PROTON NMR SPECTROSCOPY OF HUMAN BLOOD PLASMA Dallas L. Rabenstein, Kevin K. Millis, and Erin J. Strauss Department of Chemistry University of California Riverside, CA 92521
NMR spectroscopy is an important technique for the study of biological fluids and intact cells (1-8). Compared with other analytical methods, it is nondestructive and noninvasive, and it allows delicately balanced chemical and cellular processes to be observed directly at the molecular level. 13 C, 15 N, 31 P, and lH NMR spectroscopies have all been used to study biological fluids and/or intact cells. Because of the inherent low NMR sensitivities and low natural abundances of 13C and 15 N, isotopically enriched compounds generally are used in 13 C and 15N NMR studies. The advantage is a relatively simple spectrum that consists of resonances from the enriched compounds and their metabolites superimposed on much weaker background resonances. However, isotopically enriched compounds are required, and, in the case of intact cell studies, they must be incorporated into the cells. Nevertheless, 13C and 15 N NMR with isotopically enriched compounds are widely used and important methods for the study of metabolic processes in intact cells. 31 P, which has a natural abundance of 100% and a high inherent NMR sensitivity, is also widely used. Because there are few phosphorus-containing compounds at detectable levels, 31 P
NMR spectra of biological fluids and intact cells generally are relatively simple. For the same reason, however, 31 P NMR can be used to study only a limited number of compounds. Virtually all compounds in biological fluids and cells contain hydrogen, but the very abundance of hydrogen-containing compounds makes their study by XH NMR more difficult. The measurement of XH NMR spectra is further complicated by the water resonance, which obscures a large portion of the
spectrum and creates a dynamic range problem during data acquisition. However, because of its much greater sensitivity and the ubiquity of hydrogen in biological molecules, XH NMR offers significant advantages. With state-ofthe-art high-field NMR spectrometers and some simple NMR experiments (6, 9), high-resolution XH NMR spectra can be measured routinely for biological fluids and intact cells, particularly red blood cells. The purpose of this article is to re-
Figure 1. A 500-MHz 1H NMR spectrum of human plasma. The spectrum was measured by the single-pulse method using a flip angle of ~5° to avoid overloading the analog-to-digital converter. This spectrum and all the other spectra presented in this article were measured with a Varlan VXR-500S spectrometer operated in the unlocked mode.
1380 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
0003-2700/88/A360-1380/$01.50/0 © 1988 American Chemical Society
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AND RED BLOOD view some of the NMR techniques used to measure 1 H NMR spectra of human plasma and red blood cells. The Ή NMR spectroscopy of plasma and red blood cells is of interest because report ed studies indicate that information relevant to biochemical and clinical ap plications can be obtained by XH NMR. 1
H NMR spectroscopy of plasma
A 500-MHz Ή NMR spectrum of hu man plasma is shown in Figure 1. The
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INSTRUMENTATION spectrum was measured by the stan dard single-pulse method and is char acterized by a large water resonance at 4.77 ppm superimposed on a broad en velope of overlapping resonances. The broad envelope is primarily from plas ma proteins and lipoproteins. Also su perimposed on the broad envelope are
Figure 2. A 500-MHz 1H NMR spectrum of human plasma. Heparin was used as the anticoagulant. The spectrum was measured with suppression of the water reso nance by presaturatlon (pulse sequence A in Figure 3). A presaturation time of 1 s was used, and 128 transients were co-added. Resonance assignments are given In Figure 4. The resonances identified —0Η2^- and —CH 3 are for mobile fatty acid components of chylomicrons and lipoproteins; those labeled (CH3)3N— are mainly from phospholipid choline head groups.
