Rizal Technological University

College of Arts and Sciences

Department of Biology

A research paper in Immunology:

Effects of Dietary Antioxidants on the Function of the Immune System

Submitted by:

Jose, Louise Vironica D.

CAS-02-501A

Submitted to:

Professor Marita Geroy

Immunology Instructor

Introduction

The ability to interact with the world and remain healthy is dependent to a large extent on

the healthy functioning of the immune system, which is the first line defense of the human body,

since it is responsible for fighting foreign invaders that enters the system of the human body,

such as pathogenic bacteria and viruses; and also it destroys cells within the body when it shows

the signs of being cancerous. The immune system could be an orchestra, because it contains

many different instruments that works harmoniously together with one goal and that is to defend

and protecting the human body from foreign substances that can cause damage to it. And, like an

orchestra itself, the immune system has different components that are suppose to play their roles

at the same time by cell-to-cell communication. There is a vast variety of components in regards

to the immune system from the structural barriers to the specific messenger molecules to cause

the action of the other components and also to stop the action. Thus, nutritionally supporting the

immune system means supporting all the different components that comprises the whole

orchestra. The body’s zillion or so cells always faces formidable threats that ranges from the lack

of food to the infections caused by viruses, thereby making the immune system continuously

fight off these foreign invaders.

Exogenous sources such as ozone, exposure to UV radiation via sunlight, and cigarette

smoke, which stimulates the production of free-radicals. These free radicals may come in many

shapes, sizes and chemical configurations; but even though they may differ in some aspect they

also share a similarity, which is having a voracious appetite for electrons. Whereas, these free

radicals steals electrons from any nearby substances that will yield them. Thus, the electron theft

can radically alter the “loser’s” structure or function. Free radical damage can change the

encoded amino acids that are encased in a DNA strand. This damage causes certain diseases such

as heart attack, cancer and etcetera, since it results into the low-density lipoprotein molecule

more likely to get trapped in an artery wall. Or it can alter a cell’s membrane, thereby changing

the flow of what enters and leaves within the cell. Free radical molecules have also endogenous

sources wherein they are generated during normal cellular metabolism and also made

deliberately.

Free radicals are highly-reactive molecules containing one or more than unpaired election

in the outer orbit such as oxygen free radicals are superoxide, hydroxyl, peroxyl (RO2), alkoxyl

(RO), and hydroperoxyl (HO2) radicals; there are two nitrogen free radicals such as nitric oxide

and nitrogen dioxide (NO2). Reactive oxygen species, also known as ROS, is collective term that

includes not only oxygen-centered radicals but also some of which are, non-radical derivatives of

oxygen such as superoxide anion (O2-) and hydroxyl radical (OH) and hydrogen peroxide

(H2O2). In producing a hydroxyl radical, which the most reactive and damaging of the oxygen

free radicals, H2O2 plays a major role, since it can easily breakdown particularly in the presence

of transition metal ions such as iron (Fe2+)

Oxygen radicals exert critical actions such as signal transduction, gene transcription, and

regulation of soluble guanylate cyclase activity in the cells. Also, nitrogen oxide (NO) is one of

the most widespread signaling molecules and participates in virtually in every cellular and organ

function in the body. Endothelial cells that produce physiologic levels of NO, that are essential in

regulating the relaxation and proliferation of vascular smooth muscle cells, leukocyte adhesion,

platelet aggregation, angiogenesis, thrombosis, vascular tone, and hemodynamics; it is also

generation by activated macrophages that serves as an important mediator of the immune

response. Free radicals induce oxidative stress, which is balanced by the body’s endogenous

antioxidant systems with an input from the co-factors, and by the ingestion of exogenous

antioxidants. Hence, as oxidants and inhibitors of enzymes that contains an iron-sulfur center,

free radicals and other reactive species cause the oxidation of biomolecules (e.g., amino acids,

proteins, lipids and DNA), that leads into cell injury, formation of cataracts and even death. Free

radical production in the cells can significantly increased by certain toxic called ‘redox-cycling’

compounds such as various drugs and CCl4.

