Nuclear Magnetic Resonance Spectroscopy Ian C. P. Smith* and Domthea E. Blandford Institute for Biodiagnostics, National Research Council of Canada, 435 Ellice Avenue, Winnipeg, Manitoba, Canada R3B 1Y6
Nuclear magnetic resonance (NMR) spectroscopy is a powerful, nondestructive technique capable of complete structural and conformational analysis of complex molecules, quantitative analysis of complex mixtures, and noninvasive measurement of reaction rates of chemical systems in the test tube and in intact living organisms, including humans. NMR was discovered simultaneously by two independent laboratories in 1946 (Tl, T2). Subsequently, high-resolution NMR was quickly developed by analytical chemists as a powerful technique for the determination of molecular structure. The fundamentals of NMR technology are described in specialty texts to which interested readers are referred (T3-T6). In the first section of this review, we will provide a brief overview of the basics of NMR spectroscopy including theory and principles of NMR, instrumentation, technical limitations, and spectral interpretation methods. At this point we shall quickly shift to the designation MRS, magnetic resonance spectroscopy, which has been adopted by clinical proponents to avoid any negative implications of “nuclear”. The development of MRS as a clinical analytical tool has been spurred on by the widespread use of a related analytical technique, magnetic resonance imaging (MRI), in clinical medicine. The two are related since they both utilize the same physical phenomenon, NMR; MRS emphasizes spectral or chemical information, whereas MFU emphasizes spatial information. The techniques and applications of MRI will not be addressed in this review. Interested readers are referred to recent texts and articles (7’7-23). MRS is unique in its capability to provide nondestructive in vivo and in vitro chemical analyses. While significant advances have been made in in vivo MRS, this has recently been reviewed elsewhere (TI@. The present review for clinical chemistry highlights some of the research that has been directed toward in vitro chemical analysis, namely, physiological fluids, tissue specimens, and tissue extracts. This application of MRS in pharmacology and toxicology is already quite well established; the main areas of current development are metabolic disorders, organ transplantation, neurological disorders, and cancer. The review covers the time period of 1990 to the present. Our previous review covered the literature up to that time ( T l l ) . THE BASICS OF NMR SPECTROSCOPY Theory and Principles. Spectroscopy is the measurement of the frequency dependence of absorption or emission of energy by a system. NMR refers to the absorption and release of radio frequency (rf) energy by a nucleus in a magnetic field. Possession of both charge and spin renders some nuclei magnetic and confers various properties on them which affect their behavior in an external magnetic field. One such property is a magnetic moment @). In an external magnetic field (Bo),the magnetic moment of a spinning nucleus will “precess”, or describe a cone, around the direction of the field. The precessional frequency of a particular nucleus is proportional to the strength of the magnetic field. To observe resonance, the nuclei must be irradiated with electromagnetic (I$radiation, the frequency of which must match the precessional frequency of the nuclei. The rf energy is then absorbed by nuclei in the lower energy spin state, raising them to the higher energy spin state. In actual fact, upward and
downward transitions are stimulated equally, but upward ones are more prevalent due to the greater occupancy of the lower energy state. This leads to a net absorption of rf energy. Since the energy difference between the two states is proportional to the magnetic field strength, the stronger the field, the greater is the difference between the two populations, and the stronger is the MRS signal. For any particular atomic nucleus, at a constant magnetic field in a vacuum, there is only a single resonant frequency. In bulk matter, nuclei are surrounded by electronic clouds which exert a small, but significant shielding effect. The degree to which a magnetic nucleus is shielded from the applied field by the electron cloud determines its precessional frequency; the more dense is the electron cloud (increased shielding), the lower is the preces sional frequency. Different molecular environments are characterized by a parameter called the chemical shift, which is the resonance frequency measured relative to that of a suitable reference compound. The chemical shift values (6) are typically of the order of and are therefore commonly specified in parts per million @pm). These units are independent of magnetic fields, thereby allowing direct comparison of results from different instruments. In absolute frequency terms, the separation between nuclei with different chemical shifts increases with increasing magnetic field, yielding better dispersion of resonances at high field. Electronic clouds also mediate interactionsbetween nuclei that result in a characteristic splitting pattern of the MRS signal. The so-called “spin-spin” couplings (usually given the symbol ‘7) are very useful in assigning MRS resonances, but they also cause spectra to be complicated and crowded. Nevertheless, the characteristic pattern of chemical shifts and couplings usually enables the identification and quantitation of each of a number of compounds present in a mixture. Consider the ‘H MR spectrum of glucose in water, as shown in ref T12. Two major anomeric species of glucose are present, the a- and P-pyranose forms. The resonances at 4.51 and 5.10 ppm are due to the hydrogens at position 1 of the pyranose ring, B and a anomers, respectively. Proximity to the oxygen in the ring gives these hydrogens very characteristic chemical shifts. The splitting of these resonances into two is due to Jcoupling to the hydrogen species at position 2 of the ring. The magnitude of the coupling is indicative of the coniiguration at position 1, 3.8 Hz for the a-anomer and 8.0 Hz for the ,%anomer. The other patterns in the spectrum are more complex to analyze due to overlaps, but assignments of all resonances and couplings for both anomers have been made (T12). In the MRS experiment, after the rf pulse is turned off, relaxation processes occur to restore the original equilibrium. The relaxation process implies a loss of energy from the system of nuclear spins. Two different processes, both of which are essentially exponential, are characterized by time constants: TI, longitudinal relaxation, or spin-lattice relaxation, which results in a transfer of energy to the surroundings; and 7’2, transverse relaxation or spin-spin relaxation, which is a redistribution of energy among spins. Together 7’1and TZcan provide information concerning molecular conformation and dynamics. Analytical Chemistry, Vol. 67,No. 12,June 15, 1995
