42 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2016 Vol. 2 issue 2 Corresponding author: Iryna Bekus, Department of Medical Biochemistry, I. Horbachevsky Ternopil State Medical Univer- sity, 1 Maidan Voli, Ternopil, Ukraine, 46001 Phone number: +38035252433 E-mail: bekus@tdmu.edu.ua International Journal of Medicine and Medical Research 2016, volume 2, Issue 2, p. 42-46 copyright © 2016, TSMU, All Rights Reserved doI 10.11603/IJMMR.2413-6077.2016.2.7107 BIOchEmIcAL PARAmETERs Of LIPId mETABOLIsm IN ANImALs AffEcTEd BY hEAvY mETAL sALTs ANd TREATEd WITh cARNITINE chLORIdE ANd sOdIUm ALgINATE I. R. Bekus, M. V. Kyryliv, I. B. Ivanusa, O. B. Furka, I. Y. Krynytska, M. I. Marushchak I. HORBACHEVSKY TERNOPIL STATE MEDICAL UNIVERSITY, TERNOPIL, UKRAINE Background. Lipid metabolism disorders in the organism affected by environmental pollutants, including poisoning with cadmium and lead salts are of topical matter nowadays. Objective. The study was aimed to examine biochemical features of lipid metabolism in rats subjected to toxic damage by lead and cadmium salts and treated with carnitine chloride and Algigel. Methods. Experiments were carried out on white mature outbred male rats weighing 180-200 g. To cause the toxic damage the animals were administered with aqueous solution of cadmium chloride and lead acetate daily for the period of 30 days using intra-gastric lavage. The indices of lipid metabolism were detected by biochemical methods. Results. In animals treated with cadmium chloride and lead acetate the following changes were observed: Hdl-cholesterol concentrations significantly decreased, resulting in 87% of the levels in the intact animals on the third day, 84% on the fifth and 80% on the seventh day. conversely, concentrations of Hdl-cholesterol and vldl- cholesterol significantly increased during the experiment. respectively, the ratios for Hdl-cholesterol are 240%, 352%, and 388%; and for VLDL-cholesterol 108%, 116%, and 132%. Conclusions. lipids profile of the rats displayed changes in the levels of cholesterol, triglycerides and lipoproteins of low, high and very low density. KEY WORDS: blood lipid profile, heavy metal toxicity. Introduction The lipid metabolism disorders in the or- ganism affected by environmental pollutants including poisoning with cadmium and lead salts were extensively studied. Salts of these metals are assigned Class II of toxicity in the European Union's classification system, which is regulated by Directive 67/548/eeC. They have a large migration ability (in the soil to plant, and animal to person systems), and also high cu- mulativeness [4, 14]. This creates a threat of their increased uptake if an organism is ex- posed to several contaminated sources at once: inhaling the air (car exhaust), soil particles along the roadways, industrial dust, and ingest- ing fertilizer residue with vegetables and pro- duce [8, 9]. Due to their association with indus- trial waste, these compounds often occur in combination. However, although there is a large amount of the literature describing mechanisms of action of either lead or cadmi- um salts on the body, the number of studies of the combined effect of these two toxins is lim- ited [16, 17]. This study demonstrates that in rats lipid metabolism disorders caused by the combined toxic effect of Cadmium (Cd) and Plumbum (Pb) ions were effectively corrected after carnitine chloride and sodium alginate administration. This conclusion is proved by changes in the concentrations of lipid metabolism biomarkers in blood plasma. Material and Methods experiments were carried out on white mature outbred male rats weighing 180-200 g, which were kept on a standard diet and housed in the animal facility of Ternopil State Medical University. The animal maintenance, treatment and euthanasia were conducted in accordance with the internal safety and ethical regulations, as well as european convention for the protec- I. R. Bekus et al. 43 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2016 Vol. 2 issue 2 tion of vertebrate animals used for experimen- tal and other scientific purposes [15], and Ap- plied recommendations for the maintenance and work with laboratory animals [10]. To cause the toxic damage, animals were administered with aqueous solution of cad- mium chloride, dose 3.3 mg/kg (0.05 lD50), and lead acetate, dose 11 mg/kg (0.05 lD50), daily for the period of 30 days using intra-gastric lavage [2]. The treatment used for toxic damage cor- rection consisted of 2% solution of carnitine, dose 50 mg/kg, and enterosorbent (chelator) Algigel, dose 400 mg/kg [5, 12]. Both of these substances were administered daily using intra- gastric lavage. All experimental animals were divided into the following groups: Group 1, intact rats; Group 2 (control), animals treated with cad- mium chloride and lead acetate; Group 3, ani- mals treated with cadmium chloride, lead ac- etate as well as carnitine and Algigel enter- osorbent. After finishing the heavy metals and correc- tive agents administration, we tested the lipid metabolism rates on the 3rd, 5th and 7th days. To determine lipid metabolism the following meth- ods were used: triacylglycerols (TG) were ex- tracted from blood serum using isopropanol with simultaneous