In this laboratory practical, you will use a method called the ferric reducing ability of plasma (FRAP) assay to measure the ‘antioxidant power’ of a number of plasma and food samples.
The method measures the ability of antioxidants in plasma or in foods to reduce the ferric component (Fe3 ) of a ferric tripyridyltriazine (Fe3 -TPTZ) complex (which is contained in the FRAP reagent) to the ferrous form (Fe2 ). During this reaction which takes place at a low pH, the reduction of ferric iron (Fe3 ) to the ferrous form (Fe2 ) is accompanied by the formation of a blue colour which can be measured at an absorption maximum of 593nm using a spectrophotometer.
The human body is constantly under attack from free radicals and ROS which are produced endogenously in the body as by products of normal aerobic metabolism and enzyme systems as well as being supplied from sources outside the body from cigarette smoke, lipid peroxidation products in foods or from pollutants. In order to protect the body from inappropriate exposure to these free radicals and ROS, the human body has developed a powerful and complex antioxidant defence system. Enzymatic antioxidants within the cell such as superoxide dismutase function by inactivating or removing ROS from the cell before they can cause damage. Non-enzymatic protein antioxidants function by controlling the storage and release of metal ions which are needed for the enzymatic antioxidants to function but can also convert relatively unreactive radicals such as superoxide to the much more reactive hydroxyl radical. However, antioxidants such as vitamin C, ï¡-tocopherol, carotenoids and phenolic compounds such as flavonols act as hyrdrogen (and electron) atom donors, and in this way behave as reducing agents. It is this ability to act as a reducing agent that is used to measure ‘antioxidant potential’ in the FRAP assay.
When the FRAP assay is carried out with foodstuffs, antioxidants contained in the foods that behave as reducing agents convert Fe3 in the ferric complex (Fe3 -TPTZ) to Fe2 ions forming a blue colour in the same way as non-enzymatic antioxidants contained in plasma. However, whether antioxidants contained in foods can exhibit antioxidant potential when taken into the body will depend on their bioavailability i.e. absorption and incorporation into body tissues.
The FRAP assay offers a simple index of ‘antioxidant or reducing power’ and as you will see the method can be used easily with plasma and foodstuffs. Furthermore, the results are reported to be highly reproducible and the procedure is straightforward and easy to carry out.
However, there are also a number of limitations associated with the use of this assay for measuring ‘antioxidant power’. The most obvious limitation of the FRAP assay is that while it claims to measure ‘antioxidant power’, it actually only measures ‘antioxidant power’ of non-enzymatic antioxidants that act as reducing agents. While the FRAP assay can be used to measure the ‘antioxidant power’ of foodstuffs, the results to not give us any information on the bioavailability of these antioxidants in the body and thus about their actual physiological ‘antioxidant power’ in vivo. In addition, substances that bind with either Fe3 or Fe2 could in theory interfere with the results of the assay.
Aim The aim of this experiment is to use the FRAP assay to measure the ‘antioxidant power’ of a number of plasma and food samples.
Objectives: By the end of this practical session and the lecture on antioxidants you should be able toâ€¦
1. List the different type of antioxidants in the body.
2. Explain the mechanism of action of the different type of antioxidants.
3. List the food sources of antioxidants.
4. List examples of free radicals and reactive oxygen species and their sources.
5. Explain the principle on which the FRAP assay is based.
6. Explain the main limitations of the assay.
Part A Experiment This experiment involves constructing a standard curve with concentrations of 0-1.0mM ferrous sulphate (FeSO4, Fe2 ) and carrying out the FRAP assay on 6 food samples and 4 plasma samples.
1. For this practical you will need to assemble the following items on your bench space:
A tube containing the standard solution (2mM FeSO4)
6 food samples
4 plasma samples
Rack with 6 universal tubes (each containing 24 ml distilled H2O),
Rack with 6 plastic tubes for making up the standards,
Marker (for labelling)
Plastic cuvettes cuvette box
5 ml pipette
1 ml pipette
Note: Before you start the experiment, you should switch on your spectrophotometer and set the wavelength to 593nm (you can ask your demonstrator for help).
