Magnetic resonance spectroscopy in medicine: clinical impact

Magnetic resonance spectroscopy in medicine: clinical impact

Progress in Nuclear Magnetic Resonance Spectroscopy 40 (2002) 1±34 Magnetic resonance spectroscopy in medicine: clinic...

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Progress in Nuclear Magnetic Resonance Spectroscopy 40 (2002) 1±34

Magnetic resonance spectroscopy in medicine: clinical impact Ian C.P. Smith*, Laura C. Stewart Institute for Biodiagnostics, National Research Council, 435 Ellice Ave., Winnipeg, Man., Canada R3B 1Y6 Accepted 1 July 2001

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Aims of the review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1. What techniques can be applied? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2. What parameters can be measured? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.3. What types of samples can be used? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.4. What techniques are available for automated classi®cation of spectra? . . . . . . . . . . . . . . 2. First hints of success . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Skeletal muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cardiac muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Liver and kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Bioenergetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Graft rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. Hepatitis and cirrhosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Cancer of the pelvis, thyroid and breast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Correlating spectral and biochemical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. Uterine cervix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.4. Ovary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.5. Thyroid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.6. Breast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Interesting and potentially fruitful opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Winners: prostate and brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Prostate: almost there . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Brain: already a clinical tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Metabolite levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Cancer: from biopsies to in vivo tumours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.5. Dementia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * Corresponding author. Tel.: 11-204-983-7526; fax: 11-204-984-4722. E-mail address: [email protected] (I.C.P. Smith). 0079-6565/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0079-656 5(01)00038-3

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3.2.6. Miscellaneous diseases of the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. What? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. So what? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Now what? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Keywords: NMR spectroscopy; Clinical; Disease; Breast; Prostate; Brain; Muscle; Liver; Magnetic resonance spectroscopy

1. Introduction 1.1. Aims of the review Magnetic resonance imaging (MRI) has proven to be an indispensable tool for both researcher and clinician; however, although it can detect the presence and extent of various diseases, MRI is often nonspeci®c in de®ning the underlying pathology. In many cases, diagnosis must await microscopy evaluation of tissue obtained by biopsy or autopsy. In comparison, a high degree of diagnostic speci®city can be achieved with magnetic resonance spectroscopy (MRS) because it can detect the biochemical changes that accompany speci®c diseases. MRS is the medical name for NMR spectroscopy as it applies to medicine. MRS attempts to apply the rigorous and quantitative approaches of NMR to the characterization, diagnosis and monitoring of disease and conditions in humans. This is a worthy goal. Where are we at present? Over the past decade there has been a plethora of `Look at this!' studies, a large number of con¯icting conclusions, and a disappointingly small number of studies of statistical signi®cance or medical relevance. Technical dif®culties have been many. However, in our view we have reached a critical point where the demonstrated ef®cacy of the method is leading to clinical acceptance. As stressed in a recent review [1], for MR spectroscopy or imaging to be worth the expense, inconvenience and/or risk, one or more the following assumptions must be true: (1) operative and/or post-operative management must be altered by pre-operative imaging, (2) preoperative imaging must accurately predict mortality, or (3) changes in management options based on preoperative imaging must result in an improvement in mortality or morbidity.

This article does not attempt to describe all the studies reported to date. Instead, we have completed a thorough search of the literature back to 1990, assessed the impact or potential of the reports, and have chosen to describe, in detail, those which have had, or might have, a short term impact on medicine. In addition, we have included in the references a representative sample of the studies reported during the period 1990±2000. 1.2. Methodology 1.2.1. What techniques can be applied? NMR spectroscopy can be used to provide information on a number of different sample types in a number of different ways: high resolution NMR of body ¯uids; high resolution NMR of tissue extracts; lower resolution NMR of biopsies (ex vivo); and, in vivo NMR. Recent reviews [2±4] have covered body ¯uids and tissue extracts, and these will only be alluded to as necessary. For in vivo NMR application, discussion will be focused on studies of human, rather than animal, subjects. For detailed discussions of nonspeci®c measurement of relaxation times from MR images, we refer you to reports already published [5,6]. Traditionally, one might think that 1H MRS would be the ®rst to be applied to medicine in vivo. It was, in fact, 31P MRS Ð mostly because of its much greater chemical shift dispersion. 1H MRS followed along soon after, as did 13C (enriched specimens), 19F and 23 Na. Studies of relevance will be discussed in the appropriate section. A review of medical application of ªother nucleiº by Bell et al. [7] appeared recently. Specialists in the use of NMR spectroscopy on human subjects face problems that are unknown to specialists in chemical applications. For example, ethical and subject approval must be obtained before

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Nomenclature Glossary Acq acquisition AD Alzheimer's Disease Ala alanine ANN arti®cial neural networks ATP adenosine triphosphate CDR chronic ductopenic rejection Cho choline CIN cervical intraepithelial neoplasia Cit citrate COSY correlated spectroscopy Cr creatine CrP creatine phosphate CSI chemical shift imaging FBAL ¯uoro-b-alanine FFA free fatty acid 5-FU 5-¯uoruracil Fuc fucose GABA gamma aminobutyric acid Gln glutamine Glu glutamate Gp phase encoding gradient GP genetic programming GPC glycerophosphocholine GPE glycerophosphoethanolamine Gs gradients HMQC heteronuclear multiple quantum coherence HOD hydrogen deuterium oxide HPLC high performance liquid chromatography IMCL intra-myocellular lipid ISIS image selected in vivo spectroscopy Lac lactate Leu leucine Lys lysine MAS magic angle spinning MDP methylene diphosphonate mI myoinositol MRSI magnetic resonance spectroscopic imaging MRT malignant rhabdoid tumour NAA N-acetylaspartate NDP nucleoside diphosphate NT number of transients



nucleoside triphosphate outer volume saturation phosphorylcholine perchloric acid phosphocreatine phosphodiester phosphorylethanolamine inorganic phosphate phenylketonuria phosphomonoester primitive neuroectodermal tumour frequency selective pulse taurine threonine total correlation spectroscopy uniformly labeled with 13C valine

a study can begin. This is a lengthy process, which can deter those wishing to enter the ®eld. Collaboration with hospital-based colleagues is essential. Due to variability in data, a large number of subjects must be studied in order for the conclusions to be clinically relevant. The location of the region of interest within the human subject may necessitate the construction of specialized transmitter or observe coils. Finally, the large amount of data collected leads to special challenges in data analysis. For MR spectroscopic data to be clinically useful, it is necessary to know precisely what region of the body the instrument is measuring. For the most part, this involves the use of `surface coils' [8]. These are ¯at, rigid or ¯exible coils, which can be brought close to the body and the region of interest. Early in vivo MRS studies focused on skeletal muscle, liver, and brain because of the ease of access with the surface coils. Speci®city of volume may be achieved by a variety of techniques involving variable gradients superimposed on the magnetic ®eld. Early methods were image selected in vivo spectroscopy (ISIS) [9], which involves a series of subtractions to achieve localization, and STEAM [10], PRESS [11], and DRESS [12], which involve special use of the gradients. Recently a technique for shaping the voxel to match that of the organ, SLOOP, has shown promise for minimizing


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extraneous signals [13]. More details are available in a review on single voxel proton NMR [14]. A technique which has been around for some time, but is only now coming into popularity, is spectroscopic imaging [15], also known as chemical shift imaging (CSI). This technique also uses gradients for localization, but rather than focusing on a single volume element (voxel), it obtains spectra for a number of voxels in and surrounding the region of interest. This allows one a choice of spectra to use for diagnostic purposes, and offers the option of an image based on a particular chemical shift. Corrections must be made for variation in B0 and B1 across the volume of interest. Spectroscopic imaging is experiencing a resurgence of interest, probably due to the greater detection sensitivity now available on human MR instruments, improved software for data analysis, and the facile interpretations in radiological terms of the chemical shift images rather than a large number of spectra. Another dif®culty facing spectroscopic studies in vivo is optimization of homogeneity of the applied magnetic ®eld over a volume equal to or greater than the volume of interest, especially for 1H MRS. This can be dif®cult because of the size of the volume of interest, or because of heterogeneity of environment. Examples of the latter are the lung and the thyroid gland, both of which have signi®cant air/tissue interfaces in proximity. Air and tissue have widely different properties, in particular, magnetic susceptibility, and special techniques must be employed to optimize spectral resolution [16]. For most applications of MR in medicine, detection sensitivity and spectral resolution can be improved by increasing magnetic ®eld strength. For in vivo studies, sample and tissue-dependent susceptibility effects result in line broadening, which is dif®cult to eliminate. 1H MRS measurements on spectra from both humans (1.5 and 4 T) and animals (9.4 T) have shown that increasing the magnetic ®eld strength increases the spectral resolution for all metabolite resonances, and gives more than a linear increase in sensitivity [17]. For animal studies, resolution can be improved by shimming all ®rst- and second-order shim coils using a fully adiabatic FASTMAP sequence [17]. To improve spectral resolution further in semi-solid tissue samples, the solid state technique of magic

angle spinning (MAS) has been applied to the study of brain autopsy tissue in order to obtain high resolution spectra [18±20]. It is well known that, in solids or semi-solids such as tissue, interactions such as dipole couplings and chemical shift anisotropy produce angular-dependent spectral broadening. However, if a sample is spun mechanically at the `magic angle' of 54844 0 at a rate faster than the spectral broadening, the contributions from these interactions are reduced and the spectra have a high resolution appearance. 1.2.2. What parameters can be measured? MRS experiments can be designed to provide a great deal of data. Normally, chemical shift, peak heights, peak areas, or ratios of peaks are measured. For accurate quanti®cation, relaxation times must also be determined. Sometimes, the presence of metabolic (anabolic and/or catabolic) products of a drug, such as the anti-neoplastic drug, 5-¯urouracil, can be seen. Depending on the pharmacological time course and the spectral time resolution, rates of appearance and disappearance of peaks attributed to drugs (often tagged with NMR sensitive isotopes) can be measured. 2D and higher dimensional MR methods allow a wide variety of information to be garnered, which was previously unavailable from the more elementary methods. For a detailed explanation of these multidimensional techniques, see the book by Friebolin [21]. 1.2.3. What types of samples can be used? A continuum of samples is possible Ð from extracts to cell suspensions to biopsies to in vivo Ð and there are advantages and disadvantages to each type. Chemical extracts generally provide higher resolution spectra than those from cells, biopsies or tissue. Unfortunately, the extraction procedures may result in a loss of information, introduce artifacts, and liberate chemical species which were previously immobile and unobservable. Cell suspensions, biopsies (with or without use of techniques such as MAS to increase spectral resolution) have lower resolution, but greater relevance to the clinical situation. In vivo spectra are the most clinically (and physiologically) relevant, but may be so complicated as to be indecipherable. Paired in vivo and ex vivo measurements are usually critical for a ®rm understanding of the changes seen in the 1H MR spectra from human subjects.

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Fig. 1. 31P MR spectra at 1.9 T of skeletal muscle from a patient with McArdles' syndrome. The ®rst spectrum was recorded at rest, and subsequent spectra were recorded during ischemic exercise (0±3/4 min), ischemic rest, and arterial re¯ow. The spectrum acquired at rest shows normal energetics and intracellular pH; however, spectra acquired during exercise show a precipitous decrease in CrP with concomitant increase in Pi but little change in intracellular pH. These abnormal changes in energetics and pH during exercise result from the inability to break down glycogen and generate lactic acid as a consequence of a myo-phosphorylase de®ciency. As exercise time progresses, the disability is overcome. Resonances are assigned, from left to right, as: Pi, phosphocreatine, adenosine triphosphate (ATP). The NMR pH is indicated over the Pi resonance. From: Ref. [27].

1.2.4. What techniques are available for automated classi®cation of spectra? For MRS to be used routinely by clinicians, techniques must be developed for reliable automated spectral classi®cation. The problems inherent in the design of automated systems for classifying 1H spectra were reviewed in a special issue of NMR in biomedicine by a series of authors [22]. Techniques such as pattern recognition and multivariate analysis have been developed for automatic classi®cation of 1H spectra. They go under such names as linear discriminant analysis, arti®cial neural networks (ANN), genetic algorithm, among others.

