Abstract: Background and Objective: Disturbances of trace elements are intricate factors in the progress of obesity and its associated non-alcoholic fatty liver disease (NAFLD), however, the status of these elements has infrequently been studied in NAFLD. The current study aimed to evaluate the iron, zinc, copper, manganese and selenium statuses of serum and liver in control and high-fat sucrose diet-induced non-alcoholic fatty liver disease (NAFLD) as well as their relationships with metabolic risk bio-markers of NAFLD. Material sand Methods: Forty male 1.5-month-old rats were allocated into 2 groups of 20 each. One group was given a standard diet and the second nurtured a high-fat sucrose diet (HFSD) for 16 weeks. Blood was assessed for lipid profile (triglyceride, cholesterol, LDL, HDL) and liver function markers (alanine aminotransferase (ALT) activity). Hepatic histopathology was examined by hematoxylin and eosin (H and E) staining. Serum and hepatic trace element levels were evaluated by means of inductively-coupled plasma mass spectrometry. Results: The HFSD group showed significant changes in lipid profile, increased ALT activity, hepatic small and large fat globule, hepatocyte ballooning and damage indicating induction of NAFLD. Serum iron, zinc, copper and selenium concentrations were significantly decreased and minor serum manganese and selenium levels were observed in the HFSD group compared to the control group. Both hepatic iron and copper levels of the HFSD group surpassed those of the control group. Conclusion: The HFSD-induced NAFLD plays a role in iron homeostasis, thus reducing its bio-availability and increasing its hepatic peak drained from the blood. Furthermore, HFSD-induced NAFLD disturbs serum and hepatic trace elements.
INTRODUCTION
Hepatosteatosis and associated NAFLD are considered to be serious hidden problems relating to obesity and metabolic syndrome. The NAFLD is defined as micro-vesicular or macro-vesicular lipid droplets in more than 5% of hepatocytes in non-alcoholic persons. The occurrence of NAFLD is growing globally, reaching approximately 35% in some nations and it is considered to be the greatest common hepatic disease1. A high-fat diet is frequently accompanied by metabolic syndrome, obesity, hormonal, inflammatory and lipid metabolism disorders. Such a diet is thought to have a crucial effect in insulin resistance and the progress of hepatosteatosis2,3.
Attention to trace minerals has been growing progressively over the last 25 years. Trace elements are important for the biological, physiological and biochemical functioning of the body due to their roles as structural and signaling elements and as catalytic co-factors of enzymes and hormones involved in metabolic processes4. Moreover, trace elements have multi-purpose roles in biological systems, fluctuating from adapting metabolic responses to working as antioxidants and immunological roles4,5. Several investigations have proposed that changes to trace element concentrations may be related to the cause and pathophysiology of numerous psychiatric complaints6.
Some studies have shown that body conformation may be a causative factor resulting in iron insufficiency. Iron deficiency in overweight American children and adult are more predominant than in ordinary weight7. This suggests that the dietary management of obesity and associated NAFLD should similarly reflect the study and/or handling of iron deficiency.
Several experimental8-10 and clinical11 studies have revealed a close relationship between trace mineral alterations and obesity-related disorders12. Simultaneously, the majority of experimental results depend on the nutritional status of the experimental animals, while investigations into the effects of dietetic regime, specifically a high-fat diet, on trace mineral status are insufficient.
In particular, trace minerals such as chromium, zinc, manganese and selenium are the main minerals involved in glucometabolic disorders because of their antioxidant and insulin-mimetic activity13.
The effect of zinc on carbohydrate metabolism is complicated. Zinc has a vital role in the biosynthesis, storage and secretion of insulin14. Animal studies show that small doses of zinc protect against T2DM, which is involved in the physiopathology of obesity, however, in high doses zinc is toxic to β-cells15. In this respect, obese people have considerably greater insulin resistance and lower blood zinc levels than healthy individuals. A significant inverse relationship has been established between serum zinc levels and HOMA-IR16.
