AMINO ACID COMPOSITION OF RABBIT MEAT DURING EXPERIMENTAL 1, 1 -DIMETHYLGYDRAZINE TOXICOSIS

Abstract This article demonstrates the results of amino acid composition of rabbit meat upon treatment with 1.1-dimethylhydrazine (excerpt from the protocol of ethic committee № 1). The identification of amino acids has been carried out using liquid chromatograph SHIMADZU LC-20 Prominence (Japan) with fluorometric and spectrophotometric detectors. The analysis of essential and non-essential amino acid content and amino acid score of the meat showed significant difference in control and experimental samples. The amount of amino acids in experimental group was more than in control group by 2%: valine, methionine + cysteine, and phenylalanine + tyrosine were found to be limiting amino acids. Such decrease was predicted to be the result of the experimental toxicosis with 1.1-dimethylhydrazine that negatively affects protein synthesis in liver. Finally, it could be said that decrease in the level of these amino acids in blood is reflected by the nutrition value of rabbit meat.

Keywords - 1,1 dimethylhydrazine, essential amino acids, nonessential amino acids, rabbit meat.

I.     INTRODUCTION

Hydrazine and its derivatives belong to the group of highly toxic products of an industrial space-rocket activity. One of the most toxic derivatives of a hydrazine is 1,1- dimethylhydrazine [2].

1.1-dimethylhydrazine is a fuel of heavy class rocket carrier "Proton-M". Problem of polluting our environment by rocket fuel usage and its components became more vivid these days in Kazakhstan, the territory of which includes Baikonur Cosmodrome.

The metabolism of hydrazine and its derivatives in human and animal organisms is not fully studied. Therefore, this problem is defined as a high priority in recommendations of the World Health Organization (WHO), concerning the hygienic criteria of environmental conditions [11].

Currently, we possess an experimental confirmation of negative influence of rocket fuel components (heptyl) on biological objects, which explains many morbid conditions of people who live nearby contaminated area. These experiments were held by Biochemistry Research Institute Siberian Branch of the Russian Academy of Medical Science (Novosibirsk city). The highest concentration of these components could be observed in kidneys, liver and lien [12].

Heptyl contamination and its consequences on economic growth of potatoes in the Republic of Altai were examined. According to the experiments, it can be concluded that rocket fuel NDMG negatively impacts on quality and quantity of characteristics of potatoes [2]. In addition, experiments held by Muravlevoy L.E concerning influence of rocket fuel on animal spermatogenesis, showed that single dosing of NDMG with 5mg/kg leads to negative outcomes on the spermatogenesis process. One month after the injection, the amount of nonmotile and cork cells significantly increased, moreover after 3 months symptoms of as thenozoospermia and teratozoospermia were observed [8]. In other words, experiments confirm high level of 1.1-dimethylhydrazine toxicities causes potential negative consequences on health.

Currently, rabbits are the most convenient biomaterial for defining influence of poisons. It is known that biological value of the rabbit meat is necessary for assessing its quality, which depends on its amino acid constitution and digestibility of protein components. The biological value is defined by safety, its nutritional potential,

biological activity, organoleptic properties of products and meeting the needs of human

organism [8], [1]. Lack of essential amino acids in human body leads to inadequate level of protein synthesis. Rabbit meat is a dietary product with the high protein content, rich amino-acid composition, and low fat and cholesterol levels [5]. The nutritional value of a protein depends on type and amount of its amino acids. Rabbit, therefore/ should be a good source of protein, according to Grace who evaluated the composition of rabbit meat and concluded that it contained all the essential amino acids necessary for man. [6]. The latest F.A.O. recommendation made in 1973 differs considerably from the previous one issued in 1965, in that the requirements for leucine, lysine and threonine have been increased significantly .These recommendations for the essential amino acid contents of the dietary protein of man relate to a daily intake of some 40g protein now given as the minimum necessary to satisfy the nutritional needs of an adult: this level of protein intake is itself much lower than an earlier FAO/WHO recommendation, and is considered by some experts to be too low [3].