much weaker resonances from small molecules in plasma (e.g., glucose) and resonances from the CH2 and CH3 groups of "mobile" fatty acid compo nents of chylomicrons and lipopro teins. The water resonance blocks out a large and important part of the spec trum, and because the water proton concentration is ~100 M compared with millimolar concentrations for the small molecules, it is difficult to detect the much weaker resonances. Thus it is desirable to eliminate the water reso nance. Because this problem occurs in the measurement of Ή NMR spectra of most aqueous samples, a variety of water suppression methods have been developed (10). These methods include suppression by presaturation of the water resonance, suppression by use of selective excitation pulse sequences (i.e., pulse sequences that selectively excite the spectral region of interest), and suppression by taking advantage of differences between the spin-lattice (Ti) or spin-spin (T2) relaxation times of the water protons and those of the molecules of interest. Several of these methods have been used successfully to suppress the water resonance in XH NMR spectra of plasma. Suppression by presaturation of the water resonance. Figure 2 shows the spectrum obtained from the same plasma sample with suppression of the water resonance by presaturation
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988 · 1381 A
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Figure 3. Schematic representations of the pulse sequences used to measure 1 H NMR spectra of blood plasma and red blood cells. With these pulse sequences, the water resonance and/or the broad envelope of resonances can be sup pressed. In A and Β, Φ° represents a flip angle ^c4
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Figure 4. Assignments of resonances observed in 1H NMR spectra of blood plasma and red blood cells.
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Figure 5. A 500-MHz 1H NMR spectrum of human plasma. EDTA was used as the anticoagulant. The spectrum was measured with suppression of the water reso nance by presaturation. A presaturation time of 1 s was used, and 128 transients were co-added.
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ANALYTICAL CHEMISTRY, VOL. 60,NO. 24, DECEMBER 15, 1988 · 1383 A
suppression by presaturation. It con tains additional resonances from the acetate and ethylenic protons of free EDTA (EDTA(ac) and EDTA(en)) and the Ca(EDTA) complex and the ethylenic protons of Mg(EDTA); the resonance for the acetate protons of Mg(EDTA) overlaps the CaEDTA(ac) and EDTA(en) resonances (13). The concentrations of EDTA-chelatable calcium and magnesium in plasma have been determined from Ή NMR spectra of the type in Figure 5 (13). Suppression by differences in re laxation times. Several Ή NMR stud ies of human plasma have made use of differences between the T2s of the reso nances of interest and those of interfer ing resonances to selectively eliminate interfering resonances (13-17). These measurements are based on the differ ent rates of decay of the transverse magnetization from the various pro tons in a sample. Specifically, the shorter the spin-spin relaxation time, the faster the rate of decay, as illustrat ed in Figure 6. The effective spin-spin relaxation time T"2 for a particular pro ton is related to the width of its reso nance (ΤΙ = 1/τWi/2, where W1/2 is the width in hertz at resonance half height). Thus broad resonances can be selectively eliminated by delaying ac-
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Time (s) Figure 6. Dependence of the intensity of resonances having different spin-spin re laxation times on the length of the spin-spin relaxation period. The spin-spin relaxation period is equal to the time between the pulse and the start of data acquisition in pulse sequence Β (Figure 3) and is equal to 2τ in pulse sequence C and 2τη in pulse sequences D and E. These curves were calculated with the equation Ht) = HO) exp (—f/T2), which ignores the effect of diffu sion.
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quisition until their transverse magne tization has decayed to a sufficiently small value (6,9,18,19). The spin-spin relaxation time depends largely on mo lecular size and decreases as molecular size increases; thus, resonances from proteins and other macromolecules can be selectively eliminated. This experiment can be implement ed with pulse sequence Β in Figure 3; however, because of the free precession of the transverse magnetization during the spin-spin relaxation period, it is difficult to phase the spectrum. Also, the transverse magnetization that is detected with this pulse sequence, and thus the signal, can be considerably re duced in intensity because of magnetic field inhomogeneity.
