Chlorogenic acid reduces inflammation in murine model of acute pancreatitis.

BACKGROUND: The pathogenesis of acute pancreatitis (AP) initiation and progression is still unknown, and effective treatment is limited to supportive care. Many phytochemicals have the potential to alleviate AP symptoms and may be a useful and effective supplement to standard AP treatment. The objective of the study was to examine the potential role of chlorogenic acid (CGA), a polyphenol known for anti-inflammatory effect, in the treatment of experimental AP in mice. METHODS: Two intraperitoneal (ip) injections of L-arginine (dosage 400 mg/100 g BW) were given 1 h apart to generate the AP murine model. Mice were separated into two experimental groups after 12 h from the first L-arginine injection: AP mice treated with CGA (oral gavage (po) every 12 h; 20 mg/kg BW) and non-treated AP mice (po vehicle, 5% dimethyl sulfoxide every 12 h). Every 12 h, control mice were given an equivalent volume of vehicle. At 72 h, mice were slaughtered. Histology, as well as myeloperoxidase (MPO) and amylase activity assays, were performed on pancreatic tissues. RESULTS: In murine mouse model of AP po administration of CGA decreased MPO vs. AP (40.40 ± 2.10 U vs. 7.39 ± 0.34; p < 0.001) as well as amylase activity vs. AP (1444 ± 56 mU/mL vs. 3340 ± 144 mU/mL, Fig. 2B; p < 0.001). When comparing CGA mice to AP mice, histological research demonstrated that the severity of AP was reduced following CGA treatment. CONCLUSIONS: The current study found that CGA might have anti-inflammatory effect on L-arginine-induced pancreatitis. Dietary intervention with CGA may be advised as a supportive treatment for AP, according to our findings.

Versatile cationic liposomes for RIP3 overexpression in colon cancer therapy and RIP3 downregulation in acute pancreatitis therapy.

Because the induction of strong host antitumor responses plays a very important role in antitumor therapy, identifying effective approaches to elicit immunogenic cell death could have important implications. RIP3-dependent necroptotic cancer cells have been reported to release damage-associated molecular patterns and enhance antitumor immunity. In this study, hyaluronic acid-conjugated cationic liposomes (DOTAP/DOPE/PEG-DSPE/CHOL) (HA-P-LP) were prepared as a vector for mRIP3-pDNA overexpression in tumours. Compared with standard cationic liposomes, this vector markedly increased cellular gene internalisation in vitro, enhanced the tumour-targeting effect in vivo and exhibited a significant antitumor effect in combination with adjuvant chloroquine. Considering the dramatic increase in RIP3 under the pathological condition of pancreatitis and the correlation between pancreatitis and necroptosis, non-HA-conjugated liposomes with the same formulation loaded with shRNA mRIP3-pDNA effectively controlled the disease by decreasing the serum amylase concentration and inflammatory cell infiltration. The versatile cationic liposomes loaded with plasmids with opposing functions in this study provide a new concept and method for both tumour therapy and pancreatitis therapy.

Dachaihu decoction ameliorates pancreatic fibrosis by inhibiting macrophage infiltration in chronic pancreatitis.

AIM: To explore the role of macrophages in chronic pancreatitis (CP) and the effect of Dachaihu decoction (DCHD) on pancreatic fibrosis in mice. METHODS: KunMing mice were randomly divided into a control group, CP group, and DCHD group. In the CP and DCHD groups, mice were intraperitoneally injected with 20% L-arginine (3 g/kg twice 1 d/wk for 6 wk). Mice in the DCHD group were administered DCHD intragastrically at a dose of 14 g/kg/d 1 wk after CP induction. At 2 wk, 4 wk and 6 wk post-modeling, the morphology of the pancreas was observed using hematoxylin and eosin, and Masson staining. Interleukin-6 (IL-6) serum levels were assayed using an enzyme-linked immunosorbent assay. Double immunofluorescence staining was performed to observe the co-expression of F4/80 and IL-6 in the pancreas. Inflammatory factors including monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and IL-6 were determined using real time-polymerase chain reaction. Western blot analysis was used to detect fibronectin levels in the pancreas. RESULTS: Compared with the control group, mice with 20% L-arginine-induced CP had obvious macrophage infiltration and a higher level of fibrosis. IL-6 serum concentrations were significantly increased. Double immunofluorescence staining showed that IL-6 and F4/80 were co-expressed in the pancreas. With the administration of DCHD, the infiltration of macrophages and degree of fibrosis in the pancreas were significantly attenuated; IL-6, MCP-1 and MIP-1α mRNA, and fibronectin levels were reduced. CONCLUSION: The dominant role of macrophages in the development of CP was mainly related to IL-6 production. DCHD was effective in ameliorating pancreatic fibrosis by inhibiting macrophage infiltration and inflammatory factor secretion in the pancreas.

