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Sodium pyruvate improves the plasma amino acid
prole in rats with L-arginine-induced acute
pancreatitis
Anastasiia Zub ( anastasiya.yakubovska@lnu.edu.ua )
Ivan Franko National University of Lviv
Roman Ostapiv
State Scientic-Research Control Institute of Veterinary Medicinal Products and Feed Additives
Bohdan Manko
Ivan Franko National University of Lviv
Volodymyr Manko
Ivan Franko National University of Lviv
Research Article
Keywords: sodium pyruvate, amino acids, pancreatitis, L-arginine, pancreatic morphology.
Posted Date: September 16th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-2033472/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Plasma amino acid levels are altered upon many pathological conditions including acute pancreatitis. It
is unclear whether amino acids can be used as specic biomarker of acute pancreatitis severity or
recovery. Development of acute pancreatitis is associated with mitochondrial dysfunction and decreased
cytosolic ATP level. Sodium pyruvate is considered as a potential treatment of pancreatitis due to its
ability to sustain mitochondrial oxidative and ATP-productive capacity
in vitro
. In this study investigated
the effect of sodium pyruvate on pancreatic morphology and plasma amino acid levels in rats with acute
pancreatitis.
Acute pancreatitis in rats was induced by administration of L-arginine (5 g / kg) and conrmed with
histological examination of pancreas. Experimental treatment group received sodium pyruvate (1 g / kg)
for 4 days. Blood was collected on day 8 of the experiment and plasma amino acids concentration was
determined with high-performance liquid chromatography.
Sodium pyruvate administration did not improve the pancreatic morphology and ultrastructure, but
improves the plasma amino acid levels. Rats with acute pancreatitis had signicantly lower levels of
most essential and non-essential amino acids and increased glutamate and aspartate in plasma.
Administration of sodium pyruvate completely or partially restored levels of methionine, phenylalanine,
tryptophan, leucine, isoleucine, aspartate, asparagine and ornithine levels, while increasing glutamine and
serine to levels signicantly higher than control. Plasma lysine, alanine, arginine and taurine remained
unaffected remained unaffected in all experimental groups. Sodium pyruvate may be considered for use
as a maintenance therapy in acute pancreatitis.
Introduction
Plasma amino acid concentrations vary within xed limits under normal conditions. In healthy people,
plasma free amino acids composition depends on the protein pool of amino acids and is inuenced by
many factors, e.g., diet, physical activity, circadian rhythm, etc.
It is known that plasma amino acids level is changed upon pancreatitis (Roth et al. 1985; Sandstrom et
al. 2008) due to enzyme deciency (Domínguez-Muñoz 2011), intestinal malabsorption or systemic
inammation (Schrader et al. 2009). Plasma amino acids levels are attempted to be used as a biomarker
of pancreatitis or pancreatic cancer (Zhang et al. 2012; Fukutake et al. 2015). Yet it is currently not clear if
plasma amino acid level could be used as an indicator of acute pancreatitis severity or recovery.
Recently, basic amino acid-induced pancreatitis animal (rodent) models have been developed (Biczó et al.
2011; Zhang et al. 2019). A single injection of 5 g / kg L-arginine caused selective necrosis of acinar cells
(Mizunuma et al. 1984; Tani et al. 1990). Importantly, two clinical cases of L-arginine-induced pancreatitis
have been described in humans (Mendane Saka et al. 2004; Binet et al. 2018).
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Although the pathological mechanisms of L-arginine pancreatitis remain poorly understood, it is known
that excessive doses of L-arginine cause ultrastructural changes in intracellular organelles (EPR,
mitochondria) (Kishino and Kawamura 1984; Domínguez-Muñoz 2011). Ca2+-independent mitochondrial
dysfunction associated with cyclophilin D occurs early in the development of acute L-arginine-induced
pancreatitis and is thought to be the main cause of other pathologic manifestations such as decreased
ATP levels, disruption of autophagy, endoplasmic reticulum stress, necrosis and inammation of the
pancreas (Biczo et al. 2018). Interestingly, in rat model of acute pancreatitis, L-arginine mainly
accumulates in the mitochondria, apparently due to the metabolism of this amino acid (Biczo et al.
