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The liver is the second largest gland in the body, weighing between 1 and 3 kg. It is located in the upper part of the abdominal cavity occupying the greater part of the right hypochondriac region, part of the epigastric region and extending in to the left hypochondriac region. Its upper and anterior surfaces are smooth and curved to fit the under surface of the diaphragm. Its posterior surfaces are irregular in out line. The liver is enclosed in a thin elastic capsule and incompletely covered by a layer of peritoneum. The liver consists of two anatomical lobes.
1. Right lobe
2. Left lobe.
The right lobe being about six times greater, then the size of the left lobe. The right lobe has quadrate lobe on its inferior surface and a caudate lobe on the posterior surface. The right and left lobes are separated anteriorly by a fold of peritoneum called the falciform ligament, inferiorly by the fissure for the ligament tares, and posterior by the fissure for the ligament venosum. The liver is special in that it can regrow its own tissue. As much as three-quarters of the liver can be removed, and the organ can grow back in about a month. Because of this regrowth, liver for transplant can be taken from living donors. (Ross and Wilson, 2006).
The hepatic artery and the portal vein supply the blood to the liver. Venous return is by a variable number of hepatic veins that leave the posterior surface and immediately enter the inferior vena cava just below the diaphragm. Liver lobules are hexagonal in outline and are formed by cubical shaped cells, the hepatocytes arranged in pairs of columns radiating from a central vein. Between two pairs of columns of cells are sinusoids (blood vessel with incomplete walls) containing a mixture of blood from the tiny branches of the portal vein and hepatic artery .This arrangement allows the arterial blood and portal venous blood to mix and come in to close contact with the liver cells. Amongst the cells lining the sinusoids are hepatic macrophages (kupffer cells) whose function is to ingest and destroy worm out blood cells and foreign particles present in the blood flowing through the liver.
FUNCTION OF LIVER
The liver's job is to run over 500 bodily functions. It plays a role in the processing of metabolism and excretion of xenobiotics from the body. It also plays a key role in the body's defense system. It processes almost everything a person eats, breathes, or takes in through the skin. About 90% of the body's nutrients pass through the liver from the small and large intestines.
The liver changes food into energy stores this energy is used for the production of blood proteins. The livers also detoxify the bacteria and poisons from the blood. A baby's liver also makes blood cells in the womb. (Ross and Wilson, 2006)
ROLE IN DIGESTION
The liver plays an important role in the breakdown of food in the body. Liver cells make bile, a greenish-yellow fluid that helps in the breakdown of fats and in bringing nutrients into the body. Waste made by the liver in the breakdown of food is carried in the bile and excrete from the body through the kidney. Someone with a liver that is not working properly those may have less bile production. They may not process the excretion of wastes as compared to a person with a healthy liver. When this happens, the body may have trouble in processing food and other chemical substances. Liver cells also convert heme (a portion of hemoglobin that is released when red blood cells are broken down) into Bilirubin. When the liver is damaged, Bilirubin may build up in the blood causing yellowing of the skin and whites of the eyes (jaundice).
METABOLISM IN LIVER
The liver helps to provide the energy to the body by the process of metabolism. It controls the production, storage, and release of sugar, fats, and cholesterol. When food is taken, the liver changes glucose (blood sugar) into glycogen. The glycogen is then stored in the liver as an energy source for later use. The liver changes glycogen back into glucose when energy is needed at night. This process is called glucogenolysis. The liver controls the storage of fats by changing amino acids (the building blocks of protein) into fatty acids. Some fatty acids, such as triglycerides are changed by the liver into ketones. Ketones are fuel for muscles. Ketones are used when the body does not have enough sugar. The liver also makes processes and removes cholesterol from the body. Cholesterol is an important part of cell structures and certain hormones (Harsha Mohan, 2010).
Though the liver has a marked regenerative capacity and a large functional reserve Hepatic failure may develop from severe acute and fulminant liver injury with massive necrosis of liver cells (acute hepatic failure) or from advances chronic liver disease (chronic hepatic disease). Acute liver failure develops suddenly with severe impairment of the liver function where as chronic liver failure comes insidiously. Liver injury or liver dysfunction is a major health problem that challenges not only health care professionals but also pharmaceutical industry and drug regulatory agencies.
There are two types of the hepatic failure result from different causes.
Acute (fulminant) hepatic failure:
It happens mostly in severe viral hepatitis. Other causes are hepatotoxic drug reactions (Anesthetic agents, NSAIDS, Anti depressants), CCl4 poisoning, acute alcoholic hepatitis, mushroom poisoning, and pregnancy complicated with eclampsia.
