Estimation of serum cholesterol.pdf

MOOC on Biochemistry Module-34 Estimation of Serum Cholesterol by Cholesterol Oxidase Method Academic Script Introduction: Cholesterol accounts for almost all of the sterol in plasma. It exists as a mixture of unesterified (30 to 40...

MOOC on Biochemistry Module-34 Estimation of Serum Cholesterol by Cholesterol Oxidase Method Academic Script Introduction: Cholesterol accounts for almost all of the sterol in plasma. It exists as a mixture of unesterified (30 to 40 %) and esterified (60 to 70 %) forms. Fig 1. Structure of cholesterol The proportion of the two forms is constant within and between normal individuals. Various adaptations of the colorimetric and the enzymatic assays can use for cholesterol estimation. Most diagnostic and the research laboratories use the enzymatic assay method. Fig 2. Plasma cholesterol Types of serum cholesterol: Cholesterol exists in 2 forms – unesterified or “free” (UC) and esterified (CE) – and the form determines if we can absorb it or not, or store it or not (among other things). Over time, breaking down the various components that make up the serum cholesterol reading has helped physicians to identify a number of potential health problems and treat them before they become a problem. While the presence of good cholesterol is beneficial to maintaining organ health and providing the body with necessary energy, the presence of bad cholesterol can lead to blockages that may lead to problems with the heart and lungs. For this reason, the medical community has developed standards for what is considered a healthy level of good cholesterol as well as guidelines for what constitutes an excessive amount of bad cholesterol in the blood. Overall, the total serum cholesterol is not expected to be over 200 mg for each dl of blood in the system. The bad cholesterol should not make up more than half of that amount. Ideally, many doctors prefer that the ration between good and bad cholesterol be more along the lines of two to one, with the bloodstream containing twice the amount of good cholesterol when compared to the amount of bad cholesterol present. Simple blood work from time to time helps the physician to monitor cholesterol levels in the body and identify trends that can often be corrected with diet. In some cases, a doctor may prescribe medication that is formulated to lower bad cholesterol while promoting the production of good cholesterol. Because the serum cholesterol reading contains a breakdown of all classes of cholesterol present, and the current milligram per deciliter ratio present, it is possible to deal with high cholesterol before major health issues develop. Lipoprotein : Serum cholesterol is a term that includes the total level of cholesterol that is found in the bloodstream. Within the system cholesterol is associated with lipoprotein carrier. Measuring the level of total cholesterol includes identifying all types or classes of cholesterol that are found in the system. This helpful measurement makes it possible to determine if the balance between the HDL (high density lipoprotein) or good cholesterol and LDL (low density lipoprotein) or bad cholesterol is within acceptable limits. While tracking the HDL and LDL levels are part of what goes into a serum cholesterol evaluation, they are not the only factors considered. Serum cholesterol also involves identifying the current level of very low density lipoprotein (VLDL) content, as well as the intermediate density lipoprotein levels (IDL). These readings, along with the LDL and HDL cholesterol levels, help to provide a complete picture of the lipids and proteins currently present in the body. Relationship of total cholesterol with free and bound cholesterol: TC = LDL-C + HDL-C + VLDL-C + IDL-C + chylomicron-C + remnant-C (C stands for cholesterol)  Biomedical importance of serum cholesterol: 1. The pool of cholesterol in our body is essential for life. No cholesterol means no life. 2. Much of the cholesterol we eat is in the form of cholesterol esters. It is not absorbed and is excreted by our gut (i.e., leaves our body in stool). The reason this occurs is that CE not only has to be de-esterified. 3. Re-absorption of the cholesterol is used for endogenous production of cholesterol. That is, most of the cholesterol in our body is made by our body itself. 4. The process of regulating cholesterol is very complex and multifaceted with multiple layers of control. 5. Eating cholesterol has very little impact on the cholesterol levels in your body. 6. Cholesterol and triglycerides are not soluble in plasma (i.e., they can’t dissolve in water) and are therefore said to be hydrophobic. 7. To be carried anywhere in our body, say from your liver to your coronary artery, they need to  be carried by a special protein-wrapped transport vessel called a lipoprotein. 8. As these “ships” called lipoproteins leave the liver they undergo a process of maturation where they shed much of their triglyceride “cargo” in the form of free fatty acid, and doing so makes them smaller and richer in cholesterol. 