2018). A possible mediator of the negative effects of L-arginine is ornithine, an intermediate metabolite of
arginine in the urea cycle (Biczó et al. 2010; Zhang et al. 2019).
A number of preclinical studies are aimed at treatment of pancreatitis by restoring a mitochondrial
oxidative function (Shore et al. 2016; Mukherjee et al. 2016; Javed et al. 2018). Oxidative substrate
pyruvate in vitro enhances the oxidative and ATP-productive capacity of mitochondria and protects
pancreatic acinar cells from toxic substances (Peng et al. 2018; Manko et al. 2019, 2021). It is interesting
that substantial levels of pyruvate are quickly accumulated in the pancreas among other organs after
intravenous administration (Serrao et al. 2018) making this substance a very attractive choice for acute
pancreatitis management. Indeed, intravenous sodium pyruvate has a positive effect on acute cerulein-
induced pancreatitis in rats (Ziolkowski et al. 2008).
The present study thus had two aims: rst, to assess whether plasma amino acid levels could be used as
a biomarker of pancreatic damage and recovery, and second, to investigate the effects of intraperitoneal
administration of sodium pyruvate on the morphological state of the pancreas and plasma amino acid
levels in rats with L-arginine-induced acute pancreatitis.
Materials And Methods
Animal experiments
All experiments were carried out in accordance with the "European Convention for the Protection of
Vertebrate Animals used for Experimental and Other Scientic Purposes" (Council of Europe № 123,
Strasbourg 1985). All animal procedures were approved by the Committee for the Care and Use of
Animals of the Ivan Franko National University of Lviv under the protocol number 27-12-2021.
21 male Wistar rats weighing 250–300 g were used in the experiment. Animals were kept at a constant
room temperature of 23°C with a 12-hour light-dark cycle and were given free access to water and
standard diet (D-Mix, Ukraine). The experimental animals were randomly split to 3 groups: 1) the control
group, 2) the L-arginine-induced acute pancreatitis group and 3) animals with L-arginine-induced acute
pancreatitis followed by sodium pyruvate administration. L-arginine-HCl was dissolved in physiological
saline (PS), and its pH was adjusted to 7.4 NaOH. A solution of L-arginine was prepared before each
experiment. Two doses of L-arginine-HCl 2.5 g / kg were administered intraperitoneally at an interval of 1
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hour. Control animals were injected with the same volume of PS intraperitoneally instead of L-arginine-
HCl (Rajinder Dawra and Ashok K. Saluja 2012). Sodium pyruvate administration was initiated 72 hours
after the rst intraperitoneal injection of L-arginine-HCl. The animals were injected sodium pyruvate (1 g /
kg body weight / day total) intraperitoneally TID at intervals of 6, 6 and 12 hours for 4 consecutive days.
The control group of animals and animals with L-arginine-induced acute pancreatitis were administered
the same volume of saline. Animals were sacriced by decapitation on the 8th day of experiment. The
pancreas was quickly removed and cut from the fat and lymph nodes. Part of the pancreatic tissue was
selected for electron microscopy, and another part was immediately xed in 6% neutral formaldehyde
solution for histological analysis.
Blood samples were collected from the tail vein during the course of the experiment and also after
decapitation. Whole blood samples were spun at 2,000 x g in a refrigerated centrifuge (4 °C) for 10 min in
order to separate plasma.
Materials
Reagents used in experiments were purchased from Sigma-Aldrich (L-arginine, sodium pyruvate, 2,4-
dinitro-1-uorobenzene); Fluka (cacodylic acid sodium salt, propylene oxide, Epon 812); Macron
(acetonitrile); SPI-CHEM (osmium tetroxide). All other reagents were of the purest available grade.
