In vivo NMR agnetic resonance imaging
(MRI) has achieved an amaz-
spectroscopy can be ing level of success in clinical
medicine as a noninvasive diagnostic tool.
used to determine Moreover, NMR spectroscopy is favored
by chemists as a powerful technique for
drug concentrations molecular structure determination. It
therefore seems natural that these clearly
directly in tissues related techniques should merge to
yield noninvasive, spatially localized NMR
of interest and spectroscopy for determining molecular
structure and concentration in vivo.
may provide new Considerable effort has been directed
toward developing in vivo NMR spectros-
information on copy as a clinical diagnostic tool. This ef-
fort has centered largely on probing en-
drug eficacy and dogenous biochemical metabolites by 31P
and 'H NMR. Discussions of in vivo
metabolism NMR spectroscopy have appeared else-
where (1-3). Less commonly, in vivo
NMR spectroscopy has been used to mon-
itor drugs as well as other xenobiotic
agents directly in both human and animal
studies. These studies, especially the
human trials, are of great interest because
of their potential clinical applications.
Why in vivo NMR of drugs?
The magnitude of the pharmacologic or
toxic effect of a drug is expected to de-
pend on the concentration at the receptor
sites, which are generally located in the
tissue cells of the target organ (4). Of ne-
cessity, most tissue cells are usually in
close contact with extracellular fluid,
Richard A. Komoroski which is in close contact with blood.
University of Arkansas for Medical Therefore, measuring drug levels in
Sciences plasma is a reasonable method for moni-
1024 A Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
toring drug therapy. It is also relatively
noninvasive and inexpensive, and a wide
range of analytical techniques, including
high-resolution NMR spectroscopy (5),
can be used for quantitation or identifica-
tion of drug metabolites. It is also possible
to monitor drugs and toxic agents in
urine and other body fluids, although ana-
lyte concentrations in these fluids are of-
ten less reflective of drug concentration at
the active site than blood levels are. Of
course, biopsy is sometimes possible, but
it is highly invasive and not suited for
routine monitoring of therapy.
For a variety of reasons, however, the
plasma concentration of a drug may not re-
flect the concentration at the active site.
Individual variations in drug pharmaco-
kinetics and metabolism, which are com-
mon, may arise from individual differ-
ences in physiological function, disease
state, diet, and other factors. For example,
a drug may accumulate to a high level in
tissue with long-term administration,
whereas plasma levels remain lower and
relatively constant.
Therapeutic (and/or toxic) response,
which is the ultimate clinical measure of
correct dosage, may correlate better with
the drug concentration in tissue than with
that in plasma. An in vivo probe of drug
concentration in tissue may be useful in a
variety of clinical settings. For psychiat-
ric illnesses, accurate diagnosis is often
problematic. Therapeutic response can
be difficult to measure, and drug levels in
plasma are often inadequate predictors
of response. Moreover, drug metabolism
0003- 2700/94/0366-1024A/$04.50/0
01994 American Chemical Society
in the target organ may differ substan-
tially from that in the liver (which deter-
mines the concentration in the blood-
stream) and may itself be a measure of re-
sponse.
NMR spectroscopy can, in principle,
measure drug concentration in tissue in
vivo and can probe drug metabolism if me-
tabolite resonances are resolved. Be-
cause it is noninvasive, NMR spectros-
copy can be used repetitively on the same
individual, permitting pharmacokinetic
and longitudinal studies. Independent
measurement of tissue concentration of a
drug may permit testing of pharmaco-
kinetic models and pharmacodynamic
models of drug response.

Special considerations d I1
Isotopes, sensitivity, and resolu-
tion. Few isotopes are well suited for in
vivo studies of drugs. Table 1lists the iso-
topes of interest for such studies. To our
knowledge, all of these isotopes except 31P
have been used to detect drugs in vivo.
Isotopes such as 7Li and llB, and perhaps
several others not listed, are useful for
only one or a few compounds. Some, such
as 'H, may be of more widespread util-
ity. With isotopic enrichment or indirect
detection methods-some of which are
described later in this article-compounds
containing 13C and other low-abundance
nuclei may be observable, although the
need for isotopic enrichment may limit
clinical applicability.
The most widely used isotope is "F. It A
has favorable NMR properties for in vivo

Analytical Chemistry, Vol. 66, No. 20, October 15, 1994 1025 A
studies, includinga nuclear spin of 1/2 and
relatively narrow lines, high sensitivity
(83%that of 'H), and short spin-lattice re-
laxation times (TI). In NMR a shorter TI
permits more rapid signal averaging for
sensitivity enhancement. Because 19Fis
not present in biological systems to any
significant extent, there is no back-
ground signal, and any "F signals ob-
served must originate from the drug. Also,
many drugs on the market contain fluorine
as a part of their molecular structure.