2. First hints of success 2.1. Skeletal muscle 31

P MRS in medicine was pioneered by Radda and coworkers [23], and further developed by Dawson, Gadian and Wilkie [24,25]. 31P MRS provides a quantitative assessment of muscle energetics, including creatine phosphate (CrP) and ATP, as well as intra-

cellular pH, and changes in these metabolites in the exercising human arm with normal [26] and pathological [27] muscle. Unlike muscle biopsy studies, MRS is non-invasive and measurements can be made over physiological and biochemical relevant time scales, on the order of 10 s, which allows for the assessment of not only steady state, but also transient metabolic changes. Because it is non-invasive, longer term sequential measurements can be made to assess effects of training or ef®cacy of drug therapy. For an excellent review of this topic, see Ref. [28]. A good example of the biochemical information obtainable with 31P MRS comes from early studies in Radda's lab [29]. The rate of CrP recovery after exercise, determined by 31P MRS, is an estimate of net oxidative ATP synthesis. This rate of recovery was altered in the muscles of subjects with proven mitochondrial diseases such as McArdle's syndrome (myophosphorylase de®ciency), calcium ATPase de®ciency, severe mitochondrial myopathy, hyperthyroidism, and undiagnosed fatigue. This type of analysis offers a way to quantify mitochondrial function and its abnormalities in vivo. 31 P MRS, because of its ability to measure both


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Fig. 2. (A) Series of 31P MRS at 1.5 T obtained with ISIS 1 SI of chest wall muscle and cardiac muscle. Each slice was approximately 1 cm thick. The ®nal heart spectrum was obtained by adding rows 22±25. PCr/ATP ratios are shown to the right of each spectrum. (B) Summed spectra at 1.5 T obtained on the same day (top two spectra) and eight days later (bottom spectra) showing high degree of reproducibility. From: Ref. [36].

intracellular pH and high energy phosphate levels simultaneously, has also disproven some long-standing hypotheses on muscle bioenergetics. For example, the precise mechanism of muscle fatigue during exercise remains unclear, but the long-standing hypothesis has been that fatigue occurs as a result of a rise in intracellular [H 1]. DeGroot and coworkers, using sequential, rapid (2 s) 31P MRS, have shown that [H 1] decreases in the ®rst 10 s of exercise, when force is also decreasing [30]. Furthermore, changes in levels of inorganic phosphate (Pi) and H2 PO2 4 show a closer correlation than [H 1] to changes in muscle force. Thus, acidi®cation is not likely to be

the sole or even primary mechanism of muscle fatigue but an increased level of Pi or H2 PO2 4 is a major factor. Diseases of skeletal muscle have also been studied by 31P MRS. A dramatic example of the potential of the technique is the con®rmation of McArdle's syndrome, an inherited disease in which patients have dif®culty making use of their muscles [27]. Fig. 1 shows 31P MR spectra from a human subject exhibiting muscle pain on exercise. The spectrum from resting muscle is normal, but spectra from exercising muscle shows rapid loss of CrP and concomitant increase in Pi. These changes in energetics are not

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accompanied by the expected decrease in intracellular pH. This near constant pH is consistent with a myophosphorylase de®ciency, characteristic of this disease. Note that with increasing effort, the PCr level returns to near normal values. In other words, determination can overcome the biochemical disadvantage. While 31P MRS provides information on short term energetics (high energy phosphates), 13C MRS provides information on long term energetics (glycogen) in the human muscle in vivo. 13C MRS reports on both glycogen levels and rates of glycogen metabolism during exercise and recovery under both normal and pathological conditions. Repetitive 13C MRS measurements are extremely reproducible (coef®cient of variation of ^6.5) [31]. Using this technique, the kinetics of glycogen resynthesis in human muscle, established by traditional biopsy techniques, has been reassessed and found to be much faster than previously believed: the initial rate of glycogen resynthesis during the ®rst 30 min of recovery was found to be almost four times the rate determined from biopsy samples [32]. 1 H MRS complements the data obtained by 31P MRS (high-energy phosphates, re¯ecting short term energy supply) and 13C MRS (glycogen, re¯ecting long term energy supply). Two unique applications of 1H have been reported: observation of creatine and/or CrP and observation of intra-myocellular lipids (IMCL) [33]. Multiple creatine resonances suggest that creatine is somehow motionally constrained, either in small elongated spaces between the actin/ myosin chains, or temporarily bound to a macromolecule (possibly creatine kinase) that is itself bound to ordered structures within the muscle ®bre. Lipids are stored as droplets within the muscle cells and, like glycogen, are used for long term energy storage. Both intra- and extracellular lipid can be observed separately due to different susceptibility behaviors. Extracellular lipid is concentrated (signal strongly voxel-dependent) but the IMCL signal is dispersed throughout the muscle (signal seen with creatine and water) [33]. Recovery of IMCL in fasted muscle following heavy work shows recruitment within 2 h and recovery within 40 h. Thus, 1H MRS allows for the study of this metabolite which, like glycogen, is metabolically active and available as an energy source.


2.2. Cardiac muscle Obtaining optimal high resolution 31P MR spectra of the heart in vivo is complicated by several factors including the need for gated acquisition to compensate for cardiac movement, contamination with signal from adjacent tissues (liver, skeletal muscle in chest wall) and blood in ventricles and atria, air-®lled lungs, and complicated physiological structures. Concerns persist concerning reproducibility, and the ability of the technique to diagnose transplant rejection or differentiate between normal and pathological tissue. Due to technical limitations in the surface coils used for this type of study, clinicians cannot obtain signals from the posterior sections of a heart; therefore, spectra from hearts exhibiting regional heterogeneity (i.e. due to ischemic heart disease) are dif®cult to interpret. Novel approaches to coil design, such as phased-array 31 P coils [34], may solve this problem in the future. Absolute quanti®cation of CrP and ATP is technically demanding. In principle, absolute metabolite levels can be determined by comparison with simultaneous signals from a 31P standard as well as estimates of myocardial mass based on MR imaging or by calibration of 31P signal intensity relative to tissue water proton content determined by the 1H spectrum [35]. Reproducibility and accuracy of human 31P MRS data remain a serious concern [36]. Fig. 2 shows some variability within spectra obtained from a healthy subject: CrP/ATP varied from 1.24 to 1.46 in two spectra taken on the same day and a third taken eight days later [36]. Discrepancies persist in values of CrP, ATP and CrP/ATP ratios: these have been attributed to contamination from blood and skeletal muscle and/or to under or over correction for saturation effects. There is signi®cant variability in calculated relaxation times, as well. Bottomley [35] and van Dobbenburgh et al. [37] have both reviewed the literature and reported CrP/ATP values ranging from 0.9 to 1.3 and T1 values ranging from 4.0 to 6.1 s for CrP and 1.7 to 5.8 s for the beta-phosphorus atom of ATP. Can 31P MRS differentiate between normal and pathogenic tissue? 31P MRS studies have shown alterations in levels of ATP and CrP in response to a number of factors including age [38] the presence of hypertensive heart disease and cardiac workload [39],


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Fig. 3. Proton-decoupled in vitro 31P MR spectrum, 11.7 T, of a perchloric acid extract from a normal liver. (A) full spectrum and (B) phosphomonoester and phosphodiester region. From: Taylor-Robinson et al., Gut 42 (1998) 735.

coronary artery disease [40] and heart failure [41]. In addition, 31P MRS has provided valuable information on biochemical changes in a number of pathological states. For example, Neubauer reported that 31P MRS detects reduced CrP and normal or slightly reduced ATP levels in the failing human heart [41]. These ®ndings, in combination with reduced total creatine levels and creatine kinase activity determined by biochemical assays, are consistent with the theory that alterations in highenergy phosphate metabolism contribute to the contractile dysfunction that occurs in heart failure.

However, there is not always consensus on the differences in relative metabolite levels between normal and pathogenic cardiac tissue. In dilated cardiomyopathy, biochemical studies of human and animal tissue and MRS animal studies in vivo have shown a decrease in CrP and/or ATP levels. While some in vivo MRS studies support these ®ndings, most do not [35]. Apparent reductions in CrP levels may be due to either loss of creatine during ischemia/reperfusion injury following surgery, or to the presence of edema. These discrepancies between in vitro and in

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Fig. 4. 31P CSI at 2 T of localized liver spectrum of healthy subject. From: Ref. [52].

vivo results may be attributable, at least in part, to differences in corrections for various T1 values, estimation of peak areas (integration vs curve ®tting, baseline ¯attening, etc.) and/or to contamination by surrounding tissues. A third question relates to whether or not changes in myocardial metabolite ratios, such as CrP/ATP, Pi/ ATP and PDE/ATP or PDE/CrP, might index or predict histological rejection in human heart transplants. A number of 31P MRS animal studies have shown metabolic changes that precede histological changes [41,42]. Although human studies did seem to show changes in CrP/ATP, which became more pronounced as rejection progresses [43±46], the changes occurred to different extents and at different stages of rejection. New techniques. Triglycerides are known to accumulate in ischemically insulted myocardium. Thus, NMR-observable lipid may be a marker of myocardial viability. Den Hollander and coworkers used a combination of volume selection and 2D gradient phase encoding to suppress intense lipid signals from subcutaneous adipose and epicardial lipids, but allow signal from myocardial lipid to be observed in the human heart [47]. Normal and infarcted tissue can also be differentiated using 1H MRS. Bottomley and Weiss [48] measured total creatine in spectra obtained from


patients with histories of myocardial infarction using spatially localized, water-suppressed 1H MRS. Creatine levels were signi®cantly lower in regions of infarction relative to non-infarcted regions or healthy controls. The technique was validated by comparing creatine levels determined by MRS with those determined in biopsy assays in an animal model of infarction. Using this technique, it may be possible to distinguish healthy from infarcted non-viable myocardium, a valuable adjunct in assessment of damaged heart. Current 31P MRS techniques are limited by relatively low sensitivity and less than optimal spatial resolution. A mismatch between MRS voxels and anatomical features of interest leads to voxel bleed and partial volume effects. In order to better match the sensitive volumes to the patient's anatomical structures, LoÈf¯er and coworkers have developed a 3D version of SLOOP using a clinical instrument, leading to a 30% improvement in signal-to-noise ratio relative to conventional CSI [13]. 2.3. Liver and kidney 2.3.1. Bioenergetics 31 P MRS and 13C MRS of liver extracts provide information on changes in high energy phosphate and lipid metabolism in response to disease or cancer (Fig. 3). Their in vivo equivalents may, in time, develop into useful and routinely used diagnostic techniques (Fig. 4) [49±51]. A persistent problem with the use of surface coils to visualize liver is the spectral contamination from adjacent skeletal muscle, visible as a phosphocreatine peak in the 31P MR spectra. The fact that MRS samples a relatively large area is seen as an advantage, since metabolite levels are measured over a broad tissue area, minimizing sampling errors if the changes to the liver are not uniform. Another problem with the in vivo 31P studies is the discrepancy in absolute metabolite concentrations and pH values reported in studies from various researchers. Sijens and coworkers [52] compared 31P MRS liver metabolite levels, published and re-calculated values, from ten other researchers (peak area vs Gaussian vs Lorenzian, intra- vs inter-observer variation, intra- and inter-subject variation) and found that only part of the discrepancy could be caused by differences


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Fig. 5. Image-guided localized 31P MRS spectrum, 1.5 T, of a chronically rejected human kidney transplant (left) and a kidney transplant with severe acute tubular necrosis (right). The spectrum from the rejected kidney shows increased Pi/ATP ratio and decreased pH. The spectrum from the kidney with tubular necrosis shows increased Pi to ATP ratio, decreased phosphomonoester/phosphodiester ratio, and normal pH. From: Ref. [54].

in processing and integration. They caution that T1 losses must be adequately corrected for, and that the calculated metabolite concentrations should not be claimed to be absolute. 2.3.2. Graft rejection Up to 17% of liver transplant patients subsequently develop chronic graft rejection, usually

Fig. 6. 1H MR spectra at 360 MHz of urine samples obtained from patients with normal (upper spectrum) and rejecting (lower spectrum) kidneys. Despite their apparent similarity, using an automated classi®cation strategy, spectra can be automatically assigned as normal or rejecting. From: Ref. [55].

requiring re-transplantation. If the rejection is identi®ed at an early stage, altering immunosuppressive treatment may alter the course of the rejection. This emphasizes the need for early detection. Histological examination remains the diagnostic gold standard, but it is invasive, time-consuming and subject to sampling error since changes in the liver may be heterogeneous. The ability of 31P MRS to differentiate between healthy transplants and those in early stages of rejection was investigated by Taylor-Robinson and coworkers [53]. Spectral changes associated with chronic ductopenic rejection (CDR), particularly the increase in the ratio of PME to nucleoside triphosphate (NTP) and PDE to NTP, re¯ect altered phospholipid metabolism and/or accumulation of bile phospholipids [53]. Ideally, in vivo MRS examinations should be performed serially to observe spectral changes over time, but patients are often unable (if seriously unwell or uncomfortable) or unwilling (if already released from hospital). Further studies are required to correlate in vivo hepatic 31P MRS and in vitro 31P NMR analysis of liver biopsy material, plasma and bile, and to compare these with histological studies. Although the authors caution that it is unlikely that 31P MRS may be used to assess liver post-transplant and differentiate between healthy transplants and those in early stages of CDR, it may at least be able to provide a

