Magnetic Resonance in Medicine 48:255–261 (2002)
`
`Gastrointestinal Transit Times in Mice and Humans
`Measured With 27Al and 19F Nuclear Magnetic
`Resonance
`Roman Schwarz, Armin Kaspar, Joachim Seelig,* and Basil Ku¨ nnecke
`
`Gastric emptying and gastrointestinal (GI) transit times in mice
`and humans were monitored noninvasively by using 27Al and 19F
`nuclear magnetic resonance (NMR). Al3ⴙ bound to ion-ex-
`change resin and perfluorononane were administered orally as
`selective and specific markers for the stomach and the entire GI
`tract, respectively. 27Al- and 19F-MR spectroscopy (MRS) was
`employed to follow quantitatively boli of the mixed markers in
`awake, fed mice over a period of 48 hr. The selectivity of the
`markers was confirmed by whole-body 1H-, 27Al-, and 19F-MRI
`of anesthetized mice. Gastric emptying in humans was also
`monitored with 27Al-MRS of aluminum-loaded ion exchange
`resin. GI transit was assessed by 19F projection imaging of
`pharmaceutical capsules tagged with perfluorononane. Quan-
`titative analysis of the MR data revealed that gastric emptying in
`humans proceeded linearly, whereas in mice an exponential
`decay was observed. This difference is explained by the respec-
`tive feeding patterns of humans and mice. Humans usually
`achieve nearly complete gastric emptying before each meal. In
`contrast, very short delays between successive food intakes in
`small animals result in successive dilution of the stomach con-
`tents. For stomach emptying in mice the exponential decay
`constant was 74 min, whereas the half-time of the linear gastric
`emptying in humans was 30 min. Magn Reson Med 48:
`255–261, 2002. © 2002 Wiley-Liss, Inc.
`Key words: nuclear magnetic resonance; contrast agent; gas-
`trointestinal tract; transit times; perfluorononane; aluminum-
`resin
`
`Gastrointestinal (GI) dwell and transit times have been
`recognized as crucial factors in the oral administration of
`solid drugs, because precise knowledge of the anatomical
`location of drug release in situ allows the pharmaceutical
`potential of oral drugs to be maximized (1). Moreover, GI
`diseases often elicit changes in GI transit time, which are
`of diagnostic value and provide a means of following up
`therapy of various diseases (2).
`A variety of methods have been applied for assessing GI
`transit times, motility, and drug release. Most prominent
`are the X-ray (3) and scintigraphic techniques (4), which
`have been used to monitor orally ingested capsules con-
`taining radioopaque material or gamma-emitters. On the
`
`other hand, noninvasive techniques such as ultrasound
`(5), metal detectors (6), magnetic field detectors (7), and
`dyes (8) have been used to avoid the adverse effects of
`ionizing radiation. However, all these methods have been
`severely restricted due to intrinsic constraints such as low
`temporal or spatial resolution, lack of complementary an-
`atomical information, or incomplete spatial information.
`As an alternative, MRI techniques could, in principle, offer
`a convenient, noninvasive modality for monitoring GI
`transit that does not suffer from these limitations. Unfor-
`tunately, despite its success in clinical diagnosis, 1H-MRI
`has generally failed in tracing small objects in the bowel
`because of large and intricate local signal changes. Specific
`tagging of capsules with conventional contrast agents
`based on the alteration of the magnetic susceptibility has
`not mitigated this problem (9). Conversely, 19F-MRI of
`fluorinated agents has been shown to provide excellent
`selective contrast in images of the murine GI tract (10,11).
`Due to the lack of endogenous fluorine-containing metab-
`olites in soft tissues, the 19F-MR signal can be assigned
`exclusively to the fluorinated contrast agent present inside
`the GI tract. The intensity of the 19F-MR signal is therefore
`a measure of the amount of contrast agent present in the GI
`tract. By monitoring the intensity variations of the 19F
`signal, the flow of the contrast agent out of the GI tract can
`be followed noninvasively.
