Faculty of Medicine Ain Shams University Medical Biochemistry

Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department Practical Analysis Using Spectrophotometer BY ALL STAFF MEMBERS OF MEDICAL BIOCHEMISTRY DEPARTMENT 2018 1

Faculty of Medicine Ain Shams University Medical Biochemistry & Molecular Biology Department ILOs of the current topic: By the end of this topic, the student will be able to: 1. Explain the clinical significance of lipid profile. 2. List indications of lipid profile. 3. Identify different components of lipid profile. 4. Interpret the resulting data. 5. Estimate the concentration of total cholesterol in a serum sample. 6. Identify the normal values of cholesterol in blood. 7. Identify Causes of Hypercholesterolemia. 8. Estimate the concentration of HDL and LDL.

Blood lipid profile Lipid profile or lipid panel is a panel of blood tests that serves as an initial screening for abnormalities in lipids, such as cholesterol and triglycerides. The results of this test can identify certain genetic diseases and can determine approximate risks for cardiovascular disease (CVD).

Blood Lipid Profile Total cholester ol VLDL. Triglycerid es. Lipid profile analysi s HDL cholester ol. LDL cholester ol.

Indications for Performing a Lipid Profile To assess risk of developing cardiovascular disease Identify certain genetic diseases such as familial hypercholester olemia. Monitor treatment of hyperlipidemia such as statins

When is lipid profile indicated? Monitoring: • • • Hypertension Cigarette smoking Diabetes Low HDL levels Family history of CAD • Age over 45 years (men) or 55 years (women). Screening: • High-risk children (family history of heart disease or diabetes, or being overweight). To evaluate effectiveness of treatment

Cholesterol & TG are carried as lipoprotein molecules in the blood stream

TYPES OF LIPOPROTEINS Chylomicron (TAG) VLDL (TAG) LDL (Cholesterol) HDL (Cholesterol)

LDL vs. HDL • LDL = “bad” • Provide cholesterol to the peripheral tissues • Too much can clog arteries by forming plaque • Atherosclerosis can cause heart attack.

Blood Lipid Profile

Important note: • For doing any lipid profile test, fasting for 10 -12 hours before having the blood sample drawn is required; only water is permitted.

Colorimetric estimation of serum total cholesterol

Aim of practical session The aim of this practical session is to : Estimate the concentration of total cholesterol in a serum sample by colorimetric method, using the enzymatic assay.

colorimetric estimation of cholesterol

Principle of the assay - Cholesterol esters are hydrolyzed under the action of cholesterol esterase into free cholesterol and free fatty acids. Cholesterol esters + H 2 O Cholesterol esterase cholesterol + fatty acids

Principle of the assay • Then, Cholesterol oxidase oxidizes Cholesterol with the production of H 2 O 2. Cholesterol + O 2 Cholesterol oxidase 4 -cholestenone + H 2 O 2

Principle of the assay In the presence of peroxidase, the H 2 O 2 reacts with Phenol and 4 -aminophenazone to form a red dye compound. 2 H 2 O 2+ phenol + 4 -aminophenazone peroxidase quinonimine +4 H 2 O

Steps: Label 3 dry test tubes T For the test sample S For the Standard sample B For the Blank

PROCEDURE Distilled water Cholesterol Blank Standard Sample 10 µl ___ ___ ___ 10 µl 1 ml standard Serum/plasma Cholesterol reagent

T S B B Mix the contents of the tubes and let stand for 5 minutes at room temperature.

Record the absorbance of both test & standard against the blank at 540 nm.

Calculate the test concentration by the following equation: Concentration of serum cholesterol (mg/dl) = Absorbance of Test ------------ X Concentration of standard Absorbance of standard

Reference values of serum cholesterol Normal value: < 200 mg/dl Borderline value: 200 -239 mg/dl High value: 240 mg/dl or higher

Causes of hypercholesterolemia 1 - Familial hypercholesterolemia. . 2 - Diabetes mellitus. 3 - Obesity. 4 - Nephrotic Syndrome. 5 -Hypothyroidism 6 -Cholestasis 7 -Cigarette smoking 8 -Obstructive jaundice 9 -Several medicines can worsen cholesterol level: Thiazide diuretics, Oral estrogens (contraceptive pills), Some beta blockers Immunosuppressive agents.

– How many hours of fasting are required for lipid profile? A. 6 -8 hours B. 4 -6 hours C. 10 -12 hours D. 2 -4 hours

A 45 -year old male was doing a routine checkup. He is a smoker. His blood pressure was 140/90. The results of his lipid profile were: • Fasting Total cholesterol = 350 mg/dl • Fasting TAG = 300 mg/dl • Fasting HDL-cholesterol = 40 mg/dl 1. Calculate the level of the patient`s LDLcholesterol and comment on its level 2. Calculate the 10 - year risk of for coronary heart disease in this patient

Answer 1 - Calculate the level of the patient`s LDL-cholesterol • LDL-c = Total cholesterol – (HDL-c + Triglycerides/5) • Triglycerides/5 = (VLDLc) = 300/5 = 60 • LDL-c = 350 – (60 + 40) = 250 (very high)

2 -Calculate the 10 - year risk of for coronary heart disease in this patient Apply ATPIII guidelines You will be provided with the Framingham tables 29

Framingham Point Scores by Age Group Age Points 20 -34 -9 35 -39 -4 40 -44 0 45 -49 3 50 -54 6 55 -59 8 60 -64 10 65 -69 11 70 -74 12 75 -79 13 30

Framingham Point Scores by Age Group and Total Cholesterol Total Age 20 -39 Cholesterol Age 40 -49 Age 50 -59 Age 60 -69 Age 70 -79 <160 0 0 160 -199 4 3 2 1 0 200 -239 7 5 3 1 0 240 -279 9 6 4 2 1 280+ 11 8 5 3 1 31

Framingham Point Scores by Age and Smoking Status Age 20 -39 Age 40 -49 Age 50 -59 Age 60 -69 Age 70 -79 Nonsmoker 0 0 0 Smoker 8 5 3 1 1 Framingham Point Scores by HDL Level HDL Points 60+ -1 50 -59 0 40 -49 1 <40 2 32

Framingham Point Scores by Systolic Blood Pressure and Treatment Status Systolic BP If Untreated If Treated <120 0 0 120 -129 0 1 130 -139 1 2 140 -159 1 2 160+ 2 3 33

10 -Year Risk by Total Framingham Point Scores 3 + 8+ 5+ 1+ 1 = 18 Point Total <0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 10 -Year Risk < 1% 1% 1% 2% 2% 3% 4% 5% 6% 8% 10% 12% 16% 20% 25% 34