Triglycerides, cholesterol and high and low density lipoproteins affect the way the body functions; their concentrations vary amongst every individual. This experiment will determine the concentration of these four elements in a human blood sample using enzymatic reactions.
Methods: Three procedures were carried out using samples of fasting blood. The absorbency readings were recorded and calculated to compare values of triglycerides, cholesterol and HDL levels within the blood.
Results: Recorded using a spectrophotometer calculating the wave length of the enzymatic reaction. This is then calculated against the concentration of standard and the standard wave length to determine the other all concentration of triglycerides, cholesterol and high and low density lipoproteins.
Conclusion: The result obtained from the experiment show that the participant has healthy levels of cholesterol, triglycerides, HDL and LDL cholesterol, which match the optimal levels.
Fats, or lipids, are essential to the body's diet as they are an important energy source coupled with proteins and carbohydrates. There are many kinds of lipids that exist within the body; however this experiment focuses on triglycerides as these are the common store of fat in food and the body.
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Triglycerides are made up of one glycerol molecule and three fatty acid chain which are attached through dehydration synthesis creating ester bonds. They have three functions within the body; firstly they provide an energy source, when needed triglycerides are broken down by hydrolysis. Due to fat deposits under the skin, triglycerides also serve as insulation for the body, helping reduce the loss of body heat. Finally, they also provide protection to vital organs, by creating a layer of fat around a delicate organ.
Cholesterol is another form of lipid and is a major participant in helping the body function normally. It is present in the outer layer of every cell and is needed to maintain their plasma membranes, as well as promote cell grow and division. Cholesterol determines which molecules can and cannot pass through the cell membrane, as well as aiding the production of bile acids which help to breakdown ingested foods. It is obtained in two ways: through dietary intake of animal products, and by synthesis within the liver.
Lipoproteins are molecules that carry triglycerides and cholesterol within the blood; they are any complex or compound that contains both lipids and proteins. High Density Lipoproteins (HDL) and Low Density Lipoproteins (LDL) are the two major types of lipoproteins. HDLs are made up of more proteins, therefore are denser, these are known as good cholesterol as it removes cholesterol from the arteries to the liver where it is either broken down or converted into waste products for excretion. LDLs are known as bad cholesterol as they carry it to away from the liver to the peripheral tissue, however if too much is transported it builds up on the wall of the arteries which can lead to problems such as high blood pressure and heart disease. (Christian Nordqvist, 2009)
As cholesterol is taken in through the diet, the participant must fast for 12 hours to allow the measurements of cholesterol to just be the cholesterol produced by the liver. These measurements are then calculated and measured against the optimal ranges of concentrations. (See appendix 1 for optimal cholesterol levels)
One participant volunteered to take part in the study. A sample of blood was removed from the participant after a 12 hour fasting period; this sample was then spun in the centrifuge to separate the serum from the red blood cells in preparation for the lab.
Measurement of Blood Triglyceride Levels
Label four cuvettes as blank (1), standard (2), sample (3), and unknown (4), placing 1 ml of reagent solution R1 in each one. In addition to this, add 10 µl of the standard solution into cuvette 2, 10 µl of the sample (serum) into cuvette 3, and finally 10 µl of the unknown solution into cuvette 4. Once these have been mixed, by gently inverting them several times, leave at room temperature for 10 minutes to allow the reactions to take place. Ensure that the spectrophotometer is set to a wavelength of 500 nm, then place cuvette 1 into the machine and zero the value. Then place each cuvette into the spectrophotometer ensuring that it is set to aero each time using the blank cuvette. Repeat the readings for each cuvette (2, 3, 4) three times in order to gain an average absorbance reading.
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Measurement of Total Cholesterol Levels in Blood Serum
As before, label cuvettes blank (1), standard (2), sample (3) and unknown (4), placing 1000 µl of reagent solution R1 into each one. In addition add 10µl of distilled water to cuvette 1, 10 µl of cholesterol standard to cuvette 2, 10 µl of blood serum to cuvette 3 and 10 µl of unknown solution to cuvette 4. Allow these to stand at room temperature for 10 minutes, once they have been gently inverted. Ensure the spectrophotometer is set to a wavelength of 500 nm and then repeat the procedure as before, ensuring that the spectrophotometer is zeroed between each sample.