`The routine procedure for studying the gastric emptying
`of the solid phase involves labeling ion-exchange resin
`with radioactive isotopes, which are detected by ␥-scintig-
`raphy (12,13). In principle, equivalent assessments using
`MR methods should be feasible if the radioisotopes are
`substituted with magnetic active nuclei. It has been shown
`that 27Al-MRI and 27Al-MR spectroscopy (MRS) of Al3⫹
`released from aluminum-containing antiacidics can be
`used to selectively delineate the stomach (14). 27Al has a
`quadrupolar moment that leads to quadrupole splittings
`that normally broaden the resonance beyond detection.
`However, if the 27Al nucleus experiences a highly sym-
`metric environment, the quadrupolar splitting vanishes
`and the Al3⫹ resonance has a linewidth of only a few Hz.
`This is the case for Al3⫹ in aqueous solution at a pH below
`3.5—a condition typically found in the stomach but not in
`the remainder of the GI tract (14). Al3⫹ readily binds to
`cation-exchange resins in analogy to the radioisotopes
`used for ␥-scintigraphy. Interestingly, such resin-bound
`Al3⫹ has MR properties similar to those of free aluminum
`ions (15). Moreover, aluminum has a very low natural
`occurrence in biological tissues. By taking advantage of
`these properties, Al-loaded resin can be used as a selective
`marker for the gastric contents.
`255
`
`Department of Biophysics, Biocenter, University of Basel, Basel, Switzerland.
`Grant sponsor: Swiss National Science Foundation; Grant number:
`31.49758.96; Grant sponsor: Doerenkamp Zbinden Foundation.
`Presented in part at the 7th Annual Meeting of ISMRM, Philadelphia, 1999.
`B. Ku¨ nnecke’s present address is F. Hoffmann-La Roche, Magnetic Reso-
`nance Imaging and Spectroscopy, PRBD-M, Bldg. 68/05A, CH-4070 Basel,
`Switzerland.
`*Correspondence to: Prof. Joachim Seelig, Dept. of Biophysics, Biocenter,
`University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
`E-mail: Joachim.Seelig@unibas.ch
`Received 16 October 2001; revised 14 March 2002; accepted 14 March 2002.
`DOI 10.1002/mrm.10207
`Published online in Wiley InterScience (www.interscience.wiley.com).
`© 2002 Wiley-Liss, Inc.
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`256
`
`Schwarz et al.
`
`In the present work, the concept of selective 19F and
`27Al markers was applied to quantitative MR assessments
`of gastric and GI emptying in mice and a human subject.
`
`MATERIALS AND METHODS
`Chemicals
`
`Perfluorononane with a nominal purity of 97% was pur-
`chased from Aldrich Chemical Co. (Switzerland). The cat-
`ion-exchange resin used was based on a sulfonated diethe-
`nylbenzene polymer. Two different makes were employed:
`Dowex Marathon C (Sigma, Buchs, Switzerland), with a
`particle size of 30 – 40 mesh, was used for human applica-
`tion;
`for
`the mice, Resonium A (Sanofil Winthrop,
`Mu¨ nchenstein, Switzerland), in powder form, was admin-
`istered.
`
`Preparation of the Contrast Agents
`Preparation of the Combined 19F/27Al Contrast Agent for
`Animal Use
`
`Resonium A was suspended in a saturated solution of
`AlCl3*6H2O for 24 hr. Subsequently, it was washed with
`0.1 M HCl and twice with pure water, both added in at
`least 10-fold excess. For storage, the aluminum-loaded
`Resonium A was dried with air. Prior to the gavage, 100 mg
`of dry Resonium A was soaked in 500 ␮l water for at least
`24 hr. The supernatant was then discarded and 100 ␮l of
`perfluorononane (with 1% detergent) were added. The two
`components were briefly mixed with a spatula and imme-
`diately administered to the animal.
`
`Preparation of the Aluminum Contrast Agent for
`Human Use
`
`Dowex Marathon C (60 g dry weight) was suspended in a
`solution of 90 g AlCl3*6H2O in 1 liter of water for 24 hr
`under constant stirring. Prior to its administration, the
`resin was washed with 1 liter of 0.1 M HCl, and twice with
`1 liter of pure water.
`
`Preparation of the 19F-Filled Capsule for Human Use
`A capsule with a length of 22 mm and a diameter of 7 mm
`(size 0) was made of chemically stable and biologically
`inert polychlortrifluorethylene (PCTFE) and was filled
`with 350 ␮l of perfluorononane (C9F20). The capsule, in-
`cluding its filling, had a specific density of 2.0 g/ml.
