QUOTE FOR THE DAYQUOTE FOR THE DAYSometimes you just can’t Sometimes you just can’t

take things back to the way take things back to the way they used to be.. No matter they used to be.. No matter how you try regardless of how you try regardless of

how sorry you are, coz in life how sorry you are, coz in life there are no rewinds, only there are no rewinds, only plays.. So play it right!(--:) plays.. So play it right!(--:)

QUANTITATIVE ANALYSIS OF QUANTITATIVE ANALYSIS OF LIPOPROTEINSLIPOPROTEINS

• 1. ELECTROPHORESIS1. ELECTROPHORESIS

• widely used in the routine clinical widely used in the routine clinical laboratories to separate and laboratories to separate and measure LPPmeasure LPP

• has been most successfully used has been most successfully used in conjunction with other methods.in conjunction with other methods.

LIMITATIONS AND LIMITATIONS AND REALIZATIONREALIZATION

• It is not really needed for diagnosis It is not really needed for diagnosis of most dyslipoproteinemiasof most dyslipoproteinemias

• DYSLIPOPROTEINEMIAS- the DYSLIPOPROTEINEMIAS- the presence of abnormal lipoprotein in presence of abnormal lipoprotein in bloodblood

AGAROSE GELAGAROSE GEL

•Most commonly used support Most commonly used support mediummedium

ELECTROPHORETOGRAMELECTROPHORETOGRAM

• typical separation typical separation of plasma LPPof plasma LPP

• * if the chylomicrons are present it * if the chylomicrons are present it remain at the origin.remain at the origin.

• *then the other major lipoprotein *then the other major lipoprotein migrates at rates that increase in the migrates at rates that increase in the order of HDL>VLDL>LDLorder of HDL>VLDL>LDL

SEPARATED LIPOPROTEINS SEPARATED LIPOPROTEINS ACCDG. TO THEIR MOBILITIESACCDG. TO THEIR MOBILITIES

• ~HDL (alpha-lipoprotein)- moves ~HDL (alpha-lipoprotein)- moves with the alpha-globulins.with the alpha-globulins.

• ~LDL (beta-lipoproteins)- migrates ~LDL (beta-lipoproteins)- migrates with the beta globulinswith the beta globulins

• ~VLDL (pre beta lipoprotein)- ~VLDL (pre beta lipoprotein)- migrates with the alpha2- globulinsmigrates with the alpha2- globulins

LIPOPROTEIN ELECTRO LIPOPROTEIN ELECTRO PHORETOGRAM- are usually PHORETOGRAM- are usually visualized with lipid staining visualized with lipid staining dye such:dye 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 these lipid stains react primarily with the ester bonds in triglycerides and the ester bonds in triglycerides and cholesteryl esterscholesteryl esters

2. ULTRA CENTRIFUGATION OF 2. ULTRA CENTRIFUGATION OF LPPLPP PROPERTIES: PROPERTIES:• A) since lipoprotein are lipid-protein complex A) since lipoprotein are lipid-protein complex

and fats are less dense than water,it follows and fats are less dense than water,it follows that as the proportions of lipids to protein that as the proportions of lipids to protein increase, the density decreases.increase, the density decreases.

• B) LPP can be separated based on its density B) LPP can be separated based on its density by high speed centrifugation. The rate at by high speed centrifugation. The rate at which each lipoprotein floats through a which each lipoprotein floats through a solution of NaCl may be expressed in solution of NaCl may be expressed in svedbert units (sf) of flotation.svedbert units (sf) of flotation.

3. POLYANION 3. POLYANION PRECIPITATION METHODPRECIPITATION METHOD• - most commonly used to remove apo B- - most commonly used to remove apo B-

containing lipoproteins prior to the analysis of containing lipoproteins prior to the analysis of HDL cholesterolHDL 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 INFLUENCE FACTORS THAT INFLUENCE PRECIPITATIONPRECIPITATION

• Reagent concentrationReagent concentration

• PhPh

• Ionic strengthIonic strength

• Presence of proteins and anti Presence of proteins and anti coagulantscoagulants

• Duration and condition of sample Duration and condition of sample storage storage

• B)conditions have been established in which B)conditions have been established in which major classes of lipoproteins is precipitated in major classes of lipoproteins is precipitated in a step wise fashion beginning with the lower a step wise fashion beginning with the lower density,lipid rich lipoprotein.density,lipid rich lipoprotein.

• The more dissimilar the lipoproteins, the more The more dissimilar the lipoproteins, the more satisfactorily they can be separated from each satisfactorily they can be separated from each other. other.

• The more similar in composition the The more similar in composition the lipoprotein classes are, the more critically the lipoprotein classes are, the more critically the conditions of precipitation must be controlled conditions of precipitation must be controlled separate them.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