Effects of propolis, caffeic acid phenethyl ester, and pollen on renal injury in hypertensive rat: An experimental and theoretical approach.

The objective of this study was to evaluate the antioxidant effects of propolis, caffeic acid phenethyl ester (CAPE; active compound in propolis), and pollen on biochemical oxidative stress biomarkers in rat kidney tissue inhibited by Nω -nitro-L-arginine methyl ester (L-NAME). The biomarkers evaluated were paraoxonase (PON1), oxidative stress index (OSI), total antioxidant status (TAS), total oxidant status (TOS), asymmetric dimethylarginine (ADMA), and nuclear factor kappa B (NF-κB). TAS levels and PON1 activity were significantly decreased in kidney tissue samples in the L-NAME-treated group (P < 0.05). The levels of TAS and PONI were higher in the L-NAME plus propolis, CAPE, and pollen groups compared with the L-NAME-treated group. TOS, ADMA, and NF-κB levels were significantly increased in the kidney tissue samples of the L-NAME-treated group (P < 0.05). However, these parameters were significantly lower in the L-NAME plus propolis, CAPE, and pollen groups (P < 0.05) compared with rats administered L-NAME alone (P < 0.05). Furthermore, the binding energy of CAPE within catalytic domain of glutathione reductase (GR) enzyme as well as its inhibitory mechanism was determined using molecular modeling approaches. In conclusion, experimental and theoretical data suggested that oxidative alterations occurring in the kidney tissue of chronic hypertensive rats may be prevented via active compound of propolis, CAPE administration.

Semisynthetic Flavone-Derived Antimicrobials with Therapeutic Potential against Methicillin-Resistant Staphylococcus aureus (MRSA).

A new series of semisynthetic flavone-based small molecules mimicking antimicrobial peptides has been designed from natural icaritin to combat drug-resistant Gram-positive bacterial infections. Compound 6 containing two arginine residues exhibited excellent antibacterial activity against Gram-positive bacteria, including MRSA, and very low toxicity to mammalian cells, resulting in a high selectivity of more than 511, comparable to that of several membrane-active antibiotics in clinical trials. Our data show for the first time that icaritin derivatives effectively kill bacteria. Meanwhile, this is the first study deploying a biomimicking strategy to design potent flavone-based membrane targeting antimicrobials. 6 showed rapid bactericidal activity by disrupting the bacterial membrane and can circumvent the development of bacterial resistance. Importantly, 6 was highly efficacious in a mouse model of corneal infection caused by MRSA and Staphylococcus aureus.

Vitamins D3 and K2 may partially counterbalance the detrimental effects of pentosidine in ex vivo human osteoblasts.

