SOME BIOCHEMICAL CHANGES IN HIV POSITIVE PATIENTS ON HIGHLY ACTIVE ANTIRETROVIRAL THERAPY

CHAPTER ONE

INTRODUCTION

The HIV/AIDS (human immunodeficiency virus/ acquired immunodeficiency syndrome) pandemic has been terrorizing humanity over the past three decades. It has undermined the health of so many people, consequently affecting adversely, the work force and the economic stability of so many countries all over the world. World Health Organization (WHO) and United Nations Programme on AIDS (UNAIDS) (2009), estimated that about 33.4 million people world wide were living with AIDS, with 2.7 million new infections per year. Some biochemical abnormalities accompany infection with human immunodeficiency virus. These changes occur as a result of the complications of the disease itself, for example the body’s normal response to infection depletes nutritional stores. Furthermore metabolic stress responses cause catabolism of protein stores, consequently depleting protein stores (Drain et al., 2007; Zaneta et al., 2012). Studies have shown that the antioxidant system of the body is adversely affected in HIV infection, and changes in the activities of its components have been documented by several workers (Pasupathi et al., 2009). Other studies have also stated that ALT and AST activities usually increase in asymptomatic HIV sero-positive patients, signaling liver involvement in HIV infection. Patients especially at the final stage of AIDS may develop, HIV associated nephrophathy (HIVAN) which leads to an increase in their serum creatinine levels (Rudolf and Rodriguez, 2003). There are also changes in protein concentration especially reflecting a decrease in albumin and an increase in C-reactive protein (Drain et al., 2007; Sarro et al.,

2010). The current treatment for HIV infection consists of highly active anti retroviral therapy. These drugs which are classified into, Nucleoside Reverse Transcriptase Inhibitors (NRTI), Non nucleoside reverse transcriptase inhibitors (NNRTI), Protease inhibitors (PT), and Fusion inhibitors (FI) were introduced in 1996 to improve the patients’ quality of life, reduce HIV viraemia, and possibly prolong the life of the patient. They do not cure the patient of HIV or prevent the return once treatment is stopped (Drain et al., 2007).

1.1      Human Immuno-Deficiency Virus (HIV)/Acquired Immune Deficiency Syndrome

(AIDS)

Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the retrovirus called human immunodeficiency virus (HIV) (Sepkowitz, 2001). The virus and disease are often referred to together as HIV/AIDS. The illness interferes with the immune system, making people with AIDS much more likely to get infections, including opportunistic infections and tumors that do not affect people with working immune systems. This susceptibility gets worse as the disease progresses (Weiss, 1993).

HIV is transmitted in many ways, such as: vaginal, oral or anal sex, blood and blood products transfusion, contaminated hypodermic needles, exchange between mother and baby during pregnancy, childbirth and breast feeding. It can be transmitted by any contact of mucous membrane or the blood stream with a body fluid that has the virus in it, such as the blood, semen, vaginal fluid, preseminal fluid, or breast milk from an infected person (CDC, 2005). AIDS was first recognized by the U.S Centers for Disease Control and Prevention in 1981 and its cause, HIV, identified in the early 1980s (Gallo, 2006).

Highly active antiretroviral treatment, slows the course of the disease hence reduces deaths and new infections, but does not cure the disease. Due to the difficulty in treating HIV infection, preventing it is a key aim in controlling AIDS pandemic (Palella et al., 1998).

1.2      Cluster of Differentiation Four (CD4+) T Lymphocyte Count (Cells/mm3)

Human immune deficiency virus primarily infects vital organs of the human immune system suchas CD4+  T cells (a subset of T cells), macrophages and dendritic cells. It destroys CD4+T Cells (Alimonti et al., 2003). Once the number of CD4+ T cells per millimeter cubed, of blood, drops below 200, cellular immunity is lost (Alimonti et al., 2003).

White blood cells (WBC) help prevent and fight infections. A normal count ranges from 4,000-11,

000/mm3 of blood in a healthy adult. There are three main groups of WBCs including; lymphocytes, granulocytes and monocytes. Granulocytes (or poly morphonuclear cells) include; neutrophils, eosinophils and basophils. They make up 55-80% of the total leucocyte count. Monocytes, move into

tissues after about 24 hours of their production and change into macrophages. Lymphocytes are white blood cells that produce antibodies and keep the immune system working. They make up 10-

45% of the WBC count. There are the B cells, the T cells and the natural killer cells (NK cells), T cells are divided into CD4+  cells and CD8+cells. B cells are involved in the production of antibodies.They also deal with infections that are outside the cells. CD8+ cells deal with infections that are inside the cells. CD4+ cells help B cells and CD8+ cells do their job.

HIV infects and destroys the CD4+ T cells. HIV induces the lysis of infected CD4+ T cells. Also cytotoxic T cells, kill infected cells. Generalized immune activation, coupled with the gradual loss of the ability of the immune system to generate new T Cells, all appear to account for the slow decline in CD4+ T cell numbers (Hel et al., 2006). In HIV negative people, normal CD4+ cell counts are 500-

1,500/mm3 of blood. CD4+ cell counts are the best guide for when to start preventive therapy for

opportunistic infections. Antiretroviral therapy is generally recommended when CD4+ cell counts are in the range 200 to 350 cells/mm3 of blood (Siegfried et al., 2010); this range was recently raised to

350 to 500 cells/mm3 (Rathbun, 2013).

1.3      Highly Active Antiretroviral Therapy (HAART)

The advent of HAART has been dated to the 11th International Conference on AIDS in Vancouver, British Columbia, July 7-16, 1996 (Barlett, 2006). HAART has made HIV infection a manageable chronic disease  in patients who  have access to  medication (Rathbun,  2013).  Highly active antiretroviral therapy (HAART) is actually the use of multiple drugs that act on different viral targets. These drugs act on different stages of the HIV life-cycle.