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better understanding of the underlying metabolic changes. Heindel and coworkers reported on 31P MRS of transplanted human kidney in situ [54]. Transplanted kidneys undergoing rejection (both acute and chronic) were found to have elevated Pi to ATP ratios and reduced pH, in comparison with kidneys from patients with no evidence of transplant dysfunction (Fig. 5). In addition, MRS is able to differentiate between kidneys undergoing transplant rejection and those exhibiting tubular necrosis on the basis of pH: 6.93 in rejecting kidneys and 7.14 in kidneys with necrosis. These two pathological states can only be distinguished clinically by histological analysis. Using only patients' urine samples, 1H MRS is able to differentiate between healthy transplants and kidneys undergoing rejection following transplantation (Fig. 6) [55]. Somorjai and coworkers used an automated three-stage classi®cation strategy based on MR spectral features that has proven to provide a reliable indicator of transplant status. Using these classi®ers, spectra of the patient's urine could be automatically assigned as normal or rejecting, as validated by parallel histological analysis of biopsy samples. Spectroscopy of urine shows great promise as an easy, routine, fast, inexpensive alternative to conventional kidney tissue biopsy. It is possible that the MR method can detect subclinical in¯ammation preceding the manifestation in histology. 2.3.3. Hepatitis and cirrhosis Clinical diagnosis of chronic hepatitis and liver cirrhosis is usually based on patient history and laboratory tests, and con®rmed by liver biopsy. However, the information obtained from a biopsy may be insuf®cient, since liver is often morphologically heterogeneous. 31 P MRS has been used to characterize normal, hepatitis-infected and cirrhotic livers [53,56,57]. Ratios of phosphomonoester (PME) to phosphodiester (PDE) peaks (PME/PDE) were found to be signi®cantly higher …p , 0:001† during the early stage of hepatitis A infection [56], re¯ecting changes in hepatocellular phospholipid metabolism. The spectral changes of human liver observed in response to acute viral hepatitis A infection are similar to those seen in the regenerating rat liver, indicating that elevated ratios of PME/PDE re¯ect an increased


hepatic cell turnover caused by the acute viral infection. Kiyono and coworkers [57] reported a decrease in the ratio of PDE/ATP in patients with chronic active hepatitis (1.13±1.21) or liver cirrhosis (0.74) in comparison with normal (1.43). Although there was no correlation between the spectra and histopathological grading (mild, moderate or severe hepatitis) or response to therapy, a reduced ratio of PDE/ ATP correlated with poor response to therapy, indicating a transition from chronic hepatitis to liver cirrhosis. Dynamic hepatic glucose-glycogen metabolism was assessed using 13C MRS [58] with 13C -enriched glucose, a single-tuned 20 cm surface coil (no 1H decoupling) and 20 min acquisitions. Glucose and glycogen signals were clearly visible at 120 and 80 ppm, and disappeared 3 h later, demonstrating that dynamic hepatic glucose±glycogen metabolism is detectable, allowing for direct diagnosis of metabolic disorders. High resolution 1H MAS [59], conventional 1D and spin-echo, and 1D J-resolved, total correlation spectroscopy (TOCSY) and 1H± 13C HMQC techniques have been used to study kidney carcinoma biopsies. Preliminary data indicate that diseased tissues have metabolic pro®les substantially different from those of healthy tissues: tumour tissues were characterized by increased lipid content. 1H MAS, especially if combined with pattern recognition algorithms, offers scope for studying intact biological samples such as tissue, and for providing rapid diagnostic information. 2.4. Cancer of the pelvis, thyroid and breast 2.4.1. Correlating spectral and biochemical characteristics MRS has found widespread application in the ®eld of cancer medicine. The concept that water-based MRI could be useful for the detection, diagnosis and subsequent clinical management of cancer was ®rst proposed more than 20 years ago [60]. Various types of information are needed for optimal management: imaging (for pre-surgical assessment); staging (determination of the local extent of disease and the presence of metastases); disease-induced biochemical changes and/or changes due to the ef®cacy of treatment. Over the past 20 years, MR has proved increasingly


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Fig. 7. (A) 1H MR spectra at 360 MHz of biopsy samples of normal colorectal mucosa and colorectal carcinoma: (a) un®ltered, (b) T2-®ltered n2t ˆ 8 ms; and (c) T2-®ltered n2t ˆ 20 ms: Based on multiple diagnostic parameters, including intensities of choline and other metabolites, cell surface fucosylation and altered lipid pro®les, some samples that were normal according to histopathology, exhibited spectral characteristics indicative of malignancy. This suggests that tissue can be identi®ed as abnormal by MRS, prior to any morphological changes. (B) Expanded methyl-methine coupling region of symmetrized COSY spectra of: (a) MRS-normal mucosa, (b) colorectal carcinoma and (c) MRSabnormal mucosa. From: Ref. [76].

valuable in locating tumours, but has not been as useful in providing information on tumour pathology. Consequently, histological assessment of ®ne needle aspirate of biopsy is still required for an unambiguous diagnosis of cancer. 1H MR spectra, whether they are obtained from tumour extracts, cells, biopsies or in vivo, exhibit many peaks that have proven useful for characterizing tumours: over 50 resonances from invasive cancers have already been documented and the painstaking process of identifying which resonances correlate with which speci®c biochemical functions (invasion, cellular proliferation, immunosuppression) is underway. Comparison of data from MRS with data from histopathological analysis enables researchers and clinicians to assemble a database that will eventually be used for independent and objective assessment of samples ex vivo, or tissues in vivo. Stone et al. [61] summarized the ®ndings of a

workshop on correlation between methods of oxygen measurement and tumour response to therapy. Results from 1H (lactate), 31P (ATP/Pi ratio) and 123I (iodoazomycin arabinoside uptake) MRS studies of brain and squamous cell carcinomas were reported, but quanti®cation of hypoxia is not currently possible with these techniques and correlation to treatment response has not yet been made. A large number of studies have been reported on the application of MRS to various cancers, particularly those of the pelvis (prostate, colon, uterine cervix, ovary), thyroid and breast. 2.4.2. Colon Although excellent data have been obtained from biopsies of colon cancer, no in vivo studies have been reported. Localization of colon tissue in vivo is a major hurdle that has yet to be overcome. Complications arise due to the tissue/air interface,

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Fig. 7. (continued)


Fig. 8. (A) Stacked plot of sequential in vivo 19F MR spectra, 1.5 T, of the liver of a patient following a bolus infusion of 5-¯uorouracil 2 h after administering methotrexate. Increases in the metabolic product of 5-FU, F-BAL, can be clearly seen. From: Ref. [83]. (B) Typical ®t of the 19F MR pharmacokinetic data. The decreasing curve is for 5-FU; the increasing curve for F-BAL. From: Ref. [83].


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thin dimension of the colon, movement of the colon due to peristalsis, and the convoluted shape of the organ. Moreno and Arus [62] compared 1H MR spectra from PCA extracts and intact biopsies from colonic tumours and normal mucosa. The extracts showed a signi®cant increase in levels of myo-inositol and taurine (as well as many other compounds) in tumours relative to normal tissue, indicating their potential as malignancy markers, particularly when 2D spectroscopic techniques are used. Low and highly tumorigenic human malignant colorectal cell lines have been shown to be distinguishable based on differences in lipid, choline and fucose resonances. Fucose was reported in 1H COSY spectra of plasma membrane fragments shed from human malignant colorectal cells [63,64], suggesting a biological role for shed fucosylated antigens in tumour aggression. 1 H MR-visible lipids have been observed in 1H MRS ex vivo spectra from control and carcinogen (azoxymethane)-treated rats [65]. Understanding the spatial location of lipids within the colon wall is of diagnostic importance since distortion of normal tissue architecture is associated with the progression of colon cancer. In cultured cells and human tissues, the presence of MR-visible lipid has been associated with biochemical changes associated with cancer: cellular stimulation, transformation, malignancy and altered tumorigenicity. The spatial location of this MR-visible lipid in tissue from normal and carcinomatous colon has been identi®ed using CSI, 1D and 2D NMR and histochemical staining of human colorectal tissue (ex vivo) [66]. In normal tissue, the neutral lipids that give rise to these MRS signals are associated with the submucosal layers, whereas in carcinomatous tissue, the signal comes from tumour stroma, necrotic and degenerate tumour cells and macrophages. Hakumaki and Kauppinen [67] recently reviewed the literature relating to narrow lipid resonances in the 1 H MR spectra of normal and cancer tissue. These resonances are associated not just with malignancy, but with necrosis, in¯ammation, and a number of more benign processes, including cell activation and proliferation. A number of studies have associated these signals with membrane lipids in lipoproteinlike structures [68,69], with lipid bodies or intra-

cellular droplets [70,71]. The factors leading to the accumulation of lipids in these structures and their contributions to the observable MRS signal are not yet known. Relaxation time studies showed that there is either no signi®cant difference between T1 of normal and adenocarcinomatous colon [72] or that the mean values of the T1 and T2 relaxation times are statistically different, but with differences too small to allow for diagnosis based on this NMR technique alone [73]. Earlier studies have correlated long T2 values of the resonance at 1.3 ppm with metastatic potential [74]. MacKinnon et al. [75] evaluated the ability of MRS to grade malignant colorectal disease. MRS showed detectable differences between 1H NMR spectra from cell cultures derived from human colorectal cells of differing malignant potential. MRS can document changes in human colorectal cellular chemistry, which correlate with increased genetic alterations, tumorigenicity and cellular de-differentiation. These ®ndings were extended to excised biopsies [76]. Multiple diagnostic parameters, including resonances from choline and other metabolites, cell surface fucosylation and altered lipid pro®les, were used to assess malignancy in histopathologically normal and carcinomatous tissue (Fig. 7A and B). Some samples that were histopathologically normal exhibited spectral characteristics indicative of malignancy, suggesting that tissue can be identi®ed as abnormal by MRS, prior to any morphological changes [76]. 19 F is one of the most receptive nuclei, but does not naturally occur in biological compounds. However, it is used in various pharmaceuticals, and its resonance can be used to locate them spatially in vivo, and to follow pharmacokinetics. This subject has been reviewed recently by Bachert [77] and McSheehy et al. [78]. Less conventional nuclei also show promise for colon cancer. A half life greater than twenty minutes for retention within metastases of the anti-neoplastic 5-FU, commonly used in the treatment of gastrointestinal malignancies, has been associated with a positive clinical response. In vivo 19F MRS studies of 5-¯uorouracil demonstrated prolonged 5-FU retention in tumours of some patients, with half-lives of 0.3± 1.3 h [79]. Unfortunately, 5-FU metabolites and catabolites were not detectable. Anabolites and catabolites of 5-FU, increased formation and retention of

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Fig. 9. In vivo 1H MR spectra at 1.5 T from a woman with dermoid cysts on both ovaries: (A) right ovary, (B) left ovary. From: Ref. [92].

metabolic products of 5-FU in the presence of a uridine phosphorylase inhibitor, and differences in the 5-FU metabolite patterns between viable and necrotic tissues have been reported, but only in high resolution 19F MRS of murine colon tumours [80±82]. An example of the pharmacokinetic use of 19F is shown in Fig. 8. The reduction of the resonance due to 5-FU, and the growth of that due to its metabolite ¯uoro-b-alanine (FBAL) in human liver are readily seen [83]. In a mouse colon cancer model, the metabolism of 5-¯uoruracil (5-FU) has been evaluated in vitro and in vivo using 19F MRS [82]. Accumulation and metabolism of 5-FU could be seen in colon tumours imbedded into the ¯anks of mice. Furthermore, treatment with a uridine phosphorylase inhibitor in addi-

tion to the 5-FU, resulted in an increase in formation and retention of ¯uorouracil nucleotides and a reduction in formation of catabolites, which correlated with an increased anti-neoplastic effect. While still in the pre-clinical stages, 19F MRS shows promise as a technique for evaluating changes in the metabolism of ¯uoropyrimidines, in the presence and absence of biochemical modulators, and for correlating improved therapeutic response with the biochemical effects within the tumour. The use of 19F MRS would facilitate the development of new and effective chemotherapy protocols by enabling real-time monitoring of drug metabolism and tumour response in vivo [82]. 31 P MRS has been used to determine phosphate metabolites in ®ve different human tumour cell lines [84]. Signi®cant differences were seen in their


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phospholipid metabolite levels. Merchant and coworkers identi®ed 31 individual phosphatic metabolites in perchloric acid extracts from 30 malignant colon tumours [85]. A number of spectral differences were found between tumour types and these differences may provide information on tumour biochemistry or on tumour type, treatment or outcome. Recently, it has been reported that 1H MRS of stool can indicate the presence of premalignant and malignant lesions of the colon [86]. Follow up with a much larger number of subjects has con®rmed this assertion with a high degree of sensitivity and speci®city using simple 1D spectra and multivariate analysis [87]. 2.4.3. Uterine cervix Despite the effectiveness of the Papanicolaou (Pap) smear test in screening for pre-malignant lesions, cervical cancer remains a signi®cant cause of cancer-related deaths. A clinical study of 159 cervical punch biopsy specimens [88] demonstrated that MR can provide an independent and objective assessment of cervical pathology and provide a database of the chemical and MR properties of human cervical epithelium. A strong lipid signal detected by 1H MRS discriminates invasive carcinoma from preinvasive tissue with a high sensitivity and speci®city. Kuesel et al. [89] reported a convenient experimental arrangement for the study of cervical biopsies, which is semi-quantitative and has accurate chemical shift referencing. Their data also showed a clear distinction between cancer and non-cancer. Chemical shift micro-imaging provides spatial maps based on biochemical differences in a tissue sample. 1H micro-imaging using lipid signals rather than water signals, can be used not only to distinguish invasive cancer from pre-invasive tissue, but also to assess the spatial distribution of invasive cancer with excellent resolution (nanolitre voxel size). Microimaging of small (6 mm 3) biopsies [90] revealed foci of malignant cells in 500 mm slices with an inplane resolution of 40 £ 160 mm. Images are based on the characteristic lipid resonance of the malignant cells. The MR intensity maps re¯ected the local distribution of malignant cells as assessed by histopathology. What is the biochemical relevance of these hyperintense regions? The signal intensity does not correlate with size or number of lipid droplets or

presence of necrotic regions. It is therefore likely that the signal is due to changes in the plasma membrane composition and/or structure. More recently, using a specially constructed transvaginal coil, spectra have been obtained in vivo which distinguish between normal tissue and squamous and adenocarcinoma [91]. A dif®culty with the use of this coil will be in obtaining adequate signal strength and ensuring that signals other than those from cervical tissue are excluded. Accurate staging of dysplasia, the early stages of transformation from normal to malignant cervical tissue, is a worthy goal which has not to date been realized. This is an area in which considerable subjectivity is involved in conventional pathological analysis. MRS of biopsies from the uterine cervix showed a discrimination between normal, dysplastic and cancerous tissue [92]. However, studies to date are confounded by the use of arbitrary categories for dysplasia (cervical intraepithelial neoplasia (CIN) 13), and the apparent overlap between these categories [92]. The MRS data were indicative of a continuous progression through dysplasia, not amenable to division into three categories. Even with advanced multivariate methods of analysis, accuracies of only 75% could be obtained from the MRS data. 2.4.4. Ovary 1 H MRS of biopsies from human ovary showed excellent potential to detect cancer tissue at an early stage [93,94]. Differences in lipid, (phospho)creatine, lysine and fucose were seen between normal or benign tissue and carcinomatous tissue. Using multivariate analysis techniques, ovarian cancer was distinguished from normal ovarian tissue with a high degree of sensitivity (100%), speci®city (95%) and accuracy (98%) [94]. However, until recently, no spectra had been obtained in vivo. Many of the dif®culties have now been overcome and respectable signal-to-noise ratio at 1.5 T has been reported [92,95] (Fig. 9). 2.4.5. Thyroid 1D 1H MRS can distinguish normal thyroid tissue from thyroid carcinoma in biopsy specimens. However, for an accurate diagnosis, it was necessary to apply sophisticated computerized consensus diagnostic techniques. With these methods, it is possible to