1. Pipette the standards, food samples and plasma samples into the plastic cuvettes by following the instructions below:
Firstly, you need to prepare dilutions of the stock standard in distilled H2O so that you can produce a standard curve in the range of 0.1-1.0 mM.
You should have a rack with 6 plastic test tubes lids on your bench.
You should label these tubes Blank, STD 0.2, STD 0.4, STD 0.6, STD 0.8 and STD 1.0.
You should have a tube containing the standard solution (2.0 mM FeSO4)
You should have a container of distilled H2O.
You should make the dilutions of the standard as follows (see table on next page):
Standard (final Ferrous Sulphate Concentration, mM) Volume (ml) of 2 mM Ferrous Sulphate to be added Volume (ml) of distilled H2O to be added 0 (Blank)
Mix the contents of these tubes by inversion (with lid on!).
In your cuvette box, you should have 12 plastic cuvettes as you are going to prepare these standards in duplicate.
Label the cuvettes with marker up high on the cuvette on the frosted side.
Pipette 200ïl of each concentration of standard, in duplicate, into the appropriate cuvette.
You should have 6 plastic containers of food samples / beverages.
You need to make a 1 in 25 dilution of the food samples and a 1 in 50 dilution of the coffee sample and with distilled H2O. [You should also have a rack containing 6 universal tubes each containing 24 ml of distilled H2O].
Pipette 1 ml of each of the food samples and 0.5 ml coffee and into a labelled universal tube. Mix the contents using by inversion (with lid on!).
Pipette 200ïl of each of the diluted food samples into the appropriate cuvette.
You should have plasma samples from 4 individuals namely, a smoker, a healthy adult, a type 2 diabetic patients and an individual with a diet rich in fruit and vegetables. The plasma samples are labelled P1, P2, P3 and P4 but you do not know which plasma sample belongs to which individual.
Pipette 200ïl of each plasma sample into an appropriate cuvette.
2. Add 3ml FRAP solution to each cuvette in the rack. The FRAP reagent has already been prepared for you and is in the 370C water bath back on the bench.
3. Place your cuvette box into the 370C incubator for 4 min (use your watch to time).
4. Read the absorbance of each standard, food and plasma sample (against the blank) at 593nm using the spectrophotometer and write the values in the table below.
Concentration of FeSO4 Standard (mM) Absorbance @ 593 nm (reading 1) Absorbance @ 593 nm (reading 2) Average absorbance reading 0.0 (blank)
Food Samples (dilution factor) Absorbance @ 593 nm Coffee (1/50)
Green Tea (1/25)
Black Tea (1/25)
Blackcurrant Juice (1/25)
Red Wine (1/25)
White Wine (1/25)
Pomegranate Juice (1/50)
Plasma Samples Absorbance @ 593 nm P1
Part B Calculation of results 1) Using the graph paper provided: If you did not “blank” your spectrophotometer, remember to substract your blank reading from all other readings.
Draw your calibration curve.
Determine the slope of the curve (“a” in the equation y=ax)
Using the equation, work out the antioxidant power of each food sample, in Fe2 mM equivalent.
For the food samples, you also need to adjust the final concentration for the initial dilution made.
Food sample Antioxidant Power (mM) Final Antioxidant Power (mM Fe2 equivalent) adjusted for the initial dilution Coffee 0.587
Green Tea 0.350
Black Tea 0.466
Blackcurrant Juice 0.365
Red Wine 0.988
White Wine 0.091
Pomegranate Juice 0.597
Plasma sample Antioxidant Power (mM Fe2 equivalent) P1 0.777
Submit, as part of your report, a calibration curve created in Excel, showing the equation. 1. In excel, draw scatter plot with the absorbance reading on Y-axis and concentration of standards on the X-axis as follows:
Type the concentrations and absorbance values for your standards into an excel spread sheet as shown in the example below.
Concentration (mM FeSO4) 0
Absorbance Using your cursor, highlight the information you have typed and click on the chart wizard button on the bar at the top of the screen.
Choose xy (Scatter) which will draw a scatter plot of the absorbance and concentration values. Click Finish.
Hold down the control key and click on any of the points on your scatter plot.
On the menu bar, click on chart, and choose add trend line.
Under Type – choose a linear trend line
Under options and tick the following.
