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BIOCHEMICAL EFFECTS OF INGESTED CRUDE
OIL CONTAMINATED DIETS ON WISTAR ALBINO RATS
CHAPTER ONE INTRODUCTION
1.1 Background of the study
Crude oil has been described as a complex mixture of over 6,000 potentially different hydrocarbons and metals (Edwards, 1989). The behaviour of crude oil is determined by their chemistry. The main constituents of crude oil can be grouped into several broad classes of compounds: saturates (including waxes), aromatics, resins and asphaltenes. Saturates are alkanes with structures of CnH2n+2 (aliphatics) or CnH2n in the case of cyclic saturates (alicyclics).
Aromatics are compounds that have at least one benzene ring as part of their chemical structure. Resins and asphaltenes are similar in many ways such that asphaltenes can be thought of as large resins. Both groups are thought to be composed of condensed aromatic nuclei which may carry alkyl and alicyclic systems containing hetero-atoms such as nitrogen, sulphur and oxygen. Metals such as nickel, vanadium and iron are also associated with asphaltenes (Zedeck, 1980).
The petroleum industry often characterizes crude oil according to their geographical source. Crude oil from different geographical areas have their own unique properties; they can vary in consistency from a light volatile fluid to a semi-solid. Classification of crude oil types by geographical source is generally a useful classification scheme because general toxicity, physical state and changes that occur with time and weathering are primary considerations. During an oil spill incident, the classification scheme provided below is more useful in a response scenario:
- Class A: Light, volatile oil. These oils are highly fluid, often clear; they spread rapidly on solid or water surfaces, have a strong odor, a high evaporation rate and are usually flammable. They penetrate porous surfaces such as dirt and sand and may be persistent in such a matrix. They do not tend to adhere to surfaces and flushing with water generally removes them. Class A oils may be highly toxic to humans, fish and other biota.
- Class B: Non-sticky oil. These oils have waxy or oily feel. Class B oils are less toxic and adhere more firmly to surfaces than class A oils although they can be removed from surfaces by vigorous flushing. As temperature rises, their tendency to penetrate porous substances increases and they can be persistent.
- Class C: Heavy, sticky oil. Class C oils are characteristically viscous, sticky or tarry and brown or black. Flushing with water will not readily remove this material from surfaces but the oil does not readily penetrate porous surfaces. Toxicity is low, but wildlife can be smothered or drowned when contaminated.
- Class D: Non fluid oils. Class D oils are relatively non-toxic, do not penetrate porous substances and are usually black or dark brown in color.
These classifications are dynamic for spilled oils. Weather conditions and water temperature greatly influence the behaviour of crude oil and refined petroleum products in the environment. For example, as volatiles evaporate from a class B oil, it may become a class C oil. If a significant temperature drop occurs (example at night), a class C oil may solidify and resemble a class D oil. Upon warming, the class D oil may revert back to a class C oil.
Nigeria is a major petroleum producing country. One drastic effect associated with the exploration of crude oil is the contamination of the immediate environment after a spill incident (Amadi et al., 1993). Moreover, exposure of workers in their places of work and some cultural practices which involve the use of crude oil do not only contaminate the environment but also endanger lives (Orisakwe et al., 2000).
Over the years, the local population has used crude oil or the fractions for the treatment of various ailments such as gastrointestinal disorders, burns, foot rot, leg ulcers, poisoning and even witchcraft. The route of administration is mostly oral and by external application for burns and wounds (Orisakwe et al., 2000).
Oil spills pose a major threat to the environment in Nigeria where about fifty percent (50%) of oil spills is due to corrosion, twenty eight percent (28%) to sabotage and twenty one percent (21%) to oil production operations. One percent (1%) of oil spills is due to engineering drills, inability to effectively control oil wells, failure of machines and inadequate care in loading and unloading of oil vessels (Akpofure et al., 2003).
During any oil spill incident, the properties of the spilled oil must be known immediately. The experts who respond to oil spill incidents consider all the properties when evaluating spilled oils even though, sometimes not with the urgency required. These properties include to what extent or at what rate the oils will evaporate; the viscosity of the oil at ambient temperatures as it evaporates; if the oil is likely to sink or submerge; if dispersion can be enhanced with chemicals; if emulsions will form; the health hazard to on-site personnel from volatile organic compounds and the toxicity to marine or aquatic organisms.
Small saturates (< C18) are the most dispersible components of oils. Large saturates (waxes) can produce anomalous evaporation, dispersion, emulsification and flow behaviours. The small aromatics (one and two rings) are fairly soluble in water but also evaporate rapidly from spilled crude oil. Larger aromatics show neither of these behaviours to any extent. Asphaltenes and metals do not appreciably evaporate, disperse or degrade but stabilize as water-in-oil emulsions when present in quantities greater than 3% (Fingas and Brown, 2000).
