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Nuclear Magnetic Resonance Spectroscopy

Ian C. P. Smith* and Domthea E. Blandford


Institute for Biodiagnostics, National Research Council of Canada, 435 Ellice Avenue, Winnipeg, Manitoba, Canada R3B 1Y6

Nuclear magnetic resonance (NMR) spectroscopy is a powerful, nondestructive technique capable of complete structural and
conformational analysis of complex molecules, quantitative analysis of complex mixtures, and noninvasive measurement of reaction
rates of chemical systems in the test tube and in intact living
organisms, including humans. NMR was discovered simultaneously by two independent laboratories in 1946 (Tl, T2).
Subsequently, high-resolution NMR was quickly developed by
analytical chemists as a powerful technique for the determination
of molecular structure. The fundamentals of NMR technology
are described in specialty texts to which interested readers are
referred (T3-T6). In the first section of this review, we will
provide a brief overview of the basics of NMR spectroscopy
including theory and principles of NMR, instrumentation, technical
limitations, and spectral interpretation methods. At this point we
shall quickly shift to the designation MRS, magnetic resonance
spectroscopy, which has been adopted by clinical proponents to
avoid any negative implications of nuclear.
The development of MRS as a clinical analytical tool has been
spurred on by the widespread use of a related analytical technique,
magnetic resonance imaging (MRI), in clinical medicine. The two
are related since they both utilize the same physical phenomenon,
NMR; MRS emphasizes spectral or chemical information, whereas
MFU emphasizes spatial information. The techniques and applications of MRI will not be addressed in this review. Interested
readers are referred to recent texts and articles (77-23).
MRS is unique in its capability to provide nondestructive in
vivo and in vitro chemical analyses. While significant advances
have been made in in vivo MRS, this has recently been reviewed
elsewhere (TI@. The present review for clinical chemistry
highlights some of the research that has been directed toward in
vitro chemical analysis, namely, physiological fluids, tissue specimens, and tissue extracts. This application of MRS in pharmacology and toxicology is already quite well established; the main areas
of current development are metabolic disorders, organ transplantation, neurological disorders, and cancer. The review covers the
time period of 1990 to the present. Our previous review covered
the literature up to that time ( T l l ) .
THE BASICS OF NMR SPECTROSCOPY
Theory and Principles. Spectroscopy is the measurement
of the frequency dependence of absorption or emission of energy
by a system. NMR refers to the absorption and release of radio
frequency (rf) energy by a nucleus in a magnetic field. Possession
of both charge and spin renders some nuclei magnetic and confers
various properties on them which affect their behavior in an
external magnetic field. One such property is a magnetic moment
@). In an external magnetic field (Bo),the magnetic moment of
a spinning nucleus will precess, or describe a cone, around the
direction of the field. The precessional frequency of a particular
nucleus is proportional to the strength of the magnetic field.
To observe resonance, the nuclei must be irradiated with
electromagnetic (I$radiation, the frequency of which must match
the precessional frequency of the nuclei. The rf energy is then
absorbed by nuclei in the lower energy spin state, raising them
to the higher energy spin state. In actual fact, upward and

downward transitions are stimulated equally, but upward ones are


more prevalent due to the greater occupancy of the lower energy
state. This leads to a net absorption of rf energy. Since the
energy difference between the two states is proportional to the
magnetic field strength, the stronger the field, the greater is the
difference between the two populations, and the stronger is the
MRS signal.
For any particular atomic nucleus, at a constant magnetic field
in a vacuum, there is only a single resonant frequency. In bulk
matter, nuclei are surrounded by electronic clouds which exert a
small, but significant shielding effect. The degree to which a
magnetic nucleus is shielded from the applied field by the electron
cloud determines its precessional frequency; the more dense is
the electron cloud (increased shielding), the lower is the preces
sional frequency. Different molecular environments are characterized by a parameter called the chemical shift, which is the
resonance frequency measured relative to that of a suitable
reference compound. The chemical shift values (6) are typically
of the order of
and are therefore commonly specified in parts
per million @pm). These units are independent of magnetic fields,
thereby allowing direct comparison of results from different
instruments. In absolute frequency terms, the separation between
nuclei with different chemical shifts increases with increasing
magnetic field, yielding better dispersion of resonances at high
field.
Electronic clouds also mediate interactionsbetween nuclei that
result in a characteristic splitting pattern of the MRS signal. The
so-called spin-spin couplings (usually given the symbol 7)
are
very useful in assigning MRS resonances, but they also cause
spectra to be complicated and crowded. Nevertheless, the
characteristic pattern of chemical shifts and couplings usually
enables the identification and quantitation of each of a number of
compounds present in a mixture.
Consider the H MR spectrum of glucose in water, as shown
in ref T12. Two major anomeric species of glucose are present,
the a- and P-pyranose forms. The resonances at 4.51 and 5.10
ppm are due to the hydrogens at position 1 of the pyranose ring,
B and a anomers, respectively. Proximity to the oxygen in the
ring gives these hydrogens very characteristic chemical shifts.
The splitting of these resonances into two is due to Jcoupling to
the hydrogen species at position 2 of the ring. The magnitude of
the coupling is indicative of the coniiguration at position 1, 3.8
Hz for the a-anomer and 8.0 Hz for the ,%anomer. The other
patterns in the spectrum are more complex to analyze due to
overlaps, but assignments of all resonances and couplings for both
anomers have been made (T12).
In the MRS experiment, after the rf pulse is turned off,
relaxation processes occur to restore the original equilibrium. The
relaxation process implies a loss of energy from the system of
nuclear spins. Two different processes, both of which are
essentially exponential, are characterized by time constants: TI,
longitudinal relaxation, or spin-lattice relaxation, which results
in a transfer of energy to the surroundings; and 72, transverse
relaxation or spin-spin relaxation, which is a redistribution of
energy among spins. Together 71and TZcan provide information
concerning molecular conformation and dynamics.
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In the NMR experiment, data are collected by a pulse/data