`
`GI Assessments in Mice
`
`Eight well-fed female BL/6 mice (21–32 g) were used. The
`animals received a standard laboratory mice chow and
`drinking water ad libitum. Prior to the first MR examina-
`tion, each mouse was given a gavage of 0.3 ml of the
`19F/27Al contrast agent prepared as described above. Im-
`mediately after the gavage the animal was placed in prone
`position in a narrow tube, which was inserted into the
`19F/27Al resonator used for the MR examinations. The tube
`allowed the animal to move back and forth within the
`sensitive volume of the resonator, but hindered the animal
`from turning. MR spectra were routinely acquired every
`30 min and, after stomach emptying, every hour. Gastric
`
`and intestinal emptying were expressed as the time con-
`stant of mono-exponential decays fitted to the experimen-
`tal data.
`In order to assess the location of the contrast agent, two
`of the eight animals were anesthetized immediately after
`the gavage with 1.5% isoflurane in N2O/O2 (3:1) and then
`placed in a prone position in the same resonator as de-
`scribed above. Thirty minutes after the gavage, 1H-, 27Al-,
`and 19F-MR images were acquired without repositioning
`the animals. The animals were allowed to wake up and
`were reanesthetized 3 hr later for reexamination.
`
`GI Assessments in Man
`
`All measurements were performed within the guidelines
`of the local ethics committee.
`
`19F Measurements
`A male volunteer who had fasted overnight was asked to
`swallow a capsule filled with 19F contrast agent while he
`lay inside the magnet. The abdominal region was repeat-
`edly assessed with 1H- and 19F-MR in measuring periods
`of approximately 30 min duration. The first assessment
`was performed immediately before, and the subsequent
`ones at 13⁄4 hr, 31⁄4 hr, 5 hr, 101⁄2 hr, 55 hr, and 75 hr after
`ingestion of the capsule. The first 5 min of each session
`were used for 1H-MRI-guided (re)positioning of the volun-
`teer; the rest of the session was spent on capsule tracking
`by 19F-MR. While the capsule was in the stomach, the
`volunteer was assessed in the supine position. Thereafter,
`while the capsule was moving through the GI tract, the
`volunteer was examined in the prone position. Between
`subsequent assessments, the volunteer was allowed to
`stand up, walk around, and eat according to his usual
`habits.
`
`27Al Measurements
`After ingestion of 5 g wet weight of aluminum-loaded
`resin, 27Al spectra were acquired every 5 min. The volun-
`teer was in the prone position and the surface coil was
`positioned under the gastric region as verified by conven-
`tional 1H-MRI. In a separate experiment, 27Al images were
`recorded 10, 20, and 40 min after the ingestion of 49 g (wet
`weight) Al-loaded resin. After the MR measurements, stool
`was collected to recover the resin.
`
`MRI and MRS
`In Vivo MRS and MRI of Mice
`19F- and 27Al-MR spectra were acquired on a Bruker Bio-
`spec 70/20 (7 Tesla, 20-cm horizontal-bore magnet). An
`in-house-built Helmholtz coil with a diameter of 45 mm
`was used for 27Al-NMR signal excitation and detection. A
`birdcage resonator with an inner diameter of 30 mm was
`used for 19F-NMR. The birdcage coil was located between
`the pair of Helmholtz coils, with its axis orthogonal to the
`orientation of the latter coils. 19F spectra were recorded
`using the following acquisition parameters: 90° pulses
`(94 ␮s), 6000 Hz spectral width, 1024 data points, a total
`recycle delay of 2.1 s, and eight averages. Only the CF3
`group of perfluorononane was recorded, whereas the
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`27Al- and 19F-MR of Gastrointestinal Transit
`
`257
`
`folded signals of the CF2 groups were eliminated by a set of
`high-order analog and digital filters. Aluminum spectra
`were recorded using the following acquisition parameters:
`90° pulses (130 ␮s duration), 12 kHz spectral width,
`512 data points, a total recycle delay of 37 ms, and
`1500 averages.
