Intravenous glycine improves survival in rat liver
transplantation
Peter
Schemmer1,
Blair U.
Bradford1,
Michelle L.
Rose1,
Hartwig
Bunzendahl2,
James A.
Raleigh3,
John J.
Lemasters4, and
Ronald G.
Thurman1
1 Laboratory of Hepatobiology
and Toxicology, Department of Pharmacology, and Departments of
2 Surgery,
3 Radiation Oncology, and
4 Cell Biology and Anatomy,
University of North Carolina at Chapel Hill, Chapel Hill, North
Carolina 27599
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ABSTRACT |
In situ manipulation by touching, retracting,
and moving liver lobes gently during harvest dramatically reduces
survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman.
Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with
GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of
glycine, which prevents activation of Kupffer cells, was assessed here.
Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group.
Furthermore, serum transaminases and liver necrosis were elevated 4- to
12-fold 8 h after transplantation. After organ harvest, the rate of
entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the
hepatic microcirculation. Pimonidazole binding, which detects hypoxia,
increased about twofold after organ manipulation, and Kupffer cells
isolated from manipulated livers produced threefold more tumor necrosis
factor-
after lipopolysaccharide than controls. Glycine given
intravenously to the donor increased the serum glycine concentration
about sevenfold and largely prevented the effect of gentle organ
manipulation on all parameters studied. These data indicate for the
first time that pretreatment of donors with intravenous glycine
minimizes reperfusion injury due to organ manipulation during harvest
and after liver transplantation.
organ harvest; hepatic microcirculation; hypoxia
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INTRODUCTION |
GLYCINE, A NONESSENTIAL amino acid, is nontoxic and has
been shown to protect proximal tubules and hepatocytes (19) against hypoxia. Glycine also prevents nephrotoxicity caused by cyclosporin A
(31). Furthermore, glycine added to a graft rinse solution reduced
reperfusion injury and also improved initial graft function and
survival after liver transplantation (19). Moreover, glycine improved
the hepatic microcirculation and reduced liver injury in a low-flow,
reflow perfusion model (33). A diet containing glycine improved
survival of rats given endotoxin, most likely by inactivation of
Kupffer cells, since tumor necrosis factor-
(TNF-
) production was
decreased (16). Recently, gentle in situ graft manipulation by
touching, retracting, and moving liver lobes gently during harvest has
been demonstrated to be detrimental for survival after liver
transplantation via mechanisms involving microcirculation and Kupffer
cell-dependent reperfusion injury (27). Increases of intracellular
Ca2+ concentration in Kupffer
cells are essential for the release of prostanoids and inflammatory
cytokines in response to stimuli such as endotoxin
[lipopolysaccharide (LPS)]. Glycine prevented the increase
of intracellular Ca2+
concentration by activating a glycine-gated chloride channel, which
hyperpolarized the cell membrane and made
Ca2+ influx via voltage-dependent
Ca2+ channels more difficult. Thus
glycine blunted activation of Kupffer cells by LPS. This effect is
important since activated Kupffer cells are involved in regulation of
hepatic microcirculation, and several lines of evidence suggest that
microcirculatory disturbances are a key factor in enhanced donor liver
susceptibility to cold and warm ischemia (14, 15, 29).
Depletion of Kupffer cells with gadolinium chloride
(GdCl3), which is potentially
toxic, and dietary glycine for 5 days before harvest blunted
harvest-related reperfusion injury and primary nonfunction after
transplantation in rats (27). Because organ retrieval occurs in a few
hours, it is unlikely that dietary glycine would be clinically
applicable. Therefore, the aim of this study was to determine whether
brief intravenous infusion of glycine to donors would blunt
harvest-related injury developing upon reperfusion. This is important
because primary organ manipulation during harvest cannot be prevented with standard harvesting techniques, graft nonfunction is still a major
obstacle in clinical liver transplantation, and the number of donor
organs is limited.
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METHODS |
Experimental animals and treatment.
Female inbred Lewis rats (200-230 g) were allowed free access to
standard laboratory chow (Agway PROLAB RMH 3000, Syracuse, NY) and tap
water. Donor animals were given glycine by infusion (1.5 ml; 300 mM)
for 1 h through the femoral vein before harvest. To test whether
survival of manipulated livers after transplantation is dose dependent,
donors were given infusions of various concentrations of glycine before
organ harvest. Isonitrogenous controls were given valine, which does
not have an effect on Kupffer cells (17). Experimental procedures were approved by the Institutional Animal Care and Use Committee.
Harvest procedure. Livers were gently
manipulated during harvest as described previously (27). Briefly, donor
livers were harvested within 25 min before perfusion with cold
University of Wisconsin cold storage (UW) solution. Minimal dissection
was performed in a standardized fashion during the first 12 min,
including freeing the organ from ligaments and cannulation of the bile
duct. During the last 13 min, livers were either left alone or were manipulated gently. Standardized gentle manipulation was carried out by
touching, retracting, and moving the liver lobes in situ continuously.