The major limitation of in vivo NMR
drug studies is low sensitivity. In normal
use, most drugs do not reach sufficiently
high tissue concentrations for detection by
NMR in vivo. Table 1gives estimated
minimum detectable concentrations un-
der favorable circumstances for the iso-
topes listed. These values are comparable
to those given in a recent review (6).
Several factors, including intrinsic sen-
sitivity, presence of background signal,
magnetic field strength, and volume of tis-
sue sampled, determine the minimum
detectable concentration in vivo. There-
fore it is difficult to compare the sensitiv-
ity of NMR for detecting drugs in biologi-
calfluids in vitro (5)with the in vivo situ-
ation. The typical in vivo tissue concentra-
tion necessaryfor detection of a drug is 1-10
times that necessary in vitro, because the
volumes sampled, magnetic field strengths
used, and resonance linewidths vary
widely for the two situations (6).
Because of factors such as tissue het-
erogeneity, magnetic field inhomogeneity
over the relatively large sample volume,
and restricted molecular mobility, in vivo
linewidths are substantially broader than
those obtained for the pure compounds
Natural isoto
1 3 v L v p abundance ('i0i
'H 100.0
7Li 92.6
I'P 50.4
13C 1.1
I9f 100.0
31F 1O(
a NMR receptivity relative to 'H from Reference 26.
* Typical concentration of detectable endogenous metabolites.
Estimated minimum detectable concentration under favorable circumstances.
I026 A Analytical Chemistry, Vol. 66, No. 20,October 15, 1994
in solution. Of these factors, tissue hetero-
geneity probably makes the largest con-
tribution to the in vivo linewidth. In addi-
tion, in vivo studies are usually con-
ducted at lower magnetic field strengths
than in vitro studies. Thus resolution in
multicomponent spectra may be much
lower in vivo, and compounds of similar
molecular structure, such as drug metabo-
lites, may not be resolved.
In vivo concentrations. A number
of factors complicate the determination of
endogenous metabolite or drug concen-
trations in vivo by NMR spectroscopy. Pri-
mary among these factors is the hetero-
geneous nature of biological tissue. The
brain is a pertinent example-a relatively
small volume of 0.5-1 mL (the smallest
volume that can be sampled discretely by
NMR with current technology) contains a
variety of cell types (various types of neu-
ronal and glial cells), vascular space, and
extracellular space (perhaps including
cerebrospinal fluid). Of course, within the
cellular compartments is a variety of s u b
cellular structures. NMR spectroscopy
samples all NMR-visible (i.e., liquid-
state) spins of a given isotope in the active
volume.
Without additionalinformation on the
distribution of a compound among the var-
ious compartments, NMR can only be
used to provide an average concentration
over the relatively large volume sampled.
In some cases, information on the distri-
bution of a compound or an ion may be
available from previous invasive or bio-
chemical studies. In a limited way, NMR
may be able to provide information on
compartmental distribution (7).
In clinical applications, endogenous
NMR
-xeptiviL,
100.0
27.2
13.3
0.01 8
83.4
6.65
metabolite concentrations are often mea-
sured to assess the health of the tissue in-
volved. However, metabolite NMR sig-
nals are sensitive both to decreases in me-
tabolite concentration within the cell and
to loss of cells from the tissue (with un-
changed metabolite concentration in the
remaining cells), Without additionalhisto-
logical information,the two possibilities
cannot be distinguished by NMR, al-
though such a distinction may be critical
for proper diagnosis.
The issue of NMR measurement of
concentration in tissue is somewhat difEer-
ent for drugs than for metabolites. For
drugs, the compartmental distribution will
be important in that the pertinent recep-
tors probably reside in one compartment
alone. However, even if the compartmen-
tal distribution is not available, the overall
drug concentration in the tissue should
be an acceptable alternative to concentra-
tion at the active site and should be highly
superior to plasma concentration.
Measurement of absolute concentra-
tion, even with the above limitations, re-
quires calibration to a signal of known con-
centration, such as tissue water or an ex-
ternal standard phantom. To avoid the
problems of these methods, intensity ra-
tios to an internal standard are often ob-
tained. For a single-line spectrum, this
solution is not possible, and an external
standard may be necessary.