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In the NMR experiment, data are collected by a pulse/data acquisition/delay sequence. The sequence is repeated to yield an averaged free induction decay (FID) signal of adequate signal/ noise ratio, which is subsequently Fourier transformed to yield a spectrum. The positions of NMR signals are measured in Hertz from a standard reference signal and are expressed in ppm in order to be independent of the spectrometer’s field strength. The intensity of an NMR signal is proportional to the area of the signal, and this area is generally measured by electronic integration. Under appropriate conditions, peak areas are proportional to the concentration of the particular compound observed. Thus, for mixtures of compounds, direct quantitative analysis, without separation, is possible. It is evident that MRS could be a very powerful analytical tool in the clinical laboratory and is of vast potential for clinical chemical analyses. However, this has not yet happened, perhaps due to the cost of the instrumentation, certain technical limitations, and difficulties in data interpretation. Each of these issues will be discussed in the following sections. Instrumentation. A modem MR spectrometer consists of three components: (1) a magnet, capable of sustaining a strong, stable, and homogenous magnetic field; (2) a probe within the magnetic field made up of a sample cavity, transmitter coil, and receiver coil; and (3) appropriate electronic circuitry, a computer, and peripheral devices to detect, amplify and display the NMR signal. (a) The Magnet. Magnets are classified as either permanent magnets or resistive superconducting electromagnets. Because the precessional frequency of a nucleus is directly proportional to the magnetic field strength, it is advantageous to use the strongest possible magnetic field to obtain the greatest separation between signals. Recent advances in technology have resulted in increased spectral dispersion and sensitivity, simpliied spectra, and reduced interference from strong solvent signals. The strongest magnets, those which are superconducting, are now used in MR instruments to generate field strengths up to 17.63 T (750 MHz for IH); permanent magnets and electromagnets generate field strengths up to 2.1 (60 MHz) and 2.35 T (100 MHz), respectively. The superconducting magnet, a solenoid magnet, is a coil of wire (typically a niobium-titanium alloy) which is immersed in liquid helium at a temperature of 4 K (-269 “C). At this temperature, the material is superconducting and can carry a high current with no electric loss or heat generation. This requires substantial thermal insulation. The superconducting magnets are the most expensive to run; the principal running costs are in liquid gases, particularly liquid helium. (b) The Probe. At the center of the magnetic field is the probe, which contains the sample cavity and the radio frequency coil arrangement for excitation and detection of the signal. Other coils are present in the magnet to generate additional small magnetic fields. Shim coils generate fields of various shapes so that the magnetic field is homogeneous or uniform over the entire sample. The homogeneity results in narrow MR lines and thus higher spectral resolution. Further narrowing of MR lines can be achieved by spinning the sample tube, which results in an averaging of field inhomogeneities. Magnetic field stability (Le., control of the strength of the magnetic field) is achieved and maintained through an electronic technique known as fieldfrequency locking. This is accomplished by the selection of a substance with a strong MR signal separate from those of the 510R
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sample (e.g., deuterium for lH NMR). This substance can be kept physically apart from the sample (external lock) or, more commonly, can be dissolved within the sample (internal lock). The frequency of the lock signal is electronically monitored and compared to that of the magnetic field, and the magnetic field is automatically and continuously adjusted, so that proportionality between field and frequency is maintained. In a typical experiment, chemical samples of volume 0.1-10 mL are placed in a narrow test tube. The test tube is then pneumatically inserted into the center of the magnet. While only one sample can be analyzed at a time, automatic sample changers are now available. Commercially available NMR spectrometers are not inexpensive: modem computer controlled, multinuclear I Tinstruments range from $200 000 to $1500 000. Technical Limitations. The power of MRS lies in the large number of analytes potentially amenable to identification and quantitication by this method. Compounds that contain the atomic nuclei hydrogen (‘H), phosphorus C’P), carbon (13C),sodium f3Na), and fluorine (19F) are most often used for clinical MRS applications. With the exception of 19F,these nuclei are essential components of organisms and are present in almost 100%natural abundance (except W ) . Each nucleus has a different frequency of absorption (the chemical shift) in the NMR spectrum. With respect to MRS analysis of biofluids, most studies are performed using lH or 31P. The lH nucleus has several significant advantages. It has the highest sensitivity of any stable nucleus, 100% natural abundance, and is found in virtually all metabolites. In general, ‘H MRS of physiological fluids is hindered by three problems: (1) relatively low analytical sensitivity; (2) the aqueous nature of physiological fluids; and (3) the fact that physiological fluids are complex mixtures, resulting in difficulty in the resolution and assignment of the large number of proton resonances. These limitations have all been largely overcome. (a) Analytical Sensitivity. MRS has a relatively low detection sensitivity when compared with current routine clinical methods such as immunoassays, gas chromatography/mass spectroscopy, or the newer molecular diagnostic techniques. However, highresolution MRS has undergone many hardware developments within the last decade. New high-field magnet technology, radio frequency electronics, and novel probe designs have resulted in increased spectral sensitivity and dispersion. The increase in detection sensitivity over the years for ethylbenzene, the compound used to evaluate spectrometers, has been impressive. Within a period of less than 35 years, the spectroscopy frequency has increased from 6 to 750 MHz, resulting in an increase in the signal/noise ratio from 6 to 12 000. Given that time-averaging of data is often used to improve signal to noise ratios, the gain of more than 1000 in detection sensitivity translates into a decrease of more than 106 in the time required to obtain a spectrum. MR systems operating at 17.6 T, 750 MHz for ‘H NMR, have recently been delivered to customers. The sensitivity now attainable approaches approximately 1 nM for ‘H NMR (2’13). Moreover, relatively simple concentration techniques such as lyophilization or Folch or perchloric acid extraction can be used to increase the sensitivity for higher molecular weight compounds or analytes in low concentration. Of greater significance is the fact that MRS possesses the potential to detect simultaneously a wide range of compounds of biological interest, irrespective of structure or physical or chemical properties, resulting in an MRS “profile” or