At 25 min, perfusion with 8 ml of cold Ringer followed by 3 ml of cold
UW solution was performed in situ via the portal vein. Cuffs were
attached in the cold to the infrahepatic vena cava and
portal vein after explantation.
Transplantation. Both donors and
recipients were anesthetized with methoxyflurane, and orthotopic liver
transplantation was performed in rats using rearterialization (12).
After explantation, livers were stored at 0-4°C for 1 h in UW
solution. Grafts were rinsed with 10 ml of normal saline (18°C) and
were implanted by connecting the suprahepatic vena cava with a running
7/0 Prolene suture, inserting cuffs into the corresponding vessels and
anastomosing the bile duct and hepatic artery over an intraluminal
polyethylene splint. Transplantation required <35 min; during this
time, the portal vein was clamped for 13 min. After transplantation,
all recipients had free access to standard laboratory chow and tap water.
Liver perfusion. Livers were perfused
via the portal vein at 3-4
ml · min
1 · g
1
liver with oxygenated Krebs-Henseleit bicarbonate buffer (in mM: 118 NaCl, 25 NaHCO3, 1.2 KH2PO4,
1.2 MgSO4, 4.7 KCl, and 1.3 CaCl2) at pH 7.6, saturated with
95% O2 and 5%
CO2 at 37°C in a
nonrecirculating system (3).
Assessment of microcirculation. At the
time perfusion with cold preservation solution would usually have been
performed, some donor livers were perfused ex situ with fluorescein
isothiocyanate-dextran (12 µM; mol wt 70,000, catalog no. FD-70S;
Sigma) to assess microcirculation. A mercury arc lamp equipped with a
glass filter was used to produce excitation wavelengths of 430 nm.
Fluorescence of fluorescein dextran (560 nm) was measured via a light
guide (tip diameter of 2 mm) placed on the surface of the perfused
liver with a micromanipulator. The signal was amplified and recorded as
described elsewhere (8). To normalize for day-to-day variation, all
values were expressed as a percentage of basal values. Furthermore, at
reperfusion after transplantation, the time for blood to distribute
completely was recorded visually by observing when the liver turned
homogeneously red, to index the quality of reperfusion and early
microcirculation as is done in clinical transplantation (27).
Kupffer cell isolation. Kupffer cells
were isolated by collagenase digestion and differential centrifugation
using Percoll (Pharmacia, Uppsala, Sweden) as described elsewhere (17).
After organ manipulation, livers were perfused via the portal vein with Ca2+- and
Mg2+-free Hanks' balanced salt
solution containing collagenase IV (0.025%; Sigma Chemical, St. Louis,
MO) at 37°C at a flow rate of 26 ml/min. After digestion, livers
were cut into small pieces in collagenase buffer. The suspension was
filtered through nylon gauze, and the filtrate was centrifuged at 450 g for 10 min at 4°C. Cell pellets
were resuspended in buffer, parenchymal cells were removed by
centrifugation at 50 g for 3 min, and
the nonparenchymal cell fraction was washed two times with buffer.
Cells were centrifuged on a density cushion of Percoll at 1,000 g for 15 min. The Kupffer cell
fraction was collected and washed with buffer again. Viability of cells
determined by trypan blue exclusion was >90%.
TNF-
and nitrite measurement.
Isolated Kupffer cells were cultured for 24 h in 24-well culture plates
(Sarstedt, Newton, NC) at a density of 1 × 106 cells/well in DMEM
supplemented with 10% FBS and antibiotics at 37°C in the presence
of 5% CO2. Cells were incubated
with fresh media containing LPS (100 ng/ml in 5% rat serum) for an
additional 4 h. Nonadherent cells were removed after 1 h by replacing
buffer, and cells were cultured for 24 h before experiments. All
adherent cells phagocytized latex beads, indicating that they were
Kupffer cells. TNF-
concentrations were determined in the culture
medium using an enzyme-linked immunosorbent assay kit (ELISA; Genzyme, Cambridge, MA). TNF-
production after LPS (100 ng/ml) was compared with basal values. Furthermore, isolated Kupffer cells were cultured for 24 h in glycine-free medium or in medium containing glycine (10 mM). Nitrite concentration in media was measured colorimetrically by
the Griess reaction after 48 h of culture (13). Briefly, 500 µl of
medium were mixed with an equal volume of Griess reagent (1%
sulfanilamide, 0.1% naphthalene-ethylenediamine dihydrochloride in
15%
H3PO4)
and incubated for 10 min at room temperature. The resulting product,
N-(1-naphthyl)ethylenediamine, was
quantitated spectrophotometrically at 550 nm. Nitrite levels were
calculated using a standard curve generated with known concentrations
of sodium nitrite.
Trypan blue infusion and histology.