The harmful effects of oil spills on the environment are many. Oil kills plants and animals in the estuarine zone, settle on beaches and kill organisms that live there. It also settles on ocean floor and kills benthic (bottom-dwelling) organisms such as crabs. Oil poisons algae, disrupts major food chains and decreases the yield of edible crustaceans. It also coats birds’ feathers, impairing their flight or reducing the insulative properties of their feathers thus making the birds more vulnerable to cold. Oil endangers fish hatcheries in coastal waters and as well as contaminates the flesh of commercially valuable fish (Val and Almeida-Val, 1999).
Most of the land in oil producing areas of Nigeria are used for cultivation because the main occupation of people living in the region are farming and fishing (Egborge, 1991). In land used for agricultural purposes, petroleum or diesel contaminated wastes and accidental spills of crude oil at some drilling sites pose exposure risks for occupational public, livestock and wildlife (Khan et al., 2001). The petroleum hydrocarbons can eventually get into man and animals through ingestion of contaminated food or bioconcentration through the food chain (Jessup and Leighton, 1996).
The ingestion of petroleum has been reported to induce oxidative stress (Val and AlmeidaVal, 1999) through the generation of free radicals (Achuba and Osakwe, 2003). It has been established that free radical generation with subsequent oxidative modification leads to lipid peroxidation (Halliwell, 1994) that damages critical cellular macromolecules such as DNA, lipids and proteins (Breimer, 1990; Romero et al., 1998; Souza et al., 1999) and results in inactivation of antioxidant enzymes (Pigeolet et al., 1990).
1.1 Crude Oil
Petroleum (“rock oil”)is a naturally occurring, flammable liquid found in rock formations in the Earth consisting of a complex mixture of hydrocarbons of various molecular weights, plus other organic compounds (Speight, 1999).
1.1.1 Composition of Crude Oil
The proportion of hydrocarbons in the mixture is highly variable and ranges from as much as 97% by weight in the lighter oils to as little as 50% in the heavier oils and bitumens. The hydrocarbons in crude oil are mostly alkanes, cycloalkanes and various aromatic hydrocarbons while the other organic compounds contain nitrogen, oxygen and sulfur, and trace amounts of metals such as iron, nickel, copper and vanadium. The exact molecular composition varies widely from formation to formation but the proportion of chemical elements varies over fairly narrow limits as follows (Speight, 1999).
Table 1.1: Composition of crude oil
| Carbon | 83.0 – 87.0% |
| Hydrogen | 10.0 – 14.0% |
| Nitrogen | 0.1 – 2.0% |
| Oxygen | 0.1 – 1.5% |
| Sulfur | 0.5-6.0% |
| Metals | <1000 ppm |
Crude oil varies greatly in appearance depending on its composition. It is usually black or dark brown (although it may be yellowish or even greenish). In the reservoir it is usually found in association with natural gas, which being lighter forms a gas cap over the petroleum, and saline water which being heavier generally floats underneath it. Crude oil may also be found in semi-solid form mixed with sand, as in the Athabasca oil sands in Canada, where it may be referred to as crude bitumen (Glasby, 2006).
Petroleum is used mostly for producing fuel oil and gasoline (petrol), both important “primary energy” sources (Glasby, 2006). About 84% by volume of the hydrocarbons present in petroleum is converted into energy-rich fuels (petroleum-based fuels), including gasoline, diesel, jet, heating, and other fuel oils, and liquefied petroleum gas (Robison, 2006).
Due to its high energy density, easy transportability and relative abundance, crude oil has become the world’s most important source of energy since the mid-1950s. Petroleum is also the raw material for many chemical products, including pharmaceuticals, solvents, fertilizers, pesticides, and plastics; the 16% not used for energy production is converted into these other materials. Petroleum is found in porous rock formations in the upper strata of some areas of the Earth’s crust. Known reserves of petroleum are typically estimated at around 190 km3 (1.2 trillion (short scale) barrels) without oil sands, or 595 km3 (3.74 trillion barrels) with oil sands. Utilization is currently around 84 million barrels (13.4×106 m3) per day, or 4.9 km3 per year. Because the energy return over energy invested (EROEI) ratio of oil is constantly falling as petroleum recovery gets more difficult, recoverable oil reserves are significantly less than total oil-in-place. At current utilization levels, and assuming that oil will be used only from reservoirs, known recoverable reserves would be gone around 2039, potentially leading to a global energy crisis. However, there are factors which may extend or reduce this estimate, including the rapidly increasing demand for petroleum in China, India, and other developing nations; new discoveries; energy conservation and use of alternative energy sources and new economically viable exploitation of non–conventional oil sources.