“fingerprint”. This is in contrast with the majority of analytical techniques currently used, which are designed to be specific for a single analyte or a particular class of analytes. The spectral profile provided by ‘H MRS can be of great utility for clinical and diagnostic purposes, because changes in the “normal”profile can readily be detected in abnormal samples without the requirement that the compound to be analyzed be preselected. Moreover, changes in analyte patterns in various physiological fluids may be linked to pathological processes. This feature of MRS, coupled with the accompanying structural information provided, has the potential to detect novel markers of disease or toxicity without first having to predict their likely outcome. (b) Aqueous Nature of Physiological Fluids. The presence of water-derived protons in biological fluids was an impediment to studying solutes in aqueous solutions. Water-derived protons are present in most body fluids at a concentration of approximately 110 M, some 105 times the concentration of the metabolite of interest. Suppression of the water signal, which would otherwise dominate the spectrum, is therefore necessary, and a variety of methods have been described. These methods include selective saturation of the water signal by either the application of a continuous or gated secondary irradiation field at the water resonance frequency, or multipulse solvent suppression; e.g., the Dante pulse sequence (Tl4); spectral selection based on TI relaxation time (Le., the water elimination Fourier transform method, WEFT) (Tl5);approaches based on the augmentation of the water TZrelaxation time by the addition of a water proton exchange reagent or a paramagnetic agent (WATR method) and attenuation of the water signal by CPMG (Carr-Purcell-Meiboom-Gill) spin-echo methods (Tl6-Tl9);or a combination of these methods (T20). Most of these solvent suppression techniques are now straightforward in modern MR spectrometers and may result in acceptable solvent suppression. In addition, the majority of data on physiological fluids were produced after the advent of these methods. Thus, while not a recent advance, a discussion has been included in this review to highlight the fact that the water resonance is not necessarily a limitation at this time. However, it must be kept in mind that these methods may still be inadequate for very dilute solutions, and thus lyophilization of the specimen followed by redissolution in 2Hz0 is a practical method of eliminating the water problem and increasing the concentration of the metabolites of interest. Data obtained in this fashion must be interpreted with caution, since this process can result in the loss of volatile or unstable compounds. (c) Physiological Fluids as Complex Mixtures. Several different methods have been applied to the analysis of MR spectra. In simple cases, the chemical shift values and intensities are automatically obtained using peak-picking routines in the frequency domain spectrum. However, physiological fluids contain a multitude of compounds, and thus an MR spectrum contains broad resonances arising from macromolecules such as proteins and lipids which may overlap resonances from species of low molecular weight. With the recent availability of higher field instrumentation, higher frequency measurements decrease peak overlaps and give more dispersed resonances. Nevertheless, resolution and identification of the resonances can be problematic. While the enormous complexity of the spectrum is indicative of the amount of biochemical information, useful, clinically relevant information is only obtained after elimination or reduction of these
broad resonances. One approach to this is convolution of the FID with a function that discriminates against broad resonances such as a sine bell convolution function. This function minimizes the influence of the broad underlying resonances due to macromolecules and enhances those due to species of low molecular weight. Thus, use of sophisticated methods facilitates spectral assignment. In the past, a variety of multidimensional techniques were available, the most useful of which for clinical chemistry is twodimensional proton-proton shift correlation (2D-COSY) spectros copy ( T l l ). In addition, solid-phase extraction chromatography with off-line NMR detection provides a simple and efficient means of separating and detecting complex mixtures of drugs and endogenous molecules (TZl).There are drawbacks to both of these techniques; the relatively long spectral accumulation times required by the former, and the destruction of the biofluid matrix of the latter. Use of spin-echo pulse sequences or physical pretreatment of the sample (T22)may be necessary to eliminate the broad resonances arising from macromolecules. Recently, two-dimensionalJ-resolved ORES) spectroscopy has been shown to be an efficient means of extracting data on low molecular weight compounds in urine and blood plasma (723,T23). Moreover, the plethora of biochemical information that is given by MR spectroscopy has led to the use of sophisticated pattern recognition methods for data compression and biochemical classification
(2-24-T28). CLINICAL APPLICATIONS OF NMR SPECTROSCOPY The analysis of physiological fluids by high-resolution MRS is a relatively recent application of the NMR phenomenon in clinical medicine. Despite the fact that MR spectra are rich in information on endogenous biochemical processes in health and disease, and that quantitation of metabolites can be readily achieved, the diffusion of MRS methods into the clinical laboratory remains slow. While the major technical limitations have been overcome, MRS of physiological fluids competes with long-established biochemical methods that are well accepted, highly automated, comparatively inexpensive, and readily available at most clinical sites. Moreover, data collection and interpretationis limited by the scarcity of MRS trained individuals able to exploit fully the wealth of information in the spectrum of a fluid, tissue, or tissue extract. A large number of physiological fluids is accessible for MRS studies in vitro. The first medical applications showing the utility of ‘H MRS analysis of complex metabolite mixtures involved the analysis of urine and serum (224,T29,T30).A variety of other fluids including cerebrospinal fluid (CSF‘), amniotic fluid, synovial fluid, sweat, aqueous humor, seminal plasma, saliva, bile, ascites fluid, and tissue extracts have since been examined. All physiological fluids are not equally available in terms of quantity and ease of availability (technical difticulties, patient benefit, patient discomfort, ethical issues). Nevertheless, samples are drawn in a variety of clinical situations where it would seem prudent to extract as much information as possible out of as small a specimen as possible, particularly in situations where a specimen is difficult to obtain. It is here where MRS can offer distinct advantages over conventional biochemical analytical techniques: MRS analysis requires a small sample volume (0.2-0.5 mL), which generally remains intact during measurement and thus can be used for subsequent assay by other techniques; the specimen generally requires no or very little pretreatment; spectra take only a few Analytical Chemistry, Vol. 67,No. 72,June 75, 7995 511R