Antiretroviral drugs are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits.

The classes include:-

–           Entry inhibitors (or fusion inhibitors), which interfere with binding, fusion and entry of HIV I to the host cell by blocking receptors that mediate these e.g Maraviroc and Enfuvirtide (Sharon and Lieberman, 2008).

–           Nucleoside reverse transcriptase inhibitors (NRTI) and nucleotide reverse transcriptase inhibitors  (NtRTI)  are  nucleoside  and  nucleotide  analogues  which  inhibit  reverse

transcription e.g. of NRTIs are Deoxythymidine, Zidovudine, Stavudine, Didanosine, Zalcitabine, Abacavir, Lamivudine, Emtricitabine and Tenofovir (Kalyan, 2013).

–           Non-nucleoside reverse transcriptase inhibitors (NNRTI) inhibit reverse transcriptase, by binding to an allosteric site of the enzymes. NNRTIs act as non-competitive inhibitors of reverse transcriptase e.g. nevirapine, delavirdine, efavirenz, rilpivirine (Kalyan, 2013).

–          Integrase inhibitors inhibit the enzyme integrase, which is responsible for integration of viral

DNA into the DNA of the infected cells e.g. Raltegravir and Elvitegravir (Quashie, 2013).

–           Protease inhibitors block the viral protease, necessary to produce mature virus upon budding from the host membrane (Wensing, 2010). Examples are Lopinavir, Indinavir, Nelfinavir, Amprenavir and Ritonavir. Typical combinations include 2 NRTIs + 1PI or 2NRTIs +

1NNRTI (Quashie, 2013).

Fixed dose combinations are multiple antiretroviral drugs combined into a single pill. They include

Combivir        –          Zidovudine + lamivudine

Trizivir           –          abacavir + zidovudine + lamivudine

Kaletra            –          lopinavir + ritonavir Epzicon          –          abacavir + lamivudine Kivexa            –

Truvada          –          tenofovir/emtricitabine

Atripla            –          efaviranz + tenofovir/emtricitabine

Complera        –          rilpivirine + tenofovir/emtricitabine

Stribild            –          elvitegravir + cobicistat + tenofovir/emtricitabine

1.4      Antioxidant System

An antioxidant is a molecule that inhibits the oxidation of other molecules (Sies, 1997). Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals which can start chain reactions. These reactions can damage or cause the death of a cell (Sies, 1997). Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibiting other oxidation reactions. They do these by being oxidized themselves, so they are often reducing agents (Helmut, 1997).

These free radicals are reactive species, generated in the body as a result of metabolic reactions; they are called pro-oxidants. They attack macromolecules, including protein, DNA and lipids causing cellular/tissue damage. These antioxidants are produced either endogenously or received from exogenous sources. They include enzymes such as catalase, glutathione peroxidase, glutathione reductase, superoxide dismutase, minerals such as selenium, zinc, manganese, copper, and vitamins like vitamins A, C and E; also, glutathione. In the body, pro-oxidants and antioxidants maintain a ratio. A shift in this ratio towards pro-oxidants, gives rise to oxidative stress. This oxidative stress may be mild or severe depending on the extent of shift and remains the cause of several diseases such as cardiovascular diseases, neurological diseases, malignancies, renal diseases and several metabolic diseases.

1.4.1   Antioxidant Enzymes

1.4.1.1 Catalase: EC 1.11.1.6

Catalase is a common enzyme found in nearly all living organisms exposed to oxygen. It catalyzes the decomposition of hydrogen peroxide to water and oxygen (Chelikani et al., 2004). It is a very important enzyme in protecting the cells from oxidative damage by reactive oxygen species (ROS). Catalase has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of molecules of hydrogen peroxide to water and oxygen each second (Goodsell, 2004).

Catalase is a tetramer of four polypeptide chains, each over 500 amino-acids long (Boon et al.,

2007). It contains four porphyrin haem (iron) groups that allow the enzyme to react with the hydrogen peroxide. The optimum pH for human catalase is approximately, 7 (Maehly and Chance,

1954); though the enzyme is active between pH 6.8 and 7.5 (Aebi, 1984). The pH optimum for other catalases varies between 4 and 11 depending on the species (Brenda, 2009). Catalase was first noticed in 1811 when Louis Jacques Thenard, who discovered H2O2 (hydrogen peroxide), suggested that its breakdown is caused by an unknown substance. In 1900, Oscar Loew was the first to give it the name catalase, and found it in many plants and animals. The presence of catalase in a microbial or tissue sample can be tested by adding a volume of hydrogen peroxide and observing the reaction. The formation of bubbles (oxygen) indicates a positive result (Boon et al., 2007). Catalase can catalyze  the  oxidation  by  hydrogen peroxide,  of various  metabolites  and  toxins,  including

formaldehyde, formic acid, phenols, acetaldehyde and alcohols. It does so according to the following reactions:

H2O2 +H2R                             2H2O+R

The exact mechanism of this reaction is not known. Any heavy metal ion {such as copper cations in copper (II) sulfate} can act as a non competitive inhibitor of catalase. Also, the poison cyanide is a competitive inhibitor of catalase, strongly binding to the haem of catalase and stopping the enzyme’s action (Gaetani et al., 1996).

