Total Cholesterol and Triglyceride Estimation Experiment

Total cholesterol and triglyceride estimation using the Randox Total Cholesterol and Triglyceride Assay Kits and the comparison and evaluation of the class results for Controls 1 and 3, using both of these assays.

Results

The following are the absorbances (A) and lipid profile results comprising of total cholesterol, triglyceride, LDL cholesterol and HDL cholesterol concentration all given in mmol/L, obtained for the controls, calibrator and patient’s 1 to 5. These results were obtained using the Total Cholesterol Kit and Triglyceride Kit, both by Randox. The class results for Control 1 and Control 3 for both the total cholesterol and triglyceride assays are also given.

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Table 1: First absorbance reading (A), second absorbance reading (A), average absorbance (A) for Patients 1 to 5, control 1, control 3, the calibrator and the blank, using the Triglyceride Assay Kit by Randox. The absorbances were read on a spectrophotometer at 500nm.

Note: Not all samples were tested in duplicate as there was not enough of the Randox Triglyceride Reagent for all 17 tubes.

Table 2: First absorbance reading (A), second absorbance reading (A), average absorbance (A) for Patients 1 to 5, control 1, control 3, the calibrator and the blank, using the Total Cholesterol Assay Kit by Randox.The absorbances were read on a spectrophotometer at 500nm.

Table 3: Triglyceride concentration (mmol/L), using the Triglyceride Assay Kit by Randox, total cholesterol concentration (mmol/L), using the Total cholesterol Assay Kit by Randox LDL cholesterol concentration (mmol/L), calculated using the Friedewald equation and HDL cholesterol concentration (mmol/L), which was given in each case study, for patients 1 to 5. The target values are given below each of the lipid types.

Results highlighted in red are above the target value and results in yellow are below the target value.

Calculation of triglyceride concentration (mmol/L):

Average absorbance (A) of the sample / Average absorbance (A) of the calibrator x concentration of the calibrator

E.g. Patient 1 – 0.121A / 0.317A x 2.15 mmol/L = 0.82 mmol/L

Calculation of total cholesterol concentration (mmol/L):

Average absorbance (A) of the sample / Average absorbance (A) of the calibrator x concentration of the calibrator

E.g. Patient 2 – 0.237A / 0.338A x 5.27 mmol/L = 3.70 mmol/L

Friedewald equation calculation for LDL cholesterol concentration (mmol/L):

Total cholesterol (mmol/L) – HDL cholesterol (mmol/L) – Triglycerides / 2.2

E.g. Patient 3: 7.86 (mmol/L) – 3.30 (mmol/L) – 1.45 (mmol/L) / 2.2 = 3.90 (mmol/L)

Table 4: Class results for the triglyceride concentration (mmol/L), using the Randox Triglyceride Assay Kit and total cholesterol concentration (mmol/L), using the Randox Total Cholesterol Assay Kit of Control 1 and Control 3. Included also in this table are the mean (mmol/L), standard deviation and % Coefficient of variation (%CV) for each of the samples, with and without the outliers. These results are to be evaluated in terms of between-run precision to give an indication of the overall precision of both assays.

The two triglyceride concentration (mmol/L) results for the first class result are the two separate control 1 results obtained by the same person showing poor duplicates.

The two total cholesterol concentration (mmol/L) results for the seventh class result and fourteenth class results are the two separate control results obtained by each person, these results show poor duplicates.

No control 1 result was obtained for the seventh class result in the total cholesterol assay.

The individual results are highlighted in green and the result highlighted red and with an asterisk is the other own result that was also an outlier.

Results highlighted in red are outliers.

The mean and standard deviation of the pooled plasma replicates was carried out on a Casio fx-83GT Plus calculation in the STAT mode.

 %CV calculation:      

Standard deviation / Mean (mmol/L) x100 = %CV.

Table 5: Summary table of the lipid profile results obtained for patient’s 2 and 5, which are to be discussed in conjunction with the accompanying case study in the discussion section. The target value (mmol/L) for each lipid profile component is given also in this table.

Results highlighted in red are above the target value and results in yellow are below the target value.