ï¡ Set intercept = 0
ï¡ Display equation on chart
ï¡ Display R-square value on chart
2. This should produce a straight line through your points and through zero. There will be an equation displayed on the chart and an R-squared value. The R-squared value should be as close as possible to 0.999.
Use the equation of the line to calculate Antioxidant Power of the samples. 1. You now have an equation of the line that you will use to calculate the concentrations for each of your samples.
For example: Equation of line y = 0.7543x Remember y = absorbance x = concentration You have measured the absorbance values of your samples and you want to find the concentration.
x = y/0.7543 2. Calculate the concentration for each sample and write the value in the table below. For the food samples, you also need to adjust the final concentration for the initial dilution made.
Food sample Antioxidant Power (mM Fe2 equivalent) Final Antioxidant Power (mM Fe2 equivalent) adjusted for the initial dilution Red wine 0.587
White wine 0.350
Blackcurrant juice 0.466
Black tea 0.988
Green tea 0.091
Pomegranate Juice 0.597
Plasma sample Antioxidant Power (mM Fe2 equivalent) P1 0.777
Part C Questions 1. What were the FRAP values or ‘antioxidant power’ values (in Fe2 mM equivalent) of the 4 plasma samples?
2. The plasma samples that you analysed were taken from 4 individuals, namely, a smoker, a type 2 diabetic, a healthy adult and a healthy adult with a diet rich in fruit and vegetables. Which plasma sample do you think belongs to each individual and why?
3. What were the FRAP or ‘antioxidant power’ values (in mM Fe2 equivalent) for each of your food samples?
4. List the antioxidants present in each of the foods, which could contribute towards the ‘antioxidant power’ values that you have measured?
Sample number Food name / type Main antioxidants 1
5. Can you explain briefly how the FRAP assay claims to measure ‘antioxidant power’?
6. What are the main limitations of measuring ‘antioxidant power’ using the FRAP assay?
7. The ‘antioxidant power’ that you have measured is greater for some foods compared to others. What factors would influence whether these foods could exert antioxidant potential in the body?
Requirements for a Balanced Diet
A balanced diet is one that provides an adequate intake of energy and nutrients for maintenance of the body and therefore good health. A diet can easily be adequate for normal bodily functioning, yet may not be a balanced diet. An ideal human diet contains fat, protein, carbohydrates, vitamins, minerals, water and fibre all in correct proportions. These proportions vary for each individual because everyone has different metabolic rates and levels of activity.
Malnutrition results from an unbalanced diet, this can be due to an excess of some dietary components and lack of other components, not just a complete lack of food. Too much of one component can be as much harm to the body as too little. Deficiency diseases occur when there is a lack of a specific nutrient, although some diet related disorders are a result of eating an excess.
An adequate diet provides sufficient energy for the performance of metabolic work, although the energy food is in an unspecified form. A balanced diet provides all dietary requirements in the correct proportions. Ideally this would be 1/7 fat, 1/7 protein and 5/7 carbohydrate.
Energy is provided by carbohydrates, fats and proteins. Proteins are a provider of energy in an emergency, but are primarily used as building blocks for growth and repair of many body tissues. These energy providing compounds are needed in large quantities in our diet so are described as macronutrients.
We also need much smaller amounts of other nutrients, such as vitamins and minerals. Because much smaller quantities are needed for a balanced diet these are known as micronutrients. Despite the small quantities needed these are essential to provide a healthy diet as they have specific roles in metabolic reactions and as structural components.
Within the cells of our body, the nutrients ingested are converted to other compounds which are then used for metabolism and other cellular reactions. Starch, a major carbohydrate is converted to glucose which can be then synthesised into fat for storage, proteins are synthesised from amino acids, and phospholipids are made from glycerol and fatty acids. However there are some organic compounds which despite being essential for a healthy diet cannot be made by cells so must be provided by diet. These are essential amino acids, essential fatty acids and vitamins.
Carbohydrates are a rapid source of energy, they are the body’s fuel. The bulk of a balanced diet should be made from carbohydrates. If eaten in an excess of the dietary requirements carbohydrates are easily stored as fats in the cells, although carbohydrate is the first source of energy in the body.
An average adult requires about 12,000kJ of energy a day, most of this is supplied by the respiration of carbohydrates in the cells.