Osteoporosis is a metabolic multifaceted disorder, characterized by insufficient bone strength. It has been recently shown that advanced glycation end products (AGEs) play a role in senile osteoporosis, through bone cell impairment and altered biomechanical properties. Pentosidine (PENT), a wellcharacterized AGE, is also considered a biomarker of bone fracture. Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D3, are prerequisites for optimal osteoblasts functioning. Vitamin K2 is known to enhance in vitro and in vitro vitamin D-induced bone formation. The aim of the study was to assess the effects of Vitamins D3 and K2 and PENT on in vitro osteoblast activity, to convey a possible translational clinical message. Ex vivo human osteoblasts cultured, for 3 weeks, with vitamin D3 and vitamin K2 were exposed to PENT, a well-known advanced glycoxidation end product for the last 72 hours. Experiments with PENT alone were also carried out. Gene expression of specific markers of bone osteoblast maturation [alkaline phosphatase, ALP; collagen I, COL Iα1; and osteocalcin (bone-Gla-protein) BGP] was measured, together with the receptor activator of nuclear factor kappa-B ligand/osteoproteregin (RANKL/OPG) ratio to assess bone remodeling. Expression of RAGE, a well-characterized receptor of AGEs, was also assessed. PENT+vitamins slightly inhibited ALP secretion while not affecting gene expression, indicating hampered osteoblast functional activity. PENT+vitamins up-regulated collagen gene expression, while protein secretion was unchanged. Intracellular collagen levels were partially decreased, and a significant reduction in BGP gene expression and intracellular protein concentration were both reported after PENT exposure. The RANKL/OPG ratio was increased, favouring bone reabsorption. RAGE gene expression significantly decreased. These results were confirmed by a lower mineralization rate. We provided in vitro evidence that glycoxidation might interfere with the maturation of osteoblasts, leading to morphological modifications, cellular malfunctioning, and inhibition of the calcification process. However, these processes may be all partially counterbalanced by vitamins D3 and K2. Therefore, detrimental AGE accumulation in bone might be attenuated and/or reversed by the presence or supplementation of vitamins D3 and K2.

Calendula officinalis ameliorates l-arginine-induced acute necrotizing pancreatitis in rats.

CONTEXT: Calendula officinalis L. (Asteraceae) has been traditionally used in treating inflammation of internal organs, gastrointestinal tract ulcers and wound healing. OBJECTIVE: The present study investigates the effect of ethanol extract (95%) of Calendula officinalis flowers in l-arginine induced acute necrotizing pancreatitis in rats. MATERIALS AND METHODS: Rats were divided into four groups: normal control, l-arginine control, Calendula officinalis extract (COE) treated and melatonin treated (positive control), which were further divided into subgroups (24 h, day 3 and 14) according to time points. Two injections of l-arginine 2 g/kg i.p. at 1 h intervals were administered in l-arginine control, COE and melatonin-treated groups to produce acute necrotizing pancreatitis. Biochemical parameters [serum amylase, lipase, pancreatic amylase, nucleic acid content, total proteins, transforming growth factor-ß1 (TGF-ß1), collagen content, lipid peroxidation, reduced glutathione and nitrite/nitrate] and histopathological studies were carried out. RESULTS: COE treatment (400 mg/kg p.o.) was found to be beneficial. This was evidenced by significantly lowered histopathological scores (2 at day 14). Nucleic acid content (DNA 21.1 and RNA 5.44 mg/g pancreas), total proteins (0.66 mg/mL pancreas) and pancreatic amylase (1031.3 100 SU/g pancreas) were significantly improved. Marked reduction in pancreatic oxidative and nitrosative stress; collagen (122 µmoles/100 mg pancreas) and TGF-ß1 (118.56 pg/mL) levels were noted. Results obtained were comparable to those of positive control. DISCUSSION AND CONCLUSION: The beneficial effect of COE may be attributed to its antioxidant, antinitrosative and antifibrotic actions. Hence, the study concludes that COE promotes spontaneous repair and regeneration of the pancreas.

Changes in the metabolome of rats after exposure to arginine and N-carbamylglutamate in combination with diquat, a compound that causes oxidative stress, assessed by 1H NMR spectroscopy.