Chronic hepatic failure:
It is the most common due to the cirrhosis. Other cause includes chronic active hepatitis, chronic cholestasis & Wilson disease.
The diverse functions performed by the liver, the disease of acute or chronic hepatic failure produces complex manifestations. The major manifestations are as follows.
Hepatic encephalopathy (hepatic coma)
Hepato renal syndrome
Hepato pulmonary syndrome
Ascites and edema
Skin changes and
Toxic liver injury produced by drugs and chemicals may virtually mimic any form of naturally occurring liver disease. Hepatotoxicity from drugs and chemicals is the commonest form of iatrogenic disease. Severity of the hepatotoxicity is greatly increased if the drug is continued after symptoms develop.
Among the various inorganic compounds producing hepatotoxicity are arsenic, phosphorus, copper and iron. The organic compounds include certain naturally occurring plant toxins such as pyrrolizidines alkaloids, mycotoxins and bacterial toxins. The synthetic group of the organic compounds is a large number of medicinal agents. In addition exposure to hepatic compounds may be occupational, environmental or domestic that could be accidental, homicidal or suicidal ingestion.
Drug reactions affecting the liver are divided in to two main classes.
Direct or predictable :
When the drug or one of its metabolite is either directly toxic to the liver it lowers the host immune defense mechanism. The adverse effects occur in most individuals who consume them and their hepatotoxicity is dose dependent.
Example: carbon tetrachloride
Indirect or unpredictable or idiosyncratic:
When the drug or one of its metabolites act as a hapten and induces hypersensitivity in the host. The hepatotoxicity by this group does not occur regularly in all the individuals and the effects are usually not dose related. (Harsha Mohan).
Symptoms of hepatotoxicity
Loss of appetite
Enlarged liver, Etcâ€¦
DRUG INDUCED HEPATOTOXICITY
The most widely prescribed therapeutic drugs have been focused attention on the hepatic safety of drugs. A significant number of drugs has been proven, or at least suggested, to cause hepatotoxicity. There are 900-1000 drugs and chemical substances are causing the liver toxicity throughout the world wide. It must be recognized that a drug is a chemical or biologic agent that has been found to cause a therapeutically action on a symptom or disease process, has been tested or identified for safety, and then if approved, is widely available for diagnostic purpose. There are many clinical diagnostic centers has given the manifestations, which specially suggest that a liver toxicity is the result of a therapeutic drug, with a dose dependent manner. The most important one is often the temporal relationship between initiation of a drug and the presence of the injury and of equal importance is the resolution of an abnormality following withdrawal. The attention has been given to the world population for the recognization of factors, which causing the harmful effects to the liver and who are increased the risk of developing hepatic damage from number of drugs and chemical compound (Willis C.Maddrey, MD 2005).
SPECTRUM OF HEPATOTOXICITY INDUCED BY DRUGS
The spectrum of drug induced liver injury starting from initial, nonspecific significant changes in the biochemical parameters of no clinical consequences to acute hepatitis, chronic hepatitis, acute liver damage, chronic cholestatic disease and even cirrhosis and liver tumors.
Drugs withdrawn or severely limited because of hepatotoxicity.
PREDICTIVE PARAMETERS OF DRUG-INDUCED LIVER DAMAGE
There are relatively few ways by which acute and chronic liver diseases become clinically significant. The characteristics of drugs and chemicals induced liver disease are generally nonspecific and reflect more the extent of the liver injury than the cause. The signs and symptoms found in many hepatic diseases such as acute viral hepatitis, chronic viral hepatitis, or chronic cholestasis diseases.
FACTOR THAT INFLUENCING SUSCEPTIBILITY TO DRUG-INDUCED LIVER DISEASE
Age, sex and the continuous use of other medications are important factors to consider in assessing an individual patient's susceptibility to drug induced liver disease. There are few agents, among them Valproic acid and erythromycin estolate, which predominantly cause adverse hepatic reactions in children. Valproic acid-induced liver toxicity is also more prevalent in patients who have inherited mitochondrial disorders. For most of drugs females are at an increased risk of developing an adverse hepatic reaction, when compared with that found in males.
