and Two-Dimensional 1H NMR Spectroscopy of Biological Fluids

Maili Liu, Jeremy K. Nicholson, and John C. Lindon*. Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square,...
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Anal. Chem. 1996, 68, 3370-3376

High-Resolution Diffusion and Relaxation Edited One- and Two-Dimensional 1H NMR Spectroscopy of Biological Fluids Maili Liu, Jeremy K. Nicholson, and John C. Lindon*

Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London WC1H 0PP UK

A new approach to the characterization of biomolecules in whole biological fluids is presented based on simplification of 1H NMR spectra by utilizing differences in molecular diffusion coefficients alone and combinations of relaxation and diffusion parameters. New NMR pulse sequences incorporating both spectral editing features together with solvent water resonance elimination are presented. The methods are exemplified using whole human blood plasma, and it is shown that it is possible to obtain NMR spectra of the slowly diffusing species (generally large molecules) by diffusion editing, the slowly relaxing species (generally small molecules) by spin relaxation editing, or spectra showing any range of molecular mobility using a combination of the two methods. The diffusion-based editing methods are also applicable to the selection of resonances in two-dimensional NMR spectroscopy of biofluids, and we show this for the first time by the production of 1H-1H diffusion-edited TOCSY spectra of human blood plasma where the resonance intensities are weighted according to the molecular diffusion coefficient. In this case, by measuring a diffusionedited 1H-1H TOCSY NMR spectrum of plasma, it is possible to obtain signals from only the macromolecular components, and this may be of benefit in the analysis of blood lipoproteins. In complex biofluids, the combination of diffusion and relaxation editing brings about considerable spectral simplification leading to an easier resonance assignment process. We also demonstrate the production of 1H NMR spectra with intensities corresponding to diffusion coefficient rather than number of protons, and this opens up new possibilities for pattern recognition classification of samples based on altered molecular mobility features of biofluid components.

in the intact biomatrix. One major advantage of using NMR spectroscopy to study complex biomixtures is that measurements can often be made with minimal sample preparation (usually with only the addition of 5-10% D2O) and a detailed analytical profile can be obtained on the whole biological sample.1 Hence, much effort has been expended in discovering efficient new NMR pulse sequence techniques for spectral simplification and water suppression.2 A number of methods can be used for NMR spectral editing based on NMR properties of the various nuclear spin systems present (including both homo- and heteronuclear spin systems) where single or multiple quantum coherence excitation, transformation, and selection has been demonstrated to play a key role.3-5 However, it has also been shown that a pseudoseparation of different molecules in a complex mixture can be achieved using nuclear relaxation times6 and molecular diffusion coefficients.7 The two major NMR relaxation phenomena are longitudinal relaxation characterized by a relaxation time T1, and transverse relaxation, characterized by T2. High molecular weight molecules have longer rotational correlation times (τc) than small molecules, resulting in shorter T2 relaxation times and greater NMR resonance line widths. Both T1 and T2 relaxation phenomena can be used for spectral editing but in different ways,6,8 and this has been reviewed for NMR spectra of blood plasma.9 The basic pulse sequence for spectrum editing based on T1 relaxation is the inversion-recovery sequence (180°-tr-90°-FID), where the 180° pulse causes inversion of the magnetization and the FID is collected after a suitable recovery time tr. Assuming perfect inversion, the signal intensity is proportional to [1 - (2 exp(-tr/ T1))]; thus spins with T1 > 1.44tr will give rise to negative peaks and spins with T1 < 1.44tr will give rise to positive peaks, and those spins with T1 ≈ 1.44tr will have peaks at the null point. A simple pulse sequence using transverse relaxation is the spin-

A major difficulty encountered in NMR spectroscopic studies of complex biological samples such as biofluids relates to the considerable range of concentrations, molecular weights, and molecular mobility (hence signal line width) of the individual organic components. Traditionally, in bioanalysis, such problems have mainly been resolved by extensive sample preparation using physical methods such as chromatographic separations.1 However, this process may cause both biological and physicochemical property changes of the sample, and hence the measured biochemical composition may differ from that actually occurring

