ANALYTICAL APPROACH
NMR
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Research: THE PRESENT AND THE FUTURE Ian C. P. Smith Division of Biological Sciences National Research Council of Canada Ottawa, Canada K1A 0R6
Gwendolyn N. Chmurny Program Resources, Inc. NCI-FCRDC P.O. Box Β Frederick, MD 21702
Over the past 10 years, NMR spectros copy has become a valuable tool for medical diagnosis and research. 0003-2700/90/0362-853A/$02.50/0 © 1990 American Chemical Society
able information about structure and conformation of important molecules such as RNA and their interactions with drugs. Solid-state NMR spectros copy is emerging as a technique for studying very large or tightly bound molecules, or samples that exist as amorphous solids or precipitates. Can these spectroscopic techniques be used to study the more complex and heterogeneous systems encountered in medicine? In this article, we will present proposed NMR tests for can cer. They provide examples of the po tential and the pitfalls of NMR spec troscopy as a diagnostic tool in medi cine.
Through its wide use, magnetic reso nance imaging (MRI) is quickly becom ing a household word. The beauty of MRI, whether it is providing a detailed picture (image) in color of a tiny tumor tucked away in the pituitary gland or a movie of a beating human heart (se quential images gated to the heart beat), is that it can be performed noninvasively. Traditionally, high-resolution NMR spectroscopy has been used to study cells, tissues, tumors, blood, and other body fluids. Two- and three-dimen sional NMR experiments provide valu
Chemical shift (δ), scalar coupling (J), longitudinal relaxation time (ΤΊ), and transverse relaxation time (T2) are four major parameters of NMR spectrosco py (1). (See Figure 1 for an illustration of δ and J.) For biological samples, these parameters are difficult to mea sure and easy to misinterpret because broad resonances are common in the spectra of biological samples, and the compounds of interest are often present in low concentrations in aque ous solutions of high dielectric con stant. The broad lines are attributable in part to the overlap of many chemical shifts resulting from a multitude of mo lecular species or to a single species with many similar functional groups (i.e., the CH 2 groups of the long acyl chains of triglycerides). Other causes of broadening are reduced molecular mo bility, paramagnetic ions, and magnet ic susceptibility differences across the sample. Scalar (spin-spin) couplings often are not evident in NMR spectra of bio logical systems because the other mechanisms mentioned above broaden the lines. In applying 2- or 3D NMR techniques, the influence of J cou plings, whether visible or not in the simple spectra, cannot be ignored. The longitudinal and transverse re laxation times, T\ and T2, are known in the medical community because of their utility for improving the contrast of images. They are related to the rates at which nuclear spin systems return to equilibrium after disruption during the NMR experiment, and are very impor tant in designing NMR experiments and interpreting NMR data. Differ-
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990 · 853 A
ANALYTICAL APPROACH ences in ΤΊ can cause the novice to make serious errors when reporting quantitative data. The T\ of a nucleus is affected not only by the atoms bound directly to it, but also by surrounding paramagnetic species (i.e., oxygen or iron in whole blood cells). Similarly, T-i values for different nuclei in the same molecule can vary widely if only one region of the molecule is bound or re stricted in its mobility.
