[CANCER RESEARCH 61, 6524 – 6531, September 1, 2001]
Mapping Extracellular pH in Rat Brain Gliomas in Vivo by
1
H Magnetic Resonance
Spectroscopic Imaging: Comparison with Maps of Metabolites
1
Marı ´a-L. Garcı ´a-Martı ´n, Gwe ´nae ¨l He ´rigault,
2
Chantal Re ´my, Re ´gine Farion, Paloma Ballesteros, Jonathan A. Coles,
Sebastia ´n Cerda ´n, and Anne Ziegler
3
Instituto de Investigaciones Biome ´dicas, Consejo Superior de Investigaciones Cientificas, 28029 Madrid, Spain [M-L. G-M., S.C.]; Unite ´ mixte Institut National de la Sante ´ et de
la Recherche Me ´dicale/Universite ´ Joseph Fourier: U438 “RMN Bioclinique,” Laboratoire de Recherche Correspondant du Commissariat a ` l’Energie Atomique, Centre
Hospitalier Universitaire BP 217, 38043 Grenoble, France [G. H., C. R., R. F., J. A. C., A. Z.]; and Departemento de Quı ´mica Orga ´nica y Biologı ´a, Facultad de Ciencias,
Universidad Nacional de Educacio ´n a Distancı `a, 28040 Madrid, Spain [P. B.]
ABSTRACT
The value of extracellular pH (pH
e
) in tumors is an important factor in
prognosis and choice of therapy. We demonstrate here that pH
e
can be
mapped in vivo in a rat brain glioma by
1
H magnetic resonance spectroscopic
imaging (SI) of the pH buffer ()2-imidazole-1-yl-3-ethoxycarbonylpropionic
acid (IEPA).
1
H SI also allowed us to map metabolites, and, to better under-
stand the determinants of pH
e
, we compared maps of pH
e
, metabolites, and
the distribution of the contrast agent gadolinium1,4,7,10-tetraazacyclodode-
cane-N,N,N,N-tetraaceticacid (Gd-DOTA). C6 cells injected in caudate
nuclei of four Wistar rats gave rise to gliomas of 10 mm in diameter. Three
mmols of IEPA were injected in the right jugular vein from t 0 to t 60
min. From t 50 min to t 90 min, spin-echo
1
H SI was performed with an
echo time of 40 ms in a 2.5-mm slice including the glioma (nominal voxel size,
2.2 l). IEPA resonances were detected only within the glioma and were
intense enough for pH
e
to be calculated from the chemical shift of the H2
resonance in almost all voxels of the glioma.
1
H spectroscopic images with an
echo time of 136 ms were then acquired to map metabolites: lactate, choline-
containing compounds (tCho), phosphocreatine/creatine, and N-acetylaspar-
tate. Finally, T
1
-weighted imaging after injection of a bolus of Gd-DOTA gave
a map indicative of extravasation. On average, the gradient of pH
e
(measured
where sufficient IEPA was present) from the center to the periphery was not
statistically significant. Mean pH
e
was calculated for each of the four gliomas,
and the average was 7.084 0.017 ( SE; n 4 rats), which is acid with
respect to pH
e
of normal tissue. After normalization of spectra to their water
peak, voxel-by-voxel comparisons of peak areas showed that N-acetylaspar-
tate, a marker of neurons, correlated negatively with IEPA (P < 0.0001) and
lactate (P < 0.05), as expected of a glioma surrounded by normal tissue.
tCho (which may indicate proliferation) correlated positively with pH
e
(P < 0.0001). Lactate correlated positively with tCho (P < 0.0001), phospho-
creatine/creatine (P < 0.001), and Gd-DOTA (P < 0.0001). Although lactate
is exported from cells in association with protons, within the gliomas, no
evidence was observed that pH
e
was significantly lower where lactate con-
centration was higher. These results suggest that lactate is produced mainly
in viable, well-perfused, tumoral tissue from which proton equivalents are
rapidly cleared.
INTRODUCTION
pH
i
4
in tumor cells is normal or slightly alkaline; in contrast, pH
e
is usually acid compared with normal tissue and, unlike pH
i
, appears
to vary with the type of tumor (1–3) so that measurement of pH
e
is
potentially more informative than measurement of pH
i
. Knowledge of
pH
e
is important not only for diagnosis but also for choosing chemo-
therapeutic agents, because most are weak bases or weak acids, and
their accumulation within cells and, hence, their efficacy depends on
the transmembrane pH gradient (1, 4 –7). In addition, the effectiveness
of thermoradiotherapy has been reported to correlate with the extra-
cellular acidity (8). Therefore, noninvasive measurement of tumor
pH
e
might be useful for diagnosis, choice of therapy, and prognosis.
