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Sterols are often found in association with fat. All of them have a similar cyclic nucleus resembles the phenanthrene rings( rings A, B, C) to which a cyclopentanone ring is attached. The parent nucleus is better designated as cyclopentano perhydro phenanthrene .( A.C. Deb)
Cholesterol is widely distributed in all the cells of the body. It occurs in animal fats but not in plant fats. Human body contains large quantities of cholesterol in brain and nervous tissues. Other tissues like liver, kidney, spleen and skin contains cholesterol. Quantity of cholesterol will be 140 gms in the body of a man weighing 70 kgs. Greater part of the cholesterol in the body is synthesized where as 0.3 gram per day was provided by the average diet.
Normal concentration of cholesterol in the blood is 140-220 mg per 100 ml of blood. It increases with ages and during pregnancy. Plasma cholesterol levels are positively correlated to the development of atherosclerosis, a process initiated by deposition of excess insoluble sterols in the arterial wall. It exists both in free and ester forms; normally about two-third of total serum cholesterol is esterified.
It is a white, waxy, solid associated with fats and chemically different from them.
It has aparent nucleus which is said to be cuclopentanoperhydro phenanthrene nucleus
It has a hydroxyl group at C3 an unsaturated bond at C5-C6 two methyl groups at C10 and C13 and 8 carbon paraffin sidechains attached to C17
It is an alcohol
It occurs free and combined with fatty acids by ester linkage at the hydroxyl group.
Cholesterol in ester form often reffered to as 'bound' cholesterol esters. These are normally rich in linoleic acid.( A.C.Deb, pp 361)
Physiological importance of cholesterol in the body
It is the essential constituent of cells
Aids in the permeability of the cells
Protects the RBC from haemolysis
Transportation of fats to liver in the form of cholesterol esters for oxidation.
Cholesterol is a key intermediate in the biosynthesis of related sterols such as bile acids, adrenocortical hormones, androgens, estrogens and vitamin D.
It is required structurally for formation of cell membranes and myelin sheats.
It acts as antagonists to phospholipids.
Factors affecting cholesterol level in blood
Dietary fat containing higher saturated fatty acids cause increased serum cholesterol.
Dietary cholesterol increases the serum cholestrol level.
Dietary carbohydrates like sucrose, consumption in excessive amounts causes an increases in serum cholesterol level.
Hereditary persons who are prone to become obese have ahigh level. The level becomes slightly higher in persons belonging to blood group A and AB.
Caloric intake in excess causes a significant increase in plasma cholesterol.
Synthesis of cholesterol
Liver is the principle organ for synthesis. Other tissues like skin, ovary, intestine, testis, are also capable for cholesterol synthesis. The microsomal and cytosol fraction of the cell are useful in the cholesterol synthesis. One interesting point is that brain of new born child can synthesis cholesterol but adult brain cannot.
Body cholesterol is primarily of endogenous origin and its homeostasis involves the movement of cholesterol between peripheral tissues and liver. The liver regulates
1) de novo synthesis of cholesterol
2) Excretion of cholesterol into bile directly or in the form of bile salts
3) Secretion of cholesterol into blood as very low density lipoproteins(VLDL),
4) Modulation of receptor-mediated cholesterol uptake,
5) Formation of CE and the storage of cholesterol.
Intestine regulates cholesterol absorption and excretion through faeces.
Biosynthesis of cholesterol was differentiated into five stages
Six carbon compound, Mevalonate, is synthesized from acetyl - Co A
Formation of Isoprenoid units occurs from mevalonate by the loss of CO2, which are five carbon compounds
Six of these units combine to form a thirty carbon compound named Squalene (C30H50).
Cyclization of this squalene occurs and forms a parent steroid called lanosterol.
After many steps cholesterol( C27H46O) is formed from lanosterol with the loss of three methyl groups.