Carbohydrates are used principally as a respiratory substrates, i.e. to be oxidised to release energy for active transport, macromolecule synthesis, cell division and muscle contraction. Carbohydrates are digested in the duodenum and ileum and absorbed as glucose into cells.
Sources of carbohydrates such as starch are rice, potatoes, wheat and other cereals. Sugars are also carbohydrates, sources of sugars are refined sugar – sucrose, which is a food sweetener and preservative and fruit sugars – fructose.
If the diet lacks carbohydrate stores of fat are mobilised and used as an energy source.
Lipids are a rich source of energy in the diet, they can be greatly reduced in metabolic reactions and therefore release much energy. They are easily stored in the body and can form a layer beneath the skin of adipose tissue. As lipids are such a rich source of energy they are often not needed for respiration if there are adequate quantities of carbohydrate for the energy output of the body.
Meat and animal products are rich in saturated fats and cholesterol, plant oils are rich in unsaturated fats.
As lipids are digested in the intestine into fatty acids and glycerol, some fatty acids are only available in the diet and cannot therefore be synthesised in the cell in any way. These are therefore known as Essential Fatty Acids. Fatty acids are categorised according to the number of double bonds they have in their carbon chain. Saturated fatty acids have none, monounsaturated fatty acids have one, polyunsaturated fatty acids have more than one. Essential polyunsaturated fatty acids cannot be synthesised in the body from anything else as the correct enzymes to add double bonds after the ninth carbon to the carbon chain are not present. Two essential fatty acids are linoleic and linolenic acid which are found in vegetable oils such as soya, sunflower and maize.
Fatty acids are needed for the formation of cell membrane phospholipids and also for the production of steroid hormones such as prostaglandins and thromboxin which have important roles in the renal, immune and circulatory systems as signalling chemicals.
Deficiencies of essential fatty acids result in limited growth in children, poor healing of wounds, scaly skin and hair loss.
Obesity is a result of a high fat intake in the diet and lack of exercise. Obesity is in fact a form of malnutrition as the diet is not balanced. The risk of developing diseases such as diabetes, hypertension, CHD, arthritis (due to extra pressure on joints), stroke and some cancers are increased dramatically with obesity.
Protein is not a direct source of energy in the body, it is used primarily for growth and repair of body tissues although can be used as an energy source as a last resort. Proteins fulfil a wide variety of roles in the body, they are broken down in the stomach and intestines to amino acids which are then absorbed. The body can only form 8 amino acids to build proteins from, the diet must provide Essential Amino Acids (EAAs) which are synthesised into proteins which can be structural, i.e. collagen in bone, keratin in hair, myosin and actin in muscle; metabolic enzymes, haemoglobin, protective antibodies and communicative hormones.
Sources of protein include meat, fish, eggs and pulses. The diet needs to provide 8 EAAs as the body is unable to synthesis proteins without these molecules. 2 other amino acids are synthesised from EAAs so if the diet lacks the original EAAs these other two will not be present either. Phenylalanine is converted to tyrosine and methionine is converted to cysteine. Cells draw upon a pool of amino acids for protein synthesis which either come from dietary protein digested and absorbed in the gut and the breakdown of body protein such as muscle. However, unlike fats and carbohydrates there is no store of amino acids for cells to draw on, any amino acid in excess of immediate bodily requirements is broken down into urea and excreted. It is therefore important to maintain the dietary intake of protein everyday. If the body lacks protein, muscle wasting occurs as muscle is broken down .
If protein is lacked in a diet a person develops kwashiorkor which is caused when high levels of carbohydrates are eaten to overcome the lack of protein in the diet. One symptom of kwashiorkor is the abnormal collection of fluid around the abdomen due to the lack of protein in the blood. The body cannot retain water by osmosis and fluid accumulates in tissues causing them to become waterlogged.
Vitamins cannot be synthesised by the body so must be supplied by diet. Vitamins have no common structure or function but are essential in small amounts for the body to be able to utilise other dietary components efficiently.