acquisition/delay sequence. The sequence is repeated to yield
an averaged free induction decay (FID) signal of adequate signal/
noise ratio, which is subsequently Fourier transformed to yield a
spectrum. The positions of NMR signals are measured in Hertz
from a standard reference signal and are expressed in ppm in
order to be independent of the spectrometers field strength. The
intensity of an NMR signal is proportional to the area of the signal,
and this area is generally measured by electronic integration.
Under appropriate conditions, peak areas are proportional to the
concentration of the particular compound observed. Thus, for
mixtures of compounds, direct quantitative analysis, without
separation, is possible.
It is evident that MRS could be a very powerful analytical tool
in the clinical laboratory and is of vast potential for clinical
chemical analyses. However, this has not yet happened, perhaps
due to the cost of the instrumentation, certain technical limitations,
and difficulties in data interpretation. Each of these issues will
be discussed in the following sections.
Instrumentation. A modem MR spectrometer consists of
three components: (1) a magnet, capable of sustaining a strong,
stable, and homogenous magnetic field; (2) a probe within the
magnetic field made up of a sample cavity, transmitter coil, and
receiver coil; and (3) appropriate electronic circuitry, a computer,
and peripheral devices to detect, amplify and display the NMR
signal.
(a) The Magnet. Magnets are classified as either permanent
magnets or resistive superconducting electromagnets. Because
the precessional frequency of a nucleus is directly proportional
to the magnetic field strength, it is advantageous to use the
strongest possible magnetic field to obtain the greatest separation
between signals. Recent advances in technology have resulted
in increased spectral dispersion and sensitivity, simpliied spectra,
and reduced interference from strong solvent signals. The
strongest magnets, those which are superconducting, are now
used in MR instruments to generate field strengths up to 17.63 T
(750 MHz for IH); permanent magnets and electromagnets
generate field strengths up to 2.1 (60 MHz) and 2.35 T (100 MHz),
respectively. The superconducting magnet, a solenoid magnet,
is a coil of wire (typically a niobium-titanium alloy) which is
immersed in liquid helium at a temperature of 4 K (-269 C). At
this temperature, the material is superconducting and can carry
a high current with no electric loss or heat generation. This
requires substantial thermal insulation. The superconducting
magnets are the most expensive to run; the principal running costs
are in liquid gases, particularly liquid helium.
(b) The Probe. At the center of the magnetic field is the
probe, which contains the sample cavity and the radio frequency
coil arrangement for excitation and detection of the signal. Other
coils are present in the magnet to generate additional small
magnetic fields. Shim coils generate fields of various shapes so
that the magnetic field is homogeneous or uniform over the entire
sample. The homogeneity results in narrow MR lines and thus
higher spectral resolution. Further narrowing of MR lines can
be achieved by spinning the sample tube, which results in an
averaging of field inhomogeneities. Magnetic field stability (Le.,
control of the strength of the magnetic field) is achieved and
maintained through an electronic technique known as fieldfrequency locking. This is accomplished by the selection of a
substance with a strong MR signal separate from those of the
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Analytical Chemistry, Vol. 67, No. 12, June 15, 1995

sample (e.g., deuterium for lH NMR). This substance can be kept


physically apart from the sample (external lock) or, more commonly, can be dissolved within the sample (internal lock). The
frequency of the lock signal is electronically monitored and
compared to that of the magnetic field, and the magnetic field is
automatically and continuously adjusted, so that proportionality
between field and frequency is maintained.
In a typical experiment, chemical samples of volume 0.1-10
mL are placed in a narrow test tube. The test tube is then
pneumatically inserted into the center of the magnet. While only
one sample can be analyzed at a time, automatic sample changers
are now available.
Commercially available NMR spectrometers are not inexpensive: modem computer controlled, multinuclear I
Tinstruments
range from $200 000 to $1500 000.
Technical Limitations. The power of MRS lies in the large
number of analytes potentially amenable to identification and
quantitication by this method. Compounds that contain the atomic
nuclei hydrogen (H), phosphorus CP), carbon (13C),sodium f3Na), and fluorine (19F) are most often used for clinical MRS
applications. With the exception of 19F,these nuclei are essential
components of organisms and are present in almost 100%natural
abundance (except W ) . Each nucleus has a different frequency
of absorption (the chemical shift) in the NMR spectrum. With
respect to MRS analysis of biofluids, most studies are performed
using lH or 31P. The lH nucleus has several significant advantages. It has the highest sensitivity of any stable nucleus, 100%
natural abundance, and is found in virtually all metabolites. In
general, H MRS of physiological fluids is hindered by three
problems: (1) relatively low analytical sensitivity; (2) the aqueous
nature of physiological fluids; and (3) the fact that physiological
fluids are complex mixtures, resulting in difficulty in the resolution
and assignment of the large number of proton resonances. These
limitations have all been largely overcome.
(a) Analytical Sensitivity. MRS has a relatively low detection
sensitivity when compared with current routine clinical methods
such as immunoassays, gas chromatography/mass spectroscopy,
or the newer molecular diagnostic techniques. However, highresolution MRS has undergone many hardware developments
within the last decade. New high-field magnet technology, radio
frequency electronics, and novel probe designs have resulted in
increased spectral sensitivity and dispersion. The increase in
detection sensitivity over the years for ethylbenzene, the compound used to evaluate spectrometers, has been impressive.
Within a period of less than 35 years, the spectroscopy frequency
has increased from 6 to 750 MHz, resulting in an increase in the
signal/noise ratio from 6 to 12 000. Given that time-averaging of
data is often used to improve signal to noise ratios, the gain of
more than 1000 in detection sensitivity translates into a decrease
of more than 106 in the time required to obtain a spectrum. MR
systems operating at 17.6 T, 750 MHz for H NMR, have recently
been delivered to customers. The sensitivity now attainable
approaches approximately 1 nM for H NMR (213). Moreover,
relatively simple concentration techniques such as lyophilization
or Folch or perchloric acid extraction can be used to increase
the sensitivity for higher molecular weight compounds or analytes
in low concentration. Of greater significance is the fact that MRS
possesses the potential to detect simultaneously a wide range of
compounds of biological interest, irrespective of structure or
physical or chemical properties, resulting in an MRS profile or