In normal lipid metabolism, lipids in the diet are converted to triglycerides, which are used in muscle and adipose tissue. Cholesterol is used for the synthesis of steroid hormones and bile acids and by all cells as part of the cell membrane. Lipids do not dissolve easily in water. Therefore, they have to be transported in blood as lipoproteins. Lipoproteins are formed from a protein envelope, made of apoprotein, containing a variable mix of triglycerides and cholesterol. Lipoproteins vary in size,weight and density. Figure 4.8 shows a simplified version of lipid metabolism. Chylomicrons are the largest lipoproteins and they transport lipids and cholesterol absorbed from the small intestine to the liver. The liver can synthesize cholesterol if there is not enough in the diet.Very low-density lipoproteins (VLDLs) are smaller than chylomicrons but still relatively large. They transport lipids and cholesterol from the liver to adipose tissue and muscle where they unload triglycerides and become low-density lipoproteins in the process. Low-density lipoproteins (LDLs) are medium sized and rich in cholesterol. They transport lipids to any cell that needs them. Cell membranes have LDL receptors to which LDLs attach prior to being taken into the cell. The LDLs are broken down in the cell, the lipids used and the receptors temporarily disappear. Any excess LDLs in the circulation are of a size to deposit cholesterol in the walls of damaged arteries. High levels of circulating LDLs are associated with the development of atherosclerosis. High-density lipoproteins (HDLs) are the smallest lipoproteins and are formed by the removal of excess cholesterol from cells and possibly from artery walls, which is then transported back to the liver. This prevents accumulation of cholesterol in the blood. HDLs are not atherogenic because of their small size. Total plasma cholesterol (TC) is the sum of cholesterol carried in all forms of lipoproteins,with about 70% of it being in the LDL form. In the liver, cholesterol can be stored in liver cells, used to form more VLDLs, used to form bile acids or excreted as cholesterol in bile.
Transportation of cholesterol
Cholesterol transport is mainly depends on the lipoproteins. Since lipids are insoluble molecules, they must be packed into lipoproteins for efficient transportation in blood.
Transportation of lipoproteins occurs in two ways
Exogenous pathway in which intestinally derived proteins from dietary lipids.
Endogenous pathway : lipoproteins synthesized in the liver and carrying lipids from peripheral tissues to the liver ( reverse cholesterol transport, RCT).
Lipoproteins are constantly interchanging particles organized in a hydrophobic core of CE and TG surrounded by a surface amphiatic monolayer of phospholipids, free cholesterol and apolipoproteins. They are classified into five groups depending on their electrophoretic mobility and hydrated density. They are classified into five major groups.
Very low density lipoproteins( VLDL)
Low density lipoproteins(LDL)
Intermediate -density lipoproteins(IDL)
High density lipoproteins(HDL)
Triglycerides are transported by CM and VLDL, LDL and HDL carry
mainly cholesterol. Sixty to seventy-five percent of the serum cholesterol is transported by low density lipoprotein (LDL), small but significant amount 15-25% is bound by high density lipoprotein (HDL). The LDL/HDL cholesterol ratio is a commanly used predictor for the risk to develop cardiovascular disease. Surface apolipoproteins which are on the surface of the lipoproteins are used for the binding of lipoproteins to specific receptors and for the function as co-factor for enzymes.
CMs are produced post prandially in the intestine by a process which is mediated by the microsomal TG transfer protein(MTP). They enters to the circulation by entering into the lymph through thoracic duct.They are composed of 95%TG and their major apolipoprotein is apoB-48. Half life of circulating CMs is < 15 minutes.
VLDL are produced in liver via MTP and secreted into the blood.50-80% of TG is the major component which has apoB-100 and acquire apoAs, C and Ein the circulation. AVLDLR also exists in the heart ,skeletal muscle and adipose tissue, which is thought to be involved in the uptake of lipids by peripheral tissue. Half life of circulatingVLDL is 2-3 hours.
IDL are generated in blood by TG depletion of the VLDL particles. They can be taken up by the liver or by hydrolysis of TG via hepatic lipase (HL). They can be converted into LDL.
