Molde, Bryan M.

– Lipids and Lipoproteins – Assignment

Clinical Chemistry 1
Lipids and lipoprotein – Methods in testing

Introduction – Lipid Measurement

The need to measure Lipids and lipoprotein are inclined towards its correlation
and being an indicatory of Coronary Heart Disease. Due to this, standardization
programs are implemented and Decision cut points to identify CHD risk groups base
on epidemiology. Recently, standardization programs found its way to diagnostic
medicine and routine laboratory analysis to facilitate reliable classification of patients
using cut points. Therefore, accuracy and standardization of results are especially
integral with lipid and lipoprotein analyte.

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Cholesterol Measurement

• Serum or plasma specimen with at least 12 hours fasting is preferred for total
cholesterol but there is no significant change after a meal.
• In case of delays, refrigerate at 44°C for several days.
• Lipid analysis traditionally start with measurement of total cholesterol
• Early analytic method utilizes Strong Acids (Sulfuric and Acetic) often with
other chemical (Acetic anhydride or ferric chloride) to produce color with
cholesterol which is then measured
• Reference Methods:
o Past – Hexane extraction after hydrolysis with alcoholic KOH followed
by reaction with Liebermann-Burchard color reagent
o Recent – Gas chromatography-mass spectrometry measure cholesterol
but not related sterols
o Definitive – Isotope dilution mass spectrometry. good agreement with
the gold standard method developed and applied at the U.S. National
Institute of Standards and Technology
• Enzymatic Reactions – Replaced strong acid chemistry in routine labs because
enzymes are specific towards analyte of interest providing accurate
quantitation without necessity for extraction and pretreatment. One
sequence is the most common.

• Color produced is proportional to amount of cholesterol, measured

with spectrophotometer (around 500nm)
• Reducing agents are interference that interfere with peroxidase-
catalyzed color reaction (vitamin C and Bilirubin)

TAG (Triacylglyceride) Measurement

• Useful in detecting certain genetic and types of metabolic disorders as well as

risk for CVDs
• TAG value is commonly used to estimate LDL-C through Friedewald equation
• Fasting Samples because TAG sometimes increase significantly after eating.
• Enzymatic Lipase Activity – Lipases cleaves fatty acids from glycerol in a TAG
unit. Consequently, freed glycerol participates in one of several enzymatic
sequences ~ resulting to either color change or a product that is measured in
UV region. Color reaction is more convenient than UV.

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Illustration:

• TAG reaction sequence also reacts with endogenous free glycerol and is a
significant source of interference. (10-20mg/dl) contribute to TAG
overestimation. Corrected with reagents.
• Double-cuvette blank – most common correction
• TAG reference method: involve alkaline hydrolysis, solvent extraction, and
a color reaction with chromotropic acid, an assay that is tedious and poorly
characterized and was largely only done by the lipid standardization laboratory
at CDC

Lipoprotein Measurement

• Lipoprotein separation and quantitation take advantage of physical properties

such as :
o Density
o Charges
o Size
o Lipoprotein content
• Electrophoretic separations take advantage of differences in charge and size
• Chemical Precipitation methods – depend on particle size, charge, and
differences in the apolipoprotein content (This method is once most common
in clinical laboratories, but currently primarily used in research laboratories.)
• Antibodies specific to apolipoproteins can be used to bind and separate
lipoprotein classes.
• Chromatographic methods take advantage of size differences in molecular
sieving methods or composition in affinity methods
• Direct homogeneous reagents – most common in laboratories. designed for
fully automated use with chemistry analyzers, using combinations of
detergents and, in some cases, antibodies to selectively assay cholesterol in
lipoprotein classes

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HDL Methods

• Chemical Precipitation - Precipitation reagent added which is then

centrifugated at 1,500g at 10 to 30 minutes. HDL is then quantified as
cholesterol in the supernatant following enzymatic assays modified for the
lower HDL-C range.
o Heparin with manganese – earlies common precipitation method for apo
B-containing lipoprotein.
o Significant interference from elevated triglyceride levels (VLDL and
Chylomicron)
o Still used in case of unusual patients with liver or kidney condition

• Direct / Homogeneous method – Automate HDL quantification and a better suit

for modern chemistry laboratory. Specific polymers, detergents, and even
modified enzymes are used to suppress the enzymatic cholesterol reaction in
lipoproteins other than HDL.
1. first reagent is added to“block” non-HDLs,
2. second reagent with the enzymes to quantify the accessible (HDL)
cholesterol.
o Homogeneous assays, which appear to be highly precise and reasonably
accurate, have generally replaced pretreatment methods in the routine
laboratory.

Reference method for HDLC

o Three step procedure developed at the CDC
▪ Ultracentrifugation to remove VLDL, heparin manganese
prectipitaion from the 1.006 g/ml infrante to remove LDL, analysis
of supernatant cholesterol by Abell-Kendall assay.
o Simpler method / Direct method / Precipitation method
▪ Validated by CDC network Laboratory Group
▪ Designated comparison method using direct dextran sulfate (50kD)
precipitation of serum with Abell-Kendall cholesterol analysis

LDL Methods
• LDL-C – Validated as treatable risk factor for CHD, primary basis for
treatment decision in all guideline
• Beta Quantification – most common research method for LDL-C quantitation
and the basis for the reference method. Beta designation refer to
electrophoretic term for LDL
▪ Beta quantification combines ultracentrifugation and chemical
precipitation.
• Ultracentrifugation is used to float CLDL and any chylomicron
for separation.
• Chemical precipitation is used to separate HDL from either
whole serum or the infranate obtained from
ultracentrifugation.

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▪ Beta quantification is also the basis for the accepted reference

method for LDL.
• More common approach – bypass ultracentrifuge calculating LDL-C by the
Friedewald calculation.
▪ This method is commonly performed as the lipid panel and is widely
used in estimating LDL-C in routine clinical practice.
▪ Recommended by NCEP Guidelines
• NCEP panel concluded that better methods are needed for routine diagnostic
use, preferably methods that directly sperate LDL for cholesterol quantitation.
• Direct LDL-C methods – Full automation of the challenging LDL-C separation.
Have the potential to streamline the measurement while improving precision.
But not utilized because of extra cost compared to Friedewald Calculation.
Advantage of being able to be used on non-fasting sample.

Compact analyzer

• Designed for point-of care testing at bedside of patient, physician’s office,

wellness centers, and home.
• Compact device
• Measure common lipids and lipoprotein

Apolipoprotein Methods
• Apolipoprotein are commonly measured in research, specialty laboratories
supporting cardiovascular practices, or clinical studies in which they are
routine in addition to lipoproteins.
• 3 apolipoproteins are the point of interest. Considered to be emerging
markers.
o Apo B – Major protein of LDL and VLDL , indicator of combined LDL and
VLDL concentration and can be measured directly in serum
immunoassay. Proposed to be better indicator of atherogenic particle
than LDL-C
o Apo A-1, major protein of HDL measured directly in serum in place of
separation analysis of HDL-C.
o Lp(a), variant of LDL shown to be an independent indicator of CHD risk,
sometimes determined in managing patients. Lp(a) has pre-B mobility
on agarose electrophoresis and can be quantified by this technique.

• Most common in routine laboratories are turbidimetric assays for chemistry

analyzers or nephelometric assays for dedicated nephelometers.
• Enzyme-linked immunosorbent assay (ELISA), radial immunodiffusion
(RID), and radioimmunoassay (RIA) methods have also been available, but
the latter two methods are becoming less common.
o Antibodies used may be polyclonal or monoclonal.
• International efforts to develop reference materials and standardization
program for the assays are in progress.

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