fingerprint. This is in contrast with the majority of analytical


techniques currently used, which are designed to be specific for
a single analyte or a particular class of analytes. The spectral
profile provided by H MRS can be of great utility for clinical and
diagnostic purposes, because changes in the normalprofile can
readily be detected in abnormal samples without the requirement
that the compound to be analyzed be preselected. Moreover,
changes in analyte patterns in various physiological fluids may
be linked to pathological processes. This feature of MRS, coupled
with the accompanying structural information provided, has the
potential to detect novel markers of disease or toxicity without
first having to predict their likely outcome.
(b) Aqueous Nature of Physiological Fluids. The presence
of water-derived protons in biological fluids was an impediment
to studying solutes in aqueous solutions. Water-derived protons
are present in most body fluids at a concentration of approximately
110 M, some 105 times the concentration of the metabolite of
interest. Suppression of the water signal, which would otherwise
dominate the spectrum, is therefore necessary, and a variety of
methods have been described. These methods include selective
saturation of the water signal by either the application of a
continuous or gated secondary irradiation field at the water
resonance frequency, or multipulse solvent suppression; e.g., the
Dante pulse sequence (Tl4); spectral selection based on TI
relaxation time (Le., the water elimination Fourier transform
method, WEFT) (Tl5);approaches based on the augmentation
of the water TZrelaxation time by the addition of a water proton
exchange reagent or a paramagnetic agent (WATR method) and
attenuation of the water signal by CPMG (Carr-Purcell-Meiboom-Gill) spin-echo methods (Tl6-Tl9);or a combination of
these methods (T20).
Most of these solvent suppression techniques are now straightforward in modern MR spectrometers and may result in acceptable
solvent suppression. In addition, the majority of data on physiological fluids were produced after the advent of these methods.
Thus, while not a recent advance, a discussion has been included
in this review to highlight the fact that the water resonance is
not necessarily a limitation at this time. However, it must be kept
in mind that these methods may still be inadequate for very dilute
solutions, and thus lyophilization of the specimen followed by
redissolution in 2Hz0 is a practical method of eliminating the water
problem and increasing the concentration of the metabolites of
interest. Data obtained in this fashion must be interpreted with
caution, since this process can result in the loss of volatile or
unstable compounds.
(c) Physiological Fluids as Complex Mixtures. Several
different methods have been applied to the analysis of MR spectra.
In simple cases, the chemical shift values and intensities are
automatically obtained using peak-picking routines in the frequency domain spectrum. However, physiological fluids contain
a multitude of compounds, and thus an MR spectrum contains
broad resonances arising from macromolecules such as proteins
and lipids which may overlap resonances from species of low
molecular weight. With the recent availability of higher field
instrumentation, higher frequency measurements decrease peak
overlaps and give more dispersed resonances. Nevertheless,
resolution and identification of the resonances can be problematic.
While the enormous complexity of the spectrum is indicative of
the amount of biochemical information, useful, clinically relevant
information is only obtained after elimination or reduction of these

broad resonances. One approach to this is convolution of the FID


with a function that discriminates against broad resonances such
as a sine bell convolution function. This function minimizes the
influence of the broad underlying resonances due to macromolecules and enhances those due to species of low molecular weight.
Thus, use of sophisticated methods facilitates spectral assignment.
In the past, a variety of multidimensional techniques were
available, the most useful of which for clinical chemistry is twodimensional proton-proton shift correlation (2D-COSY) spectros
copy ( T l l ). In addition, solid-phase extraction chromatography
with off-line NMR detection provides a simple and efficient means
of separating and detecting complex mixtures of drugs and
endogenous molecules (TZl).There are drawbacks to both of
these techniques; the relatively long spectral accumulation times
required by the former, and the destruction of the biofluid matrix
of the latter. Use of spin-echo pulse sequences or physical
pretreatment of the sample (T22)may be necessary to eliminate
the broad resonances arising from macromolecules. Recently,
two-dimensionalJ-resolved ORES) spectroscopy has been shown
to be an efficient means of extracting data on low molecular weight
compounds in urine and blood plasma (723,T23). Moreover,
the plethora of biochemical information that is given by MR
spectroscopy has led to the use of sophisticated pattern recognition methods for data compression and biochemical classification

(2-24-T28).
CLINICAL APPLICATIONS OF NMR
SPECTROSCOPY
The analysis of physiological fluids by high-resolution MRS is
a relatively recent application of the NMR phenomenon in clinical
medicine. Despite the fact that MR spectra are rich in information
on endogenous biochemical processes in health and disease, and
that quantitation of metabolites can be readily achieved, the
diffusion of MRS methods into the clinical laboratory remains slow.
While the major technical limitations have been overcome, MRS
of physiological fluids competes with long-established biochemical
methods that are well accepted, highly automated, comparatively
inexpensive, and readily available at most clinical sites. Moreover,
data collection and interpretationis limited by the scarcity of MRS
trained individuals able to exploit fully the wealth of information
in the spectrum of a fluid, tissue, or tissue extract.
A large number of physiological fluids is accessible for MRS
studies in vitro. The first medical applications showing the utility
of H MRS analysis of complex metabolite mixtures involved the
analysis of urine and serum (224,T29,T30).A variety of other
fluids including cerebrospinal fluid (CSF), amniotic fluid, synovial
fluid, sweat, aqueous humor, seminal plasma, saliva, bile, ascites
fluid, and tissue extracts have since been examined. All physiological fluids are not equally available in terms of quantity and
ease of availability (technical difticulties, patient benefit, patient
discomfort, ethical issues). Nevertheless, samples are drawn in
a variety of clinical situations where it would seem prudent to
extract as much information as possible out of as small a specimen
as possible, particularly in situations where a specimen is difficult
to obtain. It is here where MRS can offer distinct advantages over
conventional biochemical analytical techniques: MRS analysis
requires a small sample volume (0.2-0.5 mL), which generally
remains intact during measurement and thus can be used for
subsequent assay by other techniques; the specimen generally
requires no or very little pretreatment; spectra take only a few
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minutes to acquire and, unlike some other screening techniques