LDL are formed in blood by delipidation of VLDL and HDL by LPL and HL. They account for >70% of the cholestreol present in human blood. Apo B-100 is the only apolipoprotein on the surface of LDL. A structure related to LDL is known as lipoprotein (a), Lp(a). Lp(a) has additional lipoprotein ,apo(a), bound to its apoB, which is thought to be an atherogenic and thrombogenic particle.
HDL are secreted by the liver and intestine as lipid -poor apoA-I that become immediately lipidated by the action of the ATP-binding cassette transporter A1. Peripheral cells are unable to degrade cholestrol . mature HDL particles are formed by incorporation of such cholestrol after esterification by lecithin -cholestrol acyl transferase(LCAT). CE transfer protein(CETP) in plasma is useful for the exchange of CE for TG between HDL and apoB- containing lipoproteins . HDL promote expulsion of cholesterol from peripheral cells(RCT)and indirectly, from the body, thus protecting against cardiovascular diseases.
Intestinal cholesterol absorption
Cholestrol enters the lumen of the small intestine by four routes,
The intestinal cells pumping cholesterol back to the lumen
As cell debris derived from the rapid turnover of intestinal cells.
Mostly it is absorbed in the duodenum and proximal jejenum. In humans 30-50% of cholestrol in the lumen is absorbed and returned to the liver, while the rest is eliminated with the feces.
Dietary cholesterol mixes with the biliary cholesterol and presented to the brush border of the small intestine in the form of mixed micelles. Transportation of cholesterol across the plasma membrane of the enterocyte through a pump recently identified as the niemann-Pick C1 like1protein (NPC1L1). A fraction of this cholesterol is pumped back into the intestinal lumen by ATP-binding cassettes hemi trans porters ABCG5 and ABCG8,while the remaining moves to the endoplasmic reticulum where it is esterified by the enzyme acyl-coenzyme A. Cholesterol acyl transferase 2 (ACAT2) and incorporated into nascent lipoproteins and the NPC1L1 sterol transporter has 50% homology with NPC1, which is involved in intracellular chlesterol trafficking and storage.
ABCG5 and ABCG8 actively transports cholesterol in the intestine . They are also expressed in the liver where they serve as pumps for the ABCG5 secretion of cholesterol into bile .ABCG5 and ABCG8 each encode aprotein with 6 trans membrane domains, therefore a dimerization to form a 12- transmembrane protein complex is required for transport activities.
Mutations in either hemitransporter cause sitosterolemia, a condition characterized by over absorption of plant sterols and dietary cholesterol.
Affected people absorb 15-20% of plant sterols instead of normal 1-3%. Biliary sterol excretion will be reduced. Plant sterols enter the enterocyte but, they are poor substrates for ACAT2, they remain unesterified until they pump back to the intestinal lumen by ABCG5/G8. Plant sterols may be atherogenic compounds that the body efficiently expels as general defence mechanism.
Bile acid binding resins (cholesteramine) or selective lipase inhibitors (Orlistat) reduce cholesterol absorption by interfering with the processes other than cholesterol transporters. ACAT2 and partial CETP inhibitors are currently used in clinical trials for the same purpose.
Diets containing high amount of fats or cholesterol lead to both hypercholesterolemia and hypertriglycedemia which are major prognosis for cardiovascular diseases and leading causes of death in developing and developed countries .The World Health Organization (WHO) estimates that sixty per cent of the world's cardiac patients will be Indians by 2010.
Dietary cholesterol comprised of free and esterified cholesterol.In diets rich in meats,significant percent of cholesterol is esterified. Hydrolysis of cholesteryl ester in the lumen is catalysed by cholesterol esterase ( CEase).
Cholesterol esterase is an acid lipase ,which is synthesized in the pancreas, catalyses the hydrolytic cleavage of cholesterol,sterol esters and triglycerides.