(2) Ernst, R. R.; Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance Spectroscopy in One and Two Dimensions; Clarendon Press: Oxford, UK, 1987. (3) Liu, M.; Farrant, R. D.; Nicholson, J. K.; Lindon, J. C. J. Magn. Reson. 1995, 106B, 270-278. (4) Liu, M.; Farrant, R. D.; Sweatman, B. C.; Nicholson, J. K.; Lindon, J. C. J. Magn. Reson. 1995, A113, 251-256. (5) Liu, M.; Farrant, R. D.; Nicholson, J. K.; Lindon, J. C. J. Magn. Reson. 1995, A112, 208-219. (6) Rabenstein, D. L.; Isab, A. A. J. Magn. Reson. 1979, 36, 281-286. (7) Barjat, H.; Morris, G. A.; Smart, S.; Swanson, A. G.; Williams, S. C. R. J. Magn. Reson. 1995, B108, 170-172 (8) Rabenstein, D. L.; Millis, K. K.; Strauss, E. J. Anal. Chem. 1988, 60, 1380A1391A. (9) Rabenstein, D. L.; Nakashima, T.; Bigam, G. J. Magn. Reson. 1979, 34, 669-674.

(1) Nicholson, J. K.; Wilson, I. D. Prog. NMR Spectrosc. 1989, 21, 449-501.

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echo (90°-ts-180°-ts-FID), and here the signal intensity is proportional to exp(-2ts/T2). Unlike the inversion-recovery method, the spin-echo method can allow the spectra to be phased to give positive peaks for all spins, but phase distortion is expected for homonuclear coupled spin systems when longer spin-echo times (2ts) are used or when larger coupling constants are present. In this method, the signals in the NMR spectra are attenuated to different extents according to the relative values of T2 and ts. Molecular diffusion coefficients are parameters that are not related directly to NMR spectral intensities under normal conditions. However, molecular diffusion can cause NMR signal intensity changes when pulsed field gradients are applied during the FT NMR experiment.10 The simplest pulse sequence for this experiment is the gradient spin-echo (90°-G-180°-G-FID), where G represents a pulsed magnetic field gradient, and the signal attenuation is proportional to exp[-(γgδ)2D(∆ - δ/3) 2ts/T2], where γ is the gyromagnetic ratio of the spin, g is the strength of the rectangular gradient, δ is the duration of the gradient, ∆ is the time between the starting point of the two gradients, and D is the diffusion coefficient of the molecule. It is noted that both diffusion and transverse relaxation contribute to the signal attenuation in this case. We demonstrate here the editing of 1H NMR spectra of biofluids based on diffusion alone or on a combination of spin relaxation and diffusion and present a new pulse sequence which combines the effect of molecular diffusion and transverse relaxation times on the spectra of biofluids and also allows the suppression of the solvent water NMR resonance. We have termed this the diffusion and relaxation editing (DIRE) pulse sequence. This approach is complementary to the editing of 1H NMR spectra based on differences in T1 and T2 reported by Rabenstein et al.9 One of the major approaches to the assignment of resonances in the NMR spectra of biofluids relies on the measurement of resonance connectivities using two-dimensional NMR spectroscopy, particularly COSY11 and TOCSY.12 The latter technique has two advantages in that the off-diagonal cross peaks are all in-phase and additional information on spin coupling connectivities along chains of coupled protons is obtained.12 Even so, two-dimensional correlation spectra of complex biofluids show much overlap of cross peaks,13 and further editing is often desirable. To this end, we have already demonstrated the separation of resonances into subspectra according to whether the protons arise from CH, CH2, or CH3 groups via use of maximum quantum coherence spectroscopy (MAXY)3-5 and demonstrated that it is possible to produce two-dimensional NMR spectra showing TOCSY connectivities to only the selected type of proton.5 Here, we demonstrate new methods for editing TOCSY spectra of biofluids, in this case based on differences in molecular diffusion coefficients, and this has been termed diffusion edited TOCSY (DETOCSY). This approach complements the editing of TOCSY NMR spectra based on coherence selection and promises to provide an efficient alternative strategy for assignment of resonances in complex mixtures such as biofluids and cell extracts. (10) Stejskal, E. O.; Tanner, J. E. J. Chem. Phys. 1965, 42, 288-292. (11) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1975, 64, 2229-2246. (12) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355-360. (13) Nicholson, J. K.; Foxall, P. J. D.; Spraul, M., Farrant, R. D.; Lindon, J. C. Anal. Chem. 1995, 67, 793-811.