minutes to acquire and, unlike some other screening techniques (e.g., HPLC), it is not necessary to preselect the metabolites of interest in order to detect and quantitate them. Presently, MRS has penetrated the areas of pharmacology and toxicology. Many pharmaceutical companies have implemented automated MRS screening procedures to aid the metabolic and toxicological studies of various experimental therapeutic agents. MRS has also found applications in various clinically important situations. The main areas of current development are in hereditary metabolic disorders, organ transplantation, neurological disorders, and cancer. These and other clinical applications will be discussed in the following sections. The discussions will attempt not only to highlight the recent advances but also to emphasize the advantages of MRS over conventional analysis in particular clinical situations. Hereditary Metabolic Disorders. Inborn Errors of Metabolism. Historically, one of the most successful clinical a p plications of MRS has been the detection of a wide range of inborn errors of metabolism. In these disorders, the reduction or absence of activity of a single enzyme or cofactor can have dramatic consequences for metabolism and its control. Many inherited metabolic disorders result in the accumulation of large amounts of organic intermediates, or derivatives thereof, which are produced proximal to the defective enzyme step and eventually spill into the blood and urine. 'H MRS has been used to study the urinary excretion of such compounds. The literature describing 'H, 31P,and I3C studies was extensively reviewed by us (T22) and others (T32). Most data on physiological fluids have been acquired on unmodified urine using 'H MRS. In the ensuing years, very little additional work has focused on this area. This is perhaps not surprising since this is essentially an application already suited to routine use. Research energies have been directed toward other clinical applications. However, recent investigations on unmodified urine have detected the metabolites histidine and formic acid in a patient with histidinemia (T3.2). The spectra were acquired in 15 min and did not require any pretreatment of the specimen. Similar investigations of urea cycle enzyme disorders demonstrated the presence of the diagnostic metabolites citrulline and N-acetylcitrulline in four patients with citrullinemia (2'33); argininosuccinate in three patients with argininosuccinic aciduria (deficiency of argininosuccinate lyase (T33, T34)); and orotate, uracil, and uridine in four patients with ornithine carbamoyl transferase deficiency (7'33).Other studies have essentially confirmed the detection of the diagnostic metabolites in alkaptonuria (T32),multiple acyl CoA dehydrogenase deficiency (glutaric aciduria type 11) (T35),methylmalonic aciduria (T36),and propionic aciduria (T36). Recently it was shown that deproteinizing plasma samples by centrifuging through a filter with a l@kDa molecular exclusion leads to improved quantification of metabolites, in particular those associated with 5oxoprolinuria (T37). Neonatal screening programs currently exist for a number of inborn errors of metabolism, in particular those for which treatment has been shown to prevent or ameliorate the severity of the disorder. Current test procedures routinely used include simple chemical tests to detect excessive metabolites and amino acids and various chromatographic techniques that range in purpose from the detection of abnormal amino acids to the quantitative identification of specific amino acids. For each particular disorder, a different screening test is required, followed by confirmation using yet another diagnostic test. The develop 512R
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ment of a screening program using a technology such as MRS would allow one to analyze the same blood and/or urine sample simultaneously for many markers. In fact, simultaneous measurement of such a range of components is not possible for other techniques, and this coupled with the other benefits of MRS suggests that MRS is a viable alternative to current neonatal screening programs. Moreover, there exists the possibility for the detection of some novel markers of inborn errors of metabolism, as well as insights into the underlying defects, using MRS. Organ Transplantation. High-field 'H MRS has recently been used for the rapid multicomponent analysis of urine and plasma in order to investigate the patterns of metabolic changes associated with early rejection of transplanted kidneys (T38, T39) and hearts (T40-T4.2). (a) Kidney. The assessment of renal graft dysfunction following transplantation relies on the measurement of plasma creatinine, renal biopsy, and response to therapy. Since novel markers of nephron damage, i.e., abnormal profiles of trimethylamine N-oxide (TWO), dimethylamine @MA) and dimethylglycine @MG), have been detected by MRS in urine and blood (T.2.2, T43),it seems reasonable to speculate that these or other markers may be able to detect early rejection processes requiring dialysis and/or cyclosporin toxicity or overdose. In a recent study (T38),the spectra of normal human urine showed signals for creatinine, glycine, citrate, alanine, lactate, and N-methylated metabolites in the chemical shift range of 3.1-3.3 ppm. The spectra of patients' urine collected following renal transplantation were considerably different. Compared to normal urine, the spectral pattern of urine from a patient with an immediate functioning graft showed decreased concentrations of citrate and the presence of high levels of protein. Urinary tract infection in another patient was associated with an abnormal elevation of alanine, glycine, lactate, acetate, and succinate. In a third patient, with renal tubular ischemia, elevated levels of the medullary osmolytes, DMA and myo-inositol, and glucose were observed. The spectra of urine from a patient with a nonfunctioning graft, compared to that of normal urine, was grossly distorted as a result of significant proteinuria and hematuria. Thus, profiles such as these may provide diagnostic and prognostic information. Moreover, these spectral profiles suggest that the excretion of specific renal metabolites may be associated with episodes of graft dysfunction. In this regard, a combination of parameters (e.g., TMAO, DMA, alanine, citrate, etc.) all related to creatinine concentration were recently studied. A high excretion of TMAO appeared to be associated with biopsy-confirmed acute graft rejection episodes (T38, T39). However, there was some degree of overlap, suggesting that TMAO alone may not be a reliable marker of graft dysfunction; further studies are required. Nevertheless, the results are encouraging and suggest that a combination of parameters in the proton spectra of urine could be used to improve diagnosis and management of these patients. This method could be routinely included in the evaluation of these patients with minimal inconvenience to the patient. (b) Heart Rejection of a heart transplant is similarly monitored by a combination of invasive and noninvasive techniques including repeated endomyocardial biopsy and doppler echocardiography (CDE). Recently, two parameters measured by MRS have been proposed for use in the assessment of heart transplant rejection, plasma lipoproteins and the glycosylated residues N-acetylglucosamine (NAG) and N-acetylneuraminic acid ("A)