To eliminate these effects, spectra generally are measured with pulse se quences in which one or more refocusing pulses are applied during the spinspin relaxation period (20). The sim plest pulse sequence of this type, and one that has been widely used in stud ies of biological fluids and intact red blood cells, is the Hahn spin-echo pulse sequence (C in Figure 3) (21). In the spin-echo pulse sequence, applica tion of a 180° pulse midway through the spin-spin relaxation period refocuses transverse magnetization that has dephased because of magnetic field inhomogeneity and eliminates chemi cal shift effects (19-22). The behavior of the transverse magnetization during the spin-echo pulse sequence and its
Figure 7. A portion of the 500-MHz spin-echo 1H NMR spectrum of human plasma. The spectrum was measured using a τ = 0.067 s in the spin-echo pulse sequence (C in Figure 3) and water suppression by saturation; 128 transients were co-added.
Figure 8. A portion of the 500-MHz CPMG 1H NMR spectrum of human plasma. The spectrum was measured with pulse sequence D in Figure 3 using a τ = 3.2 X 10~4 s and 2τη 0.35 s; 128 transients were co-added.
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use for spectral simplification on the basis of differential rates of spin-spin relaxation have been described previ ously (19, 22). Figure 7 shows a 500-MHz spin-echo Ή NMR spectrum of plasma. The spectrum was measured with a spinspin relaxation period of 0.134 s, and the water resonance was suppressed by presaturation. Several features of this spectrum are of interest. The broad en velope of resonances attributable to plasma proteins and lipoproteins has been eliminated by spin-spin relax ation. Also, some resonances are posi tive, some are negative, and some are out of phase. This is a characteristic feature of spectra measured by the spin-echo pulse sequence and results from phase modulation caused by homonuclear scalar coupling. The origin of this phase modulation has been dis cussed in terms of the behavior of the transverse magnetization (6, 20, 22). Although the phase modulation can be useful (e.g., in making resonance as signments), it tends to complicate spectra. For example, it is difficult to measure the intensity of resonances that are out of phase, and, depending on the length of the spin-spin relax ation period, the spin-spin coupling constant, and the nature of the multi plet pattern, strongly coupled reso nances can be greatly reduced in inten sity. The phase modulation can be eliminated with the C a r r - P u r c e l l Meiboom-Gill (CPMG) pulse sequence, in which multiple 180° refocusing pulses are applied during the spin-spin relax ation period (90x° - (τ - 1 8 0 / - τ)„ Acquisition; pulse sequence D in Fig ure 3), with the time between succes sive 180° pulses short relative to the inverse of the spin-spin coupling con stant (22-24). A 500-MHz CPMG Ή NMR spec trum of plasma, measured with a spinspin relaxation period (2τη) of 0.35 s and r of 3.2 Χ 10 - 4 s, is presented in Figure 8. As in the spin-echo spectrum, the broad envelope of resonances is eliminated; however, the resonances are all in phase and are normal, recog nizable multiplet patterns. The water resonance in Figure 8 is reduced in intensity by spin-spin re laxation. Although it can be completely eliminated by using a longer spin-spin relaxation period (>1.3 s), other reso nances are also considerably reduced in intensity by spin-spin relaxation. Al ternatively, several other approaches can be used to reduce the intensity of the water resonance. For example, the water resonance can be suppressed by selective saturation followed by mea surement of the spectrum by the CPMG pulse sequence (E in Figure 3). With this method, a much shorter spin-spin relaxation period can be used. For example, the spectrum in
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988 · 1385 A
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Figure 9 was measured with a spinspin relaxation period of 0.05 s. The advantage is that relative resonance in tensities are not so distorted by differ ential spin-spin relaxation (compare the relative intensities of the phospho lipid —CH2— and CH3 resonances and the a l , L I , and vl resonances in Figures 8 and 9). Alternatively, the spin-spin relax ation time of the water protons can be selectively decreased by adding para magnetic ions (25) or water proton ex change reagents (17,26,27) to the sam ple. For example, addition of NH4CI decreases the water proton spin-spin relaxation time as a result of exchange
of protons between ammonium ions and water molecules (26, 27). This method of water suppression, called the WATR method for Water Attenua tion by T 2 Relaxation (26), allows reso nances with exactly the same chemical shift as the water resonance to be ob served—an advantage over the presaturation method. The spectrum shown in Figure 10 was measured by the WATR method. 1 H NMR spectroscopy of red blood cells
The measurement of high-resolution 1 H-NMR spectra of intact red blood cells is complicated by the same fea-
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Figure 9. A portion of the 500-MHz CPMG 1H NMR spectrum of human plasma. The spectrum was measured with pulse sequence Ε in Figure 3, using a τ = 3.2 X 10~4 s and 2τη = 0.05 s. The decoupler was on for water suppression during a period of 1 s before the start of the CPMG pulse sequence and during the pulse sequence; 128 transients were co-added.
