ANALYTICAL APPROACH
able information about structure ana conformation of important molecules such as RNA and their interactions with drugs. Solid-state NMR spectrascopy 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 testa for cancer. They provide examples of the potential and the pitfalls of NMR spectroscopy as a diagnostic tool in medicine.
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I Research: THE PRESENT AND THE FUTURE
Ian C. P. Smith
Division of Blologlcal Sciences National Research Council of Canada Ottawa, Canada K I A OR6
Gwendolyn N. Chmumy Program Reswrces, Inc.
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Over the past 10years, NMR spectroscopy has hecome a valuable tool for medical diagnosis and research. 0003-2700/90/0382-853A/$O2.50/0 0 1990 American chemical Socie~~
Through its wide use, magnetic reaonance imaging (MRI) is quickly becoming 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 bequential images gated to the heartbeat), is that it can he performed noninvasively. Traditionally, high-resolution NMR spectroscopy has been used to study cells, tissues, tumors, blood, and other body fluids. Two- and three-dimensional NMR experiments provide valu-
NMR spectroscopy parameters Chemical shift (6), scalar coupling (J), longitudinal relaxation time (Ti), and transverse relaxation time (7'2) are four major parameters of NMR spectroscopy (I).(See Figure 1for an illustration of 6 and J.) For biological samples, these parameters are difficult to measure 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 aqueous solutions of high dielectric constant. The broad lines are attributable in part to the overlap of many chemical shifts resulting from a multitude of molecular species or to a single species with many similar functional groups (Le., the CH2 groups of the long acyl chains of triglycerides). Other causes of broadening are reduced molecular mobility, paramagnetic ions, and magnetic susceptibility differences across the sample. Scalar (spin-spin) couplings often are not evident in NMR spectra of biological systems because the other mechanisms mentioned above broaden the lines. In applying 2- or 3D NMR techniques, the influence of J couplings, whether visible or not in the simple spectra, cannot he ignored. The longitudinal and transverse reare known in laxation times, TIand Tz, 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 important in designing NMR experiments and interpreting NMR data. Differ-
ANALYTICAL CHEMISTRY, VOL. 82, NO. 15. AUGUST 1. I990
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Sound and Vibration Damping with
Polymen
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ever before has a bwh on v brauon damp,ng tontaineo so mucn information from ver) aifferent speciates. Empha sizing tne slate of the an. tPis neyr vo "me lwks at damping mmr.a.s f r o r the new. paints of poymer scenttsts. p h p t sfs. ana acoustical engineers. Presenting the newpoints of w t n mente and engmeenng. this modern t e n examines blends ana interpenetrating networks for their ability to damp over broad temperature ana frquenq ranges. Backgrom ma1er.a is pre sented. as m.1 as information on fi..ers. p.astltizers. aad tives. nm icstr,mentation ana theory. With 25 cnapters. this iniqJe t e n tov. ers these toprs: 0 0 0 0
ices in TIcan cause the novice to ake serious errors when reporting lantitative data. The TIof a nucleus affected not only by the atoms bound rectly to it, but also by surrounding vamagnetic species (Le., oxygen or on in whole blood cells). Similarly, TZ tlues for different nuclei in the same olecule can vary widely if only one ,@onof the molecule is bound or rericted in ita mobility. IuHMlmenslonalNMR better separation of species detectd e by NMR spectroscopy is often :hieved using multidimensional specoscopy (2D and 3D).Although the ssic concepts of these methods are relively simple, implementation can be )mplicated. An excellent introductory ticle on 2D NMR Spectroscopy by om Farrar has aDDeared ._ in this JURNAL (2). Two-dimensional NMR snectroscoy can be defined using the'following me domaim: preparation, evolution I), mixing, and acquisition (tz). Oneimensional NMR spectra are oblined when the evolution and mixing
times are zero. In 2D NMR spectrawopy. the evolution-mixing period is used
w perform spin gymnastics on interacting nuclei. The second dimenaion is generated by sampling this interaction as a function of time in exactly incremented times, t i . Fourier transformation in both time domains (t2 and ti) then givea the 2D NMR spectrum. Three-dimensional NMR spectroscopy develops by adding a eecond evolutionmixing period and sampling it as a function of a third time, t3. 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 t r and 1.1to demonstrate these interactions. For example, MRI is a t y p e of multidimensional NMR spectroscopy in which field gradients are changed during t r and t3. The correlation spectroscopy (COSYJ technique ia a 2D experiment that relies on the spin-spin coupling between two nuclei. Figure 2 show the proton COSY spectrum of a colon tu-
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m e treatment s we.. ba.ancea. offering material needed in ind-str).. government laoorato. nes.and academia. Aitnors are drawn tmm among the internanonal leadels n the fiela. RobDrt D. Conam. Editor. Naval Researtn Laboratory L. H. Sperliq. Eoiror, Lehign Lnwersiry