After 24 h of storage in cold UW solution, trypan blue (500 µM;
Aldrich, Milwaukee, WI) was infused in the liver to assess viability of
cells. Livers were then flushed with additional perfusate to remove
excess dye and were fixed by perfusion with 4% paraformaldehyde in
Krebs-Henseleit bicarbonate buffer at pH 7.6, embedded in paraffin, and
processed for light microscopy using an eosin counterstain. The
presence of trypan blue in the nuclei is indicative of irreversible
loss of cell viability (3). Five pericentral and five periportal fields
(×100 magnification) were selected at random from at least four
different sections per sample, and mean values of stained nuclei from
nonparenchymal and parenchymal cells were calculated. Furthermore, some
livers were evaluated for histology either before cold storage or after
removal from rats killed 8 h after transplantation. Livers were fixed
by perfusion with 4% paraformaldehyde in Krebs-Henseleit bicarbonate
buffer at pH 7.6, embedded in paraffin, and processed for light
microscopy after hematoxylin and eosin staining. Liver damage was
assessed by estimating necrotic areas as described elsewhere (30).
Briefly, five fields (×100 magnification) were selected at random
from at least four different sections per sample, and mean values were calculated.
Determination of reduced, protein-bound pimonidazole
by ELISA and immunohistochemistry. Pimonidazole is a
2-nitroimidazole that detects hypoxia in liver tissue (2). Pimonidazole
(120 mg/kg, 5 min before donor operation) was given intravenously, and
adducts were measured in tissue homogenates with a competitive ELISA
procedure previously described (26) and modified for liver tissue (2).
Protein levels in tissue homogenates were determined with the
bicinchoninic acid assay using a commercially available kit (Pierce
Chemical, Rockford, IL). Paraffin blocks of Formalin-fixed liver tissue
were sectioned at 6 µm, and pimonidazole adducts were detected with a
biotin-streptavidin-peroxidase indirect immunostaining method using
diaminobenzidine as a chromogen as described previously (2). After the
immunostaining procedure, a counterstain of hematoxylin was applied. A
Universal Imaging Image-1/AT image acquisition and analysis system
(Chester, PA) incorporating an Axioskop 50 microscope (Carl Zeiss,
Thornwood, NY) was used to capture the immunostained tissue sections at
×100 magnification (1).
Enzyme assays. Blood samples were
collected from the tail vein 8 h after transplantation. Serum was
obtained by centrifugation and was stored at
80°C until
analysis. Aspartate aminotransferase and alanine aminotransferase
activity was determined by standard enzymatic methods, whereas total
bilirubin was determined in sera by direct spectrophotometry at 454 nm
(4). To detect proteolytic activity after harvest, livers were rinsed
with 2 ml of UW solution before cold storage. Arginine-specific
proteolytic activity was measured in the effluent using
H-D-Ile-Pro-Arg-p-nitroaniline-2HCl (KabiVitrum). Activity was determined from the rate of formation of
p-nitroaniline at 405 nm
spectrophotometrically at 37°C (18).
Glycine measurements. Blood was taken
before experiments for glycine determination in serum as described
previously (4). Briefly, glycine was extracted and benzolated, and the
resulting hippuric acid was extracted and dried. Subsequently, the
concentration of hippuric acid was determined spectrophotometrically at
458 nm (22).
Statistics. Mean values ± SE for
groups were compared using Fisher's exact test or ANOVA (2-way ANOVA)
with Student-Newman-Keuls post hoc test as appropriate with
P < 0.05 selected before the study as the
criterion for significance.
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RESULTS |
Intravenous glycine prevents effects of gentle organ
manipulation on graft viability. In livers that were
not manipulated, survival was 100% after transplantation; however,
gentle manipulation decreased survival by ~50% in controls given
valine (isonitrogenous control). In contrast, rats receiving
manipulated livers from glycine-infused donors survived as well as
nonmanipulated controls (Fig. 1).
Furthermore, manipulation elevated transaminases and total bilirubin
six- to sevenfold 8 h after transplantation (Table 1) and doubled proteolytic activity in the
valine group (Fig. 2). In contrast,
manipulated livers pretreated with glycine, which increased glycine
serum levels about sevenfold (1.7 ± 0.2 mM), reduced transaminases
and proteases with values similar to unmanipulated controls (Table 1
and Fig. 2). Furthermore, survival after transplantation, defined as
living for 7 days, improved as the dose of glycine given was increased
(Fig. 1). Elevation of glycine to 1.7 mM by infusion totally prevented
the effect of manipulation on survival (Fig. 1).

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Fig. 1.
Intravenous glycine improves survival of gently manipulated livers.
Donor livers were harvested within 25 min with or without gentle in
situ organ manipulation before perfusion with cold University of
Wisconsin cold storage (UW) solution. Livers were stored in UW solution
at 0-4°C for 1 h, and transplantation was performed using
rearterialization. Top: survival of
manipulated livers is compared after transplantation. Valine, the
isonitrogenous control, or glycine (1.5 ml; 300 mM) was infused over 1 h before organ harvest. Bottom: dose
dependence. a P < 0.05 compared with no manipulation;
b P < 0.05 compared with manipulation after valine infusion by Fisher's exact
test, n = 10-16 rats. Fractions
indicate survival per total number of transplantations studied.