(e.g., HPLC), it is not necessary to preselect the metabolites of
interest in order to detect and quantitate them.
Presently, MRS has penetrated the areas of pharmacology and
toxicology. Many pharmaceutical companies have implemented
automated MRS screening procedures to aid the metabolic and
toxicological studies of various experimental therapeutic agents.
MRS has also found applications in various clinically important
situations. The main areas of current development are in
hereditary metabolic disorders, organ transplantation, neurological
disorders, and cancer. These and other clinical applications will
be discussed in the following sections. The discussions will
attempt not only to highlight the recent advances but also to
emphasize the advantages of MRS over conventional analysis in
particular clinical situations.
Hereditary Metabolic Disorders. Inborn Errors of Metabolism. Historically, one of the most successful clinical a p
plications of MRS has been the detection of a wide range of inborn
errors of metabolism. In these disorders, the reduction or absence
of activity of a single enzyme or cofactor can have dramatic
consequences for metabolism and its control. Many inherited
metabolic disorders result in the accumulation of large amounts
of organic intermediates, or derivatives thereof, which are produced proximal to the defective enzyme step and eventually spill
into the blood and urine. 'H MRS has been used to study the
urinary excretion of such compounds. The literature describing
'H, 31P,and I3C studies was extensively reviewed by us (T22) and
others (T32). Most data on physiological fluids have been acquired on unmodified urine using 'H MRS. In the ensuing years,
very little additional work has focused on this area. This is perhaps not surprising since this is essentially an application already
suited to routine use. Research energies have been directed
toward other clinical applications. However, recent investigations
on unmodified urine have detected the metabolites histidine and
formic acid in a patient with histidinemia (T3.2). The spectra were
acquired in 15 min and did not require any pretreatment of the
specimen. Similar investigations of urea cycle enzyme disorders
demonstrated the presence of the diagnostic metabolites citrulline
and N-acetylcitrulline in four patients with citrullinemia (2'33);
argininosuccinate in three patients with argininosuccinic aciduria
(deficiency of argininosuccinate lyase (T33, T34)); and orotate,
uracil, and uridine in four patients with ornithine carbamoyl
transferase deficiency (7'33).Other studies have essentially confirmed the detection of the diagnostic metabolites in alkaptonuria
(T32),multiple acyl CoA dehydrogenase deficiency (glutaric aciduria type 11) (T35),methylmalonic aciduria (T36),and propionic
aciduria (T36). Recently it was shown that deproteinizing plasma
samples by centrifuging through a filter with a l@kDa molecular
exclusion leads to improved quantification of metabolites, in
particular those associated with 5oxoprolinuria (T37).
Neonatal screening programs currently exist for a number of
inborn errors of metabolism, in particular those for which
treatment has been shown to prevent or ameliorate the severity
of the disorder. Current test procedures routinely used include
simple chemical tests to detect excessive metabolites and amino
acids and various chromatographic techniques that range in
purpose from the detection of abnormal amino acids to the
quantitative identification of specific amino acids. For each
particular disorder, a different screening test is required, followed
by confirmation using yet another diagnostic test. The develop
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ment of a screening program using a technology such as MRS


would allow one to analyze the same blood and/or urine sample
simultaneously for many markers. In fact, simultaneous measurement of such a range of components is not possible for other
techniques, and this coupled with the other benefits of MRS
suggests that MRS is a viable alternative to current neonatal
screening programs. Moreover, there exists the possibility for
the detection of some novel markers of inborn errors of metabolism, as well as insights into the underlying defects, using MRS.
Organ Transplantation. High-field 'H MRS has recently been
used for the rapid multicomponent analysis of urine and plasma
in order to investigate the patterns of metabolic changes associated
with early rejection of transplanted kidneys (T38, T39) and hearts
(T40-T4.2).
(a) Kidney. The assessment of renal graft dysfunction
following transplantation relies on the measurement of plasma
creatinine, renal biopsy, and response to therapy. Since novel
markers of nephron damage, i.e., abnormal profiles of trimethylamine N-oxide (TWO), dimethylamine @MA) and dimethylglycine @MG), have been detected by MRS in urine and blood
(T.2.2, T43),it seems reasonable to speculate that these or other
markers may be able to detect early rejection processes requiring
dialysis and/or cyclosporin toxicity or overdose. In a recent study
(T38),the spectra of normal human urine showed signals for
creatinine, glycine, citrate, alanine, lactate, and N-methylated
metabolites in the chemical shift range of 3.1-3.3 ppm. The
spectra of patients' urine collected following renal transplantation
were considerably different. Compared to normal urine, the
spectral pattern of urine from a patient with an immediate
functioning graft showed decreased concentrations of citrate and
the presence of high levels of protein. Urinary tract infection in
another patient was associated with an abnormal elevation of
alanine, glycine, lactate, acetate, and succinate. In a third patient,
with renal tubular ischemia, elevated levels of the medullary
osmolytes, DMA and myo-inositol, and glucose were observed.
The spectra of urine from a patient with a nonfunctioning graft,
compared to that of normal urine, was grossly distorted as a result
of significant proteinuria and hematuria. Thus, profiles such as
these may provide diagnostic and prognostic information. Moreover, these spectral profiles suggest that the excretion of specific
renal metabolites may be associated with episodes of graft
dysfunction. In this regard, a combination of parameters (e.g.,
TMAO, DMA, alanine, citrate, etc.) all related to creatinine
concentration were recently studied. A high excretion of TMAO
appeared to be associated with biopsy-confirmed acute graft
rejection episodes (T38, T39). However, there was some degree
of overlap, suggesting that TMAO alone may not be a reliable
marker of graft dysfunction; further studies are required. Nevertheless, the results are encouraging and suggest that a combination of parameters in the proton spectra of urine could be used to
improve diagnosis and management of these patients. This
method could be routinely included in the evaluation of these
patients with minimal inconvenience to the patient.
(b) Heart Rejection of a heart transplant is similarly monitored by a combination of invasive and noninvasive techniques
including repeated endomyocardial biopsy and doppler echocardiography (CDE). Recently, two parameters measured by MRS
have been proposed for use in the assessment of heart transplant
rejection, plasma lipoproteins and the glycosylated residues
N-acetylglucosamine (NAG) and N-acetylneuraminic acid ("A)