The name pancreatic cholesterol esterase is ascribed to the only enzyme in the pancreas that hydrolyzes cholesterol esters to unesterified cholesterol and free fatty acids. However, extensive investigations over a period of more than 30 years revealed that a protein with similar properties can also be purified from homogenates of several other tissues and body fluids and that enzyme is a nonspecific lipase capable of hydrolyzing cholesteryl esters, vitamin esters, triacylglycerol, phospholipids, and lysophospholipids. At the onset of these investigations, it was not clear whether these various enzyme activities were properties of the same protein. Thus, this enzyme was also named nonspecific lipase, phospholipase A1 lysophospholipase, bile-salt-stimulated lipase, bile salt-dependent lipase, carboxyl ester lipase, and carboxyl ester hydrolase.
Sequence comparison with other proteins also revealed that this enzyme is responsible for the lipoamidase activity in milk, which may account for its ability to hydrolyse the physiological lipoamide substrate ceramide.
Nomenclature of this enzyme was made based on the various substrates of this enzyme.
Most commonly used name Carboxy ester lipase(CEL), based on the general reactivity of with lipids containing carboxyl ester bonds.
Cholesterol esterase or cholesterol ester lipase, due to its documented physiological function as acholesteryl ester hydrolase.
Bile-salt-stimulated or bile salt dependent lipase, based on unique bile salt dependency.
Cholesterol esterase has received most attention as having a potential role in cholesterol absorption. CEase has a wide substrate specificity, hydrolyzing tri-, di- and mono glycerides and phospholipids in vitro. It also hydrolyzes cholesterol esters, which form a fraction of dietary cholesterol and cannot be engrossed without prior hydrolysis to free cholesterol. As such, it is one of the central enzymes that mediates absorption of dietary lipids through the intestinal wall into the blood stream. A number of studies have suggested a possible role for CEase in the absorption of free cholesterol at the brush border membrane of the small intestine , through a CEase gene .
Synthesis of cholesterol esterase (CEase)
Major tissues for the synthesis of this enzyme was acinar cells of exocrine pancreas and lactating mammary glands.
Enzyme synthesized by the pancreas is stored in zymogen granules and is secreted with the pancreatic juice in a process stimulated by the gastric hormones such as cholecytokinin, secretin and bombesin. CEase mixes with the bile salt in the lumen of digestive tract and becomes active enzyme which catalyzes nutrient digestion and absorption through GIT.
Enzyme produced from the lactating mammary glands secreted as a major constituent of milk proteins and reaches the digestive tract of the infants, which plays a role in nutrient digestion and absorption in them.
Low but significant levels are also synthesized in other tissues like liver, eosinophils, endothelial cells and macrophages. Physiological function of this enzyme synthesized outside digestive tract is unknown.
Structure - Function Relationship
Primary structure of CEase which is deduced from the nucleotide sequencing of its cDNA from various species, indicates that this enzyme is highly conserved and is a member of the α/β-hydrolase. CEase utilizes a catalytic triad of Ser-His-Asp/Glu to form the charge relay network required for substrate hydrolysis. Site specific mutagenesis experiments documented that Ser194 is the key residue in CEase responsible for nucleophilic attack on the substrate carboxy ester bond. The reaction is assisted by His435 through a general acid-base catalysis reaction on the substrate carbonyl. These also revealed the participation of Asp320 sin the catalytic triad serves by providing a better acid-base reaction through modulation of the pKa.
CEase enzyme is active alone in hydrolyzing carboxyl esters containing short chain fatty acids, but it requires bile salt activation for the hydrolysis of carboxyl esters with long chain fatty acyl groups. Numerous studies suggested the mechanism for bile salt activation of CEase , bile salt interacts with two sites on the protein which produce different effects.
One site is termed as nonspecific site , which has the capability to bind both di- and tri hydroxylated bile salts. Negatively charged side chain of bile salt interacts with one or more arginine residues in the enzyme. This protect CEase from proteolysis and promotes the binding of CEase to the surface of lipid emulsion prior to its hydrolysis of emulsified substrates. Bile salts binding to this site has no effect on CEase hydrolysis of water soluble substrates.