polymers
times are zero. In 2D NMR spectrosco py, the evolution-mixing period is used to perform spin gymnastics on inter acting nuclei. The second dimension is generated by sampling this interaction as a function of time in exactly incre mented times, ii. Fourier transforma tion in both time domains (Î2 and ii) then gives the 2D NMR spectrum. Three-dimensional NMR spectroscopy develops by adding a second evolutionmixing period and sampling it as a function of a third time, ta. The only requirements for successful multipledimension NMR spectroscopy are the interaction of the two nuclei of interest through one or two of the NMR parameters discussed above and selection of the appropriate spin gymnastics in each i 2 and Î3 to demonstrate these interactions. For example, MRI is a type of multidimensional NMR spectroscopy in which field gradients are changed during ti and (3. The correlation spectroscopy (COSY) technique is a 2D experiment that relies on the spin-spin coupling between two nuclei. Figure 2 shows the proton COSY spectrum of a colon tu-
Multidimensional NMR
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ever before has a book on vibration damping contained so much information from very different specialties. Empha sizing the state of the art, this new volume looks at damping materials from the view points of polymer scientists, physicists, and acoustical engineers. Presenting the viewpoints of both science and engineering, this modern text examines blends and interpenetrating networks for their ability to damp over broad temperature and frequency ranges. Background material is pre sented, as well as information on fillers, plasticizers, additives, new instrumentation, and theory. With 25 chapters, this unique text cov ers these topics: • definitions and concepts • dynamic evaluation • acoustic attenuation • polymeric materials • vibration damping • advanced materials The treatment is well balanced, offering mate rial needed in industry, government laborato ries, and academia. Authors are drawn from among the international leaders in the field. Robert D. Corsaro, Editor, Naval Research Laboratory
A better separation of species detect able by NMR spectroscopy is often achieved using multidimensional spec troscopy (2D and 3D). Although the basic concepts of these methods are rel atively simple, implementation can be complicated. An excellent introductory article on 2D NMR spectroscopy by Tom Farrar has appeared in this JOURNAL
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L. H. Sperling, Editor. Lehigh University Developed from a symposium sponsored by the Divi sion of Polymeric Materials: Science and Engineering of the American Chemical Society ACS Symposium Series No. 4 2 4 469 pages (1990) Clothbound ISBN 0-8412-1778-5 LC 90-325 $99.95
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American Chemical Society
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Figure 1. Proton NMR spectrum at 360 MHz of ethylbenzene, 0.1 % in deuterochloroform. Insets are expansions of the multiplets for the CH3 and the CH2 moieties to illustrate scalar (J) couplings: The CH2 and CH3 resonances are split into multiplets by the J coupling between them. Coupling to the three protons of the CH3 group results in a quartet structure for the CH2 resonances, whereas the two protons of the CH2 group result in a triplet for the CH3 resonances. The chemical shift (δ) is the frequency (with respect to an internal standard, in parts per million relative to the frequency of the spectrometer) characterizing the particular proton species. For further explanation of these parameters, see Reference 1. (Courtesy of J. K. Saunders, NRC, Canada.)
854 A · ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
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mor. The ID resonances lie along the diagonal of the COSY spectrum, whereas off-diagonal cross peaks dem onstrate which groups of chemical shifts are spin-coupled to one another. A good example is given by cross peaks Y and Y', which result from coupling between a -CH3 and a >CH(OH) moi ety. Possible candidates for cross peaks in this region are lactic acid, the amino acid threonine, and the sugar fucose. Careful consideration of the various parameters described above is essential for the proper design of a medical NMR experiment. This provides an ex cellent opportunity to enhance the in teraction between scientists and medi cal practitioners. The Fossel test
The recent controversy in the NMR community over the Fossel test, a pro posed blood test for cancer (3), exem plifies the need for collaboration be tween clinicians and spectroscopists. The test compares the average of the widths at half-height of the resonance envelopes at 1.3 and 0.8 ppm in the water-suppressed NMR spectrum of plasma. The test is fraught with diffi culties because a great diversity of chemical shifts, linewidths, and T2 val
DIOXINS & FURANS