borne by the plasma proteins. Changes in both these parameters have previously been shown to reflect inflammatory processes and immunological reactions. Measurement of the width at half-height of both the methyl and methylene resonances arising from lipoproteins in plasma (total line width, TLW) has been shown to be correlated to graft rejection (T41); increased TLW values were observed in patients with evidence of a rejection process. When a TLW cutoff value was set at 62 Hz, the sensitivity and specifcity of the test was 71 and 90%,respectively, with a positive predictive value (PPV) of 78%and a negative predictive value ( N w ) of 86%. If the TLW value was referred to a reference value (to minimize the effects of a wide dispersion of preoperative TLW values), and the ratio thereof was greater than 1.15,the accuracy of the TLW test for detection of graft rejection increased. Sensitivity and specifcity increased to 80 and 95%,respectively, with a P W of 90%and a N W of 91%. The second parameter that has been proposed as a noninvasive marker of rejection is the variation of the glycosylated residues of glycoproteins NAG and NANA In a recent experiment, proton NMR spectra of plasma were obtained. Resonances were assigned to total glycosylated residues (GRt) and to mobile NAG and NANA residues. Then GRt, NAG, and NANA were measured on the basis of area of MRS signals (T40). The variations of the GRt/ CH3 and (NAG NANA)/alanine ratios were analyzed singly, in combination with each other, and in combination with CDE, and compared to an endomyocardial biopsy. Sensitivity was greatest (68%)when either the GRt/CH3 or (NAG NANA)/alanine ratios were increased or CDE was positive; specifcity however was 51%. Specificity was greatest (95%) when the GRt/CHS or (NAG NANA)/alanine ratios were increased and CDE was positive; however, with this combination, sensitivity was decreased to approximately 20%. Thus, the optimal detection requires a combination of the MRS and CDE parameters. Together, these preliminary studies are very encouraging and suggest that analysis by proton MRS of blood plasma of heart transplant recipients might contribute significantly to the early diagnosis of acute cardiac graft rejection. The advantages of a less invasive detection method are obvious, not only from the patient’s point of view but also from a technical one. Biopsy, the “gold standard, may not be accurate, since the pattern of rejection within the myocardium is not necessarily uniform and may be asymmetric. Thus, the biopsy specimen may not be representative of the underlying pathology. The ultimate objective may be to limit the use of myocardial biopsy to confirmation of a previously detected rejection process. Neurological Disorders. The study of cerebrospinal fluid is a common aid to the differential diagnosis of neurological diseases. CSF reflects the cytological and biochemical basis of disorders of the central nervous system and analysis thereof increases the accuracy of the diagnosis. MRS allows the simultaneous quantitation of several metabolites that are not routinely measured in CSF and that would require several different analytical techniques to be assayed by conventional methods. By the combination of two-dimensional measurements, resonances have been assigned recently to 46 chemical species in CSF (T44). In addition, pattern recognition approaches and discriminant analyses that separate samples into different classes have been used in order to differentiate between normal controls and various neurological disorders (T45-T47). It has been reported that the spectra of CSF of normal controls and subjects with tumors or
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multiple sclerosis can be perfectly separated, whereas those from subjects with disk herniations can be separated approximately 90% of the time using principle component analysis (T46). In this study, spectra from various neurological disorders were compared. The spectrum of CSF from a normal subject showed signals for lactate, glucose, acetate, citrate and creatinine, the most important metabolites. In the spectrum of a case with an intramedullary mixed germ cell tumor, distinct differences were observed; glucose signals (3.2-4.0 ppm) were reduced, and new signals were apparent between 0.8 and 1.0 ppm and at 1.45, 1.97, and 2.39 ppm, identified as valine, a-alanine, and possibly putrescine and glutamine, respectively. Specimens from patients with disk herniations and multiple sclerosis differed from controls in the relative concentrations of acetate and a number of metabolites (including citrate, valine, a-alanine, acetate, creatinine, and glucose), respectively. Recent studies have addressed the issue of whether MRS of CSF can be used as an aid in the diagnosis of specific neurological disorders. Using high-resolution ‘H MRS of human post mortem CSF, and pattern recognition computer methods, partial separation of CSF from patients with Alzheimer’s disease (AD) and normal controls was achieved (T48); more formal statistical analysis suggested that citrate by itself was the best discriminator. Citrate levels were signiscantly reduced in the CSF of AD patients relative to control samples. These preliminary results are encouraging, since there are currently no antemortem diagnostic markers of AD. Novel markers of AD may aid in the early diagnosis of AD, allowing affected patients the benefits of earlier therapeutic intervention. In the CSF of patients with Huntington’s disease (HD), MRS analysis demonstrated a 60% increase in the pyruvic acid concentration as compared to controls; however, no unknown or unexpected metabolites were detected (T49).The significance of this increase in pyruvic acid is unknown. A preliminary study of CSF from 30 patients with definite or suspected multiple sclerosis (MS) showed that there were no significant differences between the levels of most metabolites as compared to controls, with the exception of acetate and formate, which were increased and decreased, respectively, in patients with MS. Moreover, in 93%of patients with actively progressing MS, an unknown singlet peak at 2.82 ppm was found; this peak did not appear in the spectrum of CSF from any of the control subjects (T50). The chemical shift suggested that the unknown is likely an N-methyl metabolite, which may form the basis for a new diagnostic test for MS. Such a test would be beneficial since diagnosis of MS currently depends largely on its clinical features. Laboratory tests including increased IgG levels, oligoclonal banding patterns on electrophoresis of CSF, increased levels of myelin basic protein, abnormal evoked potentials and lesions on MRI, and C T scans are useful only in support of the clinical diagnosis. A definitive marker would clearly be beneficial. The results obtained are promising and suggest that ‘H MRS spectroscopy of CSF may result in an analytical tool for diagnosis, treatment monitoring, and prognosis of neurological disorders. Further prospective studies are warranted. Prenatal Diagnosis. MRS of human amniotic fluid is a recent clinical application of the NMR technology. Analysis of amniotic fluid can provide information on fetal and fetomatemal physiology. Indeed, amniotic fluid lipid measurements are used to identify fetal lung maturity, amniotic fluid bilirubin measurements are used Analytical Chemistry, Vol. 67, No. 12, June 15, 1995
