 |
INTRODUCTION |
Because of the dependence of the chemical shift of phosphates on
pH (1-3), 31P NMR spectroscopy has progressively become
the standard method for the measurement of intracellular pH
(pHi)1 in biological
systems, mostly using inorganic orthophosphate (Pi) as a
naturally occurring pH probe (4-6). Although this technique was first
applied to measure pH in a variety of biological fluids, the
development of pulsed Fourier transform NMR and wide-bore supraconducting magnets has soon allowed the noninvasive study of cell
cultures and isolated perfused organs under physiologic or pathologic
conditions. In particular, the use of 31P NMR has
considerably increased our understanding of the dynamics of pHi
changes during ischemia-induced acidosis in the heart (7-9) and liver
(10, 11). Although Pi resonance has been successfully used
to describe the main pHi-regulatory systems in these organs
(12, 13), more subtle trans-sarcolemmal proton movements that could
occur in different pathologies may escape investigation if they are
related to extra- and intracellular pH values different by less than
0.2-0.3 pH units (4). In addition to this relative lack of resolution,
Pi levels vary with cell metabolism, and the chemical shift
of Pi has been demonstrated to be affected by ionic
strength (4-6).
The search for improved exogenous 31P NMR pH indicators as
alternatives to Pi yielded a variety of alkyl- and
aminoalkylphosphonic acids having their resonance peak distinct
from that of phosphorylated metabolites, and an NMR sensitivity

ab (defined as the mean difference between the
chemical shifts of the protonated
a and the unprotonated
b forms) in the range of Pi (i.e.
2-3 ppm). There have been a number of studies on the in
vitro properties of synthetic alkylphosphonates as 31P
NMR pH probes (14-20), but only a few have focused on more complex systems such as the isolated perfused bladder (21), heart (22, 23), or
liver (24). In these studies, phenylphosphonate was found to be a
specific extracellular pH and volume marker (21-24), but even though
it was reported to allow the monitoring of pHi in the liver
where it can reach a suitable degree of cytosolic uptake (24), no
information could be obtained on the occurrence of more acidic
organelles participating in proton transport mechanisms (25, 26).
By introducing an amino function in the
or
position of the
alkyl group of the charged phosphonate structure, suitable NMR pH
probes of lower pKa values (in the range 5.5-6.3) that entered in Dictyostelium discoideum amoebae were used
to study the pH compartmentation and the kinetics of endosomal
acidification (27) and the effect of ammonia on cytosolic and vesicular
pH (28). In view of these previous results (27, 28), we have recently
proposed that uncharged aminophosphonates that protonate at the amine
function may show enhanced properties as 31P NMR pH probes
in biological systems (29). A large series of such uncharged
- and
-aminophosphonates have been tested as pH markers in standard
perfusion buffers and found to give access to a large range of
pKa values, with a 4-fold better NMR sensitivity
than Pi and other previously reported alkyl- and aminophosphonates (29).
In the present study, the non-toxic
-aminophosphonate
diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH), having a
pKa of 6.8 at 37 °C and an extended

ab of 9.7 ppm (29), was used to probe the pH changes
occurring during ischemia/reperfusion of isolated rat hearts and
livers. It was observed that DEPMPH, which did not affect the cellular
phosphorylated metabolite content during ischemia in both organs,
allowed for the first time the simultaneous and accurate
31P NMR monitoring of extra- and intracellular pH in the
normoxic and ischemic heart and liver. In the liver, use of DEPMPH as
31P NMR pH probe offered the possibility to investigate in
real time the ischemia-induced pH changes associated with the acidic compartments (pH < 5.5).
 |
MATERIALS AND METHODS |
Chemicals--
Ultrapure DEPMPH was obtained by as described
previously (30) by using commercial 2-methylpyrroline and diethyl
phosphite as starting material (Aldrich). All other reagents were of
analytical grade. Doubly distilled deionized water was used throughout.
All solutions were filtered through a 0.2-µm filter prior to use.
Isolated Liver and Heart Preparations--
Male Wistar rats
weighing 80-100 g (liver studies) or 300-390 g (heart studies) were
anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg body
mass), and the studied organ was immediately perfused at 37 °C in a
non-recirculating mode either via the portal vein (liver studies) or
the ascending aorta (heart studies). Isolated perfused hearts were
instrumented to provide continuous monitoring of hemodynamic function
as described below. The perfusion medium (pH 7.35) used in liver
(heart) studies was a Krebs-Henseleit buffer consisting of 143 (144)
mM Na+, 5.0 (5.8) mM
K+, 2.0 (2.5) mM Ca2+, 1.2 (1.2)
mM Mg2+, 127 (129.8) mM
Cl
, 25 (25) mM HCO3
,
1.2 (0) mM
H2PO4
/HPO42
,
2.0 (1.2) mM SO42
, 0 (10)
mM glucose, and 0 (0.5) mM EDTA (throughout
referred to as "standard" buffer). The buffer was saturated with
95% O2, 5% CO2 by using a membrane oxygenator
(liver studies) or by bubbling (heart studies). Perfusion was performed
at a flow rate of 3 ml·min
1·g
1
liver wet weight or at a constant pressure of 100 cm of H2O
in hearts. For NMR experiments, organs were inserted into a 20-mm NMR
sample tube that was placed into the wide-bore 9.4-Tesla magnet of the spectrometer.