ues complicates the spectra, making in terpretation difficult. Problems arose when the test was subjected to large general populations. The NMR spectroscopy linewidths were found to be very sensitive to the distribution of plasma lipoproteins and triglycerides. Changes in these levels may result for a variety of reasons: diet, trauma, and diseases other than can cer. Many clinical assessments of this test have been reported, and most of these show that the test is not useful for screening an asymptomatic population for cancer (4-10). Although the Fossel test did not ful fill its original expectations, it stimu lated a great deal of research on the NMR spectroscopy of plasma and in creased awareness of the literature on plasma lipoprotein profiles and their relation to disease. In addition, the test prompted many collaborations among researchers from the basic and clinical sciences. Furthermore, this simple method might be useful for monitoring the response of cancer patients to ther apy (4). Malignancy-associated lipoprotein
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Mountford and co-workers have taken a very detailed approach to NMR analCIRCLE 20 ON READER SERVICE CARD ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990 . 855 A
ANALYTICAL APPROACH πκϋ**»
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ysis of serum obtained from cancer pa tients (11). Their measurements led to the discovery of a unique lipoprotein band in the serum of these patients, which they named the malignancyassociated lipoprotein (MAL). As discussed earlier, the NMR spec trum of human plasma or serum is de rived from a composite of many lipo proteins and a wide variety of small molecules. (The terms plasma and se rum refer to different fluids obtained from blood. Serum is the fluid remain ing after the blood has coagulated and the clot has been removed. Plasma is the fluid remaining after the red cells are removed by centrifugation; coagu lation of the blood is prevented by added agents.) Mountford and co workers fractionated the serum into its component lipoproteins on a density gradient of KBr by means of ultracentrifugation and found that an extra band containing MAL lay between the low-density lipoprotein (LDL) and high-density lipoprotein (HDL) bands for the cancer patients (Figure 3). The Ή NMR spectrum of this MAL band (Figure 4) looks very much like that of serum, because it contains resonances attributable to lipids and proteins. The peak at 1.3 ppm results mainly from the CH2 groups of lipid. However, one
can separate the contribution of lipid at this chemical shift from other com ponents by using a variety of tech niques. Because compounds of different mo lecular weight or rotational mobility have different T 2 values, a pulse se quence can be used that allows the spectra of the short T% components to decay before the desired spectrum is obtained (12). Thus the spectra of only the components of long T 2 can be ob tained (Figure 5). Mountford and co workers found that a component with a T 2 of ~800 ms was present at 1.34 ppm in the NMR spectrum of serum from cancer patients (11). A candidate for the long T 2 component is the methyl group of fucose, a carbohydrate that is often found in the antigenic com pounds on the surface of cancer cells. A second method for the resolution of overlapping peaks, the 2D COSY ex periment (Figure 2), provided further evidence for the presence of fucose. The cross peak Y' linking 1.3 and 4.3 ppm is consistent with fucose. Vari ous chemical and biochemical experi ments confirmed the presence of fucose in the sample, but definite proof that fucose is solely responsible for the use ful NMR parameter has not been ob tained; the amino acid threonine has
chemical structures. Contents Pesticides in the Soil Microbial Ecosystem • Effects of Long-Term Phenoxyalkanoic Acid Applications · Carbamothioate Herbicide Deg radation · Carbamothioate Herbicides · Dicarboximide Fungicides in Soil · Degradation of Insecticides · Insecticides in Soil & Microbial Activity · Degradation of S-Ethyl N.N-Dipropylcarbamothioate in Soil · Roles of Fungi & Bacteria · Influence of Metabolites on Degra dation • Molecular Genetics of Pesticide Deg radation by Soil Bacteria · Response of M i crobial Populations to Carbofuran • Adaptation of Microorganisms in Subsur face Environments · Microbial Adaptation in Aquatic Ecosystems · Coping with Soil Insec ticide Degradation · Systems Allowing Use of Carbamothioate Herbicides • Persistence of Carbamothioate Herbicides • Spectrophoto metry Methods · Detoxification of Herbicide Waste in Soil · Implications for Use & Study of Pesticides in Soil Kenneth D. Racke, Editor, DowElanco
Chylomicra + VLDL
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Joel R. Coats, Editor. Iowa State University Developed from a symposium sponsored by the Division of Agrochemicals of the American Chemical
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tients, separated on a density gradient of KBr by ultracentrifugation ( 11).
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(Courtesy of C. E. Mountford, University of Sydney, Australia.)
856 A ·
ANALYTICAL CHEMISTRY,
Figure 3 . Representation of the lipoprotein bands, f r o m the serum of cancer pa
VOL. 6 2 , NO. 15, AUGUST
1, 1990
MAL
Serum
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ovarian tissue
5
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The MAL band was isolated on a KBr gradient from serum taken from a patient with breast can cer and from a control. (Courtesy of C. E. Mountford, University of Sydney, Australia.)