Numerous factors can induce oxidative stress in animal production and lead to growth retardation, disease, and even death. Arginine and N-carbamylglutamate can alleviate the effects of oxidative stress. However, the systematic changes in metabolic biochemistry linked to oxidative stress and arginine and N-carbamylglutamate treatment remain largely unknown. This study aims to examine the effects of arginine and N-carbamylglutamate on rat metabolism under oxidative stress. Thirty rats were randomly divided into three dietary groups (n = 10 each). The rats were fed a basal diet supplemented with 0 (control), 1% arginine, or 0.1% N-carbamylglutamate for 30 days. On day 28, the rats in each treatment were intraperitoneally injected with diquat at 12 mg per kg body weight or sterile solution. Urine and plasma samples were analyzed by metabolomics. Compared with the diquat group, the arginine + diquat group had significantly lower levels of acetamide, alanine, lysine, pyruvate, tyrosine, α-glucose, and ß-glucose in plasma; N-carbamylglutamate + diquat had higher levels of 3-hydroxybutyrate, 3-methylhistidine, acetone, allantoin, asparagine, citrate, phenylalanine, trimethylamine-N-oxide, and tyrosine, and lower levels of low density lipoprotein, lipid, lysine, threonine, unsaturated lipid, urea, and very low density lipoprotein (P < 0.05) in plasma. Compared with the diquat group, the arginine + diquat group had significantly higher levels of citrate, creatinine, homogentisate, and α-ketoglutarate while lower levels of acetamide, citrulline, ethanol, glycine, isobutyrate, lactate, malonate, methymalonate, N-acetylglutamate, N-methylnicotinamide, propionate, and ß-glucose (P < 0.05) in urine. Compared with the diquat group, the N-carbamylglutamate + diquat group had significantly higher levels of allantoin, citrate, homogentisate, phenylacetylglycine, α-ketoglutarate, and ß-glucose while lower levels of acetamide, acetate, acetone, benzoate, citrulline, ethanol, hippurate, lactate, N-acetylglutamate, nicotinamide, ornithine, and trigonelline (P < 0.05) in urine. Overall, these results suggest that arginine and N-carbamylglutamate can alter the metabolome associated with energy metabolism, amino acid metabolism, and gut microbiota metabolism under oxidative stress.

Nonclinical Safety Assessment of PER977: A Small Molecule Reversal Agent for New Oral Anticoagulants and Heparins.

A new molecular entity, PER977 (di-arginine piperazine), is in clinical development as an anticoagulant reversal agent for new oral anticoagulants and heparins. The good laboratory practices (GLP)-compliant studies were conducted to evaluate the toxicity of PER977 and its primary metabolite, 1,4-bis(3-aminopropyl)piperazine (BAP). PER977 and BAP were negative for systemic toxicity in dogs and rats. PER977 was rapidly eliminated from the blood with little to no accumulation. PER977 was negative for genotoxicity and did not alter neurological, respiratory, or cardiovascular function. Maximum tolerated doses for PER977 were 40 (rat) and 35 mg/kg (dog), and greater than 80 mg/kg (rat) for BAP. The no observable adverse effect level (NOAEL) for 14-day intravenous exposure to both rats and dogs was 20 mg/kg/d. For BAP, the NOAELs for 14-day intravenous exposure to rats and dogs were 5 and 20 mg/kg, respectively. Based on these results, a safe and conservative dose level of 19.4 mg/d was used for the PER977 first in human study.

Long term exposure to L-arginine accelerates endothelial cell senescence through arginase-II and S6K1 signaling.

L-arginine supplementation is proposed to improve health status or as adjunct therapy for diseases including cardiovascular diseases. However, controversial results and even detrimental effects of L-arginine supplementation are reported. We investigate potential mechanisms of L-arginine-induced detrimental effects on vascular endothelial cells. Human endothelial cells were exposed to a physiological (0.1 mmol/L) or pharmacological (0.5 mmol/L) concentration of L-arginine for 30 minutes (acute) or 7 days (chronic). The effects of L-arginine supplementation on endothelial senescence phenotype, i.e., levels of senescence-associated beta-galactosidase, expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, eNOS-uncoupling, arginase-II expression/activity, and mTORC1-S6K1 activity were analyzed. While acute L-arginine treatment enhances endothelial NO production accompanied with superoxide production and activation of S6K1 but no up-regulation of arginase-II, chronic L-arginine supplementation causes endothelial senescence, up-regulation of the adhesion molecule expression, and eNOS-uncoupling (decreased NO and enhanced superoxide production), which are associated with S6K1 activation and up-regulation of arginase-II. Silencing either S6K1 or arginase-II inhibits up-regulation/activation of each other, prevents endothelial dysfunction, adhesion molecule expression, and senescence under the chronic L-arginine supplementation condition. These results demonstrate that S6K1 and arginase-II form a positive circuit mediating the detrimental effects of chronic L-arginine supplementation on endothelial cells.