`For 1H imaging, the 19F resonator was tuned to the
`proton frequency by the addition of a copper shield
`around the resonator (for 19F and 27Al imaging this shield
`was removed). Coronal projections for 19F and 27Al, and
`1-mm slices for proton MRI were obtained over a field of
`view (FOV) of 6 ⫻ 6 cm2 using the following parameters: a
`rapid acquisition with relaxation enhancement (RARE) se-
`quence (16) with a 128 ⫻ 128 matrix, TR/TE ⫽ 1297/75
`ms, and a RARE factor of 16 for 1H MRI; a single-shot
`RARE sequence with a 128 ⫻ 128 matrix, TR/TE ⫽ 2000/
`439 ms, and two averages for 19F-MRI; and a gradient-echo
`sequence with a 64 ⫻ 64 matrix, TR/TE ⫽ 8.2/2.5 ms, and
`1000 averages for 27Al-MRI.
`
`In Vivo 27Al MRS and MRI of Man
`27Al-MR spectra were acquired on a 1.5 Tesla Siemens
`Magnetom Vision (Siemens, Erlangen) equipped with an
`additional broadband channel. An in-house-built, two-
`turn surface coil with an inner diameter of 13.5 cm was
`used for 27Al signal excitation and detection (15). 27Al
`spectra were recorded using the following acquisition pa-
`rameters: pulses of 250 ␮s duration (adjusted in intensity
`to provide maximum signal), 1000 averages, and a total
`recycle delay of 98 ms. 27Al images were acquired by using
`a standard fast low-angle shot (FLASH) sequence with the
`following acquisition parameters: 60° flip angle, TR/TE of
`10.6/3.2 ms, 64 ⫻ 128 pixels covering an FOV of 15 ⫻
`15 cm2, and 300 averages, which resulted in a total scan
`time of 31⁄2 min.
`
`In Vivo 19F-MR Projection Imaging of Man
`
`MR measurements were performed on the same clinical
`MR scanner as described above using an in-house-built 19F
`surface coil integrated into the patient bed, and a commer-
`cial 1H flexi-coil placed opposite to the 19F coil. The 3D
`spatial location of the tracer capsule was determined by
`using projections obtained with a modified fast imaging
`with steady precession (TrueFISP) sequence (17), from
`which the slice and phase gradients had been removed. A
`total of 128 scans for each of the three spatial directions
`were averaged interleaved with a TR of 5.9 ms, resulting in
`a time resolution of 2.26 s. A spatial resolution of 2.7 mm
`was obtained by acquiring 128 data points over an FOV of
`35 cm. Conventional anatomical 1H-MR images were ob-
`tained with a multislice TrueFISP sequence. Coronal
`views with a slice thickness of 6 mm, FOV of 35 ⫻ 35 cm2,
`and TR/TE set to 4.8 ms/2.3 ms were acquired into a data
`matrix of 256 ⫻ 256 points.
`
`RESULTS
`19F and 27Al Measurements in Mice
`
`Figure 1a shows a coronal slice through the abdominal
`region of a mouse obtained with 1H-MRI. Figure 1b and c
`
`shows corresponding coronal projections made at the 27Al
`and 19F resonant frequency. All three images were ac-
`quired about 20 min after contrast agent administration,
`without repositioning the mouse between the different
`scans. Figure 1a delineates the anatomy of the abdominal
`region close to the ventral abdominal wall. Due to proton
`depletion caused by the perfluorinated contrast agent or
`the general lack of protons, the stomach and several loops
`of the bowel, as well as the lungs, were depicted as dark
`areas. 27Al-MRI (Fig. 1b) detected a bright region at the
`location of the stomach. No other signal, with the excep-
`tion of some scanner-specific noise at the upper fringe of
`the image, could be observed. Similarly, 19F-MRI (Fig. 1c)
`also detected a region of strong signal intensity at the
`location of the stomach. However, this region has a caudal
`extension, which can be assigned to fluorine that has al-
`ready left the stomach and has entered the duodenum.
`After the animal had been awake for 3 hr, it was reassessed
`with 1H-, 27Al-, and 19F-MRI using the same acquisition
`parameters as in the first examination. The anatomical
`1H-MR image (Fig. 1d) shows few changes, aside from that
`caused by a slightly different positioning of the animal
`within the probe. However, the 27Al signal has almost
`completely vanished (Fig. 1e) and the fluorine signal is
`distributed within different loops of the bowel (Fig. 1f).