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to monitor the severity of erythroblastosis fetalis, and measurements of amniotic fluid a-fetoprotein and acetylcholinesterase are useful in the prediction of open neural tube defects. Conventional biochemical tests have various technical limitations, and thus MRS may be particularly well suited to the analysis of amniotic fluid. While high-resolution 'H MRS was first used to characterize amniotic fluid for a variety of components including amino acids, lactate, and glucose (T52),recent work has generally focused on two areas, namely, 31PMRS analysis of phospholipid extracts of amniotic fluid (T52-T54) and quantitation of the constituents of amniotic fluid and their clinical correlation by 'H MRS (T55T57). (a) Fetal Lung Maturity. Testing for fetal lung maturity has traditionally been done by the measurement of the lecithin/ sphingomyelin ( W S ) ratio via numerous thin-layer chromatographic techniques. Novel techniques, such as fluorescence polarization and lamellar body number counts, have been proposed recently to decrease technical difficulties and increase turnaround time. In a preliminary study, 600-MHz 'H MR spectra of untreated amniotic fluid specimens from 66 patients were analyzed. Linear discriminant analysis was performed to determine how well the peak ratios could predict the fetal maturation category, as determined by either L/S ratio or fluorescence polarization. Of 43 third-trimester fluids, 65%were placed in the correct category-immature, transitional, or mature (T55). While this is a reasonable prediction of fetal lung maturity, it lacks the sensitivity and specificity required for a clinical test. However, the metabolites measured likely do not relate specifically to pulmonary surfactant; better agreement might be anticipated when more relevant compounds such as phosphatidylcholine and phosphatidylglycerol are analyzed. 31P MR spectra of phospholipids in human amniotic fluid have been obtained recently (T52T54), but clinical correlations were not attempted. (b) Fetomaternal Complications. MRS of human amniotic fluid yields a wealth of information on chemical content and its variation with the condition of the mother (T58). To determine how concentrations of the various metabolites of amniotic fluid detected by MRS may relate to the clinical status of the fetus and/ or the mother, a number of fetomaternal complications were studied. No differences in peak intensity ratios were observed for mothers with gestational diabetes or in cases of fetal trisomy 21 where the spectra were generally normal in appearance (T55). Amniotic fluid from mothers with preeclampsia, on the other hand, showed differences in peak intensity ratios for choline, succinate and acetate. Linear discriminant analysis correctly distinguished all cases (n = 5) of open spina bifida where the MRS spectra were markedly altered: lactate, glutamate, and acetate concentrations were significantly increased. New peaks, previously not detected in normal amniotic fluid were found, and other peaks normally present were absent (T55). Resonances observed at 6-8 ppm in the MR spectrum of amniotic fluid have also been observed in the same region in MR spectra of human urine. It has been suggested that these resonances might be useful as markers for fetal renal output (T56). In addition, many low molecular weight compounds in amniotic fluid have been reported to be of clinical importance: amino acid elevations have been reported in central nervous system disorders (T59); glucose concentrations have been used in the diagnosis of intraamniotic infection (2'60);lactic acid has been associated with fetal acidosis (7'59). Thus, the ability of MRS to provide a high-resolution spectrum with the 514R
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identification of many low molecular weight constituents makes this a powerful technique for the investigation of fetal lung maturity and also conditions of suspected fetal anomalies and in various maternal disease states. Moreover, in vitro MRS studies of amniotic fluid may lay the foundation for noninvasive MRS analysis of amniotic fluid in vivo. Infertility. Recently 'H MR spectroscopic methods have been applied to the analysis of seminal fluid and its component secretions from normal and infertile human males. The spectrum of whole seminal fluid is extremely complex with many overlapping resonances, but over 120 resonances have been assigned to various metabolites using a combination of 2-D 'H MRS methods (7'61) and a one-dimensional homonuclear polarization-transfer experiment (T62). In order to investigate whether the measurement of some biochemical markers by MRS could allow the differentiation of various types of infertility, Hamamah et al. (7'63) measured peak areas of glycerylphosphorylcholine (GPC) , glycerylphosphorylethanolamine (GPE), citrate, and lactate in seminal plasma of normal and infertile males. The peak areas for GPC, citrate, and lactate in seminal plasma were smaller for all infertile subjects as compared to controls. In addition, peak area ratios for citrate/ lactate, GPC/lactate, and GPE/GPC were found to be different between infertile subjects with spermatogenic failure (nonobstructive) or obstructive azoospermia post vasectomy. With a cutoff value of 0.12, the GPE/GPC ratio was determined to have a high sensitivity (86%)and reasonable specificity (71%)in distinguishing between these two types of infertility. Other investigations have shown that metabolite patterns differ between obstructive and nonobstructive azoospermia (T61). These results provide some quantitative markers that may have clinical applications in the evaluation of infertility in men using MRS, but more detailed studies are needed. Cancer. Due to the increased incidence of cancer in recent years, the search for an easy, accurate, and noninvasive screening test for early malignancy has inspired many investigations. In 1986, a proton MRS measurement on human plasma that was quite unlike the traditional tumor markers, such as a-fetoprotein or carcinoembryonic antigen, was reported (T64). The average methyl and methylene resonance line width of the plasma 'H NMR spectrum, below 33 Hz, showed a strong correlation with the presence of cancer. This initiated a large number of similar investigations which generally found this method to be unreliable (T65- T72), since it essentially measured hyperlipidemia. A second promising possibility for cancer screening has been the detection of a novel lipoprotein band in density gradients of plasma from cancer patients (T73, T74). This band has been identified as lipoprotein(a) (T75). While these methods may not be suitable for screening for early malignancy, they may yet develop a role for use as a tumor marker in the prognosis, monitoring of treatment, and followup of patients previously diagnosed as having a malignancy, particularly if serial measurements are made and conditions under which measurements are made are standardized. Alternatively, they may be regarded as complementary methods in screening patients at risk for cancer, followup, and monitoring of treatment (T67, T76, T77). In recent years, attention has shifted away from the search for a simple screening test of plasma for malignant conditions to the assessment of whether MRS of various tissue specimens and extracts of tissue specimens can differentiate between malignant
