24 N(CH3) CH 2 (2) 4.07 CH 2 (1) Glu 2.11 CH 2 (3) CH 2 (3)+ CH 2 (4) CH 2 (3)+CH2 (4) 2.35 CH 2 (4) CH 2 (3)+CH(2) 3.76 CH(2) Gln 2.14 CH 2 (3) CH 2 (3) +CH(2) 2.46 CH 2 (4) 3.79 CH(2) 6.13 CH(1 ) c 8.25 CH(2) 8.54 CH(8) Ins 3.27 CH(5) CH(5)+ CH(4,6) CH(5)+ CH(4,6) 3.53 CH(1,3) CH(1,3)+CH(4,6) CH(1,3)+ CH(2) 3.59 CH(4,6) CH(1,3)+ CH(2) 4.05 CH(2) Lac 1.33 CH3(3) CH 3 (3)+ CH(2) CH 3 (3)+ CH(2) 4.11 CH(2) a VS. TMS at 0.0ppm b Abbreviations are listed in Table 1 c Temperature-dependent chemical shift; in vivo? = 7.85 ppm d Sugar proton

7 a b Fig. 7. In vitro 2D MRS spectra for human brain metabolites including from 1 ppm to 4.5 ppm. The typical scale including for brain metabolites. (a) COSY spectrum (b) NOESY spectrum

8 인체 뇌 대사물질에서의 In vitro 2D-COSY와 2D-NOESY 스펙트럼 분석 평가 (Figure 7b, Figure 9b). 그리고 Figure 10에서 볼 수 있듯이 ppm 사 이에는 2.02 ppm 의 NAA 메틸 (CH3) 양성자, 2.11 ppm의 Glu 메틸렌 (CH2(3)) 양성자, 2.14 ppm의 Gln 메틸렌 (CH2(3)) 양성자들이 거의 비슷한 공명주파수 하에 겹쳐져 있 는 모습을 보여준다. Lactate는 2D-COSY 스펙트럼의 1.33 ppm에서 명확한 두 개 (doublet) 피크로 확인되었다 (Figure 11). a b Fig. 8. In vitro 2D MRS spectra for human brain metabolites including from 1.9 ppm to 2.8 ppm. (a) COSY spectrum (b) NOESY spectrum. The cross peaks were observed for neurotransmitter (Glx) around 2.5 ppm as an evidence for the closest distance between corresponding protons. a b Fig. 9. In vitro 2D MRS spectra for human brain metabolites including from 3.11 ppm to 4.2 ppm. (a) COSY spectrum (b) NOESY spectrum. The cross peaks were observed for myo-inositol (Ins) at 3.5 ppm

보았다. 인체 뇌 대사물질 중 주요 분석대상으로 선정한 총 7가지 화학물 (NAA, Cr, Cho, Gln, Glu, Ins)은 스펙트럼상에서 모 두 확인 가능하였다. 팬톰 화학물 제작 시 농도를 이미 학계 에 발표된 수치를 사용하였기에 정량분석을 이루어 지지 않았 다.

9 최보영 외 COSY와 NOESY를 직접 적용하여 인체 뇌 대사물질들간의 스칼라 짝지움 (coupling)과 쌍극자 (dipolar) 상호작용-NOE 에 대한 양성자들간의 3-bond 연결관계 및 공간거리 연결관 계에 대한 connectivity는 Table 2에 정리되어 있다. 결 론 본 연구에서는 in vitro 상태의 인체 뇌 대사물질에 2-D MR 기술, 즉 2D-COSY와 2D-NOESY 기술을 직접 적용하고, 데 이터를 획득하여 인체 뇌 대사물질들간의 스칼라 짝지움 (coupling)과 쌍극자 (dipolar) 상호작용-NOE에 대한 분석을 통하여 결합연결관계 및 공간연결관계에 대한 정보를 성공적 으로 획득하여 분석하여 보았다. 인체 뇌 대사물질 중 주요 분석대상으로 선정한 총 7가지 화학물 (NAA, Cr, Cho, Gln, Glu, Ins)은 스펙트럼상에서 모 두 확인 가능하였다. 팬톰 화학물 제작 시 농도를 이미 학계 에 발표된 수치를 사용하였기에 정량분석을 이루어 지지 않았 다. 그러나 조영제 Magnevist에 화학적 이동 (chemical shift) 및 정량에 대한 정보는 확보할 수 없었다. 이는 Magnevist의 농도가 1 ml/l. (Table 1)로서 너무 미량이라 스펙트럼상에 표 출되지 못한 가능성도 있으리라 사료된다. 예상한대로 2차원 COSY와 NOESY 스펙트럼은 1차원 스펙 트럼보다 1차원 증가한 만큼 해상도가 향상되었다. 단순한 투 시 (projection)선 만을 보여주는 1차원 스펙트럼에 비하여 2 차원 스펙트럼은 여러 가지 다양한 병리학적 변화와 직결되는 대사물질의 변화를 측정하는데 사용할 수 있는 교차피크를 보 여준다. 수분억제를 위한 CHESS 펄스에 의하여 2D 스펙트럼은 대 각선을 중심으로 정확히 대칭을 이루지 않기 때문에 일반적으 로 대각선을 중심으로 아래에 위치한 교차피크를 사용한다 (25). 물론 대각선을 중심으로 대칭성을 이용한 평균치 산출 방법으로 이차원 후처리 기법에 분석할 수 있으나 인공 (artificial) 크로스피크 발생 가능성이 있으므로 배제하였다. 결 Fig. 11. In vitro 2D-COSY spectra for human brain metabolites. The cross peaks for lactate at 1.3 ppm were observed as doublet. a b Fig. 10. In vitro 2D MRS spectra for human brain metabolites including from 1.96 ppm to 2.2 ppm. (a) COSY spectrum (b) NOESY spectrum. The peaks for neurotransmitter at 2.0 ppm were not distinguishable in 1D spectrum, but can be identified in 2D spectrum

