Anal. Chem. 1987, 59, 2885-2891
2885
Chemical-Exchange and Paramagnetic T 2 Relaxation Agents for Water Suppression in Spin-Echo Proton Nuclear Magnetic Resonance Spectroscopy of Biological Fluids S.Connor and Jeremy K.Nicholson* Department of Chemistry, Birkbeck College, University of London, London WClE 7HX, U.K. Jeremy R. Everett Beecham Pharmaceuticals, Chemotherapeutic Research Centre, Brockham Park, Betchworth, Surrey RH3 7AJ, U.K.
The WATR (water attenuation by T , relaxatlon) method was used to effect solvent Suppression In 400-MHz spin-echo 'H NMR spectra d urlne, blood plasma, Mle, and seminal plasma. Both chemlcal-exchange (urea and guanldlnlum salts) and paramagnetlc relaxatlon agents (Mn2+ and Gda+ Ions) were effectlve In attenuatlng the water signal In spin-echo spectra of urlne. However, the paramagnetlc Ions form coordlnatlon complexes wlth many blofluld metabolltes (e.g. cltrate, lactate, and hippurate) so attenuating their proton resonances. The concentratlons of these metabolites In bloflulds are sufflclent to modulate the relaxatlon effects d the paramagnetlc Ions on the water protons, hence reduclng the efficlency of the WATR method. Indeed, Mn2+ Ions were Ineffective at reducing the water T i s In Mood and seminal plasmas, unless used In amounts that slgdflcantly attenuated metabolite resonances. By contrast urea and guanldlnlum salts were very effective as water relaxatlon agents In all bloflulds studied. New resonance asslgnments for blle and seminal plasma are reporled.
Useful biochemical information on many low molecular weight compounds of both endogenous and xenobiotic origin can be obtained from high-resolution 'H NMR spectra of biological fluids such as urine, plasma, and cells or cell extracts (1-9). In general, the main obstacle to obtaining high-quality 'H NMR spectra of these materials is the large dynamic range of the spectrum resulting from the intense signal from solvent water (>lo0M in protons). This is mainly because limitations in the size of the analog-to-digital converter may result in incomplete digitization of weaker signals from the solutes present in millimolar concentrations. The use of some type of water suppression technique is therefore essential if good digitization of these weak signals and adequate signal-to-noise levels are to be obtained in lH NMR spectra of biological samples. A variety of solvent suppression methods have been used to obtain acceptable dynamic range conditions so that welldigitized high-resolution spectra of aqueous solutions, including biofluids, can be obtained without resorting to freeze drying and reconstitution with q20. Simple approaches,such as the application of a secondary irradiation field at the water resonance frequency, in order to produce selective presaturation of the signal, may be effective (10). Selective excitation methods (11,12) or spectral selection based on T1relaxation time, i.e. the water elimination Fourier transform method or WEFT' (13,14),have also been applied successfully. However, in biological fluids, where many metabolites are present at low-millimolar or submillimolarconcentrations, giving proton signals over a wide chemical-shift range, these methods may not suppress the water signal adequately or may introduce unacceptable signal intensity distortions. 0003-2700/87/0359-2885$01.50/0
When low molecular weight components present in cell suspensions and protein-rich biofluids such as plasma are being measured by 'H NMR, it is necessary to use spin-echo methods (15) to eliminate the broad lines from macromolecules which have fast transverse relaxation times (16-18). This may also result in the attenuation of the relatively broad water signal. Fbbenstein and co-workers have recently described a novel method for water suppression, based on the reduction of water T2 relaxation time by ammonium chloride or guanidinium chloride which can exchange protons with solvent (WATR method (19,20)). The resulting broad water signal is then selectively attenuated in Carr-Purcell-Meiboom-Gill (CPMG (21,22)) spin-echo spectra. With the WATR method, efficient solvent suppression can be achieved and signals close to the water resonance can be observed without intensity distortion (19,20). This approach has been shown to provide an effective means of water suppression in biological fluids (20,23,24). Paramagnetic ions such as Mn2+ can be used to shorten water T2'sin dilute solutions of biochemicals and biological fluids, and so result in the attenuation of the solvent signal in CPMG spin-echo spectra (25-27). This approach relies on the assumption that the metal ion will be freely accessible to the solvent and will not be extensively complexed or bound to solutes. The metabolite resonances will also be severely broadened if significant first coordination sphere interactions occur (26). Biological fluids contain a wide range of compounds that can coordinate with paramagnetic ions and this might resul t in unpredictableeffects on the water T2relaxation time. We have now compared the use of paramagnetic and chemical-exchange water T2relaxation agents in spin-echo water suppression experiments on biofluids. Several types of biological fluids (urine, blood plasma, bile, and seminal plasma) with markedly different physicochemicalproperties have been studied taking into considerationpossible chemical interactions between the relaxation agents and the metabolites. EXPERIMENTAL SECTION NMR Spectroscopy. 