Lipids (CC1 Lec)

Notes in CARBOHYDRATES, LIPIDS, PROTEINS, AND NPNs

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  • Lipids are composed of mostly carbon-hydrogen (C-H) bonds.

  • Lipids refer to fats and are the primary source of fuels in the body.

  • Lipids provide stability to the membrane and allow for transmembrane transport.

  • Lipids are insoluble in blood plasma but soluble in organic solvents.

  • Lipids are classified into fatty acids, triglycerides, cholesterol, phospholipids, and fat-soluble vitamins.

  • Lipids are transported by lipoproteins such as VLDL, LDL, and HDL.

  • Fatty acids are linear chains of C-H bonds that terminate with -COOH.

  • Fatty acids are mainly derived from the hydrolysis of triglycerides in adipose tissue.

  • Fatty acids are important sources of energy and provide substance for the conversion to glucose.

  • Fatty acids can be classified based on size (short, medium, long chain) and double bond (saturated, monounsaturated, polyunsaturated).

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  • Triglycerides contain three fatty acids attached to one molecule of glycerol.

  • Triglycerides are the main storage form of lipid in the body and provide insulation.

  • Triglycerides are hydrophobic and are broken down in adipose tissue by lipoprotein lipase and hormones like epinephrine and cortisol.

  • Phospholipids contain two fatty acids attached to one molecule of glycerol and have phospholipid head groups.

  • Phospholipids are amphipathic, with hydrophilic and hydrophobic head groups.

  • Phospholipids have similar structure with triglycerides and provide surface tension and act as surfactants.

  • Cholesterol is an unsaturated steroid alcohol that contains four rings and is a component of steroids.

  • Cholesterol is found on the surface lipid bilayer and is important in the assembly of cell membranes and bile acids.

  • Cholesterol is not catabolized by most cells and is recycled and transported back to the liver.

  • Cholesterol is routinely measured in the laboratory, both unesterified and esterified forms.

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  • Unesterified cholesterol is hydrophilic and constitutes about 30% of total cholesterol.

  • Esterified cholesterol is hydrophobic and constitutes about 70% of total cholesterol.

  • Esterified cholesterol is bound to fatty acids and is catalyzed by lecithin cholesterol acyl transferase (LCAT).

  • Cholesterol can be converted to bile salts, steroid hormones, vitamin D, and cell membranes.

  • Lipoproteins are composed of both lipids and proteins (apolipoproteins).

  • Lipoproteins have different densities based on the protein content.

  • Apolipoproteins maintain structural integrity, act as ligands for cell receptors, and activate or inhibit enzymes.

  • Major apolipoproteins include Apo A-I, Apo A-II, Apo A-IV, Apo B-100, Apo B-48, Apo C-I, Apo C-II, Apo C-III, Apo E, and Apo(a).

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  • Lipoproteins have free cholesterol and phospholipids on the surface and triglycerides and cholesteryl esters in the core.

  • Apolipoproteins maintain structural integrity, act as ligands for cell receptors, and activate or inhibit enzymes.

  • Major apolipoproteins include Apo A-I, Apo A-II, Apo A-IV, Apo B-100, Apo B-48, Apo C-I, Apo C-II, Apo C-III, Apo E, and Apo(a).

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Characteristics of the Major Human Lipoproteins

  • Chylomicrons

    • Density: <0.93 g/mL

    • Diameter: 80-1,200 nm

    • Total lipid: 98% by weight

    • Triglyceride: 84% by weight

    • Total cholesterol: 7% by weight

    • Major Protein: Apo B-48

  • VLDL

    • Density: 0.93-1.006 g/mL

    • Diameter: 30-80 nm

    • Total lipid: 89-96% by weight

    • Triglyceride: 44-60% by weight

    • Total cholesterol: 16-22% by weight

    • Major Protein: Apo B-100

  • LDL

    • Density: 1.019-1.063 g/mL

    • Diameter: 18-30 nm

    • Total lipid: 77% by weight

    • Triglyceride: 11% by weight

    • Total cholesterol: 62% by weight

    • Major Protein: Apo B-100

  • HDL

    • Density: 1.063-1.21 g/mL

    • Diameter: 5-12 nm

    • Total lipid: 50% by weight

    • Triglyceride: 3% by weight

    • Total cholesterol: 19% by weight

    • Major Protein: Apo A-1

Major Types:

  • Chylomicrons

    • Largest and least dense

    • Produced in the intestine

    • Delivery of dietary lipids to hepatic and peripheral cells

    • High consumption of fats leads to high chylomicron levels

    • Completely cleared from the plasma within 6-9 hours after eating

  • VLDL

    • Also known as pre-beta lipoprotein

    • Produced in the liver

    • Transfers endogenous triglycerides from the liver to peripheral tissue

  • LDL

    • Also known as beta lipoprotein or bad cholesterol

    • Formed from lipolysis of VLDL to IDL then to LDL

    • Transfers dietary cholesterol to peripheral tissues

    • Most atherogenic type, high LDL in plasma increases the risk of coronary heart disease/stroke