1.4.1.1.1         Cellular role of catalase

Hydrogen peroxide is a harmful by product of many normal metabolic processes; to prevent damage to cells and tissues, it must be quickly converted into other, less dangerous substances. To this end, catalase is used by cells to rapidly catalyze the decomposition of hydrogen peroxides into less reactive gaseous oxygen and water molecule (Gaetani et al., 1996). A catalase deficiency may increase the likelihood of developing type 2 diabetes (Laszlo et al., 2001; Laszlo, 2008). Catalase is usually located in a cellular, bipolar environment organelle called the peroxisome (Alberts et al.,

2002). Catalase contributes to ethanol metabolism in the body after its ingestion.

According to recent scientific studies, low levels of catalase may play a role in the graying process of human hair. Hydrogen peroxide is naturally produced by the body and catalase breaks it down. If catalase levels decline, hydrogen peroxide cannot be broken down as well. This allows the hydrogen peroxide to bleach the hair (Wood et al., 2009; Hitti, 2009; Science News, 2009).

1.4.1.2 Glutathione Peroxidase (GPx) (EC I.11.1.9)

Glutathione peroxidase is the general name of an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage. The biochemical function of glutathione peroxidase is to reduce lipid hydro peroxides to their corresponding alcohols and to reduce free hydrogen peroxide to water (Epp et al., 1983).

1.4.1.2.1         Isozymes of glutathione peroxidase

Several isozymes are encoded by different genes, which vary in cellular location and substrate specificity. Glutathione peroxidase 1 (GPx 1) is the most abundant version, found in the cytoplasm of nearly all mammalian tissues, whose preferred substrate is hydrogen peroxide. Gluatathione peroxidase 4 (GPx 4) has a high preference for lipid hydroperoxides; it is expressed in nearly every mammalian cell, though at much lower levels. GPx 2 is an intestinal and extracellular enzyme, while glutathione peroxidase 3 is extracellular, especially abundant in plasma (Muller et al., 2007). GPx 5 is an epididymal androgen-related protein, GPx 6 is of olfactory origin, GPx 7 and GPx 8 are putative.

1.4.1.2.2         Reaction of glutathione peroxidase

The main reaction that GPx catalyzes is:

2GSH + H2O2                   GS-SG- + 2H2O2

Where GSH represents reduced monomeric glutathione and GS-SG represents glutathione disulfide.

The mechanism involves oxidation of the selenol of a selenocyteine residue by hydrogen peroxide. This process leaves the derivative with a seleninic acid (RSe OH) group. The seleninic acid is then converted by GPx to the selenol by a two step process that begins with reaction with GSH to form the GS-SeR and water. A second GSH molecule reduces the GS-SeR intermediate back to the selenol, releasing GS-SG as the by product. A simplified representation is shown below as observed by Krishna and Bhabak (2010).

RSeH + H2O2                       RSeOH + H2O RSeOH + GSH                       GS-SeR + H2O GS-SeR + GSH              GS-SG+RSeH

Glutathione reductase then reduces the oxidized glutathione to complete the cycle. GS-SG+NADPH + H+                       2GSH + NADP+

1.4.1.2.3         Structure of glutathione peroxidase

Mammalian GPx 1, GPx 2, GPx 3 and GPx 4 have been shown to be selenium-containing enzymes;

whereas GP x 6 is a seleno protein in humans with cysteine-containing homologues in rodents. GPx

1, GPx 2 and GPx 3 are homotetrameric proteins; whereas GPx 4 has a monomeric structure. As the integrity of the cellular and subcellular membranes depends heavily on glutathione peroxidase, its antioxidative protective system itself depends heavily on the presence of selenium (Muller et al.,

2007).

Mice genetically engineered to lack glutathione peroxidase I GPx1 (knockout mice) are phenotypically normal and have normal life spans, indicating that this enzyme is not critical for life. However, they may develop cataract at an early age and exhibit defects in muscle satellite cell proliferation (Muller et al., 2007). The bovine erythrocyte enzyme has a molecular weight of

84KDA. Glutathione peroxidase was discovered by Mills (1957).

1.4.1.3 Glutathione Reductase

This is an enzyme (EC 1.8.1.7) that reduces glutathione disulfide (GSSG) to the sulfhydryl form GSH, which is an important cellular antioxidant (Meister, 1988; Mannervik, 1987). For every mole of oxidized glutathione (GSSG), one mole of NADPH is required to reduce GSSG to GSH. The enzyme forms a flavine adenine dinucleotide (FAD) bound homodimer.

In cells exposed to high levels of oxidative stress like red blood cells, up to 10% of the glucose consumption may be directed to the pentose phosphate pathway (PPP) for production ofthe NADPH needed for this reaction. In the case of erythrocytes, if the PPP is non-functional, then the oxidative stress in the cell will lead to cell lysis and anaemia (Murray et al., 1999). The activity of glutathione reductase is used as indicator for oxidative stress. The activity can be monitored by the NADPH consumption or GSH production (Smith et al., 1988). The reduction of GSSG to GSH plays an important role in the bactericidal function of phagocytes hence glutathione reductase facilitates host defense and plays an important role in immunity (Yan et al., 2012).

1.4.1.4 Superoxide Dismutase

Superoxide dismutases (SOD, EC 1.15.1.1) are enzymes that catalyse the dismutation of superoxide (02-) into oxygen and hydrogen peroxide. Thus, they are an important antioxidant defense in nearly all cells exposed to oxygen (Borgstahl et al., 1996).

