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Cadmium oxidative stress markers role of palm oil pre-treatment

Cadmium poses risk even at the slightest level of exposure to animals and plants, because the body normally does not have much capacity to degrade it to less harmful species, it is poorly excreted and is easily bio-accumulated (Waalkes, 2003). When exposed to the body, several organs and tissues are under attack thus endangering immune and metabolic stability. Long term exposure to cadmium favours its gradual build up and encourages different forms of liver and kidney related diseases (Karl-Heinz and Magnus, 2000). In acute cadmium poisoning, its symptoms usually manifests within 24 hours affecting cardiovascular functions which leads to shortness of breath, general weakness and fever. In some other severe cases, it may manifest in form of pneumonia, respiratory malfunctioning and death. (Jarup et al., 1998). Long- term exposure to high dose cadmium leads to ltai-ltai disease. This disease mostly common with women is characterized by severely impaired kidney function and generalized loss of bone minerals that results in multiple bone fractures (Inaba et al., 2005).

Palm oil constitutes majorly of palmitic and oleic fatty acids, a semi-solid fat at room temperature (Sundram et al., 2001; Sundram 2011). In its crude nature, it constitutes 1% of the carotenoids (α and β carotene), Vitamin E (in the form of tocotrienolsand tocopherol) and it is considered to be the most prevalent source of natural carotenoids, and the human body uses Carotenoids as Vitamin A (Choo et al., 1997). Although present in small quantities, these minor constituents to a certain extent are responsible for the healing or medicinal properties of palm oil. Like all oils, triglycerides (TGs) are the major constituents of palm oil, forming about 95% of palm oil (Choo et al., 1997). TG consists of glycerol molecule esterified with three fatty acids. During oil extraction from the mesocarp, the hydrophobic TGs attract other fat or oil soluble cellular components. These components form the minor components of palm oil such as phosphatides, sterols, pigments, tocopherols, tocotrienols and trace metals (Goh et al., 1983; 1985). Other components in palm oil are the metabolites in the biosynthesis of TGs and products from lipolytic activity. These include the monoacylglycerols (MAGs), diacylglycerols (DAGs) and free fatty acids (FFAs). Triacylglycerol or triglyceride

Obahaigbon, (2012) submitted that one of the major constituents of red palm oil includes phospholipids that forms the main building blocks in all living cells (including human). It further submitted that apart from being an important constituent of lipoproteins, it works hand in hand with vitamin E and other cascade of antioxidants to maintain a balance in the management of oxidative homeostasis. Several phospholipids listed to be constituents of palm oil includes phosphatidylcholine, Phosphatidylinositol, Phosphatidylethanolamine and Phosphatidyl-glycerol all of which has been advocated by researchers to contribute to brain development in developing and growing foetus as well as lactating mothers (Zeisel et al., 1986). In another development, the studies of Jager et al., (2007); Starks et al., (2008) also lend their thoughts to the relevance of the phospholipids constituents of palm oil to the improvement of hormonal status and balance for athletes as well as their memory improvement. According to these researchers, this occurs when cortisol level increase are inhibited as well generally improving the easy transportation and absorption of food nutrients relevant for energy generation and the maintenance of energy homeostasis.

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The management of cadmium poisoning has been around the use of chelating agents of which most has been identified to be expensive, having their own significant side effects and remains expensive, not easily afforded by rural dwellers mostly vulnerable to these metal exposure (Twumasi et al., 2014). However, the use of natural products for treatments of various forms of poisoning has proven to have minimal side effects and is also cost-effective. Evidences in literature have implicated the use of palm oil for poisoning management in local Nigeria (Achuba and Ogwumu, 2014) as well as its anti toxic and antihepatocarcinogenic effects (Nwokocha et al., 2011). Aside the nutritional attributes, the healing properties of palm oil have been recognized for generations (Sundram, 2011) and until modern medicine arrived, red palm oil was the remedy of choice for nearly every illness in most parts of Africa (Chandrasekharan et al., 2000). Since it has been established that antioxidant supplementation has been found to be beneficial in metal toxicity, it is conceivable that palm oil may prevent cadmium toxicity and contribute to prevention of acute cadmium poisoning side effects in rats.

Materials and Methods

Palm Fruits Collection

The purchase of oil palm fruits were from a local oil palm plantation in Obiaruku, Delta State of which the Elansaes guinensis Tenera specie was used for the research. Identification was carried out at the Department of Botany and registered with the assertion number ID/2017/16807/Tenerra Spp.

Extraction of Palm Oil

Harvested ripe palm fruits weighing 10 kg was boiled for four hours. Once it has been ascertained properly cooked, the pulp was extracted by pounding in wooden mortar and transferred into a bowl containing 10L of water with thorough stirring. The oil palm fruit fibers were further removed using a handmade basket as sieve and the collected filtrate transferred into a cooking pot of about 20 liter volume and boiled under a controlled temperature of 1500C for five hours . At the end of the boiling period, the heavily heated mixture was allowed to stand for 30 minutes and 2L of cold water sprinkled at the surface using a sprinkler. After cooling, the palm oil already set on top was collected into a fresh container and heated for ten minutes to remove any trace of water.

