Sodium pyruvate improves the plasma amino acid profile in rats with l-arginine-induced acute pancreatitis

Plasma amino acid levels are altered upon many pathological conditions including acute pancreatitis. It is unclear whether amino acids can be used as specific 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. 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) Experimental treatment group received sodium pyruvate (1 g/kg) for 4 days. On day 8 of the experiment, animals were killed, blood was collected and plasma amino acid concentration was determined with high-performance liquid chromatography. Histological examination showed large areas of fibrosis in the pancreas of animals treated with l-arginine irrespectively of sodium pyruvate administration. Sodium pyruvate improved the plasma amino acid levels. Rats with acute pancreatitis had significantly 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 the levels of methionine, phenylalanine, tryptophan, leucine, isoleucine, aspartate, asparagine and ornithine levels, while increasing glutamine and serine to levels significantly higher than control. Plasma lysine, alanine, arginine and taurine 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 fixed limits under normal conditions.In healthy people, plasma-free amino acid composition depends on the protein pool of amino acids and is influenced by many factors, e.g., diet, physical activity and circadian rhythm.
It is known that plasma amino acids level is changed upon pancreatitis (Roth et al. 1985;Sandstrom et al. 2008) due to enzyme deficiency (Domínguez-Muñoz 2011), intestinal malabsorption or systemic inflammation (Schrader et al. 2009).Plasma amino acid 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.
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).Ca 2+ -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 inflammation 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 the 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. 2019Manko et al. , 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: first, 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.

Animal experiments
All experiments were carried out in accordance with the "European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific 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-h light-dark cycle and were given free access to water and standard diet (D-Mix, Ukraine).The experimental animals were randomly split to three 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 h.Control animals were injected with the same volume of PS intraperitoneally instead of l-arginine-HCl (Dawra and Saluja 2012).Sodium pyruvate administration was initiated 72 h after the first intraperitoneal injection of l-arginine-HCl.The animals were injected sodium pyruvate (1 g/kg body weight/day total) intraperitoneally TID at 8 a.m., 2 p.m. and 8 p.m. 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 killed by decapitation on the 8th day of the experiment.The pancreas was quickly removed and cut from the fat and lymph nodes.Part of the pancreatic tissue was immediately fixed 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 2000g in a refrigerated centrifuge (4 °C) for 10 min to separate plasma.

Amino acid analysis
Sulfosalicylic acid was added to plasma 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 amino acid analysis performed.
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-fluorobenzene in a water bath at 40 °C for 40 min.

Evaluation of amylase activity in plasma
Turbidimetric kinetic method was used for measuring alphaamylase 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 a 10 mm glass cuvette.

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.Significance 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 significant effect.P < 0.05 values were considered statistically significant.

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.To assess the pancreatic damage, we measured plasma amylase activity prior to l-arginine injection, 3, 5 and 8 days afterward.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 first measurement.In rats, serum amylase activity was shown to increase at 12 h after l-arginine injection, peak at 24 h and decrease at 48 h (Tashiro et al. 2001), unlike in mice, where the highest amylase activity was observed at 72 h after l-arginine injection (Dawra and Saluja 2012).
To confirm that pancreatitis did develop, we performed histological examination of the pancreas after killing the animal.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 the pancreas of animals with l-arginine-induced acute pancreatitis, irrespectively of sodium pyruvate administration, atypical cells were found in all cases, probably a sign of fibrosis (Fig. 1b,  c).No signs of leukocyte infiltration, necrosis, edema or hemorrhage were detected, probably due to late examination (7 days after the expected pancreatitis episode).
The level of essential amino acids in plasma of rats with l-arginine-induced experimental acute pancreatitis significantly decreased compared to control (Table 1): methionine-by 27% (P < 0.05), phenylalanine-by 40% (P < 0.05), tryptophan-by 49% (P < 0.05), leucine and isoleucine-by 30% (P < 0.05).In animals with pancreatitis after sodium pyruvate administration, the levels of methionine, phenylalanine, leucine and isoleucine did not significantly 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 (Table 1): the level of asparagine significantly decreased by 29% (P < 0.05) accompanied by a slight non-significant decrease of glutamine concentration, while the level of deamination products aspartate and glutamate increased by 49% (P < 0.05) and 38% (P < 0.05), respectively.Administration of sodium pyruvate to animals with l-arginine-induced pancreatitis caused a significant decrease of aspartate compared to animals with pancreatitis and an increase of glutamine level compared to both control and the l-arginine group (Table 1).The plasma arginine concentration in rats with pancreatitis did not change, while the level of its urea cycle metabolite ornithine decreased by 45% (P < 0.05) 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 < 0.05) and 33% (P < 0.05), respectively, while alanine level remained similar to control.Administration of pyruvate significantly 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.

Discussion
In the present study, we have investigated the effects of sodium pyruvate on the morphological state of the 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 arginine, ornithine and taurine significantly increased and glutamate decreased, but other amino acid levels were unchanged (Buchmann et al. 1996) (Table 2).In contrast, 24 h after arginine dose, plasma 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 that 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 arginine administration are associated with pancreatic damage observed in our study.
The changes of plasma amino acid profile in our study generally resemble those in case of acute pancreatitis in patients (Roth et al. 1985;Sandstrom et al. 2008) (Table 2).Specifically, 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 insufficiency.However, during experimental starvation and the levels of essential amino acids do not decrease, while branched-chain amino acids increase (Adibi 1971;Holeček and Mičuda 2017) (Table 2) 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 infiltrative pancreatic immune cells.In traumatic conditions, glutamine is intensively absorbed by macrophages, neutrophils and lymphocytes (Newsholme 2001) and is involved in both energy metabolism and gene expression regulation by influencing cell proliferation, cytokine synthesis and surface receptors (Cruzat et al. 2018).Thus, the most likely cause of observed changes of plasma amino acid is inflammation and/or corollary organ damage.Moreover, such alteration of plasma amino acid profile is not specific to acute pancreatitis, but also occurs in patients with inflammatory 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) (Table 2).
Rats upon pyruvate administration displayed significantly better plasma amino acid profile, both essential and non-essential.Based on the 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 of the normal level of a number of essential (methionine, phenylalanine, isoleucine and leucine) and non-essential (ornithine, serine) amino acids, which were significantly 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, inflammation and metabolic state of organism.Thus, pyruvate injection could be beneficial as maintaining therapy of acute pancreatitis and its mechanisms of action need to be further elucidated.

Table 1
Plasma amino acids in rats measured on day 8 of the experiment Bold font indicates a statistically significant difference compared to control or arginine pancreatitis group Arginine pancreatitis group received two doses of 2.5 g l-arginine/1 kg body weight intraperitoneally.Sodium pyruvate group also received sodium pyruvate at a 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 *P< 0.05 vs. controls, # P< 0.05 vs. arginine pancreatitis