'H NMR spectra were measured at ambient probe temperature (20 & 1 O C ) on Bruker AM400 and WH400 spectrometersoperating at 400-MHz proton resonance frequency with quadrature detection and fitted with 12-bit analog-to-digital(ACD)converters. Some T2measurements were also made on a Bruker AM500 (500 MHz) spectrometer with a 16-bit ADC. For each biofluid sample, typically 64 or 72 free induction decays (FID's) were collected into 16 384 computer data points. In single pulse experiments 45' pulses were used with a repetition rate of 6.7 s. The water signal was suppressed by applying either a gated secondary irradiation field at the water resonance frequency, or spin-echo methods (see below). Prior to Fourier transformation, an exponential apodization function was applied to the FJD correspondingto a line broadening of 0.5 Hz. In most was added to each biofluid sample t o 20% (v/v), to cases 2Hz0 provide an internal field-frequency lock. Chemical shifts were 0 1987 American Chemical Society
2886
ANALYTICAL CHEMISTRY, VOL. 59, NO. 24, DECEMBER 15, 1987
""I
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I
60-
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40-
1
2
R
' PO,
Figure 1. pH dependence of the observed T , relaxation time of water in urine samples containing urea at 1.2 M measured at 400 MHz.
referenced internally to the singlet resonance of sodium, 3-(trimethylsilyl)[2H4]pr~pionate(TSP, 6 = 0 ppm) added in 'HzO. Spin-echo spectra were also acquired by using 64 or 72 repetitions of the Carr-Purcell-Meiboom-Gill (CPMG) (20, 21) pulse sequence. D - 90', - ( 7 2 - 180°+y - T 2 ) n collect FID The following parameters were used: T~ = 2 ms, a TI relaxation delay D of 5 s, and a variable number (n)of 180' pulse trains, the total spin-spin relaxation period being 2n7 ms (see text). Observed T2relaxation times (TPobsd) were calculated by using the expression Biofluid Samples. Urine samples were collected from healthy male volunteers, not undergoing any drug therapy. Freeze-dried quality control serum (Wellcomtrol 3, Wellcome Laboratories, UK) of bovine origin, collected without anticoagulant, was reconstituted to its original volume with 10% %IzO and 90% 'H20 prior to NMR measurements. Human plasma was separated from fresh whole blood collected by venepuncture using lithium heparin as the anticoagulant. Bile was obtained from the cannulated bile ducts of adult Sprague-Dawley rats. Seminal plasma (cell free) was obtained from vasectomized patients undergoing routine fertility screening procedures postvasectomy (samples courtesy of Professor M. D. Kendall, St. Thomas' Hospital, London). Paragmagnetic Ions. Analytical grade manganese(II)chloride and gadolinium(II1)chloride (purchased from Aldrich Chemicals) were used as paramagnetic Tz relaxation agents in experiments on urine, with final concentrations ranging from 56 to 416 pM (see text). In experiments on blood plasma Mn2+was added to give final concentrations of 160 p M . In experiments on seminal plasma Mn2+was titrated into samples in 200 MMincrements to a maximum of 1 mM (see text). Effect of Citric Acid on Water T, Relaxation Times in Urine Containing Mn2+. The effect of citric acid on the water T2relaxation time in urine in the presence of absence of Mn2+ was investigated. Aqueous trisodium citrate (purchased from Aldrich) was titrated into control human urine (diluted with 20% 2H20),the inital citrate concentration being 1.1mM (measured by IH NMR spectroscopy) and into urine containing416 PM Mn2+ at either pH* 2.5 or pH* 7. Measurement of pH. All pH measurements were made at 20 "C by using a Corning Model 150 ion analyzer with a long reach micro (3.5-mm diameter) combination electrode and subject t o automatic temperature correction. All biofluids samples contained 20% ,HzO and no corrections were made for the different activity coefficient of the deuterons. The pH values are quoted exactly as measured and so were designated pH*. Curve Fitting. The curve shown in Figure 1 was calculated by using an eighth-order polynominal function minimization routine (Statworks, Microsoft) on a Macintosh Plus microcomputer. RESULTS AND DISCUSSIONS Effect of Urea a n d Guaaidinium Salts on the Water T2Relaxation Times in Biofluids. Rabenstein et al. have shown that by the use of chemical exchange water T2relaxation agents, the WATR method can give very effective solvent
04
.