1.4.1.4.1         Reactions of superoxide dismutase

The SOD–catalyzed dismutation of superoxides may be written with the following half-reactions:–          M (n+1) +-SOD + 02-                 Mn+-SOD + 02

–          Mn+-SOD + 0-

+ 2H+                       M(n+1)+ SOD + H 0

2                                                        –                      2   2

Where M=cu (n=1); Mn (n=2); Fe (n=2); Ni (n=2)

In this reaction the oxidation state of the metal cation oscillates between n and n+1 (Borgstahl et al.,

1996). Irwin Fridovich and Joe McCord discovered the activity of superoxide dismutase. SODs were previously known as a group of metallo proteins with unknown function; for example, CuznSOD was known as erythrocuprein and as the veterinary antiinflamatory drug “orgotein” McCord and Fridovich (1988). Likewise, Brewer (1967) identified a protein that later became known as superoxide dismutase as an indophenol oxidase by protein analysis of starch gels using the phenazine tetrazolium technique (Brewer, 1967).

Several common forms of SOD exist. They are proteins co-factored with zinc and copper or manganese, iron or nickel. Thus there are three major families of superoxide dismutase, depending on the metal cofactor: Cu/Zn (which binds both copper and zinc), Fe and Mn types (which bind either iron or manganese) and the Ni type which binds nickel.

1.4.1.4.2   Copper and Zinc as componets of superoxide dismutase

This is most commonly seen in the cytosol of virtually all eukaryotic cells. Cu-ZnSOD is a homodimer of molecular weight 32,500 (Richardson et al., 1975). It is an eight stranded “Greek key” beta barrel, with the active site held between the barrel and two surface loops. The two subunits are tightly joined back-to-back mostly by hydrophobic and some electrostatic interactions. The ligands of the copper and zinc are six histidine and one aspartate side-chains; one histidine is bound between the two metals (Tainer et al., 1983).

Iron or manganese SOD (Fe-SOD or Mn-SOD) is seen in prokaryotes, protists and in mitochondria. E. coli and many other bacteria contain a form of the enzyme with iron (Fe-SOD); some bacteria contain Fe-SOD, others Mn-SOD), while some contain both. The 3D structures of the homologous Mn and Fe superoxide dismutases have the same arrangement of alpha-helices and their active sites contain the same type and arrangement of amino acid side-chains (Borgstahl et al., 1992).

1.4.1.4.3         Nickel–Superoxide dismutase

This is also prokaryotic; it has a hexameric structure built from right handed 4-helix bundles, each containing N-terminal hooks that chelate a Ni ion (Barondeau et al., 2004; Wuerges et al., 2004). Three forms of superoxide dismutase are present in humans, and in all other mammals and most chordates. SOD1 is located in the cytoplasm, SOD2 in the mitochondria and SOD3 is extracellular. The first is a dimer (consists of two units) whereas the others are tetramers (four sub units). SOD1 and SOD3 contain copper and zinc, whereas SOD2 the mitochondrial enzyme, has manganese in its reactive centre. The genes are located on chromosomes 21, 6, and 4. Simply stated, SOD out- competes damaging reactions of superoxide, thus protecting, the cell from superoxide toxicity. The reaction of superoxide is  spin  forbidden; this  means that  its  main reactions are with itself (dismutation) or with another biological radical such as nitric oxide (NO) or with a transition-series metal. The superoxide anion radical (O2-) spontaneously dismutes to O2 and hydrogen peroxide

(H2O2) quite rapidly (~105  M-1s-1  at pH 7). SOD is necessary because superoxide reacts with

sensitive and critical cellular targets. For example, it reacts with the NO radical, and makes toxic peroxynitrite. 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 0.1mM). In contrast, the reaction of superoxide with SOD is first order with respect to superoxide concentration. Moreover, superoxide dismutase has the largest Kcat/Km (an approximation of catalytic efficiency) of any known enzyme (-~7×109  M-1s-1) (Heinrich et al., 2006). This reaction is only limited by the frequency of collision between itself and superoxide. That is, the reaction rate is diffusion limited. Even at the subnanomolar concentrations achieved by the high concentrations of SOD within cells,

superoxide inactivates the citric acid cycle enzyme aconitase, can poison energy metabolism and releases potentially toxic iron. Aconitase is one of several iron-sulfur containing dehydratases in metabolic pathways shown to be inactivated by superoxide (Gardner et al., 1995).

1.4.1.4.4         Physiology of superoxide dismutase

Superoxide is one of the main reactive oxygen species in the cell. Consequently 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, amid massive oxidative stress (Li et al., 1995). Mice lacking SOD1 develop a wide range of pathologies, including hepatocellular carcinoma (Elchuri et al., 2005), and an acceleration of age- related muscle mass loss (Muller et al., 2006), an earlier incidence of cataracts and a reduced life span. Mice lacking SOD3 do not show any obvious defects and exhibit a normal life span, though they are more sensitive to hyperoxic injury (Sentman et al., 2006). Knockout mice of any SOD are more sensitive to the lethal effects of superoxide generating drugs, such as paraquat and diquat.

Mutations in SOD1 can cause familial amyotrophic lateral sclerosis (ALS a form of motor neuron disease) (Deng et al., 1993; Al-chalabi and Leigh, 2000; Conwit, 2006; Milani et al., 2011). Over expression of SOD1 has been linked to the neural disorders seen in Down syndrome (Groner et al.,

1994). In recent years it has become more apparent that in mice, the extra cellular superoxide

dismutase (SOD3 EC SOD) is critical in the development of hypertension (Gongora et al., 2006; Lob et al., 2010). In other studies, diminished SOD3 activity was linked to lung diseases such Acute Respiratory Distress Syndrome (Young et al., 2006; Gongora et al., 2008; Ganguly et al., 2009).