MECHANISM OF HEPATOTOXICITY
Several mechanisms have been suggested that, most of the drugs and chemical compounds have been involved in producing drug induced liver toxicity. The chemical or drug which producing the liver toxicity is referred as the hepatotoxins. Some therapeutically important drugs are produced hepatotoxins and are readily identified as such in preclinical and clinical evaluations. With many drugs intermediary products produced during metabolism have been proved to be highly reactive and toxic. Potentially toxic metabolic products may be present only transiently and are rapidly metabolized further into harmless substances, thereby avoiding injury. The cytochrome P450s, a family of enzymes largely involved in initial oxidative (phase I) reactions of drug metabolism, has established roles in the production of highly reactive intermediates as well as established roles in further metabolism and accumulation. Identification and characterization of the various cytochrome P450 subspecies involved in metabolism of a drug allow predictions to be made regarding the likelihood of production of reactive intermediates and may focus assessment of the potential for drug-drug interactions, if two or more drugs are used in combination. For a growing number of drugs, there is evidence that genetic polymorphism in metabolic pathways are important in determining which individuals are likely to have an adverse reaction.
SPECIFIC DRUGS OF SPECIAL INTEREST
Paracetanol-induced hepatic injury is the most common form of drug-induced liver damage and more particularly of acute liver toxicity in the United States, accounting for nearly 50% of all cases. Paracetanol is likely the most widely used drug in the United States and is found in a remarkable number of prescribed and over-the-counter single and combination products, including cold remedies and medications for pain (Vicodin) with names that in no way indicate paracetamol is a component. Paracetanol is established dose-related hepatotoxins. In healthy individuals, there is apparently a considerable therapeutic range between harmless and harmful doses of paracetamol. In therapeutic doses (3 g/day), the drug is usually quite safe and well tolerated. Ingestion of excessive amounts of paracetamol (10-15 g), often in suicidal attempts, predictably leads to liver injury and occasionally death. The issues lie in assessing the risk of patients receiving paracetamol of 3 to 10 g/day, and whether there are settings in which liver injury is more likely to occur when the patient has not taken a large amount of the drug with a suicidal intent (so-called "therapeutic misadventures'') Hepatic injury from paracetamol is caused by the effects of a highly reactive metabolic product, N-acetylbenzoquinone-imide (NAPQI). Paracetanol is predominantly metabolized by conjugation reactions to form sulfate and glucuronide metabolites, which are excreted in the urine. A lesser amount of the drug is metabolized by cytochrome P450 2E1 to form N-acetylbenzoquinone-imide, Which is rapidly bound to intracellular glutathione and excreted in the urine as mercapturic acid, When excessive amounts of paracetamol are ingested, the ability to conjugate is overwhelmed and metabolism by cytochrome P450 2E1 becomes of much greater importance. In these situations, the capacity of glutathione to serve as an effective hepatoprotectant may be overwhelmed, and the hepatocyte becomes relatively defenseless against attack by the reactive intermediates. Two important factors determine the production of hepatic injury by paracetamol, the amount of N-acetylbenzoquinone-imide produced by P450 2E1 and the availability of glutathione as a hepatoprotectant. The intracellular concentration of NAPQI and dose of paracetamol ingested are clearly associated. However, there is more to the story than simply dosage. Factors that affect the production of cytochrome P450 2E1 and of glutathione are of importance. With chronic ingestion of alcohol, doses of paracetamol near or even with the suggested therapeutic range may lead to liver injury, promoted by an alcohol-induced decrease in intracellular glutathione and possibly an increase (actual or relative to GSH) of cytochrome P450 2E1. The end result is overproduction of NAPQ1 relative to the dispositional pathway of GSH leading to a heightened likelihood of liver damage. It must be noted that there continues to be controversy regarding the risk of paracetamol use in patients who drink alcohol. Many discussions have occurred at the U.S. Food and Drug Administration regarding labeling and the need for increased awareness of risks by the public. There is general agreement that an overdose of paracetamol is more likely to cause liver injury in a patient who is a chronic alcoholic. The debate is the definition of overdose and the amount of alcohol ingestion needed to predispose the patient to injury. N-acetylcysteine, now available in both oral and intravenous formulations, has proven effective when given early in the course of minimizing paracetamol-induced injury
The kidneys are the bean shaped paired organs, each weighing about 150 the adult male and about 1 the adult female. The hilum of the kidney is located at the midpoint on the medial aspect where the artery, vein, lymphatics and ureter are located. The kidney is surrounded by a thin fibrous capsule which is adherent at the hilum.
Cut surface of the kidney shows three main structures well demarcated
Inner most renal pelvis.
The renal cortex forms the outer rim of the kidney and is about 1cm in thickness. It contains all the glomerular and about 85% of the nephron tubules, remaining 15% nephrons consisting of collecting tubules, collecting ducts, loop of the henle and vasarecta send their loops in to the medulla, and are therefore called Juxtamedullary nephrons.