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Figure 10. A portion of the 500-MHz CPMG 1H NMR spectrum of human plasma. Sufficient NH4CI was added to give a concentration of 0.5 M, and the pH was buffered at 7.1 with phos phate. The water resonance was completely and selectively eliminated by the WATR method (Water At tenuation by Γ2 Relaxation) using pulse sequence D in Figure 3 with τ = 3.2 X 10~4 s and 2τη = 0.5 s.
1386 A . ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
tures as those for plasma (see the 500MHz Ή NMR spectrum for packed red blood cells in Figure 11). The large water resonance is superimposed on a broad envelope of resonances, in this case from the protons of hemoglobin and the red cell membrane. The tech niques discussed above can be used to selectively eliminate the interfering resonances and obtain high-resolution Ή NMR spectra for the small intracel lular compounds (6, 9). To illustrate, a 500-MHz spin-echo X H NMR spectrum of intact red blood cells is shown in Figure 12. This spec trum was measured with a spin-spin relaxation period of 0.134 s. The hemo globin and membrane resonances are eliminated, and the water resonance is significantly reduced in intensity. By
increasing the length of the spin-spin relaxation period, the water resonance can be further reduced in intensity (6, 28). Most of the resonances in the spinecho spectrum have been assigned to small intracellular compounds; the as signments are given in Figure 4. The spin-echo method has been used ex tensively in Ή NMR studies of chemi cal and metabolic processes in intact red blood cells. Figure 13 shows a 500-MHz CPMG X H NMR spectrum of intact red blood cells. This spectrum, which is for a dif ferent sample of red blood cells, was measured with a longer spin-spin re laxation period (0.27 s) to completely eliminate the water resonance. Com pared with the spin-echo spectrum in Figure 12, the resonances in this spec-
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Figure 11. The 500-MHz 1H NMR spectrum of human red blood cells. The spectrum was measured by the single-pulse method using a flip angle of ~ 5 ° . Sample preparation involved separating the red blood cells from plasma and white cells by centrifugation.