removal of phospholipids mix from the reaction by precipitation with aluminum oxide [3]; the presence of choles- terol was determined using a colour reaction whereby in the presence of acetate anhydride and a mixture of acetic and sulphuric acid a green compound was formed [1]. levels of cholesterol were evaluated by the intensity of the colour using colorimetry. Free cholesterol was detected using digitonin precipitation [3]. Cholesterol-digitonin complex was subse- quently dissolved in chloroform, evaluated by liebermann–Burchard test. The level of high- density lipoprotein (HDl) cholesterol was de- termined in soluble fraction after low density (lDl) and very low-density (vlDl) cholesterols in blood plasma were precipitated using hepa- rin in the presence of manganese ions [4]. levels of lDl and vlDl cholesterols were evaluated by mathematical models. The results of the study were analyzed by the Department of Statistical Research of Ter- nopil State Medical University using STATISTICA software (StatSoft). Arithmetic mean of the sample (m), its variance and mean squared er- ror (MSe) were calculated for all biochemical indices. Significance of the difference between independent quantitative values was deter- mined using Student's t-test. Results and Discussion Cadmium and lead belong to a group of heavy metals; their ions have the ability to ac- cumulate in the body. Since in this study these substances were administered for 30 consecu- tive days in subthreshold doses, we looked for general toxic effect in addition to the parame- ters of lipid metabolism. Thus, for the duration of the experiment, we observed the influence of cadmium chloride and lead acetate on the overall condition of the animals, change in body mass, peripheral blood composition and certain other hematological parameters [7, 8, 11, 13]. The early symptoms of poisoning with heavy metal are: rejection of food by the animal, general depression, and weight loss. Our ob- servations indicate that on the 5th day of the experiment, the control group of animals some- times refused their food or ate smaller quanti- ties compared to the intact animals. We also observed lethargy, reduced movements, and changes in fur cover. To prove these observa- tions, we measured body mass of the intact and control animals, which allowed us to determine its changes. We determined the levels of total choles- terol, triacylglycerols (TG), HDl, lDl and vlDl cholesterol in plasma of the experimental and control animals [6]. The data presented in Table 1 proves that in plasma of the animals injected with heavy metals, the concentrations of TG significantly increased compared to intact ani- mals, for instance on the third day the it was 186%, on the fifth — 179%, and on the seventh — 166% of the levels of the intact animals. We consider that the increase of triacylglyc- erol levels in response to prolonged cadmium chloride and lead acetate intoxication occurs according to a specific mechanism. The intoxi- cation by salts of lead and cadmium causes disruption of energy processes at the mitochon- dria. This evidences the activation of tissue li- pases and mobilizing of fatty acids from fat depots, which are rapidly delivered to the liver synthesizing the surplus amounts of triacylg- lycerols. Cholesterol is an important indicator allow- ing lipid metabolism evaluation. Under physi- ological conditions, the levels of cholesterol in the body are in a state of equilibrium between HDL and LDL cholesterols: the amount of cho- lesterol incoming with food and synthesized de novo corresponds to what is excreted as bile I. R. Bekus et al. 44 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2016 Vol. 2 issue 2 acids and free cholesterol. In this study, the concentration of total cholesterol in blood of the intact animals was 2.18±0.09 mmol/l (Table 1), but it was different in the group of the animals that received heavy metal salts. The maximal differences between the two groups were recorded on the seventh day after the beginning of toxicant administration, when cholesterol concentration increased by 16% in the treated animals compared to the intact ones. Cholesterol is present in blood as its free form and its esters of fatty acids. each of these forms has specific physiological functions and diagnostic value for understanding of lipid metabolism disorders. Free cholesterol is exchanged between tissues, so its concentration in serum reflects the total content of cholesterol in the body. The results of this study prove that there are significant differences in the rates of different form of cholesterol between the intact and control groups. Thus, toxic action of heavy metal ions disrupts esterification of cholesterol decreasing concentrations of its esters in blood serum. lipids are transported in blood as complex supramolecular complexes, lipoproteins. Therefore, in this study we determined con- centrations of different lipoprotein classes. In the animals treated with cadmium chloride and lead acetate the following changes were observed: HDL­cholesterol concentrations significantly decreased, resulting 87% of the levels in the intact animals on the third day, 84% on the fifth day and 80% on the seventh day. Conversely, the concentrations of