borne by the plasma proteins. Changes in both these parameters


have previously been shown to reflect inflammatory processes and
immunological reactions. Measurement of the width at half-height
of both the methyl and methylene resonances arising from
lipoproteins in plasma (total line width, TLW) has been shown to
be correlated to graft rejection (T41);
increased TLW values were
observed in patients with evidence of a rejection process. When
a TLW cutoff value was set at 62 Hz, the sensitivity and specifcity
of the test was 71 and 90%,respectively, with a positive predictive
value (PPV) of 78%and a negative predictive value ( N w ) of 86%.
If the TLW value was referred to a reference value (to minimize
the effects of a wide dispersion of preoperative TLW values), and
the ratio thereof was greater than 1.15,the accuracy of the TLW
test for detection of graft rejection increased. Sensitivity and
specifcity increased to 80 and 95%,respectively, with a P W of
90%and a N W of 91%.
The second parameter that has been proposed as a noninvasive
marker of rejection is the variation of the glycosylated residues
of glycoproteins NAG and NANA In a recent experiment, proton
NMR spectra of plasma were obtained. Resonances were assigned
to total glycosylated residues (GRt) and to mobile NAG and NANA
residues. Then GRt, NAG, and NANA were measured on the
basis of area of MRS signals (T40). The variations of the GRt/
CH3 and (NAG NANA)/alanine ratios were analyzed singly, in
combination with each other, and in combination with CDE, and
compared to an endomyocardial biopsy. Sensitivity was greatest
(68%)when either the GRt/CH3 or (NAG NANA)/alanine ratios
were increased or CDE was positive; specifcity however was 51%.
Specificity was greatest (95%) when the GRt/CHS or (NAG
NANA)/alanine ratios were increased and CDE was positive;
however, with this combination, sensitivity was decreased to
approximately 20%. Thus, the optimal detection requires a
combination of the MRS and CDE parameters.
Together, these preliminary studies are very encouraging and
suggest that analysis by proton MRS of blood plasma of heart
transplant recipients might contribute significantly to the early
diagnosis of acute cardiac graft rejection. The advantages of a
less invasive detection method are obvious, not only from the
patients point of view but also from a technical one. Biopsy, the
gold standard, may not be accurate, since the pattern of rejection
within the myocardium is not necessarily uniform and may be
asymmetric. Thus, the biopsy specimen may not be representative
of the underlying pathology. The ultimate objective may be to
limit the use of myocardial biopsy to confirmation of a previously
detected rejection process.
Neurological Disorders. The study of cerebrospinal fluid
is a common aid to the differential diagnosis of neurological
diseases. CSF reflects the cytological and biochemical basis of
disorders of the central nervous system and analysis thereof
increases the accuracy of the diagnosis. MRS allows the simultaneous quantitation of several metabolites that are not routinely
measured in CSF and that would require several different
analytical techniques to be assayed by conventional methods. By
the combination of two-dimensional measurements, resonances
have been assigned recently to 46 chemical species in CSF (T44).
In addition, pattern recognition approaches and discriminant
analyses that separate samples into different classes have been
used in order to differentiate between normal controls and various
neurological disorders (T45-T47). It has been reported that the
spectra of CSF of normal controls and subjects with tumors or

multiple sclerosis can be perfectly separated, whereas those from


subjects with disk herniations can be separated approximately 90%
of the time using principle component analysis (T46). In this
study, spectra from various neurological disorders were compared.
The spectrum of CSF from a normal subject showed signals for
lactate, glucose, acetate, citrate and creatinine, the most important
metabolites. In the spectrum of a case with an intramedullary
mixed germ cell tumor, distinct differences were observed;
glucose signals (3.2-4.0 ppm) were reduced, and new signals
were apparent between 0.8 and 1.0 ppm and at 1.45, 1.97, and
2.39 ppm, identified as valine, a-alanine, and possibly putrescine
and glutamine, respectively. Specimens from patients with disk
herniations and multiple sclerosis differed from controls in the
relative concentrations of acetate and a number of metabolites
(including citrate, valine, a-alanine, acetate, creatinine, and
glucose), respectively.
Recent studies have addressed the issue of whether MRS of
CSF can be used as an aid in the diagnosis of specific neurological
disorders. Using high-resolution H MRS of human post mortem
CSF, and pattern recognition computer methods, partial separation
of CSF from patients with Alzheimers disease (AD) and normal
controls was achieved (T48); more formal statistical analysis
suggested that citrate by itself was the best discriminator. Citrate
levels were signiscantly reduced in the CSF of AD patients relative
to control samples. These preliminary results are encouraging,
since there are currently no antemortem diagnostic markers of
AD. Novel markers of AD may aid in the early diagnosis of AD,
allowing affected patients the benefits of earlier therapeutic
intervention.
In the CSF of patients with Huntingtons disease (HD), MRS
analysis demonstrated a 60% increase in the pyruvic acid concentration as compared to controls; however, no unknown or
unexpected metabolites were detected (T49).The significance
of this increase in pyruvic acid is unknown.
A preliminary study of CSF from 30 patients with definite or
suspected multiple sclerosis (MS) showed that there were no
significant differences between the levels of most metabolites as
compared to controls, with the exception of acetate and formate,
which were increased and decreased, respectively, in patients with
MS. Moreover, in 93%of patients with actively progressing MS,
an unknown singlet peak at 2.82 ppm was found; this peak did
not appear in the spectrum of CSF from any of the control subjects
(T50). The chemical shift suggested that the unknown is likely
an N-methyl metabolite, which may form the basis for a new
diagnostic test for MS. Such a test would be beneficial since
diagnosis of MS currently depends largely on its clinical features.
Laboratory tests including increased IgG levels, oligoclonal
banding patterns on electrophoresis of CSF, increased levels of
myelin basic protein, abnormal evoked potentials and lesions on
MRI, and C T scans are useful only in support of the clinical
diagnosis. A definitive marker would clearly be beneficial.
The results obtained are promising and suggest that H MRS
spectroscopy of CSF may result in an analytical tool for diagnosis,
treatment monitoring, and prognosis of neurological disorders.
Further prospective studies are warranted.
Prenatal Diagnosis. MRS of human amniotic fluid is a recent
clinical application of the NMR technology. Analysis of amniotic
fluid can provide information on fetal and fetomatemal physiology.
Indeed, amniotic fluid lipid measurements are used to identify
fetal lung maturity, amniotic fluid bilirubin measurements are used
Analytical Chemistry, Vol. 67, No. 12, June 15, 1995