Petroleum is a mixture of a very large number of different hydrocarbons; the most commonly found molecules are alkanes (linear or branched), cycloalkanes, aromatic hydrocarbons, or more complicated chemicals like asphaltenes. Each petroleum variety has a unique mix of molecules, which define its physical and chemical properties, like color and viscosity (Robison, 2006).
The alkanes, also known as paraffins, are saturated hydrocarbons with straight or branched chains which contain only carbon and hydrogen and have the general formula CnH2n+2 They generally have from 5 to 40 carbon atoms per molecule, although trace amounts of shorter or longer molecules may be present in the mixture. The alkanes from pentane (C5H12) to octane (C8H18) are refined into gasoline (petrol), the ones from nonane (C9H20) to hexadecane (C16H34) into diesel fuel and kerosene (primary component of many types of jet fuel), and the ones from hexadecane upwards into fuel oil and lubricating oil. At the heavier end of the range, paraffin wax is an alkane with approximately 25 carbon atoms, while asphalt has 35 and up, although these are usually cracked by modern refineries into more valuable products. Any shorter hydrocarbons are considered natural gas or natural gas liquids. The cycloalkanes, also known as napthenes, are saturated hydrocarbons which have one or more carbon rings to which hydrogen atoms are attached according to the formula CnH2n. Cycloalkanes have similar properties to alkanes but have higher boiling points.
The aromatic hydrocarbons are unsaturated hydrocarbons which have one or more planar six-carbon rings called benzene rings, to which hydrogen atoms are attached with the formula CnHn. They tend to burn with a sooty flame, and many have a sweet aroma. These different molecules are separated by fractional distillation at an oil refinery to produce gasoline, jet fuel, kerosene, and other hydrocarbons. For example 2,2,4–trimethylpentane (isooctane), widely used in gasoline, has a chemical formula of C8H18 and it reacts with oxygen exothermically (Robison, 2006).
The amount of various molecules in an oil sample can be determined in laboratory. The molecules are typically extracted in a solvent, then separated in a gas chromatograph, and finally determined with a suitable detector, such as a flame ionization detector or a mass spectrophotometer (Robison, 2006).
1.1.2 Classification of Crude Oil
The petroleum industry generally classifies crude oil by the geographic location it is produced in (e.g. West Texas, Brent, or Oman), its API (Advanced Photonix Incorporated) gravity (an oil industry measure of density), and by its sulfur content. Crude oil may be considered light if it has low density or heavy if it has high density; and it may be referred to as sweet if it contains relatively little sulfur or sour if it contains substantial amounts of sulfur (Glasby, 2006).
The geographic location is important because it affects transportation costs to the refinery. Light crude oil is more desirable than heavy oil since it produces a higher yield of gasoline, while sweet oil commands a higher price than sour oil because it has fewer environmental problems and requires less refining to meet sulfur standards imposed on fuels in consuming countries. Each crude oil has unique molecular characteristics which are understood by the use of crude oil assay analysis in petroleum laboratories (Glasby, 2006).
Barrels from an area in which the crude oil’s molecular characteristics have been determined and the oil has been classified are used as pricing references throughout the world. Some of the common reference crude oils are:
West Texas Intermediate (WTI), a very high-quality, sweet, light oil delivered at Cushing, Oklahoma for North American oil
Brent Blend, comprising 15 oils from fields in the Brent and Ninian systems in the East Shetland Basin of the North Sea. The oil is landed at Sullom Voe terminal in the Shetlands. Oil production from Europe, Africa and Middle Eastern oil flowing West tends to be priced off the price of this oil, which forms a benchmark
Dubai–Oman, used as benchmark for Middle East sour crude oil flowing to the Asia–Pacific region
Tapis (from Malaysia, used as a reference for light Far East oil)
Minas (from Indonesia, used as a reference for heavy Far East oil)
The Organization of Petroleum Exporting Countries (OPEC) Reference Basket, a weighted average of oil blends from various OPEC countries.
There are declining amounts of these benchmark oils being produced each year, so other oils are more commonly what is actually delivered. While the reference price may be for West Texas Intermediate delivered at Cushing, the actual oil being traded may be discounted Canadian heavy oil delivered at Hardisty, Alberta. For a Brent Blend delivered at the Shetlands, it may be a Russian Export Blend delivered at the port of Primorsk (Robison, 2006).
1.1.3 Uses of Crude Oil
The chemical structure of petroleum is composed of hydrocarbon chains of different lengths. Because of this, petroleum may be taken to oil refineries and the hydrocarbon chemicals separated by distillation and treated by other chemical processes, to be used for a variety of purposes. See Petroleum products (Joseph and Gordon, 2008).
The most common distillation fractions of petroleum include:
Ethane and other short-chain alkanes
Diesel fuel (petrodiesel)
Liquefied petroleum gas (LPG)
Natural gas.