Experimental Groups and Perfusion Protocols for NMR
Experiments--
In a recent study, it was found that DEPMPH did not
affect base-line cardiac function when perfused up to 10 mM
in normoxic rat hearts (30). Consequently, the selected DEPMPH
concentration for all perfusion experiments was 5 mM. Liver
perfusion at 37 °C (n = 6) included a 20-min
perfusion with plain standard buffer followed by a 20-min normoxic
perfusion with DEPMPH, 60 min of ischemia, and a final 20-min
reperfusion with plain buffer. Heart perfusion at 37 °C
(n = 6) included a 30-min normoxic control period
followed by 30 min of no-flow normothermic ischemia and a final 60-min
reperfusion. In these latter experiments, DEPMPH was added to the
perfusion medium during the last 15 min of control period and the first
5 min of postischemic reflow. In matched untreated groups, livers
(n = 8) or hearts (n = 7) underwent
perfusion with plain buffer throughout.
To test whether DEPMPH (5 mM) could be a sensitive marker
of extracellular pH variations, an additional NMR experiment was carried out in which the effects of increasing the concentration of
Pi or decreasing the pH of the perfusate were assessed. For this purpose, livers (n = 2) were perfused normoxically
at 37 °C according to the following protocol: an initial 30-min
perfusion with plain standard buffer; a 10-min perfusion with
DEPMPH-supplemented standard buffer; a 10-min perfusion with a
DEPMPH-supplemented buffer in which the concentration of
KH2PO4 was raised to 3 mM; a 12-min
perfusion with DEPMPH-, KH2PO4 (3 mM)-, and isobutyric acid (35 mM)-supplemented
buffer; a 15-min perfusion with the former buffer in which isobutyric
acid was omitted; and a final 20 min of perfusion with plain standard
buffer. In this experiment, the liver perfusate was timely collected
for pH measurement.
Perchloric Acid Extraction--
To assess whether the different
NMR resonances seen in liver experiments with DEPMPH could be related
to degradation products of the pyrrolidine, livers were subjected to
perfusion as in the untreated and DEPMPH (5 mM) groups
described above (n = 2/group), except that organs were
freeze-clamped with aluminum tongs precooled in liquid nitrogen at the
end of ischemia. The frozen tissue was weighed, grounded in liquid
nitrogen, and homogenized with 6 volumes of 0.9 M
HClO4 (w/v) at 4 °C. After centrifugation at 15,000 × g for 15 min at 4 °C, the supernatant was neutralized
by 9 N KOH as described previously (31). After a second
centrifugation at 3,500 × g for 10 min at 4 °C, the
supernatant was lyophilized and stored in 2 aliquots at
80 °C. An
aliquot was used for gas chromatography-mass spectrometry analysis (see
below) after suspension into doubly distilled deionized water. Before
use for NMR spectroscopy, the second aliquot was dissolved in 0.5 ml of
D2O containing 50 mM EDTA, and methylene
diphosphonic acid (MDPA) was added as chemical shift reference. In this
latter sample, two NMR measurements were performed by consecutively
adjusting its pH at 5.5 and 7.5 with KOH or HCl, respectively.
Mass Spectrometry on Liver Perchloric Acid Extracts--
Gas
chromatography-mass spectrometry analysis was performed using a Varian
3400 gas chromatograph (Palo Alto, CA) coupled with a Finnigan Mat
TSQ700 mass spectrometer (San Jose, CA) operating in electron impact
mode and controlled by a Dec Station 5000/33. Helium was the carrier
gas, and the column was a fused silicon 25-m capillary column OV1. Mass
spectrometer settings were as follows: electron energy, 70 eV in the
range 60-650 m/z, scan speed, 600 m/z. The column temperature was programmed from
100-280 °C (10 °C/min) with the injector temperature fixed at
220 °C.
31P NMR Spectroscopy--
31P NMR
spectra of isolated perfused organs and perchloric extracts were
recorded at 161.9 MHz using a Bruker DPX 400 spectrometer (Karlsruhe,
Germany) equipped with a 31P/13C dual 20-mm probe.
31P NMR on Perfused Livers--
A one-pulse sequence
was used to acquire spectra in 2-min blocks corresponding to 240 free
induction decays obtained using a 70° flip angle, a 0.5-s repetition
time, 4K data points, and a 10-kHz spectral width. A Lorentzian
line broadening of 15 Hz was applied prior to Fourier transformation.
All chemical shifts were expressed with respect to the resonance from a
capillary of MDPA at 18.4 ppm. The peak area of the
-NTP resonance
was measured by integrating the peak from the spectrum and by comparing the area with that of MDPA (13 µmol of 31P nuclei).
Variations in
-NTP content were expressed as percentage of the
control
-NTP level obtained during the initial perfusion period with
plain buffer. pHi was estimated from the position of the
intracellular Pi resonance.
31P NMR on Isolated Hearts--
A one-pulse sequence
was used to acquire spectra in 5-min blocks corresponding to 140 summed
free induction decays obtained using a 60° pulse, a 2.151-s
interpulse delay, a 8K data table, and a 9.4-kHz spectral width.
A Lorentzian line broadening of 15 Hz was applied prior to Fourier
transformation. The peak areas of the
-ATP and creatine phosphate
(CrP) resonances were analyzed by both planimetry and an automatic
integration procedure. Variations in
-ATP and CrP contents were
expressed as percentage of the base-line levels obtained during the
initial phase of perfusion with DEPMPH-free buffer. pHi was
measured from the chemical shift of the Pi peak relative to
CrP (32).
31P NMR Spectra of Perchloric Acid
Extracts--
31P NMR spectra were recorded in a 5-mm tube
using a reverse probe tuned at 161.9 MHz. Instrument settings were as
described in the isolated liver experiments except that acquisition
time was extended to improve the signal-to-noise ratio.