Signal visibility. The above consid-
erations of in vivo concentration assume
that all of a given species is in a liquid-like
state and is contributing to the NMR sig-
nal of interest. The compound must reside
in one of the fluid spaces in the tissue or
be in rapid chemical exchange with the so-
lution state if undergoing weak binding to
macromolecules or membranes. These
assumptions, however, are not always the
case. Low molecular weight metabolites
and drugs can bind strongly to a variety of
macromolecules or to the cell mem-
brane. This binding can greatly restrict the
molecular mobility of the small mole-
cules, broaden the NMR resonance, and
render the metabolite or the drug invisi-
ble to high-resolution NMR.
Another factor is partitioning. Drugs
are often lipophilic and may enter the cell
membrane. The fraction of the total
amount of drug that is represented by the
in vivo NMR signal remains a question
that can be answered definitively only by sensitivity. Most in vivo drug studies have brain tissue may be more relevant. Many
in vivo measurement followed by in vitro been performed with only the crudest lo- psychoactive agents contain fluorine as a
analysis of the same tissue. calization because of inadequate signal part of their molecular structure, al-
Instrumental requirements. In strength from regions of smaller size. though few reach sufficient concentration
vivo studies of drugs are carried out with Under these circumstances care must be in the brain for detection by in vivo F
the same instrumentation used for studies exercised in interpreting such signals, NMR Initial work has centered on detec-
of endogenous metabolites (1-3). Sim- which may arise from several different tion and quantitation of compounds con-
ple studies without spatial localization can anatomical regions. taining trifluoromethylgroups, including
be done on very small animals (mice and the antidepressant fluoxetine (Prozac) and
rats) by using standard high-resolution Multinuclear applications the antipsychotic agents trifluoperazine
NMR systems. For some time, research- For most in vivo drug studies, nonhydro- (Stelazine; TFP) and fluphenazine (IO).
grade systems for small animal studies gen NMR isotopes, primarily F, have Figure 2 shows the F NMR spec-
have been available with horizontal-bore been used to measure concentration, dis- trum of the head of a 13-year-old patient
-
magnets (of 30-40 cm bore size) and of- tribution, or pharmacokinetics. A number who had been on a typical dose of 20 mg/
fer the capability of generating the mag- of related biomedical applicationsof F day of fluoxetine for one month (11).
netic field gradients necessary for NMR NMR, such as imaging of perfluorocarbon The spectrum demonstrates a typical S/N,
imaging and spatially localized spectros- blood substitutes or of regional glucose although patients on fluoxetine generally
copy. Human studies are usually per- metabolism by using fluorinated glucose gave spectra with higher S/N than pa-
formed on clinical MRI scanners at a field analogues, have been reviewed by Thom- tients on the trifluorinated antipsychot-
strength of 1.5T or greater. However, as (8).The following multinuclear applica- ics (12).The patients brain had accumu-
most MRI scanners, even at 1.5T, do not tions demonstrate the advantages and lated about the minimum concentration
have spectroscopic capability. Some have limitations of the technique. detectable for fluoxetine (- 1.3 pg/mL).
the capability for H spectroscopy but lack Psychoactive drugs. In most cases, The second peak in the spectrum arises
the broadband electronics necessary for serum concentrations of psychoactive from a vial of a standard compound used
non-H studies. drugs used to treat mental illnesses pro- to estimate the in vivo drug concentration.
In our laboratory at the University of vide little useful information concerning On the basis of data from 22 patients on
Arkansas for Medical Sciences, animal clinical response and side effects (9).A di- fluoxetine treatment, we found that the
studies are performed on a General Elec- rect measure of drug concentration in brain concentration continued to increase
tric Omega CSI system with a 3 k m bore,
4.7-T magnet. Human studies are per-
formed on a General Electric Signa 1.5T
clinical MRI system with multinuclear
spectroscopic capability. For multinu-
clear studies on humans or animals, it is
usually necessary to build the tuned radio-
frequency (rf) coils in house. The fre-
quency, size, and shape of such coils are
designed for a particular experiment with
the goal of maximizing sensitivity. Fig-
ure 1 shows the birdcage coil built for
the F studies of psychoactive drugs in
human brain described below.
Spatial localization. The ability to
restrict the spatial region that gives rise to
an NMR spectroscopic signal in vivo is
critical to clinical utility. The simplest
method of spatial localization is restriction
of the rf coil size and shape so as to excite
and detect a signal from an approximate
region of interest. Spatially localized
spectroscopy using field gradients to de-
fine the region of interest has been under-
going rapid development, and many a p
proaches are now available.
Successful performance of localized Figure I.Birdcagevolume rf coil designed for F NMR studies of
spectroscopy is dependent on several fac- psychoactivedrugs in human brain.
tors, but for drugs the most important is The coil is tuned to 60.1 MHz for operation on a 1.5-T clinical MRI system.

