Biol Trace Elem Res DOI 10.1007/s12011-015-0359-7

Selenium Content in the Liver of Wistar Rats Fed Diets of Different Fatty Acid Quality Patrícia Mendonça de Castro Barra 1 & Céphora Maria Sabarense 2 & Marcelo Bonnet Alvarenga 3 & Rafael Arromba de Sousa 1 & Marcone Augusto Leal de Oliveira 1

Received: 28 January 2015 / Accepted: 28 April 2015 # Springer Science+Business Media New York 2015

Abstract The purpose of this work was to measure the amounts of selected mineral elements (sodium, calcium, iron, selenium, magnesium, zinc, copper, and manganese) in the liver of Wistar rats and evaluate possible correlations between the levels of these minerals and the lipid metabolism in the studied animals. Three experimental groups each containing six Wistar rats were designed. Each group was fed a different diet. The control group was fed a diet prepared with fresh soybean oil and named control group—CG. The second group (named experimental group B—EGB) and third group (named experimental group C—EGC) were fed a diet containing soybean oil that had been used to fry different foods for four or ten cycles, respectively. The mineral elements in Wistar rat livers were measured by inductively coupled plasma optical emission spectrometry (ICP OES). Only the elements calcium and selenium differed significantly between the control and experimental groups. There was a significant reduction of 33 % for Ca and 41 % for Se in the EGB in comparison to the control group. The reduction in mineral concentration, especially Se, is the result of interactions with fatty acid metabolism. The animals in the EGC exhibited more intracytoplasmic accumulation of fat and more intense vasodilatation, in relation to the other groups. Collectively, evidence hereby collected suggests that impaired dietary lipid

* Marcone Augusto Leal de Oliveira [email protected] 1

Chemistry Department, Institute of Exact Sciences, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil


Nutrition Department, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil


EMBRAPA Gado de Leite, Juiz de Fora, MG, Brazil

quality in otherwise balanced diets can reduce hepatic Se levels and potentially harm liver function. Keywords Selenium . Liver . Wistar rats . Fatty acids

Introduction Several nutritional studies have shown the importance of selenium (Se) as a mineral nutrient and a possible biomarker. In the present work, this element was considered in relation to the quality of fatty acids present in the diet of rats under a controlled environment. An ideal dietary biomarker should accurately reflect nutrient intake, with a specificity and sensitivity that could be applicable to any population studied. For predictable applications, the selected biomarker must be linked to the bioavailability of the micronutrients of interest. Mineral elements can be used as biomarkers if they provide different responses to different levels of depletion or appropriate nutrient supplementation [1]. The action of antioxidant enzymes such as superoxide dismutase requires the use of zinc or manganese ions, for example. In the context of lipid metabolism, selenium is used as a prosthetic group in the control of membrane lipid peroxidation [2]. In addition, mineral nutrients are absorbed from the diet only if present as bioavailable chemical species. Together with other trace elements, Se is associated with the modulation of fatty acid distribution as well as body weight and feed efficiency [3]. Low concentrations of Se have been shown to cause outbreaks of several chronic diseases, including cardiovascular diseases [4], cancer [5], diabetes [6], and inflammatory disorders [7]. Factors influencing the availability of selenium include amount and food source of selenium, the interaction of other dietary components, digestion efficiency, synthesis of absorbable selenium compounds,

de Castro Barra et al.

intestinal transition, nutritional status of the organism in relation to selenium, and diseases of the intestinal tract [8]. Generally, fried foods have high acceptance, and one of the preferential aspects of this kind of food is the sensorial features developed and their energetic value. However, the frying process induces changes in the oils’ physical-chemical properties, fatty acid [FA] profile and thus, the production of toxic compounds [9], which could be ingested from fried foods [10–12]. Since the metabolic impact of these frying oils on population fat intake is difficult to measure, it has been studied in animal models, especially in rats, using metabolic studies or histological evaluation of hepatic tissue [13]. According to Wang and colleagues [14], in vivo data from male Wistar rats indicate that diets with high levels of saturated fatty acids (and poor in unsaturated fatty acids) resulted in induction of hepatic endoplasmic reticulum (ER) stress and liver damage. Chronic ER stress induces numerous intracellular processes which can lead to systemic inflammation, hepatic fibrosis, and hepatocyte cell death. An accumulation of saturated fatty acids in the liver may play an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), and a growing amount of evidence suggests that ER stress may mediate the toxic effects of saturated fatty acids. The term NAFLD refers to any fatty infiltration in the liver that is not caused by significant alcohol abuse, which makes the pathogenesis of NAFLD multifactorial. It is a pathological condition that encompasses a wide spectrum of liver diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. Furthermore, NAFLD also increases the risk of cardiovascular disease [15]. According to Mattson and Grundy (1985), a diet rich in saturated fatty acids promotes a rise in LDL level in comparison with monounsaturated and polyunsaturated diets [16]. The presence and severity of NAFLD are closely related to risk factors for insulin resistance and the metabolic syndrome. The metabolic syndrome is characterized by the accumulation of visceral fat, dyslipidemias associated with high plasma triglycerides (TG) and low HDL, hypertension, and high concentrations of fasting plasma glucose, as well as low-grade inflammation [17]. The liver is an organ of intense metabolic activity, and changes in metabolic activity may be caused by various factors, including the quality of the diet. Specifically, lipid dietary intake and composition affect hepatic metabolism. As several metabolic processes are mediated by minerals, selenium levels in the liver were investigated alongside those of other important minerals in order to verify other possible correlations between mineral elements and lipid metabolism. Inductively coupled plasma optical emission spectrometry (ICP OES) was chosen as analytical technique given its multi-element characteristics. The results could help elucidate biological

