Anal. Chem. 1996,67,793-811
750 MHz IH and IH-l3C NMR Spectroscopy of Human Blood Plasma Jeremy K. Nicholson* and Peta J. D. Foxall Department of Chemistv, Birkbeck College, Univetsity of London, Gordon House, 29 Gordon Square, London WClH OPP, U.K.
Manfred Spraul Bruker Analytische Messtechnik GmbH, D76287-Rheinstetten, Silberstreifen, Germany
R. Duncan Farrant and John C. Lindon Department of Physical Sciences, Wellcome Research Laboratofies, Beckenham, Kent BR3 3BS,
High-resolution 750 M H z lH NMR spectra of control human blood plasma have been measured and assigned by the concerted use of a range of spin-echo, twodimensional J-resolved, and homonuclear and heteronuclear (lH-13C) correlation methods. The increased spectral dispersion and sensitivity at 750 MHz enable the assignment of numerous 'H and 13C resonances from many molecular species that cannot be detected at lower frequencies. This work presents the most comprehensive assignment of the 'H NMR spectra of blood plasma yet achieved and includes the assignment of signals from 43 low M, metabolites, including many with complex or strongly coupled spin systems. New assignments are also provided from the 'H and 13C NMR signals from several important macromolecularspecies in whole blood plasma, i.e., very-low-density,low-density, and high-densitylipoproteins, albumin, and al-acid glycoprotein. The temperature dependence of the one-dimensional and spinecho 750 M H z 'H NMRspectra of plasmawas investigated over the range 292-310 K The lH N M R signals from the htty acyl side chains of the lipoproteins increased substantially with temperature (hence also molecular mobility), with a disproportionate increase from lipids in low-density lipoprotein. Two-dimensional 'H- 13C heteronuclear multiple quantum coherence spectroscopy at 292 and 310 K allowed both the direct detection of cholesterol and choline species bound in high-density lipoprotein and the assignment of their signals and c o h e d the assignment of most of the lipoprotein resonances. During the last decade, a diverse range of 'H NMR spectroscopic studies has been performed on vertebrate biological fluids in relation to the investigation of toxicological processes, metabolic diseases, and drug Considerable effort has gone (1) Nicholson, J. IC; Wilson, I. D. Prog. Nucl. Mugn. Reson. Spectrosc. 1989,
21,449-501. (2) Nicholson, J. K, Wilson, I. D. In Drug Metabolism--from Molecules to Man Benford, D. J., Bridges, J. W., Gibson, G. G., Eds.; Taylor Francis: London, 1987; pp 189-207. (3) Malet Martino, M. C.; Martino, R Biochemie 1992,74,785-800. 0003-2700/95/0367-0793$9.00/0 0 1995 American Chemical Society
U.K.
into obtaining useful biochemical information from 'H NMR spectra of blood plasma. Early successful attempts in this area were focused on the measurement of mobile, low M,metabolites in whole blood plasma using Hahn spin-echo (HSE) experiments to attenuate the broad signals from lipoproteins and other macromolecules which dominate the one-dimensional (1-D) spectra.6 HSE experiments on blood plasma were found to be useful for the simultaneous investigation of ketone body (3-Dhydroxybutyrate,acetoacetate, and acetone) production, perturbed amino acid metabolism, and changes in mobile lipid levels in human subjects with diabetes mellitus and hypertrigly~eridemias.~ There have also been many more recent attempts to use 'H NMR spectra of plasma to provide novel diagnostic information on metastatic tumors based on measurements of composite lipoprotein signal line widths.S-lZ This has led to numerous investigations into the limitations of this approach and to a detailed evaluation of both the causes of variation in the 'H NMR signals of plasma lipoproteinsand the computer fitting of complex line shapes which arise from the long-chain CHZand terminal CH3 signals from the different lipoprotein components.'3-19 Although in principle these (4) Holmes, E.; Foxall, P. J. D.; Nicholson,J. IC]. Phann. Biomed. Anal. 1990, 8, 955-958. (5) Foxall, P. J. D.; Mellotte, G.; Bending, M.; Lindon, J. C.; Nicholson, J. K Kidney Int. 1993,43, 234-245. (6) Nicholson, J. IC; Buckingham, M. J.; Sadler, P. J. Biochem. J. 