ROS are produced in vivo, since it is majorly produced by the mammalian mitochondria

and enzymes is another endogenous source. As organisms have evolved antioxidant defense

systems are either to prevent the generation of ROS or to intercept any that is being produced.

They exist in both the aqueous and membrane compartments of the cells and can be enzymes or

non-enzymes. Catalase and glutathione peroxidase are enzymes that can safely decompose

peroxides, particularly H2O2 that are produced during the respiratory burst that involves in

microbial killing n phagocytic cells, while superoxide dismutase that intercepts or ‘scavenges’

free radicals. Most free-radical scavengers are not enzymes, and many can be obtained through

diet. a-tocopherol is the most important in cell membranes, which is a major member of the

vitamin E family. This molecule acts as a ‘chain-breaking anti-oxidant’, that intercepts lipid

peroxyl radicals and in so terminating lipid peroxidation chain reactions. Another group of lipid-

soluble compounds that can also act as antioxidants are the carotenoids, such as β-carotene,

lycopene and lutein; these compounds can be found in highly-pigment fruits and vegetables (e.g.,

mangoes, tomatoes, spinach and etcetera,). The polyene structure of these compounds allows the

molecules to quench, or to inactivate, singlet oxygen and free-radicals. Ascorbic acid, also

known as vitamin C, that play a role in ‘sparing’ vitamin E that regenerates a-tocopherol from

the oxidized tocopheroxyl radical.

As viewed from the perspective of a ‘two-faces’ with ROS as one face while antioxidants

is the other, it has been thought that tipping the balance in favor of the ROS. Due to the fact, that

ROS is a major contributor to several degenerative disorders such as cancer, chronic diseases,

arthritis and cardiovascular diseases; also to the ageing process, in general. Strong associations

between diets that are rich in antioxidant nutrients and a reduced incidence of cancer have been

observed in numerous epidemiological studies, and it has been suggested that a boost to the

body’s immune system by antioxidants might, at least may taken part in, account for this

findings. It is probably crucial to attempt to keep the balance of ROS to antioxidants levels as

possibly. Thus, ideally by the dietary means rather than by taking supplements, from as early age

as possible, to prolong, if not prevent, the onset of many-age related disorders.

The immune system is particularly sensitive to oxidative stress. As the immune cells

relies heavily on cell-cell communication, particularly via membrane- bound receptors for

working more effectively. Cell membranes that are rich in polyunsaturated fatty acids which, if

peroxidized, can lead to a loss of membrane integrity, altered membrane fluidity, and that could

result into the alterations in intracellular signaling and cell function. In addition, the production

of ROS by phagocytic immune cells can damage the cells themselves, if they are not sufficiently

protected by antioxidants.

Terminologies

Angiogenesis

The physiological process through which new blood vessels form from pre-existing

vessels.

Cytokines

Cells of the immune system communicate with one another by releasing and responding

to chemical messengers

Dismutase

is an enzyme that catalyzes a dismutation reaction

Dismutation reaction

A disproportionation reaction, especially in a biological context, in which oxidized and

reduced forms of a chemical species are produced simultaneously

Ewes

A female sheep

Glucocorticoids

A class of steroid hormones that bind to the glucocorticoid receptor, which is present in

almost every vertebrate animal cell

Glutathione peroxidase (GPx)

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 hydroperoxides to their corresponding alcohols

and to reduce free hydrogen peroxide to water.

Hydroperoxyl

Also known as the perhydroxyl radical, is the protonated form of superoxide with the

chemical formula HO2.

Hydroxyl radical

•HO, is the neutral form of the hydroxide ion. Hydroxyl radicals are highly reactive and

consequently short-lived; however, they form an important part of radical chemistry.

Immunosuppressive

Suppression of the immune response, as by drugs or radiation, in order to prevent the

rejection of grafts or transplants or to control autoimmune diseases. Also called

immunodepression.

