Clinical safety of amino acids and vitamins administered through parenteral route

The parenteral use of amino acids and vitamins has been used to improve performance in animal production. However, few studies demonstrating the toxicity of these compounds. The present investigation evaluated the toxicological and clinical safety of the injectable supplement compound by amino acids and vitamins, administered by intramuscular route in Wistar rats. 56 females (± 220g), pubescent virgins, were randomly divided into seven groups (n=8): Treated with dose of 0.2, 2 and 4 mL, sampled 24 and 96 hours post-treatment (HPT), totaling six groups and one control group (Physiology Standard). Signifi cant increase in the enzymatic activity of serum ALT, AST, LDH and ALP was observed in animals treated with 2 and 4 mL (24 HPT). However, these changes returned to baseline levels 96 HPT, except in serum AST and ALP in animals treated with 4 mL. CK-NAC showed dose-response effect, since the higher compound dose was followed by proportional increase in the circulating levels of this enzyme, associated to muscle degeneration and necrosis area in rats treat with 4mL at 96 HPT. The evaluation of hepatic function did not result in signifi cant changes in the serum levels of triglycerides, cholesterol, albumin, urea and creatinine in the comparison between rats treated with 0.2 mL and control rats, demonstrating the innocuousness of this treatment on the activity of liver and kidney. However, deleterious effects were observed in animals subjected to doses of 2 and 4 mL, including observed increased splenic and hepatosomatic response, signifi cant decrease in circulating albumin levels and signifi cant leukocytosis, which is characterized by neutrophilia, followed by lymphopenia demonstrating the compound irritating effect on muscle tissue. Research Article Clinical safety of amino acids and vitamins administered through parenteral route Alessandra Cristina de Moraes1, Marcos Roberto Scherma2, Ed Johnny da Rosa Prado1, Vanessa Pavesi de Faria1, Ives Charlie da Silva1, Wilson Gomez Manrique2 and Marco Antonio de Andrade Belo1,2* 1Department of Preventive Veterinary Medicine, College of Veterinary Medicine, São Paulo State University, Jaboticabal, SP, Brazil 2Laboratory of Pharmacology and Clinical Pathology, College of Veterinary Medicine, Camilo Castelo Branco University, Descalvado, SP, Brazil Dates: Received: 29 May, 2017; Accepted: 05 August, 2017; Published: 07 August, 2017 *Corresponding author: Marco Antonio de Andrade Belo, Av. Hilário da Silva Passos, 950, Parque Universitário, Zip Code: 13.690-000, Descalvado, SP, Brazil, E-mail:


Introduction
The amount of essential amino acids available in the metabolism must be iqual nutritional needs, seeking to supply maintenance and production requirements [1]. To improve the metabolic activity and productive performance of animals, the use of parenteral administration of amino acids and vitamins in animal production has increased mainly in ruminants (bovines, goats and sheep), due to the peculiarities of intermediary metabolism, resulting from biotransformations that foods suffer through ruminal fermentation [2].
A relevant aspect in the study of exogenous administration of organic modifi ers should be related to the compounds safety. Therefore, these formulations need to provide clinical safety for animals. According to Castro et al. [3], all substance is potentially toxic, the correct dose differentiates a remedy of a toxicant. The practice of amino acids parenteral administration presents toxicological risks as observed with most chemical compounds such as spoiled food, industrial residues, among others, which induce lipid peroxidation mediated by the presence of free radicals, resulting in damage to biomembranes, consequently, promoting cellular and tissue dysfunctions [4,5].
Peroxidative processes involving lipids present in cell membranes represent critical factors in the pathogenesis of diseases related to free radicals, including infl ammation, hepatic and renal disorders [6,7]. In the pathophysiology of these events, hepatocytes play a central role in the homeostatic balance of essential biological processes, regulating important metabolic functions involving the synthesis, activation, storage and catabolism of endogenous and exogenous chemicals [8]. According to Rivera-Huizar et al. [9] and Belo et al. [5], the occurrence of development of hepato-renal syndrome is high in cases of poisoning. Therefore severe damages to liver by different etiologies can impair the renal function and aggravate the clinical status of the animal.
Moreover, evidences indicate that the processes of hepatic regeneration depend on the availability of amino acids, vitamins, among others. Studies  rats with cirrhosis [10,11], as well as the synergistic effect of these nutrients, Kröger et al. [12] investigated the benefi cial participation of methionine supplementation when combined with nicotinamide in reducing toxic effects induced by the treatment of methotrexate.
The growing use of parenteral supplementation with amino acids and vitamins in intensive production systems, seeking to improve the animals metabolic activity and productive performance, associated with the scarce literature regarding the possible side effects of this practice, this study aimed to evaluate the toxicological and clinical safety of amino acids and vitamins administered by intramuscular route in rats.

Animals
The experiment was conducted in the Laboratory of Clinical  [13]. 56 females albino Wistar rats (± 220g), pubescent virgins, born in the same period and from UNESP-Botucatu biotherium, were kept in polypropylene boxes, suitable for the species, fed with commercial feed (Labina® -Purina ) and water ad libitum, exposed to temperature and photoperiod of about 14 hours.

Experimental design
Animals were randomly divided into seven groups (n=8): Treated with dose of 0.2, 2 and 4 mL, sampled 24 and 96 hours post-treatment (HPT), totaling six groups and one control group (Physiology Standard). The treatments with the supplement of amino acids and vitamins (Table 1) were performed by intramuscular administration in the right hind limb. Likewise, the physiological standard group was submitted to the administration of saline 0.9% of 02.mL, to standardize the stress stimulus for capture and compound application.