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Fig. 10. 1H MR spectrum of human thyroid, 3 T, taken with a multi-ring surface coil, voxel size 1.5 cm £ 1.5 cm £ 1.7 cm, centred within a thyroid nodule. Clinical diagnosis for this nodule was goitre. From: Ref. [99].

distinguish between the histological subgroups of papillary and follicular carcinomas [96]. The use of 2D 1H MRS allows for identi®cation of speci®c chemical species that have overlapping peaks

Fig. 11. 1H MR at 360 MHz of ®ndings of breast ®ne-needle biopsies from unequivocally benign versus in®ltrating carcinoma. Data are grouped on the basis of the ®nal histopathologic ®ndings in tissue specimens. From: Ref. [102].

in the 1D spectrum. 2D COSY 1H MR spectra obtained from 93 consecutive thyroid nodules ex vivo, showed an increase in the di-triglyceride lipid cross peaks in cancerous vs colloid/hyperplastic nodules (colloid/hyperplastic nodule , follicular adenoma , adenoma , carcinoma) and an increase in cross peaks attributable to cell surface fucosylation in cancerous vs benign specimens [97]. Other cross peaks were commonly seen in carcinomas (cholesterol/cholesteryl esters) and benign lesions (unassigned). Non-speci®c T1 and T2 and magnetization transfer rate constants showed some discrimination between follicular adenoma and carcinoma of the thyroid [98]. Extension of the earlier biopsy work to the thyroid gland in vivo requires a coil designed to deal with the particular location of the gland in the body. Both interior and exterior faces of the gland involve an air/tissue interface. Due to the large difference in magnetic susceptibility between tissue and air, large variations in magnetic ®eld homogeneity exist near the surface of the gland. A coil to observe spectra of the gland must therefore be capable of producing an extremely well localized voxel within the gland, with a high level of homogeneity of the static and radiofrequency ®elds. King et al. [16] recently


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Fig. 12. Localized 1H MR spectrum, 1.5 T, from a large ductal carcinoma, showing an intense signal at 3.3 ppm. From: Ref. [107].

reported a multi-ring surface coil for this purpose, with superior signal to noise and radiofrequency power characteristics. It was subsequently used to examine thyroid glands in human subjects [99]. Fig. 10 shows the spectrum from a voxel located within a thyroid nodule, diagnosed clinically as goitre. It should now be possible to accumulate a database for use of 1H MRS in vivo to diagnose thyroid abnormalities non-invasively. 2.4.6. Breast Breast cancer is the second most prevalent killer of women. The most common method of screening is X-ray mammography, which has a detection accuracy of approximately 70%. If lesions are detected, two approaches may then be followed; ®ne needle biopsy or open biopsy, the former being the less invasive. Diagnosis then follows via conventional histology. Preliminary in vitro 1H MRS studies of extracts of surgically-removed tissue showed signi®cant metabolic differences between malignant breast tissue and adjacent non-involved breast tissue [100,101], including high levels of choline-containing

compounds and increased ratios of Cho/Cr. In the spectra of invasive cancers, high levels of phosphocholine were also seen. It has been reported that 1H MRS of ®ne needle biopsies yields spectra which are signi®cantly different for normal and cancerous tissue [102]. The ratios of the areas of resonance due to choline and creatine fell into two groups, with some overlap (Fig. 11). Speci®city and sensitivity of 100 and 87% were reported, but the analysis was based on a simple comparison of ratios, with no measure of robustness. Application of advanced statistical methods, including use of the genetic algorithm to choose the most discriminating regions of the spectra, and linear discriminant analysis, led to a speci®city and sensitivity of 94 and 98%, with a much greater degree of robustness [103,104]. An extra bene®t from this sophisticated analysis is the ability to determine with high accuracy, from only a one pulse 1H MR spectrum of a biopsy of the primary tumour, the involvement of distinct lymph nodes (accuracy of 95%) and the degree of vascularization of the primary tumour (accuracy 92%).

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Fig. 13. In vitro 1H MR spectra, at 360 MHz, obtained from perchloric acid extracts of prostate biopsy samples showing: (A) benign prostatic hyperplasia (intense citrate signal; 2.5±2.7 ppm), (B) adenocarcinoma (low citrate signal); and L and C indicate resonances due to lipid and citrate, respectively. From: Ref. [118].

Single voxel 1H MR spectra of breast tumours [105±107] have been reported. Using a two-turn breast coil, Gribbestad et al. [107] identi®ed a peak at 3.3 ppm (choline-containing) present in varying intensity in the 1H MR spectra of patients with ductal or undifferentiated carcinomas, but not in the controls. They showed that Cho/Cre ratios can be measured, and that these ratios correlate with the histological diagnoses (Fig. 12). 1 H MRS with contemporary instruments allows for good spatial resolution, short acquisition times and smaller volumes of interest. Focus must be placed on the obtaining of spectra of high signalto-noise ratio in a clinically reasonable time, with a robust method of analysis. Various approaches have been reported [99,107±108]. An alternative approach to the early location of breast cancer involves the following of image intensity as a func-

tion of time after administration of paramagnetic contrast reagents [100,109,110]. This must, of course, be followed by biopsy for con®rmation. 31 P MRS of breast tissue is problematic because the relatively low concentration of phosphoruscontaining metabolites (breast tissue is predominantly water and fat) means long acquisition times and/or large volumes. Nevertheless, Hentschel and coworkers reported the development of a double-tuned breast coil that can be used for 1H MR imaging, 1H MR spectroscopy and 31P MR spectroscopy [108]. The advantage of such a coil is that 1H imaging for tumour localization and 31P spectroscopy for tumour characterization can be done without repositioning the patient or hardware. No clinical results have been reported as yet Ð data are only for phantoms.


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Fig. 14. 1H MRS at 360 MHz of prostate biopsy samples: (A) cancer and (B) benign prostatic hyperplasia. From: Ref. [120].

2.5. Interesting and potentially fruitful opportunities MRS can also be used to measure metabolites Ð both naturally occurring and the breakdown products of various labeled and unlabeled drugs. In addition to the advantage of providing invaluable clinical information, this type of analysis is rapid and inexpensive. MRS of urine has been used to assess kidney rejection and drug metabolism. The success of 1H MRS as a tool to identify kidney transplants in early stages of rejection was discussed above [55]. In another study, metabolites of a new drug, currently undergoing clinical evaluation, have been detected in urine samples. Using 1H MRS, rates of uptake and methods of metabolism and elimination of a potent HIV-1 protease inhibitor can be determined [111]. 13 C MRS has been used to trace the metabolism of [U- 13C]fructose in normal and fructose-intolerant children with the intent of using this technique as a diagnostic test for inherent abnormalities of fructose metabolism [112]. The extent of hepatic conversion of

fructose to glucose was determined by measuring 13Clabeled glucose in the blood, and was found to be signi®cantly lower in fructose-intolerant children. An impediment to more studies of this type has been the cost of the labeled material. 13 C MRS of feces has shown the presence of bene®cial bi®dobacteria in the digestive tracts of infants. Bi®dobacteria has been determined indirectly using 13 C MRS of fecal suspension, which detects the products of glucose metabolism that are unique to these bacteria: 3- 13C-glucose is metabolized primarily to 13CH3 13COOH [113]. Recently, 1H MRS of feces has been found to be predictive of the presence of neoplastic lesions of the colon (adenomas) and malignant lesions. 2D COSY yielded cross peaks between 1.3 and 4.2 ppm, currently attributed to fucose in cancer cell antigens, and distinguished neoplasia or cancer with high accuracy [86]. The much simpler 1D spectra, analyzed by multivariate analysis, yielded even more accurate values, with 100% sensitivity and 100% speci®city [87].

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Fig. 15. In vivo 1H MRS, 1.5 T, of adenocarcinoma of prostate. The spectra shown are from three voxels where cancer was demonstrated by histopathology. The resonances due to choline and the citrate anion are indicated. The article did not give a scale for the ®gure: the usual chemical shift reported for the choline N-CH3 is 3.2 ppm. From: Ref. [130]. 31

P MRS of serum from patients with acute leukemia, before and during therapy, showed an increase in resonances due to phospholipids in the responding patients, and no change in these resonances in the non-responding patients [116]. 2D TOCSY was performed on human sarcoma tissue biopsies [117]. TOCSY is similar to COSY, but it yields cross peaks to all protons coupled to a particular proton. A large number of cross peaks were identi®ed. Ratios of several cross peak volumes were found to be indicative of malignancy. The number of samples was limited (13) and a rigorous method for data analysis is required.

Fig. 16. Pulse and gradient sequences for multislice spectroscopic imaging at 1.5 T with a single spin echo. Outer volume saturation (OVS) refers to outer volume suppression by regionally selective sinc pulses used for suppression of the water resonances. The 32 gradient values used for Gp1 and Gp2 resulted in a matrix of 32 £ 32 phase encoding steps, and voxel resolution of 0.4± 0.6 cm 3. From: Ref. [132].


A fruitful approach to a problem with chemical shift imaging of hearts has been reported recently [114]. Acquisition weighted CSI, in which K-space weighting is applied during data acquisition and a different number of averages are taken per phase encoding step [115], led to improved spatial response in human heart. Signal to noise was improved due to fewer contributions from surrounding muscle (from 5.4 to 7.2 for ATP), and the PCr/ATP ratio (2.05) was considerably larger than that obtained by the conventional CSI method (1.60).

3. Winners: prostate and brain 3.1. Prostate: almost there An excellent example of an application with routine clinical potential is 1H MRS of the prostate gland. Prostate cancer affects and kills many, and is dif®cult to diagnose early. Studies of tissue extracts indicated that signi®cant differences exist between benign and malignant tissue. Normal tissue contains high amounts of citric acid, whereas adenocarcinoma contained very little [118,119] (Fig. 13). However, the content of citric acid in tissue with benign hyperplasia was variable, confounding the diagnostic utility. High resolution 1H MRS of prostate biopsies showed that cancer can be distinguished from benign disease at high levels of sensitivity and speci®city [120] (Fig. 14) including a separation of stromal and glandular benign disease, with con®rmation in a second study [121]. However, both of the above methods involve the taking of a biopsy, with concomitant trauma to the patient and sampling error. In vivo 1H studies using transrectal surface coils have demonstrated that high resolution spectra may be obtained from localized regions of the gland [122± 126], and that the ratio of intensities of various resonances may be diagnostic of disease [122]. Using an endorectal coil, it was possible to identify and quantify resonances from citrate, (phospho)choline, creatine, taurine and myoinositol, and to measure T1 and T2 values for citrate [127]. It was found that although the average citrate/choline ratio was signi®cantly lower in cancer tissue in comparison with benign prostatic hyperplasia or non-cancerous tissue, individual ratios overlapped with ratios for normal or


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Fig. 18. 1H MRS, 1.5 T, of adults with Down's Syndrome showing elevated myo-inositol and choline. NAA levels remain normal until dementia develops, at which time spectra are indistinguishable from those due to AD. From: Ref. [27].

benign hyperplasia tissue [127]. Furthermore, the levels of the polyamine, spermine, are reduced in tumour biopsy tissue (determined by high performance liquid chromatography (HPLC) and high resolution NMR) [128]. However, the spermine signals (3.0±3.3 ppm) are obscured by choline (3.2 ppm) and creatine (3.0 ppm) resonances in vivo. Most recently, MR spectroscopic imaging, MRSI, has been used to obtain chemical maps over the entire prostate gland [122,123,129±131], indicating areas where the concentration of citric acid is low and of choline is high (Figs. 15 and 17). This approach is more amenable to use by both radiologists and oncologists. It has been shown that multi-slice 1H MRSI data for prostate may be obtained using a single spin echo sequence [132]. To minimize quanti®cation errors


arising from baseline distortions caused by the outer lines from the citrate multiplet [133], van der Graaf and coworkers [134] suggested a modi®cation of the PRESS sequence using accurate pulse sequence timings. A phased array body coil was used for excitation and an endorectal coil for detection. Outer volume suppression was used to minimize signals from outside the region of interest. Fig. 16 shows the pulse and gradient sequences used. Fig. 17 shows the many voxel spectra from three planes through the prostate gland of a healthy volunteer, images of citrate within those three planes, and enlargement of the spectrum from one of the voxels. The spectrum shows the strong resonance due to citrate, and the weak resonance due to choline, expected for a healthy prostate gland. In order to establish a ®rm basis for this method, it is now necessary to perform these measurements on a large number of patients, to correlate the MRS data with that from conventional pathology, and to establish a non-subjective method of analysis. Recently, Wefer et al. [130] have compared the accuracy of MRI, MRS and sextant biopsy with respect to the diagnosis by step section histology. They concluded that MRI and MRSI have accuracy similar to that of biopsy in general, and are more accurate in the prostate apex. 3.2. Brain: already a clinical tool The application of 1H MRS to the brain has been particularly successful. The brain has a number of advantages over other tissues: it does not beat, so the signal is not degraded by motion artifacts; it ®lls virtually the entire skull, so the signal is not contaminated by other tissue types; it is large, high in fat and so is proton-rich, yields high signal to noise ratios, and voxels can be small. Recently, a book has appeared with discussion of a wide variety of protocols and procedures for brain MRS [135]. Interestingly, the concurrent use of MRI and MRS in the study of disease states in the brain has shown that there may be dissociation in space between anatomically obvious events and biochemical changes

Fig. 17. Spectroscopic imaging data, 1.5 T, for a normal human prostate, using spectra from three planes (a, 1±3). (b) is a spin echo image from Section 2 in (a) with outer volume suppression of regions outside the gland, (c) individual voxels superimposed on the anatomical MR image, (d) spectroscopic images of the citrate anion, (e) enlargement of the spectrum indicated by the white box in (c). From: Ref. [132].