1983,211, 605-615. (7) Nicholson, J. IC; O'Flynn, M. P.; Sadler, P. J.; Macleod, A. F.; Juul, S. M.; Sonksen, P. H. Biochem. J 1984,217, 365-375. (8) Fossel, E. T.; Carr, J. M.; McDonagh,J. N. Engl. J Med. 1986,315,13691376. (9) Mountford, C. E.; Tattersall, M. H. N. Cancer Surveys 1987,6,285-314. (10) Bradamante,S.; Barchesi, E.; Pilotti, S.; Borasi, G. Mugn. Reson. Med. 1988, 8, 440-449. (11) Buchthal, S. D.; Hardy, M. A; Brown, T. R Am. J. Med. 1988,85,528532. (12) Fossel, E. T. Cancer Cells 1991,3,173-182. (13) Bell, J. D.; Brown, J. C . L.; Norman, R E.; Sadler, P. J.; Newell, D. R NMR Biomed. 1988,1 , 90-94. (14) Hemng, F. G.; Phillips, P. S.; Pritchard, P. H.]. Lipid Res. 1989,30, 521528. (15) Herring, F. G.; Phillips, P. S.; Pritchard, P. H.; Silver, H.; Whittal, IC P. Mug%.Reson. Med. 1990,16, 35-48. (16) Otvos, J. D.; Jeyarajah, E. J.; Hayes, L. W.; Freedman, D. S.; Janjan, N. A; 369-376. Anderson, T. Clin. Chem. 1991,37, (17) Otvos, J. D.; Jeyarajah, E. J.; Bennett, D. W. Clin. Chem. 1991,37,377385. Analytical Chemistty, Vol. 67, No. 5, March 1, 1995 793
experiments can give useful clinical data on whole blood plasma, full exploitation of the potential of high-resolution NMR to give biochemical information is hampered by the incompleteness of the assignment data and a poor understanding of the dynamic intermolecular interactions that occur in blood plasma. All biological fluids are highly complex mixtures of metabolites, and even 1-D high-frequency 'H NMR spectra may contain many thousands of resolved NMR resonance lines with varying degrees of signal overlap, depending on the fluid type and the chemical shift range under consideration.'Qo As with all high-resolution NMR experiments, the exact observation frequency at which the measurements are performed influences signal dispersion, sensitivity, and the relaxation properties of the nuclei in molecules under study. Given the complexity of the biofluid matrix, the frequency at which 'H NMR spectra are measured has a major effect on the amount of chemical and biochemical information that can be obtained. Most biological fluids are effectively isotropic solutions and are much more magnetically homogeneous than, e.g., whole body, organ, tissue, or even cell preparations. This should make the interpretation of high-resolution NMR data more reliable and hence should lead to the discovery of new biochemical information. However, despite the powerful armory of multipulse and multidimensional NMR tools available, the complexity of the spectra and the degree of signal overlap have hindered the comprehensive assignment of the 'H NMR spectra of most biofluids. Certain biofluids have been partially characterized by the use of 600 MHz 'H NMR measurements, with many resonance assignments for the low M, components of blood plasma,2O cerebrospinal fluid,21and seminal The recent development of 750 MHz 'H NMR spectroscopy, with its signifcant improvement in spectral dispersion and sensitivity, provides the possibility to extend further the knowledge of the composition of biological fluids and the dynamic interaction of their component metabolites and macromolecular species. As many of the molecules of interest are small in size, they have short rotational correlation times and hence relatively long 'H relaxation times. For heteronuclei, there is little chance of experiencing detrimental line-broadening effects of chemical shift anisotropy relaxation, which may occur, for example, in 13C NMR spectra of '3C-labeled macromolecules measured at such high frequencies. Unlike the 'H NMR spectra of single substances (within the molecular mobility range of effective NMR observation), the total assignment of blood plasma spectra must always be incomplete because each sample is unique and subject to individual biological variation. However, the detailed assignment of the 'H NMR spectra of plasma is necessary in order to obtain precise bioanalytical and dynamic information from the whole fluid and ultimately to enable greater precision in diagnoses in clinical and toxicological investigations. 