Amino acid analysis
To plasma was added sulfosalicylic acid, stored at 4°C for 1 day to precipitate plasma proteins. After
that, the samples were centrifuged for 30 min at 5000 rpm, the supernatant was collected and performed
amino acid analysis.
The studies were performed using a Waters liquid chromatograph consisting of an Alliance separation
module and a Waters 996 diode array detector. For the separation of amino acids, a stationary phase
Luna C18 size 250 * 4.6 mm column with a particle size of 5 μm and 100 A pores was used. The mobile
phase consisted of acetonitrile (Super Gradient) and 0.05 M monosubstituted sodium phosphate
solution, pH 2.0. Gradient elution was used for separation. The eluent feed rate was 1.0 ml / min. Amino
acids were detected by absorption length (lambda) of 350 nm. Amino acid derivatization was performed
with 2,4-dinitro-1-uorobenzene in a water bath at 40° C for 40 minutes.
Evaluation of amylase activity in plasma
Turbidimetric kinetic method was used for measuring alpha-amylase activity (Virolle et al. 1990). The
method relies on the reduction in turbidity that occurs upon digestion of a 1% (w / v) of starch
suspension with plasma alpha-amylase solution. Absorbance was measured at 37°C at 300 nm using
Denovix DS-11+ spectrophotometer in 10 mm glass cuvette.
Electron microscopy
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Fixation of pancreatic tissue was performed with 1.5% OsO4 solution in 0.2 М sodium cacodylate
solution at pH 7.2 for 2–2.5 hours in the cold. The samples were dehydrated in increasing concentrations
of ethanol (30 min each). Next, the samples were incubated in propylene oxide for 10 minutes. Later, the
samples were poured into epoxy resins Epon 812 and polymerized in a thermostat at 60°C for 24 hours.
Sections were made on an ultramicrotome UMTP–6M using a diamond knife (DIATOM). Contrast
sections in 1% uranyl acetate solution and in contrast were performed according to Reynolds (Reynolds
1963). Sections with a thickness of 100 nm were examined using an electron transmission microscope
TEM - 100. Photographed using a digital camera SONY–H9.
Histological preparation
Histological samples were prepared and stained with hematoxylin and eosin (H&E). Sections of the
pancreas 5 μm thick were analyzed and evaluated using an Olympus IX73 microscope with a DP-74
camera.
Statistical analysis
Results are presented as means ± SD. Statistical analysis was performed using Origin Pro 2018.
Signicance of difference between the groups was determined with one-way ANOVA followed by a Holm-
Bonferroni corrected post-hoc t tests in case ANOVA showed a signicant effect. P < 0.05 values were
considered statistically signicant.
Results
L-arginine in high doses is known to cause acute pancreatitis in rats (Mizunuma et al. 1984). We used the
same L-arginine doses as in the latter study. In order to assess the pancreatic damage, we measured
plasma amylase activity prior to L-arginine injection, 3, 5 and 8 days afterwards. Unfortunately, we were
unable to detect any changes of amylase activity on those days (not shown). This is probably because
the peak of enzyme activity in plasma was prior to the rst measurement. In rats, serum amylase activity
was shown to increase at 12 hours after L-arginine injection, peak at 24 hours and decrease at 48 hours
(Tashiro et al. 2001), unlike in mice, where the highest amylase activity is observed at 72 h after L-
arginine injection (Rajinder Dawra and Ashok K. Saluja 2012).
To conrm that pancreatitis did develop, we performed histological examination of the pancreas and
electron microscopy after animal sacrice. In control animals, the acinar cells of the pancreas had normal
morphology, and no changes in the parenchyma and duct system were detected (Fig.1a). In pancreases
of animals with L-arginine-induced acute pancreatitis strong immune cell inltration and diffuse
reduction of acinar cells size with areas of necrosis were observed (Fig.1b). Sodium pyruvate
administration did not improve pancreatic morphology as assessed with a histological examination
(Fig.1c).