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Dwelopea from a s p p o s m sponsneo 0) Ire C . rar dPaymeM Uaterals kerre ana Ergireeiicg ofthe Amenwan Cnemra Saiety ACS Symposium Series No. 424
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Igure 1. Proton NMR spectrum at 360 MHr of ethylbenzene. 0.1% In deuterochb )form. ~)(ll(l are expansions ot ne multiplsts lor me CHsand me CHI minles lo lllu8trBls scalar (4 cwplinps:
and CY, re8onSllces are splk lntD ~lltlplelsby the J capling behresn h. Coupling to the me pmMm o f h C H 3 g a p results In a qmatnu;tue lame CHrrewnances, whereasme two rotom of lhe C& s a p result in a triplet lor me CHp resonances. The chemical shin (6) is ne lreqwncy fiih respect lo an internalatanderd. In parla per million reYlve lo frwwnoy 01 the spcmnster) laraotalzlnp me penkules pmton speclss. Fm further explanation d nSae p m w s . aee Reference (Canwof J. K. Saundera. NRC. Canada.) W
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
DEMAND THE BEST! GC/MS STANDARDS
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I rigure 2. Proton COSY spectrum of a colon rumor.
me cross peeks d Interestare labeled Y and Y'.
Assignment 01 the m e r labeled peaks has been de-
scribsdsarller(1l).
mor. The 1D resonances lie along the diagonal of the COSY spectrum, whereas off-diagonal c r w peaks demonstrate 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) moiety. 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 excellent opportunity to enhance the interaction between scientists and medical practitioners. The Fosssl tea The recent controversy in the NMR community over the Fossel test, a proposed blood test for cancer (3), exemplifies the need for collaboration between 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 difficulties because a great diversity of chemical shifts, linewidths, and 7'2 val-
ues complicatesthe spectra, making interpretation difficult. Problems arose when the test was subjected to large general populations. The NMR spectroscopy linewidths were found to he 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 cancer. 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 fulfill its original expectations, it stimulated a great deal of research on the NMR spectroscopy of plasma and increased 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 therapy (4). Malignancy-associatedlipoprotein Mountford and eo-workers have taken a very detailed approach to NMR analCIRCLE 20 ON READER SERVICE CAR0 ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
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ANALYrICA L APPROACH ysis of serum obtained from cancer patients (11).Their measurements led to the discovery of a unique lipoprotein band in the serum of these patients, wbicb they named the malignancyassociated lipoprotein (MAL). As discussed earlier, the NMR spectrum of human plasma or serum is derived from a composite of many lipoproteins and a wide variety of small molecules. (The terms plasma and serum refer to different fluids obtained from blood. Serum is the fluid remaining after the blood has coagulated and the clot has been removed. Plasma is the fluid remaining after the red cells are removed by centrifugation; coagulation of the blood is prevented by added agents.) Mountford and coworkers 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 Ley between the low-density lipoprotein (LDL) and high-density lipoprotein (HDL) bands for the cancer patients (Figure 3). The 'H NMR spectrum of this MAL band (Fqure 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 CHZgroups of lipid. However, one
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can separate the contribution of lipid at this chemical shift from other components by using a variety of techniques. Because compounds of different molecular weight or rotational mobility have different TZvalues, a pulse sequence can be used that allows the spectra of the short TZcomponents to decay before the desired spectrum is obtained (12).Thus the spectraof only the components of long TZcan be obtained (Figure 5). Mountford and coworkers found that a component with a TZof -800 ms was present at 1.34 ppm in the NMR spectrum of serum from cancer patients (11). A candidate for the long TZcomponent is the methyl group of fume, a carbohydrate that is often found in the antigenic compounds on the surface of cancer cells. A second method for the resolution of overlapping peaks, the 2D COSY experiment (Figure 2), provided fwtber evidence for the presence of fucose. The craw peak Y' linking 1.3 and 4.3 ppm is consistent with fucose. Various chemical and biochemical experiments confirmed the presence of fucose in the sample, but defiiite proof that f u m e is solely responsible for the useful N M R parameter has not been obtained; the amino acid threonine has
Cancer (breast)
,-
1.34 ppm
control
1:O 018 0.'6 PPm
1:8 1:6 1:4 1.'2
iure 5.400-MHz proton NMR spectrz of the malignancygssociated lipopro-
tein (MAL) band obtained using the CPMG pulse sequence (1)(2r = 640 ms) to eliminate most of the rapidly relaxing CHl resonances from fatty acyl chains. The MAL bard was imoialed On a KBr gradiem (mm serum taken horn a patiem with breast c ~ n CBT ard from a comroi (Courtesy of C E Mountfad. University of Sydney. Australia 1
Figure 4. 'H NMR spectra (400 MHz) of (top)the malignancyassociated lipoprot,
(MAL) band obtained from the serum of a cancer patient, (middle)serum from tho
same patient, and (bottom)an excised piece of malignant ovarian tissue. (Courtesy of C. E. MounlfoTd, University ot Sydney, Australia.)