The important fact is that, the thermal treatment does not change the amino acid composition of meat, and influences only their quantity. The nutritional value of a protein depends on the type and amount of its amino acids of rabbits [4], [5]. Therefore, there should be good source of protein. The most abundant and essential amino acids of rabbit meat are lysine, methionine and tryptophan, 10.43%, 2.37% and 1.55%, respectively. In addition, the age of animals slightly influences the content of amino acids [4].The study has shown that, the diet containing rabbit as the sole source of protein caused the growth of young 37, 38 rats, and these rats had a higher PER, weight gain, food consumption and lower feed efficiency at a 0.05 level of significance comparing to the diets based on pork, chicken or cod [8]. Biologically it is superior than beef-containing died in these following four factors, but differences are not statistically significant. Comparing to the essential amino acids of all these meats according to FAO amino acid patterns and to the reference protein (casein), only pork had higher protein content than rabbit, but rabbit contained a better ratio of amino acids than pork.

Although, chicken had a better ratio of amino acids comparing to pork, it had lower crude protein content [4], [1].

Quantitative analysis of amino acids and their ratios are the most important criteria for the assessment of nutritional value of proteins from muscular tissue. Therefore, the following paper focuses on amino-acid composition of proteins from rabbit muscular tissueduring from experimental 1.1-dimethylhydrazine toxicosis.

II.     MATERIAL AND METHODS

 The experiments had been carried out for the purpose of identifying the amino acid composition of rabbit meat during 1.1-dimethylhydrazine toxicosis. The experimental toxicosis was caused by watering rabbits with 100 ml of distilled water adding 1.1- dimethylhydrazine during 5 days period (frequent and chronic effect). According to the standards, two groups of animals had been chosen. The first group included rabbits (n=9), which have been instilled orally the compound of 98% distilled water and 1.1- dimethylhydrazine in doze of 0.075 mg/kg per body weight, while the second group consisted of (n=9) control animals. Decapitation of rabbits was made on the 6th day of the experiment. Rabbits prosection was carried out according to the Schor method. Given doze was lower than the average-toxic level and was coordinated with the scientists of the Republican state enterprise "Research-Development Centre "Garysh- Ecology" and Aerospace Committee of the Ministry Of Investment and Development of

the    Republic    of    Kazakhstan".   (The    extract   from    the    protocol   of    ethic committee №1 from 2015/02/02, "Veterinary & Livestock Technology" Faculty).

98% 1.1-dimethylhydrazine SSRS (state standard reference sample), producer SigmaAldrich, Germany. In order to work with 1.1-dimethylhydrazine doctoral candidates passed special training courses such as: "Industrial security on hazardous industrial facilities" with the qualification: "Staff was admitted to work with high toxic substances" and "An individual is responsible for safety acceptance, storage and supply of highly toxic substances". Materials used for investigations were 18 meat sample tests.

The research was based on “Veterinary-sanitary” laboratory “Kazakh Agro-technical University named after S. Seyfullin", experimental regional laboratory of the engineering profile "Scientific Center of Radio Ecological Researches" State agency of Shakarim, Semey city.The assessment of value of protein in the rabbit meat was made by total amount of amino acids, separated by essential and non-essential acids and their ratio and by the method of collection amino acid score.

 A. Determination of Amino Acids in Animal Origin Products.

Determination of amino acids was conducted by using liquid SHIMADZU LC-20 Prominence chromatograph, (Japan) with the fluorometric and spectrophotometric detector. The chromatograph column by the size of 25 cm* 4.6 mm SUPELCO C18, 5 mkm (USA) with a pre-column was used for protection of the main column from pollution. Chromatographic analysis was held in the gradient mode in consumption of eluent at 1.2 ml/min and 400C temperature. Measurements were collected using highly effective liquid chromatograph with a turned phase column and fluorometric detectors with length waves of 246 nm and 260 nm with the use of acid hydrolysis and amino acids modificated solution of carbonylin isopropyl alcohol with receiving feniltiogidantoins. The mixture of 6.0 mm sodium acetate solution with рН 5.5 (component A), 1% solution of isopropyl alcohol in an acetonitrile (components) and 6.0 mm sodium acetate solution with рН 4.05 (component C) was used as a mobile phase.

