Transcript Document

Cholesterol
• Lipid
– Soluble in organic solvents and nearly insoluble in water
– Yield fatty acids on hydrolysis
– Complex alcohols that combine with fatty acids to form
esters
• Lipoprotein
– An association of a core lipids with coat phospholipid and
protein
– For solubility
• Apolipoproteins
– Protein components of lipoprotein.
Classification of Clinically Important lipids
Structure of cholesterol
Cholesterol biosynthesis
Esterification of cholesterol mediated
by ACAT & LCA
Structure of a typical lipoprotein
particle
• apo B-100—containing lipoproteins
– VLDL, IDL, Lp(a), LDL
Contributors to total cholesterol in
normal people
• LDL
– Two thirds
• HDL
– One third
• IDL and Lp(a)
– 2 to 3 mg/dL (each)
• Total chol = VLDL chol + LDL chol + HDL chol
Classification of LDL.Total. and
HDL Cholesterol (mg/dL)
• Reference methods
• To establish the accuracy of lipid and
lipoprotein measurements
• Using the same basis (protocol ) for accuracy
that had been used in developing the
relationships between lipid and lipoprotein
concentration and CHD.
• From studies, cut points for the risk
characterization in patients were derived.
• The accuracy of existing or newly developed
methods could be assessed.
Methods and procedures commonly
used
• Reference Method (total cholesterol)
– Chemical method
•
•
•
•
•
•
•
Hydrolyze the cholesteryl esters. alcoholic KOH
extracted from the mixture with hexane
dried in vacuo
acetic acid, acetic anhydride, and sulfuric acid
Color development, 620 nm
pure cholesterol as the calibrator.
Expressed as mg/dL = mmoI/L x 38.7
HDL cholesterol
• Reference Method
– Prepare the HDL-containing fraction.
• a combination of ultracentrifugation and polyanion
precipitation
– Ultracentrifugation
» Supernate
• VLDL and any chylomicrons accumulate as a floating layer
» Infranate
• IDL, LDL, Lp(a), HDL, and the other serum proteins
• Precipitation with heparin sulfate and MnCl2
• Supernate HDL
• Precipitate IDL, LDL, Lp(a)
– The cholesterol in HDL fraction is then quantitated
V-LDL & LDL cholesterol
• VLDL chol = [Total chol] – [d > 1.006g/mL chol]
• LDL cholesterol
– Cholesterol infranate – cholesterol supernate
• (IDL, LDL, Lp(a), HDL— HDL fraction)
• The Friedewald Equation
– [LDL choI] = [Total chol] - [HDL chol]-[Triglyceride]/5
• Unacceptable at TG concentrations > 400 mgldL
Methods
• Reference methods
– are complex, time consuming, at least partially
manual, and require a high level of expertise for
reliable operation
• Routine Methods
– Enzymatic methods
Enzymatic method
• Interference
– Competition with the oxidation reaction
• bilirubin, ascorbic acid, and hemoglobin.
– adding substances such as bilirubin oxidase and dual
wavelength readings to minimize the effects of hemolysis
• β-hydroxy sterols and plant sterols (e.g., β-sitosterol)
can also react.
– are generally at very low concentrations ( so,not significant)
Sources of Variation in Lipid and Lipoprotein
Measurements
• Analytical Variation
• Physiological Variation
– Contribute about 70 to 98% of the overall variance
– Variation for triglyceride is considerably higher
+, minimal to moderate increase
++, moderate to high increase
-, minimal to moderate decrease
-, moderate to high decrease
NC, essentially no change or trend.
Recommendations
• Cholesterol
– Two serial samples obtained at least 1 wk apart be
used
– Fasting or non-fasting
• Triglyceride, HDL and LDL cholesterol
– Two to three serial specimens are recommended
– 12-h fast, or 9-h
• Specimens
– Serum or plasma (EDTA plasma)
• Sampling
– in the same position on each occasion
• Specimen storage
– Serum or plasma should be removed from cells
within 3 h
– Specimens can be stored for up to 3 d at 4 °C
– Up to several wk at —20 °C
– at —70 °C or lower for longer periods