Figure 1. Pulse sequences for editing (a) 1H NMR spectra using a combination of T1 and T2 relaxation times with solvent suppression using field gradients, (b) 1H NMR spectra based on differences in diffusion coefficients and T2 relaxation times (DIRE), and (c) twodimensional 1H-1H TOCSY NMR spectra (DETOCSY) using differences in diffusion coefficients. Sequences b and c incorporate the WATERGATE15 solvent elimination sequence. The narrow bars are 90° pulses, the open rectangles are 180° pulses, G′ is a rectangular z-direction magnetic field gradient, the vertical hatched rectangles comprise the “3-9-19-19-9-3” 180° pulse sequence used in the WATERGATE solvent suppression sequence,15 and G are sineshaped z-direction magnetic field gradients. The phase cycling for the pulse sequences in (b) and (c) is φ1 ) x, -x; φ2 ) (y)3, (-y)3; φ3 ) (x)2, (-x) 2; φ4 ) (x)4, (-x)4; φ5 ) y; φ6 ) x; φ7 ) (x)8, (-x)8; φr ) x, (-x)2, x, -x, (x)2, (-x)2, (x)2, -x, x, (-x)2, x. Te ) 50 ms, ∆ ) 400 µs, ∆′ ) 500 ms, τ ) 200 µs.

NEW NMR PULSE SEQUENCES FOR EDITING SPECTRA OF COMPLEX MIXTURES It is possible to edit 1H NMR spectra based on a combination of T1 and T2 relaxation times alone. Hence, Figure 1a shows a pulse sequence that combines the effects of transverse and longitudinal relaxation as well as water suppression which is essential for biological samples. The sequence starts with a 180° pulse to invert the magnetization. The first 90° pulse is applied after a suitable recovery time (tr) for editing on the basis of T1, to create the transverse magnetization which then experiences a spinecho period (2ts) to allow T2 editing. The second 90° pulse after a further delay (∆) converts the off-resonance magnetization to the z-axis and leaves the on-resonance magnetization of water unchanged, which then is dephased by the gradient. The last two 90° pulses, separated by the same delay (∆), are used to generate observable off-resonance magnetization and to remove the phase offset caused by the first delay (∆). The gradient also removes any phase distortions caused by spin-spin coupling during the spin-echo period. Alternatively, it is possible to take advantage of NMR resonance intensity attenuation caused by the application of magnetic field gradients, and a pulse sequence that provides spectral editing Analytical Chemistry, Vol. 68, No. 19, October 1, 1996

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based on differences in diffusion coefficients is given in Figure 1b. Here sine-shaped bipolar gradients have been used to minimize spectral artifacts.14 A spin-echo time of 2ts is used first together with a bipolar gradient for diffusion and the 180° refocusing pulse has been replaced by a “3-9-19-19-9-3” pulse train which is the same as that used in the WATERGATE sequence for solvent resonance elimination.15 Since the “3-919-19-9-3” pulse train has no effect on the on-resonance solvent magnetization and is equivalent to a 180° pulse for the offresonance magnetization, the bipolar gradient should label the spatial positions only of the off-resonance spins. The detection part starts with another spin-echo scheme in which another pair of bipolar gradients with identical strength and duration are used and the spin-echo time is kept minimal. A zero quantum filter with a delay of 50 ms, together with a fifth gradient is inserted before data acquisition to remove the phase distortion caused by spin-spin couplings and to reduce the eddy current artifact induced by the use of gradients. Figure 1c shows the novel diffusion-edited TOCSY (DETOCSY) pulse sequence with water suppression. The pulse sequence is an extension of that given in Figure 1b with an incremented precession period t1 and a spin-lock pulse using the MLEV-17 sequence16 inserted before data acquisition. TPPI phase incrementation is also applied to the last 90° pulse. EXPERIMENTAL SECTION The test material comprised control human blood plasma (obtained by venepuncture using lithium-heparin as an anticoagulant and centrifugation at 3000g for 5 min to separate plasma from red blood cells). D2O (5%) was added to the plasma samples to provide a field-frequency lock for the NMR measurements. The NMR experiments were carried out at 400.13 MHz using a Bruker DRX-400 instrument with a BGU-2 field gradient accessory capable of delivering a z-field gradient up to 590 mT/m. One-dimensional spectra were acquired into 32K data points using a spectral width of 6400 Hz and with a 1.5 s relaxation delay. Resolution enhancement of the spectra was achieved using LorentzianGaussian transformation before Fourier transformation (FT). 1H-1H DETOCSY NMR spectra were also measured on control human blood plasma. Here the acquisition and processing parameters included a relaxation delay of 1.5 s, a spectral width in F1 and F2 of 4006 Hz, 2K time domain points, states-TPPI phase cycling, 128 F1 increments, 64 transients per increment, a spinlock time of 27 ms, a gradient strength of 2 × 236 mT/m, and sine-bell apodization in F1 and F2 and with 2K by 2K points in F1 and F2 after FT. RESULTS Water Resonance Suppression. Figure 2a shows a normal one-pulse 1H NMR spectrum of control human blood plasma and Figure 2b is same as Figure 2a but with the vertical scale increased by a factor of 1000. The broad background from albumin and the broad peaks from the lipoproteins are now clearly visible as (14) Wu, D.; Chen, A.; Johnson, C. S., Jr. J. Magn. Reson. 1995, 115, 260-264. (15) Piotto, M.; Saudek, V.; Sklenar V. J. Biomol. NMR 1992, 2, 661-665. (16) Marion, D.; Wuthrich, K. Biochem. Biophys. Res. Commun. 1983, 113, 967974.