Measurement of HDL - Cholesterol Concentration in Blood Serum
Once again label 4 cuvettes blank (1), standard (2), sample (3) and unknown (4), placing 1000 µl into each one. In addition, add 100 µl of deionised water to cuvette 1, 100 µl of standard solution to cuvette 2, 100 µl of the blood serum to cuvette 3 and 100 µl of unknown solution to cuvette 4. Gently invert these and leave at room temperature for 10 minutes. Carry out the same procedure as before using the spectrophotometer to record absorbency readings on a wavelength of 500 nm.
(To view the completed absorbency tables and calculations carried out, see appendix 2)
Measurement of Triglyceride Concentration in Serum
0.132/0.262 x 195 = 98.24 mg/dl
To get the free glycerol we have to deduct 10mg/dl from the above figure bringing it to 88.24 mg/dl.
Measurement of Total Cholesterol Levels in Blood Serum
0.187/0.294 x 196.86 = 125.21 mg/dl
Measurement of HDL- Cholesterol in Serum
0.236/0.806 x 196.86 = 57.64 mg/dl
Calculation of LDL - cholesterol
125.21 - (88.24/5) - 57.64
125.21 - 17.648 - 57.64 = 49.922mg/dl
As expressed before, cholesterol is an important component in helping the body function properly, however it is essential to have the right levels of cholesterol otherwise there is risks of strokes, heart disease and atherosclerosis. Reviewing the results gathered from the experiment, the participants' triglyceride levels fit into the desirable category showing that their body is consuming sufficient amounts of carbohydrates and fats within their diet, allowing any excess sugars not used for energy to be converted to LDL cholesterol by insulin within the liver. The levels of LDL and HDL cholesterol within the blood of the participate fit into the optimal level for LDL cholesterol and the average for HDL cholesterol, presenting the participant to have a minimal build up of cholesterol around the arteries suggesting that they are fit and healthy. The levels of cholesterol effect changes in the proteins that are present on the plasma membrane, as well as glucose metabolism (Hao, Steven Head, Gunawardana, Hasty, Piston, 2007) ; if HDL is too low this can course depression in the individual (Psychosomatic Medicine 2000;62). If the individual consumes too much fatty foods rich in sugars, and does not use them for energy then there is a build up of LDL cholesterol being converted; this results in cholesterol surrounding the arteries increasing the risk of heart disease. Insulin that is released to balance out the sugars within the body can promote the storage of fat, helping to encourage rapid weight gain and elevated triglycerides (Hao, et al, 2007). The participants' total cholesterol level is at a desirable stage reinforcing that the levels of HDL and LDL are adequate within the body and do not present a risk of heart disease of stroke. If the arteries had a high build up of cholesterol then it will restrict amount of oxygen (carried by the blood) getting to the heart, resulting in chest pains and in extreme cases heart attacks (U.S Department of Health and Human Service, 2001).
Cholesterol is affected by many different factors, physical activity helps to lower LDL cholesterol and increase HDL cholesterol, therefore allowing more cholesterol to be transported away from the heart to be converted into bile and excreted, or helps with the function of the cell membrane. Carrying out regular weekly exercise that burns 800 -1200 kcal is recommended to increase HDL to a desirable amount (Heaner, M.); the participant supports this theory by carrying out regular (3-5) aerobic sessions a week, promoting the increase of HDL's. Triglycerides can also be reduced by 15 to 25 percent through regular exercise however the trigger for this is much higher, exceeding 2,500 to 3000 calories to be burnt per week. In summary this study shows that the participants levels of cholesterol, broken down to HDL, LDL and triglycerides, matches the desirable levels of a healthy person, showing that they have a healthy diet, with regular exercise to avoid the LDL cholesterol and triglyceride levels from increasing.
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In conclusion, the result obtained from the experiment show that the participant has healthy levels of cholesterol, triglycerides, HDL and LDL cholesterol, representing that their body is functioning correctly and they are maintaining a healthy balanced diet.