Clinical diagnostic biochemistry - 8 CLS 334 Dr. Maha Al-Sedik 2015

Transport of Fat: Lipoproteins Chylomicrons VLDL. IDL. LDL. HDL

Lipoproteins Lipids in plasma combined with apo lipoprotein and transferred as lipoproteins. Lipoprotein is arranged in the blood to be protein and phospholipid to the outside and triglyceride and cholesterol to the inside.

Spherical particles with a hydrophobic core (TG and esterified cholesterol), and a hydrophilic surface (apoprotein, phospholipids & free cholesterol).

Precipitated with cold ethanol Can be separated by electrophoresis Like Protein Water soluble Precipitated with cold ethanol Can be separated by electrophoresis Not like protein Less dense Float in high centrifugation

Lipoproteins lipoprotein classes: Particles found in plasma that transport lipids including cholesterol. lipoprotein classes: Chylomicrons: take lipids from small intestine to the blood. Very low density lipoproteins (VLDL): take endogenous lipid from the liver. Intermediate density lipoproteins (IDL) Low density lipoproteins (LDL) High density lipoproteins (HDL)

LIPOPROTEINS HDL IDL LDL VLDL Chylomicron Alpha α Beta Β Pre beta Density Electrophoresis LIPOPROTEINS HDL IDL LDL VLDL Chylomicron Alpha α Beta Β Pre beta Stay in its origin

Apolipoproteins: The protein moiety on the surface of lipoproteins. Major components of lipoproteins. Classified by alphabetical designation (A to E) Responsible for transport and redistribution of lipids among cells. Strengthen and maintain the lipoprotein structure. Responsible for recognition of particle by receptors. Cofactor and activator for enzymes involved in lipid metabolism.

Chylomicron Chylomicron contains the exogenous triglycerides and protein ( Apo B 48 , APO C II and APO E ). It tends to float even without centrifugation. High chylomicron leads to milky plasma. It remains in its origin in electrophoresis.

VLDL VLDL contains the endogenous synthetized cholesterol and triglycerides and protein ( Apo B 100 , APO C II and APO E ). It produces pre B band in electrophoresis. High VLDL leads to turbid plasma.

IDL IDL contains equal amounts of cholesterol and triglycerides and protein ( Apo B 100 , Apo C II and APO E ). It produces slow pre B band in electrophoresis. IDL is transformed to LDL by losing APO C II and APO E .

LDL LDL contains mostly cholesterol ester , little triglycerides and protein ( Apo B 100 ). It produces B band in electrophoresis. High LDL does not alter plasma clarity. LDL enters the tissues through LDL receptor . People with high levels of LDL are at high risk of developing atherosclerosis.

HDL HDL contains mostly 50 % protein and 20 % cholesterol , 30 % phospholipid and traces of triglycerides. It gives Apo C II and Apo E to the nascent chylomicron and nascent VLDL. It produces α band in electrophoresis. High HDL does not alter plasma clarity. High levels of HDL are good signs .

Exogenous Lipid Transport

Fatty acids are absorbed by the apical microvilli of mucosal cells Fatty acids are absorbed by the apical microvilli of mucosal cells. Apo B48 is the structural protein of the chylomicron. Now the chylomicron is called Nascent chylomicron. Then it reaches the blood where it receives Apo C II and Apo E from HDL. Apo C II stimulates lipoprotein lipase enzyme. Lipoprotein lipase digest the triglyceride in the chylomicron transforming chylomicron to chylomicron remnants. Chylomicron remnants enter the hepatocyte by Apo E receptor in the hepatocyte.

Endogenous Lipid Transport

Endogenously synthesized cholesterol and triglyceride combine with Apo B 100 to form VLDL. VLDL from liver enters plasma. VLDL combines with Apo E (binds to hepatocyte receptor) and Apo C II (activates lipoprotein lipase). LPL works on VLDL forming IDL. IDL has two pathway, it may enter the hepatocyte through Apo E. Or, IDL loses APO E and APO CII to form LDL. LDL enters the tissues through LDL receptor ( APO B 100 ). People with high level of LDL are at high risk of having diseases.

Laboratory investigations for lipids

Important pre analytical considerations: An individual's lipid and lipoprotein profile should be measured only when the individual is in a metabolic steady state. Subjects should maintain their usual diet and weight for at least 2 weeks before the determination of their lipids or lipoproteins. Repeat sampling : The diagnosis should be confirmed on at least 2 specimens 2 – 4 weeks in between.

Subjects should not perform vigorous physical activity within the 24 hours before testing. The patient should be fasting except for total cholesterol. Sampling in sitting position: Individuals should be seated for at least 5 minutes before. specimen collection: The tourniquet should not be kept on more than 1 minute during venipuncture. Either serum or plasma should be used to measure total cholesterol, triglyceride, and HDL cholesterol concentrations.

Blood specimens always should be considered potentially infectious and therefore handled accordingly. You should wait for at least 3 months after major surgery.

Laboratory investigations for lipids

Standing plasma tests: Observing plasma standing sample after 24 hours at 4oC: Turbid or cloudy ---------------------------- VLDL Layer of cream and clear below it ----------------------- Chylomicron Clear ---------------------- LDL or HDL

Lipid profile: After fasting for about 12 hours.

( plasma lipoprotein free fraction ). Ultracentrifugation: Ultracentrifuge for 16 – 18 hours at 10 oc ( plasma lipoprotein free fraction ).

This method has been used as the standard by which the accuracy of other methods are judged . Now it is only used in research.

Electrophoresis.

Chromatography: Ion exchange and gel permeation chromatography are the most common types. It is used only for research .

Total cholesterol = HDL + LDL + VLDL VLDL = Triglyceride / 5 LDL = Total cholesterol – ( HDL + Triglyceride / 5 ) Friedewald formula

Reference: Burtis and Ashwood Saunders, Teitz fundamentals of Clinical Chemistry, 4th edition, 2000.

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