Second site is specific for trihydroxylated bile salts. Binding of the bile salts like cholate and taurocholate to this site induces a conformational change in the enzyme and increases hydrolytic activity against both water soluble and lipid soluble emulsified substrates.
The size of CEase protein differs from various species primarily due to the number of proline -rich repeating sequences near the carboxyl terminus of the protein.the largest CEase protein is the human enzyme at 100 kDa, which contain 16 repeating units with consensus sequence of PVPPTDDSQ. The rat , mouse, bovine and rabbit enzymes are smaller proteins at respectively 74 kDa and contain four, three and two such repeating units. These proline rich repeating units is important for maintaining the stability of the protein and these are the sites of
O-glycosylation. the carboxyl terminus of CEase also contain a domain that is required for normal intracellular processing and secretion of protein. The truncated CEase without any proline rich repeating units or the C-terminal domain also retain hydrolytic activities against both water soluble and lipid substrates, this suggests that repeating units donot participate in the catalytic activity of the protein. Modified enzymes without the C-terminal domain and with deletion of all proline rich repeating units were more active than native enzymes at low bile salt concentration in subatrate hydrolysis. This data says that C-terminal domain and proline rich repeating units are important in regulating the substrate accessibility to the active site of the protein.
According to the recent reports from the two different laboratories of X-ray crystal structure of bovine CEase provide additional support for the importance of the C-terminal domain in regulating substrate delivery to the active site domain of CEase. It is a glycoprotein of 579 aminoacids, with a notable proline-rich region between amino acids 540 and 573, and a highly conserved six-amino acid hydrophobic sequence forming the extreme C terminus of the protein. Crystallized truncated version of bovine CEase without the C-terminal repeats with 13β- strands and 14α-helices at .28 nm resolution. Others made a contrast experiments and reported a structure with11β -strands and 15α-helices at .16 nm resolution using a full length protein without N-linked glycosylation. But both structures are similar in having the central location of the active site triad same to that observed in other lipases and esterases. Both crystal structures which are predicted from the molecular modelling of the protein indicates that CEase lacks the amphipathic helical lid domain of other lipases prominent for interfacial activation. But it contains atruncated lid with a pair of antiparellel β-strands, overlapping the N-terminal disulfide loopat residue 64-80.
Role of CEase in adult pancreatic secretion, has been debated but this was centered around the role of this enzyme in the absorption and esterification of dietary cholesterol. Hydrolysis of the cholesterol esters is the primary function of this enzyme. This function will not play a major role in cholesterol absorption because cholesteryl esters represent only 10% of dietary cholesterol. The ability of CEase to re-esterifycholesterol in vitro and its localization within the interstitial cells in immunohistochemistry studies led to the hypothesis that CEase may either serve as docking or a carrier protein for free cholesterol uptake by enterocytes. It had been postulated that CEase mediates the re- esterification of cholesterol after it transverse through the membrane bilayer and this step is required prior to the cholesterol secretion into the lymph as part of chylomicrons and very low density lipoproteins.
A natural CEase substrate present in the diet is vitamin A, which is present in the form of long chain fatty acyl esters of retinol. This absorption in natural form requires the hydrolysis of the ester in the similar way as that of cholesteryl ester absorption. Because CEase is able to recognize the retinyl esters as a substrate. So, It has been proposed that this enzyme may be important for the absorption of vitamin A.
Based on the evidence of the invivo studies done by using the rats which are fed with the tetrahydrolipstatin showed the reduced retinyl palmitate absorption.
Adequately triglyceride hydrolysis in adults was accomplished by the high concentration of pancreatic lipase. End products of this absorption are free fatty acids and monoglycerol both are efficiently absorbed by the small intestine. CEase may enhance the digestion and absorption by hydrolysing the monoacyl glycerol, But this physiological implication is not clear.