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Fractionation of Crude Palm Oil

The methods for the fractionation of the three palm oil extracts (unsaponifiable extract, silica gel extract and bleached extract) used in this study have been previously described by Twumasi et al (2014) and Osei (2013) These methods are

Unsaponifiable extract (UPE)”,

Preparation of unsaponifiable extract was done using the methods of Meloan as modified by Twumasi, et al. (2014).

Silica Gel extracts (SGE)

Silica gel extract was prepared based on the one described by Ahmad et al. (2008).

Bleached Extract

Bleaching was carried out based on a method described by Patterson (1992).

Experimental Design

Role of palm oil and palm oil extract pre-treatment were investigated by carrying out toxicity studies following acute cadmium exposures. The dose of the palm oil and palm oil extracts used was based on an established lethal dose of undiluted palm oil greater than 5gKg-1 body weight and available data in literature on the time dependent toxicological effects of cadmium intoxication in rats (Johnson and Slaga, 2000; ASTDR, 2008).

A total of seventy-two male rats with an average weight of 180-200g were obtained from the animal house of Emma Maria Research Laboratory and Consultancy Abraka Nigeria. These rats were allowed to acclimatize for two weeks and eventually distributed randomly into six groups of twelve rats each. The rats in group A served as the control and were neither treated with palm oil nor administered cadmium. The rats in group B were neither treated with palm oil but exposed to a single dose of 20mgKg-1 body weight of cadmium chloride on the 29th day of the experiment. Rats in Groups C-F were treated with 5mlKg-1 body weight of crude palm oil and the various fractions of palm oil for a period of 28 days as follows. (Group C= Crude palm oil (CPO); Group D= Silica Gel Extract (SGE); Group E= Unsaponifiable Extract (UPE) ; Group F = Bleached Extract (BE). On the 29th day of the experiment, these rats were exposed to a single dose of 20mgKg-1 body weight of cadmium chloride by gavage and four animals from each group were sacrificed at intervals of 12hrs, 24hrs and 48hrs.

Sample Collection and Preparation

Following animal sacrifice at the various study periods and timing, blood samples were collected using hypodermic syringe and needle after dissection and cardiac puncture. The tissues (liver, kidney, heart, brain and muscle) were harvested weighed and transferred immediately into labelled containers while the blood samples were transferred into labelled plain tubes. The serum was then collected by centrifugation of the clothed blood at 3″,000g and stored in the refrigerator at -40C. The tissues already collected were further homogenated using pre-chilled mortar and pestle in cold normal saline solution. These were also spurn at 5000g for 10 minutes and the supernatants collected and stored in the refrigerator at -40C.

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Determination of Lipid Peroxidation

A breakdown product of lipid peroxidation thiobarbitoric acid reactive substance (TBARS) was measured in the tissue homogenates by the method of Gutteridge and Wilkins (1982). The principle of this assay was based on the ability of Malondialdehyde (MDA) formed from the breakdown of polyunsaturated fatty acid (PUFA) to serve as a convenient index for the determination of the extent of peroxidation reaction. Malondialdehyde was identified with the barbituric acid to give a red species which was assayed at 532nm.

Determination of Reduced Glutathione

The reduced glutathione was estimated in serum and tissue homogenates using the method of Ellman (1959). To 0.5ml of tissue homogenate was added 2ml 10% trichloroacetic acid and centrifuged. The supernatant (1 ml) was treated with 0.5ml of Ellman’s reagent and 3ml of phosphate buffer. The colour developed was read at 412nm. A series of standard was treated in similar manner along with a blank containing 3.5ml of buffer.

Calculation:

The concentration of reduced GSH in μmole of GSH/0.5g of wet tissue was extrapolated from a standard curve that was plotted (See Appendix I).

Assay for Superoxide Dismutase (SOD)

The activity of SOD in the tissue homogenates was estimated spectrophotometrically using an earlier published method of Misra and Fredorich (1972). The principle of the assay was based on an indirect method that depends on the inhibitory effect of SOD in the initial rate of epinephrine autooxidation. One unit of SOD was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of epinephrine to adrenochrome within 60 seconds.

Assay for Catalase

The activity of catalase was determined in the serum and tissue homogenates by the method of Cohen et al. (1972). The principle of the assay is based on the ability of excess potassium permanganate added to the reaction mixture while the residual unreacted permangantate is measured spectrophotometrically. It has been shown that the decomposition of hydrogen peroxide by catalase follows first order kinetics (Haber and Weiss, 1934).

Method of Statistical Analysis

Data was analysed using the computer software the statistical package for social science version 21 (SPSS 21). The simple analysis of variance (ANOVA) was used while multiple comparisons across groups were done using Turkey HSD analysis at a significant level of p<0.05

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