Titration Curves--
The aim of these studies was to compare
the effects of pH on the 31P NMR chemical shift of the
resonance peak of DEPMPH in three environments having the ionic
strength representative of either the extracellular medium
(i.e. the standard Krebs- Henseleit medium described in
perfusion studies), or the protein-free, or complete cytosolic medium
(i.e. aqueous 125 mM KCl or a supernatant
solution of a rat heart homogenate (see below) diluted in aqueous 125 mM KCl, respectively). Each of these three solutions was
supplemented with KH2PO4 (1.2 mM)
and DEPMPH (5 mM) and was allowed to equilibrate at
22 °C or 37 °C. The pH was then adjusted to 30 different values in the range 4.0-10.0 with 6 N solutions of HCl or NaOH
and transferred into a 10-mm NMR tube for 31P NMR analysis
(Bruker AMX 400 NMR spectrometer) at its corresponding equilibration
temperature. The pKa values were calculated by
iterative fitting of the chemical shift
and pH data according to
the standard Henderson-Hasselbalch Equation 1,
|
(Eq. 1)
|
using a nonlinear regression (
a and
b are the protonated and unprotonated DEPMPH chemical
shifts, respectively).
To assess the influence of metal ions on DEPMPH resonance chemical
shift, increasing concentrations of Fe3+ (5 µM to 1 mM) or Fe2+ (1 µM to 1 mM) as sulfate salts were added to a
test 5 mM DEPMPH solution adjusted to pH 7.35, and
31P NMR spectra were obtained at 37 °C.
Preparation of Rat Heart Homogenates--
Heart homogenates were
obtained by a modification of the method described in Ref. 32 as
follows: a total of 25 g of freshly excised rat heart was minced
in 35 ml of 125 mM KCl at 2 °C, homogenized in a blender
for 10 min, and centrifuged at 10,000 × g for 15 min
at 2-3 °C, and the clear supernatant solution was used in titration experiments.
Measurements of Spin Lattice Relaxation Times
(T1)--
31P NMR spectra of
rat livers (n = 3) perfused normoxically for 80 min
with standard Krebs-Henseleit buffer containing 5 mM DEPMPH
were acquired at 37 °C as described above. T1
calculation was achieved using a progressive saturation experiment
consisting of a pulse sequence, (90°
Ta
]nn, with an acquisition time
(Ta) of 0.18 s and
values varying from 0.1 to 16 s. For each spectrum a total of 100 transients were
acquired. A 5-Hz line broadening was applied before Fourier transformation. To allow correct T1 estimation,
spectra were first deconvoluted into Lorentzian lines. Relaxation
parameters were evaluated using the same non-linear least squares
fitting method to the repetition time dependence of the resonance area.
Experimental Groups and Protocol for Hemodynamic Experiments in
Rat Hearts--
To assess the effect of DEPMPH on cardiac function,
additional untreated (n = 13) or DEPMPH (5 mM)-treated (n = 11) rat hearts were placed
into a small water-jacketed chamber at a controlled temperature of
37 °C and underwent the ischemia-reperfusion protocol described
above. Contractile function was assessed by means of a saline-filled
latex balloon inserted into the left ventricle and connected to a
pressure transducer (Gould Statham P23); the filling pressure was
adjusted until left ventricular end diastolic pressure (LVEDP) was in
the range 8-12 mm Hg. Throughout perfusion, LVEDP, left ventricular
developed pressure (LVDP), and its first derivative with time
(dp/dt), and heart rate were monitored at 5-min
intervals (Gould 8000S recorder). Rate pressure product, an index of
overall cardiac performance, was calculated as the product of heart
rate and LVDP. Coronary flow was measured by timely collecting the
coronary perfusate.
Statistical Analysis--
Metabolic and hemodynamic data were
expressed as mean ± S.E. Evaluation of statistical significance
was performed with two-way analysis of variance with repeated measures
over the different phases of the experimental protocols. If a
difference was established, the values in the DEPMPH-treated groups
were compared with that of the corresponding untreated groups using the
Newman-Keuls test. One-way analysis of variance was then carried out to
test for any differences among the mean values of the different groups at every time, and this was followed by the Duncan test. Significance was accepted at the p < 0.05 level. When necessary,
significance values were adjusted for multiple comparisons with use of
the Bonferroni correction. Animal care and handling conformed to the Guide for the Care and Use of Laboratory Animals (33).
 |
RESULTS |
pH Dependence of the DEPMPH 31P NMR Chemical
Shift--
The variation of the 31P NMR chemical shift of
DEPMPH (5 mM) was determined as a function of pH at 22 and
37 °C in the standard Krebs-Henseleit medium used for heart studies
which contained 1.2 mM Pi (ionic strength 164 mM). The results in Table I
show that, in this medium, the apparent pKa of
DEPMPH is in the physiological range and that the sensitivity

ab of the pyrrolidine as a pH marker is about 3.6 times higher than that of Pi at both tested temperatures.