10 1.Bottomley PA. Human in vivo NMR spectroscopy in diagnostic medicine: clinical tool or research probe? Radiology 1989;170: Ross B, Kreis R, Ernst T. Clinical tools for the 90s: magnetic resonance spectroscopy and metabolite imaging. Eur J Radiol 1992;14: Weiner MW. The promise of magnetic resonance spectroscopy for medical diagnosis. Invest Radiol 1988;23: Henriksen O. MR spectroscopy in clinical research. Acta Radiologica 1994;35: Choe BY. MR spectroscopy. J Korean Soc Magn Reson Med

11 1997;1: Choe BY, Kang SK, Chu MJ, Baik HM, Kim EN, Kim BS, Lee JM, Chung ST, Ahn CB, Oh CH, Kim JH, Kim SI, Lee KN, Lee HK, Suh TS, Lee HK, Shinn KS. High resolution MR images from 3T Active-shield whole body MRI system. J Korean Soc Magn Reson Med 2001;5: Karplus M. Contact electron-spin coupling of nuclear magnetic moments. J Chem Phys 1959;30: Karplus M. Vicinal proton coupling in NMR. J Am Chem Soc 1963;85: Wuthrich K. NMR of proteins and nucleic acids, 1986, Wiley, New York 10.Aue WP, Bartholdi E, Ernst RR. Two-dimensional spectroscopy, application to nuclear magnetic resonance. J Chem Phys 1876;64: Wagner G. Two-dimensional relayed coherence transfer spectroscopy of a protein. J Magn Reson 1983;55: Braunschweiler L, Ernst RR. Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy. J Magn Reson 1983;53: Subramanian S, Bax A. Generation of pure phase NMR subspectra for measurement of homonuclear coupling constants. J Magn Reson 1987;71: Kumar A, Ernst RR, Wuthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun 1980;95: Davis DG, Bax A. Separation of chemical exchange and crossrelaxation effects in two-dimensional NMR spectroscopy. J Magn Reson 1985;64: Thomas MA, Yue K, Binesh N, et al. Localized two-dimensional shift correlated MR Spectroscopy of human brain. Magn Reson Med 2001;46: Thomas MA, Hattori N, Umeda M, Sawada T, Naruse S. Evaluation of twp-dimensional L-COSY and JPRESS using a 3T MRI scanner: from phantoms to human brain in vivo. NMR Biomed 2003;16: Kreis R, Boesch C. Spatially localized, one- and two-dimensional NMR spectroscopy and in vivo application to human muscle. J Magn Reson B 1996;113: Thomas MA, Chung HK, Middlekauff H. Localized two-dimensional 1H magnetic resonance exchange spectroscopy: a preliminary evaluation in human muscle. Magnetic Resonance in Medicine 2005;53: Thomas MA, Yue K, Binesh N, DeBruhl N. Volume localized t- wo dimensional correlated MR spectroscopy of human breast cancer. J Magn Reson Imaging 2001;14: Schirmer T, Auer DP. On the reliability of quantitative clinical magnetic resonance spectroscopy of the human brain. NMR Biomed 2000;13: Johnston ER, Dellwo MJ, Hendrix J. Quantitative 2D exchange spectroscopy using time-proportional phase incrementation. J Magn Reson 1986;66: Barker PB, Breiterb SN, Soher BJ. Quantitative proton spectroscopy of canine brain: In vivo and in sitro correlations. Magn Reson Med 1994;32: Van Zijl PCM, Barker PB. Magnetic resonance spectroscopy and spectroscopic imaging for the study of brain metabolism. Annals New York Academy of Sciences 1997;820: Binesh N, Yue K, Fairbanks L, Thomas MA. Reproducibility of localized 2D correlated MR spectroscopy. Magn Reson Med 2002;48: Welch JWR, Bhakoo K, Dixon RM, Styles P, Sibson NR, Blamire AM. In vivo monitoring of rat brain metabolites during vigabatrin treatment using localized 2D-COSY. NMR Biomed 2003;16: Prescot AP, Dzik- Jurasz ASK, Leach MO, Sirohi B, Powles R, Collins DJ. Localized COSY and DQF-COSY 1H-MRS sequences for investigating human tibial bone marrow in vivo and initial application to patients with acute leukemia. J Magn Reson Imaging 2005;22:

12 Evaluations of Spectral Analysis of in vitro 2D-COSY and 2D-NOESY on Human Brain Metabolites Bo-Young Choe 1, Dong-Cheol Woo 1, Sang-Young Kim 1, Chi-Bong Choi 1, Sung-Im Lee 2, Eun-Hee Kim 2, Kwan Soo Hong 2, Young-Ho Jeon 2, Chaejoon Cheong 2, Sang-Soo Kim 3, Hyang-Sook Lim 3 1 Department of Biomedical Engineering, College of Medicine, The Catholic University of Korea, Seoul, Korea 2 MR Team, Korea Basic Science Institute, Choongbuk, Korea 3 Department of Molecular Genetics, College of Medicine, The Catholic University of Korea, Seoul, Korea Purpose : To investigate the 3-bond and spatial connectivity of human brain metabolites by scalar coupling and dipolar nuclear Overhauser effect/enhancement (NOE) interaction through 2D- correlation spectroscopy (COSY) and 2D- NOE spectroscopy (NOESY) techniques. Materials and Methods : All 2D experiments were performed on Bruker Avance 500 (11.8 T) with the z- shield gradient triple resonance cryoprobe at 298 K. Human brain metabolites were prepared with 10% D 2 O. Two-dimensional spectra with 2048 data points contains 320 free induction decay (FID) averaging. Repetition delay was 2 sec. The Top Spin 2.0 software was used for post-processing. Total 7 metabolites such as N-acetyl aspartate (NAA), creatine (Cr), choline (Cho), glutamine (Gln), glutamate (Glu), myo-inositol (Ins), and lactate (Lac) were included for major target metabolites. Results : Symmetrical 2D-COSY and 2D-NOESY spectra were successfully acquired: COSY cross peaks were observed in the only ppm, however, NOESY cross peaks were observed in the ppm and 7.9 ppm. From the result of the 2-D COSY data, cross peaks between the methyl protons (CH 3 (3)) at 1.33 ppm and methine proton (CH(2)) at 4.11 ppm were observed in Lac. Cross peaks between the methylene protons (CH 2 (3,H )) at 2.50ppm and methylene protons (CH 2,(3,H B )) at 2.70 ppm were observed in NAA. Cross peaks between the methine proton (CH(5)) at 3.27 ppm and the methine proton (CH(4,6)) at 3.59 ppm, between the methine proton (CH(1,3)) at 3.53 ppm and methine proton (CH(4,6)) at 3.59 ppm, and between the methine proton (CH(1,3)) at 3.53 ppm and methine proton (CH(2)) at 4.05 ppm were observed in Ins. From the result of 2-D NOESY data, cross peaks between the NH proton at 8.00 ppm and methyl protons (CH 3 ) were observed in NAA. Cross peaks between the methyl protons (CH 3 (3)) at 1.33 ppm and methine proton (CH(2)) at 4.11 ppm were observed in Lac. Cross peaks between the methyl protons (CH3) at 3.03 ppm and methylene protons (CH2) at 3.93 ppm were observed in Cr. Cross peaks between the methylene protons (CH 2 (3)) at 2.11 ppm and methylene protons (CH 2 (4)) at 2.35 ppm, and between the methylene protons(ch 2 (3)) at 2.11 ppm and methine proton (CH(2)) at 3.76 ppm were observed in Glu. Cross peaks between the methylene protons (CH 2 (3)) at 2.14 ppm and methine proton (CH(2)) at 3.79 ppm were observed in Gln. Cross peaks between the methine proton (CH(5)) at 3.27 ppm and the methine proton (CH(4,6)) at 3.59 ppm, and between the methine proton (CH(1,3)) at 3.53 ppm and methine proton (CH(2)) at 4.05 ppm were observed in Ins. Conclusion : The present study demonstrated that in vitro 2D-COSY and NOESY represented the 3-bond and spatial connectivity of human brain metabolites by scalar coupling and dipolar NOE interaction. This study could aid in better understanding the interactions between human brain metabolites in vivo 2D- COSY study. Index words : Magnetic resonance spectroscopy (MRS), 2D-MR, COSY, NOESY Address reprint requests to : Bo-Young Choe, Ph.D. Department of Biomedical Engineering, College of Medicine, The Catholic University of Korea, #505 Banpo-Dong, Seocho-Gu, Seoul , Korea. Tel Fax bychoe@catholic.ac.kr

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