Leukotriene B4

A leukotriene involved in inflammation. It is produced from leukocytes in response to

inflammatory mediators and is able to induce the adhesion and activation of leukocytes

on the endothelium, allowing them to bind to and cross it into the tissue.  In neutrophils,

it is also a potent chemoattractant, and is able to induce the formation ofreactive oxygen

species and the release of lysosome enzymes by these cells. It is synthesized

by leukotriene-A4 hydrolase from leukotriene A4.

Metallothionein

S family of cysteine-rich, low molecular weight (MW ranging from 500 to

14000 Da) proteins. They are localized to the membrane of the Golgi apparatus. MTs

have the capacity to bind both physiological (such as zinc, copper, selenium)

andxenobiotic (such as cadmium, mercury, silver, arsenic) heavy metals through

the thiolgroup of its cysteine residues, which represents nearly the 30% of its amino

acidicresidues.

Muscle proteolysis

Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. In

general, this occurs by the hydrolysis of the peptide, and is most commonly achieved by

cellular enzymes called proteases, but may also occur by intramolecular digestion, as well

as by non-enzymatic methods such as the action of mineral acids and heat.

Nitrogen free radicals

An important cellular signaling molecule involved in many physiological and

pathological processes. It is a powerful vasodilator with a short half-life of a few seconds

in the blood

Peroxidation

A type of reaction in which oxygen atoms are formed leading to the production of

peroxides. It is stimulated in the body by certain toxins and infections.

Selenoproteins

 Any protein that includes a selenocysteine (Se-Cys) amino acid residue. Among

functionally characterized selenoproteins are five glutathione peroxidases (GPX) and

three thioredoxin reductases, (TrxR/TXNRD) which both contain only one Se-Cys.

Soluble guanylate cyclase activity (sGC)

The only known receptor for nitric oxide, NO. It is soluble, i.e. completely intracellular.

Most notably, this enzyme is involved in vasodilation. In humans, it is encoded by the

genes GUCY1A2, GUCY1A3, GUCY1B2 andGUCY1B3.

Xanthise oxidase

is a form of xanthine oxidoreductase, a type of enzyme that generates reactive oxygen

species.These enzymes catalyze theoxidation of hypoxanthine to xanthine and can further

catalyze the oxidation of xanthine touric acid. These enzymes play an important role in

the catabolism of purines in some species, including humans

List of Antioxidants:

Vitamin E. Alpha-tocopherol, the most widely available isomer, has the highest biopotency, or

strongest effect in the body. Because it is fat-soluble (and can only dissolve in fats), alpha-

tocopherol is in a unique position to safeguard cell membranes -- largely composed of fatty acids

-- from damage by free radicals. Alpha-tocopherol also protects the fats in low-density

lipoproteins (LDLs, or the "bad" cholesterol) from oxidation.

- Broccoli, carrots, chard, mustard and turnip greens, mangoes, nuts, papaya, pumpkin,

red peppers, spinach, and sunflower seeds

Vitamin C, also known as ascorbic acid, is a water-soluble vitamin. As such, it scavenges free

radicals that are in an aqueous (watery) environment, such as inside your cells. Vitamin C works

synergistically with vitamin E to quench free radicals. Vitamin C also regenerates the reduced

(stable) form of vitamin E.

- Berries, broccoli, Brussels sprouts, cantaloupe, cauliflower, grapefruit, honeydew, kale,

kiwi, mangoes, nectarines, orange, papaya, red, green or yellow peppers, snow peas,

sweet potato, strawberries, and tomatoes

Beta-carotene, also a water-soluble vitamin, is the most widely studied of the 600 carotenoids

identified to date. It is thought to be the best quencher of singlet oxygen (an energized but

uncharged form of oxygen that is toxic to cells). Beta-carotene is also especially excellent at

scavenging free radicals in low oxygen concentration.