Nevertheless, there is a deficiency and a controversy in the literature concerning disorders of the mineral status of the body and their association with obesity, NAFLD and raised insulin resistance.
Trace element administration as dietary schemes are intended for NAFLD controlling17. An increasing number of investigations recommend a possible relation between HFD, obesity and changed iron metabolism18. Accordingly, some data about mineral status in NAFLD are necessary for actual monitoring of the disease. Thus, the main aim of the current work was to evaluate the trace elements level in the serum and the liver in a model of HFSD-induced NAFLD in rat using ICP-MS. Also, to measure the role of HFSD on trace element level in hepatic tissues in rats and study its relationship with lipid profile, hepatic function, leptin and resistin levels.
MATERIALS AND METHODS
Experimental animals: A total of 40 male 1.5 month-old Albino rats, weighing 85-95 g were attained from the Institute for Research and Medical Consultation (IRMC, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia). Rats were kept for 1 week before the beginning of the experiment. Entirely, rats had ad libitum contact to water and food. The rats were housed separately in elastic pens at 22±3°C and a period of 12 h light/dark in the animal house of the IRMC laboratories.
Diet: Two different food types were offered to the rats, 1st standard rat chow food and the 2nd a high-fat and sucrose diet (HFSD). Standard diet composed of protein (whey protein, soya bean and meat), corn, calcium carbonate and phosphate, magnesium oxide, sodium chloride and vitamins. The standard rat food contain of 60% carbohydrates, 8% fat, 22% basic protein, 10% dietary fiber, vitamins and minerals. The HFSD contained 65% carbohydrates, (HFSD diet contained an addition of 5% sucrose) 15% crude protein, 15% fat (The highest kind of fat used was a long chain saturated palmitic acid and stearic FA) and 5% dietary fiber, vitamins and minerals. The HFSD was used to develop the rat model of experimental fatty liver and was equivalent with the preparation previously used by Al-Muzafar and Amin19.
Chemicals
Assay kits: Colorimetric assay kits obtained from HUMAN Gesellschaft für Biochemica und Diagnostica mbH (Wiesbaden, Germany) were used to assess the serum of rats and determine their lipid profile (TG, TC, LDL and HDL) and liver function (alanine transaminase (ALT), LDH and bilirubin). The TG levels were measured by method of GPO-PAP, a colorimetric enzymatic analysis for TG with lipid clearing factor (REF # 10724 kit). Cholesterol (TC) was determined by method of CHOD-PAP, a colorimetric enzymatic test for TC with lipid clearing factor (REF 10028 kit). The LDL was determined by direct homogenous enzymatic colorimetric test (REF. 10294). The HDL was measured using, liquicolor direct homogenous enzymatic test (REF, 10284). The ALT was determined by BCG-method, photometric colorimetric test (REF. 10560). Lipid hormones, leptin and resistin were analyzed using enzyme-linked immunosorbent assay (ELISA) kits from Bertin Bioreagent (Montigny le Bretonneux, France) (cat. no. A051760 and A05179, respectively).
Trace element analysis: The attained serum and hepatic samples were organized for trace mineral examination. Hepatic tissue sections were washed with cold deionized distilled water (DW) and disconnected from connective tissue by cleaned stainless utensils.
Inductively coupled plasma mass spectrometer ICP: This method was intended for the analysis of trace element in food such as homogenate, cereals, milk, spice, blood and other kinds of foodstuff.
Principle of the method: Introduction of a determining solution into a radiofrequency blood where energy transfer routes from the plasma cause atomization, dissolution and ionization of elements. Second extraction of the ions from plasma through a differently pumped vacuum interface with integrated ion optics and separation. Third transmission of the ions through the mass isolation unit and recognition. Fourth quantitative determination after calibration with suitable calibration solutions.
Materials and equipment for ICP: Devices, ICP-MS and microwave were used for trace element analysis.