Inflammatory milieu as an early marker of kidney injury in offspring rats from diabetic mothers.

The present study investigated the early presence of inflammatory response in renal tissue of young offspring from diabetic mothers. The effect of L-arginine (L-arg) supplementation was also investigated. The offspring was divided into four groups: group CO (controls); group DO (diabetic offspring); group CA (CO receiving 2% L-arg solution) and group DA (DO receiving the 2% L-arg solution). Glycemia, arterial pressure and renal function were evaluated; gene and protein expression of pro-inflammatory cytokines were also measured. Blood pressure levels were significantly increased in 2 and 6 month-old DO rats, whereas L-arg administration caused a significant decrease in the DA group, at both ages. DO rats showed a significantly blunted glycemic response to exogenous insulin. In 2 month-old DO animals, renal protein expression of pro-inflammatory molecules was significantly increased. At six months of age, we also observed an increase in gene expression of pro-inflammatory molecules, whereas L-arg supplementation prevented this increase at both ages. Our data suggest that activation of inflammatory pathways is present early in the kidney of DO rats, and that L-arg can attenuate the expression of these markers of tissue inflammation. Our results also reinforce the concept that intrauterine environmental factors are a fundamental determinant in the development of metabolic and vascular diseases later in life.

Metabolomic analysis to discover candidate therapeutic agents against acute pancreatitis.

Novel and effective drugs against acute pancreatitis are required. Therefore, we examined the changes in the metabolite levels in the serum and pancreatic tissue of mice with cerulein- and arginine-induced pancreatitis using gas-chromatography/mass-spectrometry (GC/MS) and investigated whether these alterations affected the severity of acute pancreatitis. In the cerulein-induced pancreatitis model, 93 and 129 metabolites were detected in the serum and pancreatic tissue, respectively. In the L-arginine-induced acute pancreatitis model, 120 and 133 metabolites were detected in the serum and pancreatic tissue, respectively. Among the metabolites, the concentrations of tricarboxylic acid (TCA) cycle intermediates and amino acids were altered in pancreatitis, and in pancreatic tissue, the levels of the intermediates involved in the initial part of the TCA cycle were increased and those of the intermediates involved in the latter part of the TCA cycle were decreased. Some metabolites exhibited similar changes in both pancreatitis mouse models, e.g., the levels of glutamic acid and O-phosphoethanolamine were significantly decreased in the pancreatic tissue. Supplementation with glutamic acid and O-phosphoethanolamine attenuated the severity of cerulein-induced acute pancreatitis. Our results suggest that GC/MS-based metabolomics is capable of accurately representing the status of acute pancreatitis, leading to the discovery of therapeutic agents for pancreatitis.

Protective effect of Salvia miltiorrhizae injection on N(G)-nitro-D-arginine induced nitric oxide deficient and oxidative damage in rat kidney.