9. Special proteins, apoproteins, play an important role in moving lipoproteins around the body and facilitating their interactions with other cells. The most important of these are the apoB class, residing on VLDL and LDL particles, and the apoA-I class, residing for the most part on the HDL particles. 10. Cholesterol transport in plasma occurs in both directions, from the liver and small intestine towards the periphery and back to the liver and small intestine (the “gut”). 11. The major function of the apoB-containing particles is to traffic energy (triglycerides) to muscles and phospholipids to all cells. Their cholesterol is trafficked back to the liver. The apoA-I containing particles traffic cholesterol to steroidogenic tissues, adipocytes (a storage  organ for cholesterol ester) and ultimately back to the liver, gut, or steroidogenic tissue. 12. All lipoproteins are part of the human lipid transportation system and work harmoniously together to efficiently traffic lipids. The trafficking pattern is highly complex and the lipoproteins constantly exchange their core and surface lipids. Fig 3. Different types of lipoprotein Sources of serum cholesterol: Exogenous source: From diet meat and dairy product, it is present in abundance in animal fat and liver. Endogenous source: From intestines Fig 4. Sources of serum cholesterol Methods for serum cholesterol estimation:  Fluorometric--of cholesterol has been achieved, but simple and rapid methods are still needed for the determination of microquantities of cholesterol in biological fluids. Fluorometric methods are generally more sensitive than spectrophotometric methods, and existing fluorometric methods for cholesterol were modifications of the tedious Lieberman- Burchard reaction. The Tschugaeff reaction also has been applied to fluorometry. Although the acid ferric chloride reaction of Zlatkis et al. has been accepted as a sensitive spectrophotometric method for the determination of cholesterol, a fluorometric application of this reaction has not been investigated previously. In Leffler’s modification of the ferric chloride method, 2-propanol was used to extract cholesterol. Since this solvent has no measurable fluorescence, this method seemingly could be adapted for use with other instruments, depending upon the concentration of cholesterol in sample. The proposed fluorometric method, the method of choice for microsamples.  Spectrophotometric— a. Chemical method: By using Ferric chloride- In this experiment, acetone alcohol is used for extraction process of cholesterol. This is done by separating the cholesterol from serum by centrifugation process. The cholesterol extract can used for to determine the esterified and unesterified cholesterol. Acetone alcohols vaporized and leave behind the cholesterol. Free cholesterol is easily determined by direct precipitation. Digitonin is a substrate to differentiate free cholesterol from total cholesterol and esterified cholesterol. In digitonin precipitation method, alcoholic solution of cholesterol is mixed with an alcoholic solution of digitonin, which formed cholesterol digitonide. This complex has a low solubility and making the test is more sensitive. After precipitation is completed, digitonide is separate into its component. Ester and digitonide give a purple colour of equal intensity. Cholesterol digitonide is insoluble in water but soluble in glacial acetic acid. Ferric chloride-sulphuric acid is added to the tube when measuring the density for cholesterol standard at 575nm. b. Enzymatic method: By cholesterol oxidase— Cholesterol esters are oxidized to cholesterol and free fatty acids by the help of cholesterol esterase. This Cholesterol is then oxidized to Cholesten-3-one and H2O2 by the help of cholesterol oxidase, ultimately from this H2O2 molecular oxygen and water is getting produced where the molecular oxygen is coupled with 4- AAP to give pink colour of quinoneimine dye (pink in colour) and can be measured at 530nm spectrophotometrically. Fig 5. Enzymatic determination of cholesterol ESTIMATION OF CHOLESTEROL BY CHOLESTEROL OXIDASE METHOD: A. HISTORY:  Cholesterol estimation methodologies have been critically reviewed by Tonks, 1967.  The enzymatic method described below, and used in this assay, is a modification of that described in 1974 by Allain et.al. B.PRINCIPLE: The cholesterol esters are hydrolyzed to free cholesterol by cholesterol esterase (CE). The free cholesterol is then oxidized by cholesterol oxidase (CO) to cholesten-3-one with the simultaneous production of hydrogen peroxide. The Hydrogen peroxide (H2O2) produced then is converted to H2O and O2 in the presence of peroxidase, consequently this molecular oxygen O2 couples with 4-aminoantipyrine and phenol, to yield a chromogen (Quinoneimine dye)with maximum absorbance at 530 nm. The intensity of the color produced is directly proportional to the concentration of total cholesterol in the sample. Cholesterol esterase 1. Cholesterol Esters ------------ ---------------→ Cholesterol + Fatty Acids Cholesterol oxidase  2. Cholesterol + O2 ----------- -----------------→ Cholesten-3-one + H2O2 Peroxidase 3. 