very similar NMR parameters. In a study of a patient with ovarian cancer, both the long Tzand the cross peak had disappeared 9-12 months after successful surgery (11).The MAL band has been associated with lipoprotein(a), a component that also is formed in cardiovascular disease (13). NMR spectraswpy ol colon tumm In an NMR spectroscopy study of adenocarcinoma cells and tumors producedbythesecellsinrata (14),spectra similar to those described ahove for the MAL band were found. For cells that produced cancers that metastasized, the peak at 1.3 ppm had a long TZcomponent. On the other hand, cells that generated tumors that did not spread produced short TZvalues for the peak at 1.3 ppm. Reversion of the nonmetastatic cells toward their metastatic progenitors could be followed by the ap-
pearance of a long T2 component at 1.3 ppm. Thus the metastatic potential of the cancer could he predicted. The advantage of using this method to study cancer in humana 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 Tz at 1.3 ppm of > 300 ms. By using the criteria established in the rat study, almost all of these cancers were diagnosed as metastatic. Records kept at that time indicated that half of the patients had metastatic cancer. The other patients were followed for four years. Among patients whose tumors had shown long Tzvalues, many had suffered subsequent metastasis; a much smaller number of patients, whose tumors did not have long Tz values, did
not suffer from recurrences or metastases. Thus the comparability of the rat model was confirmed. Many more colon tumors have since been studied (16).The long T2 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 retrieved from the 'HNMR 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 resonance at 1.3 ppm becomes progressively narrower, and the intensity of spectral 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 resonance at 0.8 ppm, I (3.1/0.8) (see Table 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
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ANALYVCA L APPROACH halfway between thii region and the tumor (midpoint in Table I), showed that NMR spectroscopy was able to distinguish malignant, transitional, benign, and normal tissue. The accuracy of the method was such that when the NMR spectroscopy prediction differed from that of conventional histopathology, the NMR spectroscopy prediction was upheld in all cases by a second histological analysis. Thus, if accurate location of the tumor and spectral acquisition can be performed, 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 heen reported for small cervical biopsy samples from which positive Pap smears were obtained (17). Originally, cancerous tissue could only he distinguished from normal tissue. However, more recent work from Mountford's laboratory as weU as from our own laboratory indicates 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 1D spectra, we can characterize various abnormal but premalignant stages of the cervical tissue. This is an area in which conventional pathological methods fall short of our expectations. NMR spectroscopy may provide a precise and objective means of characterizing cervical biopsies from abnormal tissue. In vivo studies The next phase is to use NMR spectroscopy 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 provisos, but spectra are being obtained using both IH and 31P. Some techniques, 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 procedure is the length of time the patient must remain in the magnetic field. Minimizing this time, to keep the patient comfortable, conflicts with trying to visualize the smallest volume possible and obtain the best specificity. Both of these needs can he met by increasing 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 operate at a field of 1.5 T. However, three manufacturers have supplied 4-T instruments. Debate rages over the best combination of field strength and price, because it appears that the incremental gain with increasing field flattens out rapidly above 3 T. Techniques and instruments are available to perform localized spectral accumulation from humans noninvasively. 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. Studies such as those on colon biopsy sam-
Table 1. Summary of NMR parameters chara the development of colon cancer (16)" Hlstoloalcal rpeclrnen or spatlal locatlon
No. of rpeclmens examined
ples and some of the existing data on brain tumors suggest that NMR spectroscopy 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 31P spectra of the area, and then regenerate 3'P chemical shift images of the individual 31Pmetabolites (18).However, we are at a point in medical NMR spectroscopy where communication is crucial. In the past, clinicians were able to build on their knowledge of X-ray and computerized tomography techniques to familiarize themselves with MRI; no such base exists for building a comfortable relationship with NMR spectroscopy. On the other hand, NMR spectroscopists are collecting mountains of
!ing s
Y"
In resected colon Malignant (Dukes A, B) Midpointe (transitionalmucosa) Benign Normal
50
3 13
No
No -
Statistical tests 01 the Significanceof the differencesfou Pvaiues < 0.0001. boala Obtained from 20 COSY spectra. Data obtained from a 10 NMR spectrum. *Detwmined by measuring the T2 value 01 a composite filtered COSY to ascemin the relaxation rate Of cross peak 'Tissue taken from midpoint beween tumor and reYICti( nwmat.
