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0041-1337/01/7111-1515/0
TRANSPLANTATION Vol. 71, 1515–1517, No. 11, June 15, 2001
Copyright © 2001 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A.
DEUTERIUM NUCLEAR MAGNETIC RESONANCE FOR
EVALUATING THE METABOLIC STATUS OF LIVERS SUBJECTED
TO WARM ISCHEMIA
1
MARINA SCARPA,
2,5
ALESSANDRA CORAZZA,
3
FABIO VIANELLO,
3
ADELIO RIGO,
3
LUCREZIA FURIAN,
4
NICOLA BALDAN,
4
AND PAOLO RIGOTTI
4
Dipartimento di Fisica and INFM, Universita ` di Trento, Via Sommarive 14, 38050 Povo-Trento; Dipartimento di Chimica
Biologica, Universita ` di Padova, Viale G. Colombo 3, 35121 Padova; and Dipartimento, di Scienze Mediche e Chirurgiche,
Clinica Chirurgica Generale IV, Universita ` di Padova, Via Giustiniani 2, 35121 Padova, Italy
Background. The development of reliable methods
for assessing the viability of currently available livers
is expected to increase the number of successful
transplantations.
Methods.
2
H nuclear magnetic resonance (NMR) was
used to search for metabolic markers of ischemia in
explanted rat livers. Deuterium oxide (
2
H
2
O) was used
as a source of
2
H. A total of 10–80% v/v
2
H
2
O was
added to homogenates obtained from a liver biopsy
and the formation of
2
H-labeled metabolites was
monitored.
Results. Some well-resolved
2
H resonances were
found in the homogenates from biopsies of warm isch-
emic liver. Two of these were identified as [3-
2
H] lac-
tate and [2-
2
H] lactate, and a linear relationship was
found between the ratio of [[2-
2
H] lactate] to [[3-
2
H]
lactate] and the warm ischemia time. The deuterium
incorporation into lactate was explained on the basis
of the metabolic events occurring under hypoxic
conditions.
Conclusions. The experimental results support the
application of
2
H NMR for a reliable evaluation of the
metabolic status of a liver harvested from non-heart-
beating donors.
INTRODUCTION
Accurate methods for assessing organ viability are needed
to extend the use of organs from marginal donors in trans-
plantation. The viability of a liver and its postoperative func-
tion depend mainly on its metabolic status. Currently avail-
able methods for obtaining information on liver function are
based on enzymatic analysis (1) or high-pressure liquid chro-
matography (2). However, these methods are time-consum-
ing or provide only limited information on organ energetics.
31
P-NMR is the most promising technique at present, be-
cause it allows for the measurement of phosphorus-contain-
ing metabolites by a noninvasive and relatively fast proce-
dure. Many papers report on the use of this technique to
assess organ preservation and viability (3, 4). However, sev-
eral broad resonances occur in
31
P-NMR spectra of liver and
their interpretation is still a matter of debate (5, 6); in addi-
tion, conclusive evidence that high-energy phosphates are a
direct indication of organ viability has yet to be reported (7).
The advantages of monitoring the metabolic status of an
organ by NMR has stimulated the development of alternative
and more direct spectroscopic methods based on labeled com-
pounds. Among the labeled tracers, deuterated water (
2
H
2
O)
appears very promising due to its fast and free diffusion in
tissues and its low cost. Moreover,
2
H
2
O offers the advan-
tages of
2
H NMR, i.e., minimal interference from resonances
of endogenous compounds, a high real-time sensitivity due to
fast deuterium relaxation, and a relatively high frequency of
resonance (46 MHz at 7 T).
2
H
2
O has been used in metabolic
studies (8, 9) and we recently demonstrated that deuterium
incorporation in lactate in the brain of living rats supplied
with
2
H
2
O is dependent on O
2
concentration in the air for
breathing (10).
2
H
2
O has also been found to improve the
preservation quality of organs for transplantation (11). In our
study we show that the [3-
2
H lactate]/[2-
2
H lactate] ratio
produced in liver biopsies incubated in the presence of
2
H
2
O
is a very sensitive indicator of warm ischemia times before
liver explantation and we propose a novel method for estab-
lishing the viability of organs for transplantation.
MATERIALS AND METHODS
Deuterium oxide was provided by Sigma Chemical Company (St.
Louis, LO). Eurocollins buffer came from Monico (Venice, Italy).
Wistar rats (23510 g) were anesthetized using ethyl ether. Warm
ischemia was caused in situ by inducing cardiac arrest with superior
and inferior vena cava occlusion. These operations took about 5 min.
After a variable warm ischemia time, 1 g liver biopsies were collected
and homogenated in 2 ml Eurocollins buffer with a Polytron appa-
ratus (Heidolph, Diax 900) at 12,000 rpm. This step lasted 1 min.
After the addition of
2
H
2
O (10 or 80% v/v), the homogenate was
incubated at 37°C for a certain time (10 –30 min), then extracted by
perchloric acid. The extract was neutralized by 1 M KOH and cen-
trifuged, and the supernatant was collected. This step took about 20
min. The neutralized extract was frozen in liquid nitrogen and ly-
ophilized. Lyophilization was necessary to remove the excess
2
H
2
O
because the available NMR instrument did not possess the hardware
for suppressing the
2
H
2
O signal.
The lyophilized extract was resuspended in 0.5 ml of 0.1 M phos-
phate buffer at pH 7.4 and transferred to an NMR sample tube.
Pyridine-d
5
was added both as chemical shift reference and as an
internal concentration standard. Alternatively, the resonance inten-
sities were normalized with respect to
2
H
2
O, having previously
verified that phosphate solutions of the lyophilized livers have the
background
2
H
2
O content (8 mM).
2
H spectroscopy of the neutralized extracts was performed at 46
MHz in a Bruker MSL 300 wide bore spectrometer run in unlocked
1
Supported by the Istituto Superiore di Sanita ` , Grant .“Sostitu-
zioni funzionali, organi artificiali e trapianti d’organo.”
2
Dipartimento di Fisica and INFM, Universita ` di Trento.
3
Dipartimento di Chimica Biologica, Universita ` di Padova.
4
Dipartimento, di Scienze Mediche e Chirurgiche, Clinica Chiru-
rgica Generale IV, Universita ` di Padova.
5
Address correspondence to: Marina Scarpa, PhD, Dipartimento
di Fisica, Via Sommarive 14, 38050 Povo-Trento, Italy.
1515