Analytical Chemistry, Vol. 66, No. 20, October 15, 1994 1027 A
well after the clinical effects of the drug
were evident and seemed to level off after
a six- to eight-month period (11).The
drug accumulated to levels of roughly 20
times those in plasma in the same pa-
tients. Because fluoxetine exerts a thera-
peutic effect within about two weeks, there
appears to be no correlation of steady-
state brain concentration with clinical re-
sponse.
Fluoxetine metabolizes to the thera-
peutically active compound norfluoxetine
in brain. As expected, the 19F chemical
shift of norfluoxetine is very close to that
of fluoxetine, and the two compounds can-
not be resolved in vivo. In vitro "F NMR
studies of postmortem samples from a pa-
tient who had been on fluoxetine and con-
firmatory studies of brain extracts from
rats given fluoxetine demonstrated that
the in vivo signal arises in roughly equal
proportion from fluoxetine and norfluoxe-
tine. The in vivo spectra in Figure 2 were
acquired using the coil shown in Figure 1.
The coil detects drug in both brain and
surrounding tissue; however, the contribu-
tion of drug and metabolites in nonbrain
Figure 2. Chemical structure of fluoxetine and the in vivo ''F NMh
spectrum from the head of a 13-year-old.
The in vivo peak arises from fluoxetine and the metabolite norfluoxetine. The in-coil standard
peak is from a vial of 12 mmol/L 2,2,2-(trifluoroethy1)-ptoluene sulfonate in CDCI, mounted on
the side of the head with an elastic band. (Adapted with permission from Reference 10.)
1028 A Analytical Chemistry, Vol. 66, No. 20, October 75,1994
tissue to the in vivo signal was estimated though 20-40% of patients respond poorly
at < 20%by low spatial resolution localized or not at all. Serum Li concentrations
spectroscopy (10). above 2 mmol/L are often toxic, although
Antipsychotic drugs such as TFP, al- neurotoxicity can also be seen in the
though given in oral milligram doses that nominal therapeutic range in some pa-
are comparable to those for fluoxetine, do tients. These considerations suggest that
not visibly accumulate to the same ex- the Li concentration in brain may be a bet-
tent in brain and give weaker "F NMR sig- ter measure of efficacy and/or neurotox-
nals. Nevertheless, in unlocalized studies icity than the concentration in serum.
we observed signals for six responding pa- Moreover, the distribution of Li in brain
tients taking different doses of TFP (12). may help elucidate its still unknown mech-
A good correlation of brain concentration anism of action.
and daily dose was found. Interestingly, The 7Li isotope is relatively favorable
however, on four attempts we could not for in vivo NMR spectroscopy studies (Ta-
observe a signal from a nonresponding pa- ble l).Moreover, the tissue concentra-
tient who was on a very high dose of the tion necessary for successful therapy is
drug. This preliminary result suggests that sufficientlyhigh to make in vivo detection,
the drug was not accumulating in that pa- and even low spatial resolution localized
tient ' s brain at a detectable level. In vivo spectroscopy, feasible. Following the pio-
"F NMR may play a role in assessing the neering work of Renshaw and co-work-
reasons for nonresponse to antipsychotic ers (13), several groups, including our
medication. own, have pursued in vivo 7Li NMR in
Lithium, typically given in the form of humans and animals. The topic has been
Li,CO, tablets, is used to treat mania and recently reviewed (14,15).
manic-depressive illness. Serum Li con- In animals, effort has been directed to-
centrations are usually maintained in the ward developing methods for measuring
therapeutic range of 0.5-1.2 mmol/L, al- the pharmacokinetics of Li uptake in brain.
Using a stimulated echo acquisition
mode (STEM) sequence for spatial local-
ization, Ramaprasad et al. (16) found
time constants of 48-98 min for the up-
take of a single dose of Li in rat brain with
signals that were uncontaminated by
those from surrounding tissue. Because
relatively high doses and long data acquisi-
tion times can be used for anesthetized
animals, it was possible to obtain informa-
tive 7Li NMR images in the rat.
Figure 3 shows sagittal 'H and 7Li im-
ages of the head of a rat dosed with Li. The
quality of the 7Li image is such that the
overall shape of the head and neck is rea-
sonably well defined. The strongest sig-
nals come from muscle at the back of the
head and neck.
Intensities in the brain region are typi-
cally half of those for muscle. Within the
brain region the intensity is relatively uni-
form, although the intensity in the cere-
bellum is lower than that in the frontal re-
gion. Before such images can be used re-
liably to measure local Li concentrations,
issues concerning relaxation times, visi-
bility, and compartmentalization must be
resolved.
