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QUOTE FOR THE DAYQUOTE FOR THE DAY
Sometimes you just can’t takeSometimes you just can’t take
things back to the way they usedthings back to the way they used
to be.. No matter how you tryto be.. No matter how you try
regardless of how sorry you are,regardless of how sorry you are,
coz in life there are no rewinds,coz in life there are no rewinds,
only plays.. So play it right!(--:)only plays.. So play it right!(--:)

QUANTITATIVE ANALYSIS OFQUANTITATIVE ANALYSIS OF
LIPOPROTEINSLIPOPROTEINS
• 1. ELECTROPHORESIS1. ELECTROPHORESIS
• widely used in the routine clinicalwidely used in the routine clinical
laboratories to separate and measure LPPlaboratories to separate and measure LPP
• has been most successfully used inhas been most successfully used in
conjunction with other methods.conjunction with other methods.

LIMITATIONS ANDLIMITATIONS AND
REALIZATIONREALIZATION
• It is not really needed for diagnosis ofIt is not really needed for diagnosis of
most dyslipoproteinemiasmost dyslipoproteinemias
• DYSLIPOPROTEINEMIAS- the presenceDYSLIPOPROTEINEMIAS- the presence
of abnormal lipoprotein in bloodof abnormal lipoprotein in blood

AGAROSE GELAGAROSE GEL
• Most commonly used supportMost commonly used support
mediummedium

ELECTROPHORETOGRAMELECTROPHORETOGRAM
• typical separation oftypical separation of
plasma LPPplasma LPP

• * if the chylomicrons are present it remain* if the chylomicrons are present it remain
at the origin.at the origin.
• *then the other major lipoprotein migrates*then the other major lipoprotein migrates
at rates that increase in the order ofat rates that increase in the order of
HDL>VLDL>LDLHDL>VLDL>LDL

SEPARATED LIPOPROTEINSSEPARATED LIPOPROTEINS
ACCDG. TO THEIR MOBILITIESACCDG. TO THEIR MOBILITIES
• ~HDL (alpha-lipoprotein)- moves with the~HDL (alpha-lipoprotein)- moves with the
alpha-globulins.alpha-globulins.
• ~LDL (beta-lipoproteins)- migrates with~LDL (beta-lipoproteins)- migrates with
the beta globulinsthe beta globulins
• ~VLDL (pre beta lipoprotein)- migrates~VLDL (pre beta lipoprotein)- migrates
with the alpha2- globulinswith the alpha2- globulins

LIPOPROTEIN ELECTROLIPOPROTEIN ELECTRO
PHORETOGRAM- are usuallyPHORETOGRAM- are usually
visualized with lipid staining dyevisualized with lipid staining dye
such:such:
• *oil red o*oil red o
• *fat red 7B*fat red 7B
• *sudan black B*sudan black B
• *scharlach red*scharlach red

• these lipid stains react primarily with thethese lipid stains react primarily with the
ester bonds in triglycerides andester bonds in triglycerides and
cholesteryl esterscholesteryl esters

2. ULTRA CENTRIFUGATION OF2. ULTRA CENTRIFUGATION OF
LPPLPP
PROPERTIES:PROPERTIES:
• A) since lipoprotein are lipid-protein complex andA) since lipoprotein are lipid-protein complex and
fats are less dense than water,it follows that asfats are less dense than water,it follows that as
the proportions of lipids to protein increase, thethe proportions of lipids to protein increase, the
density decreases.density decreases.
• B) LPP can be separated based on its density byB) LPP can be separated based on its density by
high speed centrifugation. The rate at whichhigh speed centrifugation. The rate at which
each lipoprotein floats through a solution of NaCleach lipoprotein floats through a solution of NaCl
may be expressed in svedbert units (sf) ofmay be expressed in svedbert units (sf) of
flotation.flotation.

3. POLYANION3. POLYANION
PRECIPITATION METHODPRECIPITATION METHOD
• - most commonly used to remove apo B- containing- most commonly used to remove apo B- containing
lipoproteins prior to the analysis of HDL cholesterollipoproteins prior to the analysis of HDL cholesterol
• A)LPPA)LPP
~ precipitated by polyanions:~ precipitated by polyanions:
• *heparin sulfate*heparin sulfate
• *dextran sulfate*dextran sulfate
• *phosphotungstate*phosphotungstate
• ~ in the presence of divalent cations such:~ in the presence of divalent cations such:
• *calcium*calcium
• *magnesium*magnesium
• *manganese*manganese

FACTORS THAT INFLUENCEFACTORS THAT INFLUENCE
PRECIPITATIONPRECIPITATION
• Reagent concentrationReagent concentration
• PhPh
• Ionic strengthIonic strength
• Presence of proteins and anti coagulantsPresence of proteins and anti coagulants
• Duration and condition of sample storageDuration and condition of sample storage

• B)conditions have been established in whichB)conditions have been established in which
major classes of lipoproteins is precipitated in amajor classes of lipoproteins is precipitated in a
step wise fashion beginning with the lowerstep wise fashion beginning with the lower
density,lipid rich lipoprotein.density,lipid rich lipoprotein.
• The more dissimilar the lipoproteins, the moreThe more dissimilar the lipoproteins, the more
satisfactorily they can be separated from eachsatisfactorily they can be separated from each
other.other.
• The more similar in composition the lipoproteinThe more similar in composition the lipoprotein
classes are, the more critically the conditions ofclasses are, the more critically the conditions of
precipitation must be controlled separate them.precipitation must be controlled separate them.

-are important source of energy for a variety of tissue
-Recently, there has been an increased interest in the measurement of
FFA kinetics in vivo, using radio labeled or stable isotopic tracers.
-Standard techniques for measurement of FFA-specific activity
are relatively imprecise and have limited sensitivity.
-The authors have developed a method for determination and
specific activity. Using this method, one can measure the
kinetics of three or more long chain fatty-acids simultaneously.
-Its sensitivity is a particular advantage
if one wishes to measure low rates of
FFA turnover such as those
encountered during hyperinsulinemia.

-serum or heparinized plasma can be used and rapid separation
of serum from blood clots is suggested because presence of
lipase permits slow in vitro lipolysis
-stress must be avoided as in those encountered
before/during blood sample collection because
cathecolamines are released which enhances the activity of
hormone sensitive lipase.
-patient must be under NPO state; feeding induces
depression in plasma fatty acids

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