These variables did not significantly vary when titrations were
performed either in an aqueous solution of physiological cellular ionic
strength (i.e. aqueous 125 mM KCl; ionic
strength 125 mM) or in a KCl (125 mM)-supplemented rat heart homogenate taken as a complete
intracellular medium (Table I). These latter data showed that pH
measurement from DEPMPH 31P NMR chemical shift is rather
insensitive to the release of cytosolic proteins or to changes in ionic
concentrations within physiological limits. Since phosphates are good
ion chelators and since ischemia/reperfusion of isolated organs has
been shown to trigger the release of metal ions, including
Fe2+ or Fe3+ ions (34), the effect on the
chemical shift of DEPMPH of adding iron ions to the Krebs medium has
been examined. Supplementing the Krebs medium with concentrations of
Fe2+ or Fe3+ ions in large excess of the
physiological range (i.e. 1 µM to 1 mM) at pH 7.35 had no displacing effect on the chemical
shift of DEPMPH (data not shown).
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Table I
Effect of milieu and temperature on pH-dependent
31P NMR parameters of DEPMPH (5 mM) and Pi
(1.2 mM)
The composition of Krebs-Henseleit medium used in heart studies, the
preparation of rat heart homogenates, and the protocol for titrations
were described under "Materials and Methods." Values are means ± S.D. from three independent experiments. a, limiting
chemical shift in acidic medium; b, limiting chemical shift
in basic medium;  ab = a b.
Chemical shifts are expressed relative to 85% H3PO4 as
external reference.
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|
31P NMR Studies on the Ischemic Reperfused Liver Using
DEPMPH as pH Probe--
Fig. 1 shows
typical 31P NMR spectra recorded during normoxic perfusion
of the same liver with plain buffer (Fig. 1A), after 20 min
of normoxic perfusion with DEPMPH (5 mM)-supplemented
buffer (Fig. 1B), after 25 min of ischemia (Fig.
1C), and after 20 min of reperfusion with DEPMPH-free buffer
(Fig. 1D). Resonances in Fig. 1A have been
assigned as previously reported (13). During normoxia, when DEPMPH was
added to the perfusion medium at 5 mM, no significant
alteration of the base-line metabolic variables or pHi was
observed as compared with livers perfused with plain buffer (Table
II). This pre-ischemic perfusion of
DEPMPH had also no effect on ischemia-induced decrease of the levels of
phosphorylated metabolites and pHi, and on post-ischemic recovery of metabolic variables of livers reperfused with plain buffer
(Table II and Fig. 1, C and D).

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Fig. 1.
31P NMR spectra recorded during
the ischemia-reperfusion of the same isolated rat liver. Spectra
were acquired after 20 min of initial normoxic perfusion at 37 °C
with Krebs-Henseleit buffer (A), after 20 min of normoxic
perfusion with DEPMPH (5 mM)-supplemented buffer
(B), after 25 min of total ischemia in the presence of
DEPMPH (C), and after 20 min of reperfusion with plain
buffer (D). The expanded regions show the peaks assigned to
DEPMPH in extracellular (a), cytosolic (b), and
acidic (c) liver compartments. Spectra were acquired as
described under "Materials and Methods," and chemical shifts are
given relative to external MDPA resonance. Peak assignments are as
follows: PME, phosphomonoesters; GPE, glycerol
3-phosphorylethanolamine; GPC, glycerol
3-phosphorylcholine.
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Table II
Effect of DEPMPH on metabolic variables in perfused rat livers
DEPMPH (5 mM) was added to the Krebs-Henseleit buffer
during the last 20 min of the 40-min normoxic period. Untreated group
received plain buffer throughout. The protocols for perfusion and
31P NMR experiments are described under "Materials and
Methods." Data are presented as mean ± S.E. For -NTP, units
are percent of base-line control values (set at 100%) taken after 20 min of normoxic perfusion. NM, not measurable.
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Owing to the close similarity of the pH titration curves obtained in
standard buffer and in rat heart homogenate-containing buffer, the pH
values in all perfusion experiments were determined from the DEPMPH
calibration curve in the standard buffer at 37 °C (Table I).
Although the NMR spectrum of DEPMPH in the perfusate at 37 °C
consisted of a single peak at 30.63 ppm, corresponding to a pH of 7.35, three resonances (termed as a, b and c) were observed in the low field
portion of the NMR spectra of normoxic livers perfused in the presence
of 5 mM DEPMPH, resonance peak b appearing as a shoulder of
the large resonance peak a (Fig. 1B). The chemical shifts
(mean ± S.E., n = 6) of these new resonances were
30.49 ± 0.14, 29.57 ± 0.16, and 24.02 ± 0.08 ppm,
corresponding to pH values of 7.36 ± 0.03, 7.12 ± 0.04, and
5.65 ± 0.03 for peaks a, b, and c, respectively. The three peaks
shifted to high field during ischemia as a consequence of acidosis, the
shift of peaks a and c being less pronounced than that of peak b, which became fully separable from peak a (Fig. 1C). After 25 min
of ischemia, the pH values derived from the chemical shifts of peaks a-c were 7.00 ± 0.03, 6.28 ± 0.05, and 5.48 ± 0.08, respectively. Given that the pH changes for peak b and Pi
peak were parallel (Fig. 1), it was assumed that peak b was relative to
cytosolic DEPMPH. Analysis of the time course of pH variation during
the entire perfusion protocol for peaks a-c confirmed that peak b was
significantly correlated to the Pi peak (r = 0.995; Fig. 2) and that it could be
reasonably attributed to DEPMPH located in intracellular cytosolic
medium.

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Fig. 2.
Time course of the changes in the intra- and
extracellular pH during rat liver ischemia-reperfusion in the presence
of DEPMPH (5 mM). pH values are calculated from the
corresponding 31P NMR resonances of Pi ( )
and the resonances a ( ), b ( ), and c ( ) of DEPMPH. For
conditions of perfusion and for 31P NMR measurements, see
"Materials and Methods." Each point corresponds to the mean ± S.E. (n = 6).
