1. Lipids (Lipid Profile)
Lab. 5

2. Introduction The major lipids present in the plasma are:
fatty acids,
Triglycerides,
cholesterol
and phospholipids.
Other lipid-soluble substances, present in much smaller amounts (e.g. steroid hormones ).
Elevated plasma concentrations of lipids, particularly cholesterol, are related to the pathogenesis of atherosclerosis.

3. Lipids transport
Lipids are carried in the bloodstream by complexes known as lipoproteins.
This is because these lipids are not soluble in the plasma water.
Thus they travel in micelle-like complexes composed of phospholipids, cholesterol and protein on the outside with cholesteryl esters, and triglycerides on the inside.
The four main types of lipoproteins are chylomicrons, VLDL, LDL, and HDL

4. Clinical Significance
Cholesterol and triglycerides, like many other essential components of the body, attract clinical attention when present in abnormal concentrations.
Increased or decreased levels usually occur because of abnormalities in the synthesis, degradation, and transport of their associated lipoprotein particles.
Increased or decreased plasma lipoproteins are named hyperlipoproteinemia & hypolipoproteinemia respectively.

5. Lipids as Biochemical Markers of Disease
Clinical chemistry laboratories offer many tests for lipid disorders.
One of the most common tests is the lipid profile.
This panel of tests includes measurement of triglycerides and cholesterol in the form of lipoprotein-cholesterol molecules,
low density lipoprotein cholesterol (LDL-C)
and high-density lipoprotein cholesterol (HDL-C).
The results of testing for these lipids provide measures of risk for coronary artery disease.

6. It is therefore clear that lipid measurements should be made in all patients known to have vascular disease, and in those at increased risk.
Thus plasma lipids should be measured in the individuals with the following:
CHD (and cerebrovascular and peripheral vascular disease)
a family history of premature coronary disease (occurring at age <60 years)
other major risk factors for CHD (e.g. diabetes mellitus, hypertension)
patients with clinical features of hyperlipidaemia
patients whose plasma is seen to be lipaemic.

7. Positive Risk Factors No Risk•
Age: ≥45 years for men; ≥55 years or premature menopause for women
Family history of premature CHD
Current cigarette smoking
Hypertension (blood pressure ≥140/90 mm Hg or taking antihypertensive medication)
LDL cholesterol concentration ≥160 mg/dL , with ≤1 risk factor
LDL cholesterol concentration ≥130 mg/dL , with ≥2 risk factors
LDL cholesterol concentration ≥100 mg/dL , with CHD or risk equivalent
HDL cholesterol concentration <40 mg/dL
Diabetes mellitus = CHD risk equivalent
No Risk
HDL cholesterol concentration ≥60 mg/dL
LDL cholesterol <100 mg/dL

8. Triglycerides Sources of Triglycerides:
Glycerol backbone with FA attached by ester bonds
Sources of Triglycerides:
Exogenous source: Dietary
Endogenous : Liver and tissue storage
Glycerol
Triglyceride

9. Triglycerides
Serum triglycerides measurements are done for the following clinical reasons:
• Hypertriglyceridemia increases the risk for pancreatitis.
• Hypertriglyceridemia is associated with the following clinical findings: eruptive xanthoma, lipemia retinalis, hepatomegaly, splenomegaly, depressed HDL-cholesterol.
in characterizing risk of CVDs
• For the estimation of LDL-cholesterol, using the Friedewald equation
lipemia retinalis: that manifested by a milky appearance of the veins and arteries of the retina.
Eruptive xanthoma: The sudden eruption on the skin of crops of pink papules (firm pea-sized bumps) with a creamy center. They may appear on the hands, feet, arms, legs and buttocks.

10. Specimen Serum, Plasma (EDTA)
Certain anticoagulants, such as fluoride, citrate, and oxalate, cause large shifts of water from the red blood cells to the plasma, which result in the dilution of plasma components ( as much as 10%)
Fasting sample (from 12 to 16 h) is essential for triglyceride analysis
Samples drawn from nonfasting patients are not suitable for analysis, since elevated TG levels caused by normal absorption of food cannot be distinguished from elevated TG resulting from abnormal lipid metabolism or inborn errors of metabolism.
Storage and stability

11. Enzymatic Method
Glycerol, released from triglycerides after hydrolysis with lipoprotein lipase,
Tranformed by glycerolkinase into glycerol-3- phosphate
which is oxidized by glycerolphosphate oxidase into dihydroxyacetone phosphate and hydrogen peroxide.
In the presence of peroxidase, the hydrogen peroxide oxidizes the chromogen 4- aminophenazone/ESPT to form purple quinoneimine whose intensity is proportional to the concentration of triglycerides in the sample.