- Apricots, asparagus, beets, broccoli, cantaloupe, carrots, corn, green peppers, kale,

mangoes, turnip and collard greens, nectarines, peaches, pink grapefruit, pumpkin,

squash, spinach, sweet potato, tangerines, tomatoes, and watermelon

Selenium is a trace element. It is a mineral that we need to consume in only very small

quantities, but without which we could not survive. It forms the active site of several antioxidant

enzymes including glutathione peroxidase.

- Found in Brazil nuts, tuna, beef, poultry and fortified breads, and other grain products

Quercetin: a plant-based chemical (phytochemical) found in apples, onions, teas, red wines, and

other foods; it fights inflammation and may help reduce allergies.

Luteolin: a flavonoid found in abundance in celery and green peppers; it also fights

inflammation, and one study showed it may help protect against inflammatory brain conditions

like Alzheimer’s.

Catechins: a type of flavonoid found in tea; catechins in tea may help reduce risk of heart

disease, cancer, and Alzheimer’s disease.

Review of Related Literature

A 1999’s study that was conducted by David A. Hughes about the Effects of dietary

antioxidants on the immune function of middle-aged adults and it was said that:

The immune system is highly reliant on accurate cell-cell communication for optimal

function, and any damage to the signalling systems involved will result in an impaired

immune responsiveness. Oxidant-mediated tissue injury is a particular hazard to the

immune system, since phagocytic cells produce reactive oxygen species as part of the

body’s defence against infection. Adequate amounts of neutralizing antioxidants are

required, therefore, to prevent damage to theimmune cells themselves. Many antioxidants

can be obtained directly from the diet (e.g. ascorbic acid, a-tocopherol, carotenoids and

polyphenolic flavonoids) or require micronutrients as integral components (e.g. Se in the

metalloenzyme glutathione peroxidase (EC 1.11.1.9)). Numerous epidemiological studies

have found strong associations between diets rich in antioxidant nutrients and a reduced

incidence of cancer, and it has been suggested that a boost to the body’s immune system

by antioxidants might, at least in part, account for this. Although more striking effects

have been observed in the elderly, there is also evidence that antioxidant nutrients can

modify cell mediated immune responses in younger individuals. Indeed, it might be

essential to have an adequate intake of antioxidant nutrients from an early age in order

to help prevent the development of, or at least delay the onset of, several degenerative

disorders. The present paper will review the effects of specific nutrients on immune

function in young to middle-aged human subjects, focusing on the antioxidant vitamins C

and E, and on Se. A further review, dealing more specifically with the effects of

carotenoids on human immune function, will be presented at a forthcoming meeting of

the Nutrition Society.

A European research on the function effects of dietary antioxidants in 2003, it has been

concluded that:

The Antioxidant Hypothesis suggests that reducing agents prevent oxidative damage and

thus increased levels will also reduce the risk of chronic disease. This lead to the

suggestion that increased intake of dietary compounds, some of which are capable of

acting as chemical antioxidants, would be of benefit by augmenting cellular defenses and

protect components of the cell against oxidative damage and, ultimately disease. There is

compelling epidemiological evidence linking consumption of diets rich in antioxidants,

direct and indirect, with reduced risk of developing degenerative disease. However, the

enormous focus on this field of research worldwide has generated data that both support

and challenge the hypothesis. Observational studies in humans have demonstrated high

intakes of foods rich in dietary antioxidants (i.e. fruit and vegetable, cereals and grains,

and some specific oils and fats) and high plasma concentrations of these dietary

antioxidants are associated with reduced morbidity and mortality. However, plasma

antioxidant concentrations are a measure of intake, rather than a measure of

concentration at target sites, and an association is not necessarily causal. Human

intervention studies have shown no consistent benefit of increased intake of dietary

antioxidants, either individually or in combination as isolates or foods, in the prevention

of human diseases. Many arguments have been put forward to explain why these studies

have failed to demonstrate an effect consistent with the epidemiology, but few have

addressed the underlying and fundamental problems associated with complex nutritional

trials.