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Since peaks a and c could be related to DEPMPH metabolites that could
have been formed during normoxia and ischemia and further washed out at
reperfusion (Fig. 1D), additional perfusion experiments were
performed in which livers (n = 2) undergoing a normoxic
perfusion with DEPMPH (5 mM)-supplemented buffer and a
total ischemia were subjected to perchloric acid extraction followed by
31P NMR and gas chromatography-mass spectrometry analyses.
Fig. 3 shows a typical 31P
NMR spectrum of a DEPMPH-treated liver submitted to perchloric acid
extraction after 60 min of ischemia and recorded at pH 5.90. This
signal was similar to that obtained from an untreated ischemic liver
(with a high level of Pi and dramatically decreased NTP levels), except that the supplementary resonance peak of DEPMPH was
evident at 24.2 ppm. Raising the pH of the extract to 7.34 caused an
expected downfield shift of the DEPMPH peak to 30.53 ppm (data not
shown). Gas chromatography-mass spectrometry analysis of perchloric
extracts from DEPMPH-treated livers allowed the detection of an
m/z = 221 peak similar to that recorded from a DEPMPH
(5 mM) solution in standard buffer. No additional peak was
observed in the 31P NMR and gas chromatography-mass
spectrometry spectra of extracts obtained from control livers perfused
with plain buffer. Taken together, these data suggest that the three
peaks a-c observed in DEPMPH-treated normoxic and ischemic livers
(Fig. 1) are likely related to DEPMPH located in different extra- or
intracellular environments. Interestingly, pH values during the
normoxic period and at the end of ischemia, when calculated from the
peak a chemical shift (Fig. 2), accurately match the mean pH values
directly measured in the bath surrounding the liver during these two
phases of the perfusion protocol (i.e. 7.36 ± 0.03 and
6.81 ± 0.06, respectively). These data suggest that peak a may be
related to DEPMPH located in extracellular medium (i.e. the
perfusate itself and the liver vascular volume), the pH of which
decreased during ischemia due to a slow removing of acid equivalents
from the cytosol.

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Fig. 3.
31P NMR spectrum at pH 5.90 of
the perchloric acid extract from a normothermic 60-min ischemic rat
liver receiving DEPMPH (5 mM). The perfusion protocol,
31P NMR spectroscopy and perchloric acid extraction are
given under "Material and Methods." Peak assignments of
phosphorylated metabolites are as indicated in the legend of Fig. 1
except for phosphodiesters (PDE).
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A quite unexpected feature of the 31P NMR spectra of
normoxic livers perfused with DEPMPH-containing buffer was the presence of the high field peak c (Fig. 1B). Based on the calibration
data of Table I, this peak c was related to some DEPMPH located in an
intracellular compartment having the very low pH of 5.65 ± 0.07 during normoxia. During ischemia, a slight acidosis of this acidic
compartment was evidenced (Fig. 1C), the pH of which reached 5.37 ± 0.07 after 60 min of ischemia (Fig. 2).
In a further attempt to assign peak a seen in Fig. 1, B and
C, to DEPMPH in the extracellular environment, a load of 35 mM isobutyric acid (pKa 4.84) was added
to a DEPMPH (5 mM)- and KH2PO4 (3 mM)-supplemented Krebs buffer that was used as a perfusion
medium for normoxically perfused livers. Isobutyric acid has been
shown, in the presence of HCO3
, to
decrease selectively the extracellular pH without significantly affecting pHi in the normoxically perfused rat liver (13).
Addition of isobutyric acid led to a rapid decrease of the pH of the
perfusate to 7.10, which recovered its initial pH value of 7.35 when
isobutyric acid was removed from the perfusion buffer. Under these
conditions, very similar profiles of pH variation were obtained whether
pH was directly measured or calculated from the 31P NMR
resonance peaks of either extracellular KH2PO4
or peak a (Fig. 4), confirming that peak
a is related to extracellular pH. The 31P NMR resonance of
peak b remained unaffected by the presence of isobutyric acid
(initially at 29.92 ± 0.09 versus 29.98 ± 0.06 ppm during isobutyric acid treatment, n = 2),
corresponding to a stable pH of 7.18-7.20. As expected from the data
of Ref. 13, this result confirms that peak b was related to cytosolic
DEPMPH. During isobutyric acid infusion, a decrease of high field peak c chemical shift was observed (initially at pH 5.60 ± 0.04 versus 5.46 ± 0.09 during isobutyric acid treatment,
n = 2).

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Fig. 4.
Typical time course of the changes in intra-
and extracellular pH during normoxic perfusion of an isolated rat liver
in the presence of DEPMPH (5 mM), Pi (3 mM), and isobutyric acid (isoBu, 35 mM). pH values are calculated from the corresponding
31P NMR resonances of external Pi ( ) and the
resonances a ( ) and c ( ) of DEPMPH or directly measured into the
perfusion bath surrounding the liver ( ). For conditions of perfusion
and for 31P NMR measurements, see "Materials and
Methods."
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The appearance of three NMR peaks during DEPMPH perfusion in normoxic
livers (Fig. 1B) allowed the calculation of individual T1 values, which were found quite different as
follows: 3.94 ± 0.4, 3.5 ± 0.2, and 4.16 ± 0.3 s
for peaks a, b, and c, respectively. The T1 for
cytosolic Pi was 0.62 ± 0.2 s, in agreement with
previously published data (35).