 The standard samples of amino acids produced by Sigma Aldrich, acetonitrile A.C.S., isopropyl alcohol A.C.S., and carbonyl produced by SigmaAldrich, phosphoric acid, sodium acetate were used for a liquid chromatography. The sample preparation: for carrying out of hydrolysis, 100 mg of sample is placed into the glass ampoules with delayed end, after that we add 10 ml. of 6M solution of hydrochloric acid. Mixture was mixed carefully and blown by the nitrogen current within 2 min. Glass ampoules were soldered and placed in the thermostat. Hydrolysis was carried out at a temperature of 110oC during 24 h. After cooling, the hydrolysates were filtered through the membrane filters with a pore diameter of 0.45 mkm, and selected aliquots of 0.5 ml. Aliquots were dried up by usage of the air at 650C, 0.10 ml solution of Na OH 0.15 M was added to the dried-up aliquots and carefully mixed. Then 0.35 ml solution of carbonyl in isopropyl alcohol was mixed and added with 0.05 ml of distilled water and dried up on a water- bath at 600C, and filtered through the membrane filter with a pore diameter of 0.45 mkm. Received solutions were subjected to the chromatographic analysis. Amino acids concentration in samples was calculated by mg per 100 gr of the product.

III.     RESULTS AND DISCUSSION 

It was concluded that, there was a low level of essential and non-essential amino acids in meat protein. There were reliable differences on the content of essential amino acids in comparison with control and fluctuations from 2.1% to 5.8%.

Table 1 - Amino-acid composition of protein rabbit meat, mg / 100g. 

The results obtained during calculation are presented in the Table I. As it can be seen from the Table I and Fig. 1, essential amino acids: valine – 1042.3±1.17 mg/100g (by 2.1% less than in control group), leucine – 1711.6±1.06 mg/100g (by 1.4% less than in control group), isoleucine – 844.8±1.20 mg/100g (by 2.3% less than in control group), lysine- 2171.1±0.97 mg/100g (by 1.3% less than in control group), methionine- 483±1.19 mg/100g (by 3.2%less than in control group), threonine - 897±1.20 mg/100g (by 1.8% less than in control group), tryptophan - 308±2.25 mg/100g (by 5.8 % less than in control group), phenylalanine- 495.5±0.94 mg/100g( by 3.3% less than in control group).

From the Table I, it can be concluded that there is significant difference in non- essential amino acids from 1.2% to 7%. Non-essential amino acids: alanine - 1473.6±0.84 mg / 100g ( by 1% less than in control group), arginine- 1450.5±0.94 mg / 100g (by 1.5 % less than in control group), aspartic acid – 1848.3±0.74 mg / 100g (by 1.2 % less than in control group), histidine - 601±2.69 mg / 100g (by 4% less than in control group), glycine – 933.6±0.98 mg/ 100g (by 2.3%less than in control group), glutamic acid – 3412.3±0.79 mg / 100g (by 1.9%less than in control group), proline- 822.6±0.84 mg/100g (by 2.5% less than in control group), tyrosine-443.8±0.88 mg / 100g (by 4.4% less than in control group), cysteine – 240.8±0.83 mg/ 100g ( by 7% less than in control group), serine-821.3±0.84 mg/100g (by 2.6% less than in control group).

The analysis of the obtained data shows that the studied samples differ from the normal by 2% with the sum of amino acids – 20.001 mg / 100g. Assessment results of a protein nutrition value in meat by comparison with the standard scale of amino acids recommended by the joint committee of FAO and WHO, the UN (method of amino-acid score, i.e. index) are presented in the Table 2.