Discussion

A lipid profile consists of triglyceride, total cholesterol, HDL cholesterol and LDL cholesterol measurement. These measurements are used to diagnose and guide the treatment of disorders of lipid metabolism and endocrine disorders namely, diabetes mellitus and kidney disease. Lipids play a pivotal role in most aspects of bodily function. They provide energy and insulation in the body to prevent heat loss and to allow the conduction of nerve signals and are also involved in metabolic and hormonal pathways and are the structural components of cells.

Patient 2 was referred to the lipid clinic by her GP. This patient has a family history of coronary artery disease and so, is at higher risk of developing the disease. She presented to her GP with tendon xanthomata, which is a fatty deposition of cholesterol in the tendons. This is characteristic in patients who have familial hypercholesterolaemia, which this patient was diagnosed with on presentation. Familial hypercholesterolaemia is a lipid disorder that is autosomal dominant. It is caused by a defect in the receptor that is responsible for recognising and removing LDL from the circulation. This defect can either be due to ineffective internalisation of the LDL cholesterol or due to the receptor being defective or absent, resulting in decreased LDL binding. The clinical characteristics of familial hypercholestrolaemia include xanthomata in the elbows, knees, feet or hands. This is the subcutaneous deposition of lipids. An LDL cholesterol concentration that is increased, a slightly decreased HDL cholesterol, along with a moderately triglyceride concentration are all seen in this lipid disorder. In most individuals who have familial hypercholesterolaemia, hypercholesterolaemia will be present at birth. Heterozygous familial hypercholesterolaemia is the most common form of the disorder and is seen in 1 in 500 people in the United States, while homozygous familial hypercholesterolaemia occurs in 1 in 1 million people. Heterozygous familial hypercholesterolaemia is treated with statin therapy and or sequestrants of bile salts. Homozygous familial hypercholesterolaemia does not respond to therapy and often times, liver transplantation is required. Those who are homozygous for the disorder have a 4 to 6 increase of LDL cholesterol than normal, while heterozygotes show a 2 to 3 LDL increase. The fasting sample that was tested for the patient in this practical is from a routine check-up appointment. Her triglyceride, total cholesterol and LDL cholesterol concentration were all within the target values. However, her HDL cholesterol concentration was less than the target value. HDL cholesterol gathers up and carries excess cholesterol back to the liver. It partakes in reverse cholesterol transport and because of this, HDL is anti-atherogenic. Since her levels of this cholesterol are low, she is at greater risk of developing atherosclerosis. The LDL result obtained for this patient was below the target value. LDL cholesterol is rich in cholesterol, delivers this cholesterol to peripheral cells and facilitates the build- up of fatty deposition in arteries, in a process known as atherosclerosis. The patient was put on statin therapy as she is a high risk patient for coronary artery disease. Statins are lipid lowering medication. They are also known as HMG-coA reductase inhibitors. HMG-coA reductase is an enzyme that controls the rate at which cholesterol is synthesized in the liver. HMG-coA reductase inhibitors are a medication that blocks the rate at which cholesterol is endogenously synthesized in the liver. This inhibitor also causes the LDL receptors to be expressed in the liver, increasing plasma LDL catabolism and lowering the concentration of cholesterol in the plasma and thus, reduces the risk of atherosclerosis. As this patient’s LDL concentration was below the target value, it is clear that the statin therapy

is causing the patient’s cholesterol levels to be decreased. Statins reduce the risk of cardiovascular disease and mortality in high risk patients. As this patient has been started on statin therapy, this indicates that she is heterozygous for the disorder. She has also presented with tendon xanthomata, which manifests by 20 years of age in heterozygous individuals (Varghese, 2014).