and nonmalignant diseases. The major focus in the literature has been in cancer of breast, cervix, colon, liver, ovary, prostate, and thyroid, although investigations have looked at intracranial tumors (278, T79), endometrial carcinoma (T80),esophageal cancer (T81),lung cancer (T82),malignant melanoma (T83), and stomach cancer (T84). (a) Breast. MR spectroscopy of extracts from human breast tumors has utilized a number of nuclei. I3C NMR spectroscopy detected differences in the levels of monounsaturated and saturated fatty acids between carcinoma and noncancerous tissues (T85).The precise role of fatty acids in promoting breast carcinoma and tumor progression has not been delineated, but a potential use of this technique includes screening for changes in fatty acid composition that may predispose to development of carcinoma. The most numerous studies utilized 31PNMR spectroscopy to assess the role of phospholipid metabolites of tissue extracts. The results presently are inconclusive; some studies suggest that phospholipid metabolite levels are not useful indicators of tumor prognosis (T86), whereas others suggest the contrary (2'87, T88). Finally, proton MR spectroscopy demonstrated increased lactate content and an increased phosphocholinehaline ratio in tumor extracts, with decreased glucose and inositol, as compared to extracts of uninvolved tissue (T89). Quantitative MRS, or pattern recognition methods (vide infra), may provide even better means of correlating these findings with tumor characteristics such as grade and proliferative rate. (b) Colon. Detailed 'H MRS investigations of cancerous tissue began with colon. Early studies concentrated on the long TZvalue associated with a lipid resonance at 1.3 ppm as an indicator of metastatic potential (290). Later studies demonstrated the utility of particular peaks in the one- and two-dimensional 'H MR spectra to characterize stages of colon carcinoma (291). Recent studies confirm the utility of the method and suggest clinical screening of colorectal biopsies could provide a useful adjunct to histology for the assessment of tissue intermediate between normal and malignant (292). Detailed studies on colorectal cell lines of varying degrees of invasiveness support the earlier conclusions and indicate correlations between genetic changes and the appearance of MR resonances (293). (c) Thyroid. Early studies of thyroid lesions suggested a role for lH NMR spectroscopic analysis of thyroid tissue in characterizing normal, benign, and malignant processes from one another (294). A recent study has supported such a role, demonstrating that 'H MRS could separate thyroid neoplasms into two discrete (benign vs malignant) categories: benign follicular neoplasms, which are difficult to distinguish from their malignant counterparts by histology, produced spectra with some parameters similar to those of normal thyroid tissue, whereas malignant neoplasms produced spectra with properties in common with those of papillary, medullary, and follicular carcinomas (795). It has recently been shown, by means of consensus multivariate analysis of lH MR spectra, that benign follicular adenomas may be distinguished from carcinomas with high sensitivity and spedicity, suggesting that many surgical interventions on thyroid may be eliminated by clinical use of 'H MRS (796). (d) Cervix. 'H MR spectra of cervical biopsies allow distinction between carcinoma of the cervix and cervical dysplasia (297). Using a convenient method of specimen preparation for lH MR semiquantitative analysis, specimens could be grouped into normal, dysplastic, and invasive cancer via simple MRS parameters
(298). Multivariate analysis of these spectra in our laboratory, using methods reported in ref T96,indicates that subgrouping within the class of dysplasia is possible. It is hoped that these multivariate methods will be broadly used to extract the maximum information from the 'H MR spectra. Recently, using chemical shift imaging based on the 1.3 ppm lipid resonance in the lH MR spectrum, it has been possible to map regions of carcinoma within a cervical biopsy (Ts9). (e) Ovary. Similar studies have recently been completed on ovarian biopsies (TIOO). Malignant tissue could be distinguished from normal and benign tissue with sensitivity and specificity of 87 and 91%,respectively. Two-dimensional COSY spectra of the specimens yielded classification with sensitivity and specificity of 88 and 97%. In the two-dimensional spectra, cross-peaksindicative of cell surface fucosylation were diagnostic for malignancy. (9Liver. Extracts of diseased liver tissues, including primary and secondary tumors, and histologically normal tissue have been studied recently using both 31P and 'H MRS. The 31P MR spectrum of primary liver tumors showed an increase in phosphorylethanolamine and phosphorylcholine resonances and a decrease in glycerophosphorylethanolamineand glycerophosphorylcholiie when compared to spectra from histologically normal tissue (T101,T102).The 'H MR spectra of liver tissue show additional and complementary information. Levels of several metabolites have been shown to change significantly in tumor tissue compared with normal biopsies; citrate, alanine, lactate, taurine, and glycine are elevated,whereas creatine and threonine are decreased (T101). (g) Prostate. 31P,'H, and I3C MRS have been used in studies to differentiate between benign and malignant lesions of the prostate gland. It has been suggested that the relative levels of the phosphorylated metabolites phosphocreatine and phosphomonoesters can be used to discriminate malignant from benign tissue (T103).Other investigators were able to measure the citrate concentration in prostatic tissue using proton MRS (T104-TI06). Lower levels of citrate were not uniformly observed in cases of adenocarcinoma as compared to benign prostatic hypertrophy, in particular mixed or primarily stromal hypertrophy (T105). However, relative ratios of metabolites (citrate/lactate, citrate/ total choline, phosphocholine/total creatinine, choline/total creatine, alanine/total creatine, phosphoethanolamine/total phosphate, phosphocholine/total phosphate, and glycerophosphoethanolamine/total phosphate) were statistically different for prostate cancer specimens as compared to benign prostatic hypertrophy (2'107).It is hoped that these observations may contribute to the understanding of in vivo magnetic resonance spectra of the prostate and thus aid in the diagnosis of prostate malignancy. Monitoring of Disease Processes or Therapy. Given the potential of MRS for the diagnosis of a variety of pathological conditions, it follows that MRS would also have equal or greater value in the monitoring of either the disease processes or the response to a therapeutic regimen. (a) Inborn Errors of Metabolism. While not all inborn errors of metabolism have effective treatment, dietary restrictions have been shown to ameliorate the symptoms of some of these disorders; for example, dietary restriction of the intake of phenylalanine and branched-chain amino acids have been advocated for the treatment of phenylketonuria and branched chain ketoaciduria, respectively. These treatments require frequent and prolonged Analytical Chemistry, Vol. 67, No. 12, June 15, 1995
