In vivo NMR spectroscopy can be used to ... - ACS Publications

In vivo NMR spectroscopy can be used to determine drug concentrations directly in tissues of interest and may provide new information on drug eficacy and metabolism

agnetic resonance imaging (MRI) has achieved an amazing level of success in clinical medicine as a noninvasive diagnostic tool. Moreover, NMR spectroscopy is favored by chemists as a powerful technique for molecular structure determination. It therefore seems natural that these clearly related techniques should merge to yield noninvasive, spatially localized NMR spectroscopy for determining molecular structure and concentration in vivo. Considerable effort has been directed toward developing in vivo NMR spectroscopy as a clinical diagnostic tool. This effort has centered largely on probing endogenous biochemical metabolites by 31P and 'H NMR. Discussions of in vivo NMR spectroscopy have appeared elsewhere (1-3). Less commonly, in vivo NMR spectroscopy has been used to monitor 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?

Richard A. Komoroski University of Arkansas for Medical Sciences

The magnitude of the pharmacologic or toxic effect of a drug is expected to depend on the concentration at the receptor sites, which are generally located in the tissue cells of the target organ (4). Of necessity, most tissue cells are usually in close contact with extracellular fluid, which is in close contact with blood. Therefore, measuring drug levels in 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 identification of drug metabolites. It is also possible to monitor drugs and toxic agents in urine and other body fluids, although analyte concentrations in these fluids are often 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 reflect the concentration at the active site. Individual variations in drug pharmacokinetics and metabolism, which are common, may arise from individual differences 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 psychiatric 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 substantially from that in the liver (which determines the concentration in the bloodstream) and may itself be a measure of response. NMR spectroscopy can, in principle, measure drug concentration in tissue in vivo and can probe drug metabolism if metabolite resonances are resolved. Because it is noninvasive, NMR spectroscopy 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 pharmacokinetic models and pharmacodynamic models of drug response.

d

Special considerations Isotopes, sensitivity, and resolution. 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 utility. 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 has favorable NMR properties for in vivo

I1

A

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 relaxation 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 background signal, and any "F signals observed 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 under favorable circumstances for the isotopes listed. These values are comparable to those given in a recent review (6). Several factors, including intrinsic sensitivity, presence of background signal, magnetic field strength, and volume of tissue sampled, determine the minimum detectable concentration in vivo. Therefore it is difficult to compare the sensitivity of NMR for detecting drugs in biologicalfluids in vitro (5)with the in vivo situation. The typical in vivo tissue concentration 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 heterogeneity, 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

1 3 v L v p

'H 7Li I'P 13C I9f

31F a NMR

in solution. Of these factors, tissue heterogeneity probably makes the largest contribution to the in vivo linewidth. In addition, in vivo studies are usually conducted 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 metabolites, may not be resolved. In vivo concentrations. A number of factors complicate the determination of endogenous metabolite or drug concentrations in vivo by NMR spectroscopy. Primary among these factors is the heterogeneous 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 neuronal 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., liquidstate) spins of a given isotope in the active volume. Without additionalinformation on the distribution of a compound among the various compartments, NMR can only be used to provide an average concentration over the relatively large volume sampled. In some cases, information on the distribution of a compound or an ion may be available from previous invasive or biochemical studies. In a limited way, NMR may be able to provide information on compartmental distribution (7). In clinical applications, endogenous