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.
,
6
.
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8
.
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10
.
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Figure 2. pH dependence of observed T , relaxation time of water in aqueous solutions contalnlng 1 M guanidinium sulfate (M) and 1 M guanldinium chloride ( 6 )at 400 MHz.
suppression in dilute solutions of biochemicals (19,ZO). We have investigated further the effects of urea and other guanidinium salts on water Tz relaxation times in urine and other biofluid samples. The effect of pH on the observed T2relaxation time of water in urine samples containing 1.2 M urea is shown in Figure 1. These data extend our previous study of acid catalyzed exchange (24). Two T2relaxation "troughs" occur around pH* 3.2-3.4 and 10.5-10.7. These correspond, respectively, to acid and base catalyzed urea-water proton exchange processes (28, 29). In the approximate pH* ranges 2.5-4.5 and 10-11.8 the water signal is sufficiently broad (i.e. >13 Hz) to allow effective solvent suppression in CPMG spin-echo spectra of urine samples (24). Solvent suppression factors of greater than lo4 can be obtained with relatively short total relaxation delays (ca. 250 ms) when the water line width is >25 Hz (24). With urine samples the use of urea as a chemical-exchange line broadening agent has an additional attraction in that it is an abundant endogenous component of the fluid and may require no supplementation in order to effect a significant line broadening of the water signal after pH* optimization (24). However, in practice, it is expedient to add urea in the 2Hz0 to give shorter water T i s over a wider pH range. There appears to be no simple relationship between pH* and T%wover the pH* range 5-9, where the water line widths vary between 6 and 9 Hz. This probably reflects a multitude of overlapping proton exchange processes between the solvent and a variety of endogenous urinary solutes including, for example, ammonium and phosphate ions, which occur in urine a t 10-50 mM (30)and also exchange protons with water and so affect the line width (19, 20, 23). The effects of pH on the observed T2 of water in 1 M aqueous solutions of three guanidinium salts are shown in Figures 2 and 3. Since this work was completed Rabenstein et al. have reported (20)similar data on 0.25 M solutions of guanidinium chloride although for a given pH the water TzoM were considerably larger. All guanidinium salts broadened the water signal over a greater pH range than did urea at similar concentrations. For guanidinium sulfate and guanidinium chloride at 400 M H z (Figure 2) the water TzoMminima occur at pH* 8.7, but differ in magnitude from ca. 10 (h:. drochloride) to ca. 5 ms (sulfate). The effect of guanidiniua sulfate on the water Tzobd in urine samples was virtually identical with that in simple aqueous solution, i.e. 4.2 ms at pH* 8.7. Effective Tz-based solvent suppression (corresponding to water vl,a)s > 13 Hz (24)in the CPMG spin-echo experiment can be achieved in the pH* range 8-10 by using the sulfate or pH* 8-9.5 by using the hydrochloride. The presence of carbonate counterions produced markedly different effects on the water pH/T2 curves. The effect of pH on the T b of~water in the presence of guanidinium carbonate
ANALYTICAL CHEMISTRY, VOL. 59,NO. 24, DECEMBER 15, 1987
:
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of water in 1 M guanidinium carbonate at 200 MHz (0)400 MHz (+), and 500 MHz (D).
I ,
a'
-
10 20 [citrate] mM
30
Flgure 5. Effect of citrate concentration on the water T , relaxation time in urine in the absence of Mn2+ (a) at pH' 7 (D) and in the presence of 416 pM Mn2+ (b) at pH' 7 (0)and (c)at pH' 2.5 (+).