Other derivatives:-
Certain types of resultant hydrocarbons may be mixed with other non-hydrocarbons, to create other end products:
Alkenes (olefins) which can be manufactured into plastics or other compounds
Lubricants (produces light machine oils, motor oils, and greases, adding viscosity stabilizers as required).
Wax, used in the packaging of frozen foods, among others.
Sulfur or Sulfuric acid. These are useful industrial materials. Sulfuric acid is usually prepared as the acid precursor oleum, a byproduct of sulfur removal from fuels.
Petroleum coke, used in speciality carbon products or as solid fuel.
Aromatic petrochemicals to be used as precursors in other chemical production.
1.1.4 Incidents and Environmental Impact/Effect
Crude oil and refined fuel spills from tanker ship accidents have damaged natural ecosystems in Alaska, the Galapagos Islands, France and many other places. The quantity of oil spilled during most accidents has ranged from a few hundred tons to several hundred thousand tons (Atlantic Empress, Amoco Cadiz). Oil spills have already proven to have a great impact on ecosystems such as the Exxon Valdez oil spill. Oil spills at sea are generally much more damaging than those on land, since they can spread for hundreds of nautical miles in a thin oil slick which can cover beaches with a thin coating of oil. This can kill sea birds, mammals, shellfish and other organisms it coats. Oil spills on land are more readily containable if a makeshift earth dam can be rapidly bulldozed around the spill site before most of the oil escapes, and land animals can avoid the oil more easily.
Control of oil spills is difficult; requires ad hoc methods, and often a large amount of manpower. The dropping of bombs and incendiary devices from aircraft on the Torrey Canyon wreck produced poor results (Speight, 1999). Modern techniques would include pumping the oil from the wreck, like in the Prestige oil spill or the Erika oil spill (Speight, 1999).
1.2 BIOCHEMICAL END-POINT MARKERS
Biochemical biomarkers are increasingly being used in ecological risk assessment of the ecosystem to identify the incidence of exposure and effects caused by xenobiotics (Olsen et al., 2001). The most powerful tools for the investigation of pollutants ―in situ‖ are biomarkers. Ideally, biomarkers will identify effects at sub-cellular level before they manifest at higher levels of biological organization (McCarthy and Shugart, 1990). The use of biomarkers in environmental monitoring is now becoming a routine method for examining toxicity of chemicals (Onwurah, 1999; Shertzer et al., 1994; Bronk and Gores, 1999; Anozie and Onwurah, 2001; Khan et al., 2001).
Biomarkers can provide information on the potential adverse impacts of contaminants and also act as early warning signals of impending environmental damage. Such biomarkers include enzymes such as acetyl cholinesterase (ACHE), glutathione S-transferase (GST), superoxide dismutase (SOD) and several others (Olsen et al., 2001). In the past, a variety of cellular biochemical parameters have been related to occurrence of specific diseases resulting from exposure to pollutants or to various classes of pollutants. For example, a rise in mixed function oxidase (MFO) in the liver of experimental animals taken from a polluted area may signify pollution exposure (Depledge, 1993).
Recently, the biomarker concept has been extended from purely biochemical measurement to include those of cellular pathology, physiological processes and even behaviour of organisms exposed to varying concentrations of pollutants (Sanders et al., 1991). Biomarkers may also have an important role in unraveling the interactions between natural environmental stressors (e.g. hypoxia, thermal and salinity stress) and pollutants as well as the effect of mixtures of contaminants in areas exposed to several pollutants (Livingstone et al., 1988).
1.3 Lipid peroxidation
Lipid peroxidation refers to the oxidative degradation of lipids. It is the process whereby free radicals “steal” electrons from the lipids in cell membranes, resulting in cell damage. This process proceeds by a free radical chain reaction mechanism. It most often affects polyunsaturated fatty acids, because they contain multiple double bonds which lie in between the methylene -CH2– groups that possess especially reactive hydrogens. As with any radical reaction the reaction consists of three major steps: initiation, propagation and termination (Sies, 1997).
1.3.1 Mechanism of Lipid Peroxidation
1.3.1.1 Initiation
Initiation is the step whereby a fatty acid radical is produced. The initiators in living cells are most notably reactive oxygen species (ROS), such as OH·, which combines with a hydrogen atom to make water and a fatty acid radical (Aruoma, 1993).
1.3.1.2 Propagation
The fatty acid radical is not a very stable molecule, so it reacts readily with molecular oxygen, thereby creating a peroxyl-fatty acid radical. This too is an unstable species that reacts with another free fatty acid producing a different fatty acid radical and a hydrogen peroxide or a cyclic peroxide if it had reacted with itself. This cycle continues as the new fatty acid radical reacts in the same way.