750 MHz lH NMR spectroscopy clearly offers the best means so far for completing the signal assignment of whole blood plasma. Blood plasma is a complex multicompartmental or multiphasic system with many different types of simultaneously occurring (18) Hiltunen, Y.; Ala-Korpela, M.; Jokisaari, J.; Eskelinen, S.; Kiviniitty, Y. A Magn. Reson. Med. 1992,26, 89-99. (19) Ala-Korpela, M.; Heitvenen, Y.; Jokisaari, J.; Eskelinen, S.; Kiviniitty, IC; Savolainen, M. J.; Kesaniemi, Y. A NMR Biomed. 1993,6,225-233. (20) Foxall, P. J. D.; Parkinson, J.; Sadler, I. H.; Lindon, J. C.; Nicholson, J. K J. Pharm. Biomed. Anal. 1993,11, 21-31. (21) Sweatman, B.; Farrant, R D.; Holmes, E.; Ghauri, F. Y. IC; Lindon, J. C.; Nicholson, J. I C J Pharm. Biomed. Anal. 1993,11, 651-664. (22) Lynch, M.; Masters, J.; Prior, J.; Spraul, M.; Foxall, P. J. D.; Lindon, J. C.; Nicholson, J. IC J. Pharm. Eiomed. Anal. 1994,12 (l),5-19.
794 Analytical Chemistw, Vol. 67,No. 5,March 7, 7995
intermolecular interactions, including metal complexation and chemical exchange reactions, micellar compartmentation of metabolites, enzyme-mediated biotransformations, and the binding of small molecules by macromolecules.' 'H NMR measurements made at very high observation frequencies should confer a number of benefits, because along with the sensitivity and dispersion factors, there is also a reduction in the number of observed second-order coupling effects and a change in the time scale of NMR-monitored events which may be particularly important for the observation of chemically exchanging species. In a preliminary study, we reported the use of standard one-dimensional, spin-echo, and two-dimensional j-resolved 600 and 750 MHz 'H NMR spectroscopy of blood plasma to extend the known assignments of metabolite signals in the plasma of a normal individual, compared the metabolite profile with that of a patient with chronic renal failure, and indicated some of the advantages of 750 MHz measurement^.^^ In the present study, we have comprehensively assigned the spectra of normal blood plasma using a range of one- and two-dimensional 750 MHz 'H NMR and 'H-13C NMR techniques together with different data processing methods and investigated the effects of temperature change on the 750 MHz 'H NMR spectra of human plasma. MATERIALS AND METHODS
Blood from seven adult male volunteers was collected by venipuncture into lithium heparinized vacutainers, and the plasma was separated by centrifugation, snapfrozen, and stored at -40 "C prior to NMR measurement. Each subject had fasted overnight, and the blood was collected in the morning preprandially. The samples were thawed immediately before use, and 0.7 mL of each was diluted by 10%with DzO to provide a field-frequency lock. 'H chemical shifts were referenced internally to the a-glucose HI resonance at 6 5.233, measured relative to the primary internal chemical shift reference trimethylsilyl [2,2,3,32H4]propionate at 6 0.00. 'H NMR spectra were measured on otherwise untreated biofluid samples at 750.14 MHz on a Bruker AMX 750 spectrometer at ambient probe temperature (292 IC), and selected samples were also remeasured at 298,304, and 310 K. As the temperature readings on standard NMR spectrometer variable temperature control units may be subject to calibration errors, the exact internal temperature of the samples (TplasmJwas measured from the chemical shift difference24 between the 'H NMR signal of H2O and the HI signal of a-glucose using the following equation:
Tplasma = 84.17Aa
+ 17.23A: + 256.87
where & = 6 (Hl,a-glucose) - 6 (320). Selected plasma samples were also measured on a Bruker AM 400 spectrometer operating at 400.13 MHz using a 5 mm inverse geometry broadband probe. In order to suppress the large water signal, all 1-D 750 MHz 'H NMR spectra were acquired using a pulse sequence based on the two-dimensional NOE experimentz5called NOESYPRESAT, comprising the following pulse sequence: RD-90°-t,
-90"-tm-9O0-acquire
FID
where RD is a relaxation delay of 1-3 s, during which the water (23) Foxall, P. J. D.; Spraul, M.; Farrant, R D.; Lindon, J. C.; Neild, G. H.; Nicholson, J. IC/. P h a m . Eiomed. Anal. 1993,11, 267-276.