Flgwe 3. pH and field dependence of the observed T , relaxation time
0
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2887
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Figure 4. Dependence of the observed spin-spin relaxation rate (l/T2w) of water on manganese concentration in urine samples at pH' 3 ( + ) and pH' 7 (D) at 400 MHz.
at three field strengths is shown in Figure 3. There was a strong field dependence of the water TZ0w, particularly above pH* 8.5. Between pH* 7 and 8.5 water line broadening sufficient to effect solvent suppression could be obtained at any field strength. In the pH* range 7-10 efficient solvent suppression can be achieved at 400 or 500 MHz by using the WATR method. In general, water suppression factors of at least IO3 are needed to allow adequate digitization of signals from low molecular weight metabolites in biofluids (24). Suppression factors of greater than lo4 are needed if signals close to the water resonance are to be observed in biofluids such as urine (24). This degree of suppression can only be attained with acceptably short total spin-spin relaxation periods (i.e. 2nr 5 300 ms), when water line widths are >20 Hz. The lowest value of 2nr that allows adequate water suppression should be used in order to minimize metabolite signal intensity distortions from differential T2 relaxation. Effect of Mn2+ Ions on Water T 2 Relaxation in Aqueous Samples. The dependence of the observed water T2 relaxation rate on the concentration of Mn2+ in urine samples at pH* 3 and 7 is shown in Figure 4. The spin-spin relaxation rate, 1 / T2,of a nucleus in chemical exchange with a paramagnetic species in solution is given by
+ l/T2D + 1/TZ,OS where TZobsd is the observed T2 relaxation time, P, is the 1/T2obsd = Prn/(T2M +
Tex)
probability of the observed spin interacting directly with the
paramagnetic site, T 2is~the relaxation time of the nucleus in the paramagnetic center, rex is the average time of the species under observation in the paramagnetic environment, 1/Tm is the diamagneticcontribution, and l/Tz,osis the outer sphere relaxation rate (26). The probability, P,, of the observed spin sampling the paramagnetic site is directly proportional to the concentration of the paramagnetic species in solution, assuming that the interacting species are not constrained or immobilized. Under such conditions, there will be a linear dependence of the spin-spin relaxation rate of an interacting species, e.g. water, on the concentrationof the paramagnetic ion, i.e. Mn2+(Figure 4). In urine samples, the water T2is also strongly dependent on pH, i.e. smaller T2'sbeing attained for a given Mn2+concentration at lower pHs. This suggests that the paramagnetic relaxation effect on the water protons in urine must be mediated by at least one other interacting chemical species with ionizable groups. The most likely explanation of this is that endogenous chelating agents are in competition with water protons for Mn2+binding, thus limiting the availability of free Mn2+at higher pH's. We investigated the effect of an abundant urinary chelating agent, citric acid (30),on the Mn2+-enhancedT2relaxation of the water protons in urine at pH* 2.5 and at pH* 7. Although there was a strong dependence of the water Tbbd on the concentration of added citrate at pH* 7, there was little effect a t pH* 2.5 (Figure 5 ) . Mn2+forms moderately strong complexes with citric acid under pH and ionic strength conditions similar to those found in normal urine, the equilibrium constant (log K'Mn-citrate) being 3.72 at pH 7.2 (31). Coordination of citrate with the metal ion is normally via two of the carboxylate groups and the hydroxyl function (32). The pK,'s for the three carboxylate functions of citric acid are 6.396, 4.761, and 3.128, respectively (33). At pH* 2.5 the association between Mn2+and citrate is relatively weak as the carboxylic acid groups are largely protonated, so Mn2+ions are free to interact more strongly with solvent water. Addition of citrate had no detectable effect on the T2relaxation time of water in urine in the absence of Mn2+ ions (Figure 5 ) . Efficient paramagnetically induced relaxation of the solvent water is dependent on rapid chemical exchange of relaxed protons between the paramagnetic centers and bulk solution. If the paramagnetic ion is extensively complexed with a chelating species, such as citrate, the paramagnetic center is less accessible to solvent. This has two important implications for the use of paramagnetic species in effecting T2based water suppression in biological fluids. Firstly, the strength of the paramagnet-water interaction will be modulated by endogenous metal chelating or coordinating species, the concentrations of which will vary unpredictably from sample to sample. Secondly, the protons of the chelating or metal binding solutes may relax rapidly through sampling the first coordination sphere of the paramagnetic ion. Their resonances will then
