Lipids and Lipoproteins

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Lipids and Lipoproteins : 

Lipids and Lipoproteins DR.OKINDA N.A.

Classification of lipids : 

Classification of lipids Fatty acids (palmitic, linoleic, prostagladins etc) Glycerol esters (mono/di/triglycerides) Sterols (cholesterol, steroid hormones, vitamin D, bile acids) Terpenes (vitamins A, E, K) Sphingosine derivatives (sphingomyelin, gylcosphingolipids)

Fatty Acids : 

Fatty Acids Major constituent: triglycerides and phospholipids. Different types: short chains<8, medium chains 8-12, large chains 16-24. Saturated vs. unsaturated: based on the number of double bonds and body’s ability to breakdown the fats. No double bonds (saturated); one double bond (monounsaturated) and two or more double bonds (polyunsaturated).

Fatty acids : 

Fatty acids Even-numbered fatty acids predominate The most common saturated fatty acids are palmitic (16:0) and stearic (18:0), but unsaturated fatty acids are more common in nature Lauric acid (C12, 12:0, n-dodecanoic acid)

Unsaturated fatty acids : 

Unsaturated fatty acids Double bonds in fatty acids are nearly always cis Palmitoleic acid (16:19, 9-hexadecanoic acid)

Essential fatty acids : 

Essential fatty acids Mammals can synthesize saturated and mono-unsaturated fatty acids. Linoleic (18:2) and linolenic (18:3) fatty acids cannot be synthesized, and therefore must be obtained from the diet (plants). Both are required for the biosynthesis of prostaglandins

Triglycerides : 

Triglycerides Composed of the one molecule of glycerol and 3 fatty acids. Saturated fats: triglycerides packed closely together and solidify at room temperature, most come from animal source. Unsaturated fats: not packed together and are liquid at room temperature, most come from plants and are polysaturated.

Source of Triglyceride : 

Source of Triglyceride Exogenous form (diet intake) Endogenous (synthesized in the liver and tissue.) Form of fats that allow the body to store long carbon chains (fatty acids) for energy used during fasting states or between meals. 95% of stored fats in tissue are the high energy triglyceride molecules.

Triglycerides : 

Triglycerides Transported in the plasma in the form of large triglyceride rich particles called: Chylomicrons and VLDL. Metabolism of triglyceride: fatty acids are released to the cells and converted into energy. Glycerol of the triglyceride is recycled into triglyceride. Portions of the triglycerides are deposited into adipose cells for insulation.

Slide 10: 

Breakdown of triglyceride facilitated by hormones such as: lipase, lipoprotein lipase (LPL), epinephrine and cortisol. LPL attach to haparan sulfate stalks in the capillaries as the triglyceride rich lipoproteins (chylomicrons and VLDL) circulate- triglycerides are hydrolyzed as they come into contact with LPL. Supply of free fatty acids available to be used in TCA cycle for energy production depends on LPL interaction with the chylomicrons and VLDL.

Slide 11: 

Lipase: Acts inside adipose (fat) cells to release free fatty acids from triglyceride stores for energy when supplies are low. (diet or fasting states) Epinephrine and cortisol: promotes breakdown of triglyceride when glucose stores are low or depleted.

Triglyceride synthesis : 

Triglyceride synthesis Triglyceride: synthesis begins with the activation of fatty acids to acyl-CoA ester. Requires input of energy from ATP. 2 Different routes: Final stage of both involves removal of phosphoryl group followed by acylation to form the triglyceride molecule.

Clinical importance of fatty acids : 

Clinical importance of fatty acids Fecal fatty acids are sometimes measured to detect malabsorptive and pancreatic disorders—the test is mostly considered obsolete Serum free fatty acids help distinguish between hyperinsulinemic hypoglycemia (FFA normal) and disorders of fatty acid oxidation (FFA elevated and negative ketones)

Glycerol esters (acylglycerols) : 

Glycerol esters (acylglycerols) Triglycerides are the most abundant family of lipids in plant and animal cells, and are major components of the the human diet Glycerol Triglyceride

Measuring triglycerides (enzymatic method) : 

Measuring triglycerides (enzymatic method) Triglycerides Dihydroxyacetone + H2O2 Glycerophasphate oxidase Peroxidase Quinoneimine dye max 500 nm

Measuring triglycerides (reference method) : 

Measuring triglycerides (reference method) Triglycerides are extracted into chloroform prior to analysis Triglycerides KOH fatty acids + glycerol Periodate formic acid + formaldehyde chromotropic acid chromogen =570 nm

Slide 19: 

Most enzymatic reaction react with free glycerol which is present in significant amount. To correct for interference, use a double blanking method and parallel measurements using reagent with and without lipase.

