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Antioxidants are the substances that protect the body from oxidation reaction. Antioxidants consist of vitamin A, C, E and selenium. Antioxidants have its ability to reduce or prevent oxidative stress in the body. Oxidative stress occurs when there are harmful molecules called free radicals. Free radicals are developed in our body and environmental factors such as pollution, cigarette smoke, sunlight, stress and lack of sleep and exercise etc. Antioxidant helps to neutralize and against free radicals in the body.
The aim of the study is to investigate the effect of antioxidant supplements on oxidative stress level and estimate cumulative effect of oxidant stress in the body. This study will prove the antioxidant effect on oxidative stress level in the body.
Atoms consist of neutrons, protons and electrons. Electrons are negative charge, protons are positive charge and neutrons have no charge. The role of electrons is to bond the atoms to form molecules. The outer shell will gain, lose or share the electrons to fill its outer shell to reach the maximum stability of the molecules. When the outer shell is not complete filled, the atoms will become unstable.
Free radicals are atoms or molecules which had unpaired electrons n the outer shell. Free radicals will form when weak bonds split. They are highly unstable and reactive. Free radicals will take the electrons from another molecule and paired with the unpaired electrons in the outer shell. That molecule will become a free radical due to the missing electrons and then the free radical chain will continue. The reaction is known as oxidative stress. Oxidative stress cause cell and tissue damage, DNA damage, ageing and develop diseases like cancer and heart disease which are harmful to the body. Types of the common free radicals are superoxide anion (O2-), the hydroxyl radical (OH ·), singlet oxygen (1O2 ), and hydrogen peroxide (H2O2) which produce in our bodies.
Figure 1 shows how the free radical forms
Sources of free radical http://health.howstuffworks.com/food-nutrition/antioxidant1.htm
Free radicals come from endogenous sources and exogenous sources. Endogenous refers to produce within the cells in the body. Free radicals are form during aerobic respiration, metabolism, and inflammation etc. Exogenous sources refers to external factors outside the body. Free radicals form from environmental factors like pollution, radiation, ultraviolet(UV)light, cigarette smoke, alcohol and pesticides etc.
Figure 2 shows the formation of free radicals
The effects of free radical damage
Free radicals can destroy the cells in the body. It can increase by the process of free radical chain reaction. Types of damage caused by free radical are lipid peroxidation, membrane damage, protein damage and DNA damage. For example, when the DNA is being damaged by free radicals, it will lead to mutation in cells which cause cancer. When the cells are being destroyed, the body's system will unable to function properly and lead to varies diseases such as cancer, premature aging and heart disease.
Antioxidant is a substance that protects the body against oxidation reactive. It is known as enzyme. It can be found in our body and extracted from food such as fruits and vegetables. Antioxidant consists of vitamin A, C, E and selenium. Antioxidant helps to neutralise free radicals during oxidation. During the process, antioxidant will become oxidized. Antioxidant will break the chain of free radical reaction by giving their electrons to free radical. When a free radical gains the electron from antioxidant, it will not attack the healthy cells, thus the free radical chain reaction is broken. It also prevent oxidation by stabilize the metal radicals such as copper and iron and reduce the rate of the free radical chain reaction.
Figure 3 shows how the antioxidants work
Types of antioxidants
Vitamin A is a fat soluble vitamin. It is important for visual function especially in the dark. It also helps to maintain healthy skin, immune function, against infections and bone growth. Vitamin A keeps the cells healthy which resistant to cell damage. Vitamin A can be found in both animal and plant food such as eggs, milk, cheese, fish oil, carrots, sweet potatoes and pumpkin. Retinol is natural form of vitamin A which found in animal's liver. Carotenoids are compounds found in plants which convent to vitamin A in the body. Beta-carotene is member of carotenoids. Beta-carotene consists of eight isoprene units that cyclised at each end. Besides being vitamin A of beta-carotene, it is also an antioxidant. It able to destroy singlet oxygen, neutralise free radicals and protect the cell membrane lipids from degradation. Singlet oxygen can cause free radical chain reaction. It prevents and against cancer, heart disease and strengthens immune system. Sources of beta-carotene are yellow, orange and green leafy fruits and vegetables such as carrots, spinach, lettuce and tomatoes.