Natural isoto abundance ('i0i

NMR -xeptiviL,

100.0 92.6

100.0 27.2 13.3 0.01 8 83.4 6.65

50.4 1.1 100.0 1O(

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

metabolite concentrations are often measured to assess the health of the tissue involved. However, metabolite NMR signals are sensitive both to decreases in metabolite concentration within the cell and to loss of cells from the tissue (with unchanged metabolite concentration in the remaining cells), Without additionalhistological information,the two possibilities cannot be distinguished by NMR, although such a distinction may be critical for proper diagnosis. The issue of NMR measurement of concentration in tissue is somewhat difEerent for drugs than for metabolites. For drugs, the compartmental distribution will be important in that the pertinent receptors probably reside in one compartment alone. However, even if the compartmental distribution is not available, the overall drug concentration in the tissue should be an acceptable alternative to concentration at the active site and should be highly superior to plasma concentration. Measurement of absolute concentration, even with the above limitations, requires calibration to a signal of known concentration, such as tissue water or an external standard phantom. To avoid the problems of these methods, intensity ratios to an internal standard are often obtained. For a single-line spectrum, this solution is not possible, and an external standard may be necessary. Signal visibility. The above considerations of in vivo concentration assume that all of a given species is in a liquid-like state and is contributing to the NMR signal of interest. The compound must reside in one of the fluid spaces in the tissue or be in rapid chemical exchange with the solution 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 membrane. This binding can greatly restrict the molecular mobility of the small molecules, broaden the NMR resonance, and render the metabolite or the drug invisible 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 in vivo measurement followed by in vitro analysis of the same tissue. Instrumental requirements. In vivo studies of drugs are carried out with the same instrumentation used for studies of endogenous metabolites (1-3). Simple studies without spatial localization can be done on very small animals (mice and rats) by using standard high-resolution NMR systems. For some time, researchgrade systems for small animal studies have been available with horizontal-bore magnets (of 30-40 cm bore size) and offer the capability of generating the magnetic field gradients necessary for NMR imaging and spatially localized spectroscopy. Human studies are usually performed on clinical MRI scanners at a field strength of 1.5T or greater. However, most MRI scanners, even at 1.5T, do not have spectroscopic capability. Some have the capability for ‘H spectroscopy but lack the broadband electronics necessary for non-’H studies. In our laboratory at the University of Arkansas for Medical Sciences, animal studies are performed on a General Electric Omega CSI system with a 3 k m bore, 4.7-T magnet. Human studies are performed on a General Electric Signa 1.5T clinical MRI system with multinuclear spectroscopic capability. For multinuclear studies on humans or animals, it is usually necessary to build the tuned radiofrequency (rf) coils in house. The frequency, size, and shape of such coils are designed for a particular experiment with the goal of maximizing sensitivity. Figure 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 define the region of interest has been undergoing rapid development, and many a p proaches are now available. Successful performance of localized spectroscopy is dependent on several factors, but for drugs the most important is

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brain tissue may be more relevant. Many psychoactive agents contain fluorine as a part of their molecular structure, although few reach sufficient concentration in the brain for detection by in vivo ”F NMR Initial work has centered on detection and quantitation of compounds containing trifluoromethylgroups, including the antidepressant fluoxetine (Prozac) and the antipsychotic agents trifluoperazine Multinuclear applications (Stelazine; TFP) and fluphenazine (IO). For most in vivo drug studies, nonhydroFigure 2 shows the ‘’F NMR specgen NMR isotopes, primarily ”F, have trum of the head of a 13-year-old patient been used to measure concentration, distribution, or pharmacokinetics. A number who had been on a typical dose of 20 mg/ day of fluoxetine for one month (11). of related biomedical applicationsof ”F NMR, such as imaging of perfluorocarbon The spectrum demonstrates a typical S/N, although patients on fluoxetine generally blood substitutes or of regional glucose gave spectra with higher S/N than pametabolism by using fluorinated glucose tients on the trifluorinated antipsychotanalogues, have been reviewed by Thomas (8).The following multinuclear applica- ics (12).The patient’s brain had accumulated about the minimum concentration tions demonstrate the advantages and detectable for fluoxetine (- 1.3 pg/mL). limitations of the technique. Psychoactive drugs. In most cases, The second peak in the spectrum arises from a vial of a standard compound used serum concentrations of psychoactive to estimate the in vivo drug concentration. drugs used to treat mental illnesses proOn the basis of data from 22 patients on vide little useful information concerning clinical response and side effects (9).A di- fluoxetine treatment, we found that the brain concentration continued to increase rect measure of drug concentration in

sensitivity. Most in vivo drug studies have been performed with only the crudest localization because of inadequate signal strength from regions of smaller size. Under these circumstances care must be exercised in interpreting such signals, which may arise from several different anatomical regions.