Figure 12. A portion of the 500-MHz spin-echo 1H NMR spectrum of intact red blood cells. The spectrum was measured with pulse sequence C in Figure 3, using r = 0.067 s; 128 transients were co-added. The red blood cells used in the measurement of this spectrum were washed once with an equal volume of isotonic saline solution after separation from the plasma. To obtain good resolution, the blood was bubbled with oxygen to ensure that the hemoglobin was in the diamagnetic oxygenated form.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988 · 1387 A
trum are not phase modulated. Conse quently the multiplet patterns are more easily recognized, and more reso nances can be observed in the CPMG spectrum (e.g., the broad, relatively weak multiplet patterns in the 1.7-2.1ppm region). Some of the powerful two-dimen sional Ή NMR techniques can also be applied to red blood cells by incorpo rating a spin-spin relaxation period in the pulse sequence to eliminate broad interfering resonances {29, 30). For ex ample, 1 H COSY spectra have been measured for human and sheep red blood cells with the delayed COSY ex periment (29). Resonance connectivi ties established from the COSY spectra were used to assign resonances in the X H NMR spectrum of transport-defi cient sheep red blood cells and to es tablish the presence of compounds whose resonances were hidden by over lap in one-dimensional spectra. Experimental considerations
The spectra in the figures were ob tained with pulse sequences that gener ally are included in the standard soft ware supplied with state-of-the-art pulse/Fourier transform NMR spec trometers. However, several experi mental considerations should be men tioned. J H NMR methods for the non invasive study of metabolism and other processes involving small molecules in intact red blood cells have also been described in detail elsewhere (6). Normally, NMR spectrometers are operated in a "locked" mode, using a lock signal derived from a deuterium resonance to maintain field/frequency stability. Because plasma and red blood cells do not contain sufficient natural-abundance deuterium for a lock signal, either D2O must be added to the sample or the spectrometer must be run in the unlocked mode. Fortu
nately, the superconducting magnets in many high-field spectrometers are sufficiently stable, or, as is the case with the spectrometer used to measure the spectra presented here, they are equipped with drift correction hard ware so that high-resolution spectra can be obtained without a field/fre quency lock, even when signal averag ing is required to achieve an adequate signal-to-noise ratio (S/N). The deuterium field/frequency lock signal is also generally used as a mea sure of magnetic field homogeneity when shimming the magnet. However, as long as sample volumes are carefully reproduced, we can obtain high-resolu tion spectra for plasma and red blood cells by using the same shim settings that give good spectra for a D2O solu tion of amino acids. Thus Ή NMR spectra can be obtained for intact plas ma or red blood cells, without the need to add D2O or other reagents. Sample heating can occur when mea suring spectra of plasma and red blood cells by the CPMG pulse sequence. Sample heating can affect the quality of the spectrum if the spectrometer is run in the locked mode. The effect of the sample heating on the quality of the spectrum can be eliminated by run ning an appropriate number of dummy scans to achieve a steady-state sample temperature before starting data ac quisition or by running in the unlocked mode. Another important consideration is that it is advantageous to make mea surements of the type described here on the highest field spectrometer avail able, not only for greater dispersion and sensitivity, but also because the spin-spin relaxation rate of H2O in creases as the field strength increases because there is an exchange contribu tion to the spin-spin relaxation rate of H 2 0 protons in protein solutions and
cell suspensions. The larger the field, the shorter the relaxation delay needed for elimination of the water resonance. The spectra presented in Figures 8,10, 12, and 13 demonstrate that at 500 MHz the water resonance can be selec tively eliminated with a spin-spin re laxation period sufficiently short that a good S/N is obtained for resonances from the low molecular weight com pounds. With the high sensitivity of state-ofthe-art spectrometers, spectra of the type presented here can be obtained quickly, making it possible to follow relatively fast chemical processes (e.g., metabolic reactions) as a function of time directly in intact cells. With our spectrometer a reasonable 500-MHz CPMG Ή NMR spectrum of intact red blood cells can be obtained from four transients, which takes a total instru ment time of 8 s. To achieve this high sensitivity, how ever, some basic data acquisition con siderations should be kept in mind. With the large computer memory of state-of-the-art spectrometers, it may seem desirable to acquire large free in duction decays (FIDs). However, ac quisition of a large FIDs can have a deleterious effect on the spectrum, as illustrated by the series of spectra in Figure 14. These spectra are for the methyl protons of alanine and lactate in intact red blood cells. They were measured by the CPMG pulse se quence with a spin-spin relaxation pe riod of 0.27 s. Each spectrum was ob tained from four transients, no resolu tion or sensitivity enhancement was applied, and each FID was zero-filled as necessary to give a total of 32,768 points before Fourier transformation. This series of spectra shows the effect of the length of data acquisition on the resolution and S/N. Acquisition of data for 0.103 s, which corresponds to the
Figure 13. A portion of the 500-MHz CPMG 1H NMR spectrum of intact red blood cells. The spectrum was measured with pulse sequence D in Figure 3, using τ = 3.2 Χ 1 0 - 4 s and 2τη = 0.27 s; 200 transients were co-added. The red blood cells used in this measurement were not washed after separation from plasma.