Micro-pipettes, plastic tubes, ICP-MS vials, plastic volumetric flasks and wash bottles, moreover gases as argon, helium and hydrogen purity of at least 99.9%.
Standards: Fe, Zinc, Cu, Mn and Se, each standard was stored at room temperature.
Stock standard solutions
Preparation of standard for ICP-MS: Intermediate-1-multi-element standard contain all single standard into 1 plastic tube 50 mL with a mass concentration 10 mg L–1. About 10 mL of filling solution was added into a 50 mL plastic tube and 500 μL of 1,000 mg L–1 standard solutions from each single standard Fe, Zn, Cu, Mn and Se and add de-ionized water up to 50 mL. Then store at room temperature.
Experimental design and animal grouping: About 40 male rats were randomly allocated into two groups (n = 20/group). The 1st control group was administered a standard chow diet during the study period (16 weeks). The 2nd group was fed HFSD for 16 weeks to induce NAFLD. The total experimental time was 16 weeks. Rats were weighed each weekend their body weights were recorded and weight gains were calculated.
Blood and tissue sampling: Blood was assembled from the medial canthus of the eye of all rats during the overnight fasting time before blood sampling by using a micro-hematocrit capillary tube and stored in dry glass centrifuge tubes. The blood was stored at 24±3°C and permitted to coagulate. Coagulated samples were centrifuged at 1400 rpm for 20 min at a temperature 20°C and the clear supernatant serum were stored at -80°C prior to further biochemical analysis. Immediately after blood sampling, the rats were sacrificed by decapitation while still under anesthesia. Part of the liver was extracted, washed with saline and fixed in 10% formalin at 25°C and for 48 h. The tissue samples were embedded in paraffin and cut using a rotary microtome into 3 mm thick sections and transferred to slides prior to hematoxylin and eosin staining for histopathological analysis.
Histopathological examinations: Tissue samples underwent hematoxylin and eosin staining. A microscope was used to inspect the structure and characteristics of hepatic cells, whether, fat globules or inflammation were present and whether, the hepatocytes had undergone any degenerative alterations. The grade of NAFLD was assessed at low (x40) and high (x100) magnifications. The level of NAFLD, the presence of minor/moderate fat droplets, micro and macro-vesicular NAFLD, hepatoglobular inflammation and the presence of inflammatory cells were recorded and used to determine the steatosis score as previously described19.
Statistical analysis: The results were evaluated by Student t-test analysis. The data were displayed as the Mean±Standard Error (SE). The p<0.05 indicated a statistically significant variations. Statistical examination was achieved by Graph Pad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA).
RESULTS
NAFLD induction: The attained data showed that HFSD had significantly increased the body weight gain in rats compared to control group (Table 1). At the similar period, HFSD-fed rats were categorized by greater adiposity and steatosis as indicated by hepatic gross photograph (Fig. 1).
A significant increase in serum lipid profile of TG, cholesterol and LDL, while decrease in HDL level were observed in the HFSD group (Fig. 2 a-d) compared to the control group. A significant elevation in the serum total and direct bilirubin levels, in addition to LDH and ALT activities as liver function markers was observed in the HFSD compared with the control group (p<0.05, Table 1 and Fig. 3). The increased levels of bilirubin and ALT activities showed damages to the hepatocytes. In addition, a significant rise was detected in serum leptin level (Fig. 4a) and resistin (Fig. 4b) in HFSD matched with the control group. These changes indicated steatosis and NAFLD induction associated with liver injuries.
These liver affection confirmed by histopathological and gross inspection of hepatic tissues that established proliferation in micro and macro-globules in HFSD-fed . The microscopic examination showed: (a) Hepatic centrilobular section in HFSD-fed rats characterized by normal hepatic cells, (b) Hepatic periportal area characterized by normal hepatic construction in control group and (c) Hepatocytes are described by the existence of small (micro-vesicular) and large (macro-vesicular) lipid droplets and marks of ballooning (Fig. 5).