`In Fig. 2, representative in vivo 27Al- (left stack) and
`19F-NMR spectra (right stack) acquired in an awake animal
`after administration of a bolus of contrast agent are plotted
`against time. Both time courses show distinct decreases of
`signal intensities over time. Since the 27Al signal can only
`be observed while the contrast agent is in an acidic envi-
`ronment, the decay of the 27Al signal (left stack) represents
`the stomach emptying. On the other hand, perflu-
`orononane is metabolically inert and is not resorbed. The
`only way it can leave the body is by rectal excretion.
`Therefore, the decay of the 19F signal represents the time
`course of emptying of the entire GI tract.
`For a more quantitative analysis of gastric and GI emp-
`tying, the 27Al- and 19F-MR signals were integrated and
`plotted against time. Figure 3 shows the time courses of
`these signals in the same animal as used in Fig. 2. The
`circles (aluminum resonances) show the stomach empty-
`ing, and the diamonds (fluorine signal) indicate the emp-
`tying of the entire GI tract. The stomach emptying follows
`an exponential decay starting immediately after adminis-
`tration of the contrast agent. The release of the fluorine has
`a pronounced lag phase, which is also followed by an
`exponential decay. The 19F-NMR signal does not return to
`zero because a small amount of perfluorononane remains
`in the stomach (verified by 19F-MRI). This rest is com-
`pletely eliminated after approximately 3 days (not shown).
`The experiment was repeated with a total of six animals.
`For stomach emptying, the exponential decay constants
`averaged ␶ ⫽74 ⫾ 17 min, whereas for the GI transit the lag
`time until perfluorononane excretion started was 154 ⫾
`24 min. However, the rates of gastric emptying, as well as
`the lag times, showed considerable interindividual varia-
`tions. Large variations were also detected for repetitive
`measurements of
`the same animal. Nevertheless,
`the
`shapes of the curves were the same for all mice, except for
`two individuals that refused to eat. In those two cases, no
`decrease of the 19F-MR signal could be observed. On the
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`258
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`Schwarz et al.
`
`FIG. 1. Upper trace: (a) 1H-, (b) 27Al-, and (c) 19F-MR images of the abdominal region of an anesthetized mouse acquired approximately
`20 min after the gavage of 19F/27Al contrast agent. Lower trace: (d) 1H-, (e) 27Al-, and (f) 19F-MR images of the abdominal region of the same
`mouse as above, but obtained from the reanesthetized animal, which had been allowed to move freely for 3 hr.
`
`other hand, stomach emptying had taken place, but at a
`significantly slower rate.
`Apart from signal decay over time, the 19F resonances
`showed intricate line-shape variations that were attributed
`to macroscopic magnetic field inhomogeneities in the an-
`imal’s bowel. There was a trend toward a shift to higher
`frequencies at later time points, suggesting that deconvo-
`lution of the observed line shape may provide additional
`information as to the current location and/or the environ-
`ment of the perfluorinated contrast agent. However, be-
`cause of its complexity this potential source of information
`was not further assessed in the present study.
`
`27Al Measurements in Man
`
`GI assessments akin to those carried out with mice were
`also performed in the human subject. Figure 4 shows a
`coronal projection obtained by 27Al-MRI of the abdominal
`region in man. The image was acquired 10 min after the
`ingestion of aluminum-loaded resin. A bright area is visi-
`ble at the location of the stomach (as verified by 1H-MRI;
`not shown); no signals were detected in other regions.
`For a quantitative analysis of the gastric emptying, the
`27Al-MR signal was integrated and plotted against time.
`
`FIG. 2. Time course of in vivo 27Al- (left) and 19F-NMR spectra (right)
`obtained from an awake mouse after gavage of 27Al/19F contrast
`agent. The aluminum signal intensity represents pure gastric emp-
`tying, whereas the fluorine signal arises from both the stomach and
`the intestinal tract and represents the course of emptying of the
`entire GI tract. The decrease of the aluminum signal is significantly
`faster than that of the fluorine signal, which, in addition, shows a
`typical lag phase.