The renal medulla is composed of 8-18 cone shaped renal pyramids. The base of a renal pyramid lies adjacent to the outer cortex and forms the cortico-medullary junction; while the apex of each called the renal papilla contains the opening of each renal pyramid for passage of urine collected from collecting ducts and goes down in to minor calyces.The renal pelvis is the funnel shaped collection area of the urine for drainage in to the ureter.
FUNCTIONS OF THE KIDNEY
In general the kidney performs the following vital functions in the body.
Excretion of waste products resulting from protein metabolism
Regulation of the acid-base balance by excretion of H+ ions (acidification) and bicarbonate ions.
Regulation of salt water balance by hormones secreted both intra and extra renally.
Formation of renin and erythropoietin and there by playing a role in the regulation of the blood pressure and erythropoietin respectively. (Ross and willson).
PATHOPHYSIOLOGY OF RENAL DISEASE
Diseases of the kidney are divided in to 4 major groups according to the predominant involvement of the corresponding morphologic components.
Renal failure is one of the major complications of myeloma, found at presentation in 20% of patients and occurring in 50% of patients during the cause of disease. Only 20% of the adults with a nephritic syndrome have minimal change nephropathy and for that reason a renal biopsy is necessary to establish the type of glomerulonephritis. Idiopathic glomerulonephritis accounts for 90% of childhood cases of nephritic syndrome and 80% in adult patients. In patients presenting with acute renal failure the proportion with acute interstitial nephritis varies from 6.5-1.5%. A recent year study (2000) from renal units and ICU in a defined geographical area of Scotland found that 131 patients per million per year required renal replacement therapy for acute renal failure.
The incidence of chronic kidney disease leading to dialysis varies worldwide: the number of patients per million population starting dialysis each year is 110 in the UK.
In acute renal failure causes untold suffering among patients leading to complications that sometimes lead to death It is estimated that in India, there are estimated 80, 000 people with severe renal failure. About fifteen percent of diabetics succumb to some renal complication or renal failure
In India, the Chandigarh study showed that 30 years ago diarroheal disease and obstetric complications each accounted for about 25% of cases of ARF in north India but more recently each has accounted for about 10%.
Intravascular haemolysis is a common feature of many cases, being found in over 20% of 325 patients receiving dialysis for ARF in Chandigarh. ARF develops in 5- 30% of the victims of severe snake viper poisoning and is the cause of between 2-3 % of cases of ARF. In Europe renal biopsy, performed for the indication of unexemplified acute renal impairment, shows interstitial nephritis (Harsha Mohan).
Water and sodium homeostasis and their disorders
Total body water accounts for about 60% of the body weight of a healthy adult: two third is intracellular and one- third is extracellular. The extracellular fluid compartment is divided into vascular (blood volume) and interstitial fluid compartments in the ratio 1:2 for a 75kg adult, the total volume of body water is approximately 45 liters, with intracellular and extracellular volumes of 30 and 15 liters respectively, the later comprising the blood (5 liters) and interstitial (10 liters) compartments. Sodium is the main intracellular cation, which with its anion chloride contributes 95 percent of the extracellular solute. By contrast the major intracellular cation is potassium. Many cell membranes are freely permeable to water, but not to most electrolyte, which results in the extracellular and intracellular compartments.
Body water and therefore solute concentrations are regulated mainly by vasopressin (ADH) mediated alteration of renal water excretion, but also to some extent by thirst as a motivation for drinking. The secretion of vasopressin and thirst are influenced principally by changes in circulating concentration of sodium, but also in past by significant falls in blood volume or pressure. The volume of extracellular compartments is determined by its total sodium content, which is regulated by numerous mechanisms. Sodium intake is poorly controlled in humans, although some animals do demonstrated a specific sodium appetite. The kidney is the major effector organ influencing sodium homeostasis.
Complex intrarenal mechanisms contribute to the maintenance of sodium homeostasis, in addition to which are endocrine factors that either tend to reduce excretion of sodium by the kidney for example renin-angitensin-aldosterone system or which produce a natriursis. Blood volume and pressure are also influenced by a variety of vasaoactive substances that act locally or systemically (ex: catecholamines, prostaglandins, nitric oxide, Endothelin). It can therefore be appreciated that there is an intricate network of homeostatic mechanisms controlling both sodium and water balance.
Disorders of water and salt homeostasis
Cranial diabetes insipidus
Nephrogenic diabetes insipidus.