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[136]. This has been con®rmed in a number of disease states including stroke, tumours, multiple sclerosis and degenerative diseases. Similarly, there may be MRS-detectable biochemical changes in the absence of any MRI-detected anatomical abnormalities. The use of MRS can thus increase the window of therapeutic opportunity by providing an early and accurate identi®cation. 3.2.1. Metabolite levels 1 H and 31P MRS has been used for the identi®cation and non-invasive assay of energetics, osmolytes, biochemical markers, and neurotransmitters associated with disorders of the brain. In particular, the presence, concentration and distribution of N-acetylaspartate (NAA), a neuronal marker, has provided an enormous amount of diagnostic information. NAA is reduced or absent in stroke, brain tumour (glioma), ischemia, degenerative disease, inborn metabolic errors and trauma. MRS has proven extremely useful in the study of neonatal neurology. Not only can it be used to identify many neurological disorders that could not otherwise be diagnosed (inborn errors of metabolism, hereditary diseases, neonatal hypoxia, cerebral palsy, neuroAIDS, dementias, stroke, epilepsies, infections and many encephalopathies), but it can also be used to identify developmental changes in normal infant brain [135]. Example of 1H MR spectra of adults with Down's Syndrome and dementia are shown in Fig. 18. To address the question of the precision of cerebral 1 H MRS measurements, a comprehensive two year study of metabolite peak area ratios and water-referenced metabolite peak areas for long echo time PRESS spectra from healthy volunteers was performed [137]. They attributed their excellent precision to the use of highly automated techniques for voxel shimming, water suppression and peak area measurements. A potential problem lies in the variation of relaxation times between different regions of the healthy brain Ð corrections may be arbitrary and prone to error [138]. The advantage of this method is that MR-visible water does not vary much in the brain; hence, the water signal can be used as a concentration reference [139]. Only a small fraction (,5%) of the tissue water in brain is MR-invisible. Thus, the use of the uncorrected water signal for calibration introduces an error

of ,10% Ð comparable to expected errors from using other calibration procedures. The quanti®cation of metabolite concentration in the brain using the 1H water signal is well reviewed by Henriksen [140]. Metabolite levels obtained from in vitro (perchloric acid extracts of biopsy samples) and in vivo spectra were not always comparable: NAA concentrations in cancerous tissue were three-fold higher in vivo than in vitro. This was attributed to partial volume effects, spectra overlap, or hydrolysis effects during extraction [138,139,141]. 3.2.2. Stroke 31 P MRS of adult stroke patients mirrored earlier ®ndings in newborns with hypoxic-ischemic disease: intracerebral pH and the Pi/ATP ratio are good predictors of clinical outcome [136]. By means of 1H MRS, increased Lac/Cr and decreased NAA/Cr were observed in cerebral edema, associated with tumours or ischemic stroke [142]. Post-treatment recovery from edema is accompanied by a normalized NAA/Cr ratio and the disappearance of lactate. T1 values are unchanged by the presence of edema, but T2 values are signi®cantly shorter (data not given) in edematous tissue and normalized with reduction of the edema [142]. This study provides a reminder that accurate quanti®cation requires careful consideration of relaxation times. 2D 1H MRS also showed differences between normal and subacutely to acutely infarcted (stroke) brains: increased lactate and decreased NAA, creatine and choline levels were seen in the infarcted areas in comparison with controls [143,144]. This metabolic pro®le is indicative of increased anaerobic glycolysis, but decreased cell density. In a comparison study of MRS, MRI and MR angiography in patients following an acute middle cerebral artery stroke, brain regions with hyperintensity in T2weighted MRI corresponded to elevated lactate and reduced NAA levels as determined by 1H MRS [145]. Lactate levels were also found to be elevated in regions peripheral to the infarcted tissue, perhaps indicating ischemic regions at risk of infarction. The same authors found that in the early stages of stroke, MRS identi®ed regions with elevated lactate but no other spectroscopic or imaging abnormality, indicating ischemic zones at risk of infarction.

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Fig. 19. Localized in vivo 1H MRS spectra, 1.5 T, from: (A) healthy control subject, and patients with (B) low grade tumour, (C) anaplastic astrocytoma and (D) glioblastoma multiforme. From: Ref. [152].

3.2.3. Cancer: from biopsies to in vivo tumours How can MRS be clinically useful? Possible areas include diagnosis (differentiate normal and cancer tissue, different cancer types, and neoplastic from non-neoplastic), design of the most favourable treatment regimens for each patient, and post-treatment monitoring.

Differentiating normal and cancer tissue. The possibility that tumours have unique metabolic ®ngerprints was supported by the results from a number of studies of extracts from cultures of various tumour cell lines. Signi®cant differences in alanine, glutamate, creatine, phosphorylcholine and threonine levels were found among tumour types [146].


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Fig. 20. 1H MR spectra at 1.5 T from two patients with bilateral thalamic glioma: patient 1, normal (A) and tumoral (B) areas; patient 2, normal (C) and tumoral (D) areas. The abnormal patterns differ from that observed in a low-grade glioma (E). From: Ref. [159].

Similar ®ngerprint patterns obtained from HR 1H MRS studies (1D and 2D) of various cancer cell cultures obtained from biopsies have lead to the assembly of a database on various cancer cell types: metabolites, metabolite ratios and T1 values. A high degree of variability in metabolite levels between cell cultures from different tumour types was reported [147]. The choline signal varies among spectra of different tumours, among tumors with similar degree of malignancy and within the same tumour. Differences in levels of aspartate, alanine, inositol, and glutamine/glutamate were found between normal cells and various tumour types. Lactate levels vary between individual spectra, but do not correlate with tumour types or degree of malignancy. This high degree of variability in metabolite levels suggests that some differences observed in vivo spectra may be attributable to secondary macroscopic structural changes (hypoxia, necrosis) and not to tumour-speci®c characteristics. Early 31P MRS studies also showed signi®cant differences between normal and cancerous tissue (various tumour types): metabolite levels were

found to be reduced by 20±70%, CrP/Pi ratio is significantly reduced and pH (7.12) is more alkaline in cancerous tissue relative to normal (pH 6.99) [148,149]. Peak area ratios, absolute metabolite levels, and T1 values for a variety of tumour types in vivo are summarized in these articles. 1 H 2D and T2 data for biopsies were used to classify tumours according to histopathological diagnosis Ð glioblastomas were distinguished from astrocytomas and normal brain, and the spectra appear to be indicative of malignant potential [149]. Using 1D 31P spectroscopic imaging, the ratios of PDE/NTP and PME/NTP were found to be higher in glioblastomas and astrocytomas than in normal brain, and ratios of Pi/NTP and CrP/NTP were higher in astrocytomas compared with glioglastomas and normal tissue [149]. In addition, the pH values of brain tumours range from alkaline to neutral. These techniques show promise for the development of non-invasive diagnosis of tumour lineage by 31P and 1H MRS; however, there is a need for a multi-site clinical trial with a rigorously controlled protocol to establish clinical criteria for diagnosis.

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In other studies of brain in vivo, differences in metabolic concentrations and their ratios, and differences in relaxation times between normal and cancerous tissue were reported: NAA and creatine levels are reduced, cholines are increased, and ratios of creatine to choline-containing compounds (Cr/Cho) and Nacetyl-aspartate to cholines (NAA/Cho) are signi®cantly reduced in all tumour types relative to control tissue [139,150]. Metabolites speci®c to astrocytomas (lactate) and meningiomas (lactate and alanine) were reported [150]. Differences in metabolite concentrations and ratios between different types of tumours (gliomas vs meningiomas vs glioblastomas vs noncerebral tumours) have also been reported [151]. Metabolite T2 values exhibit a high degree of scatter, but are not signi®cantly different between tumour type or stage. T2 scatter was attributed to variation in tissue oxygen pressure, pH, tissue structure and/or protein concentration [139]. T1 and T2 values were obtained for various metabolites in white/grey matter, cerebellum and different tumour types [150]. Once again, it was emphasized that differences in T2 values can be an important factor in calculations of metabolite concentrations. The relative concentrations of metabolites were found by single voxel 1H MRS to be dependent on whether the tissue is neoplastic or non-neoplastic, rapidly proliferating or slow growing (Fig. 19) [152] although this claim has been disputed by Dillon [153]. In addition, spectral differences were detected between some tumour types: for example, lactate signals are clearly present in gliomas with a high grade of malignancy [151]. Even different types of glioma can be differentiated based on 1H MRS metabolic pro®les. The Cr peak intensity is greater than that of the Cho peak in bilateral thalamic gliomas, whereas Cho is more intense than Cr in spectra from more common gliomas. Using 3D spectroscopic techniques, a clear response within the targeted region, and progression of disease outside the targeted region in patients with brain tumours has been observed [154±156]. Lin and coworkers proved the usefulness of single voxel 1H MRS in clinical decision making by assessing 15 patients (16 regions of interest) with lesions suggestive of primary brain tumours [157]. NAA, choline, creatine, lactate, myo-inositol levels relative to creatine were calculated before and after (mean


follow-up time 12.5 months) treatment. MRS accurately predicted the pathological nature and clinical outcome in 15 of the 16 lesions (96%), in¯uenced clinical decision making in 12 cases and altered surgery planning in 7 patients. Thus, MRS showed itself to be an important guide for clinical decisionmaking. Recently, MRS has been shown to be useful in differentiating between two pediatric tumours of the central nervous system that are radiologically indistinguishable: malignant rhabdoid tumour (MRT) and primitive neuroectodermal tumour (PNET) [158]. MRT is a rare, highly malignant tumour and PNET is the most common malignant central nervous system tumour occurring during the ®rst decade of life. Correct and early diagnosis of these two conditions is essential for appropriate therapy. Bilateral thalamic gliomas, in which a large tumour appears symmetrically in both thalami, is considered to be anatomically different from other gliomas. EsteÁve and coworkers [159] studied two patients with this condition using in vivo 1H and 31P MRS. The 1H spectra indicated increased creatine/phosphocreatine peaks in these tumours relative to normal brain tissue in the same subject, Fig. 20. The conclusion was consistent with the 31P data for one patient which showed an elevated phosphocreatine level. These data lend support to the concept of subgroups of brain gliomas. Pattern recognition and multivariate analysis. In order for MRS to be used routinely by clinicians, reliable automated classi®cation methods, which can be fully validated, need to be developed. The problems inherent in the design of automated systems for classifying 1H spectra and the progress to date was reviewed recently [160]. It was found that automated classi®cation could be achieved using a simple pattern recognition programme based on pair-wise comparisons, trained with a group of previously acquired spectra. ANN and genetic programming (GP) are automated pattern recognition techniques for evolving computer programs to solve problems. The potential of ANN analysis to differentiate brain tumours and nonneoplastic brain disorders was assessed on in vivo 1 H MR spectra from a relatively large cohort (138 subjects) [161]. The speci®city of ANN diagnosis was 98, 92, 99 and 100% for low- and high-grade