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Figure 2. 400 MHz 1H NMR spectra of control human blood plasma: (a) normal spectrum, (b) with increased vertical scale (×1000), and (c) using the pulse sequence given in Figure 1a with a spin-echo time, ts, of 5 ms, showing attenuation of the resonance from the solvent water. Albumin resonances: A1 and A2, methyl and lysyl -CH2 resonances, respectively. Lipoprotein resonances: L1, CH3; L2, (CH2)n; L3, CH2‚CH2.CO; L4, CH2‚CH2‚CHd; L5, CH2‚CHd; L6, dCH‚CH2‚CHd; L7, CHd Region A, amino acids and carbohydrates, mainly R- and β-glucose, N-acetyl, N-acetyl resonances from carbohydrate units of glycoproteins, principally R1-acid glycoprotein.

are sharp peaks from a number of small molecule endogenous metabolites. Many of the resonances have been assigned,13 and some key assignments are given on the figure. Figure 2c is obtained using the pulse sequence in Figure 1a with minimal spinecho (5 ms) and recovery times (1 ms); a gradient strength of 177 mT/m (30%) and excellent water suppression (by a factor of about 105) is achieved. The improved dynamic range allows better characterization of the small peaks. Even though the spin-echo time was kept short, some attenuation of the relatively fast relaxing lipoprotein and chylomicron signals can still be observed in comparison to the intensity of the lactate methyl resonance. The assignments of the resonances in 750 MHz 1H NMR spectra of human blood plasma have been published recently,13 and much work has been carried out on the deconvolution of the NMR signals from lipoproteins.17 The resonances from the small molecules in the sample are somewhat enhanced relative to the macromolecule resonances, and this may aid the assignment process. NMR Spectral Editing of Biofluids Based on Diffusion Alone. The 1H NMR spectra of human blood plasma shown in Figure 3 were acquired using the pulse sequence given in Figure 1b. When a total gradient strength of only 59 mT/m (10%) is used, the spectrum as shown in Figure 3a is similar to that in the (17) Ala-Korpela, M. Prog. NMR Spectrosc. 1995, 27, 475-554.

Figure 3. 400 MHz 1H NMR spectra of control human blood plasma with solvent water elimination and edited on the basis of differences in diffusion coefficients using the pulse sequence of Figure 1b: (a) normal spectrum with application of 10% gradient strength, (b) spectrum with gradient application at 50%, and (c) the difference between (a) and (b). Assignments are as for Figure 2, plus L8, choline and glycerol protons of phospholipids; N+Me3, N-trimethyl group of choline in phospholipids; Me(ω-3), CH3 resonance from CH3‚CH2‚ CHd containing fatty acids in lipoproteins.