mechanisms of inflammatory liver diseases, which in turn can help their prevention (mainly under the nutritional perspective) and clinical diagnosis via chemical element analysis.

Materials and Methods Instrumentation A Perkin-Elmer inductively coupled optical emission spectrometer (model Optima 7000DV, Norwalk, USA) was used to measure mineral contents. The instrument was equipped with a peristaltic pump, and a concentric nebulizer coupled to a cyclonic spray chamber. Briefly, experimental conditions were as follows: auxiliary argon flow rate of 0.2 L min−1, nebulization argon flow rate of 0.6 L min−1, and radiofrequency plasma power of 1300 W. The elements analyzed and each wavelength (nm) were as follows: sodium (Na) 588.995, calcium (Ca) 317.933, iron (Fe) 238.204, magnesium (Mg) 285.213, zinc (Zn) 213.857, selenium (Se) 196.026, copper (Cu) 324.752, and manganese (Mn) 257.610. In relation to the plasma view, sodium and calcium were determined in the radial view, due to their high concentration in samples, while the more sensitive axial view was employed for the other analytes. Chemicals and Materials The gas used in the spectrometer was pure argon (99.996 %, White Martins, Sao Paulo, Brazil). Standard solutions for each metal were used to prepare a multi-elemental analytical curve, in 2 % v/v HNO3. All glassware used was cleaned with a solution of 10 % v/v HNO3 and then washed with deionized water (Quimis, São Paulo, Brazil) prior to use. For the acid digestion of the sample tissues (sample preparation), concentrated HNO3 (Vetec, Sao Paulo, Brazil) was used. Experimental Design The study was conducted after approval by the Local Commission of Animal Experiment Ethics at the Federal University of Juiz de Fora, Brazil (protocol number 032/ 2011). The rats were kept in the animal facility of the Institute of Biology at the University of Juiz de Fora, three animals per box, two boxes per group, giving 18 animals in total, with filtered water and food (for each group) supplied ad libitum. The temperature was maintained between 22 and 26 °C. The light and dark cycles lasted 12 h each. The animals were fed with a diet formulated with high purity ingredients, closely following the guidelines outlined by the American Institute of Nutrition, in which soybean oil is the only source of dietary fat recommended [18, 19].

Selenium Content in the Liver of Wistar Rats Fed Diets of Different Fatty Acid Quality

The animals were randomly divided into three groups containing six rats each. The control group was fed with the AIN93G [18] diet, the fat in which was fresh soybean oil (labeled SOY OIL A) from the same lot that was used in the frying process that produced the other two oils. Animals in the second group (EGB) were also fed the AIN-93G diet, whereas soybean oil (labeled SOY OIL B) had been used for four frying cycles (an intermediate point of collection). The animals in the third group (EGC) were fed with the AIN-93G diet, but the soybean oil (called SOY OIL C) had been used for the Bmaximum time^ (10 cycles) before its disposal, according to undesirable sensory parameters such as viscosity and darkening. It should be noted that the soybean oil was used to fry various foods over a variable number of days. However, the percentage of each dietary component did not differ between the groups, except for the quality of the soybean oil used, i.e., fresh or successively deep-fried. Forty-five days after starting the experiment, all animals involved were euthanized according to the Guide for the Care and Use of Laboratory Animals [20]. The entire liver was excised and stored in suitable containers at −80 °C until analysis, according to the flowchart in Fig. 1. Liver samples for the histology analyses were fixed in 10 % v/v buffered formaldehyde immediately after collection and subsequently subjected to histological processing. The remainder of the biological material was disposed as medical waste. Sample Preparation (Liver Acid Digestion) The liver samples were first ground and then stove-dried at 70 °C for three consecutive days. The dried samples were then digested using nitric acid and moderate heating under atmospheric pressure [21]. All digestion processes were performed

Fig. 1 Experimental design and basic protocols—dietary manipulation to liver analysis by ICP OES. Fatty acid analysis is explained in detail by Barra et al. [26]

in a chamber with adequate dissipation apparatus for harmful vapors. In this procedure, ca. 300 mg of each liver sample was weighed separately and mixed with 2.0 mL of concentrated HNO3. The mixture was heated in a thermostatically controlled water bath at 90–95 °C for 10 h, under a reflux system connected to the top of glass flasks. The digested samples were then diluted with deionized water to 25.00 mL, according to Fig. 2. ICP OES Analytical Performance Although the instrumental parameters used were the ones recommended by the instrument factory for aqueous samples, the quality of the selenium determinations was evaluated by considering the analytical curve and other analytical parameters, such as linear range, slope, linear coefficient, r2, limit of quantification, and accuracy. Data Evaluation (Statistical Analysis) Statistical tests for normality, homoscedasticity, and independence as well as box plots were performed in SPSS 8.0 for Windows software.