Metabolism of vitamin A:
90% of vitamin A is being absorbed in the intestine. Vitamin A is stored within the body. Maximum absorption is two to six hours after digestion. Retinol-binding protein (RBP) is the carrier of vitamin A which transports all-trans in plasma. Estimate 50 to 85% of the body retinol is stored in the liver. Beta-carotene is stored in adipose cells of fat. Kidneys are the excretion of vitamin A.
RDA (recommended daily allowance): 15-50mg per day is recommended for general health
Overdose, toxicity and deficiency of beta-carotene (vitamin A):
Overdose of intake will cause temporary symptom of skin discoloration which gives yellowish colour of skin. Toxicity symptoms are nausea, vomiting, blurring version etc. Toxicity of beta carotene will lead to Hypervitaminosis A - cause birth defects, liver problems, osteoporosis, hair loss etc and Carotenodermia - skin disorder. It is also toxic for smokers as it will increase the risk of lung cancer and heart disease. Lack of beta-carotene intake will lead to increase risk of cancers and heart disease due to the increase of free radicals, dry skin, reduce the effectiveness of visual and immune system.
Vitamin C is ascorbic acid and water-soluble vitamin. Its molecular formula is C6H8O6 and molecular mass of 176.12. It helps to develop and maintain bones, blood vessels and skin and also strength immune system. Vitamin C is being absorbed by intestines. It builds collagen in connective tissue, regenerate reduced E back to stable state and work as antioxidant in water components tissues. It neutralizes and destroys free radicals in watery parts of the body such as blood and inside the cells. It helps to protect healthy cells against free radical damage. When taking with vitamin E, vitamin C protect LDL(low-density lipoprotein) cholesterol from oxidation. It also prevents heart disease, cancer, strengthen immune system and cataracts. The body does not produce Vitamin C, so it has to be taken from diet or supplements. Vitamin C is found mainly in fruits and vegetables like oranges, tomatoes, broccoli, papaya and berries.
Metabolism of vitamin C:
Ascorbic acid (AA) is easily oxidized to from dehydroascorbic acid (DHAA), oxidation is reversible. When AA is being oxidized to DHAA, it transported across to cell membrane is more quickly. Once it is inside the tissue, it will turn back to AA. Overdose of vitamin c will lead to excess of AA which causes gastrointestinal problems. Active transport is the main transporter of vitamin C within the body. Vitamin C is stored in body tissues and blood. It is being stored in water soluble tissue. Water soluble vitamin C will excreted in urine. Less than 1500mg vitamin C level in the body will reabsorb by kidney, thus no vitamin C concentration present in the urine.
RDA (recommended daily allowance): non-smoking adults is 75mg for women and 90mg for men per day. Smokers are 110mg for women and 125mg for men per day.
Overdose, toxicity and deficiency of vitamin C:
Overdose of vitamin C can cause diarrhea, stomach upset and risk of develop kidney stones. Vitamin C will increase the absorption of iron absorption; it can lead to toxicity of iron in people with iron overload diseases. Deficiency of vitamin c will cause breakdown of protein collagen, increase infections, slower wound-healing and scurvy. Scurvy is a condition which lack of vitamin C severely. Smoking cigarettes lowers the amount of vitamin C, so smokers have a high risk of deficiency. Smokers have to take higher intake of vitamin c than non-smokers. Signs of deficiency are tiredness, rashes, bleeding gums and muscle weakness.