Table 2 - Amino-acid score of rabbit meat ,%

I.                CONCLUSION

Thus, the sum of amino acids in experimental group was lower by 1.7% in comparison with control group. Such decrease was caused by diminution of essential and non- essential amino acids. Given decline is caused by decrease in level of essential and non- essential amino acids. 1.1-dimethylhydrazine experimental toxicosis leads to liver function abnormality and amino acids synthesis as well. In our opinion, this could be due to detoxicant roles of some amino acids. It is important to mention that decline in index of food value of protein meat consists of methionine+ cysteine (64%), phenylalanine + tyrosine (39.1%) and valine (98.6%). It is known that up to 80-89% of methionine could be transformed into cysteine and 70-75% of phenylalanine into tyrosine [13].

Thus, the amount of methionine essential amino acids must be greater, since their most part is used in cysteine and tyrosine creation. Existence of cysteine and tyrosine in food may lead to decline in essential amino acids of methionine and phenylalanine as well as burning of nutrient materials. Methionine + cysteine is sulfur-containing amino acids.

Sulphuric acid is a product of fermentative transformations of sulfur-containing amino acids. It produces paired ether sulfur acids which remove toxins from an organism by bonding with toxic products of liver functioning.

REFERENCES

[1] Artemov V.S., The biological value of chicken meat with ligfola/ В.С. Artemov V.S., Evsukov M.P., Alegin M.V. // The role of science in improving the sustainability of agro-industrial complex functioning of the Tambov region – т.2. collection of scientific papers, Michurinsk – Naukagrad, 2004. pp. 258-260.

[2] Belov A.A. On the question of the toxicity and hazards of hydrazine and its derivatives (review) // Industrial Toxicology 1999, № 5, pp 3-15.Liquid propellants. Directory-M.: Institute of Biophysics,1991. — pp.263.

[3] Blaxster K.//Food Chains and human nutrition pp:137, 1979.Nelson S.D., Gardon W.P. «Metabolic activation of hydrazines» // Abv. exp. med. Biol. —1982. —V. 136.—P. 971—981.

 [4] Colman Y., Rem K.G. Transparent biochemistry. - M.: Mir, 2000. pp.469.Grace, R.I. The significance of meat for nutrition. Food Science and Technology Abstracts. 1971. pp:131- 138,

[5] Dalle Zotte A Main factors influencing the rabbit carcass and meat quality. In: Proceedings of the 7th World Rabbit Congress (Valencia, Spain), 2000 pp. 1–32.

[6] Grace, R.I. The significance of meat for nutrition. Food Science and Technology Abstracts. 1971. pp:131- 138.

[7] Liquid propellants. Directory-M.: Institute of Biophysics,1991. — pp.263.

[8] Muravleva L.E., Kultanov B.Zh.,Medvedev V.I., Tankibayeyva N.U., Mustafina F.H., Britko V.V.,Dyusekeeva B.N., Klyuev D.A./ Influence unsymmetrical dimethyl spermatogenesis growing animals\\Journal of Basic.№ 12, 2007.pp-525-527.

[9] Nelson S.D., Gardon W.P. «Metabolic activation of hydrazines» // Abv. exp. med.Biol. —1982. —V. 136. —P. 971—981.

 [10]   Smith E.B., Clark D.A. «Absorption of hydrazine throygh canine skin» // Toxicol.appl. Pharmacol. —1972. —V. 21. —P. 186—193.

 [11]   The World Health Organization //Hydrazine. Hygienic criteria on environment.Joint publication of the programme. United Nations, Geneva 1991.

 [12]   Zabolotnyh M.V. The usefulness of the protein of broiler meat in the diet in the application of sapropel extract / Kuricina V.M, Malceva N.M // poultry о.-2007.-№12. pp.32-33.

[13] Zapadnyuk V.I., Kuprash L.P., Zaika M.U., Bezverhaya I.S., The textbook "The amino acids in medicine", Kiev,1982 г.,pp.97.