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monitoring of the patient’s serum, which could be done by MRS quickly and efficiently. Whether this has additional benefits over conventional methods is unclear at present; however, use of this technology may serve to provide novel markers of the disease or a greater understanding of the disease process. lH MRS has been used to study metabolic perturbations in patients with disorders of propionyl-CoA metabolism (propionic acidemia and methylmalonic aciduria) during the administration of carnitine therapy. Administration of carnitine resulted in an increase in the excretion of propionylcarnitine and acetylcamitine coincident with an improvement in clinical condition (T108). (b) Premature and Sick Infants. The investigation of sick babies is often complicated by the small sample volume that can be collected for biochemical analysis. Often insufficient sample is available to yield a complete picture of the biochemical derangement. MRS has the advantage that the technique is rapid and nondestructive yet requires a small sample to give diverse biochemical information. Conditions of clinical relevance that have been previously monitored include drug metabolism, fasting, inherited metabolic disorders, birth asphyxia, necrotizing enterocolitis, and ketosis (T109).Recently, nutrient intake in premature infants was measured using MRS. An oral dose of D20 was administered, and urine samples were analyzed for DzO by ‘H MRS. The results correlated well with the those of conventional methods, with the additional advantages of speed, accuracy, and ease of sample preparation (Tll0). Thus, MRS investigations are potentially useful in the diagnosis and monitoring of disease processes or response to treatment in sick or premature infants. (c) Transplanted Organs. As mentioned earlier, MRS has been used to investigate early rejection processes and cyclosporin toxicity or overdose in patients with transplanted hearts or kidneys, and this area of investigation shows considerable promise (T38- T42). (d) Cancer Followup. While MRS of plasma and tissue specimens has not given a specific tumor marker for diagnosis, recent work suggests that there may be suflicient differences in the spectra for the effects of therapy to be monitored. This area is worthy of immediate investigation. (e) Renal Function. MRS studies on physiological fluids have provided much information on the biochemical and toxicological effects of many compounds, as well as on endogenous biochemical processes in health and disease. The application of MRS urinalysis to the study of the effects of region-specific nephrotoxins uncovered distinct abnormal patterns of metabolites that were associated with different sites of nephrotoxic action: renal proximal tubular toxins caused glycosuria, lactic aciduria, and aminoaciduria; renal papillary toxins produced different abnormal excretions patterns in terms of both time course and composition; and renal papillary necrosis produced early increases in TMAO and DMA excretion, followed by subsequent increases in N,N-dimethylglycine, succinate, and acetate and decreases in TMAO and 2-oxoglutarate (Till). This knowledge of “novel” markers of site-specific renal damage has been applied to the diagnosis of tubular and papillary distortions in glomerulonephritis. Glomerulonephritis is characterized by an increase in the excretion of amino acid, ketone bodies, lactate, TMAO, and DMA and a decrease in the excretion of citrate and a-ketoglutarate (T212),indicating that tubular interstitial changes and isolated tubular or papillary distortions develop with the disease (T113).Moreover, these changes can develop at any stage of the disease. Thus, MRS urinalysis is 516R
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suitable for diagnosing latent tubular interstitial changes, which are not readily detected by traditional techniques and which result in a poorer prognosis for the patient. This approach would enable identification of patients at risk of rapid deterioration and would enable more aggressive treatment. Moreover, the effects of the treatment could easily be monitored using this noninvasive method. Similar studies using plasma samples from patients with chronic renal failure demonstrated markedly elevated plasma creatinine levels as well as an increase in lactate, TMAO, and DMA (TZZ,T43). The role of TMAO and DMA in the progression of renal failure has not been evaluated but may prove to be a marker of renal damage. Measurement of plasma creatinine, on the other hand, is used clinically to assess glomerular filtration rate (GFR) and thereby evaluate the progression of renal disease or nephrotoxicity. Although plasma creatinine and creatinine clearance measurements are convenient to measure, they likely do not reflect GFR in patients with renal insufficiency. The clearance of gandolinium (Gd) -diethylenetriaminepentaacetic acid (DTPA) , an approved NMR contrast agent, has recently been evaluated as a novel marker of glomerular filtration. The results of the clearance of Gd-DTPA closely approximated the clearance of technetium [99MT~] DTPA, an accurate method for determining GFR (2214). These results indicate that Gd-DTPA is a safe, nonradioactive indicator of GFR that may provide an alternative method for clinical studies of progressive renal disease. (f) Strength Recovery after Surgery. Watters et al. (2‘115) have followed the recovery of subjects from abdominal surgery and correlated the degree of success with body composition and nitrogen balance. Pivotal to the study was the estimation of total body water by ingestion of D20 and analysis of its concentration in urine by 2H MRS. SUMMARY AND CONCLUSIONS The range of problems in clinical chemistry that can be addressed by MRS is wide. The number of applications reported in the literature is growing steadily, particularly since the study of the composition of physiological fluids and tissues, and the changes thereof in disease, are well suited to study by MRS. Moreover, the major technical limitations that have impeded its progress into the clinical laboratory in the past have been addressed. Recent hardware and software developments have further improved and simpliied MRS analysis. Thus, it would be surprising if MRS of physiological fluids and tissues does not become an essential technique for clinical chemists and pathologists. In practice, three main obstacles remain to be overcome: a greater availability of instruments, a larger data base of spectral changes correlated with pathological conditions, and an enhanced supply of MR-trained individuals in the clinical environment. Ian C.P . Smith is Director General of the Institute for Biodiagnostics, National Research Council of Canada, in Winnipeg, Canada. He received B.Sc. (1961) and M.Sc. (1962) degrees in physacal chemistry from the University ofManitoba and a Ph.D. (1965) an theoretical chemistryfiom the University of Cambridge, England. A j e r postdoctoral research in biophysics at Stanford University and the Bell Laboratories, he joined the Division of Chemzstly, Natzonal Research Counczl %,Canada, Ottawa. He became the Director General of the Institute for aologacal Scaences, also in Ottawa, in 1987. His research interest is a plication of hysical techniques to the diagnosis, management, and un$rstanding ojhuman disease. Dorothea Blandford is a Scientific Associate of the Institute or Biodiagnostics, Nataonal Research Council of Canada, Winni eg. sfhe received a B.Sc. de ree (1985) om the Unavers?ty of Waterio and a Ph.D. degree (19 9 8 from the dave+y of Manatoba and Tom.leted a postdoctoral residency program an clznacal chemzstry (1993) an dnnipeg,
Manitoba. Her research interests include the a plication of both hysical and chemical techniques to the diagnosis an! management ojhuman disease, as well as the pharmacokinetics and pharmacodynamics of its treatment.
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