Results and Discussion Initial box plots (data not shown) for iron analysis indicated no difference in median values between the EGC and control groups; yet, there was a small reduction in its content in the EGB. There was also one outlier, indicating a sample with a different profile response. For manganese, the median values were similar in all three groups, and there were no outliers. For zinc, the median value of the EGB was lower than that of the control and final groups, which themselves had similar median values. In addition, there was slightly more dispersion of the data for the final group, in comparison to the control group. This variation may be indirectly related to liver damage

Fig. 2 Schematic sample preparation (liver) before analysis by ICP OES

de Castro Barra et al. Table 1 Statistical results and elemental concentrations (mg of metal/100 g) for iron, magnesium, sodium, zinc, calcium, copper, selenium, and manganese as determined in prepared liver samples Animals









1 2 3 4

13.4 19.2 21.3 18.5

111 54.5 55.0 63.1

68.5 127 131 72.2

15.6 7.11 8.97 9.53

194 46.5 45.7 42.8

0.907 0.739 0.884 1.24

0.265 0.243 0.278 0.255

0.586 0.465 0.407 0.553


Median Normality p value Homogeneity p value

15.4 18.5 13.9 12.0 19.4 14.7 13.9 13.3 13.9 13.8 15.3 17.2 19.9 19.5 23.6 18.3 0.687 0.506

60.5 60.5 69.8 49.7 50.24 61.1 61.4 58.8 60.0 52.0 58.3 46.7 66.6 58.0 51.0 55.0 0.002 0.080

97.8 97.8 76.6 98.0 112 105 90.9 86.8 94.4 70.9 77.2 97.6 107 93.9 125 95.7 0.553 0.123

9.55 9.53 6.97 7.40 8.36 8.32 8.16 9.18 8.24 9.27 9.91 8.39 9.84 9.54 7.16 9.40 0.009 0.100

39.6 45.7 65.9 39.0 37.2 33.8 31.6 30.1 35.5 30.5 25.2 29.8 41.9 44.2 30.0 30.2 0.000 0.015

0.827 0.884 0.783 0.681 0.780 0.975 0.929 1.01 0.856 0.884 1.02 0.811 0.971 0.888 0.817 0.886 0.229 0.363

0.258 0.258 0.221 0.230 0.204 0.220 0.224 0.219 0.220 0.168 0.178 0.132 0.192 0.155 0.0850 0.162 0.059 0.030

0.419 0.465 0.452 0.388 0.344 0.514 0.491 0.490 0.471 0.400 0.483 0.274 0.516 0.475 0.3610 0.438 0.255 0.644

ANOVA p value










Median Intermediate

Median Final

6 7 8 9 10 11 12 13 14 15 16 17

and should be investigated in future work. Overall, the zinc profile among groups was similar to that observed for iron, suggesting that there is a direct correlation between these elements. For copper, the median value did not differ significantly between groups. However, as for zinc, there was an increase in the dispersion of the values for the EGB and EGC groups. Calcium content was apparently lower in the final group in comparison to the intermediate and control groups. In addition, there was a gradual increase in the dispersion from the intermediate to the final group. There was also an outlier in the control group and another in the EGB. Fig. 3 Percentage variation in selenium and polyunsaturated FA in the evaluated groups: control, experimental group B (ECB), and experimental group C (EGC)

Finally, for selenium, there was a considerable decrease in the median from the EGB to the EGC group. There was only one outlier, belonging to the EGB. Table 1 presents all analytical data obtained for the liver tissues. To assess zinc, calcium, and selenium analytical data, nonparametric statistical analysis was used, since zinc and calcium did not follow a normal distribution (Shapiro-Wilk test), while selenium data showed lack of homogeneity. Although a number of mineral elements were assessed, only calcium and selenium showed significant differences between the control and EGC groups, at a 95 % confidence level

Selenium Content in the Liver of Wistar Rats Fed Diets of Different Fatty Acid Quality Table 2 ICP OES analytical performance for Se determination

ICP OES parameters


Linear range Slope Linear coefficient r2 Limit of quantification Accuracy

0.100–5.00 mg L−1 1141 221 0.999 0.100 mg L−1 94 %a

a Estimated from the measurement of a standard reference blinded analyzed during experimental analytical determinations

(p value

Selenium Content in the Liver of Wistar Rats Fed Diets of Different Fatty Acid Quality.

The purpose of this work was to measure the amounts of selected mineral elements (sodium, calcium, iron, selenium, magnesium, zinc, copper, and mangan...
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