Vitamins fall into two categories, fat soluble vitamins such as vitamin A, D, E and K which are ingested with fatty foods and water soluble vitamins such as the B group vitamins and vitamin C. Vitamins are known as micronutrients because only small quantities are required for a healthy diet, in fact fat soluble vitamins can be toxic in high concentrations, for example the body stores vitamin A, or retinol, in the liver as it is toxic if kept in high concentrations in the blood stream, a dose of more than 3300mg of vitamin A can be considered toxic. Water soluble vitamins such as vitamin C and B groups vitamins can be excreted in the urine if in excess in the diet.
Vitamins carry out a wide range of functions and prevent specific deficiency diseases. A diet that lacks a certain vitamin is not a balanced diet, vitamins have vital roles in the maintenance of a healthy body.
An example of a deficiency is when the diet does not contain enough, or any vitamin A.
Vitamin A is found in some animal foods such as milk, eggs, liver and fish liver oils, related compounds such as carotenoids e.g. b carotene, are in a wide variety of vegetables such as cabbages, carrots and spinach.
Vitamin A is essential to the proper functioning of the retina in the eye and the epithelial tissues. A lack of vitamin A results in dry, rough skin, inflammation of the eyes, a drying or scarring of the cornea – xerophthalmia, which occurs when the secretion of lubricating tears is stopped, the eyelids become swollen and sticky with pus. Mucous surfaces of the eye may become eroded allowing infection to set in, leading to ulceration and destruction of the cornea. Night blindness – an inability to see in dim light can also occur. Rod cells in the retina of the eye detect light of low intensity, they convert vitamin A into a pigment, rhodopsin, which is bleached when light enters the eye. Rod cells resynthesis rhodopsin, but if there is a deficiency of the vitamin, rod cells can no longer function and the result is night blindness. Epithelial cells use retinol to make retinoic acid, an intracellular messenger used in cell differentiation and growth. Without retinoic acid epithelial cells are not maintained properly and the body becomes susceptible to infections, particularly measles and infections of the respiratory system and gut.
Xenophthalmia is common among children who’s diets consist of mainly cereals with little meat or fresh vegetables, this is common in Indonesia, Bangladesh, India and the Philippines.
Vitamin D, or calciferol, is another fat soluble steroid vitamin which functions to stimulate calcium uptake from the gut and its deposition in bone. Vitamin D acts as a hormone when converted by enzymes in the gut and liver into an active form “active vitamin D”, which stimulates epithelial cells in the intestine to absorb calcium. Vitamin D is therefore essential in growing children’s diets to enable the growth of strong bones. Without adequate amounts of vitamin D children can develop rickets, which is the deformation of the legs caused when they lack calcium to strengthen the bones. In adults a lack of vitamin D in the diet can lead to osteomalacia, a progressive softening of the bones which can make them highly susceptible to fracture.
Vitamin D is made by the body when exposed to sunlight and is stored in the muscles, however, if the skin is rarely exposed to the sunlight or is dark little vitamin D is produced. Foods such as eggs and oily fish are all rich in vitamin D.
Vitamin K, phylloquinone, is found in dark green leafy vegetables such as spinach and kale. It is a fat soluble vitamin which is involved in the clotting process of blood. In the intestines bacteria synthesise a number of important clotting factors which need vitamin K. Without vitamin K cuts can fail to heal and internal bleeding can occur.
Vitamin C is a water soluble vitamin, known chemically as ascorbic acid. It is found in citrus fruits such as oranges and lemons, and also in potatoes and tomatoes. The main function of vitamin C is the formation of connective tissues such as collagen. It is also known to be an antioxidant which helps to remove toxins from the body and aids the immune system. A lack of vitamin C leads to Scurvy, a condition experienced by sailors on long journeys when they did not have fruit in their diets. Scurvy causes painful, bleeding gums. As vitamin C is water soluble, it is not toxic in high doses as it can be excreted in the urine, very high doses can however cause diarrhoea.
B group vitamins have a wide range of roles acting as co-enzymes in metabolic pathways. They are found in most plant and animal tissues involved in metabolism, therefore foods such as liver, yeast and dairy products are all rich in B group vitamins. Deficiency of B group vitamins include dermatitis, fatigue and malformation of red blood cells.
Some minerals are considered to be macronutrients as they are required in fairly large amounts in the diet to maintain a healthy body. Minerals are required in their ionic state in the diet.