31P NMR and Hemodynamic Studies on Ischemic Reperfused
Hearts Using DEPMPH as pH Probe--
When DEPMPH (5 mM)
was added to the perfusate of isolated normoxic rat hearts
(n = 7), base-line hemodynamic variables did not
significantly differ from that recorded in untreated hearts (n = 12), although developed pressure was not
significantly increased (Table III).
Contractile activity rapidly ceased in both groups immediately
following induction of ischemia, and only 2 hearts in each group showed
systolic oscillations of less than 2 mm Hg in amplitude at 10 min of
ischemia; after 15 min of ischemia all hearts remained asystolic.
Diastolic ventricular contracture, as assessed by LVEDP measurements,
progressively increased in both groups during ischemia, and no
significant intergroup difference was found with respect to contracture
development over the entire period of ischemia (Table III). Due to the
severity of ischemia, LVEDP values peaked within the first 5 min of
post-ischemic reflow in both groups, DEPMPH appearing significantly
protective on the development of contracture. At the end of
reperfusion, mechanical performance recovery was similar in both
groups, coronary flow and diastolic pressure being significantly
improved in the DEPMPH-treated group (Table III).
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Table III
Effect of DEPMPH on hemodynamic variables during ischemia-reperfusion
of isolated rat hearts
DEPMPH (5 mM) was added to the Krebs-Henseleit buffer
during the last 15 min of equilibration period and the first 5 min of
reperfusion (n = 7). Untreated hearts received buffer
throughout perfusion (n = 12). The protocol for heart
perfusion is described under "Materials and Methods." Data are
presented as mean ± S.E. The abbreviations used are: RPP, rate
pressure product; CF, coronary flow. Statistics (two-way analysis of
variance followed by Student's t test).
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Fig. 5 shows representative
31P NMR spectra from the same heart at the end of normoxic
perfusion in presence of 5 mM DEPMPH (Fig. 5A),
after 10 min of global ischemia (Fig. 5B), after 30 min of
global ischemia (Fig. 5C), and after 5 min of reflow (Fig. 5D). As for hemodynamic variables, pre-ischemic application
of DEPMPH did not affect metabolic variables and pHi values, when compared with untreated hearts (Table
IV). DEPMPH had also no effect on
ischemia-induced decrease of the levels of phosphorylated metabolites
and pHi. Although DEPMPH induced no effect on post-ischemic
pHi values, an improved recovery of metabolic variables was
obtained (Table IV). Two resonances (termed as a and b) were observed
in the low field portion of the 31P NMR spectra of normoxic
hearts treated with DEPMPH (Fig. 5A). Due to the relatively
low catabolic activity of the heart, it was considered that both peaks
a and b were relative to DEPMPH in two different compartments having
the respective pH values of 7.36 ± 0.03 and 7.12 ± 0.04 (n = 6). The two peaks shifted to high field during
ischemia as a consequence of acidosis, the shift of peak a being less
pronounced than that of peak b (Fig. 5, B and C).
After 30 min of ischemia, the pH values derived from the chemical
shifts of peaks a and b were decreased to 7.23 ± 0.03 and
6.41 ± 0.02, respectively. Again, the pH values calculated from
peaks b and Pi chemical shifts were similar (Table IV).
Analysis of the time course of pH variation during the entire perfusion protocol for peaks a and b confirmed that peak b was significantly correlated to the Pi peak (r = 0.997; Fig.
6) and that it could be reasonably
attributed to DEPMPH internalized into the cytosol. Direct measurements
of the pH of the external perfusate (data not shown) showed that peak a
could be attributed to external medium, which slightly acidified during
ischemia (Fig. 6). A third peak (termed as peak d in Fig.
5B) appeared as a shoulder of peak a after 10 min of
ischemia, becoming fully separable from peak a when the duration of
ischemia increased to 30 min, corresponding to a mean pH of 7.05 (Fig.
5C). A similar pH value was directly measured from the
coronary effluent driven from the atrial cavity during ischemia. With
the onset of reperfusion, peaks a, b, and d returned back to downfield
(Fig. 5D and Fig. 6) and rapidly disappeared as DEPMPH
perfusion stopped.

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Fig. 5.
31P NMR spectra recorded during
the ischemia-reperfusion of the same isolated rat heart. Spectra
were acquired after 30 min of initial normoxic perfusion at 37 °C
with DEPMPH (5 mM)-supplemented Krebs-Henseleit buffer
(A), after 10 min of total ischemia (B), after 30 min of total ischemia (C), and after 5 min of reperfusion
with DEPMPH (5 mM)-supplemented Krebs-Henseleit buffer
(D). The expanded spectra B-D show the peaks
assigned to DEPMPH in extracellular (a and c) and
cytosolic heart compartments. Spectra were acquired as described under
"Materials and Methods," and chemical shifts are given relative to
CrP. Other peak assignments are follows: phosphomonoesters
(PME); Pi; and ATP.
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Table IV
Effect of DEPMPH on metabolic variables in perfused rat hearts
DEPMPH (5 mM) was added to the Krebs-Henseleit buffer
during the last 15 min of equilibration period and the first 5 min of
reperfusion (n = 6). Untreated group received buffer
throughout perfusion (n = 7). The protocols for
perfusion and 31P NMR experiments are described under
"Materials and Methods." Data are presented as mean ± S.E.
Units are percent of base-line control values after 10 min of normoxic
perfusion (set at 100%), except for pHi. The abbreviation used
is: NM, not measurable. Statistics (two-way analysis of variance
followed by Student's t test).
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Fig. 6.