N(G)-nitro-D-arginine (d-NNA) could convert into N(G)-nitro-L-arginine (l-NNA) in vivo, and kidney is the major target organ. In the chiral inversion process, a number of reactive oxygen species (ROS) were generated and NOS activity was inhibited, which may cause renal damage. Salvia miltiorrhiza (SM), a traditional Chinese drug, was used in the treatment of cardiovascular diseases and chronic renal failure. The aim of the present study was to investigate the kidney damage caused by D-NNA administration for 12 weeks and to evaluate the effects of treatment with SM on D-NNA-induced kidney damage. The rats, induced with D-NNA for period of 12 weeks, showed significant elevation of Blood Urea Nitrogen (BUN), Creatinine (Crea) and MDA levels, and significant decrease of SOD and GSH-Px activities, as compared with control group. In addition, the kidney of rats induced with D-NNA only showed remarkable histopathology, including severe mononuclear cell infiltration, mild tubular dilatation and congestion, and moderate interstitial desmoplasia. After 4 weeks SM treatment, the activity of SOD, GSH-Px and iNOS and the production of NO were significantly higher (P<0.05), and the levels of BUN, Crea and MDA were significantly lower than that of D-NNA only group (P<0.05). In addition, treatment with SM showed histopathological protection in tubular dilatation, congestion, mononuclear cell infiltration and interstitial desmoplasia. The present results indicate that the toxicity of D-NNA relates to its ability to generate oxidative stress and upregulate NOS activity in rat kidney. SM probably ameliorates D-NNA-induced nephrotoxicity in rats according to scavenging free radical and upregulating NOS activity.

Protective effect of antioxidants on cerebrum oxidative damage caused by arginine on pyruvate kinase activity.

We have demonstrated that acute arginine administration decreases antioxidant defenses and compromises enzymes of respiratory chain in rat brain. In this study we evaluated in vivo and in vitro effect of arginine on pyruvate kinase activity, as well as its effect on an important parameter of oxidative stress namely thiobarbituric acid-reactive substances (TBA-RS) in cerebrum of rats. We also tested the influence of antioxidants, namely alpha -tocopherol plus ascorbic acid on the effects elicited by arginine in order to investigate the possible participation of free radicals on the effects of arginine on these parameters. Results showed that arginine acute administration inhibited pyruvate kinase activity in cerebrum of rats, as well as increased TBA-RS. By the other hand, arginine added to the incubation medium, in vitro studies, did not alter these parameters in rat cerebrum. In addition, pretreatment with antioxidants prevented the reduction of pyruvate kinase activity and the increase of TBA-RS caused by arginine. The data indicate that acute administration of arginine induces lipid peroxidation in rat cerebrum and that the inhibition of pyruvate kinase activity caused by this amino acid was probably mediated by free radicals since antioxidants prevented such effect. It is presumed that these results might be associated, at least in part, with the neuronal dysfunction of patients affected by hyperargininemia. Finally, we suggest that the administration of antioxidants should be considered as an adjuvant therapy to specific diets in hyperargininemia.

The protective effects of green tea extract against L-arginine toxicity to cultured human mesangial cells.

The aim of this study was to investigate whether green tea extract (GTE) has the protective effects on excess L-arginine induced toxicity in human mesangial cell. Human mesangial cells treated with L-arginine were cultured on Dulbecco's modified eagle medium in the presence and absence of inducible nitric oxide synthase (iNOS) inhibitor and GTE. The cell proliferation was determined by 3 (4,5-dimethylthiazol-2-yl)-2, 5-diphengltetrqzolium bromide, a tetrazole assay. The iNOS mRNA and its protein expression were detected by reverse transcription polymerase chain reaction and Western blot, respectively. The concentration of nitric oxide (NO) was measured by NO enzyme-linced immuno sorbent assay kit. L-arginine significantly inhibited the proliferation of human mesangial cells, and induced the secretion of NO to the media. NO production by L-arginine was significantly suppressed by GTE and iNOS inhibitor (p<0.01). The expression level of iNOS mRNA and its protein that was significantly increased by L-arginine was decreased by iNOS inhibitor but not by GTE. GTE protected the mesangial cells from the NO-mediated cytotoxicity by scavenging the NO rather than by iNOS gene expression. Therefore, we conclude that GTE has some protective effect for renal cells against oxidative injury possibly by polyphenols contained in GTE.

Temporal metabonomic modeling of l-arginine-induced exocrine pancreatitis.