resonance is selectively irradiated; tl represents the first increment in a NOESY experiment and is set to 3 ps; and fm, the mixing time in the NOESY sequence, has a value of 100-150 ms, during which the water resonance was again selectively irradiated. Typically 128 transients were collected into 64K data points with a spectral width of loo00 Hz. The NOESYPRESAT pulse sequence results in attenuation factors of 105 or more for water signals in biofluid samples,thus eliminating the potentially severe dynamic range problem. For samples measured at lower frequencies, spectral widths were reduced and pulse recycle times adjusted to maintain the digital resolution and the relaxation delays equal to those for the 750 MHz measurements. Prior to Fourier transform 0, exponential line-broadenings of 0.2-0.5 H z were applied to the FIDs, which were also zero-filled by a factor of 2. Resolution enhancement of spectra was obtained through the application of the Lorentzian-Gaussian transformation method. Selected 1-D spectra were also analyzed using the maximum entropy method incorporated into Bruker UXNMR software (Memsys 5, Maxent Solutions Ltd., Cambridge, U.K.). Carr-Purcell-Meiboom-Gill (CPMG) spin-echo spectraz6 were measured on all seven samples and the FIDs collected into 64K data points with a total spin-spin relaxation delay (2nt) of 87.8 ms and a total delay between pulse cycles of 4.28 s. For two of the plasma samples measured at 750 MHz, standard 1-D and CPMG spectra were remeasured at 292 K under comparable chemical shift range and relaxation conditions on a Bruker ARX 400 at 400.13 MHz, again with solvent presaturation. Variable temperature experiments were performed on two control plasma samples, which were measured successively using consecutive standard 1-D and CPMG experiments at 292,298,304, and 310 K and then remeasured at 292 K Two-dimensional ]-resolved URES) spectraz7were measured with solvent presaturation on each plasma sample at 292 or 304 K. The transients were collected into 8192 data points with a spectral width of 8064 Hz, and the F1 (/-coupling) domain spectral width covered 63 Hz with 64 increments of fl and eight transients were collected for each fl increment. Prior to the double FT and magnitude calculation, the F1 data were zero-filled to 1024 computer points and apodized by means of a sinebell function in f2 and a sine-bell-squared function in tl. The spectra were tilted by 45" to provide orthogonality of the chemical shift and coupling constant axes and subsequently symmetrized about the F1 axis. Spectra were displayed both in the form of contour plots and as skyline F2 projections. 750 MHz 'H-lH correlationz8spectroscopy (COSY45 version) was performed on three plasma samples with water presaturation. Transients were acquired into 4096 data points with 16 scans per increment, a spectral width of 10 000 Hz,and 512 increments in the F1 axis, which was zero-filled to 2048 prior to FT. The relaxation delay between successive pulse cycles was 2.7 s. The data sets were weighted using a sinebell function in fl and fz prior to FT. 750 MHz IH-IH total correlation spectroscopy (TOCSrZg) was performed on two samples to confirm the 'H NMR assign(24) Fanant, R D.; Nicholson, J. K; Lindon, J. C. NMR Biomed. 1994, 7,243247. (25) Jeener, J.; Meier, B. H.;Bachmann, P.; Emst, R R]. Chem. Phys. 1979, 71, 4546-4553. (26) Meiboom, S.; Gill, D. Reu. Sci. Instnrm. 1958, 29, 688-691. (27) Aue, W. P.; Karham, J.; Emst, R R J Chem. Phys. 1976, 64,4226-4227. (28) Nagayama, K; Kumar, A; Wuthrich, IC;Emst, R RJ. Mqn. Reson. 1980, 40, 321-334. (29) Bax, A; Davis, D. G. /. Magn. Reson. 1985, 65, 355-360.