Anesthesia and blood collection
The animals were anesthetized with an association of ketamine 10% (Vetaset® -Fort Dodge) and xylazine 2% (Rompun® -Bayer) in the proportion of 6:1, administered by intraperitoneal route at a dose of 1mL/kg of b.w., to collect blood samples from the posterior vena cava. Blood collection was realized in two sets of 5 mL syringes (needles 25x8), the fi rst set was heparinized to collect plasma and second without anticoagulant to obtain serum.

Hematology
For determination of erythrogram was used automatic blood cell counter (Model CC510, Celm). The Leukocyte and platelet counts were performed manually in Neubauer chamber. The percentage of cell volume was determined in micro-hematocrit capillary tubes, centrifuged 5 minutes at 3000 rpm. Blood extensions were prepared for differential leukocyte count, stained with May-Grünwald-Giemsa-Wright. 200 cells were counted by extension to determine the percentage of each cell type [14].

Morphometric evaluation
At necropsy, liver, kidney and spleen were colleted for morphometric evaluation which was carried out according to Weibel et al. [15], using the ratio between the organ weight (OW) and the body weight (BW). These were expressed as liver somatic index (LSI), kidney somatic index (KSI) and spleen somatic index (SSI), and were calculated using the formula: Somatic Index (SI) = OW x 100 / BW.

Histological examinations
For histopathologic assessment, samples of liver, kidney, spleen and skeletal striated muscle from the right hind limb (administration site of the medicinal compound) were collected from all animals and fi xed in 10% buffered formalin. After routine processing, the tissue was embedded in paraffi n, sectioned at 5μm, and stained with hematoxylin and eosin (H&E) for photomicroscopic assessment. A blinded experienced pathologist performed histopathologic analyses and the histological fi ndings related to tissues injury.

Statistical analysis
All data was statistically analyzed using a completely randomized split-plot design, with the main treatments (four doses 0, 0.2, 2 and 4 mL) and the subplots (two harvest times).
Comparison of the different experimental groups was carried out by applying a PROC MIXED procedure, using the Statistical Analyses System [16]. Signifi cant differences (P< 0.05) were estimated on the basis of Tukey´s test, according to Snedecor and Cochran [17].

Results
In the biochemical serum (Table 2) The study of serum CK-NAC showed dose-response effect, since the higher compound dose was followed by proportional increase in the circulating levels of this enzyme, corroborating this fi nding the Figure 1 presents the muscle degeneration and necrosis area in rats treat with 4mL at 96 hours after compound inoculation. Therefore the analysis of CK-MB did not present signifi cant (p>0,05) changes in rats submitted to different treatments (  increase in blood levels of urea after 96 HPT in rats subjected to a dose of 4 mL, and no signifi cant changes were found in the study of creatinine. In the study of the red blood cells (Table 3), there was no signifi cant change (P> 0.05) in the number of erythrocytes, percentage values of hematocrit, mean corpuscular volume (MCV), hemoglobin concentration and mean corpuscular hemoglobin (MCHC) when comparing animals treated with the dose of 0.2 mL and control rats. However, there was a signifi cant decrease in the erythrocyte global counts, hematocrit values and the amount of hemoglobin concentration in rats subjected to a dose of 4 mL.
The liver is essential for the homeostasis of proteins, and the primary site for synthesis of the majority of plasma proteins. Albumin is the main protein of hepatic export and represents about 50-60% of plasma proteins and about 75% of the oncotic pressure [19]. In this study, there were no changes in the circulating albumin values in rats treated with a dose of 0.2 mL, suggesting the hypothesis that treatment with the organic moderator did not impair the metabolic activity of the protein hepatic synthesis. However, deleterious effects were observed in animals submitted to doses of 2 and 4 mL which showed a signifi cant decrease in circulating albumin levels.
On the other hand, 90% of ammonia released in the liver is converted into urea which becomes the main metabolic product in liver detoxifi cation protein catabolism [9]. The study of blood urea and creatinine levels showed normal values when comparing animals treated with saline and 0.2 mL. Despite the kidney somatic study did not vary signifi cantly, congestive changes and tubular cell swelling were observed in renal histopathology of animals treated with 4 mL.
The erythrocyte and platelet study of rats treated with 0.2 mL showed no signifi cant changes compared with control animals throughout the experimental period, with the mean values observed within the normal range for Wistar rats described by Fukuda et al. [28]. However, there was signifi cant decrease in the global number of erythrocytes, hematocrit values and in the amount of circulating hemoglobin in rats treated with 4 ml, characterizing a normocytic / normochromic anemia, since there were no alterations in the values of MCV and MCHC.
However, there was a signifi cant leukocytosis in animals treated with the amino acid compound, which is characterized by neutrophil, followed by lymphopenia, demonstrating the compound irritant action in the muscle tissue. In acute infl ammatory reactions occurs signifi cant increase in the number of neutrophils that are recruited by chemotaxis to act in the infl amed site [29,30].
Animals with liver lesions may present functional changes in the number and function of platelets. In cases of intra and extrahepatic circulatory deviations, patients may present with congestive splenomegaly, resulting from portal hypertension, leading to conditions of thrombocytopenia [19]. This correlation was evident in this study, since animals treated with a dose of 4 mL showed increased splenic and hepatic somatic index, as well as congestive changes in liver and kidney tissue. toxic effects, and the recommended dose (0.2 mL) demonstrated to be clinically safe.