Figure I.“Birdcage”volume rf coil designed for “F NMR studies of psychoactivedrugs in human brain. The coil is tuned to 60.1 MHz for operation on a 1.5-T clinical MRI system.


though 20-40% of patients respond poorly tissue to the in vivo signal was estimated at < 20%by low spatial resolution localized or not at all. Serum Li concentrations above 2 mmol/L are often toxic, although spectroscopy (10). neurotoxicity can also be seen in the Antipsychotic drugs such as TFP, alnominal therapeutic range in some pathough given in oral milligram doses that are comparable to those for fluoxetine, do tients. These considerations suggest that the Li concentration in brain may be a betnot visibly accumulate to the same extent in brain and give weaker "F NMR sig- ter measure of efficacy and/or neurotoxicity than the concentration in serum. nals. Nevertheless, in unlocalized studies we observed signals for six responding pa- Moreover, the distribution of Li in brain may help elucidate its still unknown mechtients taking different doses of TFP (12). anism of action. A good correlation of brain concentration The 7Li isotope is relatively favorable and daily dose was found. Interestingly, for in vivo NMR spectroscopy studies (Tahowever, on four attempts we could not observe a signal from a nonresponding pa- ble l).Moreover, the tissue concentration necessary for successful therapy is tient who was on a very high dose of the drug. This preliminary result suggests that sufficientlyhigh to make in vivo detection, and even low spatial resolution localized the drug was not accumulating in that patient ' s brain at a detectable level. In vivo spectroscopy, feasible. Following the pioneering work of Renshaw and co-work"F NMR may play a role in assessing the reasons for nonresponse to antipsychotic ers (13), several groups, including our own, have pursued in vivo 7Li NMR in medication. humans and animals. The topic has been Lithium, typically given in the form of recently reviewed (14,15). Li,CO, tablets, is used to treat mania and In animals, effort has been directed tomanic-depressive illness. Serum Li conward developing methods for measuring centrations are usually maintained in the 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 localization, Ramaprasad et al. (16) found time constants of 48-98 min for the uptake 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 acquisition times can be used for anesthetized animals, it was possible to obtain informative 7Li NMR images in the rat. Figure 3 shows sagittal 'H and 7Li images 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 reasonably well defined. The strongest signals come from muscle at the back of the head and neck. Intensities in the brain region are typically half of those for muscle. Within the brain region the intensity is relatively uniform, although the intensity in the cerebellum is lower than that in the frontal region. Before such images can be used reliably to measure local Li concentrations, issues concerning relaxation times, visiFigure 2. Chemical structure of fluoxetine and the in vivo ''F NMh bility, and compartmentalization must be spectrum from the head of a 13-year-old. resolved. The in vivo peak arises from fluoxetine and the metabolite norfluoxetine. The in-coil standard Most 7Liwork in vivo has centered on peak is from a vial of 12 mmol/L 2,2,2-(trifluoroethy1)-ptoluene sulfonate in CDCI, mounted on humans. Initial unlocalized studies meathe side of the head with an elastic band. (Adapted with permission from Reference 10.)

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 patients. Because fluoxetine exerts a therapeutic effect within about two weeks, there appears to be no correlation of steadystate brain concentration with clinical response. Fluoxetine metabolizes to the therapeutically active compound norfluoxetine in brain. As expected, the 19F chemical shift of norfluoxetine is very close to that of fluoxetine, and the two compounds cannot be resolved in vivo. In vitro "F NMR studies of postmortem samples from a patient who had been on fluoxetine and confirmatory studies of brain extracts from rats given fluoxetine demonstrated that the in vivo signal arises in roughly equal proportion from fluoxetine and norfluoxetine. 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 contribution of drug and metabolites in nonbrain

