NMR Spectroscopy Techniques for Application to …

1 The Handbook of Metabonomics and MetabolomicsJohn C. Lindon, Jeremy K. Nicholson and Elaine Holmes (Editors) 2007 Published by Elsevier 3 NMR Spectroscopy Techniques for Applicationto MetabonomicsAlfred Ross, Goetz Schlotterbeck, Frank Dieterle, and Hans SennPharma Research, F. Hoffman La-Roche AG, Basel IntroductionSince its discovery in the 1940s,NuclearMagneticResonance (NMR) Spectroscopyhas become a powerful, interdisciplinary method. A brief historical review wouldreveal as many as nine Nobel Prize laureates since the time when Isador I. Rabi devel-oped resonance methods for recording the magnetic properties of atomic nuclei andwas awarded the Nobel prize in physics (1944). The NMR phenomenon was soon laterdemonstrated for protons. After years of continuous development, Fourier Transform(FT) NMR entered the scene in the 1960s, followed by the evaluation of ,three-dimensional(3D) structure elucidation of proteins at atomic resolution in aqueous environment wasdeveloped [1].

2 This breakthrough was made possible also by the availability of super-conducting materials and stable and robust electronic has since been used in an almost unlimited variety of ways in physics,chemistry and biology. For the investigation of biological systems it is convenientto distinguish between three types of applications [2]: (1) to study structure andfunction of macromolecules, (2) to study metabolism, and (3) to obtainin vivoimages of anatomical structure and functional (physiological) use of1H NMR for metabolic studies was described as early as 1977 whenit was shown that1H signals could be observed from a range of compounds in asuspension of red blood cells, including lactate, pyruvate, alanine and creatine [3].A great deal of metabolic information can be derived from such metabolic studiesand it was soon recognized that1H NMR of body fluids has a considerable role to5556 NMR Spectroscopy Techniquesplay in areas of pharmacology, toxicology and the investigations of inborn errors ofmetabolism [4 6].

3 Since these early applications ,1H NMR of biofluids and cell extracts includinghigh-resolution magic angle spinning (HR-MAS) NMR of soft tissues have beensuccessfully applied to investigate numerous diseases and toxic processes [7 9].This chapter is meant to give an introduction and overview on NMR underthe view point of its Application in metabolic profiling (metabonomics). It is notmeant to review all the different applications of NMR in this field but rather toconcentrate on the essentials and prerequisites of its successful implementation asa metabolite profiling tool. To this end, specific NMR hardware requirements formetabolite profiling are reviewed and compared as well as automation and roboticscontrol of the work flow, which are crucial for achieving high throughput andconsistent quality of results. It is then shown that sample preparation and handlingis of utmost importance for meaningful comparison of hundreds of samples andthousands of spectral variables in a metabolite profiling study.

4 Therefore all aspectsknown to us which may change the sample property of biofluids are included andextensively discussed. The handling and preparation of urine samples is especiallydemanding in order to control and handle the wide variability of conditions thatinfluence the1H NMR spectrum. These problems are less severe with blood serum,plasma orcerebrospinalfluid (CSF) where homeostasis ensures a much narrowerrange of sample variability. In the section on NMR Experiments and Processing,the information which is necessary to obtain high-quality one-dimensional (1D) andtwo-dimensional (2D) NMR spectra of biofluids is summarized. Finally, the state-of-the-art of data pre-processing, which is the intermediate step between recordingNMR raw spectra and applying uni- or multivariate data analysis and modelingmethods, is discussed in great detail. In metabonomics, this is an important step tomake subsequent analysis and modeling easier, more robust and more Principles of NMRThe theory of NMR is highly developed and the dynamics of nuclear spin-systemsfully understood [10, 11].

5 A first-principle quantum mechanical description is avail-able, but beyond the scope of this introduction. What is missing to complete thetheoretical picture is a reliable and accurate prediction of the chemical shift of , recourse to spectral databases is needed if a chemical interpretation ofmetabonomics data is present a phenomenological description of magnetic resonance needed forthe understanding of the metabonomic literature. It has to be stressed, however, thatnearly any aspect of modern NMR Spectroscopy is of importance for the acquisitionof high-quality data needed for a reliable biological interpretation of results. After anAlfred Ross, Goetz Schlotterbeck, Frank Dieterle, and Hans Senn57introduction into the principles of NMR (magnetization, chemical shift, relaxation,J-coupling) we touch upon more advanced concepts involving chemical exchange,2D and heteronuclear experiments.