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Fig. 2.
Intravenous glycine prevents increase of proteolytic activity in rinse
solution by gentle organ manipulation. Livers were rinsed with cold UW
solution after explantation, and proteolytic activity was measured in
rinse effluent. Valine or glycine was infused as described in
METHODS. Values are means ± SE
(P < 0.05 by 2-way ANOVA with
Student-Newman-Keuls post hoc test, n = 10-14 rats).
a P < 0.05 for
comparison with no manipulation;
b P < 0.05 compared with manipulation after valine infusion.
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Intravenous glycine blunts liver necrosis after
reperfusion. In all groups studied, tissue injury was
undetectable before cold storage. In addition, nonmanipulated grafts
developed <1% necrosis 8 h after transplantation (Table 1 and Fig.
3). However, after graft
manipulation of controls, ~14% of the tissue was necrotic 8 h after
transplantation (Table 1 and Fig. 3). Fourteen percent necrosis is
considerable after 8 h since animals do not begin to die until 1 day
after transplantation. This damage was largely prevented by infusion of
glycine before the donor operation (Table 1 and Fig. 3). Moreover,
necrosis progressed rapidly in the manipulated valine group, yielding
values of >60% (P < 0.05) on the
first day after transplantation, whereas <5% of the cells were
necrotic in the glycine group, values not different from unmanipulated controls.

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Fig. 3.
Pattern of necrosis after transplantation. Conditions as described in
Fig. 1. Rats were killed 8 h after transplantation, and liver tissue
was processed for light microscopy by hematoxylin and eosin staining.
Top: no manipulation;
middle, manipulated livers from donors
infused with valine; bottom, manipulation + glycine. Typical
photomicrographs are shown. Magnification = ×100.
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Intravenous glycine prevents disturbances to the
hepatic microcirculation. Livers were perfused with
fluorescein isothiocyanate-dextran (12 µM) for 3 min to index the
hepatic microcirculation. Because fluorescein isothiocyanate-dextran is
confined to the vascular space in liver, the rate of fluorescence wash
in and washout as well as the percent increase of surface fluorescence
over basal are indicative of microcirculation and vascular space,
respectively (8). Surface fluorescence was maximal and stable within 1 min. Indeed, microcirculation was disturbed significantly when grafts were perfused after manipulation, reflected by a two- to fourfold decreased rate for fluorescein isothiocyanate-dextran entry and exit
from the vascular space (Figs. 4 and
5). Moreover, manipulation decreased
surface fluorescence nearly twofold (P < 0.05; Figs. 4 and 5). Intravenous glycine infusion before
manipulation totally prevented the effects of manipulation on surface
fluorescence and rates of changes in fluorescence (Figs. 4 and 5). To
determine the influence of glycine in manipulated livers on
microcirculation at reperfusion after transplantation, the time for the
organ to turn uniformly red due to the hemoglobin pigment upon
completion of implantation was recorded. In unmanipulated controls,
blood was distributed completely in 30 ± 2 s. This time was
prolonged more than fivefold by gentle manipulation in the valine
group. However, when glycine was infused before manipulation, blood
distributed rapidly and was not different from unmanipulated controls
(Fig. 6).

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Fig. 4.
Intravenous glycine prevents effects of gentle organ manipulation on
surface fluorescence. Conditions are as described in Fig. 1. Livers
were perfused ex situ with fluorescein-dextran via the portal vein
before cold storage as indicated by vertical lines. Top, no
manipulation; middle, fluorescence after manipulation in
valine group; bottom, fluorescence in
manipulated livers from glycine-infused rats. Typical experiments are
shown.
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Fig. 5.
Intravenous glycine prevents effect of gentle organ manipulation on
microcirculation. Fluorescein isothiocyanate-dextran (12 µM) was
infused for 3 min after removal of the liver, and percent increase over
basal fluorescence (top) and the
rate of change in basal fluorescence
(bottom) were recorded for each
liver as described in METHODS. Values are means ± SE
(P < 0.05 by 2-way ANOVA with
Student-Newman-Keuls post hoc test, n = 5-8 rats).
a P < 0.05 for comparison with no
manipulation; b P < 0.05 compared with
manipulation after valine infusion.
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Fig. 6.
Intravenous glycine prevents effects of gentle organ manipulation on
blood distribution. Donor livers were harvested as described in
METHODS. Some donors were infused with valine or glycine.
After transplantation, the time for blood to distribute homogeneously
was recorded during reperfusion as described in METHODS.