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gliomas, tubuculomas and abscesses, respectively. GP has been used to classify meningiomas and nonmeningiomas using 1H MRS of extracts [162]. The advantage of GP over the neural network method is that it uses simple combinations of a small group of metabolites (glutamine, glutamate and alanine). In vivo 1H MR spectra of a number of brain tumours have been analyzed using linear discriminant analysis. A high level of agreement with histopathology was found. However, data were not partitioned into training and test sets to demonstrate how robust the classi®cation algorithm would be towards the n 1 1th sample [163]. A number of multivariate methods have been applied to analysis of 1H MR spectra of brain biopsies [164]. Careful pre-processing of the spectra, including optimal regional selection, greatly improved correlation with histopathological analysis. Accuracy could be improved even further by applying the results of a linear discriminant analysis and an arti®cial neural net analysis through a consensus classi®er. Overall accuracies of greater than 90% were realized in distinguishing epileptic tissue from that of the meningiomas and astrocytomas. The need for training and validation data sets was stressed, in order to assess the robustness of the classi®er. Regrettably, this approach is still applied only rarely in contemporary publications. A thorough discussion of the application of spectroscopic imaging (MRSI) to human brain was presented recently [165]. HR 1H MAS MRS. Metabolite concentrations, metabolic ratios and T2 values obtained by high resolution 1 H MAS MRS have been used to differentiate normal tissue from low-grade anaplastic astrocytomas, glioblastomas or meningiomas [18]. MAS offers a number of advantages over the use of perchloric acid extracts: it preserves tissue structure and minimizes artifacts, it requires less sample preparation time, and it requires much smaller tissue samples (,50 vs 200±500 mg for extracts). Although MAS MRS has the potential to provide biochemical information inaccessible by classic histopathology techniques, and can be completed quickly and automatically, it has yet to prove itself to be clinically useful. Millis and coworkers found potentially useful differences in lipid concentrations between lipomas and liposarcomas using this method [19]. However, more patients must be studied, and a

method for robust data analysis developed, before this technique can be used in the clinic. Effect of chemo or radiation therapy. Radiation therapy plays an important role in the treatment of brain tumours. MRS shows promise as a clinical tool to optimize individual therapy and to predict both irradiation effects in tumours and adverse effects in healthy tissues. Radiation-induced changes in brain metabolites have been reported in vivo by 1H MRS (PRESS). NAA, elevated in cerebral tumours, was reduced following radiation treatment, indicative of neuronal damage [166]. Concentrations of creatine and choline-containing compounds were unaffected by the therapy. The ability of MRS to predict clinical post-surgical outcome has been demonstrated [167]. In 10 pediatric patients treated with a combination of surgery, chemotherapy and/or radiation therapy, the choline signal decreased following treatment in patients who responded to surgery, but increased or remained unchanged in patients whose condition was unresponsive to treatment. The ef®cacy of brain tumour treatments is traditionally assessed in terms of variations in tumour mass by serial MRI. In pediatric glioma, signi®cant tumour shrinkage may not always correlate with modi®cations of tumour behavior. Factors other than tumour volume are therefore needed for designing treatment protocols. Single voxel vs 3D spectroscopic MR imaging. Caution has been raised that single voxel studies [152] may have only limited clinical usefulness [153]. The reason for this is that in spite of the good separation between three selected tumour categories [152], tumours containing a mixture of diagnoses, non-neoplastic lesions and treated neoplasms may demonstrate overlap and heterogeneity in MR spectra. It was further noted that single voxel techniques generally sample regions within the contrastenhanced tumours whereas 3D spectroscopic imaging indicates that the most metabolically active portion of a tumour is at the leading edge, beyond the margin of the contrast enhancement. This was also seen in 31P spectra of tumours [168]. 3.2.4. Epilepsy Multinuclear MRS has been used to detect and localize metabolic changes associated with seizures [169]. Biochemical pro®les of epileptogenic lesions

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could help distinguish them from other types of lesions and could be useful as monitors of therapeutic ef®cacy. MRS-determinable metabolic changes include acidosis, reduced CrP and elevated Pi (and reduced CrP/Pi), elevated lactate, but essentially constant ATP. Although there are some discrepancies among human studies, they are generally consistent with previous animal studies. Reduced NAA, a common and possibly early chemical abnormality of chronically epileptogenic brain tissue, has also been detected [169]. Increased free fatty acid (FFA) has been detected by 1H MRS in the brain in epilepsy and after electroconvulsive therapy [170]. Gamma aminobutyric acid (GABA) can be detected in human brain by 1H MRS [171]. In vitro 1H MRS of PCA extracts from biopsies can quantitate creatine, NAA, glutamate and GABA content [172]. Paired in vivo and ex vivo measurements are essential for a ®rm understanding of the changes seen in the 1H spectra from patients with epilepsy. Neural network analysis [173] has been used to differentiate spectra of normal from epileptic patients, disregarding from which side of the brain the spectra were recorded. In addition, the networks could be trained to recognize whether the spectra were recorded from the ipsilateral or contralateral side of the epileptic focus, showing promise as a pre-surgical tool. 3.2.5. Dementia 1 H and 31P MRS have been used for the identi®cation and non-invasive assay of speci®c neuronal (NAA) and glial (myo-inositol) markers, energetics (including PME and PDE) and osmolytes, neurotransmitters (glutamate, GABA) associated with dementia [134]. A number of disease states associated with dementia, including Alzheimer's disease (AD) and birth injury, can be recognized from their associated spectra. One area that shows particular promise is the study of metabolic changes in patients with AD. 1H MRS in AD shows increased myo-inositol/Cr and decreased NAA/Cr. Both sets of peaks must be evaluated, since cerebral myo-inositol (mI) has been shown to be affected in other common diseases of the elderly: it is increased in renal failure, diabetes mellitus, chronic hypoxic encephalopathy and hypernatremia; and decreased in hepatic encephalopathy and hypo-


natremia [134]. Elevated levels of myo-inositol, seen in natural abundance 1H and 13C MRS, may be attributed to increased intracellular levels of mI (via increased uptake, increased anabolism or decreased catabolism) or it may be a metabolic marker for a speci®c cell type or AD-associated plaque or tangle. Increased levels of mI are consistent with abnormalities in cerebral inositol metabolism that have been observed in AD using conventional biochemical tests. In principle, 1H MRS may be used to determine: (a) whether drugs currently used for the treatment of AD result in decreased levels of mI or (b) whether mI levels can be decreased therapeutically, and whether such a decrease correlates with improved clinical outcome [134]. Some time ago, interest was shown in the use of MR of 7Li to study the distribution in brain of lithiumcontaining drugs used for the treatment of bipolar disorders [174±176]. Serial measurements indicated that Li concentrations in the brain increased markedly during manic episodes, while serum concentrations were unchanged. Little work appears to have been done on this subject recently. 3.2.6. Miscellaneous diseases of the brain 1 H MRS has detected low levels of myo-inositol in brain in hepatic encephalopathy [177]. Brain abscesses can be differentiated from tumours based on signals from acetate, succinate or amino acids (not seen in tumours) [178]. Phenylketonuria (PKU), elevated blood serum and cerebral phenylalanine levels resulting from a enzyme de®ciency, is the most common treatable disorder of amino acid metabolism in man [179]. Since the severity of neurologic complications does not correlate well with blood phenylalanine levels, a rapid, simple assay for cerebral phenylalanine levels is required. 1H MRS showed elevated cerebral phenylalanine levels (and, possibly, its metabolites) in all subjects with PKU at expected concentrations (comparable to those measured previously in post mortem samples using classical biochemical assays). The brain concentrations (determined by MRS) were signi®cantly lower than blood serum levels (determined biochemically) and there was a poor correlation between brain and blood concentrations. 1 H MAS MRS of brain tissue obtained by biopsy or autopsy yields high resolution spectra of unprocessed


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tissue of a quality comparable to those observed with liquid samples of brain extracts, but without solvent artifacts [180]. Pick's Disease, a neurodegenerative disorder accounting for 5% of dementias, can currently be diagnosed only by histopathological analysis of the postmortem brain. A correlation between neuronal loss (shown by traditional neurohistopathology) and decrease of the neuronal marker Nacetylaspartate (NAA) measured by MRS was found [180]. The effects of blood lead levels on the development of children is usually determined only through behavioral and psychological evaluations. Using 1H MRS, signi®cant alterations in brain metabolites, with a reduction in the NAA/Cr ratio in both grey and white matter, were found in a 10 year old exposed to lead by reference to his 9 year old cousin (essentially, a `matched control') [181]. MRI spectra showed no structural abnormalities. 19 F MRS has been used to characterize the elimination of ¯uvoxamine, a ¯uorinated psychotropic compound used for the treatment of obsessivecompulsive disorder. Drugs are sometimes withdrawn to alleviate speci®c side effects. Whole brain ¯uvoxamine can be detected in the micromolar range with relatively short data acquisition periods (,10 min). MRS quanti®cation of brain ¯uvoxamine and chromatographic determination of plasma ¯uvoxamine were performed following drug withdrawal [182]. The elimination half-life was found to be substantially longer (2.4 times) for the brain than for the plasma and the cerebral time-course of drug elimination correlated well with the onset of withdrawal symptoms.

4. Prognosis 4.1. What? MRS itself has shown to be a versatile technique to look at biochemical changes in organs in response to a variety of disease states. These MRS studies re¯ect a continuum from `look at this' to providing data that has positive effect on the prognosis for a speci®c clinical disorder. The impact of MRS on clinical medicine is dependent on the organ studied: some studies have proven

to be clinically relevant and useful, and some are as yet merely promising. For example, MRS has had a major impact on both the science and the diagnosis and therapeutic monitoring of the brain. The same is about to be true for MRS of the prostate gland. For other organs, MRS has completed its ®rst descriptive phase and has proved capable of identifying both normal tissue and the biochemical changes characteristic of a myriad of disease states. 4.2. So what? A technique is only truly useful in the clinical setting when it provides novel biochemical information or when it changes the outcome for the patient. This information must also be obtained at reasonable cost, and with a minimum of technical complexity. The onus is on both researchers and instrument manufacturers to address these points. In our view, the regular use of MRS in the clinic will be realized within the next ®ve years. 4.3. Now what? Improvements in coil design, localization, techniques and possibly ®eld strength should allow for better S/N and/or smaller voxels and/or better localization. Studies should involve larger numbers of subjects, and clinical trials should be performed. These experiments are best performed in teams with complementary expertise in medicine, physics and data analysis. Acknowledgement The authors gratefully acknowledge Racquel Baert for her hard work in ®nalizing this article. References [1] B.R. Stotland, E.S. Siegelman, J.B. Morris, M.L. Kochman, Colorectal Carcinoma 11 (1997) 635. [2] I.C.P. Smith, D.E. Blandford, Anal. Chem. 34 (1995) 509R. [3] I.C.P. Smith, D.E. Blandford, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, Wiley, Chichester, 2000, p. 181. [4] I.C.P. Smith, T. Bezabeh, in: I.R. Young, D.M. Grant, R.K. Harris (Eds.), Methods in Biomedical Magnetic Resonance Imaging and Spectroscopy, Wiley, Chichester, 2000, p. 891.

I.C.P. Smith, L.C. Stewart / Progress in Nuclear Magnetic Resonance Spectroscopy 40 (2002) 1±34 [5] J.D. De Certaines, O. Henrikson, A. Spisni, M. Cortsen, P.B. Ring, Magn. Reson. Imag. 11 (1993) 841. [6] O. Henrikson, J.D. De Certaines, A. Spisni, M. Cortsen, R.N. Muller, P.B. Ring, Magn. Reson. Imag. 11 (1993) 851. [7] J. Bell, E.L. Thomas, K.K. Changani, in: J.C. Lindon, G.E. Tranter, J.L. Holmes (Eds.), Encyclopedia of Spectroscopy and Spectrometry, Academic Press, New York, 2000, p. 857. [8] J.J.H. Ackerman, T.H. Grove, G.G. Wong, D.G. Gadian, G.K. Radda, Nature 283 (1980) 167. [9] R.J. Ordidge, A. Connelly, J.A.B. Lohman, J. Magn. Reson. 66 (1986) 283. [10] J. Frahm, K.-D. Merboldt, W. Hanicke, J. Magn. Reson. 72 (1987) 502. [11] P.A. Bottomley, Ann. NY Acad. Sci. 508 (1987) 333. [12] P.A. Bottomley, T.B. Foster, R.D. Darrow, J. Magn. Reson. 59 (1984) 338. [13] R. LoÈf¯er, R. Sauter, H. Kolem, A. Haase, M. von Kienlin, J. Magn. Reson. 134 (1998) 287. [14] J. Frahm, W. Hanicke, in: D.M. Grant, R.K. Harris (Eds.), Encyclopedia of Nuclear Magnetic Resonance, Wiley, New York, 1996, p. 4407. [15] T.R. Brown, B.M. Kincaid, K. Ugurbil, Proc. Natl. Acad. Sci. 79 (1982) 3623. [16] S.B. King, Evolution of methodology for obtaining 1H magnetic resonance spectra of thyroid nodules in vivo, PhD thesis, Department of Physics and Astronomy, University of Manitoba, Canada, 1999. [17] R. Gruetter, S.A. Weissdorf, V. Rajanayagan, M. Terpstra, H. Merkle, C.L. Truwit, M. Garwood, S.L. Nyberg, K. Ugurbil, J. Magn. Reson. 135 (1998) 260. [18] L.L. Cheng, I.-W. Chang, D. Louis, R.G. Gonzalez, Cancer Res. 58 (1998) 1825. [19] D. Moka, R. Vorreuther, H. Schicha, M. Spraul, E. Humpfer, M. Lipinski, P. Foxall, J.K. Nicholson, J.C. Lindon, J. Pharm. Biomed. Anal. 17 (1999) 125. [20] K. Millis, P. Weybright, N. Campbell, J.A. Fletcher, D.C. Fletcher, D.G. Cory, S. Singer, Magn. Reson. Med. 41 (1999) 257. [21] H. Friebolin, Basic One-and Two-Dimensional NMR Spectroscopy, Wiley-VCH, New York, 1998. [22] G. Hagberg, NMR Biomed. 11 (1998) 147±156. [23] D.I. Hoult, S.J.W. Busby, D.G. Gadian, G.K. Radda, R.E. Richards, P.J. Seeley, Nature 252 (1974) 285. [24] M.J. Dawson, D.G. Gadian, D.R. Wilkie, J. Physiol. 258 (1976) 82. [25] M.J. Dawson, D.G. Gadian, D.R. Wilkie, Nature 274 (1978) 861. [26] B. Chance, J.S. Leigh, B. Clark, J. Maris, J. Kent, D. Nioka, D. Smith, Proc. Natl. Acad. Sci. USA 82 (1985) 8384. [27] B.D. Ross, G.K. Radda, D.G. Gadian, G. Rocker, M. Esiri, J. Falconer-Smith, New Engl. J. Med. 304 (1981) 1338. [28] P. Cerretelli, T. Binzoni, Int. J. Sports Med. 18 (1997) S270. [29] G.L. Kemp, D.J. Taylor, G.K. Radda, NMR Biomed. 6 (1993) 66. [30] M. Degroot, B.M. Massie, M. Boska, J. Gober, R.G. Miller, M.W. Weiner, Muscle Nerve 16 (1993) 91.