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To study the pancreatic tissue in more detail, electron microscopy was performed. In control animals the
ultrastructure of acinar cells was completely normal with heterochromatic nuclei, normal mitochondria,
correctly located parallels of the rough endoplasmic reticulum and zymogenic granules located near the
apical part of acinar cells (Fig.2a). In animals with pancreatitis, the cytoplasm and nuclei of some
pancreatic acinar cells were completely disintegrated, in other cases mitochondria with degenerated
cristae, autophagosomes accumulation or non-apical zymogenic granules localization was observed
(Fig.2b). Sodium pyruvate administration apparently did not improve the ultrastructure of acinar cells
(Fig.2c).
The level of essential amino acids in plasma of rats with L-arginine-induced experimental acute
pancreatitis signicantly decreased comparing to control (Table1): methionine – by 27% (P = 0.002),
phenylalanine – by 40% (P < 0.0001), tryptophan – by 49% (P < 0.0001), leucine and isoleucine – by 30%
(P = 0.03). In animals with pancreatitis after sodium pyruvate administration the levels of methionine,
phenylalanine, leucine and isoleucine did not signicantly change compared to control. Moreover,
tryptophan and phenylalanine increased and valine decreased compared to the pancreatitis group
without treatment. Interestingly, lysine plasma level did not change in any animal groups.
Plasma non-essential amino acid concentration also changed in rats with experimental pancreatitis
(Table1): the level of asparagine signicantly decreased by 29% (P = 0.01) accompanied by a slight non-
signicant decrease of glutamine concentration, while the level of deamination products aspartate and
glutamate increased by 38% (P = 0.0001) and 49% (P = 0.0002), respectively. Administration of sodium
pyruvate to animals with L-arginine-induced pancreatitis caused a signicant decrease of aspartate
compared to animals with pancreatitis and an increase of glutamine level compared to both control and
the L-arginine group (Table1). The plasma arginine concentration in rats with pancreatitis did not
change, while the level of its urea cycle metabolite ornithine decreased by 45% (P = 0.0002) in animals
with pancreatitis. Sodium pyruvate restored the level of ornithine in the plasma to control level. In animals
with pancreatitis, among the amino acids known to be associated with pyruvate metabolism only serine
and dipeptide cystine decreased by 42% (P < 0.0001) and 33% (P = 0.006), respectively, while alanine level
remained similar to control. Administration of pyruvate signicantly increased serine concentration in
plasma of animals with L-arginine-induced pancreatitis to a higher than control level while slightly
decreasing cysteine level. Plasma taurine did not change in all animal groups.
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Table 1
Plasma amino acids in rats measured on day 8 of the experiment. Arginine pancreatitis group received 2
doses of 2.5 g L-arginine / 1 kg body weight intraperitoneally. Sodium pyruvate group also received
sodium pyruvate at total dose of 1 g / kg body weight / day for 4 consecutive days, starting 3 days after
L-arginine injection. Results are presented as absolute data (µmol / L, mean ± SD), n = 6
Amino acids Controls
(µmol /
L)
Arginine pancreatitis group
