14.2.2. One- and two-dimensional NMR spectroscopy techniques

The number of observed metabolites in body fluids is largely dependent on the magnetic field strength of the NMR spectrometer. At higher fields, the spectral dispersion and sensitivity increase, allowing assignment of many metabolites that cannot be detected at lower fields. Therefore, working at the highest field available will provide the most complete metabolic information. Currently, most studies are performed on 400, 500 or 600 MHz spectrometers, but higher field strengths up to 800 MHz have been used [23–25]. Additionally, low frequency spinning of the sample can be used to improve spectral resolution. Reported measurement temperatures are found between 20 and 37 °C.

A wide range of one-dimensional (1D), two-dimensional (2D) and even higher dimensional NMR experiments are available to the modern NMR operator. Especially in the field of structural biology, multidimensional experiments are important for successful determination of protein or nucleic acid structures [26, 27].

For studying inborn errors of metabolism, 1D 1H NMR spectra are most frequently employed. Additionally, several homo- and heteronuclear 2D experiments can be recorded, which are particularly helpful in assisting the assignment of resonances. The most commonly used experiments for diagnosing metabolic diseases will now be discussed. The information content of the spectra will be demonstrated using spectra recorded on a sample containing pure methionine (Figure 14.3). A schematic overview of the experiments is in Table 14.1. Figure 14.3.. Six different 500 MHz NMR spectra of methionine. A: 1D 1H-NMR spectrum S-CɛH 3 : The methyl protons on Cɛ give a singlet resonance at 2.13 ppm. This singlet corresponds to the three equivalent protons of the methyl group. The fact that it is a singlet illustrates that the neighboring atom (the S atom) does not carry any proton(s). In body fluids the 2.13 ppm resonance is often used to quantify methionine. CγH 2 : Due to the J-coupling with the two neighboring protons on Cβ, the protons on Cγ give a triplet resonance at 2.63 ppm (γJ γ,β = 7.3 Hz). CβH 2 : The protons on Cβ give two multiplets at 2.10 and 2.20 ppm. This is explained by the fact that Cα is an asymmetrical carbon atom, and the two methylene protons on Cβ are therefore diastereotopic; consequently, they give separate resonances. Due to the J-coupling with the proton on Cα and the protons on Cγ, we find two multiplets. CαH: Due to the J-coupling with the two diastereotopic methylene protons on Cβ, the proton on Cα gives a disturbed triplet (doublet/doublet) resonance at 3.95 ppm. The peak area for the various resonances in methionine is proportional to the number of protons attached to the various C atoms. B: 2D 1H-1H COSY spectrum Chemical shift and scalar coupling can be obtained simultaneously for methionine by detecting the off-diagonal cross peaks, which are symmetric with respect to the diagonal. The diagonal and cross peaks connected by dashed lines indicate which protons of methionine have a scalar coupling. The diagonal peak of the two protons on Cβ forms a corner of two squares, as these protons are coupled both to the proton on Cα and to those on Cγ. C: 2D 1H-1H TOCSY spectrum Compared with the COSY spectrum (Figure 14.3b) two new signals have now appeared. These provide evidence of a correlation between the protons on Cα and Cγ. D: 1D 1H-decoupled 13C spectrum In the 1D 1H-decoupled 13C NMR spectrum, Cα, Cβ, Cγ and Cɛ from methionine give resonances at 56.7, 32.5, 31.7 and 16.7 ppm, respectively. Note: This 1D 1H-decoupled 13C spectrum was recorded at pH 7.0. E: 2D 1H-13C HSQC spectrum This type of NMR spectroscopy reveals which carbons and protons in methionine are chemically bonded. For methionine, the 1H/13C shift values for the correlation peaks are 3.86/56.7 (α), 2.20; 2.10/32.5 (β), 2.65/34.7 (γ) and 2.13/16.7 (α). Note: This HSQC spectrum of methionine was recorded at pH 7.0. F: 2D 1H J-resolved spectrum The J-multiplets are displayed along the F 1 dimension, while the chemical shifts are shown in the F 2 dimension. This minimizes overlap and allows better determination of coupling constants, which also are detectable in 1D 1 H-NMR. The protons on C7 give a triplet resonance at 2.63 ppm with a J-coupling of 7.3 Hz (γJ γ,β = 7.3 Hz) Table 14.1. Overview of different experiments used in the diagnosis of inborn errors of metabolism Experiment Description 1D 1H (Figure 14.3a) Single pulse with solvent presaturation. Routinely used to determine chemical shifts, J-values and metabolite quantities (only for well-resolved peaks). 1H-1H COSY (Figure 14.3b) Used to establish which protons are spin-coupled. Helpful in resolving overlap and to assign crowded regions. 1H-1H DQF-COSY COSY variant with greater resolution. Cross peaks near the diagonal can be resolved. Sensitivity is 50% less compared to COSY. 1H-1H TOCSY (Figure 14.3c) COSY variant displaying relayed connectivities. Used as an aid in assignment. 1H J-resolved (Figure 14.3f) Separates J-splitting and chemical shifts on to two orthogonal axes. Reduced overlap and more accurate determination of J-values and chemical shifts. 1H 1D Spin-echo Spectral simplification based on difference in relaxation times. Not suitable for quantification. 1D 1H-decoupled 13C (Figure 14.3d) More resonances resolved due to larger 13C spectral width than 1H. Suffers from inherent low sensitivity and low natural abundance. 1H-13C HSQC (Figure 14.3e) Displays connectivities between chemically bonded 1H and 13C nuclei. Helpful when assigning complicated spectra.