Several researches in the National Research Center on Giza, Egypt conducted a study about the

Effects of Dietary Antioxidants Supplementation on Cellular Immune Response and Evaluation

of their Antimicrobial activity against some Enteric Pathogens in Goats (2013). That stated:

Oxidant- mediated tissue injury is a particular hazard to the immune system, since

phagocytic cells produce reactive oxidants, as a part of the body’s defense against

infectious agents. Therefore, adequate amounts of neutralizing antioxidants are required

to prevent damage in the immune cells themselves [2].The results of the present study as

shown in Table (4), indicated that BCS and multi-antioxidants significantly (P<0.05)

enhanced lymphocyte proliferative responses.

Similarly, our recorded data demonstrated significant (P<o.o5??) elevation of

phagocytic % and phagocytic index in both groups received antioxidants supplement

compared with non- supplemented group. Many effects have been reported for the seeds

of N. sativa and Zn- Met. With Vit. E and Se including their antioxidant roles and

immune potentiating effects [2, 3]. This explained the improvement of cellular immune

response in the two groups of animals received antioxidants in the current study.

Recently, Haman and Abou- Zeina [7] reported that ewes supplemented with

vitamin E and Se has better antioxidants status and greater immunoglobulins. It was

concluded that vitamin E and Se supplementation improve the status of these

micronutrients and humoral immune response in buffalo calves.

Meanwhile, Abou- Zeina et al., [16] demonstrated that Zn, Se and Vit E

administered to buffalo cows under heat stress condition, induced better reproduction

performance and increase in immunoglobulin levels.

How Se and Vit E enhance the immune response is not fully understood. It is

possible that Se as a co-factor of the GPx family of enzymes, either through a direct effect

or via destruction of inhibitory peroxides, contributes to a better immune response [10].

As a constituent of selenoproteins, Se is needed for the proper function of neutrophils,

macrophages, natural killer cells and T lymphocytes [5]. Mild Se deficiency appears to

increase susceptibility to oxidant stress [50] and risk of infections. It was documented by

many authors that deficiency of Se also affects the ability of the macrophages to release

to leukotriene B4 which signals the white blood cells to aid in the destruction of harmful

microorganisms [51]. The antioxidant activity of vitamin E in preventing lipid oxidation

may be one of the mechanisms by which vitamin E enhances immunity particularly

phagocytosis [11]. Besides, Vitamin E causes a reduction of glucocorticoids, which are

known to be immunosuppressive. Researchers suggested that relatively higher levels of

vitamin E supplementation may improve animal performance, which may be due to

enhanced immunity p[15, 52].

Likewise, Zinc is crucial for normal development and function of cells mediating

non-specific immunity such as neutrophils and natural killer cells [53]. Zinc also,

regulates the expression in lymphocytes of metallothionein and metallothionein-like

proteins with antioxidant activity. In vitro intracellular killing by macrophages is very

sensitive to zinc deficiency adversely affects the secretion and functions of cytokines, the

basic messengers of the immune system [13].

Conclusion:

Free radicals have been implicated in the etiology of varying large number of major

diseases. In which, they can adversely alter many crucial biological molecules leading to loss of

form and function. Such undesirable changes in the body can lead to diseased conditions.

Antioxidants can protect the body against the damaged induced by the free radicals that acts in

various levels, and it may also enhance the function of the immune response thereby,

strengthening the components that of which make-up the immune system. Dietary and other

components of pants form major sources of antioxidants.

The relation between free radicals, antioxidants and function of the various organs and

organ systems is highly complex and the discovery of the ‘redox- signaling’ has been a milestone

in this crucial relationship. Recent research centers on various strategies to protect crucial tissues

organs against oxidative damage induce by the free radicals. Many novel approaches are made

and significant finding have come to light in the last few years. The higher intake of food with

functional attributes including high level of antioxidant sin functional foods is one strategy that is

gaining importance in advanced countries and is making its appearance.

Co-ordinate research involving biomedical scientists, nutritionist and physicians can

make significant differences to human health in the coming decades. Research on free radicals

and antioxidants involving theses is one such effort in the right direction.

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