1388 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
Figure 14. Dependence of line shape and sensitivity on number of data points acquired and thus on the length of data acquisition. The data acquisition times were (from left to right): 0.103, 0.205, 0.110, 0.819, 1.638 and 3.277 s; four transients were collected for each spectrum, and the FIDs were zero-filled as necessary to give a total of 32,768 points before Fourier transformation; no resolution or sensitivity enhancement was applied. The reso nances are a1 (left) and L1 (right) of alanine and lactate, respectively; the spectra were measured for the red blood cell sample described in the legend of Figure 13.
1024 point FID, is not long enough, as indicated by the truncation effects at the base of the lactate resonance. The truncation effects are eliminated by doubling the number of data points and thus the length of data acquisition. However, as the length of data acquisi tion is increased further, there is a de crease in S/N because, after a certain length of data acquisition, only noise is being acquired. Applications
The spectra in the figures demonstrate that with simple NMR experiments, high-resolution ! H NMR spectra can easily be obtained for small molecules in human plasma and red blood cells. A major disadvantage of XH NMR is its low sensitivity; detection limits for small molecules in plasma and erythro cytes are on the order of 0.01-0.1 mmol/L, depending on the number of equivalent protons giving the reso nance and its multiplicity. However, Ή NMR offers several advantages for clinical and biochemical studies, as il lustrated by the following examples. Most of the reported Ή NMR stud ies of human plasma have been by Sadler, Nicholson, and co-workers (1317). They have assigned many of the resonances to the protons of mobile, low molecular weight metabolites in plasma, serum, and urine samples from fasting and diabetic subjects, including resonances for the ketone bodies 3-Dhydroxybutyrate, acetone, and aceto-
acetate {14). In a particularly interesting study that demonstrates the potential of high-resolution J H NMR for clinical applications, lH NMR spectra were measured for plasma and urine from subjects who had taken acetaminophen as a therapeutic dose or in self-poison ing episodes (both fatal and nonfatal) (16). The Ή NMR spectra of plasma from overdose patients suffering from acute liver failure showed gross eleva tion of lactate and the amino acids ala nine, glutamine, proline, valine, methi onine, serine, lysine, phenylalanine, ty rosine, and histidine. The patterns of plasma metabolite concentrations indicated by Ή NMR reflected characteristic and severe liver dysfunction, suggesting that Ή NMR spectroscopy of body fluids may be valuable in cases of drug overdose and perhaps in the diagnosis of various other diseases. For such applications, the advantages of NMR are that the spectrum can be obtained quickly and a wide range of metabolites present at millimolar concentrations often can be detected simultaneously rather than individually or in selected classes, as with conventional methods of clinical analysis. This capability was used in the diagnosis of D-lactic acidosis in a patient with jejuno-ileal bypass who developed a neurological syndrome as sociated with metabolic acidosis (31). L-lactate, as determined by an enzy matic procedure, was only 2.2 mmol/L, whereas XH NMR gave a total lactate
(D- and L-lactate are indistinguishable by *H NMR) concentration of 10 mmol/L. The diagnosis of D-lactic aci dosis was subsequently confirmed by a specific enzyme measurement of D-lactate, which gave a concentration of 7.5 mmol/L. A report that the average widths of the resonances for the CH 2 and CH 3 groups of mobile fatty acid components of lipoproteins in the 0.8-1.3-ppm re gion correlated with the presence of malignant tumors in human subjects (32) stimulated considerable interest in the use of 1 H NMR for the diagnosis of cancer. It was reported that the aver age width of the CH2 and CH 3 reso nances was 29.9 ± 2.5 Hz in the pres ence of and 39.5 ± 1.6 Hz in the absence of malignant tumors. A subsequent de tailed study of these resonances showed that each contained several overlapping components from chylo microns, very low density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) (15). It also was found that av erage line widths of