[31] T.B. Price, D.L. Rothman, M. Avison, P. Buonamico, R.G. Shulman, J. Appl. Physiol. 70 (1991) 1836. [32] T.B. Price, D.L. Rothman, R. Taylor, M. Avison, G.I. Shulman, R.G. Shulman, J. Appl. Physiol. 76 (1994) 104. [33] C. Boesch, R. Kreis, Int. J. Sports Med. 18 (1997) S310. [34] C.J. Hardy, P.A. Bottomley, K.W. Rowling, P.B. Roemer, Magn. Reson Med. 28 (1992) 54. [35] P.A. Bottomley, Radiology 191 (1994) 593. [36] H.J. Lamb, J. Doornbos, J.A. den Hollander, P.R. Luyten, H.P. Beyerbacht, NMR Biomed. 9 (1996) 217. [37] J.O. van Dobbenburgh, C. Lekkerkerk, C.J.A. van Echteld, R. de Beer, NMR Biomed. 7 (1994) 218. [38] M. Okada, K. Misunami, T. Inubushi, M. Kinoshita, Magn. Reson. Med. 39 (1998) 772. [39] H.J. Lamb, H.P. Beyerbacht, A. vander Laarse, B.C. Stoel, J. Doornbos, E.E. ven der Wall, A. der Roos, Circulation 99 (1999) 2261. [40] T. Yabe, K. Misunami, T. Inubushi, M. Kinoshita, Circulation 92 (1995) 15. [41] S. Neubauer, Heart Failure Rev. 4 (1999) 269. [42] L.D. Shorr, R.T. Thompson, A.A. Driedger, T. Morell, F.N. McKenzie, M. Shkrum, C. Guiraudon, Transplant. Proc. 20 (1988) 842. [43] R.J. Herfkens, H.C. Charles, R. Negro-Volar, P. van Tright, Proc. Soc. Magn. Reson. Med. (1988) 827. [44] W.T. Evanochko, A. Bouchard, J.K. Kirklin, R.C. Bourge, D. Luney, Proc. Soc. Magn. Reson. Med. (1990) 246. [45] C.L. Wolfe, G. Caputo, W. Chew, et al., Proc. Soc. Magn. Reson. Med. 1 (1991) 574. [46] P.A. Bottomley, R.G. Weiss, C.J. Hardy, W.A. Baumgartner, Radiology 181 (1991) 67. [47] J.A. den Hollander, W.T. Evanochko, G.M. Pohost, Magn. Reson. Med. 32 (1994) 175. [48] P.A. Bottomley, R.G. Weiss, Lancet 351 (1998) 714. [49] R.A. Iles, I.J. Cox, J.D. Bell, L.M.S. Dubowitz, F. Cowan, D.J. Bryant, NMR Biomed. 3 (1990) 90. [50] R. Gruetter, I. Magnusson, D.L. Rothman, M.J. Avison, R.G. Shulman, Magn. Reson. Med. 31 (1994) 583. [51] I. Magnusson, D.L. Rothman, B. Jucker, G.W. Cline, R.G. Shulman, G.I. Shulman, Am. J. Physiol. 266 (1994) E796. [52] P.E. Sijens, P.C. Dagnelie, S. Halfwerk, P. van Dijk, K. Wicklow, M. Oudkerk, Magn. Reson. Imag. 16 (1998) 205. [53] S.D. Taylor-Robinson, E.L. Thomas, J. Sargentoni, C.D. Marcus, B.R. Davison, J.D. Bell, Biochim. Biophys. Acta 1272 (1995) 113. [54] W. Heindel, H. Kugel, F. Wenzel, D. Stippel, R. Schmidt, K. Lackner, J. Magn. Reson. Imag. 7 (1997) 858. [55] R.L. Somorjai, B. Dolenko, A. Nikulin, P. Nickerson, D. Rush, A. Shaw, M. de Glogowski, J. Rendell, R. Deslauriers, Vibrational Spectrosc. (2001) in press. [56] Y. Yamane, M. Umeda, T. O'Uchi, T. Mitsushima, K. Nakata, S. Nagataki, Dig. Dis. Sci. 39 (1994) 33. [57] K. Kiyono, A. Shibata, S. Sone, T. Watanabe, M. Oguchi, N. Shikama, T. Ichijo, K. Kiyosawa, T. Sodyama, Acta Radiol. 29 (1998) 209. [58] M. Ishihara, H. Ikehira, S. Nishikawa, T. Hashimoto, K. Yamada, F. Shishido, T. Ogino, K. Cho, S.K. Yashi, M.



[60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71]

[72] [73] [74] [75] [76] [77] [78]

[79] [80] [81]

[82] [83]

[84] [85]

I.C.P. Smith, L.C. Stewart / Progress in Nuclear Magnetic Resonance Spectroscopy 40 (2002) 1±34 Kawana, T. Matumuto, T.A. Iinuma, N. Arimizu, Y. Tateno, Am. J. Phys. Imag. 7 (1992) 32. D. Moka, R. Vorreuther, H. Schicha, M. Spraul, E. Humpfer, M. Lipinski, P.J.D. Foxall, J.K. Nicholson, J.C. Lindon, J. Pharm. Biomed. Anal. 17 (1999) 125. R. Damadian, Science 171 (1971) 1151. H.B. Stone, J.M. Brown, T.L. Phillips, R.M. Sutherland, Radiat. Res. 136 (1993) 422. A. Moreno, C. Arus, NMR Biomed. 8 (1996) 33. C.L. Lean, W.B. Mackinnon, C.E. Mountford, Magn. Reson. Med. 20 (1991) 306. C.L. Lean, W.B. Mackinnon, E.J. Delikatny, R.H. Whitehead, C.E. Mountford, Biochemistry 31 (1992) 11,095. K.M. BrieÁre, A.C. Kuesel, R.P. Bird, I.C.P. Smith, NMR Biomed. 8 (1995) 33. D. Ende, A. Rutter, P. Russell, C.E. Mountford, NMR Biomed. 9 (1996) 179. J.M. Hakumaki, R.A. Kauppinen, Trends Biol. Sci. (TIBS) 25 (2000) 357. C.E. Mountford, L.C. Wright, Trends Biol. Sci. (TIBS) 13 (1988) 172. E.J. Delikatny, C.M. Lander, T.M. Jeitner, R. Hancock, C.E. Mountford, Int. J. Cancer 65 (1996) 238. R. Callies, R.M. Sri-Pathmanathan, D.Y.P. Ferguson, K.M. Brindle, MRM 29 (1993) 546. C. ReÂmy, N. FouilheÂ, I. Barba, E. Sam-LaõÈ, H. Laahrech, M.G. Cucurella, M. Izquierdo, A. Moreno, A. Ziegler, R. Massarelli, M. DeÂcorps, Carles AruÂs, Cancer Res. 57 (1997) 407. P. Fantazzini, A. Sarra, MAGMA 4 (1996) 157. P. Kowalski, P. Skupin, J. Sowier, K.H. Okszewski, Physiol. Chem. Phys. Med. NMR 29 (1997) 51. I.C.P. Smith, E.J. Princz, J.K. Saunders, J. Can. Assoc. Radiol. 41 (1990) 32. W.B. MacKinnon, K. Huschtscha, R. Dent, C. Hancock, Int. J. Cancer 59 (1994) 248. C.L. Lean, R.C. Newland, D.A. Ende, E.L. Bokey, I.C.P. Smith, Magn. Reson. Med. 30 (1993) 525. P. Bachert, Prog. NMR Spectrosc. 33 (1998) 1. P.M.J. McSheehy, L.P. Lemaire, J.R. Grif®ths, in: D.M. Grant, R.K. Harris (Eds.), Encyclopedia of Nuclear Magnetic Resonance, vol. 2, Wiley, New York, 1996, p. 2048. C.H. Blesing, D.J. Kerr, J. Drug, Targeting 3 (1996) 341. Y.J.K. Kamm, A. Heerschap, G. Rosenbusch, I.M.C.M. Rietjens, J. Vervoort, D.J.T. Wagener, Magn. Res. Med. 36 (1996) 445. A.W. Blackstock, L. Kwock, C. Branch, E.M. Zeman, J.E. Tepper, Int. J. Radiat. Oncol. Biol. Phys. 36 (1996) 641. S.K. Holland, A.M. Bergman, Y. Zhao, E.R. Adams, Magn. Res. Med. 38 (1997) 907. P. Mohankrishnan, L. Hutchins, S. Nauke, J. Sprigg, D. Cardwell, M.R. Williamson, R.A. Komoroski, N.R. Jagannathan, Curr. Sci. 76 (1999) 677. S.E. Franks, A.C. Kuesel, N.W. Lutz, W.E. Hull, Anticancer Res. 16 (1996) 1365. T.E. Merchant, P.M. Diamantis, G. Lauwers, T. Haida, J.N.

[86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103]


[105] [106] [107]

Kasimos, J. Guillem, T. Glonek, B.D. Minsky, Cancer 76 (1995) 1715. T. Bezabeh, B. Levin, C. Johnson, I.C.P. Smith, Proc. Int. Soc. Magn. Reson. Med. 1 (1999) 267. T. Bezabeh, B, Levin, C. Johnson, I.C.P. Smith, C. Bernstein, patent pending. E.J. Delikatny, P. Russell, J.C. Hunter, R. Hancock, K. Atkinson, C. van Haaften-Day, C.E. Mountford, Radiology 188 (1993) 791. A.C. Kuesel, T. Kroft, J.K. Saunders, M. PreÂfontaine, N. Mikhael, I.C.P. Smith, Magn. Red. Med. 27 (1992) 349. B. Kunnecke, E.J. Delikatny, R.P. Russell, J.C. Hunter, C.E. Mountford, J. Magn. Reson. Ser. B 104 (1994) 135. J.H. Lee, K.S. Cho, Y.-M. Kin, S.-T. Kim, C.-W. Mun, J.-H. Na, J.-E. Mok, T.-H. Lim, Am. J. Radiol. 170 (1998) 1279. L.J. Friesen, Magnetic Resonance Imaging and Spectroscopy in the Female Pelvis, PhD Thesis, University of Manitoba, Canada, 1999. W.B. Mackinnon, P. Russell, G.L. May, C.E. Mountford, Int. J. Gynecol. Cancer 5 (1995) 211. J.C. Wallace, G.P. Raaphorst, R.L. Somorjai, C.E. Ng, M. Fung Kee Fung, M. Senterman, I.C.P. Smith, Magn. Reson. Med. 38 (1997) 569. L.J. Friesen-Waldner, I.C.P. Smith, unpublished results. R.L. Somorjai, A.E. Nikulin, N. Pizzi, D. Jackson, G. Scarth, B. Dolenko, H. Gordon, P. Russell, C.L. Lean, L. Delbridge, C.E. Mountford, I.C.P. Smith, Magn. Reson. Med. 33 (1995) 257. W.B. Mackinnon, L. Delbridge, P. Russell, C.L. Lean, G.L. May, S. Doran, S. Dowd, C.E. Mountford, World J. Surg. 20 (1996) 847. C. Callicott, A.W. Goode, Phys. Med. Biol 43 (1998) 627. S.B. King, L.N. Ryner, B. Tomanek, J.C. Sharp, I.C.P. Smith, Magn. Reson. Med. 42 (1999) 655. I.S. Gribbestad, S.B. Petersen, H.E. FjoÈsne, S. Kvinnsland, J. Krane, NMR Biomed. 7 (1994) 181. I.S. Gribbestad, B. Sitter, S. Lundgren, J. Krane, D. Axelson, Anticancer Res. 19 (1999) 1737. W.B. MacKinnon, P.A. Barry, P.L. Malycha, D.J. Gillette, P. Russell, C.L. Lean, S.T. Doran, B.H. Barroclough, M. Bilous, C.E. Mountford, Radiology 204 (1997) 661. C. Mountford, R. Somorjai, L. Gluch, P. Malycha, C. Lean, P. Russell, M. Bilous, B. Barraclough, D. Gillett, U. Himmelreich, B. Dolenko, A. Nikulink, I. Smith, Proc. Soc. Magn. Reson. Medicine Workshop on MR of Cancer, Geiranger, Norway, 2000. C. Mountford, R. Somorjai, L. Gluch, P. Malycha, C. Lean, P. Russell, M. Bilous, B. Barraclough, D. Gillett, U. Himmelreich, B. Dolenko, A. Nikulink, I. Smith, Brit. J. Surg. 88 (2001) 1234±1240. N.R. Jagannathan, M. Singh, V. Govindaraju, P. Raghunathan, O. Coshic, P.K. Julka, G.K. Rath, NMR Biomed. 11 (1998) 414. N.R. Jagannathan, M. Kumar, P. Raghunathan, O. Coshic, P.K. Julka, G.K. Rath, Curr. Sci. 76 (1999) 777. I.S. Gribbestad, T.E. Singstad, G. Nilsen, H.E. FjoÈsne, T. Engan, O.A. Haugen, P.A. Rinck, J. Magn. Reson. Imag. 8 (1998) 1191.