1.4.2   Antioxidant Molecule

1.4.2.1 Glutathione

The international union of pure and applied chemists (IUPAC) name of glutathione is (25)- 2 amino

-4-{[(IR)-1- [(Carboxymethyl) carbamoyl -2- Sulfanyl ethyl] carbamoyl} butanoic acid

1.4.2.1.1         Other names of glutathione

R-L-glutamyl –L-Cysteinylglycine

(25)-2- Amino-5- [[(2R) -1- (Carboxymethyl amino) -1- oxo -3- Sulfanyl propan-2-yl] amino] -5- oxopentanoic acid.

Molecular formular – C10H17N3O6S Molar Mass    – 307.32g mol-1

Glutathione (GSH) is a tripeptide with a gamma peptide linkage between the amine group of cysteine (which is attached by normal peptide linkage to glycine) and the carboxyl group of the glutamate side-chain. It is an antioxidant, preventing damage to important cellular components caused by reactive oxygen species such as free radicals and peroxides (Pompella et al., 2003). Thiol groups are reducing agents existing at a concentration of approximately 5mM in animal cells. Glutathione reduces disulphide bonds formed within cytoplasmic proteins to cysteines byserving as an electron donor. In the process glutathione is converted to its oxidized form glutathione disulphide (GSSG), also  called  L-(-)  glutathione. Once  oxidized, glutathione can  be  reduced back by glutathione reductase, using NADPH as an election donor (Couto et al., 2013). The ratio of reduced glutathione to oxidized glutathione within cells is often used as a measure of cellular toxicity (Pastore, 2003). Since glutathione can be synthesized in the body; it is not an essential nutrient. It can be synthesize from the amino acids L-cysteine, L-glutamic acid and glycine. The sulphhydryl (thiol) group (SH) of cysteine is the rate-limiting factor in cellular glutathione synthesis, since this amino acid is relatively rare in food stuffs.

Cells make glutathione in two adenosine triphosphate (ATP) dependent steps:

–        First, gamma-Glutamyl cysteine is synthesized from L-glutamate and cysteine using the enzyme gamma-glutamyl cysteine synthetase (glutamate cysteine ligase, GCL). This reaction is the rate limiting step in glutathione synthesis (White et al., 2003)

–        Second, glycine is added to the C-terminal of gamma –glutamyl cysteine by the enzyme glutathione synthetase.

1.4.2.1.2         Functions of Glutathione

Glutathione exists in both reduced (GSH) and oxidized (GSSG) States. In the reduced state, the thiol group of cysteine is able to donate a reducing equivalent (H++ e-) to other unstable molecules such as reactive oxygen species (ROS). In donating an electron, glutathione itself becomes reactive, but readily reacts with another reactive glutathione, to form glutathione disulphide (GSSG) probablydue to the relatively high concentration of glutathione in cells. GSH can be regenerated from GSSG by the enzyme glutathione reductase (GR) (Couto et al., 2013) and for every GSSG, and NADPH, two reduced GSH molecules are gained. In healthy cells, more than 90% of the total glutathione pool is

in the reduced form (GSH) and less than 10% are in the disulphide form (GSSG). An increased

GSSG-GSH ratio is indicative of oxidative stress. Some functions of glutathione are:-

–        It is a major endogenous antioxidant and participates directly in the neutralization of free radicals and reactive oxygen species, as well as maintaining exogenous antioxidants such as vitamins C and E in their reduced (active) forms (Scholz et al., 1964)

–        It regulates the nitric oxide cycle, which is critical for life but can be dangerous if unregulated (Clementi et al., 1999)

–        It is used in metabolic and biochemical reactions such as DNA Synthesis and repair, protein synthesis, prostaglandin synthesis, amino acid transport, and enzyme activation. Thus every system in the body can be affected by the state of the glutathione system, especially the immune system, the nervous system, the gastrointestinal system and the lungs.

–        It also participates in detoxification and leukotriene synthesis. Low glutathione is strongly implicated in wasting and negative nitrogen balance, (Droge and Holm, 1997) as seen in cancers, AIDS, sepsis, trauma, burns and even athletic overtraining. Glutathione supplementation can oppose this process, and in AIDs for example, results in improved survival rates (Herzenberg et al., 1997)

1.4.3   Antioxidant Elements

1.4.3.1 Selenium

This is a chemical element with symbol, Se and atomic number, 34. It is a non metal with properties that are intermediate between those of its periodic table column-adjacent chalcogen element sulfur and tellurium. Selenium (Greek selene meaning “Moon”) was discovered in 1817 by Jons Jacob Berzelius and Johan Gottlieb Gahn who noted the similarity of the new element to the previously known tellurium (Berzelius, 1818). Selenium salts are toxic in large amounts, but trace amounts are necessary for cellular function in many organisms, including all animals. Selenium is a component of the antioxidant enzyme glutathione peroxidase and thioredoxin reductase (which indirectlyreduce certain oxidized molecules in animals and some plants). It is also found in three deiodinase enzymes, which convert one thyroid hormone to another (Ruyle, 2009).

Selenium came to medical notice because of its toxicity to human beings working in industries. In

1954 the first hint of specific biological functions of selenium were discovered in micro organisms (Pinset, 1954; Stadman, 2002). Its essentiality for mammalian life was discovered in 1957 (Schwarz and Foltz 1957; Old field, 2006). In living systems, selenium is  found in the amino  acids selenomethionine, selenocysteine and methyl selenocysteine. Although, selenium is toxic in large doses, it is an essential micronutrient for animals. In humans, selenium is a trace element nutrient that functions as cofactor for reduction of antioxidant enzymes such as glutathione peroxidase (Ruyle, 2009) which catalyze certain reactions that remove reactive oxygen species such as hydrogen peroxides and organic hydroperoxides.