1028 A Analytical Chemistry, Vol. 66, No. 20, October 75,1994

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muscle peaked after 2 h, whereas the level in brain peaked after - 4 h. Thus, although there is some delay for Li to cross the blood-brain barrier, the delay is short and cannot account for the wellknown delay of 1week for Li to exert clinical efficacy. Figure 4b shows serial results for the same subject after he was taken off of Li treatment because he developed a tremor (19). Initially the Li levels dropped r a p idly. At six days after terminating Li therapy, 7Li in vivo NMR no longer detected Li in muscle but did detect it in brain. At 10 days, no signal was detected from brain. This result is of interest because the toxic symptoms took about a week to s u b side, a result that suggests that in vivo 7Li NMR could be used to relate toxic effects to residual brain concentration. Preliminary spectroscopic imaging of the spatial distribution of Li in human brain has also been performed (19) and may shed light on the mechanisms of the therapeutic and toxic effects of Li. Antineoplastic agents. A widely used cytotoxic drug for the treatment of colorectal and breast cancer is 5fluorouraFigure 3. NMR images of the head cil(5FU). The cytotoxic effect of 5FU is of a rat dosed with lithium. attributed to the anabolic formation of flu(a) Midline sagittal 'H NMR image used for localization of 7Li imaging. (b) 7Li image after oronucleosides and fluoronucleotides a multidose protocol of intraperitoneal LiCI. In that interfere with DNA and RNA metabothis image, the outer line defines the shape lism. The drug, which is used in combiand location of the rat head, and the inner line the brain and spinal cord. The pulse repetition nation with other compounds that modutime 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, - 200?of patients. Clinicians still cannot with a 7-mm slice thickness. (Adapted with permission from Reference 16.) predict who will respond to the drug, despite its long history of use. Lack of response 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 0.1a-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 inLi is eliminated from the body relatravenous 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 chemonths and was considered to be at steady motherapy for breast cancer (20).Bestate (18).After a single 300-mg dose of cause they are quite different chemically, Li,CO,, the concentrations in brain and 5-FU and its catabolite FBAL are well recalf muscle (by 7Li NMR) and in serum solved spectroscopically. Under these (by flame photometry) were monitored conditions of drug administration, sensitivfor 6 h. At the low time resolution of these ity is sufficient to acquire a spectrum in experiments, the levels in serum and several minutes, permitting pharmacoki-

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netic analysis. Unfortunately, the therapeutically important metabolites are usually not seen, except with extensive signal averaging (21). Of course, more important than the metabolism in healthy liver is the metabolism in tumors. Wolf (22),Presant (23,24), and co-workers have been using 19FNMR in vivo to monitor the pharmacokinetics of 5-FU elimination from human tumors as a predictor of therapeutic response to the drug. They observe only the disappearance of 5-FU, not FBAL or therapeutic metabolites, in tumors. Their hypothesis, which is supported by human and animal data, is that responding tumors pool 5-FU and display a long t1,2 for elimination; non-

Figure 4. Li concentrations in serum, brain, and calf muscle. (a) Concentration profiles for a subject at steady state after a 300-mg oral dose of Li,CO,. (Adapted with permission from Reference 18.) (b) Plots of Li concentrations for a subject removed from Li therapy. The serum values are plotted to the lowest level reported in the serum Li assay. (Adapted with permission from Reference 19.)


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 probably arises from factors such as vascularization of the tumor and transmembrane transport of the drug into tumor cells. At last report (24),this trapping behavior was proving true for the 57 patients studied to that point, although many are still being evaluated for clinical response. Of 9 patients with tumors that demonstrated trapping, 8 had partial responses to 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.