Disorders of potassium homeostasis
Potassium is the most abundant cation in the body. Total body potassium ranges from 37 and 52mmol/kg body weight, and of this 98% is found in cells, where its concentration is in the range of 150-160 mmol/kg. The maintenance of intracellular and extracellular gradient is largely dependent on the ubiquitous Na+, K+ATP ASE enzyme which pumps potassium anions into the cell for every three sodium ions extruded.
Hyperkalaemia (Harsha Mohan)
Acute renal failure
It is defined as a significant decline in renal excretory function occurring over hours or days. This is usually detected clinically by arise in the plasma concentration of the urea or creatinine. Acute renal failure may arise as an isolated problem, but much more commonly occurs in the setting of circulatory disturbance associated with severe illness, trauma, or surgery; transient renal dysfunction.
Vascular causes of acute renal failure:
Acute cortical necrosis
Large vessel obstruction
Small vessel obstruction
Accelerated phase hypertension
Glomerulonephritic and vasculitic causes of ARF
Hanta virus disease. (Europe)
Chronic renal failure
It is the clinical syndrome of the metabolic and systemic consequences of a gradual, substantial and irreversible reduction in the excretory and homeostatic functions of the kidneys. It can be difficult to recognize because the symptoms and clinical manifestations are non- specific.
Causes of chronic renal failure
The most important causes of chronic kidney disease are diabetes, glomerulonephritis, hypertension and other vascular disease.
Arteriopathic renal disease and hypertension
Infective, obstructive and reflux nephropathies
Familial or hereditary kidney disease, e.g. polycystic kidneys
Connective tissue diseases
Renal bone disease is a major cause of disability in patients with terminal renal failure (Arthur. C. Guyton).
MECHANISM OF NEPHROTOXICITY BY PARACETAMOL
Paracetamol induced kidney failure occurred as a result of severe hepatic failure. In the presence of severe hepatotoxicity that precludes further hepatic metabolism of the parent paracetamol, there may be spillover of paracetamol to the kidney where it will be metabolized. Nephrotoxicity then results when there is an insufficient glutathione in the renal parenchyma. This was initially interpreted as a hepatorenal syndrome. When paracetamol is metabolized in both liver and kidney, nephrotoxicity may occur independently of hepatotoxicity depending on the balance of metabolism and glutathione stores with bin the kidney. Biotransformation in the kidney of paracetamol to a reactive electrophile contributes to covalent binding and subsequent nephrotoxicity. As a cytochrome p-450 is the terminal oxygenase controlling most drug oxidation in the kidney, liver, and other tissues and because of this enzyme system is concentrated primarly in the renal cortex, it is likely that metabolic deactivation of paracetamol to an arylating metabolite might be responsible for the renal lesion just as it is for the hepatic injury. Compared to the liver, the cytochrome p-450 enzymes in the kidney are less able to detoxify paracetamol. Thus the renal glutathione stores are depleted more rapidly and kidney cell injury occurs as acute tubular necrosis.
In human life oxygen is very essential, without oxygen we cannot survive. Our evolutionary ancestors developed defense mechanisms that can minimize the toxic effects of oxygen, without this protection causes the end of life. Natural defenses are imperfect; the damage of the cells caused by oxygen can be minimized by using antioxidants. A lot of research works are made in this past decades. In this study the metabolites produced by the oxygen species and with research works they have learned how to prevent the diseases caused by the reactive oxygen species. Now research works are doing for improving the antioxidant activity (Lillian, 1995).
Free radicals of different forms are constantly generated for specific metabolic requirement and quenched by an efficient antioxidant network in the body. When the generation of these species exceeds the levels of antioxidant mechanism, it leads to oxidative damage of tissues and biomolecules, eventually leading to disease conditions, especially degenerative diseases. Many plant extracts and phytochemicals have been shown to have antioxidant/free radical scavenging properties and it has been established as one of the mechanisms of their action (Dubey et al., 2009).
Reactive oxygen species (ROS) such as O2·, H2O2 and OH· are highly toxic to cells. Cellular antioxidant enzymes and the free radical scavengers normally protect a cell from toxic effects of the ROS. When generation of the ROS overtakes the antioxidant defense of the cells, oxidative damage of the cellular macromolecules (lipids, proteins and nucleic acid) occurs, leading finally to various pathological conditions (Bandyopadhyay et al., 1999).