The NAFLD hepatic score in the control group was 0 for 17 rats and 1 for 2, 2 for 1 rat and 3 for 0 cases, while the score in HFSD group was 1 for 13 rats and 2 for 6 rats and 3 for 1 rat Table 2. This postulated that the NAFLD scores were apparently intensified in the HFSD in comparison with control group.
HFSD decreased the level of serum iron: A significant decrease was noticed in the serum level of Fe, Zn, Cu and Se Table 3 compared to the control group (p<0.04, 0.03, 0.02 and 0.04, respectively), these changes indicated increased fat accumulation and the development of NAFLD. The HFSD resulted in significant decline in serum Zn concentration, while hepatic content was not affected.
| Fig. 1: | Gross photograph on fatty liver associated with adipose tissues |
| Table 1: | Changes in serum T Bilirubin, bilirubin and LDH in control and HFSD group |
| The data are displayed as the Mean±Standard Error. *Indicate a significant difference between groups at p>0.05 | |
| Table 2: | NAFLD score in comparison of control (n = 20) and HFSD-fed (n = 20) rats |
| aThe percentage part affected in the examined hepatic photographs. HFSD, high-fat, high-sucrose diet | |
| Table 3: | Changes in serum trace element in normal and NAFLD group |
| The data are displayed as the Mean±Standard Error. *Indicate a significant difference between groups | |
| Table 4: | Changes in hepatic trace element in normal and NAFLD group |
| The data are displayed as the Mean±Standard Error. *Indicate a significant difference between groups at p>0.05 | |
Table 4 demonstrated the changes in hepatic trace element in control and NAFLD group and there was non-significant overload and elevation in Fe and Cu hepatic level and Zn, Mn and Se were non-significantly changed.
| Fig. 2(a-d): | Changes in lipid profiles (a) TG, (b) TC, (c) LDL and (d) HDL in control and HFSD groups |
| Fig. 3: | Changes in liver function in normal and HFSD groups |
DISCUSSION
The weight gain, lipid profile, the liver function, hormones associated with lipid accumulation, including leptin and resistin were disrupted in the HFSD group compared to the control group. These data specified that a variety of biochemical bio-markers were involved in hepatic fat accumulation and subsequently, the NAFLD progress. These results indicated that HFSD consumption leads to a significant weight gain, were in agreement with Minematsu et al.20.
Serum Fe level decreased significantly while hepatic Fe was non-significantly at its highest peak in the HFSD group as compared to the control group. These results indicated that blood iron deficiency and iron overload in hepatic tissues have existed in this study Table 3 and 4.
| Fig. 4(a-b): | Influence of HFSD on serum leptin and resistin hormones in rats |
| Fig. 5(a-c): | Hepatic histopathology in experimental rats (hematoxylin and eosin staining, ×100), (a) Hepatic centrilobular section in HFSD-fed rats characterized by normal hepatic cells, (b) Hepatic periportal area characterized by normal hepatic construction in control group and (c) Hepatocytes are described by the existence of small (micro-vesicular) and large (macro-vesicular) lipid droplets and marks of ballooning as indicated by arrows |
The possible mechanisms of hypoferremia remain indefinite and may be as follows: Owing to a dietary iron deficiency, there may be elevated iron requirements because of raised blood volume as a consequence of extraordinary adipose tissue mass and/or general inflammation from adiposity21. Therefore, decreased iron grade in people who are overweight and have NAFLD may arise from a combination of dietary (low absorption) and functional (raised confiscation) iron insufficiency22.
Disorders in iron homeostasis or iron deficiency are repeatedly linked to obesity, overweight kids, teenagers and adults23 which caused reduced study ability, exhaustion and anemia.
The NAFLD is frequently accompanied by disturbances in iron homeostasis, where the risk of iron shortage is increased in obese individuals. The principal mechanism for this may be via increased hepcidin expression. Hepcidin is a protein that acts as an important controller for iron metabolism in mammals by inhibiting its intestinal absorption, regulating its entry into the circulation24,25 and sequestration of iron within the hepatic cells and macrophages leading to insufficient serum iron for red blood cells production and consequently anemia occurred. It is also known to be a mediator of inflammation26 and may account for obesity-related iron insufficiency.