ments, particularly on lipids with chains of coupled protons. The spectra were collected in the phase-sensitive mode using timeproportional phase incrementation (TPPI), and the M W 1 7 pulse sequence was used for the spin-lockamThe spectral width was loo00 Hz, with data collected into 4096 time domain points. "Qpically 512 increments were measured with 16 transients per increment, the data set being zero-filled to 1024 in t l , and a sinebell-squared apodmtion function was applied prior to FT. 750 MHz IH-13C phase-sensitive (I"PI) heteronuclear multiple quantum correlation (HMQC) spectra31were collected for two of the plasma samples with lH detection using an inverse geometry triple nucleus probe at both 292 and 310 K A relaxation delay of 2.1 s was used between pulses, and a refocussing delay equal to 1 / 2 1 / ~ -(3.57 ~ ms) was employed. Composite pulse broadband 13Cdecoupling (globally alternating optimized rectangular pulses, GARP) was used during the acquisition period.32 Typically 2048 data points with 64 scans per increment and 400 experiments were acquired with spectral widths of 8064 Hz in F2 and 27.7 kHz in F1. The FIDs were weighted using a shifted sinebell-squared function in F2 and exponential linebroadening of 0.3 H z with forward complex linear prediction33to 800 data points in F1 prior to FT and two-dimensional phasing. To aid signal assignments for blood plasma, a series of 1-D lH NMR and lH-l3C HMQC spectra were measured at 310 K on a Bruker AMX 600 spectrometer operating at 600.13 MHz on the following substances (all from Sigma U.K): human serum albumin (HSA, 40 g/L) , lowdensity lipoprotein (LDL, 5 g/L) , highdensity lipoprotein (HDL, 10 g/L) , human al-acid glycoprotein (100 g/L), and cholesterol linoleate (2 g/L), all made up in H20/D20 (lO:l), except for cholesteryl linoleate, where a 1:2:1 mixture of CD30D/DzO/ DMSO-& was used. Two-dimensional lH-13C HMQC data sets were enhanced using the technique of forward linear prediction on the real data points to twice the number of time domain points in the 13C dimension. RESULTS AND DISCUSSION Assignment of One and Wo-Dimensional750MHz lH and lH-13C NMR Spectra of Blood Plasma. Through the increased spectral dispersion of 750 MHz measurements and via the employment of appropriate 2-D NMR methods, we have been able to extend considerably the assignment of resonances in blood plasma spectra in normal individuals undergoing a standard overnight fast. Assignments are based on comparison of chemical shifts and spin-spin coupling constants with those of model compounds measured in phosphate buffer at pH 7.4, with the signals detected in the various 1-and 2-D experiments performed on plasma. In general, resonances have not been assigned unless all of the 'H NMR signals for a given molecule were resolved. In addition, proof of spin-spin coupling connectivity withii a given molecule was obtained via the use of 2-D correlation spectroscopy (see below). In summary, a comprehensive list of the 43 lH NMRdetectable low M,metabolites and the assigned macromolecular signals observed in human plasma, together with their spin systems and their 'H chemical shifts (and 13C shifts where available), is given in Table 1. A detailed consideration of the contributionsof each of the major NMR experiments used in the (30) Bax,A; Subramanian, S. J Magn. Reson. 1986, 67, 565-569. (31) Bax, A; Griffey, R H.; Hawkins, B. L]. Mugn. Reson. 1983,55,301-315. (32) Shaka, A J.; Barker,P. B.;Freeman, R J Magn.Reson. 1985, 64, 547552. (33) Stephenson, D. S. Prog, Nucl. Magn. Reson. Spectrosc. 1 9 8 8 , 2 0 , 515-626.
Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
795
Table I.Resonance Assignments with Chemical Shifts and Spin-Spin Coupling Patterns of Metabolites Identified in 750 MHz and iH-i3C NMR Spectra of Normal Human Blood Plasma.
lH shift (6) 0.66 0.70 0.84 0.84 0.87 0.91 0.93 0.93 0.95 0.97 0.97 1.00 1.02 1.13 1.20 1.22 1.25 1.26 1.28 1.29 1.30 1.31 1.32 1.32 1.33 1.46 1.47 1.48 1.57 1.57 1.68 1.69 1.69 1.71 1.86 1.91 1.91 1.91 1.96 1.97 1.99 2.00 2.00 2.00 -2.04 2.05 2.08 2.09 2.13 2.14 2.22 2.23 2.24 2.31 2.36 2.36 2.36 2.38 2.39 2.41
2.47 2.52 2.54 2.54 2.68 2.69 2.69 2.71 2.71 2.72 2.81 2.83 2.84 2.89 2.94
multiplicity m m m t t m t d d d d d d d m m m m m m d d m d d m m m m m m m m m m m S
m m m
m m m S
m m m S
m S
m spt of d m m S
m m ABX m t d S
ABX dd d m S
m m dd S
dd t dd
molecule cholesterol cholesterol cholesterol lipid (mainly LDL) lipid (mainly VLDL) cholesterol lipid isoleucine leucine leucine valine isoleucine valine isobutyrate 3-hydroxybutyrate lipid lipid (mainly LDL) lipid isoleucine lipid (mainly VLDL) lipid fucose threonine lipid lactate alanine isoleucine lysine lipid (mainly VLDL) citrulline arginine lysine lipid leucine citrulline lysine arginine acetate isoleucine lipid proline lipid lipid glutamate glycoproteinb (acetyls) proline glutamine glutamine methionine glutamate acetoacetate lipid valine 3-hydroxybutyrate glutamate pyruvate proline 3-hydroxybutyrate u1c glutamine 2-oxoglutarate citrate methylamine u1c aspartate citrate lipid dimethylamine lipid lipid aspartate trimethylamine asparagine albumin lysyl asparagine
796 Analytical Chemistry, Vol. 67,No. 5, March 1, 1995
assignment
C18 (in HDL) C18 (in VLDL) C26 and C27 CH3(CHdn CH3CHzCHzC= c21 CH3CHz 6-CH3 6-CH3 6-CH3 CH3 P-CH3 CH3 CH3 yCH3 CH3CHzCHz (CHdn CH~CHZ(CHZ)~ half y-CHz CHzCHzCHzCO CHz CH3 Y-CHB CHzCHzCHzCO CH3 CH3 half y-CH2 y-CHz CHzCHzCO y-CHz Y-CHZ 6-CHz CHzCHzC-C P-CHz, YCH P-CHz P-CHz P-CHz CH3 /3-CH CHzC=C Y-CHZ CHzC=C CHzC=C half P-CHZ NHCOCH3 half P-CHz half P-CHz half P-CHz SCH3 half P-CHZ CH3 CHzCO P-CH half a-CHz half y-CHz CHz half P-CHz half a-CH2 CHz half y-CHz Y-CHZ half CHZ CH3 CHz half P-CHz half CHz C-CCHzC=C CH3 C=CCHzC=C C=CC HzC=C half P-CHz CH3 half P-CHz E-CH~ half P-CHZ
observed
13C shift (6)