absence of gradients. Figure 3b was acquired using strong gradients, the total strength being 295 mT/m (50%), and the resonances from the small molecules are reduced substantially due to their relatively fast diffusion compared to those of the larger molecules which give rise to the broad peaks in the spectrum. The assignment of the lipoprotein resonances follow those given in Figure 2c, and these arise from different positions within the fatty acid chains. In addition, the signal from the choline methyl groups of the phospholipid content of the lipoproteins can be seen at δ 3.2 now clearly resolved from the resonance of the H2 proton of β-glucose.13 The relatively sharp peaks near δ 2 arise from the N-acetyl groups of the carbohydrate component of glycoproteins, and their appearance in this edited spectrum confirms that they are from macromolecular systems. Other peaks are observed between δ 3.4 and 3.9, and these have been assigned to the glycerol protons and to the methylene groups of the choline group in phosholipids in lipoproteins based on the measurement of NMR spectra of model compounds. Elimination by diffusion editing of the many resonances that normally occur in this region of the spectrum arising mainly from amino acids and carbohydrates has allowed the observation of these phospholipid peaks for the first time in the 1H NMR spectra of intact plasma. A small peak is also observed to high frequency of the lipoprotein methyl resonance at about δ 0.9, and this arises from the methyl group of ω-3 fatty acid components of the lipids in the lipoproteins. The assignment has been confirmed by careful examination of twodimensional DETOCSY spectra (see later) where a cross-peak

Figure 4. 400 MHz 1H NMR spectra of control human blood plasma with solvent water elimination and edited on the basis of differences in T1 and T2 relaxation times using the pulse sequence of Figure 1a: (a) normal spectrum with solvent water elimination, spin-spin relaxation delay of 20 ms, and recovery delay of 1.0 ms. As the recovery time is so short, this spectrum has been plotted inverted to give the appearance of full recovery. (b) as with (a) but with a recovery delay of 450 ms, (c) the difference of (a) and (b), and (d) the sum of (a) and (b).

connectivity can be observed to a resonance characteristic of a methylene group adjacent to a double bond. In addition, evidence can be seen in the DETOCSY spectra of the presence of ω-4 fatty acid components. Figure 3c is the difference of Figure 3a and Figure 3b, where nearly all resonances are from small, fastdiffusing molecules and assignments are as shown. Thus, this approach provides a simple method to obtain edited NMR spectra of either the large or small molecular weight components. The ability to remove the resonances from the small molecules may be important in view of the increasing number of studies reporting lipoprotein analyses in whole plasma by using line-shape fitting algorithms.17 NMR Spectral Editing of Biofluids Based on a Combination of T1 and T2 Relaxation. Figure 4 shows the 1H NMR spectra of human blood plasma obtained using the pulse sequence given in Figure 1a, but manipulating the effects of both the spinecho and recovery times. Spectra a and b of Figure 4 have the same spin-spin relaxation delay of 20 ms but with inversionrecovery times of 1.0 and 450 ms, respectively. For the spectrum with the short recovery time shown in Figure 4a, all resonances will be essentially still be completely inverted and this spectrum has therefore been plotted inverted to appear as though there has been full recovery. Thus in Figure 4b, resonances with long T1 values appear inverted and these in general arise from small molecules. The macromolecular components have short T2, Analytical Chemistry, Vol. 68, No. 19, October 1, 1996