Most 7Liwork in vivo has centered on
humans. Initial unlocalized studies mea-
 -
muscle peaked after 2 h, whereas the netic analysis. Unfortunately, the thera-
level in brain peaked after - 4 h. Thus, al- peutically important metabolites are usu-
though there is some delay for Li to ally not seen, except with extensive signal
cross the blood-brain barrier, the delay is averaging (21).
short and cannot account for the well- Of course, more important than the me-
-
known delay of 1week for Li to exert tabolism in healthy liver is the metabolism
clinical efficacy. in tumors. Wolf (22),Presant (23,24),
Figure 4b shows serial results for the and co-workers have been using 19FNMR
same subject after he was taken off of Li in vivo to monitor the pharmacokinetics
treatment because he developed a tremor of 5-FU elimination from human tumors as
(19). Initially the Li levels dropped r a p a predictor of therapeutic response to the
idly. At six days after terminating Li ther- drug. They observe only the disappear-
apy, 7Li in vivo NMR no longer detected Li ance of 5-FU, not FBAL or therapeutic
in muscle but did detect it in brain. At 10 metabolites, in tumors. Their hypothesis,
days, no signal was detected from brain. which is supported by human and animal
This result is of interest because the data, is that responding tumors pool 5-FU
toxic symptoms took about a week to s u b and display a long t1,2 for elimination; non-
side, a result that suggests that in vivo
7Li NMR could be used to relate toxic ef-
fects to residual brain concentration. Pre-
liminary spectroscopic imaging of the spa-
tial distribution of Li in human brain has
also been performed (19) and may shed
light on the mechanisms of the thera-
peutic and toxic effects of Li.
Antineoplastic agents. A widely
used cytotoxic drug for the treatment of
Figure 3. NMR images of the head
colorectal and breast cancer is 5fluoroura-
of a rat dosed with lithium. cil(5FU). The cytotoxic effect of 5FU is
(a) Midline sagittal 'H NMR image used for attributed to the anabolic formation of flu-
localization of 7Li imaging. (b) 7Li image after oronucleosides and fluoronucleotides
a multidose protocol of intraperitoneal LiCI. In that interfere with DNA and RNA metabo-
this image, the outer line defines the shape
and location of the rat head, and the inner line lism. The drug, which is used in combi-
the brain and spinal cord. The pulse repetition nation with other compounds that modu-
time was 7 s, and the image took 4 h to late its metabolism, is effective in only
acquire. The resolution was 4 mm in-plane,
with a 7-mm slice thickness. (Adapted with - 200?of patients. Clinicians still cannot
permission from Reference 16.) predict who will respond to the drug, de-
spite its long history of use. Lack of re-
sponse may be related to the effectiveness
sured the pharmacokinetics of uptake and of the competitivecatabolic pathway, in
elimination and compared serum and which 5FU is metabolized in the liver in
brain concentrations (14,15,17, 18).Sev- the manner of endogenous pyrimidines.
eral 7Li studies in vivo confirmed that Both 5-FU and its major catabolite,
brain Li concentration is typically 0.1- a-fluoro-palanine (FBAL), are readily o b
0.6 mmol/L, which is 0.4-0.6 times the s e servable in human liver by "F NMR in
rum concentration. vivo with a surface coil after a typical in-
Li is eliminated from the body rela- travenous bolus injection of 1g of the drug
tively rapidly (t1,2=0.5-1 day). Figure 4a has been delivered over 10 min. Figure 5
shows the results for a subject who had shows a typical pharmacokinetic assay in
been on Li therapy for more than six the liver for a patient on prophylactic che- Figure 4. Li concentrations in
months and was considered to be at steady motherapy for breast cancer (20).Be- serum, brain, and calf muscle.
state (18).After a single 300-mg dose of cause they are quite different chemically, (a) Concentration profiles for a subject at
steady state after a 300-mg oral dose of
Li,CO,, the concentrations in brain and 5-FU and its catabolite FBAL are well re- Li,CO,. (Adapted with permission from
calf muscle (by 7Li NMR) and in serum solved spectroscopically. Under these Reference 18.) (b) Plots of Li concentrations
(by flame photometry) were monitored conditions of drug administration, sensitiv- for a subject removed from Li therapy. The
serum values are plotted to the lowest level
for 6 h. At the low time resolution of these ity is sufficient to acquire a spectrum in reported in the serum Li assay. (Adapted with
experiments, the levels in serum and several minutes, permitting pharmacoki- permission from Reference 19.)

Analytical Chemistry, Vol. 66, No. 20, October 15, 1994 1029 A
responding tumors tend to display a short
tl,, like that in blood or liver.