6 We will see that all effects are of relevance formetabonomic research. We do not seek for a complete review of literature of thetopic. If possible we provide examples from our own MagnetismMagnetism and spin:Matter is composed of molecules built of atomic nuclei witha characteristic proton/neutron composition. Nuclei are surrounded by electronic clouds . Besides charge and mass, a further property of protons, neutrons andelectrons is an angular momentum Iknown as spin. Because of the magnitude ofthis angular momentum I I=h2 34the aforementioned particles are known asspin-1/2 total spin of a nucleus depends on its nucleon content. Somenuclei carry a total spin (1H,2H,13C,15N,19F,31P, )2resulting in a magneticmoment M= X Iof different magnitude and sign, others ( ,14C,16O, )do not; Xis the gyromagnetic ratio of the atomic NMR- Spectroscopy samples of liquid or solid material3are exposed to anexternal static and homogeneous magnetic field referred to asB0.

7 The direction ofthis field is usually defined alongz. The magnetic moments in the sample align alongB0according to a Boltzmann distribution. In contrast to classical physics, quantummechanics shows that magnetic moments due to spin-1/2 particles can only alignparallel (called up) or anti-parallel (called down) with respect to this external field,these two states have a difference of energy given by E= X B0. Due to statisticaveraging, the magnetic moment of any macroscopic sample can be treated in manyrespects like a classical macroscopic magnetic moment M. In accordance with thedefinitions above, the thermal equilibrium magnetization of a sample aligned alongzis calledM0. For the detection of the NMR signal,M0is flipped orthogonal toB0by use of a high-frequency magnetic fieldB1applied for a defined time also applied orthogonal toz(see ). This is called aB1-pulse.

8 Classical physicsshows that M, now aligned without loss of generality along thex-direction, willprecess with a (resonance) frequency given byf0= X2 B0( )1his known as Planck s is a spin-1 particle. To date there is no Application for2H and other higher spin nuclei reported for are metabonomic applications for tissues. Tissues are not real solids, but more gel-like and soft structureswhere the spin-physics can be transformed to liquid-like behavior by use of the so-called HR-MAS Spectroscopy TechniquesThis is the reason why NMR spectrometers are normally classified in magnetization of a sample of hydrogen atoms experiencing a magnetic fieldof T will, for example, rotate with 600 MHz. Such a spectrometer is called a600 MHz apparatus. Other nuclei will rotate in the same field with another spin will precess in the same 600 MHz magnet at about 150 of the signal:The sample is positioned inside a detection coil (seeFigure ).

9 According to the law of inductivity the precessing magnetization willinduce a voltageUindmodulated withf0. The amplitude of this voltage is directlyproportional to Mand thus with the number of spins rotating withf0located insidethe observe volume of the apparatus. In the NMR literature the signal detected iscalled aFreeInductionDecay or to diamagnetism of the electron clouds of the molecule the local magneticfield experienced by a spin is changed by a small amount compared toB0. Thus thefrequencyfAmeasured for spin A directly reports the electronic/chemical neighbor-hood of the nucleus observed. The value fA f0 f0 106, measured in parts per million(ppm) in respect toB0, is called the chemical shift of this spin. This definition isindependent ofB0 thus data taken at different field strength can be comparedeasily. The chemical shift of different spin species covers different ranges:1 Hnuclei resonate in most cases within a width of 15 ppm;13C nuclei cover more thanB1(t ) = Bx (cos( 0 t ) + sin( 0 t ))Uind(t ) ~ Mx (t ) + i My (t )Figure Detection of an NMR Signal: (left) a sample with many spins (>1012) is placed inside astrong external magnetic fieldB0(orange) oriented along the z-direction.

10 The sample is enclosed in adetection coil (shown in green). At thermal equilibrium about one spin in every 10,000 contributes toa macroscopic magnetizationM0shown in transparent red. After short Application of a high-frequencyB1field (90 pulse: black bar and equation at the left panel) the magnetization is aligned along thex-axis of the rotatingB1field, and starts to precess aroundB0. The voltage,Ui, induced in the detectioncoil (dark-blue and equation at the right panel) can be described by the equation shown on the bottomof the right Ross, Goetz Schlotterbeck, Frank Dieterle, and Hans Senn59200 ppm. For the definition off0normally a reference compound is added to [12]:Each spin creates a small dipolar magnetic field spread over magnitude of this additional magnetic field experienced by another spin speciesin the neighborhood ( the same molecule) is dependent on the angle and thelength of the vector connecting both spins with respect toB0.