1
ppm
Figure 4.1H NMR spectra (400 MHz) of (top) the malignancy-associated lipoprotein (MAL) band obtained from the serum of a cancer patient, (middle) serum from the same patient, and (bottom) an excised piece of malignant ovarian tissue. (Courtesy of C. E. Mountford, University of Sydney, Australia.)
very similar NMR parameters. In a study of a patient with ovarian cancer, both the long T 2 and the cross peak had disappeared 9-12 months af ter successful surgery (11). The MAL band has been associated with lipoprotein(a), a component t h a t also is formed in cardiovascular disease (13). NMR spectroscopy of colon tumors In an NMR spectroscopy study of ade nocarcinoma cells and tumors pro duced by these cells in rats (14), spectra similar to those described above for the MAL band were found. For cells that produced cancers that metastasized, the peak at 1.3 ppm had a long T2 com ponent. On the other hand, cells that generated tumors that did not spread produced short T 2 values for the peak at 1.3 ppm. Reversion of the nonmetastatic cells toward their metastatic pro genitors could be followed by the ap
Figure 5. 400-MHz proton NMR spectra of the malignancy-associated lipopro tein (MAL) band obtained using the CPMG pulse sequence ( 1) (2τ = 640 ms) to eliminate most of the rapidly relaxing CH2 resonances from fatty acyl chains.
pearance of a long T 2 component at 1.3 ppm. Thus the metastatic potential of the cancer could be predicted. The advantage of using this method to study cancer in humans is obvious. Treatment could be far better planned if the metastatic potential of the tumor were known in advance. The first such study, performed on 51 human biopsy samples of ovarian or colon cancer (15), showed well-resolved spectra and a T 2 at 1.3 ppm of > 300 ms. By using the criteria established in the rat study, al most all of these cancers were diag nosed as metastatic. Records kept at that time indicated that half of the pa tients had metastatic cancer. The other patients were followed for four years. Among patients whose tumors had shown long T 2 values, many had suf fered subsequent metastasis; a much smaller number of patients, whose tu mors did not have long T 2 values, did
not suffer from recurrences or metasta ses. Thus the comparability of the rat model was confirmed. Many more colon tumors have since been studied (16). The long T 2 value at 1.3 ppm is the most common marker, implying that most adenocarcinomas of the colon are metastatic. However, detailed studies have revealed that much more information can still be re trieved from the 1 H NMR spectra. Figure 6 shows the spectra of tumors at different stages of invasiveness. From Dukes stages A to D, the tumors are progressively more invasive. Two differences in these spectra are evident as a function of the stage: The reso nance at 1.3 ppm becomes progressive ly narrower, and the intensity of spec tral components in the region of 3.1 ppm increases, up to and including Dukes C. The intensity at 3.1 ppm can be expressed relative to the methyl res onance at 0.8 ppm, / (3.1/0.8) (see Ta ble I). Furthermore, 2D COSY spectra reveal a strong cross peak, denoted Y' in Table I and Figure 2, between 1.3 and 4.3 ppm. Comparison of these data with those from samples taken far from the tumor (normal in Table I), and
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990 · 857 A
ANALYTICAL APPROACH
halfway between this region and the tumor (midpoint in Table I), showed that NMR spectroscopy was able to distinguish malignant, transitional, be nign, and normal tissue. The accuracy of the method was such that when the NMR spectroscopy pre diction differed from that of conven tional histopathology, the NMR spec troscopy prediction was upheld in all cases by a second histological analysis. Thus, if accurate location of the tumor and spectral acquisition can be per formed, one should be able to use NMR spectroscopy to diagnose the degree of tumor invasion within the patient, without resorting to surgery. A similar approach has been report ed for small cervical biopsy samples from which positive Pap smears were obtained (17). Originally, cancerous tissue could only be distinguished from normal tissue. However, more recent work from Mountford's laboratory as well as from our own laboratory indi cates that NMR spectroscopy can yield an even greater wealth of information. By using 2D techniques, relaxation times, and the relative intensities of a variety of resonances in ID spectra, we can characterize various abnormal but premalignant stages of the cervical tis sue. This is an area in which conven tional pathological methods fall short of our expectations. NMR spectrosco py may provide a precise and objective means of characterizing cervical biop sies from abnormal tissue. In vivo studies The next phase is to use NMR spec troscopy noninvasively. By using MRI it is usually possible to locate a tumor (Figure 7). Obtaining a spectrum from