If the correlation between drug trapping and therapeutic response holds for a sufficiently large number of patients, this technique should be useful for developing and optimizing treatment protocols. It could also be considered for application to real-timeclinical decision making in individual chemotherapy (25).For example, 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 pharmaceutical containing boron is administered to a cancer patient prior to irradiation of tumorcontaining regions with thermal neutrons. Collision of the neutrons with O ' B nuclei releases alpha particles, which have relatively short trajectories and hence only damage cells in the immediate vicinity of the collision. A technique to probe the distribution of such pharmaceutical agents in tissue would be valuable for their development and perhaps 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 pharmacokinetics of BNCT agents in laboratory animals. 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 feasibility 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 indirect detection of O ' B via spincoupled 'H NMR, whereby the 'H signal is detected with and without modulation (by heteronuclear decoupling or insertion of 180" Figure 5. Stacked plot of sequential pulses) of the 'H-"B coupling. Upon s u b in vivo ''F NMR spectra. traction, only the protons coupled to O 'B The patient received a bolus injection remain and are detected at the higher 'H (600 mg/m2 of body surface area 2 h after administering methotrexate) of 5-FU over sensitivity. The indirect detection of het10 min (-5 to +5 min). Each spectrum is the eronuclei using spincoupled protons is baseline-corrected result of 200 acquisitions an approach that could see increasing use (- 3.5 min/spectrum). The chemical shift scale is in ppm from 5-FU. in a variety of drug studies in vivo.

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1030 A Analytical Chemistry, Vol. 66, No. 20, October 15, 1994

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 surgery is commonly maintained using fluorine-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 anesthetics could be observed in animal tissues using in vivo 'F NMR spectroscopy, although there was considerable controversy concerning the pharmacokinetics of elimination, the spatial origin of the signals, and the number of signal components (2S31). Particular care is required in unlocalized studies of such lipophilic molecules because of the possibility of contamination from components in fat; the multiple time constants for elimination; the short T2s,which complicate observation of the total 'F signal; and variable physiological factors. It appears that volatile fluorinated anesthetics may exist in more than one environment in brain, and measurable concentrations may persist for a relatively long time. Menon et al. (32) have demonstrated the possibility of observing cerebral halothane in patients up to 90 min after surgery by using 'F NMR in vivo. More work is necessary to determine the spatial origin of the signals, the elimination pharmacokinetics, and the metabolism of these anesthetics before such studies can have clinical utility.

Measurement of oxygen tension. Because of their biochemical inertness and ability to dissolve large amounts of oxygen, liquid perfluorocarbon compounds have shown increasing utility as respiration and blood substitutes. "F MRI and NMR spectroscopy have been used to monitor these compounds in vivo (8). One particularly interesting spectroscopic property of these compounds is that their 19Fspin-lattice relaxation rates (l/Tl) depend linearly on the partial pressure of dissolved oxygen (Po,>. In a novel application, Wilson and coworkers (33)have used "F NMR to measure Po, at the surface of the retina in a human eye. Such a measurement is important 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 measured 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, longitudinal measurement of Po, in the human eye to gain further insight into the evolution of ischemic ocular diseases.

lyzed for "F NMR and thus also make 'H NMR less desirable for in vivo detection of drugs. It may be possible to monitor drugs with a low therapeutic index (drugs that require administration of relatively large amounts to achieve therapeutic efficacy), drugs that accumulate in the organ of interest, or drugs that reach h g h local concentrations for short periods (e.g., 5FU). Many drugs contain one or more aromatic

rings in their molecular structure, and this region in the 'H NMR spectrum in vivo is relatively free of potentially interfering background resonances. Ethanol in brain. Ethanol has been detected by localized 'H NMR spectroscopy in human and animal brain in vivo (35-38). Because ethanol freely penetrates the blood-brain barrier and is typically ingested in larger amounts than ther-

Localized 'H NMR of drugs

in vivo

Given that most drugs do not contain an NMR label such as "F, it might be expected that the more generally applicable and sensitive nucleus 'H would be preferable for detection of drugs in vivo. Applications of gradient-localized in vivo 'H NMR spectroscopy are becoming widespread, and the technique is beginning to have clinical impact (34).However, the presence of signals from endogenous metabolites at 1mmol/L or greater substantially raises the minimum concentration of a drug that can be detected. The relatively small range of chemical shifts and the necessity of suppressing the large H,O peak make it more difticult to use the very large volumes commonly ana-