Values are means ± SE (P < 0.05 by 2-way ANOVA with Student-Newman-Keuls post hoc test, n = 5-10 rats). a P < 0.05 for
comparison with no manipulation; b P < 0.05 compared with manipulation after valine infusion.
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Effect of intravenous glycine on hypoxia in
manipulated grafts. Pimonidazole, a 2-nitroimidazole
hypoxia marker, binds to hypoxic liver cells in vivo (2). Gentle liver
manipulation increased hypoxia about twofold in the valine control
group before cold storage (P < 0.05;
Figs. 7 and
8). As expected, pimonidazole binding
predominated in pericentral regions where O2 supply is naturally low (Fig. 7). Binding of pimonidazole in manipulated livers
from donors given glycine intravenously was not different from
unmanipulated controls (Figs. 7 and 8).

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Fig. 7.
Effect of intravenous glycine on pattern of pimonidazole binding after
manipulation. Conditions are as described in Fig. 1. Photomicrographs
depict patterns of pimonidazole binding in livers after harvest.
Immunohistochemistry using antibodies to bound pimonidazole is
described in METHODS. Top:
unmanipulated livers after harvest;
middle: donors undergoing graft
manipulation during harvest were infused with valine;
bottom: glycine before donor
operation. Typical photomicrographs are shown. Magnification = ×100.
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Fig. 8.
Intravenous glycine prevents hypoxia due to gentle organ manipulation.
Conditions are as described in Fig. 1. Pimonidazole was given to donors
before harvest as described in METHODS, and pimonidazole
binding was detected using competitive ELISA. Some donors were infused
with valine or glycine before manipulation. Results are means ± SE;
n = 5 rats.
a P < 0.05 for comparison with no
manipulation; b P < 0.05 compared with manipulation after valine infusion.
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DISCUSSION |
Glycine infusion to donors minimizes harvest-related
reperfusion injury. Previous studies have shown that
addition of amino acid mixtures to the perfusate of isolated kidneys
preserves tubular integrity and prolongs renal function (10). Weinberg
et al. (32) were the first to relate this protective effect to the simplest amino acid, glycine. Glycine has been demonstrated to be
protective in vitro against tissue damage caused by ischemia, ischemia-reperfusion, or toxicants in a variety of species (19, 20, 32, 33). Furthermore, Carolina rinse solution containing glycine
prevents reperfusion injury in livers in both experimental and clinical
liver transplantation (19). Moreover, dietary glycine prevents
cyclosporin A-induced nephrotoxicity after transplantation (31).
Injury developing at reperfusion, mediated by activated Kupffer cells,
is one of the most important events leading to early dysfunction or
nonfunction of grafts, which remains a major obstacle in
transplantation. Most recently, gentle in situ graft manipulation by
touching, retracting, and moving liver lobes gently during harvest has
been demonstrated to be detrimental for survival after liver
transplantation via mechanisms involving microcirculation and Kupffer
cell-dependent reperfusion injury. The effects of manipulation were
prevented by GdCl3, a rare earth
metal that depletes Kupffer cells, and with glycine, which prevents
activation of Kupffer cells (27). GdCl3 cannot be used
clinically because of its potential toxicity, and it would be difficult
to give dietary glycine to human donors before organ retrieval;
therefore, in this study, donors were given a brief intravenous
infusion of glycine before organ harvest. Gentle in situ graft
manipulation during harvest dramatically decreased survival (Fig. 1)
and elevated serum transaminases, bilirubin, and necrosis (Table 1 and
Fig. 3) after transplantation (27). Furthermore, proteolytic activity was elevated after harvest in the graft rinse solution (Fig. 2), and
microcirculation was disturbed by manipulation (Figs. 4-6). This
effect is important since manipulation of the liver during harvest with
standard techniques cannot be prevented completely in clinical liver
transplantation. Glycine given intravenously to donors before harvest
prevented all of the detrimental effects of manipulation (Table 1 and
Figs. 1-8). Therefore, it is concluded that intravenous glycine
prevents harvest-related reperfusion injury after liver transplantation.