Reactive nitrogen species (RNS) are the products of normal cellular metabolism. NOâ€¢ is a small molecule that contains one unpaired electron on the antibonding (2Ï€*y) orbital and is, therefore, a radical. NOâ€¢ is generated in biological tissues by specific nitric oxide synthases (NOSs). Overproduction of reactive nitrogen species is called nitrosative stress. This may occur when the generation of reactive nitrogen species in a system exceeds the system's ability to neutralise and eliminate them. Nitrosative stress may lead to nitrosylation reactions that can alter the structure of proteins and so inhibit their normal function (Valko et al., 2006).
Free radicals are fundamental to any biochemical process and represent an essential part of aerobic life and our metabolism. They are continuously produced by body's normal usage of oxygen such as respiration and some cell mediated immune functions. The oxygen consumption inherent in cell growth leads to the generation of reactive oxygen species (ROS) (Gulcin et al., 2007).
Most harmful effects are produced by the reactive oxygen species (ROS) in our body, ROS are act as oxidants. Free radical is a chemical species which have a lone pair of electrons at its outer orbit, so it is unstable and more reactive. Examples are hydroxyl radical, nitric oxide radical, superoxide radical, lipid peroxyl radical and non radicals are hydrogen peroxide, singlet oxygen, hypochlorous acid and ozone.
General features of a free radical reaction
Free radical reactions take three distinct identifiable steps
Initiation step : Formation of radicals
Propagation step: It is the heart of a free radical reaction. In this step, the required free radical is regenerated repeatedly, which would take the reaction to complete.
Termination step: It involves the destruction of free radical intermediates (Manavalan and Ramasamy, 2001)
I. Initiation step
II. Propagation step
R· + O2 ® RO2·
III. Termination step
Where HI and X are chain inhibitors.
SOURCES OF FREE RADICALS
Exogenous free radicals
Endogenous free radicals
Exogenous sources of free radicals are automobile exhaust fumes, UV radiation, electromagnetic radiation, cosmic radiation, interaction with chemicals, cigarette smoke, burning of organic matter during cooking, forest fires, volcanic activities, radioactive decay-¡, ¢ and radiation, lightening particularly oxides of nitrogen, byproduct of oxygen metabolism (Illness causes the body to produce greater amounts of harmful radicals than in healthy condition). Industrial effluents, excess chemicals, alcoholic intake, certain drugs, asbestos, certain pesticides and herbicides, some metal ions and fungal toxins inflict oxidative stress (Irshad and Chaudhuri, 2002).
These are internally generated sources of free radicals; they include cyclo oxygenation, lipooxygenation, lipid peroxidation, arachidonate pathways, peroxisomes, xanthine oxidase, inflammation, mitochondria, cytochrome-P450, phagocytes, reaction involving iron and other transition metals, neutrophils stimulated by exposure to microbes, re-perfusion of ischaemic organs ( Singh et al., 2004).
TYPES OF FREE RADICALS
Superoxide radical (O2·)
Superoxide anion is a reduced form of molecular oxygen created by receiving one electron. Superoxide anion is an initial free radical formed from mitochondrial electron transport systems. Mitochondria generate energy using four electron chain reactions, reducing oxygen to water. Some of the electrons escaping from the chain reaction of mitochondria directly react with oxygen and form superoxide anions. The superoxide anion plays an important role in the formation of other reactive oxygen species such as hydrogen peroxide (H2O2), hydroxyl radical (OH·) or singlet oxygen (O·) in living systems. The superoxide anion can react with nitric oxide (NO·) and form peroxynitrite (ONOO·), which can generate toxic compounds such as hydroxyl radical and nitric oxide.
Hydrogen peroxide (H2O2)
Hydrogen peroxide is the most stable reactive oxygen species. H2O2 is the primary product of the reduction of oxygen by various oxidase such as xanthine oxidase, uricase, D-amino acid oxidase and ¡-hydroxy acid oxidase localized in peroxisome. Research shows that the H2O2 is the most effective species for cellular injury. The well known Fenton reaction is initiated when Fe2+ comes in contact with H2O2. Ions of Cu, Co, and Ni can also participate in a similar reaction.
Fe2+ + H2O2 Fe3+ + ·OH + OH-
H2O2 also reacts with O2· to initiate with Haber-Weiss reaction producing ·OH in presence of Fe2+.
O2· + H2O2 O2 + ·OH + OH-
The most important function of H2O2 is its role as an intracellular signaling molecule (Ray et al., 2000).
Hydroxyl radicals (OH·)
Hydroxyl radical is highly reactive. It may react with any molecule present in the cells and hence they are short lived. The life span of OH· at 37°C is 10-9 sec. The hydroxyl radical is formed from hydrogen peroxide in a reaction catalyzed by metal ions (Fe+ or Cu+), often bound in complex with different proteins or other molecules. This is known as the Fenton reaction.
