научная статья по теме COMBINED APPLICATION OF 2D CORRELATED SPECTROSCOPY AND 2D NUCLEAR OVERHAUSER ENHANCEMENT SPECTROSCOPY TO THE BRAIN METABOLITES Медицина и здравоохранение

Текст научной статьи на тему «COMBINED APPLICATION OF 2D CORRELATED SPECTROSCOPY AND 2D NUCLEAR OVERHAUSER ENHANCEMENT SPECTROSCOPY TO THE BRAIN METABOLITES»

НЕЙРОХИМИЯ, 2014, том 31, № 1, с. 71-78

= МЕТОДЫ =

УДК 577

COMBINED APPLICATION OF 2D CORRELATED SPECTROSCOPY AND 2D NUCLEAR OVERHAUSER ENHANCEMENT SPECTROSCOPY

TO THE BRAIN METABOLITES © 2014 г. S. Y. Kim", D. C. Woo*, E. Bangc, S. S. Kimd, H. S. Limd, B. Y. Choe", *

aDepartment of Biomedical Engineering and Research Institute of Biomedical Engineering, College of Medicine,

the Catholic University of Korea, Seoul, Korea bMR Core Lab., Asan Institute for Life Science, Asan Medical Center, Seoul, Korea cMetabolome Analysis Team, Korea Basic Science Institute dDepartment of Molecular Genetics, College of Medicine, the Catholic University of Korea, Seoul, Korea

Abstract—The 2D NMR has great potential for routine use in organic chemistry, biochemistry and structural biology. The 2D correlated spectroscopy (COSY) and 2D nuclear Overhauser enhancement (NOE) spectroscopy are particularly powerful techniques for studies of scalar coupling connectivity and spatial connectivity. Here we present the 3-bond spatial connectivity of brain metabolites using in vitro 2D COSY and 2D NOE spectroscopy. The 2D spectra were obtained using 500 MHz NMR from solution of brain metabolites. In this study, we showed the 3-bond and spatial connectivity of brain metabolites detected by scalar coupling and dipolar NOE interaction. Our results may be helpful in promoting better understanding of the interactions in corresponding protons in each of metabolite and provide basic information for structure determination of neurochemicals.

Keywords: 2D NMR, correlation spectroscopy (COSY), nuclear Overhauser enhancement (NOE), brain metabolites.

DOI: 10.7868/S1027813314010087

INTRODUCTION

The interpretation of one-dimensional XH magnetic resonance spectroscopy (MRS) is often hampered due to the complexity of overlapping resonances in the limited chemical shift range (about 5 ppm). Attempts have been made to quantify various metabolites concentration more clearly. Spectral editing method often lead to the unambiguous detection of one specific compound, such as y-aminobutyric acid (GABA) [1], taurine (Tau) [2], and glutamate (Glu) [3], much of the information on other compounds is lost in the process.

A more intelligible method to detect simultaneously brain metabolites is two-dimensional (2D) NMR spectroscopy techniques separating the contributions of the different metabolites along orthogonal axes. 2D-NMR spectroscopy, originally proposed by Jeener

[4], has been considerably developed over the past decade. Homonuclear correlated spectroscopy (COSY)

[5] has proved an effective method to obtain the information about homonuclear coupling connectivities for the identification of scalar coupled spin system. A large number of different 2D-NMR experiments such as relayed coherence transfer spectroscopy [6], multi-

* Corresponding author, address: #505 Banpo-Dong, Seocho-Gu, Seoul 137-040, Korea, phone: 82-2-590-2427, fax: 82-2590-2425, e-mail: bychoe@catholic.ac.kr.

ple-quantum filtered COSY [7], total correlation spectroscopy (TOCSY) [8] yield COSY-type through-bond 1H-1H connectivities. The nuclear overhauser enhancement (NOE) spectroscopy can provide information about a network of through-space proton-proton connectivities which extend over the entire molecular structure [9]. Recently 2D NMR spectroscopy plays a fundamental role in the determination of the structures of proteins [10—12], DNA fragments [13], and polypeptides [14]. 2D NMR has also become a valuable screening tool for the binding of ligands to protein target, and has key advantages of being able to detect and quantify interactions with high sensitivity without requiring prior knowledge of protein function [15, 16].

Extensive documentation over at least 50 years proves that inducing impairments in brain metabolism cause abnormalities in memory, judgment, and other brain function [17—20]. Thus, studying for the structure of brain neurochemical including neurotransmit-ters which are important role in intercellular signal transduction pathway is necessary. There were few reports for three-dimensional spatial information of brain metabolites using 2D NMR spectroscopy. The aims of the present study were to investigate the

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Fig. 1. (a) Typical pulse sequence diagram for 2D correlated spectroscopy (COSY). This consists of preparation, evolution, mixing, and detection periods. (b) Typical pulse sequence diagram for 2D-rotating frame NOE spectroscopy. The spin-locking element consists of a 90 °, 180 °, 90 ° pulse train executed for removal of unwanted coherences.