lD, HMQC HMQC HMQC lD, JRES, COSY, HMQC l D , JRES, COSY HMQC COSY lD, JRES, COSY lD, JRES, COSY l D , JRES, COSY lD, JRES, COSY, HMQC lD, JRES, COSY, HMQC lD, JRES, COSY CPMG, JRES l D , CPMG, JRES HMQC lD, CPMG, JRES, COSY, HMQC COSY, HMQC COSY lD, CPMG, JRES, COSY COSY, HMQC CPMG, JRES, COSY JRES, COSY COSY JRES, COSY, HMQC l D , CPMG, JRES, COSY, HMQC JRES, COSY CPMG, COSY, HMQC JRES, COSY, HMQC ID, COSY ID, CPMG, COSY lD, COSY lD, JRES, HMQC COSY, HMQC COSY JRES, COSY, HMQC COSY JRES, CPMG COSY lD, COSY COSY COSY, HMQC COSY JRES, COSY lD, JRES, HMQC COSY lD, JRES, COSY lD, JRES, COSY CPMG, JRES lD, JRES, COSY, HMQC CPMG, JRES COSY, HMQC COSY CPMG, JRES, COSY COSY, HMQC ID, CPMG, JRES, CPMG COSY JRES, COSY COSY CPMG, JRES, COSY, HMQC
12.6 23.3 14.7 19.4
19.6 14.6
32.7 30.6 23.2 19.7
20.9 16.8 25.6
27.4 40.7 30.3
27.8 23.0
30.1 34.6 34.5
31.9
JRES JRES, COSY CPMG, JRES COSY JRES JRES, COSY COSY CPMG, JRES lD, COSY lD, COSY, HMQC JRES lD, CPMG, JRES JRES lD, COSY, HMQC JRES
26.2
40.3
Table 1 (Contlnued)
lH shift (6) 2.96 3.01 3.04 3.05 3.06 3.12 3.14 3.15 3.16 3.21 3.24 3.24 3.25 3.25 3.26 3.26 3.26 3.28 3.34 3.40 3.41 3.42 3.45 3.47 3.48 3.48 3.54 3.54 3.54 3.56 3.56 3.57 3.60 3.63 3.64 3.66 3.68 3.69 3.70 3.71 3.72 3.75 3.76 3.76 3.83 3.84 3.87 3.90 3.93 3.94 3.97 3.98 3.98 4.05 4.06 4.06 4.11 4.12 4.13 4.24 4.25 4.29 4.53 4.64 5.20 5.23 5.23 5.26 5.27 5.29 5.31 5.33 6.87 7.01 7.02 7.05
multiplicity t t
molecule
t dd dd t
albumin lysyl albumin lysyld creatine creatinine tyrosine phenylalanine histidine citrulline tyrosine choline arginine P-glucose histidine taurine
S
TMAO
t dd t m t t t m ddd dd t dd dd
u2c phenylalanine myc-inositol proline P-glucose taurine a-glucose proline P-glucose threonine /3-glucose a-glucose u2c glycine myo-inositol glycerol valine threonine myc-inositol glycerol choline (lipid) glutamine leucine citrulline a-glucose a-glucose u1c a-glucose alanine a-glucose a-glucose glycerol P-glucose creatine tyrosine phenylalanine histidine u2c creatinine glyceryl of lipids myo-inositol lactate proline 3-hydroxybutyrate threonine glyceryl of lipids choline (lipid) P-galactose P-glucose glyceryl of lipids a-glucose unsaturated lipid unsaturated lipid unsaturated lipid unsaturated lipid unsaturated lipid unsaturated lipid tyrosine 3-methylhistidine histidine 1-methylhistidine
S S
dd dd dd t dd S
S
dd dd d d dd dd m t dd m t dd m m q
ddd m
m dd S
dd dd
ABX S
m t q m m m m m d d m d m m m m m m
m S S
S
observed
assignment E-CH~ E-CH~ CH3 CH3 half &CH2 half P-CHz half P-CHz Y-CHZ half P-CHz N (CH3) 3 6-CHz H2 half P-CH2 CHzNH CH3
ABX