Mechanisms by which glycine reduces harvest-related
reperfusion injury. Nichols et al. (21) found that
glycine inhibited nonlysosomal
Ca2+-dependent proteases and
concluded that this action was responsible for protection of
hepatocytes from anoxic injury. Furthermore, proteolysis has been shown
to contribute to graft injury after transplantation of livers (7) and
protease inhibitors improve graft function (19). Indeed,
in this study, glycine prevented increased proteolytic activity by
manipulation (Fig. 2). Glycine activates a glycine-gated chloride
channel in Kupffer cells, and influx of chloride hyperpolarizes the
cell membrane, making Ca2+
channels more difficult to open. Therefore, glycine blunts the increase
of intracellular Ca2+, thus
minimizing activation of Kupffer cells and increases in Ca2+-dependent proteases (17). Kupffer cell-dependent
reperfusion injury after cold storage, a key event in primary
nonfunction, is also characterized by death of endothelial lining cells
(19). Furthermore, apoptosis of sinusoidal endothelial cells occurs (11). Indeed, gentle organ manipulation dramatically increased the
number of dead sinusoidal lining cells per field after 24 h of cold
storage more than fivefold (P < 0.05), which was prevented by glycine
(P < 0.05; no manipulation, 4 ± 1; manipulation + valine, 23 ± 3; manipulation + glycine,
6 ± 1), as assessed by nuclear staining with trypan blue in
perfused livers. Because Kupffer cells and stellate cells
initially retain viability (19), the exacerbated damage in manipulated
livers corresponds to death of the endothelium. Qu et al. (25) showed
that activated Kupffer cells release prostaglandin
E2, which then stimulates
parenchymal cell O2 consumption,
most likely creating hypoxia, and Bradford et al. (6) demonstrated that
GdCl3 blocks elevated
O2 uptake due to Kupffer cell activation with ethanol. In
addition, Qu et al. (24) demonstrated that O2 uptake is
nearly doubled after liver transplantation, and this effect is also
blocked by GdCl3. Because
increased O2 uptake leads to
hepatic hypoxia (1), it was evaluated in this study. Indeed, binding of
the hypoxia marker pimonidazole was increased more than twofold after
manipulation, an effect blunted by infusion of glycine (Figs.
7-9). Thus the rapid development of hypoxia
in the liver due to gentle in situ manipulation can be prevented by
intravenous glycine (Fig. 9).

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Fig. 9.
Diagram of proposed mechanism by which glycine prevents harvest-related
injury. Gentle manipulation of the liver during harvest causes
intrahepatic vasoconstriction, disturbances in microcirculation, and
hypoxia, which is followed by "priming" or activation of Kupffer
cells, which play a principal role in reperfusion injury. Activated
Kupffer cells then release vasoactive substances, free radicals,
cytokines, and proteases that contribute to reperfusion injury
developing upon transplantation, further impairing microcirculation.
The production of some mediators released from Kupffer cells requires
an increase of intracellular Ca2+.
When glycine is given to the donor, a glycine-gated chloride channel
(GlyR) in the membrane of Kupffer cells is activated, leading to
chloride influx and to subsequent hyperpolarization of the membrane
( ). This prevents activation of Kupffer cells, disturbed
microcirculation, hypoxia, and proteolytic activity caused by gentle
graft manipulation during harvest. PTK, protein tyrosine kinase; PLC,
phospholipase C; IP3, inositol
trisphosphate; LPS, lipopolysaccharide;
PIP2, phosphoinositol phosphate-2;
LBP, liposaccharide binding protein.
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Ozaki et al. (23) recently demonstrated that glycine could also protect
livers in situ from reperfusion injury by reducing lipid peroxidation,
an effect that was not due to alteration of glutathione in the liver
tissue. Furthermore, Schilling et al. (28) found that glycine
stabilized the cell membrane by inhibition of phospholipase
A2, which releases arachidonic
acid, leading to eicosanoid production. It is possible that
vasoconstrictive eicosanoids lead to the altered microcirculation
observed here (Figs. 4-6). Indeed, both during harvest and at
reperfusion after transplantation, microcirculation was improved in
manipulated grafts from donors given glycine intravenously before the
donor operation. Furthermore, glycine did not increase the production of nitric oxide (NO), a potent vasodilator, by Kupffer cells; in fact,
it caused a small but significant reduction of NO production (unpublished data). A number of studies have shown that hepatic microcirculation plays an important role in development of reperfusion injury. Several lines of evidence suggest that microcirculatory disturbances are a key factor in enhancing donor organ susceptibility to both cold and warm ischemia in livers (14, 15, 29). Kupffer cells may be responsible for reducing survival and graft viability after transplantation as a result of manipulation. This idea is supported by the fact that GdCl3,
a selective Kupffer cell toxicant, reduced harvest-related reperfusion
injury, most likely via mechanisms involving the hepatic
microcirculation (27). Taken together, these data clearly indicate that
glycine improves survival in manipulated grafts by prevention of
Kupffer cell activation via mechanisms involving hepatic microcirculation.
Possible site of action of glycine. It
is known that destruction of Kupffer cells, the major source of
eicosanoids in the liver (9), reduces reperfusion injury (33).
Moreover, Kupffer cells release proteases and TNF-
upon activation
(5). A recent study has shown that glycine reduces TNF-
production
and minimizes death induced by endotoxin, a known activator of Kupffer
cells (16). Indeed, TNF-
production after LPS (100 ng/ml), expressed as percentage of increase over basal, increased significantly in
Kupffer cells from manipulated livers; however, this effect was blunted
when glycine was infused to the donor before manipulation (P < 0.05; no manipulation, 11 ± 0.5;
manipulation + valine, 34 ± 4; manipulation + glycine, 21 ± 3).