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Figure 7. #undus photograph of a human eye and "F Tl measurement. (a) After repair of a retinal tear and detachment with the use of intraoperative FTBA; residual 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, 600, 800, 1000, 1200, and 4000 ms, front to back) and was used to measure T,. (Adapted with permission from Reference 33.)


the distribution of silicone in various organs in animals and humans. Observation of silicone is seemingly straightforward given the fact that the six equivalent Si(CH,), protons resonate upfield of common in vivo resonances. However, these workers found it necessary to mod* existing pulse sequences to generate efficient suppression of both water and fat resonances. Figure 9a shows a STEAM localized 'H spectrum of a voxel from the liver of a patient who had silicone implants for 18 years. A strong resonance attributed to both hydrolyzed and chemically unchanged 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 clinically important cases.

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Hgure 8. In vivo spectroscopic localizationof water, NAA, and ethanol plus lipids in human brain. Inversion time, 200 ms; echo time, 272 ms; repetition time, 2 s;1.5 x 1.5 x 1.5 cm voxels; 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 permission from Reference 37.)

apeutic drugs, it is relatively straightforThe image of ethanol distribution shows 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 estiand correlation with blood alcohol concen- mated as 23.0,16.6,and 8.3 mmol/L, r e tration (BAC). However, issues surspectively. If the issues of signal visibility rounding signal visibility and interference and multiple pools of ethanol can be clarifrom lipid signals have been raised, parfied 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 physiologto be only 23%of BAC when N-acetylaspar- ical effects to brain concentration and tate (NAA) was used as an internal referdistribution. The interaction of ethanol ence. The reduced visibility was attributed with membranes may be related to an indito 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 contromanner to the phospholipid cell memversy regarding the Sde@of Silicone gelbranes in brain tissue. filled breast imolants. Concerns include More recent work (37),in which spec- the possible rupture of the siliconetroscopic imaging (SI) was used at a sparubber membrane encasing the gel and tial resolution of 1.5 cm, demonstrated that the bleeding of free silicone through the the distribution of ethanol in the brain is intact envelope into the surrounding tisnonuniform. 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

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

Future prospects

NMR spectroscopy and imaging are novel, noninvasive techniques for measuring the concentration, distribution, and pharmacokinetics of certain drugs in vivo in humans and animals. A potentially powerful feature of in vivo NMR is the ability to measure simultaneously the tissue con-

,

Figure 9. Localized 'H spectrum of a voxel in the liver for (a) a patient with and (b) a subbCt without silicone implants. The spectra were acquired using the STEAM SeQuencecombined with inversion iecovery and chemical shift-selectivesatura-

permission from Reference'40.)

'

centration of a drug and related changes in brain metabolism, for example, by 'H or 31PNMR monitoring of endogenous metabolites (41). Although NMR techniques have significant limitations, in particular low sensitivity, their noninvasive character and unique information content ensure that new applications and methodological improvements will continue to appear. Higher magnetic fields, more sensitive detection coils, and postprocessing methods such as Bayesian statistical estimation of spectral parameters (42) should substantially reduce the minimum detectable drug concentration in vivo and/or improve spatial resolution. Very recently, Albert et al. (43) obtained magnetic resonance images of mouse lungs using 12%egas (a safe general anesthetic) that had been magnetically hyperpolarized by spin exchange with optically pumped Rb vapor. This procedure produced 105-fold enhancement 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-polarized Xe in tissue may be a sensitive probe of the local environment or of 0, concentration via the ',%e chemical shift or spin relaxation times.

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I thank J.E.O. Newton for his careful reading of the manuscript.

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Richard A. Komoroski received his B.S. degree from St. Louis University in 1969 and his Ph.D. in physical chemistryfiom Indiana University in 1973. After completing 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 University of Arkansas for Medical Sciences (Departments 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.