CHOLESTEROL: : 

Unsaturated steroid alcohol Consist of a perhydrocyclopentanthroline ring and side chain of 8 carbon atoms. Esterified form contains 1 fatty acid molecule Used to manufacture and repair cell membrane. Synthesis of bile acids and Vitamin D Precursors of 5 major steroid hormones. CHOLESTEROL:

Slide 21: 

Dietary intake is between 100 – 700 mg Liver and other tissue produce 500-1000mg daily. 600-1000mg of biliary cholesterol is secreted into the intestine daily (50% reabsorbed through EHC) 70% of cholesterol is located in skin, adipose tissue and muscle (stationary pool); 30 % circulating in form of lipoproteins and circulates through liver.

Metabolism of Cholesterol : 

Metabolism of Cholesterol Liver major site for synthesis of cholesterol- estrified form. Formed from actyl-CoA: 3 molecules of actyl –CoA condensed to produce 3 hydroxy-3-methyl-glutaryl coenzyme A, which is converted to melalonic acid through reductase. Synthesis is regulated by intracellular cholesterol concentration and reductase activity. In the liver cholesterol degraded to bile acids: colic acid and chenodeoxycholic acid which are needed to remove cholesterol from the body.

Cholesterol biosynthesis : 

Cholesterol biosynthesis Acetyl-CoA 3-Hydroxy-3-methylglutaryl-CoA Mevalonate HMG-CoA reductase Squalene Cholesterol + Lecithin Cholesterol ester LCAT

Cholesterol biosynthesis : 

Cholesterol biosynthesis About 2% (approximately 1 g) of total body cholesterol is replenished each day Dietary sources account for less than half Cholesterol is synthesized from Acetyl CoA 90% of in vivo synthesis occurs in the intestine and liver (although all cells have the capability) Absorption of dietary cholesterol appears to have a maximum of approximately 1 g/day

Slide 25: 

Esterification of cholesterol is important as it enhances the lipid carrying capacity of lipoprotein and prevents intracellular toxicity by unesterified cholesterol. Acyl-CoA + cholesterol>ACAT>>>CE+ CoASH Lecithin + cholesterol >LCAT>>CE+ lysolecithin

Cholesterol measurements : 

Cholesterol measurements Measurement involves assay of total cholesterol and lipoproteins Early analytical method: used strong acid (sulfuric and acetic anhydride or ferric chloride to produce a color in the presence of cholesterol) Reference method: use hexane extraction after hydrolysis with alcoholic KOH followed by reaction with Leiberman-Burchard color reagent (composed of sulfuric and acetic acids/anhydrides)

Common Enzymatic Sequence : 

Common Enzymatic Sequence ⅔ of circulating cholesterol occurs as cholesterol ester, which is hydrolysed by enzyme cholesteryl ester hydrolase. Free cholesterol then reacts with cholesterol oxidase producing hydrogen peroxide- substrate for color reaction. Intensity of the color is proportional to amount of cholesterol @ 5OO nm.

Measuring cholesterol by L-B : 

Measuring cholesterol by L-B The Liebermann-Burchard method is used by the CDC to establish reference materials Cholesterol esters are hydrolyzed and extracted into hexane prior to the L-B reaction

Enzymatic cholesterol methods : 

Enzymatic cholesterol methods Enzymatic methods are most commonly adapted to automated chemistry analyzers The reaction is not entirely specific for cholesterol, but interferences in serum are minimal Cholesterol esters

Lipoproteins : 

Lipoproteins In order to be transported in blood, lipids must combine with water-soluble compounds, such as phospholipids and proteins.

Lipoproteins : 

Lipoproteins Classified by: Density electrophoretic mobility 4 major classes: Chylomicrons VLDL HDL LDL

Lipoprotein classes : 

Lipoprotein classes

Lipoprotein Metabolism : 

Lipoprotein Metabolism Occurs in 3 separate and interacting pathways: Exogenous Endogenous Reverse cholesterol pathway LDL receptor pathway

Lipoprotein electrophoresis : 

Lipoprotein electrophoresis LEP is no longer a common laboratory test Standing plasma test for chylomicrons Total cholesterol, TG, HDL, and LDL can be measured directly Chylomicrons LDL VLDL Lp(a) HDL Migration IDL

Major lipoprotein classes : 

Major lipoprotein classes Chylomicrons (derived from diet) density <<1.006 diameter 80 - 500 nm dietary triglycerides apoB-48, apoA-I, apoA-II, apoA-IV, apoC-II/C-III, apoE remains at origin in electrophoretic field

Chylomicron : 