H2O2 + Cu+/ Fe2+ OH·+OH- + Cu+/Fe3+ â€¦â€¦â€¦â€¦.. (1)
Superoxide also plays an important role in connection with reaction 1, by recycling the metal ions.
Cu2+ / Fe3+ + O2· Cu+ / Fe2+ + O2 ..........................(2)
The sum of reaction 1 and 2 is the Haber-Weiss reaction; transition metals thus play an important role in the formation of hydroxyl radicals. Lipid is very sensitive to OH· attack and initiates LPO (Lipid per oxidation). The hydroxyl radical is responsible for DNA damage and LPO.
Nitric oxide (NO·)
Nitric oxide is an inorganic free radical containing odd number of electrons and can form a covalent link with other molecules by sharing a pair of electrons. It is synthesized by an enzyme nitric oxide synthase located in various tissues and plays an active role in free radical tumor biology. NO is synthesized enzymatically from L-arginine by NO syntheses
L-arginine + O2 + NADPH ® L-citrulline + NO + NADP+
Nitric oxide is another free radical that has an important biological role. NO· produced in the body relaxes muscles in blood vessels, and lowers blood pressure. But, excess NO· produced in cases of severe infection can be harmful. Unlike HO· or O2·, NO· is a much slower reacting radical and it combines with other free radical and inhibits further reaction (Hussain, 2002).
Xanthine oxidase (XO)
Xanthine oxidase (XO) is a source of oxygen derived free radicals. XO derives from xanthine dehydrogenase (XD), an initial translational product by proteolysis. In diseased condition, large amount of XO and XD are released into the circulation to produce significant amount of ROMs. XO catalase the oxidation of hypoxanthine to uric acid reducing O2 by one or two electrons resulting in the formation of O2· or H2O2. During reoxygenation (i.e. reperfusion phase) it reacts with molecular oxygen, thereby releasing superoxide anion radicals, hydrogen peroxide, and further hydroxyl radicals (Hussain, 2002).
Hypoxanthine + O2 ® Xanthine + O2-. + H2O2
Xanthine + O2 ® Uric acid + O2-. + H2O2
MECHANISM OF ACTION OF FREE RADICALS OR ROS FORMATION
Oxygen in the atmosphere has two unpaired electrons and these unpaired electrons have parallel spins. Oxygen is usually non reactive to organic molecules that have paired electrons with opposite spins. This oxygen is considered to be in a ground (triplet or inactive) state and is activated to a singlet (active) state by two different mechanisms.
Absorption of sufficient energy to reverse the spin on one of the unpaired electrons
Monovalent reduction (accept a single electron)
Superoxide is formed in the first monovalent reduction reaction which undergoes 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).
DISEASES CAUSED BY THE FREE RADICALS
Free radicals arise normally during metabolism sometimes the body's immune system cells purposefully create free radicals to neutralize virus and bacteria. However environmental factors such as pollution, cigarette smoke and herbicide can also produce free radicals. Normally the body can handle free radicals but if antioxidants are unavailable or if the free radical production is excess then damage can occur.
The formation of free radicals and the occurrence 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).
REACTIVE OXYGEN SPECIES AND DISEASE
Cancer and other malignancies
Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells compared with normal cells; the redox imbalance thus may be related to oncogenic stimulation. Permanent modification of genetic material resulting from "oxidative damage" incidents represents the first step involved in mutagenesis and carcinogenesis. DNA mutation is a critical step in carcinogenesis (Valko et al., 2006).
The increased Lipidperoxidation by HOâ€¢ radical and hydroperoxides results in acute and chronic alcoholic liver diseases. The ROS and lipid peroxidation may play role in pathogenesis of hepatic fibrosis with loss of normal liver function. The plasma levels of antioxidant vitamins are low in patients with chronic cholestatic liver diseases (Irshad and Chaudhuri, 2002)
ROS through oxidative changes exert a number of toxic effects in different biological systems. Such ROS induced event in lung disease is arachidonic acid (AA) release and metabolism of active products. In the lung, the release of arachidonic acid from membrane bound phospholipids is by phospholipase A2. This reaction occurs due to specific stimuli by hormones such as epinephrine or bradykinin (Ray and Hussain, 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).