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`27Al- and 19F-MR of Gastrointestinal Transit
`
`259
`
`FIG. 3. The integrated signal intensities of the 27Al- and 19F-NMR
`resonances depicted in Fig. 2 as a function of time. The aluminum
`data (circles) show the stomach emptying, and the emptying of the
`entire GI tract is represented by the fluorine data (diamonds). The
`stomach emptying as described by 27Al-NMR follows an exponen-
`tial decay beginning immediately after administration of the contrast
`agent. The fluorine intensity remains approximately constant for the
`first 2 hr and then starts decreasing exponentially. The offset in the
`fluorine curve arises from a small amount of perfluorononane that
`remains in the stomach for approximately 3 days.
`
`Figure 5 exemplifies a typical time course. A linear signal
`decrease can be observed. After 60 min the amount of resin
`inside the stomach was below the limit of detection. In
`light of the pH dependence of the 27Al signal originating
`from ingested aluminum resin (as outlined above for
`mice), this decrease reflects the gastric emptying. If a linear
`decay is fitted to the time course, an emptying rate of
`
`FIG. 5. The signal integral of 27Al in a human stomach vs. time after
`ingestion of Al-loaded resin.
`
`1.64%/min is obtained, which corresponds to a half-time
`of about 30 min.
`
`19F Measurements in Man
`
`Emptying of the entire GI tract in man was assessed with
`19F-MR of perfluorononane in analogy to the measure-
`ments performed in mice. However, the perfluorononane
`was not given as a liquid; instead it was enclosed in an
`inert capsule. Figure 6 shows a typical coronal view of the
`abdominal region obtained shortly after ingestion of such a
`capsule. Due to the complete absence of protons, the cap-
`sule could be identified as a black bar (arrow) in the
`stomach, the latter being partially filled with gastric juice.
`
`FIG. 4. Coronal projection of the abdominal region of a man, ac-
`quired by 27Al-MRI. The bright region represents the stomach,
`which was filled with Al resin.
`
`FIG. 6. Coronal view of the abdominal region of a volunteer ob-
`tained shortly after ingestion of a pharmaceutical capsule filled with
`perfluorononane. Due to complete proton depletion, the capsule
`could be identified as a black bar (arrow) in the stomach.
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`260
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`Schwarz et al.
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`and GI transit times in humans and small animals. Re-
`cently, new GI contrast agents to selectively delineate the
`stomach and the bowel were developed (10,14). These
`contrast agents are based on the use of X-nuclei, i.e., 27Al
`and 19F, which are directly imaged by non-proton MRI
`(10,14). In the present study, this concept of active MRI of
`the contrast agent itself has been adapted for MRS assess-
`ments of the GI tract. 27Al- and 19F-containing contrast
`agents were employed as specific markers for the gastric
`and GI contents, respectively.
`In analogy to the well-established procedure using scin-
`tigraphy in combination with radioactively-labeled ion-
`exchange resin, the rate of gastric emptying of solids was
`measured by 27Al-MRS after oral administration of alumi-
`num-loaded resin. In mice the gastric emptying followed
`an exponential time course, whereas in man a rather linear
`decay was observed. This latter finding is in excellent
`agreement with the gastric-emptying behavior of solids in
`man reported previously (5,18). A linear emptying rate has
`also been postulated for rats, and experimental data have
`been analyzed accordingly (19). However, reevaluation of
`these data also suggest an exponential rate of stomach
`emptying similar to that observed in our experiments.
`Simple modeling may explain the distinct differences ob-
`served for gastric emptying in man and mice. In contrast to
`humans, small animals tend to have feeding patterns with
`very short delays between successive food uptake. Based
`on the premise that the freshly eaten food matter is mixed
`with the gastric contents, an exponential dilution of the
`bolus of contrast agent is expected. In humans, a nearly
`complete gastric emptying is usually achieved before the
`next meal, and thus no continuous dilution of the solid
`contrast agent takes place.