Vitamin E is fat-soluble antioxidant. Alpha-tocopherol is the most active component in vitamin E. Vitamin E helps to maintain body's intracellular membrane and against free radical damage. It breaks the chain of lipid peroxidation and against oxidation in fatty acids. It helps to stabilize cell membrane and protect fats in LDL from oxidation. Vitamin E prevents heart diseases, cataracts, aging and cancer by strengthen immune system. Vitamin E is found in vegetable oils such as soybean, corn and sunflower, wheat germ, nuts and sunflower seeds.
Metabolism of vitamin E:
Absorption of vitamin E is utilized during fatty acid digestion. Vitamin E absorption are micelle and chylomicron formation. Bile acids are important for vitamin E absorption as it helps in fat absorption. 15 to 45% of vitamin E intake is absorbed by the cells. Vitamin E is lipid soluble vitamin so it is found in adipose tissue and muscles. Vitamin E is excreted in urine, feces and skin. Due to poor intestinal absorption of vitamin E, fecal excretion is the main route of vitamin E excretion.
RDA (recommended daily allowance): 15mg for both sexes per day.
Overdose, toxicity and deficiency of vitamin E:
Vitamin E overdose may increase bleeding which is a risk for people who are taking anticoagulant. Vitamin E deficiency will damage to nerves system. It will lead to neuromuscular disease, heart disease, cancer and anemia.
Selenium is a mineral which is needed to maintain good health but only need very small amount. It is an antioxidant and maintains the function of thyroid gland. Selenium helps to against free radical damage by working together with vitamin C and E. It protect the body against free radical damage by forming active site of antioxidant enzyme called glutathione peroxidise and combine with proteins to make selenoproteins which is also an antioxidant enzyme in the body. Antioxidant enzymes are chemical substances that neutralize free radicals by stopping the free radicals from forming, minimize the free radical damage from oxidizing chain reaction and stabilize free radicals. It helps to prevent the development of cancer, heart disease and arthritis. Selenium can be found in meats and seafood such as beef, tuna, chicken and cod.
Metabolism of selenium:
Selenium is part of amino acids within proteins. Selenium is being absorbed by duodenum. There is no absorption in stomach and little percentage of being absorb in small intestine. Selenomethionine and selenocysteine are amino acid containing selenium. It is being transport by proteins, selenoprotein. Selenium will become transport and storage of proteins after being absorbed. The heart, kidney, lungs, liver, pancreas and muscle contain higher level of selenium as glutathione. Selenium is excreted through urinary and fecal.
RDA (recommended daily allowance): 55 μg/day for both sexes.
Overdose, toxicity and deficiency of selenium:
Overdose of vitamin E called selenosis. Symptoms of selenosis are hair loss, fatigue, gastrointestinal upsets, nerve damage and white blotchy nails. Deficiency of selenium may develop heart disease, hypothyroidism and weaken immune system. Keshan disease( poor heart function and an enlarged heart), kashin-beck disease(osteoarthropathy) and Myxedematous Endemic Cretinism(mental retardation) are diseases of selenium deficiency.
Age as a factor?
When we were young, our bodies are able to produce antioxidant enzyme to against free radicals. As we age, the production of antioxidant enzyme will reduce. Insufficient of antioxidant enzyme helps to against oxidative stress. So we need to increase antioxidant from the intake of foods and supplements to reduce the oxidative stress in our bodies.
To investigate the effect of antioxidant supplements on oxidative stress level
To estimate the cumulative effect of the oxidant supplement on baseline oxidative stress level.
Inclusion and exclusion for subjects
Age 18-25 both sexes
Able to swallow tablets.
Agree and sign the informed consent
Smokers (Concentration of beta-carotene cause toxicity for smokers)
Sportsmen( Exercise can increase levels of free radicals and increase the risk of oxidative stress level)
Consuming other health supplements or on medication( may affect or interact with the antioxidant activity)
High intake of alcohol, vegetables and fruits( may affect or interact with the antioxidant activity)
Requirements of human subjects
Complete the monthly antioxidant supplements
Filled up the dietary diary monthly
Willing to give 10 toe nails and urine as sample for this study.