half P-CHz H5 half 6-CHz H4 CH2S03 H4 half 6-CHz H5 a-CH H3 H2 CH CHz H1, H3 half CHz a-CH a-CH H4, H6 half CH2 NCHz a-CHz a-CH a-CH H3 half CHzC6 a-CH half CHrC6 a-CH H5 half CHzC6 C2-H half CHzC6 CHz a-CH a-CH a-CH CH CHz CHzOCOR H2 CH a-CH P-CH P-CH CHzOCOR OCHz H1 H1 CHOCOR H1 CHaCHCHzCH-CH CH--CHCH2CH=CH =CHCHzCHz CH=CHCHzCH-CH 4HCH2CH2 -CHCH2CHz H3, H5 H4 H4 H4
lD, COSY, HMQC lD, JRES, COSY, HMQC CPMG, JRES lD, CPMG, JRES JRES, COSY
JRES JRES JRES JRES, COSY JRES, HMQC COSY, HMQC lD, JRES, COSY, HMQC
13C shift (6) 40.3 40.3
55.0 41.3 75.1
JRES JRES JRES COSY COSY
JRES COSY lD, JRES, COSY, HMQC
70.6
JRES JRES, COSY, HMQC
70.6
COSY
JRES, COSY, HMQC JRES, COSY JRES, COSY, HMQC JRES, COSY, HMQC
76.7 76.7 72.3
COSY, HMQC CPMG, JRES
JRES lD, JRES, COSY JRES, COSY, HMQC JRES JRES lD, JRES, COSY COSY, HMQC JRES, COSY, HMQC JRES, COSY, HMQC COSY JRES, COSY, HMQC 1RES. COSY. HMQC
Cos?
JRES, COSY, HMQC IRES. COSY jRES; COSY, HMQC JRES, COSY, HMQC JRES, COSY JRES, COSY, HMQC JRES JRES JRES, COSY JRES COSY, HMQC lD, JRES HMQC
JRES lD, JRES, COSY, HMQC JRES. COSY lD, JRES, COSY lD, JRES, COSY HMQC HMQC
63.5 64.2 63.5 66.7 55.4 55.1 73.6 61.6 61.4 72.3 61.4 72.6 61.6
62.5 69.2
62.5 60.2
JRES
lD, CPMG, JRES, COSY, HMQC COSY lD, CPMG, JRES, COSY, HMQC l D , COSY lD, COSY, HMQC lD, COSY lD, COSY l D , COSY, HMQC lD, COSY lD, CPMG, HMQC lD, CPMG, JRES ID, CPMG, JRES ID, CPMG, JRES
92.9 128.6 128.6 128.6 130.1 116.7
Analytical Chemistry, Vol. 67, No. 5,March 1, 7995
797
Table 1 (Contlnued) 'H shift (6)
multiplicity
molecule
7.17 7.33 7-38 7.43 7.61 7.73 7.77
m m m m
tyrosine phenylalanine phenylalanine phenylalanine Smethylhistidine histidine 1-methylhistidine formate
S S S
8.45
S
assignment
observed
13c shift
(6)
CPMG, JRES, COSY lD, CPMG, COSY lD, CPMG, COSY lD, CPMG, COSY lD, CPMG,JRES ID, CPMG CPMG,JRES lD, CPMG
H2, H6 H2, H6 H4 H3, H5 H2 H2 H2
CH
Abbreviations and Key: s, singlet; d, doublet; t, triplet; quartet' spt, septet; m, complex multiplet; dd, doublet of doublets; ddd, doublet of doublets of doublets. Chemical shifts all referenced to H-1 an%'C-1of a:glucose at 5.233 for lH and at 92.9 ppm for 13C.* Mainly al-acidglycoprotein. U1 and U2 refer to unidentified metabolites. d Indicates overlap of free lysine with albumin lysyl and al-acid glycoprotein signals. NfCl
B
threonine
J
\lyy
I
lactate
Region A
leucine
glucose-@i
/
A
..
albumin lysyl DP.
4 5
4 0
3 5
30
-2 8
2 D
1 5
1 0
0 5
O