513R

to monitor the severity of erythroblastosis fetalis, and measurements of amniotic fluid a-fetoprotein and acetylcholinesterase are
useful in the prediction of open neural tube defects. Conventional
biochemical tests have various technical limitations, and thus MRS
may be particularly well suited to the analysis of amniotic fluid.
While high-resolution 'H MRS was first used to characterize
amniotic fluid for a variety of components including amino acids,
lactate, and glucose (T52),recent work has generally focused on
two areas, namely, 31PMRS analysis of phospholipid extracts of
amniotic fluid (T52-T54) and quantitation of the constituents of
amniotic fluid and their clinical correlation by 'H MRS (T55T57).
(a) Fetal Lung Maturity. Testing for fetal lung maturity has
traditionally been done by the measurement of the lecithin/
sphingomyelin ( W S ) ratio via numerous thin-layer chromatographic techniques. Novel techniques, such as fluorescence
polarization and lamellar body number counts, have been proposed recently to decrease technical difficulties and increase
turnaround time. In a preliminary study, 600-MHz 'H MR spectra
of untreated amniotic fluid specimens from 66 patients were
analyzed. Linear discriminant analysis was performed to determine how well the peak ratios could predict the fetal maturation
category, as determined by either L/S ratio or fluorescence
polarization. Of 43 third-trimester fluids, 65%were placed in the
correct category-immature, transitional, or mature (T55). While
this is a reasonable prediction of fetal lung maturity, it lacks the
sensitivity and specificity required for a clinical test. However,
the metabolites measured likely do not relate specifically to
pulmonary surfactant; better agreement might be anticipated when
more relevant compounds such as phosphatidylcholine and
phosphatidylglycerol are analyzed. 31P MR spectra of phospholipids in human amniotic fluid have been obtained recently (T52T54), but clinical correlations were not attempted.
(b) Fetomaternal Complications. MRS of human amniotic
fluid yields a wealth of information on chemical content and its
variation with the condition of the mother (T58). To determine
how concentrations of the various metabolites of amniotic fluid
detected by MRS may relate to the clinical status of the fetus and/
or the mother, a number of fetomaternal complications were
studied. No differences in peak intensity ratios were observed
for mothers with gestational diabetes or in cases of fetal trisomy
21 where the spectra were generally normal in appearance (T55).
Amniotic fluid from mothers with preeclampsia, on the other hand,
showed differences in peak intensity ratios for choline, succinate
and acetate. Linear discriminant analysis correctly distinguished
all cases (n = 5) of open spina bifida where the MRS spectra were
markedly altered: lactate, glutamate, and acetate concentrations
were significantly increased. New peaks, previously not detected
in normal amniotic fluid were found, and other peaks normally
present were absent (T55). Resonances observed at 6-8 ppm in
the MR spectrum of amniotic fluid have also been observed in
the same region in MR spectra of human urine. It has been
suggested that these resonances might be useful as markers for
fetal renal output (T56). In addition, many low molecular weight
compounds in amniotic fluid have been reported to be of clinical
importance: amino acid elevations have been reported in central
nervous system disorders (T59); glucose concentrations have
been used in the diagnosis of intraamniotic infection (2'60);lactic
acid has been associated with fetal acidosis (7'59). Thus, the
ability of MRS to provide a high-resolution spectrum with the
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Analytical Chemistry, Vol. 67, No. 12, June 15, 1995

identification of many low molecular weight constituents makes


this a powerful technique for the investigation of fetal lung
maturity and also conditions of suspected fetal anomalies and in
various maternal disease states. Moreover, in vitro MRS studies
of amniotic fluid may lay the foundation for noninvasive MRS
analysis of amniotic fluid in vivo.
Infertility. Recently 'H MR spectroscopic methods have been
applied to the analysis of seminal fluid and its component
secretions from normal and infertile human males. The spectrum
of whole seminal fluid is extremely complex with many overlapping resonances, but over 120 resonances have been assigned to
various metabolites using a combination of 2-D 'H MRS methods
(7'61) and a one-dimensional homonuclear polarization-transfer
experiment (T62).
In order to investigate whether the measurement of some
biochemical markers by MRS could allow the differentiation of
various types of infertility, Hamamah et al. (7'63) measured peak
areas of glycerylphosphorylcholine (GPC) , glycerylphosphorylethanolamine (GPE), citrate, and lactate in seminal plasma of
normal and infertile males. The peak areas for GPC, citrate, and
lactate in seminal plasma were smaller for all infertile subjects as
compared to controls. In addition, peak area ratios for citrate/
lactate, GPC/lactate, and GPE/GPC were found to be different
between infertile subjects with spermatogenic failure (nonobstructive) or obstructive azoospermia post vasectomy. With a cutoff
value of 0.12, the GPE/GPC ratio was determined to have a high
sensitivity (86%)and reasonable specificity (71%)in distinguishing
between these two types of infertility. Other investigations have
shown that metabolite patterns differ between obstructive and
nonobstructive azoospermia (T61). These results provide some
quantitative markers that may have clinical applications in the
evaluation of infertility in men using MRS, but more detailed
studies are needed.
Cancer. Due to the increased incidence of cancer in recent
years, the search for an easy, accurate, and noninvasive screening
test for early malignancy has inspired many investigations. In
1986, a proton MRS measurement on human plasma that was quite
unlike the traditional tumor markers, such as a-fetoprotein or
carcinoembryonic antigen, was reported (T64). The average
methyl and methylene resonance line width of the plasma 'H NMR
spectrum, below 33 Hz, showed a strong correlation with the
presence of cancer. This initiated a large number of similar
investigations which generally found this method to be unreliable
(T65- T72), since it essentially measured hyperlipidemia. A
second promising possibility for cancer screening has been the
detection of a novel lipoprotein band in density gradients of plasma
from cancer patients (T73, T74). This band has been identified
as lipoprotein(a) (T75).
While these methods may not be suitable
for screening for early malignancy, they may yet develop a role
for use as a tumor marker in the prognosis, monitoring of
treatment, and followup of patients previously diagnosed as having
a malignancy, particularly if serial measurements are made and
conditions under which measurements are made are standardized.
Alternatively, they may be regarded as complementary methods
in screening patients at risk for cancer, followup, and monitoring
of treatment (T67, T76, T77).
In recent years, attention has shifted away from the search
for a simple screening test of plasma for malignant conditions to
the assessment of whether MRS of various tissue specimens and
extracts of tissue specimens can differentiate between malignant