It was proved in some in vitro studies that CEase significantly enhanced the rate and extent of pancreatic lipase-catalysed hydrolysis of triglycerides containing long chain poly unsaturated fatty acids, such as arachidonate, eicosapentaenoate and docosahexaenoate. In the absence of CEase,pancreatic lipase was unable to digest 1,2-diacyl glycerol containing these fatty acid absorption. Thus CEase may play arole in poly unsaturated fatty acid absorption. This has to be tested in in vivo studies by using CEase knock out mice.
Physiological importance of the phospholipase A1 and lyophospholipase activities of CEase has not been addressed in literature. Thus this process is poorly understood. This process is mediated by the tye 1 phospholipase A2 secreted from the pancreas or phospholipase B synthesized by small intestine.the products generated from these enzyme activities, lysophospholipids and freefatty acids are efficiently taken up by enterocytes. Thus, it is unlikely that CEase plays any significant role in phospholipid absorption.
One exception to this hypothesis is that the cod lacks both the enzymes that are useful in phospholipid digetion . thus CEase is the only lipolytic enzyme in the intestinal lumen of the cod and may function in lipd absorption in this species. Hence CEase in other species may also serve a backup function to other lipolytic enzymes for transport and uptake of these vital nutrients.
Considerable amount of direct and indirect evidence has accumulated to support the hypothesis that milk- derived CEase plays an important role in neonatal nutrition, especially in the digestion and absorption of milk fat and fat-soluble vitamins . comparision between very low birth weight infants fed either raw, pasteurized, or boiled human milk. Fecal fat output doubled when the infants were fed heat-treated milk and their fat absorption rates were decreased by 30% to 40%. It was also found that the amount of free fatty acids in the fecal fat was significantly greater after feeding the infants with raw milk, including that malabsorption was a reflection of reduced digestion of the milk fat. Weight gain was greater during the period the infants were being fed the raw milk. These findings suggested that a heat labile factor in the milk was important for proper digestion and absorption of fat by preterm infants. Alternate explanation for this is that heat treatment may alter the structure of the milk globule so that it is less digestible by either the gastric or the panceatic lipase.
Diseases caused due to high cholesterol levels
Hyperlipidemia is a major cause of atherosclerosis and atherosclerosis-associated conditions, such as coronary heart disease (CHD), ischemic cerebrovascular disease, and peripheral vascular disease. Although the incidence of these atherosclerosis related events has declined in the United States, these conditions still account for the majority of morbidity and mortality among middle-aged and older adults. The incidence and absolute number of annual events will likely increase over the next decade because of the epidemic of obesity and the aging of the U.S. population.
Dyslipidemias, including hyperlipidemia (hypercholesterolemia) and low levels of high-density-lipoprotein cholesterol (HDL-C), are major causes of increased atherogenic risk; both genetic disorders and lifestyle (sedentary behavior and diets high in calories, saturated fat, and cholesterol) contribute to the dyslipidemias seen in developed countries around the world. Drug therapy like,
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, the statins which are the most effective and best tolerated drugs currently in use for treating dyslipidemia, bile acid-binding resins, nicotinic acid (niacin), fibric acid derivatives, and the cholesterol absorption inhibitor ezetimibe.
Despite the efficacy of drug therapy, alterations in lifestyle have a far greater potential for reducing vascular disease risk and at a lower cost.
In view of the adverse effects of synthetic lipid-lowering drugs, the search for natural products with lipid-lowering potential and with minimal or no side effect is recommended . In recent times research interest has focused on various herbs that possess hypolipidemic property that may be useful in reducing the risk of cardiovascular disease . Because of their perceived effectiveness, minimal side effects and, relative low cost, herbal drugs are prescribed widely even when their biologically active compounds are unknown.
Reactive oxygen species (ROS) or oxidants can be defined as oxygen-containing molecules that are more reactive than the oxygen molecule present in air. ROS include free radicals as well as reactive compounds without unpaired electrons in their outer orbit. Such non-radical oxidants include peroxynitrite (ONOO-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl).