Figure 6 shows the 5FU elimination
curves for tumors from responding and
nonresponding patients that demonstrate
this behavior and compares them with a
control. The ability of some tumors to
trap 5FU is not well understood; it proba-
bly arises from factors such as vascular-
ization of the tumor and transmembrane
transport of the drug into tumor cells.
At last report (24),this trapping behav-
ior was proving true for the 57 patients
studied to that point, although many are
still being evaluated for clinical response. tered to a cancer patient prior to irradia-
Of 9 patients with tumors that demon-
strated trapping, 8 had partial responses to mal neutrons. Collision of the neutrons
5FU chemotherapy, whereas only 2 of 25
whose tumors did not trap the drug were
considered responsive. The patients had
primary tumors in a wide variety of sites,
so the approach appears to be generally
applicable, although with surface coils
only tumors at least 2 cm in diameter and
-
located within 8 cm of the skin surface
can be studied.
Figure 5. Stacked plot of sequential pulses) of the 'H-"B coupling. Upon s u b
in vivo ''F NMR spectra.
The patient received a bolus injection
(600 mg/m2 of body surface area 2 h after
administering methotrexate) of 5-FU over
10 min (-5 to +5 min). Each spectrum is the
baseline-corrected result of 200 acquisitions
(- 3.5 min/spectrum). The chemical shift
scale is in ppm from 5-FU.
1030 A Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
If the correlation between drug trap-
ping and therapeutic response holds for a
sufficiently large number of patients,
this technique should be useful for devel-
oping and optimizing treatment protocols.
It could also be considered for applica-
tion to real-timeclinical decision making in
individual chemotherapy (25).For exam-
ple, alternative chemotherapy could be
considered more quickly for patients
whose tumors do not trap 5FU.
Neutron capture agents. In boron
neutron capture therapy (BNCT), a phar-
maceutical containing boron is adminis-
tion of tumorcontaining regions with ther-
with O ' B nuclei releases alpha particles,
which have relatively short trajectories
and hence only damage cells in the imme-
diate vicinity of the collision. A tech-
nique to probe the distribution of such
pharmaceutical agents in tissue would be
valuable for their development and per-
haps even for guidance of BNCT in the
clinic.
Of the two boron isotopes available for
NMR, "B has the more favorable NMR
properties (26).Kabalka and co-workers
(27)have developed "B MRI and NMR
spectroscopy, optimized for the short
spin-spin relaxation time (T,)of 'lB, to
monitor the distribution and pharmacoki-
netics of BNCT agents in laboratory ani-
mals. A rat was administered the BNCT
agent B,,H,,Sg- (BSSB) by a surgically
implanted osmotic pump that delivered
224 pg B/g body weight over seven days.
A day after stopping infusion, the feasi-
bility of the technique was demonstrated
by performing "B NMR spectroscopy and
MRI at 1cm in-plane resolution.
Because the compounds administered
to patients are highly enriched in 'OB, the
"B approach may not be feasible in the
clinic. Bendel et al. (28)have used indi-
rect detection of O' B via spincoupled 'H
NMR, whereby the 'H signal is detected
with and without modulation (by heteronu-
clear decoupling or insertion of 180"
traction, only the protons coupled to O 'B
remain and are detected at the higher 'H
sensitivity. The indirect detection of het-
eronuclei using spincoupled protons is
an approach that could see increasing use
in a variety of drug studies in vivo.
Figure 6. Clearance of 5-FU from
tumors of responding (m) and
nonresponding (A)patients, and
from the blood of a control patient
(0)-
The clearance times (ti,& were 41.3 f 5.5,
15.5 k 2.9, and 9.6 k 1.4 min, respectively.
(Adapted with permission from Reference 23.)
Anesthetics. Anesthesia during sur-
gery is commonly maintained using fluo-
rine-containing inhalation agents such as
halothane and isoflurane. The mechanism
of action of these anesthetics and their
interactions with brain tissue are not well
understood. Of particular interest are the
distribution of anesthetic in the brain and
the pharmacokinetics of elimination.Early
studies demonstrated that such anesthet-
ics could be observed in animal tissues us-
ing in vivo 'F NMR spectroscopy, al-
though there was considerable contro-
versy concerning the pharmacokinetics of
elimination, the spatial origin of the
signals, and the number of signal compo-
nents (2S31).
Particular care is required in unlocal-
ized studies of such lipophilic molecules
because of the possibility of contamina-
tion from components in fat; the multi-
ple time constants for elimination; the
short T2s,which complicate observation
of the total 'F signal; and variable physio-
logical factors. It appears that volatile flu-
orinated anesthetics may exist in more
than one environment in brain, and mea-
surable concentrations may persist for a
relatively long time. Menon et al. (32)
have demonstrated the possibility of ob-
serving cerebral halothane in patients up
to 90 min after surgery by using 'F NMR
in vivo. More work is necessary to deter-
mine the spatial origin of the signals, the
elimination pharmacokinetics, and the me-
tabolism of these anesthetics before such
studies can have clinical utility.