The time-related metabolic responses to l-arginine (ARG)-induced exocrine pancreatic toxicity were investigated using single ip doses of 1,000 and 4,000 mg/kg body weight over a 7 day experimental period in male Sprague-Dawley rats. Sequential timed urine and plasma samples were analyzed using high resolution (1)H NMR spectroscopy together with complementary clinical chemistry and histopathology analyses. Principal components analysis (PCA) and orthogonal projection on latent structures discriminant analysis (O-PLS-DA) were utilized to analyze the (1)H NMR data and to extract and identify candidate biomarkers and to construct metabolic trajectories post ARG administration. Low doses of ARG resulted in virtually no histopathological damage and distinct reversible metabolic response trajectories. High doses of ARG caused pancreatic acinar degeneration and necrosis and characteristic metabolic trajectory profiles with several distinct phases. The initial trajectory phase (0-8 h) involved changes in the urea cycle and transamination indicating a homeostatic response to detoxify excess ammonia generated from ARG catabolism. By 48 h, there was a notable enhancement of the excretion of the gut microbial metabolites, phenylacetylglycine (PAG), 4-cresol-glucuronide and 4-cresol-sulfate, suggesting that compromised pancreatic function impacts on the activity of the gut microbiota giving potential rise to a novel class of surrogate extragenomic biomarkers of pancreatic injury. The implied compromise of microbiotal function may also contribute to secondary hepatic and pancreatic toxic responses. We show here for the first time the value of metabonomic studies in investigating metabolic disruption due to experimental pancreatitis. The variety of observed systemic responses suggests that this approach may be of general value in the assessment of other animal models or human pancreatitis.

A new severe acute necrotizing pancreatitis model induced by L-ornithine in rats.

OBJECTIVE: Intraperitoneal administration of large doses of L-arginine is known to induce severe acute pancreatitis in rats. We therefore set out to determine whether metabolites of L-arginine (L-ornithine, L-citrulline, and nitric oxide) cause pancreatitis. DESIGN: The authors conducted an in vivo animal study. SETTING: This study was conducted at a university research laboratory. SUBJECTS: Study subjects were male Wistar rats. INTERVENTIONS: Dose-response and time course changes of laboratory and histologic parameters of pancreatitis were determined after L-arginine, L-ornithine, L-citrulline, or sodium nitroprusside (nitric oxide donor) injection. MEASUREMENTS AND MAIN RESULTS: Intraperitoneal injection of 3 g/kg L-ornithine but not L-citrulline or nitroprusside caused severe acute pancreatitis; 4 to 6 g/kg L-ornithine killed the animals within hours. Serum and ascitic amylase activities were significantly increased, whereas pancreatic amylase activity was decreased after intraperitoneal injection of 3 g/kg L-ornithine. The increase in pancreatic trypsin activity (9-48 hrs) correlated with the degradation of IkappaB proteins and elevated interleukin-1beta levels. Oxidative stress in the pancreas was evident from 6 hrs; HSP72 synthesis was increased from 4 hrs after L-ornithine administration. Morphologic examination of the pancreas showed massive interstitial edema, apoptosis, and necrosis of acinar cells and infiltration of neutrophil granulocytes and monocytes 18 to 36 hrs after 3 g/kg L-ornithine injection. One month after L-ornithine injection, the pancreas appeared almost normal; the destructed parenchyma was partly replaced by fat. Equimolar administration of L-arginine resulted in lower pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, and histologic damage compared with the L-ornithine-treated group. L-ornithine levels in the blood were increased 54-fold after intraperitoneal administration of L-arginine. CONCLUSIONS: We have developed a simple, noninvasive model of acute necrotizing pancreatitis in rats by intraperitoneal injection of 3 g/kg L-ornithine. Interestingly, we found that, compared with L-arginine, L-ornithine was even more effective at inducing pancreatitis. Large doses of L-arginine produce a toxic effect on the pancreas, at least in part, through L-ornithine.

Risk assessment for the amino acids taurine, L-glutamine and L-arginine.