(µmol / L) Sodium pyruvate group
(µmol / L)
Essential amino
acids:
Methionine 45.4 ± 3.8 33.2 ± 5.2 * 39.5 ± 4.1
Phenylalanine 82.7 ± 5.8 49.6 ± 5.0 * 77.5 ± 8.9 #
Tryptophan 192.4 ±
18.2 97.7 ± 13.7 * 150.4 ± 22.2 * #
Leucine + Isoleucine 302.2 ±
75.7 211.6 ± 26.7 * 245.4 ± 45.1
Valine 96.7 ± 7.3 85.7 ± 6.8 80.8 ± 9.9 *
Lysine 158.0 ±
22.7 143.0 ± 13.8 139.0 ± 11.2
Non-essential amino
acids:
Transamination / deamination
:
Alanine 220.7 ±
32.7 263.5 ± 56.0 276.2 ± 48.0
Glutamine 711.8 ±
32.2 662.1 ± 62.3 942.6 ± 125.9 * #
Glutamate 114.5 ±
11.6 158.4 ± 12.2 * 150.0 ± 21.1 *
Asparagine 92.3 ±
12.2 65.5 ± 14.5 * 73.7 ± 8.4
Aspartate 126.0 ±
17.9 187.4 ± 14.8 * 131.4 ± 10.3 #
Other
:
Arginine 311.8 ±
19.1 342.2 ± 12.2 309.5 ± 49.1
Ornithine 267.5 ±
18.8 148.2 ± 42.8 * 232.1 ± 46.9 #
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Amino acids Controls
(µmol /
L)
Arginine pancreatitis group
(µmol / L) Sodium pyruvate group
(µmol / L)
Serine 173.9 ±
11.2 101.5 ± 5.1 * 207.4 ± 24.1 * #
Cysteine 373.3 ±
57.9 312.2 ± 48.9 277.5 ± 30.0 *
Cystine 70.9 ±
11.3 47.2 ± 10.1 * 64.5 ± 16.6
Taurine 131.9 ±
16.4 111.5 ± 19.2 119.2 ± 16.3
* − P < 0.05 vs. controls # − P < 0.05 vs. arginine pancreatitis
Discussion
In present study we have investigated the effects of sodium pyruvate on the morphological state of
pancreas and plasma amino acids level in rats with L-arginine-induced acute pancreatitis. Since in our
study we measured plasma amino acids 7 days after L-arginine injection we believe all the direct effects
of its metabolism have ceased. Indeed, our results differ from the data obtained shortly after L-arginine
injection in rats (Buchmann et al. 1996; Trulsson et al. 2004). 30 min after L-arginine injection plasma L-
arginine, ornithine and taurine signicantly increased, glutamate decreased but other amino acid levels
were unchanged (Buchmann et al. 1996) (Table 2). In contrast, 24 h after L-arginine dose plasma L-
arginine and ornithine dropped below control (Trulsson et al. 2004) (Table 2). In addition, the
concentration of most other essential (methionine, phenylalanine, valine, lysine) and non-essential
(glutamine, aspartate, serine and alanine) amino acids was lower than in control, partially resembling the
results of our study (Table 2). These changes of plasma amino acids were also accompanied by
pancreatic damage (Trulsson et al. 2004). Thus, the changes of plasma amino acid levels 8 days after L-
arginine administration are associated with pancreatic damage observed in our study.
Table 2Plasma amino acids level changes in pancreatitis, some inammatory diseases and starvation.
Low – decreased comparing to control, high – increased comparing to control, N – close to normal
(control) levels, “-” – not studied.
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a– inammatory bowel disease;
b– 97 of 175 patients also had diabetes mellitus
The changes of plasma amino acid prole in our study generally resemble those in case of acute
pancreatitis in patients (Roth et al. 1985, Sandstrom et al. 2008) (Table2). Specically, three main effects
are observed in patients: 1) the concentrations of most essential amino acids decrease; 2)
transamination / deamination processes increase as evident from asparagine and glutamine decrease
and glutamate increase; 3) the level of other non-essential amino acids (alanine, serine, arginine,
ornithine) decreases.