  • HDL

    • Also known as alpha lipoprotein or good cholesterol

    • Produced in the liver and intestine

    • Transfers cholesterol from peripheral cells back to the liver

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Minor Types:

  • Lipoprotein(a) or Lp(a)

    • LDL lipoprotein-like particle

    • Confers increased risk for premature coronary heart disease and stroke

    • Competes with plasminogen for fibrin clot formation

    • Accumulation on the blood vessel wall can lead to blockage and increase the risk of coronary heart disease or stroke

    • Also known as sinking pre-beta lipoprotein

  • Intermediate Density Lipoprotein (IDL)

    • Product of VLDL catabolism

    • Converted to LDL, subclass of LDL

    • Migrates in the pre-beta or beta region during electrophoresis

  • Lipoprotein X

    • Abnormal lipoprotein found in obstructive jaundice and LCAT deficiency

    • Specific and sensitive indicator of cholestasis

    • Contains mostly phospholipid and free cholesterol

    • Known as the "abnormally migrating Beta-VLDL"

    • Found in type 3 hyperlipoproteinemia or dysbetalipoproteinemia

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Adult Reference Ranges for Lipids

  • Total cholesterol: 140-200 mg/dL

  • HDL cholesterol: 40-75 mg/dL

  • LDL cholesterol: 50-130 mg/dL

  • Triglyceride: 60-150 mg/dL

Lipoprotein Physiology and Metabolism

  • Lipid absorption

  • Exogenous pathway

  • Endogenous pathway

  • Reverse cholesterol transport pathway

Lipid Absorption

  • Conversion of dietary lipids into more polar compounds

  • Pancreatic Lipase converts triglycerides, cholesterol esters, and phospholipids into more polar compounds for absorption

Exogenous Pathway

  • Chylomicrons are synthesized in the intestine and carry dietary lipids to the circulation

  • Lipoprotein Lipase (LPL) hydrolyzes triglycerides in chylomicrons into fatty acids and glycerol

  • Chylomicrons are converted into chylomicron remnant particles

Endogenous Pathway

  • Triglycerides in the liver are packaged into VLDL and carried to the circulation

  • VLDL is converted into VLDL remnants by LPL and taken up by the liver

  • Half of VLDL is transformed into LDL for delivery of exogenous cholesterol to peripheral cells

Reverse Cholesterol Transport Pathway

  • HDL removes excess cholesterol from peripheral cells and delivers it to the liver

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Lipid Disorders

  • Arteriosclerosis

  • Hyperlipoproteinemia

    • Hypercholesterolemia

    • Hypertriglyceridemia

    • Combined hyperlipidemia

  • Hypolipoproteinemia

Arteriosclerosis

  • Deposition of esterified cholesterol in artery walls

  • Leads to the formation of plaques and can cause thrombosis, blocking circulation

  • Examples: Coronary Artery Disease (CAD), Peripheral Vascular Disease (PVD), Cerebrovascular Disease (CVD)

Hyperlipoproteinemia

  • Hypercholesterolemia

    • Increased cholesterol levels

    • Increased LDL, decreased receptors

  • Hypertriglyceridemia

    • Increased triglyceride levels

    • Decreased LPL or Apo C-II

  • Combined Hyperlipoproteinemia

    • Increased triglycerides and cholesterol levels

    • Increased VLDL and chylomicron remnants

Other Lipid Disorders

  1. Familial Hypercholesterolemia (Type 2-a)

  2. Familial Dysbetalipoproteinemia (Type 3 hyperlipoproteinemia)

  3. Abetalipoproteinemia (Bassen-Kornzweig Syndrome)

  4. Hypobetalipoproteinemia

  5. Niemann-Pick Disease (Lipid Storage Disease)

  6. Tangier's Disease

  7. Lipoprotein Lipase (LPL) Deficiency

  8. Lecithin Cholesterol Acyl Transferase (LCAT) Deficiency

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  • Cholesterol Measurement

    • Plasma or serum can be used

    • Color developer mixture: glacial acetic acid, acetic anhydride, concentrated sulfuric acid

  • Patient preparation: 2 weeks prior to testing, person should be in usual diet

  • Precautions:

    • Avoid hemolyzed specimens

    • Avoid icteric specimens with bilirubin

    • Avoid water concentration

    • Precise and accurate timing is necessary

  • General methods:

    1. One-step method: direct colorimetry

    2. Two-step method: extraction followed by colorimetry

    3. Three-step method: extraction, saponification, and colorimetry

    4. Four-step method: precipitation, saponification, extraction, and colorimetry

  • Non-Enzymatic Methods: Abell Kendall

    • Principle: dehydration and oxidation of cholesterol to form a colored compound

    • Cholesteryl esters: hydrolyzed with alcoholic KOH

    • Unesterified cholesterol: extracted with hexane

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  • Cholesterol Measurement (continued)

    • Measured using Liebermann-Burchard reaction

    • Enzymatic Method: Cholesterol oxidase

      • Total Cholesterol: cholesteryl ester and cholesterol + H2O - Cholesteryl esterase -> cholesterol + fatty acid

      • Cholesterol + O2 - Cholesterol oxidase -> 4-cholestenone + H2O2

      • H2O2 + Dye - Peroxidase -> Color (Quinoneimine dye)

    • Reference method: Abell Levey & Brodie method

  • Triglyceride Measurement

    • Prior to venipuncture, strict fasting of 12 hours

    • Plasma or serum can be used for measurement

  • Non-Enzymatic Method

    • Triglyceride - Alcoholic KOH -> glycerol + fatty acid

    • Glycerol + Periodic acid -> formaldehyde

    • Formaldehyde + Chromotropic acid -> Blue solution

  • Hantzch condensation: (Fluorometric Method)

    • Formaldehyde + Diacetyl acetone + NH3 -> Yellow solution

  • Enzymatic Methods (Glycerol kinase)

    • Triglyceride - Lipase -> Glycerol + Fatty Acid

    • Glycerol + ATP - Glycerokinase -> Glycerophosphate + ADP

    • ADP + PEP - Pyruvate kinase -> ATP + Pyruvate

    • Pyruvate + NADH + H+ - LD -> Lactate + NAD+ (340 nm)

    • NAD+ is measured at 340 nm

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  • Glycerol-phosphate oxidase:

    • Glycerophosphate + O2 - GO -> Dihydroxyacetone + H2O2

    • H2O2 + Dye - Peroxidase -> Color

    • Reference method: modified von handle and silversmith

  • Lipoprotein Measurement

    • Preferred sample: collected using serum separator tube (SST)

    • For research purpose: EDTA

    • Non-fasting sample: total cholesterol and HDL can be measured but not LDL and VLDL

  • General methods:

    • Ultracentrifugation: reference method for quantitation of lipoprotein

      • Solution used: potassium bromide with specific gravity fixed at 1.063

      • Based on molecular density

      • Order from lightest to heaviest: chylomicrons, VLDL, LDL, HDL

    • Electrophoresis: based on migration in an electric field

      • Lipid stains: Oil red O, Fat Red 7B, Sudan black

      • 4 bands with fat stain: Alpha-lipoprotein (HDL), Pre-Beta, Beta, Chylomicrons

      • Most common medium: agarose gel (Sensitive type)

  • High-Density Lipoprotein (HDL) Methods

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  • Polyanion Precipitation

    • Goal: to precipitate lipoproteins other than HDL

    • Measurement: measures HDL in supernate using total cholesterol method

    • Lipoproteins (LDL, VLDL) are precipitated with polyanions (e.g., Heparin, dextran sulfate or Na phosphotungstate) in the presence of divalent cations (e.g., Mg or Mn), which are sedimented by centrifugation

    • HDL is quantified in the supernate by Abell-Kendal method

  • Low-Density Lipoprotein (LDL) Methods

    • LDL = Total cholesterol - (HDL + VLDL)

    • Friedewald: VLDL = Triglyceride / 5 = mg/dL or VLDL = Triglyceride / 2.175 = mmol/L

    • Ultracentrifugation of serum at native density gradient of 1.006 g/L to float VLDL

  • Apolipoprotein Methods

    • Apo B: for determination of LDL and VLDL concentration; directly proportional to LDL and VLDL with Apo B

    • Apo A-1: for determination of HDL concentration

  • Methods:

    1. Immunoassay/immunonepholometric method

    2. Immunoturbidimetric method

    3. Immunochemical method

  • Positive Risk Factors:

    • Age: ≥45 years for men; ≥55 years or premature menopause for women

    • Family history of premature CHD

    • Current cigarette smoking

    • Hypertension: BP ≥140/90 mmHg or taking antihypertensive medication

    • LDL cholesterol concentration: ≥160 mg/dL, with ≤1 risk factor

    • LDL cholesterol concentration: ≥130 mg/dL, with ≥2 risk factors

    • LDL cholesterol concentration: ≥100 mg/dL, with CHD or risk equivalent

    • HDL cholesterol concentration: <40 mg/dL

    • Diabetes mellitus = CHD risk equivalent

    • Metabolic syndrome (multiple risk factors)

  • Negative Risk Factor:

    • HDL cholesterol concentration ≥60 mg/dL & LDL cholesterol <100 mg/dL