GSH –Px

2GSH + H2O2                         –GSSG  +  2H2O

Selenium also plays a role in the functioning of the thyroid gland and in every cell that uses thyroid hormone, by participating as a cofactor for the three of the four known types of thyroid hormone deiodinases. These use selenium as the otherwise rare amino acid selenocysteine (Hatfield and Gladyshev, 2009).

Selenium may inhibit Hashimoto’s disease in which the body’s own thyroid cells are attacked as alien. A reduction of 21% on thyroid peroxidase antibodies (TPO antibodies) was reported with the dietary intake of 0.2mg of selenium. Increased dietary selenium intake, reduces the effect of mercury toxicity (Ralston et al., 2008) and it is now recognized that the molecular mechanism of mercury toxicity involves irreversible inhibition of selenoenzymes that are required to prevent and reverse oxidative damage in brain and endocrine tissues (Carvalho et al., 2008; Ralston and Raymond,

2010). Dietary selenium comes from nuts, cereals, meat, mushrooms, fish and eggs. Also Brazil nuts, kidney, tuna, crab and lobster (Barclay et al., 1995; Office of Dietary Supplement). The human body content of selenium is believed to be in the 13-20 mg range (Schroeder et al., 1970) selenium deficiency is rare in healthy, well nourished individuals. It can occur in patients with severely compromised intestinal function, those undergoing total parenteral nutrition (Ravaglia et al., 2000) and in those of advanced age (over 90). Also, people dependent on food grown from selenium deficient soil are at risk. Selenium deficiency as defined by low (<60% of normal) seleno enzyme activity levels in brain and endocrine tissues, only occurs when a low selenium status is linked with

an additional stress, such as high exposure to mercury (Ralston and Raymond 2010). A number of correlative epidemiological studies have implicated selenium deficiency (as measured by blood levels) in a number of serious or chronic diseases, such as cancer, diabetes, (IP, 1998) HIV/AIDs (Rayman, 2000) and tuberculosis (Kassu et al., 2005).

1.4.3.2 Zinc

Zinc is an essential mineral that is naturally present in some foods, added to others and available as a dietary supplement. It is involved in numerous aspects of cellular metabolism. It is required for the catalytic activity of over 100 enzymes (Sandstead, 1994; U.S Institute of Medicine, 2001) and it plays a role in immune function (Solmons, 1998; Prasad, 1995), protein synthesis, wound healing (Heyneman, 1996), DNA synthesis, (U.S Institute of Medicine, 2001; Prasad, 1995) and cell division (Prasad, 1995). Zinc also supports normal growth and development during pregnancy, child hood, and adolescence (Simmer and Thompson, 1985; Fabris and Mocchegiani, 1995; Maret  and Sandstead, 2006) and it is required for proper sense of taste and smell (Prasad et al., 1997). A daily intake of zinc is required to maintain a steady state because the body has no specialized zinc storage system (Rink and Gabriel, 2000).

A wide variety of foods contain zinc, including, oysters, red meat, poultry, beans, nuts. Crabs, lobster, whole grains, cereals, dairy products (U.S Institute of Medicine, 2001) (U.S Department of Agriculture, 2011). Phytates, which are present in whole grain breads, cereals and legumes bind zinc and inhibits its absorption (Wise, 1995; Sandstrom, 1997; U.S. Institute of Medicine, 2001). Thus, the bioavailability of zinc from grains and plant foods is lower than that from animal foods; although many grain and plant based foods are still good sources of zinc (U.S. Institute of Medicine, 2001). The (daily value) DV of zinc in children aged 4 and older and in adults is 15mg (US Department of Agriculture, 2011).

1.4.3.2.1         Zinc Deficiency

This is characterized by growth retardation, loss of appetite and impaired immune function. In more severe cases, zinc deficiency causes hair loss, diarrhea, delayed sexual maturation, impotence, hypogonadism in males, and eye and skin lesions (US Institute of Medicine, 2001; Prasad, 2004; Wang and Busbey, 2005 Maret and Sandstead, 2006), weight loss, delayed healing of wounds, taste abnormalities, and mental lethargy can also occur (Hambidge, 1989; Heyneman, 1996; Krasovec and

Frank, 1996; Nishi, 1996; Ploysangam et al., 1997; King and Cousins, 2005; Maret and Sandstead,

2006). Groups at risk of zinc inadequacy or deficiency need to include good sources of zinc in their daily diets. Supplemental zinc might also be appropriate in certain situations. Risk groups include people with gastro intestinal disorders e.g. after gastrointestinal surgery or those suffering from digestive disorders (such as ulcerative colitis, Crohn’s disease, and short bowel syndrome), which can   undermine zinc  absorption and  increase endogenous zinc  losses (Valberg et  al.,1986; Hambidge, 1989; Naber et al., 1998; US Institute of Medicine, 2001) other diseases associated with zinc deficiency include, malabsorption syndrome, chronic liver disease, chronic renaldisease, sickle cell disease, diabetes, malignancy, and other chronic illnesses (Prasad, 2003) chronic diarrhea also leads to excessive loss of zinc (Prasad, 2004). Vegetarians (Hunt, 2003; American Dietetic Association (2003), pregnant and lactating women are also at risk of zinc deficiency(Caulfield et al.,

1998; Krebs, 1998). Older infants who are still being exclusively breastfed, (US Department of Medicine, 2001) (Hambidge and Krebs, 2007); people with sickle cell disease (Leonard et al., 1998; Prasad, 2002) and alcoholics; this is because ethanol consumption decreases intestinal absorption of zinc and increases urinary zinc excretion (Prasad, 2002).