It is a syndrome characterized by a loss of glucose hemostasis. Hyperglycemia can increase oxidative stress and change the redox potential of glutathione. Decreased uptake of glucose into muscle and adipose tissue leads to chronic extracellular hyperglycemia resulting in tissue damage and pathophysiological complications. Increased oxidative stress is one of the major causes of the hyperglycemia-induced trigger of diabetic complications (Irshad and Chaudhuri, 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.
The brain is particularly exposed to oxidative damage because of its high oxygen utilization, its high content of oxidisable polyunsaturated fatty acids and the presence of redox-active metals (Cu, Fe). Oxidative stress increases with age and therefore it can be considered as an important causative factor in several neurodegenerative diseases like Alzheimer's and Parkinson's disease especially for older individuals (Valko et al., 2006).
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 invitro, include protein immunomodulatory substances such as granulocyte migratory factors PGs, cyclic nucleotides, as well as formed elements such as platelets (Irshad and chaudhuri, 2002).
Aging in humans is associated with changes in physical characteristics and the decline of many physiological functions. Increased accumulation of free radicals increases the risk of variety of oxidative stress in older individuals. These radicals are capable of causing apoptosis, necrosis and cell death.
Exposure of exogenous sources of oxidants is high; body's antioxidant defenses may be unable to cope, this condition called oxidative stress. An imbalance between proxidants and antioxidants. (Lillian, 1999)
Fig: 3 The balance of oxidants and antioxidants
Antioxidants are the substances which scavenges the oxidation process. Antioxidants are a type of complex compounds found in our diet that act as a protective shield for our body against certain disastrous diseases such as arterial and cardiac diseases, arthritis, cataracts and also premature ageing along with several chronic diseases.
The above definition gives a brief idea about the antioxidants and their function. Recent researches on free radical made revolution a great change in health and life styles.
Types of antioxidants
Enzymatic and Non-enzymatic.
Antioxidant derived from natural and dietary sources.
Antioxidants based on defense mechanism.
Superoxide dismutase (SOD):- SOD is an endogenously produced intracellular enzyme presents every cell in the body. SOD appears in 3 forms according to the catalytic metal present in the active site.
Glutathione peroxidase (GSH):- It is commonly found in mitochondria and cytosol. Its function is removal of Hydrogen peroxide and organic hydro-peroxide.
Catalase (CAT):- It is found in mitochondria and cytosol. Its main function is the removal of hydrogen peroxide (H2O2).
Carotenoids:- It is a lipid soluble antioxidants, commonly seen in membrane tissue. The main function is the removal of reactive oxygen species.
Bilirubin:- It is produced by heme metabolism found in blood. The main function is to act as extracellular antioxidants.
Glutathione:- It is a non protein thiol and found in cells. It has the property of cellular oxidant defense.
Alpha-lipoic acid: - It is endogenous thiol. Its property is by serving substitute for glutathione, recycling vitamin C.
Vitamin C: - It is found in aqueous phase of cell. Its property is act as free radical scavenger and also acts in the recycle of vitamin E.
Vitamin E: - It is found in cells. Function is chain breaking antioxidant.
Uric acid: - It is a product of purine metabolism. Its property is scavenging of hydroxyl (OH) radical.
Antioxidant derived from natural and dietary sources
Plants are good source of antioxidants. The leaves of the most medicinal plants have the antioxidant property. The chemical constituents contained in the leaves are causative for the antioxidant property (Eg :- Flavanoids, carotnoids, alkaloids, phenolic alcohols etc.). Daily diet contains full of antioxidants like vegetables, fruits, tea, wine etc. Secondary products of plants which are functioning as antioxidant are,
Phenolics - coumarines, flavanoids.
Poly phenolics - tannins, proanthocynidine.
Nitrogen containing compounds - alkaloids, indoles.
Antioxidant based on defense mechanism
Preventive antioxidants:- It will suppress the free radical formation, enzymes such as peroxidase, catalase, lactoferrin, carotenoids etc.
Radical scavenging antioxidants: - It will suppress the chain initiation reaction, like Vitamin C and carotenoids.
Repair and de novo antioxidant: - It consist of proteolytic enzymes, it also repairs the DNA of enzymes and genetic materials.
Enzyme inhibitor antioxidants: - It induces production and reaction of free radicals and transport of appropriate antioxidants to appropriate active site.
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), Tocopherol (Vit E), uric acid, glutathione, flavonoids (Benzie, 2003).
Interaction between antioxidants
Synthetic ways antioxidants are interacting one antioxidant protect another against oxidative destruction. Vitamin E can protect beta carotene molecule from oxidation and sparing effect is taking place.