2 H2O2 + 4-aminoantipyrine + Phenol ----- ----------------→ Quinoneimine + 4 H2O C. REACTION SCHEME: Fig 6. Reaction scheme for enzymatic cholesterol determination. Commercial cholesterol assay kits employ all the enzyme systems (cholesterol esterase, cholesterol oxidase, peroxidase) to produce a Schiff’s base (quinoneimine dye) which is a red dye, and can be determined by adsorption spectrometer at 530nm. D. REQUIREMENTS: 1. Equipments:  The equipments needed for the experiments are as follows: 1. Test tube 2. Cuvette. 3. Centrifuge machine 4. Micro pipette Fig 7. Colourimeter 5. Photoelectric colourimeter / spectrophotometer 6. Incubator 7. Test tube rack. 2. Reagents : a. The list of reagents available in kits and other than kit: 1. Cholesterol Phenol Reagent (as reagent 1): A solution containing 40 mmol/L phenol, a surfactant, and a stabilizer. 2. Cholesterol Colour Reagent (as reagent 2): A solution containing  Phosphate buffer (pH 6.7 at 25°C) containing  1.6 mmol/L 4-aminoantipyrine or 4- aminophenezone, > 5560 U/L.  Peroxidase (botanical), > 400 U/L  Cholesterol esterase (mammalian), > 400 U/L  Cholesterol oxidase (microbial), a preservative, and a stabilizer. 3. Cholesterol Calibrator (standard cholesterol): 1 x 15 ml solution containing 100 mg/dl cholesterol, and 1x 15ml solution containing 500 mg/dl cholesterol standards, a surfactant, and a preservative. Sometimes in kit upper limit standard (as reagent 3) and lower limit standard (as reagent 4) both are supplied. 4. Alternative calibration material (if kit calibrator is not appropriate for use). Fig 8.Kit for estimation of serum cholesterol by enzymatic method b. Reagent preparation: WORKING REAGENT PREPARATION:  To make working reagent : required volume of working reagent is prepared by mixing equal volumes of colour reagent and phenol reagent.  Gently and thoroughly mixing will be done. c. Reagent deterioration:  The reagent solutions should be clear. Turbidity would indicate deterioration. The reagents included are stable until the expiry date stated on the labels at 2-8°C. The prepared working reagent is stable for 4 weeks at 2-8°C or for 5 days at 18-26°C, - 20°C for 4 weeks. Stability claims are based on real time studies. d. Reagent disposal: Reagents must be disposed of in accordance with all Federal, Provincial, State, and local regulations. e. Reagent storage: Samples should be stored at 2-8°C for 1 week or - 20°C for 4 weeks. PREPARATION OF STANDARD AND BLANK:  1. A series of cholesterol standards can be made or one standard can be taken supplied by the kit (taking 500mg/dl as master stock). 2. Standard may be diluted with acetone-alcohol. 3. Series of standards includes 200mg/dl=S2, 300mg/dl= S3, 400 mg/dl=S4, made from the master stock 500 mg/dl=S5 and 100mg/dl=S1 supplied by the kit) 4. The reagent blank is made by taking water. PREAPARATION OF SPECIMEN/SAMPLE: a. Preparation of total serum sample for cholesterol estimation:  Serum: Fresh, clear, unhemolysed serum. The specimen should be drawn in the morning following a 12 hour fast. 1. 0.5 ml plasma was added into a graduated centrifuge tube. 2. 9.5 ml of acetone-alcohol were used as solvent. 3. The tubes are placed in a water-bath at 60ºC for 15 min.  4. The mixture is cooled and appropriate amount of the acetone-alcohol is added so that the final volume is 10 ml. 5. The tubes are covered and centrifuged for 5 min at 3000 rpm. 6. The supernatant obtained is used for the following analysis. Fig 9. Preparation of serum b. Storage of sample: The collected and processed serum sample must be stored at 4 °C. E. METHOD: Three tubes are taken and marked them as regent blank (B), Standard (ST), Sample (S). Then following the working table mentioned below the reagents are mixed and incubate at 37ºC for 10min. Then the O.D is measured by spectrophotometer at 530nm by spectrophotometer. Water is taken as reagent blank. Working table: Assay Wate Standard Seru Workin system r (µl) cholester m g ol (µl) (µl) Reagen t (µl) Standard 10 1000 (ST) Blank (B) 10 1000 Test/samp 10 1000 le (S) Fig 10. Preparations of assay mixture.  Inspite of taking one standard the series of standard of cholesterol can be made for preparation of standard calibration curve. With each standards (ST=S1, S2, S3, S4, S5) the same amount of reagent will added followed by spectrophotometric analysis). F. PARAMETRES FOR ANALYSIS: 1. Wave length : 530 mm (the wavelength scanning range is 500-546 nm) 2. Optical pathway : 1 cm 3. Temperature : 37ºC 4. Measurement control: Against reagent blank one hour. G. WARNINGS and PRECAUTIONS: 1. Ingestion must be avoided. 2. Contact with skin and eyes must be avoided. 3. Material Safety Data Sheet must be taken care of for additional information. 