Accordingly, it is proposed that glycine prevents activation of Kupffer
cells at harvest, thereby minimizing reperfusion injury after
transplantation. This effect is most likely related to actions on
glycine-gated chloride channels of Kupffer cells (Fig. 9).
Clinical implications. Reperfusion
injury is linked to primary graft nonfunction, which is still a major
problem after clinical liver transplantation. Harvest-related
reperfusion injury and primary nonfunction were prevented by
intravenous glycine in this study. This is important since gentle organ
manipulation cannot be prevented with standard harvesting techniques.
Based on the data presented here, clinical trials are warranted to
determine if a similar effect of glycine occurs in human liver transplantation.
 |
ACKNOWLEDGEMENTS |
This work was supported, in part, by grants from the National
Institute on Alcohol Abuse and Alcoholism and by the Deutsche Forschungsgemeinschaft.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other corrspondence: R. G. Thurman,
Lab. of Hepatobiology and Toxicology, Dept. of Pharmacology, CB no.
7365, Mary Ellen Jones Bldg., The Univ. of North Carolina, Chapel Hill,
NC 27599-7365 (E-mail: thurman{at}med.unc.edu).
Received 20 August 1998; accepted in final form 7 December
1998.
 |
REFERENCES |
1.
Arteel, G. E.,
Y. Iimuro,
M. Yin,
J. A. Raleigh,
and
R. G. Thurman.
Chronic enteral ethanol treatment causes hypoxia in rat liver tissue in vivo.
Hepatology
25:
920-926,
1997[Medline].
2.
Arteel, G. E.,
R. G. Thurman,
J. M. Yates,
and
J. A. Raleigh.
Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde perfusion of rat liver.
Br. J. Cancer
72:
889-895,
1995[Medline].
3.
Belinsky, S. A.,
J. A. Popp,
F. C. Kauffman,
and
R. G. Thurman.
Trypan blue uptake as a new method to investigate hepatotoxicity in periportal and pericentral regions of the liver lobule. Studies with allyl alcohol in the perfused liver.
J. Pharmacol. Exp. Ther.
230:
755-760,
1984[Abstract].
4.
Bergmeyer, H. U.
Methods of Enzymatic Analysis. New York: Academic, 1988.
5.
Bouwens, L.
Structural and functional aspects of Kupffer cells.
Rev. Biol. Cell.
16:
69-94,
1988.
6.
Bradford, B. U.,
U. K. Misra,
and
R. G. Thurman.
Kupffer cells are required for the swift increase in alcohol metabolism.
Res. Commun. Subst. Abuse
14:
1-6,
1993.
7.
Calmus, Y.,
L. Cynober,
B. Dousset,
S. K. Lim,
O. Soubrane,
F. Conti,
D. Houssin,
and
J. Giboudeau.
Evidence for the detrimental role of proteolysis during liver preservation in humans.
Gastroenterology
108:
1510-1516,
1995[Medline].
8.
Conway, J. G.,
J. A. Popp,
and
R. G. Thurman.
Microcirculation in periportal and pericentral regions of lobule in perfused rat liver.
Am. J. Physiol.
249 (Gastrointest. Liver Physiol. 12):
G449-G456,
1985[Medline].
9.
Decker, K.
Biologically active products of stimulated liver macrophages (Kupffer cells).
Eur. J. Biochem.
192:
245-261,
1990[Medline].
10.
Epstein, F. H.,
J. T. Brosnan,
J. D. Tange,
and
B. D. Ross.
Improved function with amino acids in the isolated perfused kidney.
Am. J. Physiol.
243 (Renal Fluid Electrolyte Physiol. 12):
F284-F292,
1982[Abstract/Free Full Text].
11.
Gao, W.,
R. C. Bentley,
J. F. Madden,
and
P. A. Clavien.
Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation.
Hepatology
27:
1652-1660,
1998[Medline].
12.
Gao, W.,
J. J. Lemasters,
and
R. G. Thurman.
Development of a new method for hepatic rearterialization in rat orthotopic liver transplantation: reduction of liver injury and improvement of surgical outcome by arterialization.
Transplantation
56:
19-24,
1993[Medline].
13.
Green, L. C.,
D. A. Wagner,
J. Glowgowski,
P. L. Skipper,
J. S. Wishnok,
and
S. R. Tannenbaum.
Analysis of nitrate, nitrite, and (15N)nitrate in biological fluids.
Anal. Biochem.
126:
131-138,
1982[Medline].
14.
Hui, A.,
S. Kawasaki,
M. Makuuchi,
J. Nakayama,
T. Ikegami,
and
J. Miyagawa.
Liver injury following normothermic ischemia in steatotic rat liver.
Hepatology
20:
1287-1293,
1994[Medline].
15.
Husberg, B. S.,
Y. S. Genyk,
and
G. B. Klintmalm.
A new rat model for studies of the ischemic injury after transplantation of fatty livers: improvement after postoperative administration of prostaglandin.
Transplantation
57:
457-458,
1994[Medline].