1.3.1.3 Termination
When a radical reacts it always produces another radical, which is why the process is called a “chain reaction mechanism.” The radical reaction stops when two radicals react and produce a non-radical species. This happens only when the concentration of radical species is high enough for there to be a high probability of two radicals actually colliding. Living organisms have evolved different molecules that speed up termination by quenching free radicals and therefore protect the cell membrane. One such important antioxidant is alpha–tocopherol, also known as vitamin E. Other anti-oxidants made within the body include the enzymes superoxide dismutase, catalase, and peroxidase (Sies, 1997).
1.3.1.4 Hazards
Lipid peroxidation if not terminated fast enough will damage the cell membranes which consist mainly of lipids. Phototherapy may cause haemolysis by rupturing red blood cell membranes in this way (Sies, 1997). In addition, end products of lipid peroxidation may be mutagenic and carcinogenic. For instance, the end product malondialdehyde reacts with deoxyadenosine and deoxyguanosine in DNA, forming DNA adducts to them (Sies, 1997).
1.3.1.5 Measurement or Assay for Lipid Peroxidation
Free radicals have a very short half-life, which makes them very hard to measure in the laboratory. Multiple methods of measurement are available today, each with their own benefits and limits. Radicals can be measured using electron spin resonance and spin trapping methods. Exogenous compounds with a high affinity for free radicals (i.e. xenobiotics) are utilized in the spin techniques. The compound and radical together form a stable entity that can be easily measured. This indirect approach has been termed “fingerprinting.” (Karlsson, 1997). However, this method is not 100% accurate. Spintrapping collection techniques have poor sensitivity, which can skew results (Acworth and Bailey, 1997). Commonly used alternate approach measures markers of free radicals rather than the actual radical. These markers of oxidative stress are measured using a variety of different assays. These assays are described below. When a fatty acid is peroxidized it is broken down into aldehydes, which are excreted. Aldehydes such as thiobarbituric acid reacting substances (TBARS) have been widely accepted as a general marker of free radical production (Clarkson, 1995). The most commonly measured TBARS is malondialdehyde (MDA) (Karlsson, 1997). The TBA test has been challenged because of its lack of specificity, sensitivity, and reproducibility. The use of liquid chromatography instead of spectrophotometer techniques help reduce these errors (Wong et al., 1987). In addition, the test seems to work best when applied to membrane systems such as microsomes (Halliwell and Chirico, 1993). Gases such as pentane and ethane are also created as lipid peroxidation occurs. These gases are expired and commonly measured during free radical research (Karlsson, 1997). Dillard et al. (1978) was one of the first to determine that expired pentane increased as Vmax increased. Kanter et al. (1988) has reported that serum MDA levels correlated closely with blood levels of creatine kinase, an indicator of muscle damage. Lastly, conjugated dienes (CD) are often measured as indicators of free radical production. Oxidation of unsaturated fatty acids results in the formation of CD. The CD formed is measured and provide a marker of the early stages of lipid peroxidation (Halliwell and Gutteridge, 1985). A newly developed technique for measuring free radical production shows promise in producing more valid results. The technique uses monoclonal antibodies and may prove to be the most accurate measurement of free radicals. However, until further more reliable techniques are established it is generally accepted that two or more assays be utilized whenever possible to enhance validity (Halliwell and Gutteridge, 1985).
1.4 ANTIOXIDANT DEFENCE
Gutheridge (1994) defined antioxidant as any substance that when present in low concentrations compared to those of oxidizable substrate, significantly delays or inhibits oxidation of that substrate. They are a complex and diverse group of molecules that protect key biological sites from oxidative damage. They usually act at several stages in an oxidative sequence and this can be illustrated by considering lipid rich products.
There is major evidence that our bodies are protected from free radical-induced oxidative damage by various antioxidant enzymes and/or nutrients e.g. glutathione S-transferase, catalase, superoxide dismutase, etc. These enzymes constitute a defense system independently, co-operatively or even synergistically (Etsuo, 1993).
According to Gutheridge (1994), antioxidants can act in many ways, namely:
- By removing oxygen or decreasing local oxygen concentrations.
- By removing catalytic metal ions.
- Removing key reactive oxygen species (ROS) such as superoxide and hydrogen peroxide.
- Scavenging the initiation of free radicals such as hydroxyl, alkoxyl and peroxyl species.
- Breaking the chain of an initiated sequence.
- Quenching or scavenging singlet oxygen (1O2) which is an example of a reactive non-radical.