Free radicals are molecules or molecular fragments containing one or more unpaired electrons in atomic or molecular orbits, which considerably increase their reactivity. The radical group includes species such as nitric oxide (NO.), superoxide(O2- ) and hydroxyl radical (HO.).
Oxidative stress has been defined as a disturbance of the equilibrium between antioxidants and oxidants in favour of oxidants. Oxidative stress might occur when the antioxidant defence system is overwhelmed by an increased oxidant burden or a reduced antioxidant supply.( Nathalie et al.,2007)
Reactive nitrogen species (RNS) have been defined as a sub group of oxidants deriving from nitric oxide (NO.) and the term nitrosative stress is used . The term ''oxidants'' will include both ROS and RNS.
Antioxidants are included in the defence systems against oxidants, which imply
(1) systems that prevent ROS generation,
(2) antioxidant systems that inactivate oxidants.
(3) systems able to limit the deleterious effects of oxidants by allowing repair of oxidative damage.
Free radical is an atom with atleast one unpaired electron in the outer most shell, this is having capability of independent existence. It is easily formed when breakage of covalent bond occurs between entities and remaining of one unpaired electron in each new atom. They are highly reactive due to the presence of unpaired electrons. Free radical in which an oxygen is present is referred as (ROS). Free radical with oxygen as center atom contain two unpaired electrons in the outer shell. Formation of new free radical occurs when they steal an electron from the surrounding compound or a molecule.
Causes of Free radicals
Food preservatives and pesticides.
Sources of free radicals
They are automobile exhausts, chemical interaction, UV radiation, cigarette smoke, forest fires, radioactive decays, volcanic activities, burning of organic matter,volcanic activities, radioactive decays,by product of oxygen metabolism, alchoholic intake, effluents from industries, some pesticides and metal ions etc. ( Nagendrappa, 2005)
Some sources like cyclooxygenation, lipo oxygenation, reperfusion of ischemic organs, lipid peroxidation etc.
Mechanism of action of free radicals or ROS formation
Oxygen has two unpaired electerons with parallel spins. Generally, oxygen is non reactive to organic molecules having paired electrons with opposite spin. Activation of this oxygen molecule from ground to singlet state can be brought by two mechanisms.
By reversal of the spin of an unpaired electron, absorption of sufficient energy is required.
b) Monovalent reduction (accept a single electron)
Superoxide is produced in the first monovalent reduction reaction which undergo further reduction to form H2O2. H2O2 further gets reduced to hydroxyl radicals in the presence of ferrous salts (Fe2+). This reaction was first described by Fenton and later developed by Haber and Weiss (Daniel et al., 1998). This reaction is known as fenton reaction.
PATHWAYS OF ROS FORMATION
Describes the pathways of ROS generation, the lipid peroxidation process and the task of glutathione (GSH) and other antioxidants (Vitamin E, Vitamin C, lipoic acid) in the management of oxidative stress (Valko et al., 2006 ).
Pathways of ROS formation
Schematic representation of various activators and inhibitors of ROS
DISEASES CAUSED BY THE FREE RADICALS
The free radicals are generated during the normal metabolic reaction in the body. Free radicals are extremely unstable and react rapidly with other compounds, trying to capture the needed electron to gain stability.
Some free radicals arise in general through metabolism sometimes the body's immune system cells tenaciously create free radicals to neutralize virus and bacteria. Normally the body can handle free radicals but if antioxidants are unavailable or if the free radical production is surplus then damage can occur (Fig
The formation of free radicals and the incidence of oxidative stress is a common component of Parkinson's disease. Patients with Parkinson's disease have reduced glutathione levels and free radical damage is found in the form of increased lipid peroxidation and oxidation of DNA bases (Benzie, 2003).