14.2.2.1. One-dimensional NMR spectroscopy Since proton NMR is relatively sensitive and because protons are present in almost every metabolite, a 1D, single pulse 1H NMR experiment with solvent presaturation is a good starting point when diagnosing a possible inborn error of metabolism. When overlap is not too severe and metabolite concentrations are high enough (>2–40 μmol/L), the chemical shifts and J-coupling constants can be determined directly and subsequent quantification of these metabolites is possible. The singlet resonance at 2.13 ppm from the ɛ methyl protons of methionine is most suited to quantify this metabolite in body fluids (Figure 14.3a). However, assignment can be rather difficult when high order spin coupling patterns are present or when spectral regions are crowded. Furthermore, presaturation of the water signal can result in loss of nearby peaks.

14.2.2.2. Two-dimensional NMR spectroscopy Two-dimensional NMR experiments can provide additional information to solve overlap problems and allow identification of metabolites that otherwise remain undetected. They are based on the couplings between magnetic nuclei. These can be dipolar (through space) or scalar (through bond) couplings. Here, only experiments from the latter category will be discussed. 14.2.2.3. 1H-1H correlation spectroscopy The first homonuclear 2D experiment is correlation spectroscopy (COSY). The underlying concept is coherence transfer from one spin to another via J-coupling [28]. It reveals the network of spin–spin couplings in each molecule and can therefore be used to aid in spectral assignment. The peaks under the diagonal of a COSY spectrum correspond to the peaks normally observed in a 1D spectrum. The off-diagonal peaks (cross peaks) provide the information on which protons are spin coupled. From the COSY spectrum of methionine, for instance, it is clear that the α -proton is J-coupled to the β-protons (Figure 14.3b). Unfortunately, there is a 90° phase difference between the diagonal and the cross peaks, which makes it impossible to correct the phase of a COSY spectrum in such a way that all peaks have absorption phase. When the phase of the cross peaks is corrected to pure absorption, the diagonal peaks display dispersive line shapes in both dimensions, and their long dispersion tails can conceal nearby cross peaks. A solution is to record a Double Quantum Filtered COSY (DQF-COSY) experiment, in which both diagonal and cross peaks have absorption line shapes. Consequently, the spectral resolution is improved and cross peaks close to the diagonal can be readily detected. Also, chemical shifts and coupling constants can be determined more easily. However, a disadvantage is that the sensitivity is reduced compared to a regular COSY, because only two out of four scans lead to cross peaks and the other two lead to diagonal peaks [28]. Another solution to the phase problem is displaying the conventional COSY spectrum in magnitude mode. All peaks appear absorptive, although cross peaks near the diagonal are still likely to be obscured. In the literature, COSY spectra of body fluids have been widely used in patients with IEM, for example in fucosidosis [8], 3-hydroxy-3-methylglutaryl CoA lyase deficiency [29], ribose 5-phosphate isomerase deficiency [30] and Salla disease [8, 31].

14.2.2.4. Total Correlation Spectroscopy The COSY spectra of complex mixtures like body fluids can still show a substantial amount of overlap. Resulting ambiguities in the spectral assignment may be resolved by detecting multiple relayed connectivities using a Total Correlation Spectroscopy (TOCSY) experiment. For instance, in an AMX spin system cross peaks are observed between A and X, where both spins are coupled to spin M, but not to each other. This allows detection of long range interactions (>3 bonds), that are usually too weak in normal COSY spectra. Other advantages are a higher sensitivity for larger molecules and absorption line shapes for both diagonal and cross peaks. The number of observed cross peaks depends on the length of the applied TOCSY mixing sequence. A spectrum recorded with a short mixing period mainly contains cross peaks of directly coupled spins, whereas a long mixing time gives rise to relayed cross peaks. In the case of methionine, the TOCSY spectrum (Figure 14.3c), for instance, shows cross peaks between the α-proton and the γ-protons, which were not present in the regular COSY (cf. Figure 14.3b). A 1D version of TOCSY spectra at 750 MHz has been used to assign peaks in spectra from seminal fluid by Spraul et al. [32]. Furthermore, 1D TOCSY has been used to identify 3-ureidoisobutyric acid as an accumulating compound in ureidopropionase deficiency [33]. To our knowledge there are no examples of successful application of 2D TOCSY to the field of inborn errors of metabolism.

14.2.2.5. J-resolved spectroscopy (JRES) Another useful homonuclear 2D experiment is the J-resolved (J-res) experiment, which separates the J fine structure from the chemical shifts. In the resulting spectrum, the J-multiplets are displayed along the F 1 dimension, while the chemical shifts are shown in the F 2 dimension. This minimizes overlap and allows better determination of coupling constants. Furthermore, a proton-decoupled 1D spectrum can be obtained by projection of the 2D J-resolved spectrum onto the F 2 axis. The JRES spectrum of methionine is shown in Figure 14.3f [30]. The 600 MHz 2D J-res 1H-NMR spectra from urine and blood plasma samples was published by Foxall et al. [34]. In the literature, JRES 1H-NMR spectra of body fluids have been used in patients with IEM, for example ribose 5-phosphate isomerase deficiency [30], maple syrup urine disease [24] and 2-methylacetoacetyl CoA thiolase deficiency [35].

14.2.2.6. Spin-echo spectra Proton spectra can be also simplified by making use of the differences in relaxation behavior between different molecules. This principle forms the basis of spin-echo experiments. In spin-echo spectra, signals arising from relatively large molecules (i.e. protein signals in intact plasma) are attenuated due to their shorter relaxation times. Furthermore, even-numbered multiplets are displayed as negative peaks, whereas singlets and odd-numbered multiplets appear positive [35, 36]. Spin-echo experiments can therefore be helpful when assigning complex spectra. However, the method is less suited for quantification due to signal loss during long echo times. Since plasma samples are routinely deproteinized, spin-echo experiments are not recorded often nowadays when trying to diagnose an inborn error of metabolism.