`In the present study, GI emptying was assessed with
`19F-MRS by observing the clearance of the GI marker from
`the body. For mice, the lag phase between administration
`and the start of the elimination of perfluorononane was
`considered a measure of the GI transit time. Since perflu-
`orononane has a low viscosity and a low surface tension, it
`could be argued that perfluorononane passes through the
`GI tract faster than fecal matter. However, it has been
`shown that the inhibition of peristalsis by anesthesia or
`lack of food input completely prevents perfluorononane
`excretion (10). It can therefore be postulated that the lag
`phase recorded with 19F-MRS of perfluorononane is pro-
`portional to the emptying rate of the GI tract. The relatively
`large inter- and intra-individual variations of the emptying
`rates may at first appear to be a matter of concern. How-
`ever, such variations have also been reported by others for
`GI transit in both humans and small animals (18,19), and
`thus do not represent a problem inherent in the MR meth-
`ods applied in the present study. Our observations indi-
`cate that the animals’ current attitude toward food intake
`is a major determinant for gastric and GI transit.
`In conclusion, we have shown that 27Al- and 19F-MR
`provide a novel noninvasive modality for assessing gastric
`and GI transit in awake animals and humans. By taking
`advantage of the pH sensitivity of Al3⫹ in an aqueous
`environment, 27Al-MRS in combination with aluminum-
`loaded resin could be used as a selective marker for gastric
`emptying of solids. In contrast to analogous scintigraphic
`methods, which use nonselective radioactive markers, the
`
`FIG. 7. 3D plot of the capsule’s passage through a part of the small
`intestine as observed by 19F-MR 208 –213 min after ingestion of a
`pharmaceutical capsule filled with perfluorononane. The black bars
`connect the positions of the capsule at adjacent time points (time
`resolution ⫽ 2.26 s).
`
`However, as soon as the capsule had left the stomach and
`entered the intestine, 1H-MRI failed to unambiguously de-
`pict the small capsule because of the very heterogeneous
`contrast and intricate intensity distribution in the intes-
`tine. In contrast, 19F-MR projection imaging provided un-
`equivocal spatial information on the location of the GI
`probe, and allowed the capsule to be followed throughout
`the entire GI tract, with a temporal and spatial resolution
`of 2.26 s and 2.7 mm, respectively.
`Figure 7 shows a representative 3D plot of the capsule’s
`passage through a part of the small intestine as observed by
`19F-MR 208 –213 min after ingestion of the capsule. Over
`this period, the capsule was pushed along one of the many
`loops of the intestine, resulting in an L-shaped movement
`from right to left and head to foot (dots connected with
`bars represent the capsule’s position at adjacent points in
`time). The path of the capsule was superimposed with
`jitters, which was ascribed to the rhythmic movement of
`the intestine due to respiration. The most extensive move-
`ments of the capsule, however, were observed after each
`period of light physical exercise which the volunteer was
`asked to perform outside the magnet. Complementary
`1H-MR images ensured a nearly perfect repositioning of
`the volunteer for subsequent scans, and provided a means
`of correlating the position of the capsule with the anatom-
`ical structures of the GI tract. After its ingestion, the cap-
`sule remained in the stomach for 2 hr before the gastric
`emptying occurred. Thereafter, the capsule could be fol-
`lowed along its path through the small intestine and colon.
`The capsule was ultimately excreted after 57 hr.
`
`DISCUSSION
`
`The aim of this study was to implement a novel MR-based
`modality for quantifying noninvasively gastric emptying
`
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`

`27Al- and 19F-MR of Gastrointestinal Transit
`
`261
`
`specificity of 27Al-MRS excludes spuriously slow rates of
`gastric emptying that may arise due to the spatial overlap
`of the bowel and stomach. On the other hand, 19F-MRS of
`perfluorononane provides a fast and easy method for quan-
`tifying GI transit. Perfluorononane is neither resorbed nor
`metabolized, and is therefore an ideal marker for the GI
`tract. Finally, 19F-MR projection imaging of 19F capsules in
`combination with 1H-MRI proved to be a robust method
`for assessing GI transit, and provided high temporal and
`spatial resolution in three dimensions together with func-
`tional and anatomical information. The concepts of mag-
`netic labeling with biologically rare X-nuclei can be
`readily extended to the simultaneous observation of sev-
`eral chemically different probes. Moreover, nontoxic con-
`trast agents, such as perfluorononane, also allow the loca-
`tion of pharmaceutical capsule disintegration to be evalu-
`ated in situ.