Mammalian cells posses elaborated defense mechanisms to detoxify radicals. The key metabolic steps are SOD catalysis of the dismutation of superoxide to hydrogen peroxide and oxygen radical and the conversion of H2O2 to 2H2O by glutathione peroxidase or to O2 and H2O by catalase. The following reactions depict the protective activities of some antioxidant enzymes:
2O2 + 2H+ Superoxide dismutase O2 + H2O2
2H2O2 Catalase O2 + 2H2O2
H2O2 + AH 2H2O + A
Finally, radical scavenging antioxidants and/ or nutrients, (e.g. Vitamin E), interrupt the chain reactions by capturing the radicals; the Vitamin E radical is relatively stable and it can be enzymatically converted back to its non-radical form. Radical scavengers thus terminate the chain reaction of radical damage. The potential significance of these ROS defense mechanism is apparent from considerations of the whole body and sub-cellular distribution of the different components. The enzymes (SOD, Catalase, GSH-peroxidase and GST) and the nutrients/substrates (GSH) tend to be in higher concentrations in locations where ROS damage is more likely and potentially more damaging (Moslen, 1994).
Lactoferrin Radical Scavengers
2O H2O + O2
Fig. 1.1:Defense Mechanism Against Cellular Damage by ROS
1.4.1 Superoxide dismutase (SOD)
The enzyme superoxide dismutase (SOD, EC 1.15.1.1), catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. One of the exceedingly rare exceptions is Lactobacillus plantarum and related lactobacilli, which use a different mechanism (Corpas et al., 2001).
1.4.2 Reactions of Superoxide dismutase
SOD was discovered by Irwin Fridovich and Joe McCord which, prior were known as several metalloproteins with unknown function. For example, CuZnSOD was known as erythrocuprein (Corpas et al., 2001). Several common forms of SOD exist: they are proteins cofactored with copper and zinc, or manganese, iron, or nickel.
Superoxide dismutase has been identified as an indophenol oxidase by protein analysis of starch gels using the phenazine-tetrazolium technique. This enzyme was detected in several human tissues as indophenol oxidase A (IPO-A). After that Brewer observed an electrophoretic variant of IPO-A, which he called ‘Morenci,’ in 3 generations of a family with presumed male-to-male transmission. Using a RT-PCR analysis Brewer has identified 5 splice variants of SOD1. The variants were expressed in brain, a region involved in amyotrophic lateral sclerosis (Peskin and Winterbourn, 2000).
The cytosols of virtually all eukaryotic cells contain an SOD enzyme with copper and zinc (Cu-Zn-SOD). For example, Cu-Zn-SOD available commercially is normally purified from the bovine erythrocytes: PDB 1SXA, EC 1.15.1.1. The CuZn enzyme is a homodimer of molecular weight 32,500. The two subunits are joined primarily by hydrophobic and electrostatic interactions. The ligands of copper and zinc are histidine side chains.
Chicken liver (and nearly all other) mitochondria, and many bacteria (such as E. coli) contain a form with manganese (Mn-SOD). (For example, the Mn-SOD found in a human mitochondrion: PDB 1N0J, EC 1.15.1.1). The ligands of the manganese ions are 3 histidine side chains, an aspartate side chain and a water molecule or hydroxy ligand depending on the Mn oxidation state (respectively II and III) (Seguí et al., 2004).
E. coli and many other bacteria also contain a form of the enzyme with iron (Fe-SOD); some bacteria contain Fe-SOD, others Mn-SOD, and some contain both. (For the E. coli Fe-SOD: PDB 1ISA, EC 1.15.1.1). Fe-SOD can be found in the plastids of plants. The active sites of Mn and Fe superoxide dismutases contain the same type of amino acid side chains. In higher plants, SOD isozymes have been localized in different cell compartments. Mn-SOD is present in mitochondria and peroxisomes. Fe-SOD has been found mainly in chloroplasts but has also been detected in peroxisomes, and CuZn-SOD has been localized in cytosol, chloroplasts, peroxisomes and apoplast (Corpas et al., 2001).
In humans, three forms of superoxide dismutase are present. SOD1 is located in the cytoplasm, SOD2 in the mitochondria and SOD3 is extracellular. The first is a dimer (consists of two units), while the others are tetramers (four subunits). SOD1 and SOD3 contain copper and zinc, while SOD2 has manganese in its reactive centre. The genes are located on chromosomes 21, 6 and 4, respectively (21q22.1, 6q25.3 and 4p15.3-p15.1) (Seguí et al., 2004).
1.4.3 Biochemistry of Superoxide dismutase
Simply-stated, SOD outcompetes damaging reactions of superoxide, thus protecting the cell from superoxide toxicity. The reaction of superoxide with non-radicals is spin forbidden. In biological systems, this means its main reactions are with itself (dismutation) or with another biological radical such as nitric oxide (NO). The superoxide anion radical (O2–) spontaneously dismutes to O2 and hydrogen peroxide (H2O2) quite rapidly (~105 M-1 s-1 at pH 7). SOD is biologically necessary because superoxide reacts even faster with certain targets such as NO radical, which makes peroxynitrite. Similarly, the dismutation rate is second order with respect to initial superoxide concentration. Thus, the half-life of superoxide, although very short at high concentrations (e.g. 0.05 seconds at 0.1mM) is actually quite long at low concentrations (e.g. 14 hours at 0.1 nM). In contrast, the reaction of superoxide with SOD is first order with respect to superoxide concentration. Moreover, superoxide dismutase has the fastest turnover number (reaction rate with its substrate) of any known enzyme (~109 M-1 s-1), this reaction being only limited by the frequency of collision between itself and superoxide. That is, the reaction rate is “diffusion limited” (Seguí et al., 2004).