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 24, DECEMBER 15, 1987
be eliminated from spin-echo spectra, hence defeating the object of the experiment. Comparison of the Effects of Various Water T2 ReA laxation Agents on ‘H NMR Spectra of Biofluids. Urine. As shown in Figures 1-4, both paramagnetic ions such as Mn2+ and compounds such as urea and guanidinium salts, which exchange protons with the solvent, can greatly reduce the water T2relaxation time of water in urine samples. ‘H NMR spectra of human urine, obtained with water suppression by using gated secondary irradiation or CPMG spin-echoes after treatment with urea (pH* 3.4) or 45 FM Mn2+(pH* 5.6), are compared in Figure 6. In all cases, the degree of water suppression was >lo3and therefore sufficient to allow adequate digitization of the metabolite signals. However, the CPMG -creatinineC spectrum of the sample containing Mn2+ is significantly 1 citrate different from the others in that many metabolite resonances I have also been attenuated, most notably from glycine, citrate, rv” lactate, alanine, valine, and P-hydroxybutyrate. Furthermore, the water line width after treatment of the urine with Mn2+ 40 30 20 10 00 was only 12 Hz, which is the minimum value for this type of PPM water suppression to be effective (24). Flgure 6. 400-MHz ‘H NMR spectra of control urine (with 20 % ,H20), The addition of 2 mM EDTA (pH* 7) to urine containing the effect of three water suppression methods on the resonances in Mn2+caused a reduction in the water line width to 8 Hz, the 0-4.5 ppm region of the spectra: (A) single pulse spectrum (72 rendering efficient spin-echo-based water suppression imscans), acquired with a secondary irradiation field applied at the water resonance frequency during the delay between observation pulses, practical(24), but did not result in the reappearance of sample pH’ 5.6, water line width 7 Hz; (B) CPMG spectrum (72 scans) paramagnetically broadened metabolite resonances in the of same urine sample (pH’ 5.6) Containing 45 added MnCI, (water spin-echo spectrum. Under these conditions, the bulk of Mn2+ line width 12 Hz) 2177 = 256 ms; (C) CPMG spectrum (72 scans) pH’ might be expected to be in the form of an Mn-EDTA2- com3.5 containing 1.2 M urea (water line width 21 Hz) 2n7 = 256 ms. plex, the calculated conditional stability constant, log KM~-EDTA’= 10.47. However, urine also contains Ca2+and of the spectra, thus aiding interpretation. We have found it Mg2+a t high concentration, typically 5-10 mm (29). Both possible to manipulate the Gd3+concentration and the CPMG metals form strong complexes with EDTA at pH 7 (with conditions to selectively eliminate the citrate resonances in equilibrium constants, log Kca-EmA’ = 7.43 and log K M ~ - E ~ A ’ ‘H NMR spectra of urine (data not shown). A similar effect = 5.37 (34)),and it is likely that these ions will compete with within Mn2+is shown in Figure 6 although Mn2+appears to Mn2+for binding to EDTA in urine samples. Acidification be less selective than Gd3+. With Gd3+,as with Mn2+,it was of urine samples containing Mn2+(but no EDTA) to pH* 3 not possible to adjust the sample conditions, i.e. Gd3+conresulted in a reduction in the water T z relaxation time, as centration, sample pH, or the spin-spin relaxation periods would be predicted from Figure 5, but the citrate and amino used in the CPMG sequence, to obtain adequate solvent acid resonances were still very broad and not detectable in suppression without spectral distortion due to the enhanced spin-echo spectra. This shows that at low pHs or even in the T z relaxation of solutes. With urea used as a chemical-expresence of EDTA both solvent water and the urinary chechange broadening agent, it was possible to suppress the water lating solutes can sample the inner Mn2+coordination sphere. signal by factors of 103-104 in CPMG spectra (24) without Consequently, it was not possible to optimize the Mn2+consignificantly distorting the intensities of urinary metabolite centration, EDTA concentration, or the pH conditions such resonances (Figure 6). However, the adjustment of the sample that the spin-echo-based water suppression could be achieved pH* to 3.4 to obtain acid-catalyzed urea-water proton exwithout causing intensity distortions of the metabolite signals. change (Figure 1) resulted in minor changes in the chemical In human urine samples containing very low endogenous shifts of protons that were close to acidic functional groups. concentrations of citrate (