and nonmalignant diseases. The major focus in the literature has


been in cancer of breast, cervix, colon, liver, ovary, prostate, and
thyroid, although investigations have looked at intracranial tumors
(278, T79), endometrial carcinoma (T80),esophageal cancer
(T81),lung cancer (T82),malignant melanoma (T83), and
stomach cancer (T84).
(a) Breast. MR spectroscopy of extracts from human breast
tumors has utilized a number of nuclei. I3C NMR spectroscopy
detected differences in the levels of monounsaturated and saturated fatty acids between carcinoma and noncancerous tissues
(T85).The precise role of fatty acids in promoting breast
carcinoma and tumor progression has not been delineated, but a
potential use of this technique includes screening for changes in
fatty acid composition that may predispose to development of
carcinoma. The most numerous studies utilized 31PNMR spectroscopy to assess the role of phospholipid metabolites of tissue
extracts. The results presently are inconclusive; some studies
suggest that phospholipid metabolite levels are not useful indicators of tumor prognosis (T86), whereas others suggest the
contrary (2'87, T88). Finally, proton MR spectroscopy demonstrated increased lactate content and an increased phosphocholinehaline ratio in tumor extracts, with decreased glucose and
inositol, as compared to extracts of uninvolved tissue (T89).
Quantitative MRS, or pattern recognition methods (vide infra),
may provide even better means of correlating these findings with
tumor characteristics such as grade and proliferative rate.
(b) Colon. Detailed 'H MRS investigations of cancerous tissue
began with colon. Early studies concentrated on the long TZvalue
associated with a lipid resonance at 1.3 ppm as an indicator of
metastatic potential (290). Later studies demonstrated the utility
of particular peaks in the one- and two-dimensional 'H MR spectra
to characterize stages of colon carcinoma (291). Recent studies
confirm the utility of the method and suggest clinical screening
of colorectal biopsies could provide a useful adjunct to histology
for the assessment of tissue intermediate between normal and
malignant (292). Detailed studies on colorectal cell lines of
varying degrees of invasiveness support the earlier conclusions
and indicate correlations between genetic changes and the
appearance of MR resonances (293).
(c) Thyroid. Early studies of thyroid lesions suggested a role
for lH NMR spectroscopic analysis of thyroid tissue in characterizing normal, benign, and malignant processes from one another
(294). A recent study has supported such a role, demonstrating
that 'H MRS could separate thyroid neoplasms into two discrete
(benign vs malignant) categories: benign follicular neoplasms,
which are difficult to distinguish from their malignant counterparts
by histology, produced spectra with some parameters similar to
those of normal thyroid tissue, whereas malignant neoplasms
produced spectra with properties in common with those of
papillary, medullary, and follicular carcinomas (795). It has
recently been shown, by means of consensus multivariate analysis
of lH MR spectra, that benign follicular adenomas may be
distinguished from carcinomas with high sensitivity and spedicity,
suggesting that many surgical interventions on thyroid may be
eliminated by clinical use of 'H MRS (796).
(d) Cervix. 'H MR spectra of cervical biopsies allow distinction between carcinoma of the cervix and cervical dysplasia (297).
Using a convenient method of specimen preparation for lH MR
semiquantitative analysis, specimens could be grouped into
normal, dysplastic, and invasive cancer via simple MRS parameters

(298). Multivariate analysis of these spectra in our laboratory,


using methods reported in ref T96,indicates that subgrouping
within the class of dysplasia is possible. It is hoped that these
multivariate methods will be broadly used to extract the maximum
information from the 'H MR spectra. Recently, using chemical
shift imaging based on the 1.3 ppm lipid resonance in the lH MR
spectrum, it has been possible to map regions of carcinoma within
a cervical biopsy (Ts9).
(e) Ovary. Similar studies have recently been completed on
ovarian biopsies (TIOO). Malignant tissue could be distinguished
from normal and benign tissue with sensitivity and specificity of
87 and 91%,respectively. Two-dimensional COSY spectra of the
specimens yielded classification with sensitivity and specificity of
88 and 97%. In the two-dimensional spectra, cross-peaksindicative
of cell surface fucosylation were diagnostic for malignancy.
(9Liver. Extracts of diseased liver tissues, including primary
and secondary tumors, and histologically normal tissue have been
studied recently using both 31P and 'H MRS. The 31P MR
spectrum of primary liver tumors showed an increase in phosphorylethanolamine and phosphorylcholine resonances and a
decrease in glycerophosphorylethanolamineand glycerophosphorylcholiie when compared to spectra from histologically normal
tissue (T101,T102).The 'H MR spectra of liver tissue show
additional and complementary information. Levels of several
metabolites have been shown to change significantly in tumor
tissue compared with normal biopsies; citrate, alanine, lactate,
taurine, and glycine are elevated,whereas creatine and threonine
are decreased (T101).
(g) Prostate. 31P,'H, and I3C MRS have been used in studies
to differentiate between benign and malignant lesions of the
prostate gland. It has been suggested that the relative levels of
the phosphorylated metabolites phosphocreatine and phosphomonoesters can be used to discriminate malignant from benign tissue
(T103).Other investigators were able to measure the citrate
concentration in prostatic tissue using proton MRS (T104-TI06).
Lower levels of citrate were not uniformly observed in cases of
adenocarcinoma as compared to benign prostatic hypertrophy,
in particular mixed or primarily stromal hypertrophy (T105).
However, relative ratios of metabolites (citrate/lactate, citrate/
total choline, phosphocholine/total creatinine, choline/total creatine, alanine/total creatine, phosphoethanolamine/total phosphate, phosphocholine/total phosphate, and glycerophosphoethanolamine/total phosphate) were statistically different for
prostate cancer specimens as compared to benign prostatic
hypertrophy (2'107).It is hoped that these observations may
contribute to the understanding of in vivo magnetic resonance
spectra of the prostate and thus aid in the diagnosis of prostate
malignancy.
Monitoring of Disease Processes or Therapy. Given the
potential of MRS for the diagnosis of a variety of pathological
conditions, it follows that MRS would also have equal or greater
value in the monitoring of either the disease processes or the
response to a therapeutic regimen.
(a) Inborn Errors of Metabolism. While not all inborn errors
of metabolism have effective treatment, dietary restrictions have
been shown to ameliorate the symptoms of some of these
disorders; for example, dietary restriction of the intake of phenylalanine and branched-chain amino acids have been advocated for
the treatment of phenylketonuria and branched chain ketoaciduria,
respectively. These treatments require frequent and prolonged
Analytical Chemistry, Vol. 67, No. 12, June 15, 1995