Calcium, Ca2 , is a major constituent of bones and teeth and is required to keep bones strong. It is required in blood clotting as an activator of various plasma proteins and is also involved in muscle contraction. Calcium is used in synapses and also as an enzyme activator. A good source of calcium is in dairy products, eggs and green vegetables, the RDA for calcium is 800mg.
Chlorine, Cl-, is required to maintain the osmotic anion / cation balance of the body and the formation of HCl in the stomach. It is found in table salt and is rarely deficient in the diet as it is used as a preservative to may foods. Sodium, Na , is also found in table salt as well as dairy foods, meat, eggs and vegetables. Sodium is used in conjunction with chlorine in the maintenance of the osmotic anion / cation balance. It is also needed in nerve conduction and muscle action. Potassium, K , is yet another mineral required in nerve and muscle action, it also has a role in protein synthesis. It is found in meat, fruit and vegetables.
Phosphorus, in the form of phosphate, PO43- is a constituent of nucleic acids, ATP, phospholipids in cell membranes, bones and teeth. It is present in dairy foods, eggs, meat and vegetables.
Magnesium, Mg2 , is an important component of bones and teeth and is also an enzyme activator. It is found in meats and green vegetables.
Micronutrients are minerals needed in trace quantities. Despite the small quantity required, they are still essential to a healthy balanced diet.
Iron, in the forms of Fe2 and Fe3 , are required in the formation of haemoglobin and myoglobin. Iron is a constituent of many enzymes as a prosthetic group and also as an electron carrier in mitochondria. Red meat, liver and green vegetables are all sources of iron. Iron supplements are taken by people who suffer from anaemia.
Iodine, I-, is a component of the growth hormone thyroxine. A lack of iodine in the diet can cause hypothyroidism which results in weight gain and in extreme cases a lack of physical and mental development known as cretinism. A swelling of the neck can occur which is called goitre if iodine is deficient in the diet. Iodine can be found in seafood such as shellfish, seaweed and fish. Iodine has also been added to water supplies in areas where it is deficient in the main water system.
Copper, Cu2 , manganese, Mn2 and cobalt, Co2 , are all needed in the diet to form co-factors for enzymes. Copper is also needed for bone and haemoglobin formation and cobalt is needed for the production of red blood cells, manganese is also a growth factor in bone development. They are found in meat and liver as well as some dairy products.
Fibre is not digested by the body, but is involved in maintaining the health of the gut and is therefore an essential part of a balanced diet. Fibre is mostly made up of cellulose from plant cell walls and is indigestible as the stomach and gut do not contain the correct enzymes. Fibre aids the formation of faeces, preventing constipation. It also aids the peristaltic movement in the intestine and has been linked to the prevention of bowel cancer. Fibre also removes some saturated fats and cholesterol therefore protecting the body a little from the build up of plaques in blood vessels. Fruit, vegetables and cereals are a good source of dietary fibre.
The diet must provide water which is required as a solvent, a transport medium, a substrate in hydrolytic reactions and for lubrication. Water in fact makes up about 70% of the total body weight of humans. Water is needed as it is lost constantly from our bodies in urine, sweat, evaporation from lungs and in faeces. An average person requires 2-3 litres of water a day which is supplied through drinks and liquid foods. Without water or food the longest anyone has ever survived is 17 days, however, with water the longest anyone has survived is 70 days, this illustrates the importance of water in the diet.
As you can see a balanced diet is imperative to maintaining a healthy body. People who choose to be vegetarians and vegans therefore must make sure that their diet contains all the correct nutrients to avoid any deficiencies that may occur, as well as people living in countries where their diet lacks certain important food groups. A diet can easily be adequate without being a properly balanced diet and since everyone has different metabolic rates everyone’s ideal diet is unique, therefore generalised guidelines have been established to aid people in obtaining a good diet. Vitamins and minerals are required in small amounts to carry out a variety of essential specific functions, fat and carbohydrates are the main fuel that the body runs on, whilst protein is needed in large amounts for growth and repair. The diet must also provide adequate quantities of essential fatty acids and amino acids which are required for the body to metabolise into proteins and are fundamental for health. Over eating of one food group is considered to be a form of malnutrition because the diet is not balanced.