only the tumor, with no interference from surrounding tissue, is the next problem. A variety of image-guided, volume-localized methods exist, each with its own set of limitations and pro visos, but spectra are being obtained using both Ή and 3 1 P. Some tech niques, such as chemical shift imaging, allow spectra from many regions to be obtained simultaneously, and one can move from volume to volume to study their spectra, retroactively (18). A prime consideration in this proce dure is the length of time the patient must remain in the magnetic field. Minimizing this time, to keep the pa tient comfortable, conflicts with trying to visualize the smallest volume possi ble and obtain the best specificity. Both of these needs can be met by in creasing the detection sensitivity of the method (e.g., by improved electronic design, finer tuned pulse sequences, and increased magnetic field strength). The last point is the subject of some current interest. Most instruments used for spectroscopy of humans oper ate at a field of 1.5 T. However, three manufacturers have supplied 4-T in struments. Debate rages over the best combination of field strength and price, because it appears that the incre mental gain with increasing field flat tens out rapidly above 3 T. Techniques and instruments are available to perform localized spectral accumulation from humans noninva sively. Protocols must be established for the careful accumulation of NMR parameters to assess their utility for diagnosis and management of cancer. We are past the initial interest stage and into the consolidation phase. Stud ies such as those on colon biopsy sam
Table I. Summary of NMR parameters characterizing stages in the development of colon cancer ( 16)a γ'£>
Histological specimen or spatial location In resected colon
No. of specimens examined
/ (3.1/0.8) >1c
Malignant (Dukes A, B) Midpoint" (transitional mucosa) Benign Normal
50 6
Yes Yes
Yes No
Yes Yes
3 13
No No
Yes No
No No
T2 at δ 1.3 ppm resonance > 450 ms"
'' Statistical tests of the significance of the differences found for the various types of tissue show Pvalues< 0.0001. b Data obtained from 2D COSY spectra. c Data obtained from a 1D NMR spectrum. " Determined by measuring the Γ2 value of a composite resonance at 1.3 ppm or using a T2 filtered COSY to ascertain the relaxation rate of cross peak Y'. " Tissue taken from midpoint between tumor and resection point in all cases was histologically normal.
858 A · ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
ples and some of the existing data on brain tumors suggest that NMR spec troscopy of tumors in situ will become a very valuable technique in cancer medicine. Future and summary Magnetic resonance has a permanent niche in medicine. MRI is producing images in steadily shorter times, with greatly improved contrast. It is now possible to obtain an anatomical representation of an area by proton MRI, take incremental 31 P spectra of the area, and then regenerate 3 1 P chemical shift images of the individual 31 P metabolites (18). However, we are at a point in medical NMR spectrosco py where communication is crucial. In the past, clinicians were able to build on their knowledge of X-ray and com puterized tomography techniques to familiarize themselves with MRI; no such base exists for building a comfort able relationship with NMR spectros copy. On the other hand, NMR spectroscopists are collecting mountains of
Normal
Dukes A
Dukes B
Dukes C
Dukes D
5.0
4.0
3.0 2.0 ppm
1.0
Figure 6.1H NMR spectra (360 MHz) of a series of colon biopsies at various stages of invasiveness, progressing from Dukes stages A to D, where D is the most advanced. "Normal" refers to tissue from the edge of a large section of bowel containing the cancer. It is the closest to normal colon tissue that could be obtained for comparison ( 16).
potentially valuable medical data with out necessarily having the expertise to interpret it. The NMR spectroscopy community must strive to store, calcu
late, and present data in a form that is intelligible to the medical community. Together, spectroscopists and oncolo gists must work harder to bridge the
communication gap. We believe that the future is indeed promising.
We are indebted to Carolyn E. Mountford, De partment of Cancer Medicine, University of Syd ney, Australia, for her collaboration and advice on much of our research. We are very grateful to John K. Saunders, Carolyn E. Mountford, and Len Avruch for Figures 1, 3-5, and 7, respectively. This project has been funded in part by the Department of Health and Human Services under contract no. NO1-CO-74102. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Hu man Services, nor does mention of trade names, commercial products, or organizations imply en dorsement by the U.S. government.
References
Figure 7. View of a brain tumor taken by MRI on a Siemens Magnetom 1.5 Τ MRI instrument. (Courtesy of L. Avruch, Ottawa General Hospital.)