ROS and disease
Lipid peroxidation and atherosclerosis
A crucial step in pathogenesis of atherosclerosis isbelieved to beoxidative modification of low densty lipoproteins. Oxidation of LDL is a free radical driven lipid peroxidation process. The aldehyde products of lipid hydro peroxide break down are responsible for the modification of LDL apoprotein. Aldehyde modified apo B protein has altered receptor affinity causing it to be scavenged by macrophages in an uncontrolled manner with the development of foam cells and the initiation of atherosclerotic lesion. The oxidation of LDL may be prevented by its endogenous antioxidant compounds, most prominent of which is α-tocopherol( daniel et al., 2002).
Rheumatoid arthritis is an autoimmune disease that causes chronic inflammation of the joints and tissue around the joints with infiltration of macrophages and activated T cells. The pathogenesis of this disease is linked predominantly with the formation of free radicals at the site of inflammation. T cells isolated from the synovial fluid of patients with rheumatoid arthritis show signs of decreased intracellular GSH level (Valko et al., 2006).
ROS through oxidative changes exert a number of toxic effects in different biological systems. Such ROS induced event in lung disease is arachidonic acid release and metabolism of active products. In the lung, the release of arachidonic acid from membrane bound phospholipids is by phospholipase A2. This reaction occur due to a specific stimuli by hormones such as epinephrine or bradykinin (Ray and Hussain, 2002).
The ROS-induced oxidative stress in cardiac and vascular myocytes has been linked with cardiovascular tissue injury. Regardless of the direct evidence for a link between oxidative stress and cardiovascular disease, ROS-induced oxidative stress plays a role in various cardiovascular diseases such as atherosclerosis, ischemic heart disease, hypertension, cardiomyopathies, cardiac hypertrophy and congestive heart failure.
Inflammatory cells (granulocytes, macrophages, some T-lymphocytes) produce active species of oxygen as part of the microbicidal or citocidal systems. The active oxygen species can modulate specific elements of the inflammatory response in vitro, include protein immunomodulatory substances such as granulocyte migratory factors PGs, cyclic nucleotides, as well as formed elements such as platelets (Irshad and chaudhuri, 2002).
The term oxidative stress is a shift towards the pro-oxidant in the pro-oxidant/antioxidant balance that occurs as a result of increase in oxidative metabolism. The improper balance between ROMs production and antioxidant defences results in oxidative stress. Its increase at cellular level can come as a consequence of several factors, including exposure to alcohol, cold, medication, trauma, infections, toxins, radiation, strenuous physical activity, and poor diet (Ray and Hussain, 2002).
The balance of oxidants and antioxidants
Antioxidants are any substance, present at lower concentration compared to that of oxidizable substance that delay or inhibits oxidative damage to a target molecule. Antioxidants neutralize free radicals by donating one of their own electrons, ending the carbon-stealing reaction. They act as scavengers, helping to prevent cell and tissue damage that could lead to cellular damage and disease. They are substances that protect other chemicals of the body from damaging oxidation reactions by reacting with free radicals and other reactive oxygen species within the body. One antioxidant molecule can only react with single free radical. Therefore there is a constant need to replenish antioxidant resources, whether endogenously or through supplementation.
Model of a balance between pro-oxidants and anti-oxidants
Under normal conditions anti-oxidants out balance pro-oxidants. But under oxidative conditions , pro-oxidants prevail over anti-oxidants which may lead to many diseases.
The body has developed several endogenous antioxidant systems to deal with the production of reactive oxygen intermediates (ROI). These systems can be divided into
MECHANISM OF ACTION OF ANTIOXIDANTS
Physical barriers preventing ROS generation or ROS access to important biological sites.
E.g. UV filters, cell membranes
Chemical traps / sinks 'absorb' energy and electrons quenching ROS.
E.g. Carotenoids, anthocyanidins
Catalytic systems neutralize or divert ROS.
E.g. SOD, catalase and glutathione peroxidase
Binding / inactivation of metal ion prevents generation of ROS by Haber-Weiss reaction.
E.g. Ferritin, catechins
Sacrificial and chain breaking antioxidants scavenge and destroy ROS.
E.g. Ascorbic acid (Vit.C), tocopherols (Vit E), uric acid, glutathione, flavonoids (Benzie, 2003).