Tumor pH
e
has been measured mainly by invasive techniques that
measure it at a single point, namely microelectrodes (9, 10) and
miniature optical probes (11). The value of these measurements de-
pends on how uniform pH
e
is throughout the tumor. Mean pH
e
within
tumors has also been measured noninvasively by NMR using extra-
cellular probe molecules containing
31
P or
19
F (6, 12–14). Spatial
variations in pH
e
have been measured over distances in the order of
100 m in the tissue between blood vessels in the exposed superficial
layers of a s.c. tumor by optical techniques (15). In the present work,
we have made maps of pH
e
on a larger scale throughout sections of C6
gliomas in rat brain. The tool we used is a new probe molecule, which
has pH-dependent
1
H resonances detectable by
1
H NMR spectros-
copy. This molecule is IEPA. It has been shown that IEPA does not
enter erythrocytes (compound 9 in Ref. 16), and it appears to remain
extracellular in a tumor model in a mouse mammary fat pad where the
1
H signal was sufficient for SI (17). Preliminary results had shown
that systemically delivered IEPA infiltrates the extracellular space of
C6 gliomas in brain and allows mapping of pH
e
(18).
In addition to providing a signal:noise ratio sufficient to allow
imaging of pH
e
rather than just measurement of the average value in
a volume including the tumor, detection by
1
H magnetic resonance
spectroscopy has an additional advantage: using the same radiofre-
quency coil, the distributions of various endogenous compounds with
1
H resonances can be readily imaged in the same experiments. These
include compounds of which the concentrations might be causally
related to the value of pH
e
, notably lactate. Hence, in this paper, we
not only report the use of IEPA to image the distribution of pH
e
within
C6 gliomas, but we have compared this distribution with the distri-
bution of lactate and also of tCho, NAA, and tCr. The results lead us
to consider the reasons why pH
e
in C6 gliomas should be acid. Both
normal brain tissue (19) and tumor cells in particular (20, 21) produce
lactate even under aerobic conditions. Lactate is exported from cells
in association with H
+
(22) and in this way is expected to contribute
to the extracellular acidity. However, Yamagata et al. (23) and Newell
et al. (24) have found that even tumor cells lacking lactate dehydro-
genase, which produced very little lactate in vitro, nevertheless cre-
ated an acid extracellular environment when grown as tumors. By
using IEPA, we have been able to see whether the distribution of pH
e
correlated with that of lactate in vivo.
Received 2/27/01; accepted 6/29/01.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported in part by an Institut National de la Sante ´ et de la Recherche Me ´dicale-
Consejo Superior de Investigaciones Cientificas Collaborative Grants and grants 08.1/
0023/1997 and 08.1/0046/1998 (to S. C.); a Strategic Group Grant (to P. B.) from the
community of Madrid; and grants from La Ligue contre le Cancer, l’Association pour la
Recherche sur le Cancer, and the Re ´gion Rhone-Alpes (to C. R.).
2
Present address: Department of Radiology, Box 8131, Washington University School
of Medicine, 660 South Euclid, St. Louis, MO 63110.
3
To whom correspondence should be addressed, at INSERM U438, CHU Pavillon B,
BP 217, 38043 Grenoble Cedex 9, France.
4
The abbreviations used are: pH
i
, intracellular pH; NMR, nuclear magnetic resonance;
pH
e
, extracellular pH; NAA, N-acetylaspartate; SI, spectroscopic imaging; tCho, total
choline-containing compounds; tCr, creatine and creatine phosphate; CHESS, chemical-
shift selective excitation; OVS, outer volume saturation; TE, echo time; TR, repetition
time; T
2
: transverse relaxation time; pH
a
, arterial pH; Gd-DOTA, gadolinium1,4,7,10-
tetraazacyclododecane-N,N',N'',N'''-tetraaceticacid; IEPA, ()2-imidazole-1-yl-3-
ethoxycarbonylpropionic acid; ppm, parts per million; WSI, water spectroscopic imaging.
6524
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