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Flgure 6. 'H NMR spectra (360 MHz) 01 a series of colon biopsies at various stages of invasiveness, progressing from Dukes stages A to D. where D is the most advanced. "NWmal" refers to tissue from the ed@ of a large section of bowel containing h e cancer. It is the ci-1 to normal colon tissue that cwm be obtained for cmparison ( 16)
potentially valuable medical d a t a witho u t necessarily having the expertise to interpret it. The NMR spectroscopy community must strive to store, calcu-
late, a n d present data in a form that is intelligible t o t h e medical community. Together, spectroscopists and oncologists must work harder to bridge t h e
communication gap. We believe t h a t the future is indeed promising.
We are indebted to Carolyn E. Mountford. Department of Cancer Medicine, University of Sydney, Australia, for her collaboration and advice on much of our research. We are very grateful to John K. Saunders. Carolyn E. Mountford, and Len Avrueh 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. N01-CO-74102. The content of this publication does not necessarily reflect the v i e w or policies of the Department of Health and Human Services, nor does mention of trade names. commercial products. or organizations imply endorsement hy the U.S. government.
References (1) Bovey, F. A. Nuclear Magnetic Resonance Spectroscopy, 2nd ed.; Academic Press: New York. 1988. (2) Farrar, T. C. 'Anal. Chern. 1987, 59,
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740 A-7fil
(3)Fossel. E.T.; Cam, J. M.: McDonagh. J. N. Engl. J . Med. 19R6.315.1369-76. 14) Chrnurnv. C . N.: Hilton. B. D.; Halver-
Flgure 7. View of a brain tumor taken by MRI on a Siemens Magnetom 1.5 T MRI instrument. (Courtesy of L. Avruch. Ottawa General Hospital.)
11.
(6)Verdary, R. B.; Benham, D. F.; McLennan, I.; Busby, N. J.; Wehrle, J. P.; Glickson, J. Biochim. Biophys. Acto 1989, 1006,287-90. (7) Shulman. R. N. Engl. J. Med. 1990,322, 1002-1003. (8) Engan, T.; Krane, J.; Klepp, 0.;Kvinns.. S. N. Engl. J. Med. 1990,322,949land, 53
i-
I a n C. P. S m i t h is director of t h e Division of Biological Sciences, National Research Council, Ottawa. H e received B S c . (1961) and M S c . (1962) degrees i n physical chemistry from t h e University of Manitoba and a Ph.D. (1965) in theoretical chemistry from Cambridge University. His research interest is the application of magnetic resonance techniques t o the diagnosis, management, a n d understanding of h u m a n 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 S t a t e University and an M.S. degree ( 1 9 6 1 ) and Ph.D. (1965)from the University of Illinois, Champaign-Urbana. Her research interests include the application of NMR techniques t o s t u d y natural products used i n cancer a n d AIDS research and NMR structural assignments in peptides and proteins.
(9)Okunieff. P.: Zietman. A.; Kahn. J.: Singer.5.: Neuringer, L. J.: Levine. R. A.; Evans, F.N. Enpl. J. Med. 1990,322,95358. (10) Pr€fontaine, M.; Kraft, T.; Monck, M.; Saundera. J. K.; Mikhael, N. 2.; 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 Loneet 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.; Mountf0rd.C. E . M . J. Cancer 1989,43,24144. (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.; Saundera, J. K.; Holmes, K. T.; Fox, cetM.; R. 1986,65143, Barr, J. R.; Smith, I.C.P. The Lan(16) Princz, E. J., MSe. Thesis, University of Ottawa, 1988. (17) Mountford, C. E.; Delikatny,,E. J.; Dyne, M.; Holmes, K.T.; MacKmnon, W. 8.; 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 1989,2,29M04.
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