1.4.4 Physiology of Superoxide dismutase
Superoxide is one of the main reactive oxygen species in the cell and as such, SOD serves a key antioxidant role. The physiological importance of SODs is illustrated by the severe pathologies evident in mice genetically engineered to lack these enzymes. Mice lacking SOD2 die several days after birth, amidst massive oxidative stress (Seguí et al., 2004). Mice lacking SOD1 develop a wide range of pathologies, including hepatocellular carcinoma, an acceleration of age-related muscle mass loss, an earlier incidence of cataracts and a reduced lifespan.. Mice lacking SOD3 do not show any obvious defects and exhibit a normal lifespan (Campana, 2004).
1.4.5 Role of Superoxide Dismutase in Disease
Mutations in the first SOD enzyme (SOD1) have been linked to familial amyotrophic lateral sclerosis (ALS, a form of motor neuron disease). The other two types have not been linked to any human diseases, however, in mice inactivation of SOD2 causes perinatal lethality (Seguí et al., 2004) and inactivation of SOD1 causes hepatocellular carcinoma. Mutations in SOD1 can cause familial ALS, by a mechanism that is presently not understood, but not due to loss of enzymatic activity or a decrease in the conformational stability of the SOD1 protein. Overexpression of SOD1 has been linked to Down’s syndrome (Campana, 2004). The veterinary antiinflammatory drug “Orgotein” is purified bovine liver superoxide dismutase.
SOD has proved to be highly effective in treatment of colonic inflammation in experimental colitis. Treatment with SOD decreases reactive oxygen species generation and oxidative stress and thus, inhibits endothelial activation and indicate that modulation of factors that govern adhesion molecule expression and leukocyte-endothelial interactions, such as antioxidants, may be important, new tools for the treatment of inflammatory bowel disease (Vozenin-Brotons et al., 2001).
1.5 Blood Protein
Blood proteins, also called serum proteins, are proteins found in blood plasma (Anderson and Anderson, 1977). They serve many different functions including circulatory transport molecules for lipids, hormones, vitamins and metals. They also serve as enzymes, complement components, protease inhibitors, and kinin precursors. Serum proteins play an important role in the regulation of acellular activity and functioning and in the immune system. Separating serum proteins by electrophoresis is a valuable diagnostic tool as well as a way to monitor clinical progress (Jacobs, 2005).
1.5.1 Types of Blood Proteins:
Albumins– Normal level 3.5-5.0g/dl
Fibrinogens – Normal level 200-450mg/dl
Regulatory
All the plasma proteins are synthesized in liver except gamma globulins. Globulins are of three types- alpha,beta and gamma (Anderson and Anderson, 1977).
alpha1-antiproteinase alpha2-macroglobulin
Other types of blood proteins include:
- Lipoproteins (chylomicrons, VLDL, LDL, HDL)
- Transferrin
- Prothrombin
Sixty percent (60%) of plasma proteins are made up of the protein albumin, which are major contributors to osmotic pressure of plasma which assists in the transport of lipids and steroid hormones. Globulins make up 35% of plasma proteins and are used in the transport of ions, hormones and lipids assisting in immune function. 4% is fibrinogen and this is essential in the clotting of blood and can be converted into insoluble fibrin (Anderson and Anderson, 1977). Regulatory proteins which make up less than 1% of plasma proteins are proteins such as enzymes, proenzymes and hormones (Jacobs, 2005). Current research regarding blood plasma proteins is centered on performing proteomics analyses of serum/plasma in the search for biomarkers. These efforts started with twodimensional gel electrophoresis (Anderson and Anderson, 1977) efforts in the 1970s and in more recent times this research has been performed using LC–tandem MS based proteomics (Adkins, 2002).
1.6 Cholesterol
Cholesterol is a lipid found in the cell membranes of all animal tissues, and is transported in the blood plasma of all animals. Cholesterol is also a sterol (a combination of steroid and alcohol). Because cholesterol is synthesized by all eukaryotes, trace amounts of cholesterol are also found in membranes of plants and fungi. The name originates from the Greek chole- (bile) and stereos (solid), and the chemical suffix -ol for an alcohol. Researchers first identified cholesterol in solid form in gallstones by François Poulletier de la Salle in 1769. However, it was only in 1815 that chemist Eugène Chevreul named the compound “cholesterine” (Olsen, 2001).