I.C.P. Smith, L.C. Stewart / Progress in Nuclear Magnetic Resonance Spectroscopy 40 (2002) 1±34 [108] M. Hentschel, J. Oellinger, C. Siewert, H. Wieder, N. Hosten, O. Wendt, T. Luth, U. Boenick, R. Felix, Biomed. Technik 44 (1999) 272. [109] G.P. Liney, P. Gibbs, C. Hayes, M.O. Leach, L.W. Turnbull, J. Magn. Reson. Imag. 10 (1999) 945. [110] H. Degani, V. Gusis, D. Weinstein, S. Fields, S. Strano, Nature Med. 3 (1997) 780. [111] S.K. Balani, B.H. Arison, L. Mathai, L.R. Kauffman, R.R. Miller, R.A. Stearns, I.-W. Chen, J.H. Lin, Drug Metabol. Disposition 23 (1995) 266. [112] A. Gopher, N. Vaisan, H. Mandel, A. Lapidot, Proc. Natl. Acad. Sci. USA 87 (1990) 5449. [113] M.J. Wolin, Y. Zhang, S. Bank, S. Yerry, T.L. Miller, J. Nutr. 128 (1998) 91. [114] R. Pohmann, M. von Kienlin, Magn. Reson. Med. 45 (2001) 817. [115] S.L. Ponder, D.B. Twieg, J. Magn. Reson. B 104 (1994) 85. [116] M. Kuliszkiewicz-Janus, S. Baczynski, Biochim. Biophys. Acta 1360 (1997) 71. [117] B. Jayashree, S. Deshmukh, T. Rajkumar, Curr. Sci. 77 (1999) 587. [118] M.L. Schiebler, K.K. Miyamoto, M. White, S.J. Maygarden, J.L. Mohler, Magn. Reson. Med. 29 (1993) 285. [119] A. Heerschap, G.J. Jager, M. van der Graaf, J.O. Barentsz, S.H.J. Ruijs, Magn. Reson. Med. 37 (1997) 204. [120] P. Hahn, I.C.P. Smith, L. Leboldus, C. Littman, R. Somorjai, T. Bezabeh, Cancer Research 57 (1997) 3398. [121] D. Ende, A. Rutter, P. Russell, C.E. Mountford, NMR Biomed. 9 (1996) 179. [122] J. Kurhanewicz, A. Thomas, P. Jajodia, B. Weiher, T.L. James, D.B. Vigneron, P. Narayan, Magn. Reson. Med. 22 (1991) 404. [123] J. Kurhanewicz, D.B. Vigneron, S.J. Nelson, H. Hricak, J.M. McDonald, B. Konety, B.P. Narayan, Urology 45 (1995) 459. [124] F. Schick, H. Bongers, S. Kurz, W.-I. Jung, M. Pfeffer, O. Lutz, P. Narayan, J. Kurhanewicz, The Prostate Suppl. 4 (1992) 43. [125] G.P. Liney, L.W. Turnbull, M. Lowry, L.S. Turnbull, A.J. Knowles, A. Horsman, Magn. Reson. Imag. 15 (1997) 1177. [126] G.P. Liney, L.W. Turnbull, A.J. Knowles, NMR Biomed. 12 (1999) 39. [127] A. Heerschap, G.J. Jager, M. van der Graaf, J.O. Barentsz, J.J.C.C.H. de la Rosette, G.O.N. Oosterhof, E.T.G. Ruijter, S.H.J. Ruijs, Anticancer Res. 17 (1997) 1455. [128] J.M. Garcia-Segura, M. Sanchez-Chapado, C. Ibarburen, J. Viano, J.C. Angulo, J. Gonzalez, J.M. Rodriguez-Vallejo, Magn. Reson. Imag. 17 (1999) 755. [129] M. van der Graaf, R.G. Schipper, G.O.N. Oosterhof, J.A. Schalken, A.A.J. Verhofstad, A. Heerschap, Magn. Reson. Mater. Phys. Biol. Med. 10 (2000) 153. [130] A.E. Wefer, H. Hricak, D.B. Vigneron, F.V. Coakley, Y. Lu, J. Wefer, U. Mueller-Lisse, P.R. Carroll, J. Kurhanewicz, J. Urol. 164 (2000) 400. [131] J. Kurhanewicz, D.B. Vigneron, S.J. Nelson, Neoplasia 2 (2000) 166.


[132] M. van der Graaf, H.I. van den Boogert, G.J. Jager, J.O. Barentsz, A. Heerschap, Radiology 213 (1999) 919. [133] A.H. Wilman, P.S. Allen, J. Magn. Res. 107 (1995) 25. [134] M. van der Graaf, G.J. Jager, A. Heerschap, MAGMA 5 (1997) 65. [135] E.R. Danielsen, B.D. Ross, Magnetic Resonance Spectroscopy Diagnosis of Neurological Diseases, Marcel Dekker, New York, 1999. [136] B.D. Ross, S. Blume, R. Cowan, E. Danielsen, N. Farrow, R. Gruetter, Biophys. Chem. 68 (1997) 161. [137] A. Simmons, M. Smail, E. Moore, S.C.R. Williams, Magn. Reson. Imag. 16 (1998) 319. [138] J. Frahm, H. Bruhn, M.L. Gyngell, K.D. Merboldt, W. Hanicke, R. Sauter, Magn. Reson. Med. 111 (1989) 47. [139] P. Christiansen, P.B. Toft, P. Gideon, G.E.R. Danielsen, P. Ring, O. Henriksen, Magn. Reson. Imag. 12 (1994) 1237. [140] O. Henriksen, NMR Biomed. 8 (1995) 139±148. [141] J.-P. Usenius, R.A. Kauppinen, P.A. Vainio, J.A. Hernesniemi, M.P. Vapalahti, L.A. Paljarvi, S. Soimakallio, J. Computer, Assist. Tomogr. 18 (1994) 705. [142] K. Kamada, K. Houkin, Y. Iwasaki, H. Abe, T. Kashiwaba, Neurol. Med. Chir. (Tokyo) 34 (1994) 676. [143] J.H. Duijn, G.B. Matson, A.A. Maudsley, J.W. Hugg, M.W. Weiner, Radiology 183 (1990) 711. [144] W. Heindel, H. Kugel, H. Lanfermann, P. Landevehr, T. Krahe, K. Lackner, Nervenarz 66 (1995) 895. [145] J.H. Gillard, P.B. Barket, P.C.M. van Zijl, R.N. Bryan, S.M. Oppenheimer, Am. J. Neuroradiol. 17 (1996) 873. [146] C.L. Florian, N.R. Preece, K.K. Bhakoo, S.R. Williams, M. Nobel, NMR Biomed. 8 (1996) 253. [147] K. Kotitschke, H. Jung, S. Nekolla, A. Haase, A. Bauer, U. Bogdahn, NMR Biomed. 7 (1994) 111. [148] B. Hubesch, D. Sappey-Marinier, K. Roth, D.J. Meyerhoff, G.B. Matson, M.W. Weiner, Radiology 174 (1990) 401. [149] A. Rutter, H. Hugenholtz, J.K. Saunders, I.C.P. Smith, J. Neurochem. 64 (1995) 1655. [150] D.J. Manton, M. Lowry, S.J. Blackband, A. Horsman, NMR Biomed. 8 (1995) 104. [151] H. Kugel, W. Heindel, R.-I. Ernestus, J. Bunke, R. du Mesmil, G. Friedmann, Radiology 183 (1990) 701. [152] M.E. Meyerand, J.M. Pipas, A. Mamourian, T.D. Tosteson, J.F. Dunn, Am. J. Neuroradiol. 20 (1999) 117. [153] W.P. Dillon, S. Nelson, Am. J. Neuroradiol. (Ed.) 20 (1998) 2. [154] E.E. Graves, S.J. Nelson, D.B. Vigneron, L. Verhey, M. McDermott, D. Larson, S. Chang, M.D. Prados, W.P. Dillon, Am. J. Neuroradiol. 22 (2001) 613. [155] S.J. Nelson, S. Huhn, D.B. Vigneron, M.R. Day, L.L. Wald, M. Prados, S. Chang, P.H. Gutin, P.K. Sneed, L. Verhey, R.A. Hawkins, W.P. Dillon, J. Magn. Res. Imag. 76 (1997) 1146. [156] L.L. Wald, S.J. Nelson, M.R. Day, et al., J. Neurosurg. 87 (1997) 525. [157] A. Lin, S. Biumi, A.N. Mamelak, J. Neuro-Oncol. 45 (1999) 69. [158] T.A.G.M. Huisman, S. Brandner, F. Niggli, D.R. Betts, E. Biotshauser, E. Martin, Neuropediatrics 31 (2000) 159. [159] F. EsteÁve, S. Grand, C. Rubin, D. Hoffmann, B. Pasquier, D.



[161] [162] [163] [164]

[165] [166] [167] [168] [169] [170] [171]

I.C.P. Smith, L.C. Stewart / Progress in Nuclear Magnetic Resonance Spectroscopy 40 (2002) 1±34 Graveron-Demilly, R. Mahdjoub, J.-F. Le Bas, Am. J. Neuroradiol. 20 (1999) 876. A.R. Tate, J.R. Grif®ths, I. Martinez-Perez, A. Moreno, I. Barba, M.E. Cabanas, D. Watson, J. Alonso, F. Bartumeus, F. Isamat, I. Ferrer, F. Vila, E. Ferrer, A. Copdevlia, C. Arus, NMR Biomed. 11 (1998) 177. H. Poptani, J. Kaartinen, R.K. Gupta, M. Neimitz, Y. Hiltunen, R.A. Kauppinen, J. Cancer, Res. Clin. Oncol. 125 (1999) 345. H.F. Gray, R.J. Maxwell, I. Martinez-Perez, C. Arus, S. Cerdan, NMR Biomed. 11 (1998) 217. M.C. Preul, A. Caramanos, R. Leblanc, J.G. Villemure, D.L. Arnold, NMR Biomed. 11 (1998) 192. R.L. Somorjai, B. Dolenko, A.K. Nikulin, N. Pizzi, G. Scarth, P. Zhilkin, W. Halliday, D. Fewer, N. Hill, I. Ross, M. West, I.C.P. Smith, S.M. Donnelley, A.C. Kuesel, J. Magn. Reson. Imag. 6 (1996) 437. S.J. Nelson, D.B. Vigneron, W.P. Dillon, NMR Biomed. 12 (1999) 123. T. Usenius, J.-P. Usenius, M. Tenhunen, P. Vainco, R. Johannson, S. Soimakallio, R. Kauppinen, Int. J. Radiat. Oncol. 33 (1995) 719. J.A. Lazareff, R.K. Gupta, J. Alger, J. Neuro-Oncol. 41 (1999) 291. A. Rutter, H. Hugenholtz, J.K. Saunders, I.C.P. Smith, Investigat. Radiol. 30 (1995) 359. J.W. Pritchard, Epilepsia 35 (Suppl. 6) (1994) 14. B.T. Woods, T.M. Chiu, Adv. Exp. Med. Biol. 318 (1992) 267. D.L. Rothman, O.A. Petroff, K.L. Behar, R.H. Mattson, Proc. Natl. Acad. Sci. USA 90 (1993) 5662.

[172] O.A.C. Petroff, L.A. Pleban, D.D. Spencer, Magn. Reson. Imag. 13 (1995) 1197. [173] I.J. Bakken, D. Axelson, K.A. Kvistad, E. Brodtkrob, B. Muller, J. Aasly, I.S. Gribbestad, Epilepsy Res. 35 (1999) 245. [174] T. Kato, S. Takahaski, T. Inubusi, Psychiatr. Res.: Neuroimag. 45 (1992) 53. [175] R.A. Komoroskik, J.E.O. Newton, J.R. Sprigg, D. Cardwell, P. Mohanakrishnan, C. Karson, Psychiatr. Res.: Neuroimag. 50 (1993) 67. [176] T. Kushnir, Y. Itzchak, A. Valevski, M. Lask, I. Modai, G. Navon, NMR Biomed. 6 (1993) 39. [177] D. Haussinger, J. Laubenberger, S. von Dahl, T. Ernst, S. Bayer, M. Langer, W. Gerok, J. Hennig, Gastroenterology 107 (1994) 1475. [178] C. ReÂmy, S. Grand, E. Sam Lai, V. Belle, D. Hoffman, F. Berger, F. Esteve, A. Ziegler, J.-F. Le Bas, A.L. Benabid, M. Decorps, C.M. Segebarth, Magn. Reson. Med. 34 (1995) 508. [179] E.J. Novotny, M.J. Avison, N. Herschkowitz, O.A.C. Petroff, J.W. Prichard, M.R. Seashore, D.L. Rothman, Pediatr. Res. 37 (1995) 244. [180] L.L. Cheng, M.J. Ma, L. Becerra, T. Ptak, I. Tracey, A. Lackner, R.G. Gonzalez, Proc. Natl. Acad. Sci. USA 94 (1997) 6408. [181] I. Trope, D. Lopez-Villegas, R.E. Lenkinski, Pediatrics 101 (1998) 1066. [182] W.L. Strauss, M.E. Layton, S.R. Dager, Am. J. Psychiatr. 155 (1998) 380.