12. glycerolphosphate oxidase
Enzymatic Method
Triglycerides Glycerol + 3 fatty acids
Glycerol + ATP Glycerol-3 phosphate + ADP
Glycerol-3 phosphate dihydroxyacetone + H2O phosphate
H2O2 + 4-aminophenazone/ESPT Quinoneimine
Lipoprotein lipase
glycerolkinase
glycerolphosphate oxidase
peroxidase

13. Procedure: Sample Standard Blank 1ml Reagent A - 10ul 10 ul
Distilled water
Mix and incubate for 10 min at room temperature or 5min at 37 .
Read the absorbance at 550( ) nm against blank.
Colour is stable for 60min,ptotected from light.

14. Cholesterol
Cholesterol is a sterol compound that is found in all animal tissues
Serves many important physiological functions including:
synthesis of bile acids,
steroid hormones,
and cell membranes.
Cholesterol also appears to be involved in atherosclerosis; thus cholesterol measurement is one of the most common laboratory tests used today.

15. Specimen Serum, Plasma (EDTA, Heparin)
Certain anticoagulants, such as fluoride, citrate, and oxalate, cause large shifts of water from the red blood cells to the plasma, which result in the dilution of plasma components.
Storage and Stability
7 days at 4 – 8 °C
3 months at -20 °C

16. Enzymatic Reaction
Determination of cholesterol after enzymatic hydrolysis and oxidation.
The colorimetric indicator is quinoneimine which is generated from 4-aminoantipyrine and hydroxybenzoate by hydrogen peroxide under the catalytic action of peroxidase
Cholesterol Esterase
Cholesterol oxidase
Cholesterol-3-one
Peroxidase

17. Procedure: Sample Standard Blank 1ml Reagent - 10ul 10 ul
Distilled water
Mix and incubate for 20 min at room temperature or 10 min at 37.
Read the absorbance at 500 nm.
Colour is stable for 60min against reagent blank.

18. HDL HDL is a fraction of plasma lipoproteins It is composed of:
25% phospholipid,
20% cholesterol,
and 5% triglycerides
Evidence suggests that high-density lipoprotein (HDL) cholesterol is a primary coronary heart disease (CHD) risk factor.

19. Analysis Methods Ultracentrifugation
Polyacrylamide Gel Electrophoresis
Precipitation
based on the ability of various agents to precipitate selectively the major lipoprotein fractions, except HDL
Immunological
Antibodies against human lipoproteins are used to form antigen-antibody complexes with LDL, VLDL and chylomicrons in a way that only HDL- cholesterol is selectively determined by an enzymatic cholesterol measurement

20. Precipitation Method
In the plasma, cholesterol is transported by three lipoproteins: high density lipoprotein, low density lipoprotein, and very low density lipoprotein
HDL lipoproteins are assayed, after precipitation of LDL and VLDL lipoproteins with polyethylene glycol (PEG) 6000.
HDL is left in the supernatant solution for cholesterol quantitation.

21. Low Density Lipoprotein
[LDL-chol] = [Total chol] - [HDL-chol] - ([TG]/2.2)
Where all concentrations are given in mmol/L
(note that if calculated using all concentrations in mg/dL then the equation is:
[LDL-chol] = [Total chol] - [HDL-chol] - ([TG]/5)
The quotient ([TG]/5) is used as an estimate of VLDL-cholesterol concentration.
It assumes, first, that virtually all of the plasma TG is carried on VLDL, and second, that the TG:cholesterol ratio of VLDL is constant at about 5:1

22. Limitations of the Friedewald equation
The Friedewald equation should not be used under the following circumstances:
when chylomicrons are present
when plasma triglyceride concentration exceeds 400 mg/dL
in patients with dysbetalipoproteinemia (type III hyperlipoproteinemia