Most of the cholesterol in the body is synthesized by the body and some has dietary origin. Cholesterol is more abundant in tissues which either synthesize more or have more abundant densely-packed membranes, for example, the liver, spinal cord and brain. It plays a central role in many biochemical processes, such as the composition of cell membranes and the synthesis of steroid hormones (Stryer, 1995).
Since cholesterol is insoluble in blood, it is transported in the circulatory system within lipoproteins, complex spherical particles which have an exterior composed mainly of water-soluble proteins; fats and cholesterol are carried internally. There is a large range of lipoproteins within the blood, generally called, from larger to smaller size, chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL). The cholesterol within all the various lipoproteins is identical.
According to the lipid hypothesis, abnormally high cholesterol levels (hypercholesterolemia), or, more correctly, higher concentrations of LDL and lower concentrations of functional HDL are strongly associated with cardiovascular disease because they promote atheroma development in arteries (atherosclerosis). This disease process leads to myocardial infarction (heart attack), stroke and peripheral vascular disease. Since higher blood LDL, especially higher LDL particle concentrations and smaller LDL particle size, contribute to this process, more than the cholesterol content of the LDL particles (Brunzell, 2008), LDL particles are often termed “bad cholesterol” because they have been linked to atheroma formation. On the other hand, high concentrations of functional HDL, which can remove cholesterol from cells and atheroma, offer protection and are sometimes referred to colloquially as “good cholesterol”. These balances are mostly genetically determined but can be changed by body build, medications, food choices and other factors (Durrington, 2003).
Fig. 1.2: Structure of cholesterol.
1.6.1 Functions of Cholesterol
Cholesterol is required to build and maintain cell membranes; it regulates membrane fluidity over a wide range of temperatures. The hydroxyl group on cholesterol interacts with the polar head groups of the membrane phospho– and sphingolipids, while the bulky steroid and the hydrocarbon chain is embedded in the membrane, alongside the nonpolar fatty acid chains of the other lipids. Some research indicates that cholesterol may act as an antioxidant (Smith, 1991). Cholesterol also aids in the manufacture of bile (which is stored in the gallbladder and helps digest fats), and is also important for the metabolism of fat soluble vitamins, including vitamins A, D, E and K. It is the major precursor for the synthesis of vitamin D and of the various steroid hormones (which include cortisol and aldosterone in the adrenal glands, and the sex hormones progesterone, the various estrogens, testosterone, and derivatives). Recently, cholesterol has also been implicated in cell signaling processes, where it has been suggested that it assists in the formation of lipid rafts in the plasma membrane. It also reduces the permeability of the plasma membrane to hydrogen ions (protons) and sodium ions (Haines, 2001).
1.6.2 Excretion
Cholesterol is excreted from the liver in bile and reabsorbed from the intestines. Under certain circumstances, when more concentrated, as in the gallbladder, it crystallises and is the major constituent of most gallstones, although lecithin and bilirubin gallstones also occur less frequently (Brown and Goldstein, 1997). Cholesterol is minimally soluble in water; it cannot dissolve and travel in the water-based bloodstream. Instead, it is transported in the bloodstream by lipoproteins—protein “molecular-suitcases” that are water-soluble and carry cholesterol and triglycerides internally (Van der Steeg, 2008). The apolipoproteins forming the surface of the given lipoprotein particle determine from what cells cholesterol will be removed and to where it will be supplied (Brown and Goldstein, 1997).
1.6.3 Food sources
Cholesterol is mostly found in animal fats: all food-containing animal fats contains cholesterol. However, food not containing animal fats also contains cholesterol in varying amounts. Major dietary sources of cholesterol include egg yolks, beef, poultry, and shrimp (Ostlund et al., 2003). Human breast milk also contains significant quantities of cholesterol (Behrman and Gopalan, 2005).
Plants have trace amounts of cholesterol, so even a vegan diet, which includes no animal foods, can have a high amount of cholesterol depending on the diet and oils used. For example, to ingest the amount of cholesterol in one egg yolk, one would need to drink about 9.6 litres (2.1 imp gal/2.5 US gal) of pure peanut oil. However unlike peanut oil, which is considered to contain “good” cholesterol, other oils like coconut oil and palm oil contain as much as, if not more cholesterol than animal fats (Behrman and Gopalan, 2005). Plant products (e.g. flax seeds, peanuts), also contain cholesterol-like compounds, phytosterols, which are suggested to help lower serum cholesterol (Ostlund et al., 2003).
1.7 AIM OF RESEARCH
This present study was undertaken to investigate the risk effects of crude oil on albino rats using biochemical end-point markers such as lipid peroxidation products, superoxide dismutase (SOD) activity, total protein and cholesterol
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