We assumed that a decrease in essential amino acids in pancreatitis might be associated with
indigestion and decreased absorption of amino acids in animals due to pancreatic insuciency. However,
during experimental starvation the levels of essential amino acids do not decrease, while branched-chain
amino acids increase (Adibi 1971; Holeček and Mičuda 2017) (Table2) due to changes in muscle
metabolism (Holeček 2020). The increase of glutamate level might be caused by the elevation of plasma
aspartate aminotransferase activity observed in L-agrinine pancreatitis model (Yenicerioglu et al. 2013;
Gulturk et al. 2021). Decreased plasma glutamine might be associated with the use of this amino acid by
inltrative pancreatic immune cells. In traumatic conditions glutamine is intensive absorbed by
macrophages, neutrophils and lymphocytes (Newsholme 2001) and is involved in both energy
metabolism and gene expression regulation by inuencing cell proliferation, cytokine synthesis and
surface receptors (Cruzat et al. 2018). Thus, the most likely cause of observed changes of plasma amino
acid is inammation and / or corollary organ damage. Moreover, such alteration of plasma amino acid
prole is not specic to acute pancreatitis, but also occurs in patients with inammatory bowel diseases
(Hisamatsu et al. 2012) or pneumonia (Ikeda 2021), but not so much upon chronic pancreatitis (Schrader
et al. 2009, Girish et al. 2011, Adrych 2010, Kawaguchi et al. 2012) (Table2).
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Rats upon pyruvate administration displayed signicantly better plasma amino acid prole, both
essential and non-essential. Based on levels of glutamine, glutamate, asparagine and aspartate we
postulate that changes of deamination / transamination observed at the pancreatitis model were
ameliorated by pyruvate administration. This was accompanied by restoration the normal level of a
number of essential (methionine, phenylalanine, isoleucine and leucine) and non-essential (ornithine,
serine) amino acid which were signicantly depleted upon pancreatitis. Such positive effects of pyruvate
administration on plasma amino acids are enigmatic, apart from the increase of its metabolite serine.
Due to the apparent lack of histological evidence of pancreatitis improvement upon the action of
pyruvate, it is thus unclear if plasma amino acids may be used as a marker of recovery from acute
pancreatitis. Despite this, pyruvate may have improved some aspects of pancreatic physiology,
inammation and metabolic state of organism. Thus, pyruvate injection could be benecial as
maintaining therapy of acute pancreatitis and its mechanisms of action need to be further elucidated.
Declarations
Competing interests
The authors have no competing interests to declare that are relevant to the content of this article.
Ethical approval
All experiments were carried out in accordance with the "European Convention for the Protection of
Vertebrate Animals used for Experimental and Other Scientic Purposes" (Council of Europe № 123,
Strasbourg 1985). All animal procedures were approved by the Committee for the Care and Use of
Animals of the Ivan Franko National University of Lviv under the protocol number 27-12-2021.
Consent to participate: Not applicable.
Consent for publication: Not applicable.
Availability of data and material: Not applicable.
Author contributions:The authors conrm contribution to the paper as follows: study conception and
design: Bohdan O. Manko, Volodymyr V. Manko, Anastasiia M. Zub; data collection: Anastasiia M. Zub,
Roman D. Ostapiv; analysis and interpretation of results: Anastasiia M. Zub, Roman D. Ostapiv, Bohdan O.
Manko; draft manuscript preparation: Anastasiia M. Zub, Bohdan O. Manko, Volodymyr V. Manko. All
authors reviewed the results and approved the nal version of the manuscript.
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Figures
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Figure 1
Histological examination of rat pancreas. Normal tissue in the control group (a1, a2); immune cell
inltration (star) and necrosis (arrowhead) with areas of intact tissue (arrow) in arginine pancreatitis (b1,
b2) and sodium pyruvate groups (c1, c2). Scale bar = 100 μm and 50 μm
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Figure 2
Electron micrograph of pancreatic acinar cells. Control cells demonstrate normal morphology with
zymogen granules (Z) around acinar lumen (L), rough endoplasmic reticulum (RER) and mitochondria
(M) (a1, a2). Arginine pancreatitis group and sodium pyruvate group show undamaged cell areas with
normal mitochondria and zymogen granules (b1, c1) and damaged areas with irregular shape of nuclei
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(N) (b2, c2, b4), autophagosome (AP) accumulation (b3, c4) and extensively injured mitochondria (c3).
Scale bar = 500 nm