The hepatic iron overload in the present data was in agreement with that of Yamano et al.27, who found that iron overload may lead to oxidative stress and is therefore, a risk factor for T2DM in the obese group. Prominent hepatic iron peaks had been identified as clinically important in some disorders such as NAFLD, alcoholic hepatic disease, prolonged hepatitis C and final stage hepatic illness28.
Iron is an element that may interact with oxygen free radicals to produce hepatic injury/fibrosis and insulin resistance. Hence, liver iron overload may increase the danger of NAFLD, non-alcoholic steatohepatitis (NASH) and its progression to hepatocellular carcinoma. Therefore, iron-reducing treatments, such as phlebotomy and iron-limiting regimes could have a favorable effect on patients with NAFLD/NASH by decreasing liver damage in addition to insulin resistance29.
The principal mechanisms of iron overload in NAFLD are strongly connected to diminish iron transfer from the hepatocytes due to low manifestation or breakdown of the iron transfer molecule FPN (a protein that transfers iron from enterocytes and reticuloendothelial macrophages) by increasing hepcidin gene expression8. In addition, iron may accumulate in fat globules in NAFLD and resulted in oxidative stress and inflammation30, that specified by TNF-α and IL-6 and changes in adipokine release of leptin and resistin considered a powerful indicator from unhealthy adipose tissue related to NAFLD to dysregulate iron besides lipid or glucose homeostasis8. In precise, it has been revealed that both serum iron insufficiency and hepatic excess could be accompanied with increasing liver lipogenesis 31 and related NAFLD. In this case supplementary trace element had a role in gluco-metabolic improvement and retain important effect on the disease progress32.
The reciprocal interaction between NAFLD-associated pathogenesis and iron homeostasis resulted in hypoferremia and increased iron hepatic content being deposited either in hepatic fat globules or in hepatic tissue macrophages. In progress to raised iron content, iron- mediated toxicity, affected the primary features of NAFLD. Therefore, both systemic hypoferremia and local iron hepatic tissue overload linked to NAFLD pathogenesis appeared to interrelate reciprocally these events in accordance with Nikonorov et al.10. These excess amount produce free radicals and oxidative stress, so aggravated the NAFLD status. Recent works had specified that iron loading is a danger factor for T2DM and nutritional iron limitation or chelation of iron improved the symptoms of T2DM in mouse model27.
Zinc is one of the best vital trace mineral in the body. Zinc insufficiency appeared to show a role in the progress of age-associated diseases and weakening of the life quality33.
HFSD produced a significant decrease in serum zinc level, while hepatic content not affected. The differences between tissues and blood concentrations was may be due the changes in element in the serum was more than that in tissues.
The mechanism of Zinc effect was to rise the stimulation of enzymes and antioxidant proteins as catalase and glutathione through maintenance of the protein sulfhydryls from oxidation and antagonizing transition metal-catalyzed reactions34. Zinc can interchange redox active element, like iron and copper. Therefore zinc deficiency leads to oxidative stress and consequence progress of the NAFLD.
Decreased hepatic Zn, Mn and Se, levels in rats suffering from NAFLD in accordance with Gatiatulina et al.35, however rats fed a HFSD had lower blood Fe, Zn, Cu and Se levels, whereas Mn exceeded the control level. The mechanism of that rearrangement may be due to definite infections and the associated inflammation linked with an alteration of trace minerals between the blood and the hepatic and other internal organs36,37. Also it may compensate for lipid disturbances in NAFLD disorders.
The interaction between the impaired trace mineral metabolism and the disturbance of adipokine in the leptin balance is key and preceded further metabolic disorders associated with obesity such as NAFLD38.