16.
Ikejima, K.,
Y. Iimuro,
D. T. Forman,
and
R. G. Thurman.
A diet containing glycine improves survival in endotoxin shock in the rat.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G97-G103,
1996[Abstract/Free Full Text].
17.
Ikejima, K.,
W. Qu,
R. F. Stachlewitz,
and
R. G. Thurman.
Kupffer cells contain a glycine-gated chloride channel.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G1581-G1586,
1997[Abstract/Free Full Text].
18.
Koyama, S.,
S. I. Rennard,
G. D. Leikauf,
R. F. Ertl,
and
R. A. Robbins.
Antiproteases attenuate the release of neutrophil chemotactic activity from bronchial epithelial cells induced by smoke.
Exp. Lung Res.
22:
1-19,
1996[Medline].
19.
Lemasters, J. J.,
and
R. G. Thurman.
Reperfusion injury after liver preservation for transplantation.
Annu. Rev. Pharmacol. Toxicol.
37:
327-338,
1997[Medline].
20.
Marsh, D. C.,
P. K. Vreugdenhil,
V. E. Mack,
F. O. Belzer,
and
J. H. Southard.
Glycine protects hepatocytes from injury caused by anoxia, cold ischemia and mitochondrial inhibitors, but not injury caused by calcium ionophores or oxidative stress.
Hepatology
17:
91-98,
1993[Medline].
21.
Nichols, J. C.,
S. F. Bronk,
R. L. Mellgren,
and
G. J. Gores.
Inhibition of nonlysosomal calcium-dependent proteolysis by glycine during anoxic injury of rat hepatocytes.
Gastroenterology
106:
168-176,
1994[Medline].
22.
Ohmori, S.,
M. Ikeda,
S. Kira,
and
M. Ogata.
Colorimetric determination of hippuric acid in urine and liver homogenate.
Anal. Chem.
49:
1494-1496,
1977[Medline].
23.
Ozaki, M.,
H. Ozasa,
S. Fuchinoue,
S. Teraoka,
and
K. Ota.
Protective effects of glycine and esterified-glutamylcystein on ischemia/reoxygenation injury of rat liver.
Transplantation
22:
753-755,
1995.
24.
Qu, W.,
E. Savier,
and
R. G. Thurman.
Stimulation of monooxygenation and conjugation following liver transplantation in the rat: involvement of Kupffer cells.
Mol. Pharmacol.
41:
1149-1154,
1992[Abstract].
25.
Qu, W.,
Z. Zhong,
M. Goto,
and
R. G. Thurman.
Kupffer cell prostaglandin E2 stimulates parenchymal cell O2 consumption: alcohol and cell-cell communication.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G574-G580,
1996[Abstract/Free Full Text].
26.
Raleigh, J. A.,
J. K. La Dine,
J. M. Cline,
and
D. E. Thrall.
An enzyme-linked immunosorbent assay for hypoxia marker binding in tumours.
Br. J. Cancer
69:
66-71,
1994[Medline].
27.
Schemmer, P.,
R. Schoonhoven,
J. A. Swenberg,
H. Bunzendahl,
and
R. G. Thurman.
Gentle in situ liver manipulation during organ harvest decreases survival after rat liver transplantation: role of Kupffer cells.
Transplantation
65:
1015-1020,
1998[Medline].
28.
Schilling, M.,
G. den Butter,
D. C. Marsh,
F. O. Belzer,
and
J. H. Southard.
Glycine inhibits phospholipolysis of hypoxic membranes.
Tex. Med.
6:
140-143,
1994.
29.
Teramoto, K.,
J. L. Bowers,
J. B. Kruskal,
and
M. E. Clouse.
Hepatic microcirculatory changes after reperfusion in fatty and normal liver transplantation in the rat.
Transplantation
56:
1076-1082,
1993[Medline].
30.
Thurman, R. G.,
I. Marzi,
G. Seitz,
J. Thies,
J. J. Lemasters,
and
F. A. Zimmermann.
Hepatic reperfusion injury following orthotopic liver transplantation in the rat.
Transplantation
46:
502-506,
1988[Medline].
31.
Thurman, R. G.,
Z. Zhong,
M. V. Frankenberg,
R. F. Stachlewitz,
and
H. Bunzendahl.
Prevention of cyclosporin-induced nephrotoxicity with dietary glycine.
Transplantation
63:
1661-1667,
1997[Medline].
32.
Weinberg, J. M.,
J. A. Davis,
M. Abarzua,
and
T. Rajan.
Cytoprotective effects of glycine and glutathione against hypoxic injury to renal tubules.
J. Clin. Invest.
80:
1446-1454,
1987[Medline].
33.
Zhong, Z.,
S. Jones,
and
R. G. Thurman.
Glycine minimizes reperfusion injury in a low-flow, reflow liver perfusion model in the rat.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G332-G338,
1996[Abstract/Free Full Text].
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