Chylomicron formed through extrusion of resynthesized triglycerides from the mucosal cells into the intestinal lacteals flow through the thoracic ducts into the suclavian veins degraded to remnants by the action of lipoprotein lipase (LpL) which is located on capillary endothelial cell surface remnants are taken up by liver parenchymal cells due to apoE-III and apoE-IV isoform recognition sites

Slide 38: 

Standing Plasma Test for chylomicrons Plasma is placed in refrigerator (4°C) overnight Chylomicrons accumulate as floating “cream” layer Chylomicrons in fasting plasma are abnormal

Slide 39: 

Chylomicron metabolism

VLDL : 

VLDL density >1.006 diameter 30 - 80nm endogenous triglycerides apoB-100, apoE, apoC-II/C-III prebeta in electrophoresis formed in the liver as nascent VLDL (contains only triglycerides, apoE and apoB) 12-16% of phospholipid content is in VLDL form. Excess dietary intake of carbohydrate enhances hepatic synthesis of triglyceride, which increases VLDL production.

VLDL : 

VLDL nascent VLDLs then interact with HDL to generate mature VLDLs (with added cholesterol, apoC-II and apoC-III) mature VLDLs are acted upon by LpL to generate VLDL remnants (IDL) IDL are further degraded by hepatic triglyceride lipase (HTGL) to generate LDLs

Slide 42: 

VLDL metabolism

Slide 43: 

IDL (intermediate density lipoproteins) density: 1.006 - 1.019 diameter: 25 - 35nm cholesteryl esters and triglycerides apoB-100, apoE, apoC-II/C-III slow pre-beta

Slide 44: 

LDL (low density lipoproteins) density: 1.019 - 1.063 diameter: 18-25nm cholesteryl esters apoB-100 beta (electrophoresis) < 130 LDL cholesterol is desirable, 130-159 is borderline high and >160 is high 50% of cholesterol; most cholesterol rich lipoprotein. Synthesized in the liver and responsible for transporting cholesterol from liver to peripheral tissue. 22-26% phospholipids content LDL have inverse relationship to triglyceride; increase LDL, decreased triglycerides; decreased LDL, increased Triglycerides.

LDL method- B-quantification : 

LDL method- B-quantification Beta-quantification: combination of ultracentrifugation and chemical precipitation. Chylomicrons are removed by preliminary low speed centrifugation. Plasma or serum is centrifuged overnight at d=1.006g/ml to spin up VLDL. The apolipoprotein B containing lipoproteins in the bottom fraction (mainly LDL) are precipitated using heparin and manganese chloride, leaving HDL in solution. LDL cholesterol is calculated by subtracting VLDL-C and HDL-C from total cholesterol. Alternatively, multiple fractions can be isolated by gradient ultracentrifugation.

Direct LDL cholesterol : 

Direct LDL cholesterol Older direct methods for LDL involved precipitation with heparin or polyvinyl sulfate Newer methods involve precipitation of VLDL, IDL, and HDL with polyvalent antibodies to Apo A and Apo E LDL is almost exclusively Apo B-100

Indirect LDL cholesterol : 

Indirect LDL cholesterol Friedewald formula assumes that all cholesterol is VLDL, LDL, and HDL lipoproteins Chylomicrons are usually low in normal, fasting subjects, and IDL and Lp(a) are usually insignificant contributors to total cholesterol Since VLDL is 55% TG and 12% Chol: [LDL Chol] = [Tot Chol] – [HDL Chol] – [TG]/5

Direct vs. Indirect LDL : 

Direct vs. Indirect LDL The Friedewald equation assumes that chylomicrons, IDL, and Lp(a) are not significant Non-fasting specimens can have chylomicrons TG > 400 mg/dL indicates the presence of chylomicrons (or remnants) Type III hyperlipidemia is characterized by high -VLDL, which has a 3:1 TG:C ratio

Lipoprotein a : 

Lipoprotein a Are LDL- like; have one additional apo linked to Apo-B-100 by a disulfide bond. Presence of LP(a) risk factor for developing premature CHD, Myocardial infarction, cerebrovascular disease. Influencing factors: Age, hormone status, liver and renal function. unusual in that it contains a kringle protein motif/domain (tri-looped structure with 3 intramolecular disulfide bonds – resembling plasminogen)

HDL : 

HDL HDL (high density lipoproteins) density: 1.063-1.210 diameter: 5-12nm cholesteryl esters and phospholipids apoA-I, apoA-II, apoC-II/C-III and apoE alpha (electrophoresis) Smallest lipoproteins Synthesized by liver and intestines Represents 25-30% phospholipids content Carries 20-35% of plasma cholesterol Takes excess cholesterol from tissue and returns to the liver.