4. The acetone-alcohol solvent was not allowed to over-boil. I. ANALYTICAL PROCEDURE: a. Test condition: Studies are performed on an automated analyzer using an endpoint test mode, with a sample to  reagent ratio of 1:100 and a wavelength reading of (primary/secondary) 505/600 nm. Fig 11. Analysis of serum cholesterol. b. Calibration: Calibration material should be used to calibrate the procedure. The frequency of calibration, if necessary, using an automated system is dependent on the system and the parameters used. c. Quality control: A normal and abnormal concentration control should be analyzed as required in accordance with local, state, and federal guidelines. The results should fall within the acceptable range as established by the laboratory. Fig 12. Quality control of serum cholesterol estimation.  H. SPECIFICITY:  Cross contamination studies have not been performed on automated instruments. Certain reagent/ instrument combinations used in sequence with this assay may interfere with reagent performance and test results. The existence of, or effects of, any potential cross contamination issues are unknown.  Interferences in the measurement of cholesterol using cholesterol oxidase procedures have been studied by different groups of scientists.  A summary of the influence of drugs on clinical laboratory tests may be found. J. CALCULATIONS: The analyzer automatically calculates the total serum cholesterol concentration of each sample. By comparing with the standard cholesterol data the test (serum) cholesterol data can be obtained. a. If the series of standards are used then from the standard curve by putting the value of unknown O.D the unknown serum cholesterol concentration can be obtained. Fig 13. Calibration (standard curve for cholesterol analysis) b. Otherwise by taking one/two standard calibrator (with known concentrations supplied by the kit) O.Ds that can be compared with the unknown O.D of the sample to detect the serum cholesterol concentration. K. LIMITATIONS: A sample with a total serum cholesterol concentration exceeding the linearity limit (>500 mg/dl) should be diluted with 0.9% saline and reassayed incorporating the dilution factor in the calculation of the value. L. RESULT/REPORT Presentation of results: Total serum cholesterol concentration is reported as mg/dL (mmol/L) by comparing with one standard or from standard curve of series of standards. Interpretation of results: High serum cholesterol is strongly implicated in the development of cardio vascular diseases. The relationships between ABO blood groups and cholesterol levels have been established but differ in many studies. Age ranging between 18-35 year were taken to assess such correlation where blood groups and total serum cholesterol were determined. A chi square test among the ABO blood groups and cholesterol levels is statistically significant at p < 0.05. Fig 14. Reports for clinical diagnosis of serum cholesterol M. PERFORMANCE CHARACTERISTICS: Reportable range :  The linearity of the procedure described is upto 500 mg/dL (12.93 mmol/L) if any standard curve is made. Precession: Run to run precision was established by assaying two control sera twice a day for 10 days. Within run precision was established by assaying 2 pools of human sera thirty times each.  Mean Standard Deviation: Cholesterol data expressed in: mg/dL, mmol/,L.  Coefficient of Variation (% ): With different serum and standards can be calculated. N. ADVANTAGES The advantages of the method are: (1) The stability of the optical density over a period of hours, (2) The stability of the reagent for 2 or 3 days, (3) The high degree of reproducibility of the results, and O. ACCURACY: Linear regression analysis with different standard cholesterol and standard serum will give the accurate results.  P. STANDARD REFERENCE INTERVALS: Values for selecting adults at moderate and high risk of coronary artery disease who require treatment. Age (Years) Moderate Risk High Risk 20-29 >200 mg/dL (5.17 mmol/L) >222 mg/dL (5.74 mmol/L) 30-39 >220 mg/dL (5.69 mmol/L) >240 mg/dL (6.21 mmol/L) 40 and over >240 mg/dL (6.21 mmol/L) >260 mg/dL (6.72 mmol/L) *From the NIH Consensus panel recommendation. These values are suggested guidelines. It is recommended that each laboratory establish the normal range for the area in which it is located. c. Clinical evaluation: Below are the information ranges for the serum cholesterol:  3.6-5.2 mmol/L (normal)  5.3 -6.2 mmol/L (borderline high)  6.2 mmol/L (hypercholesterolemic). If the serum cholesterol that obtained from this experiment is 7.0 mmol/L. This means the serum sample is hypercholesterolemic. Q. COMMENTS/CONCLUSION: According to the data obtained from the method as well as by comparing with the standard data the comments or conclusion will be drawn and accordingly the therapies will be suggested by the nutritionist. Remedy against high cholesterol: Nutrition therapy: The remedy against high serum cholesterol includes proper diet. Certain foods will avoided for hyperchoelerolemic patients like cheese, yoghurt, ice-cream, mutton, any sort of animal fat, butter etc. They are called as forbidden food by nutritionist. But some of the foods are called recommended foods to lower blood/serum cholesterol level like white portion of egg only, fruits and vegetables including grapes and black berry, whole grain bread. Fig 15a. Diet for hypercholesterolemia, Fig 15b. Forbidden food for hypercholesterolemic patient