Things to recruit people and prepared before clinical trial:
Survey is serving as the guideline for researchers to select the most suitable candidates to be human subjects in the research. As exclusion of human subjects are smokers, on medication, consume other health supplements, high intake of alcohol, vegetables and fruits and sportsmen which will affect the oxidative stress levels.
Email blast is one of the ways to recruit people in the school. 100 volunteers are needed to be human subjects in the research. It is a brief introduction of the study. Email blast includes the objective of the project, requirements and benefits of antioxidant.
Personal information of the human subjects
It is detail information for human subjects. Personal information includes the medical condition, family's medical history, other supplements consumed, height, weight and blood pressure. It is a reference for us to know more about human subjects' health.
Presentation slide is for human subjects to know more details about antioxidant and oxidative stress. Human subjects should know objective of research, the requirements they need to meet, risk of assessment and expectation of research. It will be shown to the human subjects before clinical trial.
A dietary record is a list of foods and beverages which contain antioxidants consume over time. It helps to keep track of what antioxidant contains foods and beverages being consume. Human subjects are required to fill up the dietary diary so that researchers are able to compare the results. It is because there are foods and beverages contain antioxidant which affects oxidative stress. It is a monthly dietary diary. It will be given to the human subjects monthly when they come for each session. There will be a reflection section of consuming antioxidant supplements at the end of the dietary diary.
Informed consent is needed for any clinical trials. It protects the humans from harm and researchers being sued in biomedical study. Informed consent includes the details of the study and risks and benefits. It has to be signed by human subjects before proceed to clinical trials. Human subjects must fully understand before signing. Informed consent will be given to the human subjects to sign before proceed to any stages of clinical trials. Human subjects who are below 21 have to get their parents consent.
Notes for the subjects
Notes will be given to the human subjects about the overview of the study, instructions on the antioxidant supplements and dietary diary, timeline of the study and requirements of human subjects. Human subjects are able to refer back to the notes for the uses of dietary diary and supplement.
Calendar is needed for the supplement intake and reminder for human subjects. Human subjects will be given 1 month supply of antioxidant supplements. It is required to be taken once daily. It will paste on the supplement bottle given to human subjects. Human subjects are required to cross out the number of days which they have taken the supplements.
Problems encountered during the preparation
The problems that encountered are doing dietary diary and finding literature reviews. Dietary diary is consisting of antioxidant foods and beverages consume over time. A list of common antioxidant foods and beverages are needed to be in the dietary diary. Not only finding the list of antioxidant foods and beverages, serving is also important in the dietary diary. There are different kinds of serving examples. It is difficult to find the universal serving standards. Dietary diary instruction has to be clear and understandable for human subjects. Next, finding literature reviews related to antioxidant. Literature reviews are mostly summarized or abstract. Seldom literature reviews are completed that include the methods and results etc. Time consuming was used to research on literature reviews and editing dietary diary.
Using toe nails and urine tests to determine the effect of supplements on oxidative stress and level of oxidative stress in the body. There will be total of 8 session's collection of samples. It will be taken monthly. Besides the toe nails and urinary tests, dietary diary also determines the oxidative stress in the body. At the end of experiments, results of the tests will be compared.
Toe nails test
10 toe nails are required to be taken as sample monthly. No nail polish is allowed. The bio marker of toe nails is selenium concentration. Selenium is a type of antioxidants. Atomic Absorption Spectroscopy will be used to detect the selenium concentration in the toe nails.
The bio-markers of the urinary test are glutathione and 8-hydroxy-2-deoxyguanosine. Glutathione is a tri-peptide consists of three amino acids - cysteine, glutamic acid and glycine. It is produces by the cells. It serves as an antioxidant, detoxifier and strengthen immune system. 8-hydroxy-2-deoxyguanosine is a product of oxidative damage of DNA by oxidative stress. It can be found in urine, plasma and tissues. Glutathione Assay Kit and 8-hydroxy-2-deoxy Guanosine EIA Kit will be used to detect the bio-markers of oxidative stress.
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