1.4.4   Antioxidant Vitamins

1.4.4.1 Vitamin C (Ascorbic Acid)

Vitamin C or L-ascrobic acid or ascorbate (anion of ascorbic acid) is an essential nutrient for humans and certain other animal species. Vitamin C refers to a number of vitamers that have vitamin C activity in animals, including ascorbic acid and its salts, and some oxidized forms of the molecule like dehydroascorbic acid. Vitamin C is a cofactor in at least 8 enzymatic reactions, including several collagen synthesis reactions that when dysfunctional, cause the most severe symptoms of scurvy (Food Standard Agency, 2007). In animals these reactions are especially important in wound healing and in preventing bleeding from capillaries. Ascorbate may also act as an antioxidant against oxidative stress (Padayatty et al., 2003). Ascorbic acid is widely used as food additive, to prevent oxidation. It is a weak sugar acid, structurally related to glucose. In biological systems, ascorbic acid can be found only at low pH, but in neutral solutions above pH 5 it is predominantly found in the ionized form, ascorbate.

1.4.4.1.1         Biological Significance of Vitamin C

Vitamin C acts as a reducing agent, donating electrons to various enzymatic and a few non- enzymatic reactions. The one and two-electron oxidized forms of vitamin C, Semidehy droascorbic acid and dehydro ascorbic acid, respectively can be reduced in the body byglutathione and NADPH- dependent enzymatic mechanisms (Meister, 1994; Michela and Frei, 2002). The presence of glutathione in cells and extracellular fluids helps maintain ascorbate in a reduced state (Gropper et al., 2005). Many plants and animals are able to synthesize vitamin C, through a sequence of enzyme–driven steps, which convert monosaccharides to vitamin C. In plants mannose and galactose are converted to ascorbic acid (Wheeler et al., 1998). In some animals, glucose extracted from glycogen is  utilized (Banhegyi and  Mandi,  2001).  Animals  of the  major  primate  suborder Haplorrhini, which include humans, have lost the ability to synthesize ascorbic acid. These animals all lack the L-gulonolactone oxidase (GULO) , which is required in the last step of Vit C synthesis, because they have a differing non-synthesizing gene for the enzyme (pseudogene GULO) (Harris, 1996).

Although, the body’s maximal store of vitamin C is largely determined by the renal threshold for blood, which is 1.5mg/dl in men and 1.3mg/dl in women, many tissues of the body, maintain Vitamin C concentrations far higher than in blood. Biological tissues that accumulate over 100 times the level in blood plasma of vitamin C are the adrenal glands, pituitary, thymus, corpus luteum and retina. Those with 10 to 50 times the concentration present in blood plasma include the brain, spleen, lung, testicle, lymph nodes, liver, thyroid, small intestinal mucosa, leukocytes, pancreas, kidneyand salivary glands. Ascorbic acid can be oxidized (broken down) in the body by the enzyme L- ascorbate oxidase.

1.4.4.1.2 Deficiency of vitamin C (a) Scurvy

This is an avitaminosis resulting from lack of vitamin C, since without this vitamin; the synthesized collagen is too unstable to perform its function. Brown spots are seen on the skin, spongy gums result, there is also bleeding from all mucous membranes. The patient looks pale and feels depressed. In advanced cases open suppurating wounds, loss of teeth and death may result. The human body

can store only a limited amount of vitamin C (Johnston et al., 1996); hence body stores are easily depleted if fresh supplies are not consumed.

It has been shown that smokers who have diets poor in vitamin C are at a higher risk of developing lung borne diseases, than smokers who have higher concentrations of vitamin C in the blood (BBC News and Cambridge University, 2000; Zieve et al., 2011). Nobel prize winner Linus Pauling and Canadian researcher G.G. Willis have asserted that chronic long term low blood levels of Vitamin C (“Chronic Scurvy”) is a cause of atherosclerosis (Rath and Pauling, 2000). Moderately higher blood levels of vitamin C measured in healthy persons have been found to be prospectively correlated with decreased risk of cardiovascular disease and ischemic  heart disease and an increase in life expectancy (Khaw et al., 2001).

1.4.4.1.3         The Role of the Vitamin in Mammals

Vitamin C is a co-factor for several enzymses. Infact it is an electron donor for eight different enzymes (Levine et al., 2000). It is involved in the synthesis of collagen, carnitine, neurotransmitters, also in the synthesis and catabolism of tyrosine and metabolism of microsome (Gropper et al., 2005). During biosynthesis, ascorbate acts as a reducing agent, donating electrons and preventing oxidation to keep iron and copper atoms in their reduced states.

The enzymes for which vitamin C is a cofactor are:

–        Three enzymes (Prolyl-3-hydroxylase, Prolyl-4-ydroxylase, and Lysyl hyroxylase) that are required for hydroxylation allows the collagen molecule to assume its triple helix structure, and thus Vitamin C is essential to the development and maintenance of scar tissue, blood vessels and cartilage.

–        Two  enzymes  (E-N-  trimethyl-L-  Lysine  hydroxylase  and  gamma-butyrobetaine hydroxylase) that are necessary for the synthesis of carnitine (Dunn et al., 1989; Rebouche, 1991). Carnitine is essential for the transport of fatty acids into mitochondria for ATP generation.

–        The remaining three enzymes have the following functions in common but have other functions as well.

–        Dopamine betahydroxylase participates in the biosynthesis of norepinephrine from dopamine (Kaufman, 1974; Levine et al., 1992)

–        Peptidylglycine alpha-amidating monoxygenase adds amide groups to peptide hormones greatly increasing their stability (Eipper et al., 1992; Eipper et al., 1993).

–         4-hydroxy phenylpyruviate dioxygenase modulates tyrosine metabolism (Lindblad et al., 1970; Englard and Seifter, 1986).