Time course of the changes in the intra- and
extracellular pH during rat heart ischemia-reperfusion in the presence
of DEPMPH (5 mM). pH values are calculated from the
corresponding 31P NMR resonances of Pi ( )
and the resonances a ( ), b ( ) and d ( ) of DEPMPH. For
conditions of perfusion and for 31P NMR measurements, see
"Materials and Methods." Each point corresponds to the mean ± S.E. (n = 6).
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DISCUSSION |
The widespread use of 31P NMR for the noninvasive
measurement of pH in biological systems suggests a need to control
better the requirements that have to be met by any compound intended to
be used as an improved auxiliary pH indicator. The possibility of
measuring pH by 31P NMR has been first demonstrated using
endogenous Pi as a probe (1-3), but although it is
actually an accurate indicator of the cytosolic pH, some metabolic
conditions may exist where the signal/noise ratio of cytosolic
Pi can be too low to allow a good pH determination (4-6).
The NMR visibility of the cytosolic Pi resonance can also be reduced by the external Pi signal arising from
KH2PO4-containing buffers commonly used in
perfusion experiments. The 
ab value for
Pi is only of 2.6 ppm, making cellular compartments not
distinguishable if their pH values differ by less than 0.3 pH units
(4-6). This has been for long an important limitation for the use of
Pi as a probe of trans-sarcolemmal proton movements in
ischemic situations. By using phenylphosphonate as an extracellular NMR
probe, it was shown that the pH gradient across the sarcolemma
collapses in early ischemia in isolated perfused hearts (22). Although
these data (22) and other results from perfusion studies using
phenylphosphonate (21, 24) or, in a lesser extent, methylphosphonate
and 3-aminopropylphosphonate (24) have shown the usefulness of charged
phosphonates as external pH markers, none of these molecules could be
used to probe simultaneously pHi due to their weak

ab range of 2.1-3.9 ppm (27).
The data of the present study provide experimental support to our
assumption (29) that using the uncharged pyrrolidine DEPMPH as
31P NMR pH marker in the perfused heart and liver,
simultaneous information on ischemia-induced or chemically induced weak
intra- and extracellular pH modifications can be directly obtained
which previously escaped from investigation with other charged alkyl- and aminophosphonates. This outstanding property of DEPMPH clearly appears as a conjunction of all the basic requirements that have to be
fulfilled by a potential NMR pH probe (5). Table I shows that the
pKa value of DEPMPH is in the physiological range
and exhibited a low sensitivity to changes in composition and ionic
strength in several biologically relevant milieu. In standard Krebs
buffer, a satisfactory correlation between the experimental
pKa values and the structure (i.e. the
nature of substituents linked to the nitrogen atom) has been obtained for a wide series of cyclic
-aminophosphonates such as DEPMPH (29),
suggesting that this pyrrolidine has an ideal structure for
applications in biological studies at a pKa close to
that of Pi. The use of DEPMPH as an NMR pH probe also takes advantage of the low variation of the pKa with ionic strength for compounds having a non-dissociable protonation site such
as the amino group of pyrrolidines (29). Another important feature of
DEPMPH is that it did not affect the main metabolic parameters such as
ATP and pHi of hearts and livers during normoxic perfusion and
ischemia (Tables II-IV) when added to the perfusate at a concentration
(5 mM) that permitted a good NMR detection. As pHi
profiles were not modified by the presence of DEPMPH during heart and
liver ischemia, the DEPMPH resonance peak b can therefore be considered
as a reliable index for intracellular pH monitoring in these organs
(Figs. 1 and 5). In agreement with other heart studies on
phosphorylated compounds (30, 36, 37), a slight cardioprotection was
exerted by DEPMPH on reperfused hearts (Table III), whereas this aspect
has not been investigated in detail in previous studies on
alkylphosphonate NMR probes (21-24). As a tentative explanation for
the cardioprotective effect of DEPMPH on the reperfused heart, it has
been hypothesized that pro-oxidant transition metal ions and/or
Ca2+ released from the post-ischemic heart may have been
inactivated by chelation on the diethoxyphosphoryl group of the
pyrrolidine (30). The data from Figs. 1 and 5 show that DEPMPH, even
perfused at the lowest concentration (5 mM) among those
used in previous NMR studies with charged alkylphosphonates (21-24),
reasonably penetrates heart and liver tissue, distributing into extra-
and intracellular compartments to provide a satisfactory NMR detection. In cell studies where aminoalkylphosphonates have been used as 31P NMR pH probes, it was found that compounds having a low
dipole moment (i.e. aminomethylphosphonate and
2-aminoethylphosphonate), by easily entering the cell, behave as intra-
and extracellular pH reporters (27, 28), whereas
3-aminopropylphosphonate, which is more polar, is restricted to the
extracellular compartment (38, 39). In recent 31P NMR study
on rat isolated livers, 2-aminoethylphosphonate was found to penetrate
the cytosol, but although its pKa was of 6.45, it
did not provided simultaneous information on the acidic and
extracellular compartments (40). In light of these results (27, 28,
38-40), it is therefore likely that the greatest ability of DEPMPH to
allow simultaneous NMR investigation of extra- and intracellular
compartments results from the low polarity of its diethoxyphosphoryl
moiety. This assumption appears in accord with the results of mass
spectrometry studies on liver perchloric extracts (Fig. 3) showing that
DEPMPH was internalized into several intracellular compartments, with
no significant metabolism or degradation. In agreement was the finding,
in normoxic livers perfused with DEPMPH, of three different
T1 values having the same order of magnitude as
those previously reported for charged phosphonates in heart or liver
perfusion (23, 24) and which are short enough to render this
pyrrolidine suitable for use in in vivo NMR experiments.