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values and their resonances have been attenuated in both Figure 4a and Figure 4b. Parts c and d of Figure 4 are the difference and sum of Figure 4a and Figure 4b, respectively. It can be seen that by using a spin-spin relaxation delay of 20 ms, the intensities of the broadest peaks from the fastest T2-relaxing protons have been reduced and the intensities of the sharp peaks of the small molecules are enhanced by the inversion resulting from the short recovery period. The assignments of the endogenous metabolite resonances are given in Figure 3. In Figure 4b a long T1-relaxing component gives a sharp singlet at δ 0.85, and this has not yet been assigned. This must arise from a low molecular weight molecule because it also has a fast diffusion coefficient. Good line shapes are obtained for all peaks even those with large coupling constants which could result in phase modulation. When a 40 ms spin-spin relaxation delay is used, the resonances of spins with large coupling constants become negative because the magnetization is modulated by a term cos(πJHH2ts), although the peaks still retain their normal line shapes (results not shown). The relaxation difference spectrum of Figure 4c is comparable to the diffusion difference spectrum in Figure 3c but with the lipoprotein peaks at δ 0.8 and glycoprotein N-acetyl peaks at δ 2.02 slightly larger. Again the spectrum in Figure 4d is similar to that in Figure 3b, although the suppression of the smallmolecule peaks is not as efficient. NMR Spectral Editing of Biofluids Based on a Combination of Diffusion and Relaxation. So far we have shown that it is possible to edit NMR spectra of biofluids to remove resonances from small rapidly diffusing molecules using diffusion editing and from rapidly relaxing molecules by relaxation editing. It is possible to combine these two approaches in the DIRE pulse sequence such that molecules in a given window of mobility give rise to NMR resonances. In general, for small rigid molecules, the NMR relaxation time is inversely related to the molecular rotational correlation time which in turn is related to r-3 where r is the molecular radius.18 Similarly, the molecular translational diffusion coefficient (D) can be inversely related via the StokesEinstein equation to the molecular radius.19 Thus, for a rigid small molecule tumbling freely in solution, the NMR relaxation time is inversely related to D3. Hence by deriving these parameters for all resonances in an NMR spectrum of a biofluid, any values that fall above the line of a plot of log(D) vs log(relaxation time) will have a relaxation time relatively too long, and this is likely to be caused by internal conformational flexibility. On the other hand, points that fall below the line will arise from resonances from molecules where the diffusion coefficient is relatively too small, and this is likely to be caused by restricted translational motion such as protein binding. Hence a simple plot could provide initial evidence for binding of small biochemical molecules to macromolecules such as serum albumin. This analysis would be more complicated if applied to large molecules with rotational correlation times and hence relaxation times outside the motional narrowing limit. It is possible, for example, to use diffusion editing to remove the peaks of the smallest, fastest diffusing components and relaxation editing to remove peaks from the fastest relaxing macromolecular components. Thus Figure 5a shows the 1H NMR spectrum of control human blood plasma obtained using the DIRE (18) Doddrell, D; Glushko, V; Allerhand, A. J. Chem. Phys. 1972, 56, 36833689. (19) Atkins, P. W. Physical Chemistry; Oxford University Press: Oxford, UK, 1978; p 843.

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Figure 5. 400 MHz 1H NMR spectra of control human blood plasma with solvent water elimination and edited on the basis of differences in both diffusion coefficients and relaxation times using the pulse sequence of Figure 1b: (a) the use of a 10% gradient strength and a 30 ms spin-echo period to remove resonances from molecules with short T2 and (b) the use of 100% gradient strength and a 30 ms spinecho period to remove the resonances of small, fast-diffusing molecules and molecules with short T2 values.

pulse sequence of Figure 1b with an applied 10% gradient and with a spin-echo period of 30 ms. This results in the attenuation of the resonances with the shortest T2 values, these being the largest molecules such as albumin and certain of the lipoproteins. The main resonance assignments are given in Figures 2 and 3. If the gradient strength is increased to 100%, then the spectrum shown in Figure 5b is obtained, and in addition to the loss of resonances from the short T2 components, the peaks from the fastest diffusing molecules are also attenuated, leaving a window of detectibility of intermediate-sized molecules. In this case, the major peaks arise from a component of the lipoprotein mixture that has the longest T2 but the slowest diffusion rate. From the relationship between T2 and molecular correlation time, and between D and molecular, size this is the largest particle, VLDL. In addition, peaks can be seen from the N-acetyl groups of the carbohydrate residues of glycoproteins and this in plasma is principally R1-acid glycoprotein. These latter peaks are retained in the spectrum because they arise from a molecule with a slow diffusion coefficient but whose T2 values are relatively long because of the flexibility of the carbohydrate sidechains. Generation of “Diffusion-Weighted” NMR Spectra of Biofluids. It is possible to monitor the intensity of every data point in an NMR spectrum as a function of the square of the applied field gradient and determine, on the assumption of a singleexponential decay, an apparent diffusion coefficient for every data point. If the apparent diffusion coefficient is then plotted as an alternative to the usual spectral intensity, a “diffusion-weighted” NMR spectrum results. Unlike the conventional NMR spectrum, the intensities now relate to metabolite molecular diffusion rather than concentration. We have previously demonstrated how NMRderived metabolite concentrations can be used as input to pattern recognition methods in order to classify biofluid samples in terms of toxic insult20,21 or disease,22,23 and we propose here the future

Figure 6. 400 MHz 1H NMR spectra of control human blood plasma with solvent water elimination: (a) conventional NMR spectrum and (b) generation of a diffusion NMR spectrum where the resonance intensity is now proportional to the molecular diffusion coefficient and not the metabolite concentration

Figure 7. 400 MHz 1H-1H TOCSY NMR spectrum of control human blood plasma with solvent water elimination and edited on the basis of differences in diffusion coefficients using the pulse sequence of Figure 1c: (a) normal spectrum and (b) with application of field gradients at 80% showing retention of principally lipoprotein resonances.