Ascorbic acid is also well known for its antioxidant activity, acting as a reducing agent to lessen oxidative stress. It has an impact on cardiovascular diseases, hypertension, chronic inflammatory disease, diabetes (Kelly, 1998; Tak et al., 2000; Good Year-Bruch and Pierce 2002; Mayne, 2003). Individuals with oxidative stress have lower blood levels of ascorbate (lower than 45µmol/L) compared to healthy individuals (61-80µmol/L) (Schorah et al., 1996).

1.5      Immune System

It is found in high concentration in immune cells and is consumed quickly during infections. The mechanism of its interaction with the immune system is not yet certain, but it has been hypothesized that it modulates the activities of phagocytes, the production of cytokines and lymphocytes and the number of cell adhesion molecules in monocytes (Preedy et al., 2010).

1.6      Antihistamine

Ascorbic acid is a natural antihistamine, because it both prevents histamine release and increases the detoxification of histamine (Clemetson, 1980; Johnston et al., 1992; Johnston et al., 1996). Dietary sources of ascorbic acid are fruits and vegetables including Kakadu plum, camu camu fruit, meat grape, apricot, water melon, Banana, onion, cherry, peach and carrot. Animal sources include calf liver, beef liver, oysters, codroe, and human breast milk.

1.7       Vitamin E

Vitamin E refers to a group of fat-soluble compounds that include tocopherols and tocotrienols (Brigelius-Flohe and Traber, 1999). As a fat soluble antioxidant, it stops the production of reactive oxygen species formed when fat undergoes oxidation (National Institute of Health, 2009; Herrera,

2001; Packer et al., 2001).

1.7.1   Forms of Vitamin E

The eight forms of vitamin E are divided into two groups, four are tocopherols and four are tocotrienols. They are identified by prefixes alpha-(α -), beta-(β-), gamma-(-), and delta – (ѕ-).

1.7.1.1     Tocopherol

α – Tocopherol is an important lipid-soluble antioxidant, which performs as an antioxidant in the glutathione peroxidase pathway (Wefer and Sics, 1988). It protects cell membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction (Herrera, 2001; Traber and Atkinson, 2007). The oxidized α – tocopheroxyl radicals produced in this process may be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol (Wang, and Quinn, 1999). Gamma-tocopherol is a nucleophile that can react with electophilic mutagens (Brigelius-Flohe and Traber, 1999).

1.7.1.2     Tocotrienols

These are sparsely studied, (Traber and Packer, 1995; Traber and Sics, 1996; Senand Roy, 2004) but they seem to be the more potent antioxidants in the vitamin E family. Studies have suggested that they protect neurons from damage (Sen and Roy, 2006) and that they reduce cholesterol (Das et al.,

2008). They do this by inhibiting the activity of HMG-CoA reductase. Delta-tocotrienol blocks processing of sterol regulatory element-binding proteins (SREBPs). Oral consumption of tocotrienols is also thought to protect against stroke-associated brain damage in vivo (Khanna et al.,

2005).

1.7.1.2.1         Functions of Vitamin E

Vitamin E has many biological functions, the antioxidant function being the best known (Bell, 1987).

–           It acts as a peroxyl radical scavenger, preventing the propagation of free radicals in tissues, by reacting with them, to form a tocopheryl radical, which will then be reduced by a hydrogen donor (such as vitamin C) and thus returning it to its reduced state (Traber et al.,

2011). As it is fat soluble, it is incorporated into cell membranes which protects them, from oxidative damage.

–           It is an enzymatic activity regulator, for example protein kinase C (PKC), which plays a role in smooth muscle growth, can be inhibited by α -tocopherol. α -Tocopherol has a stimulatory effect on the dephosphorylation enzyme, protein phosphatase 2A, which in turn cleaves phosphate groups from PKC, leading to its deactivation, bringing the smooth muscle growth to a halt (Schneider, 2005).

–           Vitamin E has an effect on gene-expression. Macrophages rich in cholesterolare found in the atherogenetic tissue. Scavenger receptor CD36 is a class B scavenger receptor found to be up-regulated by oxidized low density lipoprotein (LDL) and binds it: (Devaraj et al, 2001).

Treatment with ą- tocopherol was found to down regulate the expression of the CD36 scavenger receptor gene and the scavenger receptor class A (SR-A) (Devaraj et al., 2001) and modulates expression of the connective tissue growth factor (CTGF) (Azzi and Stocker, 2000; Villacorta et al., 2003).

The CTGF gene, when expressed, is responsible for the repair of wounds and regeneration of the extracellular tissue lost or damaged during atherosclerosis (Villacorta et al., 2003). Vitamin E also plays a role in neurological functions (Muller, 2010) and inhibition of platelet aggregation (Dowd and Zheng, 1995; Brigelius-Flohe and Davies, 2007; Atkinson et al., 2008). Vitamin E also protects lipids and prevents the oxidation of poly unsaturated fatty acids (Whitney and Sharon, 2011).

1.7.1.2.2         Deficiency of Vitamin E

This results in spinocerebellar ataxia, myopathies, peripheral neuropathy, ataxia, skeletal myopathy, retinopathy, impairment of the immune response and red blood cell destruction (Knowdley et al.,

1992; Brigelius-Flohe and Traber, 1999).

1.7.1.2.3         Dietary Sources of Vitamin E Wheat germ oil, nuts e.g. hazel nuts, palm oil, green leafy vegetables (spinach, turnips, beet greens), avocados, sweet potatoes, mangoes, tomatoes, papayas and lettuce are the dietary sources of tocopherols.

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