`The clinical utility of the presented method remains to
`be proven with a larger number of subjects. The feasibility
`of monitoring gastric and GI transit times noninvasively in
`humans with MR, however, has been clearly demonstrated
`herein.
`
`ACKNOWLEDGMENTS
`
`The authors are grateful to Dr. Klaus Scheffler for writing
`the 19F projection imaging sequence.
`
`REFERENCES
`
`1. Hirtz J. The gastrointestinal absorbtion of drugs in man: a review of
`current concepts and methods of investigation. Br J Clin Pharmacol
`1985;19:77S– 83S.
`2. Camilleri M, Hasler WL, Parkman HP, Quigley EM, Soffer E. Measure-
`ment of gastrointestinal motility in the GI laboratory. Gastroenterology
`1998;115:747–762.
`3. Hinton JM, Lennord-Jones JE, Young AC. A new method for studying
`gut transit times using radioopaque markers. Gut 1969;10:842– 847.
`
`4. Camilleri M, Colemont LJ, Phillips SF, Brown ML, Thomforde GM,
`Chapman N, Zinsmeister AR. Human gastric emptying and colonic
`filling of solids characterised by a new method. Am J Physiol 1989;257:
`G284 –G290.
`5. Amend M, Greiner L. The ultrasound capsule: a new method for mea-
`surement of gastrointestinal motility. Ultraschall Med 1996;17:274 –
`276.
`6. Ewe K, Press AG, Dederer W. Gastrointestinal transit of undigestible
`solids measured by metal detector EAS II. Eur J Clin Invest 1989;19:
`291–297.
`7. Weitschies W, Wedemeyer J, Stehr R, Trahms L. Magnetic markers as a
`noninvasive tool to monitor gastrointestinal transit. IEEE Trans Biomed
`Eng 1994;41:192–195.
`8. Leng-Peschlow E. Effect of sennosides and related compounds on in-
`testinal transit in the rat. Pharmacology 1988;36:40 – 48.
`9. Patak MA, Weishaupt D, Frohlich JM, Debatin JF. Sequential fast 3D
`MRI following oral ingestion of Gd-DOTA: a new means to assess
`intestinal transit time. J Magn Reson Imaging 1999;10:474 – 476.
`10. Schwarz R, Schuurmans M, Seelig J, Ku¨ nnecke B. 19F-MRI of perflu-
`orononane as a novel contrast modality for gastrointestinal imaging.
`Magn Reson Med 1999;41:80 – 86.
`11. Anie SJ, Fell JT, Waigh RD, Wood B. Gastrointestinal transit studies in
`the rat using 19F-NMR. Int J Pharmacol 1991;76:183–185.
`12. Thomforde GM, Camilleri M, Phillips SF, Forstrom LA. Evaluation of
`an inexpensive screening scintigraphic test of gastric emptying. J Nucl
`Med 1995;36:93–96.
`13. Breiter N, Sany T. Development of methods for measuring the emptying
`of solid and liquid test meals in rat stomach. Arzneimittelforschung
`1993;43:694 – 698.
`14. Kaspar A, Bilecen D, Scheffler K, Seelig J. Aluminium-27 nuclear
`magnetic resonance spectroscopy and imaging of the human gastric
`lumen. Magn Reson Med 1996;36:177–182.
`15. Kaspar A. 27Al in vivo NMR Spektroskopie und Bildgebung. Ph.D.
`thesis, University of Basel, Switzerland, 1996.
`16. Hennig J, Nauerth A, Friedburg H. RARE imaging: a fast imaging
`method for clinical MR. Magn Reson Med 1986;3:823– 833.
`17. Oppelt A, Graumann R, Barfuss H, Fischer H, Hartl W, Schajor W. FISP:
`eine neue schnelle Pulssequenz fu¨ r die Kernspintomographie. Electro-
`medica 1986;54:15.
`18. Brophy CM, Moore JG, Christian PE, Egger MJ, Taylor AT. Variability of
`gastric emptying measurements in man employing standardized radio-
`labeled meals. Dig Dis Sci 1986;31:799 – 806.
`19. Chang F, Lee S, Yeh G, Wang PS. Comparison of two orogastric feeding
`markers for measuring gastrointestinal motor functions in rats. Phar-
`macology 1994;49:151–158.
`
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