Ian C. P. Smith is director of the Division of Biological Sciences, National Re search Council, Ottawa. He received B.Sc. (1961) and M.Sc. (1962) degrees in physical chemistry from the University of Manitoba and a Ph.D. (1965) in theo retical chemistry from Cambridge University. His research interest is the applica tion of magnetic resonance techniques to the diagnosis, management, and under standing of human disease. Gwendolyn N. Chmurny, a scientist at the National Cancer Institute, Frederick Cancer Research and Development Center, received a B.A. degree (1959) from Memphis State University and an M.S. degree (1961) and Ph.D. (1965) from the University of Illinois, Champaign-Urbana. Her research interests include the application of NMR techniques to study natural products used in cancer and AIDS research and NMR structural assignments in peptides and proteins.
(1) Bovey, F. A. Nuclear Magnetic Reso nance Spectroscopy, 2nd éd.; Academic Press: New York, 1988. (2) Farrar, T. C. Anal. Chem. 1987, 59, 749 A-761 A. (3) Fossel, E. T.; Carr, J. M.; McDonagh, J. N. Engl. J. Med. 1986,315,1369-76. (4) Chmurny, G. N.; Hilton, B. D.; Halverson, D.; McGregor, G. N.; Klose, J.; Issaq, H. J.; Muschik, G. M; Urba, W. J.; Mellini, M. L.; Costello, R.; Papadapoulos, N. M.; Caporaso, N.; Smith, I.C.P.; Czuba, M.; Kroft, T.; Monck, M.; Saunders, J. K. NMR in Biomedicine 1988,1,136-50. (5) Wilding, P.; Senior, M. B.; Inubushi, T.; Ludwick, M. Clin. Chem. 1988, 34, 50511. (6) Verdary, R. B.; Benham, D. F.; McLennan, I.; Busby, N. J.; Wehrle, J. P.; Glickson, J. Biochim. Biophys. Acta 1989, 1006, 287-90. (7) Shulman, R. N. Engl. J. Med. 1990,322, 1002-1003. (8) Engan, T.; Krane, J.; Klepp, O.; Kvinnsland, S. N. Engl. J. Med. 1990, 322, 94953. (9) Okunieff, P.; Zietman, Α.; Kahn, J.; Singer, S.; Neuringer, L. J.; Levine, R. Α.; Evans, F. N. Engl. J. Med. 1990,322,95358. (10) Préfontaine, M.; Kroft, T.; Monck, M.; Saunders, J. K.; Mikhael, N. Z.; Smith, I.C.P. Cancer, in press. (11) Mountford, C. E.; May, G. L.; Wright, L. C.; MacKinnon, W. B.; Dyne, M.; Holmes, K. T.; van Haaften-Day, C.; Tattersall, M.H.N. The Lancet 1987,829-33. (12) Rabenstein, D. L.; Millis, K. K.; Strauss, E. J. Anal. Chem. 1988, 60(24), 1380 A-1391 A. (13) Wright, L. C; Sullivan, D. R.; Muller, M.; Dyne, M.; Tattersall, M.H.N.; Mountford, C. E. Int. J. Cancer 1989,43,241-44. (14) Mountford, C. E.; Mackinnon, W. B.; Bloom, M.; Burnell, E. E.; Smith, I.C.P. J. Biochem. Biophys. Methods 1984, 9, 323-30 (15) Mountford, C. E.; May, G. L.; Williams, P. G.; Tattersall, M.H.N.; Russell, P.; Saunders, J. K.; Holmes, K. T.; Fox, R. M.; Barr, J. R.; Smith, I.C.P. The Lancet 1986,651-53. (16) Princz, E. J., M.Sc. Thesis, University of Ottawa, 1988. (17) Mountford, C. E.; Delikatny, E. J.; Dyne, M.; Holmes, K. T.; MacKinnon, W. B.; Ford, R.; Hunter, J. C; Truskett, I. D.; Russell, P. Magn. Reson. Med. 1990, 13, 324-31. (18) Buchthal, S. D.; Thoma, W. J.; Taylor, J. S.; Nelson, S. J.; Brown, T. R. NMR in Biomedicine 1989,2, 298-304.
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