Patient 5 has just recently been diagnosed with Type 2 diabetes and was described as being a smoker, overweight and drinking greater than 20 units of alcohol per week, which are all risk factors in the development of type 2 diabetes. Elevated levels of free fatty acids in the plasma play a principal role in insulin resistance. Dyslipidaemia is secondary to diabetes mellitus. Insulin plays a pivotal role in controlling lipid metabolism. Type 1 and type 2 diabetes both have an association with plasma lipid abnormalities. Hypertriglyceridaemia due to an increase in very low density lipoprotein (VLDL) is seen in type 1 diabetes, upon presentation or when glycaemic control begins to deteriorate. Chylomicronaemia may also be present due to lipoprotein lipase (LPL) activity being decreased. LPL is stimulated by insulin. Hormone sensitive lipase is increased, which insulin would normally inhibit, facilitating free fatty acids to be released from adipose tissue. This occurs due to poor insulin activity. These fatty acids are involved in the synthesis of triglyceride in the liver, where they act as a substrate. This is commonly seen in type 2 insulin resistance diabetes, causing an increase in the secretion of VLDL, which this patient has. Insulin treatment reverses both of these effects. Good glycaemic control reduces the occurrence of hypertriglyceridaemia. Hypertriglyceridaemia is also seen in type 2 diabetes mellitus. Increased hepatic synthesis is the cause of the hypertriglyceridaemia, but is less severe than that in type 1 diabetes mellitus. LDL cholesterol concentration is not greatly increased. However, the LDL particles tend to be more atherogenic, small and dense. The concentration of HDL is also decreased in this type of diabetes. Lipids regulate the production of apolipoprotein B. Increased synthesis of apolipoprotein B is seen when there is insulin resistance. Apolipoprotein B may become glycated in both types of diabetes mellitus, enhancing the atherogenic characteristic of the lipoprotein. This occurs by LDL having a reduced affinity for the receptors of LDL. The LDL then begins to be increasingly taken up by the scavenger receptors of macrophages, which lead to the accumulation of fatty streaks in damaged epithelial tissue in arteries, facilitating atherosclerosis. Even with good glycaemic control, the lipid abnormalities may still persist. Lipid lowering drugs should be used to treat these abnormalities due to the significantly higher risk of cardiovascular disease due to diabetes. There is increased synthesis of apolipoprotein B, which is the major constituent of VLDL. This patient’s total cholesterol is elevated and is greater than the target value. While the LDL, HDL and triglyceride concentration for this patient are all below the target values. The treatment options that are beneficial for this patient is firstly dietary intervention. The patient should reduce their alcohol consumption, give up smoking, exercise regularly, reduce their fat intake per day and aim for their total fat intake to be less than 30% of their total daily calorie intake. Drug therapy could also be administered to this patient. Drugs such as statins, fibrates and nicotinic acid could be taken by this patient to lower their total cholesterol, VLDL and LDL cholesterol concentration. Fibrates are peroxisome proliferator activated receptor (PPAR) alpha agonists that target the PPAR to lower triglyceride and raise HDL concentrations. They target lipid pathways by reducing the synthesis of VLDL and ApoC3, which inhibits lipoprotein lipase (LPL), increasing the activity of LPL, causing the VLDL and chylomicron remnants to be removed quickly from the circulation and increasing the synthesis of apoA1, which is associated with HDL. Nicotinic acid reduces LDL concentration and the secretion of VLDL. These forms of drug therapy will aid in the control of treating this patient’s hypertriglyceridaemia and lower the risk of them developing cardiovascular disease due to atherosclerosis formation by increased LDL cholesterol concentration (Schofield et al., 2016).