 Measurement of oxygen tension. lyzed for "F NMR and thus also make rings in their molecular structure, and this
Because of their biochemical inertness 'H NMR less desirable for in vivo detection region in the 'H NMR spectrum in vivo is
and ability to dissolve large amounts of drugs. It may be possible to monitor relatively free of potentially interfering
of oxygen, liquid perfluorocarbon com- drugs with a low therapeutic index (drugs background resonances.
pounds have shown increasing utility as that require administration of relatively Ethanol in brain. Ethanol has been
respiration and blood substitutes. "F MRI large amounts to achieve therapeutic effi- detected by localized 'H NMR spectros-
and NMR spectroscopy have been used to cacy), drugs that accumulate in the organ of copy in human and animal brain in vivo
monitor these compounds in vivo (8). interest, or drugs that reach h g h local con- (35-38). Because ethanol freely pene-
One particularly interesting spectroscopic centrations for short periods (e.g., 5FU). trates the blood-brain barrier and is typi-
property of these compounds is that Many drugs contain one or more aromatic cally ingested in larger amounts than ther-
their 19Fspin-lattice relaxation rates
(l/Tl) depend linearly on the partial pres-
sure of dissolved oxygen (Po,>.
In a novel application, Wilson and co-
workers (33)have used "F NMR to mea-
sure Po, at the surface of the retina in a
human eye. Such a measurement is impor-
tant in that retinal hypoxia may play a role
in diseases such as diabetic retinopathy
and retinal venous occlusion. In this study,
a patient ' s retinal tear was repaired in a
surgical operation that involved the use of
a vitreous substitute of high specific
gravity, perfluorotributylamine (FTBA) , to
flatten and position the retina. Although
the FTBA was removed to the maximum
extent possible after surgery, small
(- 3.1 pL) but visible droplets remained
(Figure 7a), as is common but apparently
inconsequential. These workers then mea-
sured the "F T,s of the droplets in vivo
(Figure 7b), and estimated the Po, to be
6 9 mm Hg from a calibration curve. This
method should permit noninvasive, lon-
gitudinal measurement of Po, in the hu-
man eye to gain further insight into the
evolution of ischemic ocular diseases.

Localized 'H NMR of drugs

in vivo
Given that most drugs do not contain an
NMR label such as "F, it might be ex-
pected that the more generally applicable
and sensitive nucleus 'H would be prefer-
able for detection of drugs in vivo. Applica-
tions of gradient-localized in vivo 'H NMR
spectroscopy are becoming widespread,
and the technique is beginning to have
clinical impact (34).However, the pres-
ence of signals from endogenous metabo-
-
lites at 1mmol/L or greater substan-
tially raises the minimum concentration of
a drug that can be detected. Figure 7. #undus photograph of a human eye and "F Tl measurement.
The relatively small range of chemical (a) After repair of a retinal tear and detachment with the use of intraoperative FTBA; residual
shifts and the necessity of suppressing the FTBA droplets are shown. (b) In vivo 'F T, of FTBA droplets in the eye. The intensity of the CF,
peak (arrow) varied with predelay time (time between successive excitation pulses: 400, 500,
large H,O peak make it more difticult to 600, 800, 1000, 1200, and 4000 ms, front to back) and was used to measure T,. (Adapted with
use the very large volumes commonly ana- permission from Reference 33.)

Analytical Chemistry, Vol. 66, No. 20, October 75, 1994 1031 A
 the distribution of silicone in various or-
gans in animals and humans. Observation
of silicone is seemingly straightforward
given the fact that the six equivalent
Si(CH,), protons resonate upfield of com-
mon in vivo resonances. However, these
workers found it necessary to mod* exist-
ing pulse sequences to generate efficient
suppression of both water and fat reso-
nances.
Figure 9a shows a STEAM localized 'H
spectrum of a voxel from the liver of a pa-
tient who had silicone implants for 18
years. A strong resonance attributed to
both hydrolyzed and chemically un-
changed silicone was observed at 0 -
ppm. No such resonance was seen for a
subject without implants (Figure 9b). A
silicone peak was present in localized 'H
spectra of liver for half of the six patients
examined. Clearly, the above approach
could be useful for detecting leakage and
migration of silicone from implants in clin-
ically important cases.