Changes in serum trace minerals may lead to alterations in serum thyroid hormones (T4), which are considerably affected in youngsters identified with exogenous obesity, though the alteration in serum T4 concentrations was not linked to variations in trace element levels39.
The main molecular mechanism of trace element such as Cu, Zn and Se is that they are involved in several biochemical processes supportive to the healthy life. The most vital of these biochemical processes are cellular respiration, oxygen utilization, replication and synthesis of DNA and RNA, maintaining the integrity of the cell membrane and scavenging of reactive oxygen species. Cu, Zn and Se are essential to the removal of free radicals via cascading enzymatic schemes. In particular superoxide free radicals are reduced to H2O2 by superoxide dismutases (SOD) in the existence of Zn and Cu as cofactors. H2O2 was then reduced to H2O by the Se-glutathione peroxidase pair40. On the other hand, excessive intake of these trace elements leads to disease and toxicity, therefore, a fine balance is essential for health.
The attained results revealed that: (a) NAFLD is accompanied by reduced total hepatic Zn, Mn, Se, levels but increased Fe and Cu level and b) NAFLD was characterized by significantly lesser serum Fe, Zn, Cu and Se levels, while the level of serum Mn exceeded the control values. In summary, the results of the current work revealed that NAFLD was significantly disturbed the trace element status in experimental rats given HFSDs.
It was also remarkable that serum and liver were suitable indicators for trace mineral homeostasis in NAFLD and additional bio-markers should be measured preceding the control of the trace mineral status. It was advisable to consider the data from changed trace mineral status in NAFLD throughout the progress of dietary strategies for NAFLD supervision. Specifically, exceptional care should be provided for intense metabolic modification of Fe, Zinc Cu and Se in NAFLD. Putatively, the adaptation of the Fe, Se, Mn and Zn status may recover the metabolic bio-markers in NAFLD. Nevertheless, further studies in-vivo and in-vitro are necessary to evaluate the mode of action of trace mineral disturbances in NAFLD and the underlying correlation among these conditions.
CONCLUSION
It could be concluded that, the development of NAFLD produced by HFSDs was characterized by alterations lipid profile, hepatic dysfunction, hepatic histopathology and its score, leptin and resistin levels. Furthermore HFSD resulted in a serum Fe deficiency via its potential hepatic accumulation and a decrease in its serum level. Also, serum Zn, Cu and Se levels decreased significantly while hepatic Cu may increase in the HFSD group, indicating an association with NAFLD and possible hepatic damage.
All sides of the current evaluation should be painstakingly examined to design novel therapeutic strategies for the management of disturbed trace elements in NAFLD. Philosophies for the best protection against NAFLD which retain a good and balanced nutritive state should be the focus of forthcoming research. Both nutritional management and treatment of NAFLD are necessary for handling this global problem. It is suggested that the general public have regular medical inspections which include their lipid profiles, liver function evaluations and trace mineral biomarkers levels and that they modify their lifestyles by avoiding HFSDs and ingesting healthy food to control NAFLD and mineral disturbances.
ETHICS APPROVAL
The animal experiments were carried out in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. We certify that this work was performed in accordance with local ethical guidelines, with the approval of Imam Abdulrahman Bin Faisal University’s Institutional Review Board (IRB). The IRB number for this study was IRB-2104-90-66.
SIGNIFICANCE STATEMENT
Both hepatic iron overload and serum iron deficiency in HFSD group, disturbed iron metabolism that results in hepatic score and bio-marker alterations and may be iron deficiency anemia. These trace element disturbance in HFSD represents a novel approach for NAFLD therapy and advance the concept of fat and mineral instabilities. Moreover, HFSD-induced NAFLD were able to produce serum Fe, Zn and Cu insufficiency associated with leptin and resistin imbalance.
ACKNOWLEDGMENT
We acknowledge Deanship of Scientific Research at the Imam Abdulrahman Bin Faisal. Also, we appreciate the assistance of the staff member of the chemistry department and university for kind support in this study.