HDl-cho- lesterol and vlDl-cholesterol significantly increased during the experiment. Respectively, the rates for HDl-cholesterol were 240%, 352%, and 388%; and for vlDl-cholesterol — 108%, 116%, and 132% (Table 1). Treatment of exogenous intoxication invol- ves the use of antioxidants, metabolic factors, 90 95 100 105 110 115 120 3 day 5 day 7 day % Intact Treated w ith heavy metal salts * * Figure 1. Total cholesterol content in blood plasma of the animals. Notes: * — significant difference if compared to the control animals. Table 1. Concentrations of total cholesterol, triacylglycerols, HDL, LDL and VLDL cholesterol in rats’ blood serum during 30-day administration of cadmium chloride and lead acetate (n=6) Intact animals, group 1 Animals treated with cadmium chloride and lead acetate, group 2 3rd day 5th day 7th day 0.86±0.06 1.60±0.09 р1<0.01 1.54±0.08 р1<0.01 1.43±0.08 р1>0.05 Total cholesterol, blood plasma, mmol/l 2.18±0.09 2.21 ±0.17 р1>0.05 2.46±0.18 р1>0.05 2.54±0.18 р1>0.05 Esterified cholesterol, blood plasma, mmol/L 1.52±0.07 0.91±0.07 р1<0.05 0.96±0.09 р1<0.05 1.27±0.08 р1<0.05 Free cholesterol, blood plasma, mmol/l 0.66±0.04 1.30±0.11 р1<0.05 1.50±0.11 р1<0.05 1.27±0.08 р1<0.05 HDl-cholesterol, blood plasma, mmol/l 1.54±0.02 1.34±0.11 р1>0.05 1.29±0.04 р1<0.01 1.24±0.04 р1<0.001 lDl-cholesterol, blood plasma, mmol/l 0.25±0.07 0.60±0,03 р1<0.01 0.88±0.13 р1<0.01 0.97±0.10 р1<0.01 vlDl-cholesterol, blood plasma, mmol/l 0.39±0.01 0.27±0.03 р1<0.02 0.29±0.01 р1<0.001 0.33±0.04 р1>0.05 Notes: р1 — significant difference if compared to the control animals. I. R. Bekus et al. 45 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2016 Vol. 2 issue 2 cell membrane protectors and also reducing systemic concentrations of toxic components in the body. The use of so-called efferent the- rapies, including enterosorbtion is one of the approaches to removing foreign substances and metabolic products. In this study, we used the combination of carnitine and enterosorbent sodium alginate. The changes of total cholesterol in blood plasma of the experimental animals were as follows. During the whole experiment we observed significant and linear decrease of cholesterol contents in the experimental animals compared to the control group. On the third day cholesterol levels were by 75% lower in experimental group, on the fifth — by 62%, and on the seventh — by 56%, which was lower than in the intact animals (Table 1). This decrease was in the free cholesterol, while the concentration of esterified forms significantly increased for the period of the experiment. In this study, we evidenced the positive effect of combined use of carnitine and sodium on the concentrations of HDl, lDl and vlDl cholesterol. The combined effect of toxicants caused a marked reduction in the concentration of HDl cholesterol at all stages of the experiment compared to the intact animals (Figure 2). Subsequent treatment with carnitine and Algigel caused a significant increase in these rates. Thus, on the 3rd day it increased by 12%; on the 5th by 22%; and on the 7th by 26% compared to the control animals. These rates, however, were significantly different from the corresponding rates in the intact group. When treated with the corrective factors, carnitine and Algigel, the animals demonstrated significant decrease of LDLC concentration. In particular, on the 3rd day this rate decreased in 3.6 times in comparison with the control ani- mals. On the 5th day the concentration decreased more and was in 5.8 times lower than in the control animals, and on the 7th day it was in 8 times lower. In the experimental group, the concentration of vlDl cholesterol on the 3rd day increased by 79% compared to the animals of the control group, and on the 5th and 7th days by 50% and 36% respectively. Conclusions This study proved that carnitine in combi- nation with sodium alginate had a clear positive effect on some parameters of lipid metabolism in blood plasma of rats. The proposed means of correcting biochemical changes caused by poisoning with lead and cadmium salts in subthreshold doses demonstrated the need for further studies to develop possible use in clini- cal settings for correction of lipid metabolism and reducing toxic effects of xenobiotics. HDL cholesterol 0 0,5 1 1,5 2 3 day 5 day 7 day m m ol /l * * * *# *# *# LDL cholesterol 0 0,5 1 1,5 2 2,5 3 day 5 day 7 day m m ol /l * * * # # # VLDL cholesterol 0 0,1 0,2 0,3 0,4 0,5 3 day 5 day 7 day m m ol /l Intact Treated w ith heavy metal salts Treated w ith heavy metal salts, plus carnitine and Algigel * * * * # * Figure 2. Changes in the concentrations of HDl, lDl and vlDl cholesterol in blood plasma of the animals. Notes: * — significant difference if compared to the control animals; # — significant difference if compared to the affected animals. I. R. Bekus et al. 46 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2016 Vol. 2 issue 2 References 1. Harbarets BO, Wysocki IJ, Kachanova A. Workshop on biological chemistry. Sumy 1997; 28. 2. Gerasimenko TI, Domnin SG, Roslyi OF, Fedoruk AA. The evaluation of combined effects of lead- copper and lead-zinc binary mixtures. Med Tr Prom Ekol 2000; 8: 36–39. 3. Horyachkovskyy A. 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