Because of the very important 
ab value of 9.6 ppm
observed with DEPMPH between pH 4 and 10 (Table I), probing pH using 31P NMR on intact organs perfused with this pyrrolidine
appears considerably easier than with other alkyl- and
aminophosphonates since several peaks were clearly separated in the
normoxic and ischemic liver and heart (Figs. 1 and 5) under conditions
where phenylphosphonate yielded only one peak (22-24). Moreover, due to the large pH window of 5.0-8.5 where the DEPMPH titration curve is
steeper (Table I), a very accurate investigation of acidic compartments
can be considered, and it was one major aim of the present work to use
DEPMPH to study acidic compartments having pH values below 6.
During the last recent years, much work has been performed on the
characterization of the pHi-regulatory systems in hepatocytes
(25, 41). At plasma membrane sites, the
Na+-HCO3
symport was
found to play a major role in maintaining hepatocytic pH homeostasis
over a large pH range (7.5-6.5), whereas the
Na+-H+ antiport becomes preferentially
activated during systemic acidosis (13, 42, 43). Moreover, the presence
of acidic cytoplasmic organelles, including endocytic and secretory
vesicles, lysosomes, and portions of the trans-Golgi complex and of the
endoplasmic reticulum, has also been demonstrated (25, 26). In
hepatocytes, acidic organelles were found to contribute to pHi
regulation via active vacuolar ATP-driven proton pumps, which induced
pHi acidification as follows: (i) ATP depletion due to exposure
to metabolic inhibitors, (ii) hypotonic stress in K+-free
buffer that resulted in proton leakage from acidic compartments without
significantly affecting cellular ATP content (44). In both situations,
a concomitant alkalinization of cytosolic vesicles resulting in a near
equilibration of cytosolic and vesicular pH was evidenced (44).
However, since (a) the characterization of acidic
compartments in the liver has exclusively derived from studies on
cellular systems exposed to external agents that either stimulated or
inhibited the activity of vacuolar ATP-driven proton pumps, and
(b) the analytical method used (i.e.
fluorescence) did not allow the simultaneous study of all cytosolic and
organellar compartments (44, 45), the pertinence of the conclusions to the situation encountered in the whole organ may be questioned.
The present study provides the first direct evidence of the involvement
of acidic compartments in pHi regulation during ischemia and
reperfusion of the intact liver. If the DEPMPH resonance peak c in the
different hepatic cytosolic organelles cannot be precisely assigned, it
however corresponds to a pH range of 5.2-5.6 (Fig. 2) similar to that
attributed to acidic vesicles (44-46). During the normoxic period, the
pH determined from the peak c resonance remained stable, and thus a pH
gradient of 1.47 pH units between acidic vesicles and cytosol could be
determined. Upon ischemia, the cytosol and the acidic vesicles
acidified by about 0.9 and 0.3 pH units, respectively, leading to a
decrease of the pH gradient reaching a minimum at 0.70-0.80 pH units
from 35 to 60 min of ischemia (Fig. 2). This demonstrates that, in liver ischemia, acidic vesicles participate in pHi regulation possibly by removing protons from the cytosol, an opposite mechanism to
that reported in the case of chemical hypoxia of hepatocytes where
protons are released from acidic vesicles to the cytosol (44). The
exact stimuli (i.e. decrease in cytosolic pH or in ATP)
responsible for this acidification of liver vesicles during ischemia
remains to be determined since the membranes of the acidic vesicles are
highly permeable to protons (25) and vacuolar ATP-driven proton pumps
are sensitive to ATP changes (44).
Another interesting finding of the present study was that liver
vesicles acidified upon exposure to isobutyric acid (Fig. 4). This
acid-load technique, which results in a selective acidification of the
extracellular compartment by the freely membrane-permeable isobutyric
acid, has been developed to study the mechanisms of proton transport
from the cytosol (47). In a recent study using this technique in
isolated rat livers, the role of the
Na+-HCO3
symport has been
found predominant in maintaining cytosolic pH homeostasis (13). The
present data suggest that vacuolar proton pumps could also actively
remove protons from the cytosol during acid load, therefore explaining,
at least in part, the maintenance of the constant pHi
observed in Ref. 13.
In perfused hearts and livers, a peak a assigned to DEPMPH located in
the extracellular environment (i.e. the interstitial and
vascular fluids, and the bath surrounding the organ) was observed (Figs. 1 and 5). When ischemia was induced, the environment directly surrounding the liver and the heart acidified (Figs. 2 and 6), indicating, as expected, the occurrence of mechanisms that actively remove protons from the cytosol to the extracellular space. In heart
experiments, however, another interesting property of DEPMPH as NMR pH
probe was to permit the monitoring of pH changes within the ventricular
and atrial cavities using resonance peak d (Fig. 6).
In conclusion, DEPMPH was found to be an outstanding 31P
NMR pH marker in two ex vivo models of ischemia and
reperfusion, particularly able to specifically allow
intracellular pH monitoring that was not possible with any of all
previously proposed phosphonate pH markers. In liver perfusion
experiments, the use of DEPMPH has provided several new insights into
potential mechanisms of pH regulation during ischemia involving acidic
vesicles. It is therefore reasonable to speculate that DEPMPH will be a
tool of considerable interest in further studies on pH gradient changes
between vesicles and cytosol aiming at a better understanding of pH
homeostasis in various ischemic- or non-ischemic-induced
physiopathological situations.