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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 (Ad) and proton (Bd). The characters 'd' and 'x' represent diagonal and cross peaks, respectively. The < 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.

3-bond spatial connectivity of human brain metabolites using 2D NMR spectroscopy.

MATERIALS AND METHODS Basic Principle of Two-Dimensional NMR Spectroscopy

Compared to conventional one-dimensional spin-decoupling experiments, 2D NMR spectroscopy is much more powerful and more efficient method for delineation of spatial connectivities in metabolites. Through the introduction of additional spectral dimensions, the process can be simplified, and extra information can be obtained.

2D COSY uses the following sequence of two nonselective 90° pulses (Fig. 1a): [90o-t1-90°-t2]n. In addition to the preparation and detection periods, the 2D NMR experiment has an evolution time (t1) and a mixing time. After the preparation period, the various magnetization components are labeled with their characteristic precession frequencies during evolution time (t1). The second 90° pulse induces coherence transfer of magnetization components. The data are acquired during the detection period (t2), when the magnetization is labeled with the chemical shift of the second nucleus. To obtain an adequate signal-to-noise

ratio, n transients are accumulated for each value of t1. Fig lb shows rotating frame NOE sequence. After frequency labeling of the various magnetization components during t1, cross-relaxation leads to exchange of magnetization between nearby protons during the mixing time. The mixing interval is kept fixed and the signal is recorded immediately after spin-locking pulse.

Two-dimensional Fourier transform (FT) yields the 2D spectrum with two frequency axes. Figure 2 shows the simplest homonuclear 2D spectrum of two protons (HA and HB). These consist of diagonal peaks (Ad and Bd) and cross peaks (ABx). The 2D spectrum is symmetrical to the diagonal. The diagonal peaks results from contribution of magnetization that has not changed during the mixing time. The cross peaks originate from nuclei that exchanged magnetization during the mixing time. They indicate an interaction of these two nuclei. Therefore, the cross peaks contain the important information in 2D MR spectra.

Figure 3a shows a schematic drawing of the amino acid backbone structure. The 2D-COSY spectrum of the amino acid backbone structure is shown in Fig. 3b. This 2D-COSY spectrum shows the 3-bond coupling connectivity of NH, Ha, Hp, and Hy through diagonal

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Fig. 3. (a) The general amino acid backbone structure and (b) the typical 2D-COSY spectrum of the amino acid backbone. The Ca exists between the amino group (NH3) and carboxyl (COOH) group. The Ha sent protons attached to Ca character R' represents the carbon chain, which can be continuous.

, Hß, and Hy repre-Cß, and Cy, respectively. The

and cross peaks. These 3-bond coupling connectivities reveal that the magnetization is transferred through scalar coupling. The structures of the main brain metabolites that can be detected by proton MR spectroscopy are shown in Fig. 4.

Sample Preparation

We made a diluted solution containing compounds similar to the brain metabolites. GE brain MRS phantom was used as a reference [21]. The concentrations of the ten metabolites in the brain phantom were: (1) 12.5 mM, N-acetyl aspartate (NAA); (2) 12.5 mM, glutamate (Glu); (3) 12.5 mM, glutamine (Gln); (4) 10.0 mM, creatine (Cr); (5) 3.0 mM, choline (Cho); (6) 7.5 mM, myo-inositol (mI); (7) 5 mM, lactate (Lac); (8) 10 mM, alanine (Ala); (9) 10 mM, y-aminobutyric acid (GABA); (10) 6 mM, taurine (Tau) (Table 1).

MRS Acquisition and Post-Processing

All NMR experiments were performed at 298K on Unity Inova 500 or 600 (Varian Inc.) equipped with a triple resonance probe head with z-shield gradient. Human brain metabolites were prepared with 10% D2O. 2D NMR spectra, including COSY and rotating frame NOESY, were acquired with 4096 complex data points in t2 with 32 or 64 number of scan and 128 or 256 increments in the t1 dimension. Rotating frame NOE spectroscopy was performed for a mixing time of 200 ms. The 9.8 ^s pulse width (90° pulse angle)

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Fig. 4. Chemical structures of the major brain metabolites listed in Table 1. Proton number is assigned with respect to carbon.

Table 1. Chemical compounds of brain metabolites and solutions

Comp

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