Taurine, glutamine and arginine are examples of amino acids which have become increasingly popular as ingredients in dietary supplements and functional foods and beverages. Animal and human clinical research suggests that oral supplementation of these amino acids provides additional health and/or performance benefits beyond those observed from normal intake of dietary protein. The increased consumer awareness and use of these amino acids as ingredients in dietary supplements and functional foods warrant a comprehensive review of their safety through quantitative risk assessment, and identification of a potential safe upper level of intake. The absence of a systematic pattern of adverse effects in humans in response to orally administered taurine (Tau), l-glutamine (Gln) and l-arginine (Arg) precluded the selection of a no observed adverse effect level (NOAEL) or lowest observed adverse effect level (LOAEL). Therefore, by definition, the usual approach to risk assessment for identification of a tolerable upper level of intake (UL) could not be used. Instead, the newer method described as the Observed Safe Level (OSL) or Highest Observed Intake (HOI) was utilized. The OSL risk assessments indicate that based on the available published human clinical trial data, the evidence for the absence of adverse effects is strong for Tau at supplemental intakes up to 3 g/d, Gln at intakes up to 14 g/d and Arg at intakes up to 20 g/d, and these levels are identified as the respective OSLs for normal healthy adults. Although much higher levels of each of these amino acids have been tested without adverse effects and may be safe, the data for intakes above these levels are not sufficient for a confident conclusion of long-term safety, and therefore these values are not selected as the OSLs.

Pharmacokinetics and safety of arginine supplementation in animals.

Anticipating the future use of arginine to enhance fetal and neonatal growth as well as to treat diabetes and obesity, we performed studies in pigs, rats, and sheep to determine the pharmacokinetics of orally or i.v. administered arginine and the safety of its chronic supplementation. Our results indicate that all 3 species rapidly catabolized the supplemental arginine. The elevated circulating concentrations of arginine generally returned to baseline levels within 4-5 h after administration, with the rates varying with the age and physiological status of the animals. The clearance of arginine was greater in pregnant than in nonpregnant animals, in young than in adult animals, in lean than in obese animals, and in type-1 diabetic than in nondiabetic animals. I.v. administration of arginine-HCl to pregnant ewes (at least 0.081 g arginine.kg body weight-1.d-1) did not result in any undesirable treatment-related effect. Neonatal pigs, growing-finishing pigs, pregnant pigs, and adult rats tolerated large amounts of chronic supplemental arginine (e.g. 0.62, 0.32, 0.21, and 2.14 g.kg body weight-1.d-1, respectively) administered via enteral diets without the appearance of any adverse effect. On the basis of the comparative studies and a consideration of species differences in food intake per kilogram body weight, we estimate that a 70-kg human subject should be able to tolerate long-term parenteral and enteral supplemental doses of 6 and 15 g/d arginine, respectively, in addition to a basal amount of arginine (4-6 g/d) from regular diets.

Diverse effects of L-arginine on cardiac function of rats subjected to myocardial ischemia and reperfusion in vivo.

In vivo administration of L-arginine at different time points during the course of myocardial ischemia and reperfusion (MI/R) has been shown to differentially regulate postischemic apoptosis. Cardiac function is one of the most important indexes used to judge the degree of myocardial injury. The present study attempted to determine whether in vivo administration of L-arginine at different stages of MI/R has a diverse influence on cardiac function of ischemic reperfused hearts and, if so, to investigate the mechanisms involved. Male adult rats were subjected to 30 min myocardial ischemia followed by 5 h reperfusion. An intravenous L-arginine bolus was given either 10 min before and 50 min after reperfusion (early treatment) or 3 h and 4 h after reperfusion (late treatment). Early treatment with L-arginine markedly increased the left ventricular systolic pressure (LVSP) and dP/dt(max), and decreased myocardial nitrotyrosine content. In strict contrast, late treatment with L-arginine resulted in a significant decrease in LVSP and dP/dt(max) from 4 h to 5 h after reperfusion, and increase in toxic peroxynitrite formation as measured by nitrotyrosine. These results suggest that the administration of L-arginine at different time points during the course of MI/R leads to diverse effects on cardiac dysfunction. Early supplementation decreased the nitrative stress and improved left ventricular function. However, late treatment with L-arginine increased the formation of peroxynitrite and aggravated cardiac functional injury.