possibility of using “diffusion-weighted“ NMR spectra for classifying biofluid samples where the classification will be based on differences in molecular mobility rather than concentration; we are currently investigating this possibility. Figure 6 shows an (20) Anthony, M. L.; Sweatman, B. C.; Beddell, C. R.; Lindon, J. C.; Nicholson, J. K. Mol. Pharmacol. 1994, 46, 199-211. (21) Holmes, E.; Bonner, F. W.; Sweatman, B. C.; Lindon, J. C.; Beddell, C. R.; Rahr, E.; Nicholson, J. K. Mol. Pharmacol. 1992, 42, 922-930. (22) Holmes, E.; Foxall, P. J. D.; Nicholson, J. K.; Neild, G. H.; Brown, S. M.; Beddell, C. R.; Sweatman, B. C.; Rahr, E.; Lindon, J. C.; Spraul, M.; Neidig, P. Anal. Biochem. 1994, 220, 284-296.

example of such a “diffusion-weighted“ NMR spectrum of blood plasma. Figure 6a is a conventional 1H NMR spectrum of human blood plasma where the vertical axis represents the normal NMR intensity (i.e., concentration, if the spectrum is acquired appropriately), and Figure 6b is a “diffusion” NMR spectrum of the same sample. Thus, the resonances from molecules with large diffusion coefficients (i.e., freely moving small molecules) now dominate the spectrum. It can be seen that the serum albumin (23) Ghauri, F. Y. K.; Nicholson, J. K.; Sweatman, B. C.; Beddell, C. R; Lindon, J. C. NMR Biomed. 1993, 6, 163-167.

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and lipoprotein resonances have been heavily attenuated. The uneven background is caused by the differences in the values of the diffusion coefficients for the broad albumin and lipoprotein components and is consistent with albumin having faster diffusion than some of the lipoprotein components. The relatively sharp peak at δ 2.05, which arises from N-acetyl groups in flexible oligosaccharide side chains of glycoproteins,23 is also attenuated because of a relatively low diffusion coefficient. Editing of Two-Dimensional NMR Spectra of Biofluids Based on Diffusion. The approach can also be extended to multidimensional NMR spectroscopy of complex mixtures, and the diffusion-editing sequence has also been incorporated into the TOCSY pulse sequence with water suppression as shown in Figure 1c. This results in a total correlation two-dimensional NMR spectrum in which editing of both diagonal and cross peaks can be achieved on the basis of the molecular diffusion coefficients. Thus, Figure 7 shows the DETOCSY spectrum of control human blood plasma with (a) no diffusion editing and (b) with application of field gradients as in Figure 1c in order to attenuate resonances from faster diffusing molecules. Figure 7a shows resonances from both large and small molecules, many of which have been assigned previously.13 The key assignments are as given in Figures 2 and 3. However, on applicaton of the DETOCSY pulse sequence (Figure 1c), the resonances from the fast-diffusing small molecules are attenuated, leaving principally resonances from the lipoproteins. The assignment of these resonances are given in Figure 2, and the coupling connectivities of the resonances from the choline and glycerol protons of phospholipids between δ 3.6 and 4.0 can now be analyzed in detail as they are no longer

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obscured by resonances from small molecules that appear in this region, such as R- and β-glucose and amino acids. SUMMARY We show for the first time that it is possible, without sample pretreatment such as dialysis, to obtain a 1H NMR spectrum of the high molecular weight components of blood plasma, and this has possible advantages for lipoprotein analyses. It is also possible to edit NMR spectra of biofluids to distinguish the causes of broad lines where the line width is caused either by small molecules undergoing chemical exchange (short T2 but fast diffusion) or high molecular weight (short T2 and slow diffusion). This work shows the first application of editing of highresolution 1H NMR spectra of a complex biological fluid on the basis of differences in diffusion coefficient. A higher degree of spectral simplification can be achieved by using nuclear relaxation properties in addition to diffusion weighting. The excellent line shapes achieved as well as the use of efficient water suppression will ensure the application of the methods to other biofluids and complex biosystems such as cell and tissue extracts. We are currently applying these methods to extend the assignment of resonances in both normal and pathological biofluids in studies of toxicity and disease. Received for review April 29, 1996. 1996.X

Accepted July 9,

AC960426P X

Abstract published in Advance ACS Abstracts, August 15, 1996.