Table 4 displays the class results for the triglyceride assay. The between-run precision of these results was evaluated to measure the random error in the two assays carried out. For control 1, two outliers were identified. These are highlighted red in this table. The first class result had two values obtained for control 1. The first result obtained by this person was significantly higher than the other class results, while the other result was in line with class results. The reason for their first result being elevated is perhaps that twice the required volume of 20µl of the control was pipetted into the eppendorf rather than just the required 10µl. All other class results for this control were similar in value. For control 3 in this same assay, three outliers were identified. The first class result again was twice the triglyceride concentration of the rest of the class. The reason for this most likely being that double the control volume was pipetted into the eppendorf. The fourth class result, being the individual result, was also an outlier and is highlighted in red. It is lower compared to the rest of the class results. A reason for this may be that the sample was not vortexed sufficiently, causing a non-homogenous solution of control and reagent, resulting in the absorbance reading to be low and thus, the triglyceride concentration to be low Another reason could be that not the exact 10µl was added to the eppendorf, leading to a low absorbance reading as this is such a minute volume to be pipetted accurately. Control 1 for this individual result was within that of the class results and is highlighted green. The fifth class result for this control was also higher than the other class results. It again, is almost double of the other results, indicating that more than 10µl was added to the eppendorf. The mean, standard deviation and % coefficient of variation (%CV) was calculated for the class results including and without the outliers. A significant difference is seen with both the standard deviation and %CV for these results. The mean is the average of the results obtained. The standard deviation gives an indication of the variation within the results obtained and how spread out they are from the mean of the results. A standard deviation that is low signifies that the results are close to the mean obtained. The %CV gives the ratio of the standard deviation to the mean of the results obtained. A %CV less than 5% indicates good precision. The %CV obtained for both controls with the outliers is almost twice that of the %CV without the outliers. Both %CV’s calculated without the outliers are greater than the ideal %CV of 5, showing imprecision of the replicates. In a clinical setting if the controls were not within the target range, they cannot be accepted and the assay would have to be again repeated. The patient results, as a result of this can also not be released. Table 4 also displays the class results for controls 1 and 3 used in the total cholesterol assay. There are many outliers for both controls 1 and 3 in this assay. Again, the first class result for both of these controls is significantly higher than the other class results. These results are greater than double the value of the other class results, emphasising that perhaps they added twice the required volume of control to each eppendorf, causing the total cholesterol concentration to be so high. Similarly the sixth class result for control 1 is double the concentration of the other class results, indicating they made the same error as the previous person. The fourteenth class result shows poor duplicate samples for both controls that are again a result of twice the volume of control being added to the eppendorfs, due to poor adherence to the assay procedure. The %CV including the outliers showed an extremely

high level of imprecision. The ideal %CV of 5 was greatly exceeded due to a high mean and standard deviation. Without the outliers, a vast difference was seen in the %CV. However, they were still above 5 for both controls. The precision of both of the control results could have been improved if the class adhered to the procedure of each assay correctly and added the correct volume of control to each eppendorf and vortexed the eppendorf’s sufficiently prior to the incubation period. The precision for these results could be increased if each of the controls were read 20 to 50 times as this amount of repeatability is a good estimate of precision. The variables such as different students reading the controls, the two different reagents used and difference in adherence to procedure all contributed to the imprecision of the control results (Westgard and Westgard, 2013). 

In all, the lipid profile consisting of total cholesterol, triglyceride, LDL cholesterol and HDL cholesterol were carried out on both control 1 and 3 and the five patient samples. Both patient’s 2 and 5 were discussed in terms of the case studies given and the lipid profile results obtained for each. The cholesterol and triglyceride concentrations obtained for controls 1 and 3 by the class and the individual results were discussed and compared to each other in terms of precision.

References

• Schofield, J., Liu, Y., Rao-Balakrishna, P., Malik, R. and Soran, H. (2016). Diabetes Dyslipidemia. Diabetes Therapy [Online] 7, pp.203–219. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900977/pdf/13300_2016_Article_167.pdf  [Accessed on: 2^nd November 2018]
• Varghese, M. (2014). Familial hypercholesterolemia: A review. Annals of Pediatric Cardiology [Online] 7(2), pp.107–117. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070199/ [Accessed on: 1st November 2018]
• Westgard, J. and Westgard, S. (2013). Total Analytic Error. AACC. [Online] Available at: https://www.aacc.org/publications/cln/articles/2013/september/total-analytic-error  [Accessed on: 2nd November 2018]

Practical Risk Assessment Form

Practical title: Total cholesterol and triglyceride estimation using the Randox Assay Kits and the comparison and evaluation of the class results for Controls 1 and 3, using both of these assays.

Practical description: Give a brief description of work to be undertaken and the nature of the materials and techniques to be used.

Pipetting reagent and patient plasma, controls and calibrator samples.

Vortexing samples.

Using a spectrophotometer to read the absorbance of each sample.