Hgure 8. In vivo spectroscopic localizationof water, NAA, and ethanol Future prospects
plus lipids in human brain. NMR spectroscopy and imaging are novel,
Inversion time, 200 ms; echo time, 272 ms; repetition time, 2 s;1.5 x 1.5 x 1.5 cm voxels; noninvasive techniques for measuring
17-min data acquisition; 16 x 16 spectroscopic images interpolated to 256 x 256 and shown with
an edge-detected overlay of the water image to provide anatomical landmarks. (Adapted with the concentration, distribution, and phar-
permission from Reference 37.) macokinetics of certain drugs in vivo in hu-
mans and animals. A potentially powerful
feature of in vivo NMR is the ability to
apeutic drugs, it is relatively straightfor- The image of ethanol distribution shows measure simultaneously the tissue con-
ward to observe its CH, signal in vivo. considerable nonuniformity; the apparent
Initial studies (35) centered on mea- concentrations in cerebrospinal fluid,
surement of brain alcohol concentration gray matter, and white matter were esti-
and correlation with blood alcohol concen- mated as 23.0,16.6,and 8.3 mmol/L, r e
tration (BAC). However, issues sur- spectively. If the issues of signal visibility
rounding signal visibility and interference and multiple pools of ethanol can be clari- ,

from lipid signals have been raised, par- fied satisfactorily, such measurements
ticularly with the work of Moxon et al. may be useful for studying the relation-
(36),in which signal visibility was found ship of ethanol's behavioral and physiolog-
to be only 23%of BAC when N-acetylaspar- ical effects to brain concentration and
tate (NAA) was used as an internal refer- distribution. The interaction of ethanol
ence. The reduced visibility was attributed with membranes may be related to an indi-
to a combination of factors, including the vidual's tolerance to the drug (38).
suggestion of a pool of NMR-invisible Silicone gel implants. Recently
ethanol, which might be bound in some there has been considerable public contro-
manner to the phospholipid cell mem- versy regarding the Sde@of Silicone gel- Figure 9. Localized 'H spectrum of
branes in brain tissue. filled breast imolants. Concerns include a voxel in the liver for (a) a patient
More recent work (37),in which spec- the possible rupture of the silicone- with and (b) a subbCt without
silicone implants.
troscopic imaging (SI) was used at a spa- rubber membrane encasing the gel and
The spectra were acquired using the STEAM
tial resolution of 1.5 cm, demonstrated that the bleeding of free silicone through the SeQuencecombined with inversion
the distribution of ethanol in the brain is intact envelope into the surrounding tis- iecovery and chemical shift-selectivesatura-
nonuniform. Figure 8 shows the SI results
for a subject who had ingested 0.849 g
-
of ethanol 40-60 min before scanning. voxel localized spectroscopy to monitor permission from Reference'40.) '

1032 A Analytical Chemistry, Vol. 66, No. 20,October 15, 1994

centration of a drug and related changes
in brain metabolism, for example, by 'H or
31PNMR monitoring of endogenous me-
tabolites (41). Although NMR techniques
have significant limitations, in particular
low sensitivity, their noninvasive charac-
ter and unique information content ensure
that new applications and methodologi-
cal improvements will continue to appear.
Higher magnetic fields, more sensitive
detection coils, and postprocessing meth-
ods such as Bayesian statistical estima-
tion of spectral parameters (42) should
substantially reduce the minimum detect-
able drug concentration in vivo and/or
improve spatial resolution.
Very recently, Albert et al. (43) ob-
tained magnetic resonance images of
mouse lungs using 12%egas (a safe gen-
eral anesthetic) that had been magneti-
cally hyperpolarized by spin exchange
with optically pumped Rb vapor. This pro-
-
cedure produced 105-fold enhance-
ment of the ',%e NMR signal, permitting
rapid imaging of the distribution of Xe
gas in the lungs and surrounding tissue.
Beyond the obvious imaging applications
in vivo, NMR spectroscopy of laser-polar-
ized Xe in tissue may be a sensitive probe
of the local environment or of 0, concen-
tration via the ',%e chemical shift or spin
relaxation times.
I thank J.E.O. Newton for his careful reading of
the manuscript.
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Richard A. Komoroski received his B.S. de-
gree from St. Louis University in 1969
and his Ph.D. in physical chemistryfiom In-
diana University in 1973. After complet-
ing a postdoctoral Position at Florida State
University, he spent 10 years in industry
at Diamond Shamrock and B.F. Goodrich,
applying advanced NMR techniques to
polymers and other industrial materials. In
1986 he joined the faculty of the Univer-
sity of Arkansas for Medical Sciences (De-
partments of Radiology and Pathology,
NMR Laboratory, Little Rock, AR 72203,
where his researchfocuses on applications of
in vivo NMR spectroscopy and imaging for
biomedicine and materials science.