515R

monitoring of the patients serum, which could be done by MRS


quickly and efficiently. Whether this has additional benefits over
conventional methods is unclear at present; however, use of this
technology may serve to provide novel markers of the disease or
a greater understanding of the disease process. lH MRS has been
used to study metabolic perturbations in patients with disorders
of propionyl-CoA metabolism (propionic acidemia and methylmalonic aciduria) during the administration of carnitine therapy.
Administration of carnitine resulted in an increase in the excretion
of propionylcarnitine and acetylcamitine coincident with an
improvement in clinical condition (T108).
(b) Premature and Sick Infants. The investigation of sick
babies is often complicated by the small sample volume that can
be collected for biochemical analysis. Often insufficient sample
is available to yield a complete picture of the biochemical
derangement. MRS has the advantage that the technique is rapid
and nondestructive yet requires a small sample to give diverse
biochemical information. Conditions of clinical relevance that have
been previously monitored include drug metabolism, fasting,
inherited metabolic disorders, birth asphyxia, necrotizing enterocolitis, and ketosis (T109).Recently, nutrient intake in premature
infants was measured using MRS. An oral dose of D20 was
administered, and urine samples were analyzed for DzO by H
MRS. The results correlated well with the those of conventional
methods, with the additional advantages of speed, accuracy, and
ease of sample preparation (Tll0). Thus, MRS investigations are
potentially useful in the diagnosis and monitoring of disease
processes or response to treatment in sick or premature infants.
(c) Transplanted Organs. As mentioned earlier, MRS has
been used to investigate early rejection processes and cyclosporin
toxicity or overdose in patients with transplanted hearts or
kidneys, and this area of investigation shows considerable promise
(T38- T42).
(d) Cancer Followup. While MRS of plasma and tissue
specimens has not given a specific tumor marker for diagnosis,
recent work suggests that there may be suflicient differences in
the spectra for the effects of therapy to be monitored. This area
is worthy of immediate investigation.
(e) Renal Function. MRS studies on physiological fluids have
provided much information on the biochemical and toxicological
effects of many compounds, as well as on endogenous biochemical
processes in health and disease. The application of MRS urinalysis
to the study of the effects of region-specific nephrotoxins uncovered distinct abnormal patterns of metabolites that were associated
with different sites of nephrotoxic action: renal proximal tubular
toxins caused glycosuria, lactic aciduria, and aminoaciduria; renal
papillary toxins produced different abnormal excretions patterns
in terms of both time course and composition; and renal papillary
necrosis produced early increases in TMAO and DMA excretion,
followed by subsequent increases in N,N-dimethylglycine, succinate, and acetate and decreases in TMAO and 2-oxoglutarate
(Till). This knowledge of novel markers of site-specific renal
damage has been applied to the diagnosis of tubular and papillary
distortions in glomerulonephritis. Glomerulonephritis is characterized by an increase in the excretion of amino acid, ketone
bodies, lactate, TMAO, and DMA and a decrease in the excretion
of citrate and a-ketoglutarate (T212),indicating that tubular
interstitial changes and isolated tubular or papillary distortions
develop with the disease (T113).Moreover, these changes can
develop at any stage of the disease. Thus, MRS urinalysis is
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Analytical Chemistry, Vol. 67, No. 72,June 75, 7995

suitable for diagnosing latent tubular interstitial changes, which


are not readily detected by traditional techniques and which result
in a poorer prognosis for the patient. This approach would enable
identification of patients at risk of rapid deterioration and would
enable more aggressive treatment. Moreover, the effects of the
treatment could easily be monitored using this noninvasive
method.
Similar studies using plasma samples from patients with
chronic renal failure demonstrated markedly elevated plasma
creatinine levels as well as an increase in lactate, TMAO, and DMA
(TZZ,T43). The role of TMAO and DMA in the progression of
renal failure has not been evaluated but may prove to be a marker
of renal damage. Measurement of plasma creatinine, on the other
hand, is used clinically to assess glomerular filtration rate (GFR)
and thereby evaluate the progression of renal disease or nephrotoxicity. Although plasma creatinine and creatinine clearance
measurements are convenient to measure, they likely do not
reflect GFR in patients with renal insufficiency. The clearance of
gandolinium (Gd) -diethylenetriaminepentaacetic acid (DTPA) ,
an approved NMR contrast agent, has recently been evaluated as
a novel marker of glomerular filtration. The results of the
clearance of Gd-DTPA closely approximated the clearance of
technetium [99MT~]
DTPA, an accurate method for determining
GFR (2214). These results indicate that Gd-DTPA is a safe,
nonradioactive indicator of GFR that may provide an alternative
method for clinical studies of progressive renal disease.
(f) Strength Recovery after Surgery. Watters et al. (2115)
have followed the recovery of subjects from abdominal surgery
and correlated the degree of success with body composition and
nitrogen balance. Pivotal to the study was the estimation of total
body water by ingestion of D20 and analysis of its concentration
in urine by 2H MRS.
SUMMARY AND CONCLUSIONS
The range of problems in clinical chemistry that can be
addressed by MRS is wide. The number of applications reported
in the literature is growing steadily, particularly since the study
of the composition of physiological fluids and tissues, and the
changes thereof in disease, are well suited to study by MRS.
Moreover, the major technical limitations that have impeded its
progress into the clinical laboratory in the past have been
addressed. Recent hardware and software developments have
further improved and simpliied MRS analysis. Thus, it would
be surprising if MRS of physiological fluids and tissues does not
become an essential technique for clinical chemists and pathologists. In practice, three main obstacles remain to be overcome:
a greater availability of instruments, a larger data base of spectral
changes correlated with pathological conditions, and an enhanced
supply of MR-trained individuals in the clinical environment.
Ian C.P . Smith is Director General of the Institute for Biodiagnostics,
National Research Council of Canada, in Winnipeg, Canada. He received
B.Sc. (1961) and M.Sc. (1962) degrees in physacal chemistry from the
University ofManitoba and a Ph.D. (1965) an theoretical chemistryfiom
the University of Cambridge, England. A j e r postdoctoral research in
biophysics at Stanford University and the Bell Laboratories, he joined the
Division of Chemzstly, Natzonal Research Counczl %,Canada, Ottawa.
He became the Director General of the Institute for aologacal Scaences,
also in Ottawa, in 1987. His research interest is a plication of hysical
techniques to the diagnosis, management, and un$rstanding ojhuman
disease.
Dorothea Blandford is a Scientific Associate of the Institute or
Biodiagnostics, Nataonal Research Council of Canada, Winni eg. sfhe
received a B.Sc. de ree (1985) om the Unavers?ty of Waterio and a
Ph.D. degree (19 9 8 from the dave+y of Manatoba and Tom.leted a
postdoctoral residency program an clznacal chemzstry (1993) an dnnipeg,

Manitoba. Her research interests include the a plication of both hysical


and chemical techniques to the diagnosis an! management ojhuman
disease, as well as the pharmacokinetics and pharmacodynamics of its
treatment.

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