HDLs : 

HDLs Several subfamilies exist Discoidal HDL : contains cholesterol, phospholipid, apoA-I, apoA-II, apoE and is disc shaped; it is formed in liver and intestine It interacts with chylomicra remnants and lecithin-cholesterol acyl transferase (LCAT) to form HDL3

HDLs : 

HDLs HDL3 composed of cholesterol, cholesterol ester, phospholipid and apoA and apoE interacts with the cell plasma membranes to remove free cholesterol reaction with LCAT converts HDL3 to HDL2a (an HDL with a high apoE and cholesterol ester content) cholesterol ester-rich HDL2a is then converted to triglyceride-rich HDL2b by concomitant transfer of HDL cholesterol esters to VLDL and VLDL triglycerides to HDL

Slide 54: 

HDL metabolism

Functions of HDL : 

Functions of HDL transfers proteins to other lipoproteins picks up lipids from other lipoproteins picks up cholesterol from cell membranes converts cholesterol to cholesterol esters via the LCAT reaction transfers cholesterol esters to other lipoproteins, which transport them to the liver (referred to as “reverse cholesterol transport)

Measuring HDL cholesterol : 

Measuring HDL cholesterol Ultracentrifugation is the most accurate method HDL has density 1.063 – 1.21 g/mL Routine methods precipitate apolipoprotein B with a polyanion/divalent cation Includes VLDL, IDL, Lp(a), LDL, and chylomicrons Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol

Apolipoproteins : 

Apolipoproteins The protein composition differs from one lipoprotein class to another, and the protein constituents are called Apolipoproteins

Functions of apolipoproteins : 

Functions of apolipoproteins Activate enzymes involved in lipid metabolism (LCAT, LPL) Maintain structural integrity of lipid/protein complex Delivery of lipids to cells via recognition of cell surface receptors

Apolipoprotein content of LPs : 

Apolipoprotein content of LPs

Apolipoprotein Measurements : 

Apolipoprotein Measurements Apo levels depends on genetics and determines amount of cholesterol levels of individuals. Measure: ApoB, ApoA-1, Apo-LD; all assist in determining CHD risk because of their relationship with LDL, VLDL and HDL. Measured by immunoassay utilizing turbidmetric or nephelometric.

Phospholipids : 

Phospholipids 3 major types of membrane lipids includes phospholipids, glycolipids and cholesterol Are amphipathic (contain polar hydro-philic head group and non-polar hydrophobic fatty acid side chains. They act as detergents because of the hydrophilic and hydrophobic groups, major constituent of biological interfaces such as cell membranes.

Slide 62: 

Formed by the conjunction of two fatty acids and a phosphorylated glycerol. Two fatty acids consist of 14-24 carbon atoms and phosphorus group, which can be complexed to choline to form phosphatidylcholine (lecithin) or ethanolamine, inostol or serine to form cephalins. Different fatty acids results in lecithin and cephalin.

Phospholipids measurement : 

Phospholipids measurement Quantitative method used for research methods Enzymatic Method: utilizes phospholipase D choline-oxidase and horseradish peroxidase. Measures choline containing phospholipids (lecthin, lysolecithin, sphingomyelin). Method is useful in determining fetal lung maturity. (L/S measurement)

Glycolipids : 

Glycolipids Sugar containing lipids Simplest glycolipids are: galactosylceramide and glucosylceramide

Prostaglandins : 

Prostaglandins Called eicosanoids (fatty acid): 20 carbon atom. Synthesized in almost all tissues from arachidonate and polyunsaturated fatty acids. Paracrine- autocrine action. Linoleic and linolenic acid are precursors.

Effects of hormones on lipids : 

Effects of hormones on lipids Insulin: major function is the inactivating of hormone sensitive lipase. Results in build up of adipose tissue. This results in the suppression release of free fatty acids causing a reduction of free fatty acid delivered to the liver. Glucose in conjunction with insulin: promotes the esterification of free fatty acids into triglycerides.

Slide 69: 

Insulin regulates VLDL synthesis in the liver. Diabetic patients often have elevated plasma triglycerides, reduction of HDL concentration. Growth hormone: increases the production of VLDL by increasing production of Apo-E and Apo-B48 along with the stimulation of lipolysis in adipose tissue and triglyceride synthesis in the liver.

Slide 70: 

Sex hormone estrogen increases VLDL levels in conjunction with increasing the # of LDL receptors, decreases LDL levels an increase HDL levels. Thyroid hormone: has multiple effects; hypothyroid see increase in plasma LDL cholesterol.

Fredrickson classification : 

Fredrickson classification

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