Lipids and Lipoproteins Midterms PDF

# Lipids and Lipoproteins ## MIDTERMS ## Components of Lipoproteins ### 1. SURFACE - PL & NEC AKA amphiphatic lipids with polar and nonpolar groups. - This would explain the characteristic orientation of PL & NEC in the surface of lipoproteins - Polar groups are oriented outward towards the wa...

# Lipids and Lipoproteins ## MIDTERMS ## Components of Lipoproteins ### 1. SURFACE - PL & NEC AKA amphiphatic lipids with polar and nonpolar groups. - This would explain the characteristic orientation of PL & NEC in the surface of lipoproteins - Polar groups are oriented outward towards the watery environment. - Nonpolar groups are oriented in the core with cis hydrophobic. - All neutral lipids have no polar group, meaning they are strictly hydrophobic. - This means they are water insoluble, which is the reason why they are hiding at the very core of lipoproteins. - **How does NEC become esterified cholesterol?** - When fatty acid is attached to the portion of the molecule, the polarity is lost. - When the FA is attached to cholesterol, it becomes esterified because of its esterified bonds. - CE = cholesterol with FA attached. ### 2. CORE - Triacylglycerol - Esterified cholesterol - Free-fatty acids ### Apolipoproteins - Only protein moieties/components in lipoproteins (There are no other proteins in lipoproteins except Apolipo) - They serve three major roles: - **Structural:** They maintain the structural integrity of lipoprotein structure. - **LP Metabolism:** They serve as activator, inhibitor, and cofactor of enzymes needed in lipoprotein metabolism. - **Ligands:** They serve as ligands for cell receptors. ## Significant Apolipoproteins | Apo | Plasma Conc. (mg/dL) | Main distribution | Function | |:--------|:----------------------|:-----------------|:--------------------------------------------------------------------------| | A-I | 100-200 | HDL | Structural, LCAT activator | | A-II | 20-50 | HDL | Structural | | A-IV | 10-20 | CM, VLDL, HDL | Structural | | B-100 | 70-125 | VLDL, IDL, LDL | Structural, LDL-R ligand | | B-48 | <5 | CM | Structural, remnant receptor ligand | | C-I | 5-8 | CM, VLDL, HDL | Structural | | C-II | 3-7 | CM, VLDL, HDL | Structural, LPL cofactor | | C-III | 10-12 | CM, VLDL, IDL, remnants and HDL | Structural, LPL inhibitor | | E | 3-15 | CM, VLDL, IDL, remnants and HDL | Recognition factor that targets chylomicron and VLDL remnants to hepatic receptor | - **Lecithin cholesterol acyltransferase (LCAT):** Catalyzes the esterification of cholesterol. - **LDL Receptor (LDL-R):** Mediates the endocytosis of lipoproteins especially LDL. It requires interaction with ApoB-100 via the LDL-ligand receptor on the cell surface. - **Lipoprotein lipase (LPL):** Catalyzes the hydrolysis of TG in lipoproteins releasing free fatty acids and glycerol to tissues. It requires the presence of C-II to function. - **Chylomicron remnants:** It must have Apo-E to enable entry into the cell. ## Major classes of lipoproteins | | Density | Electrophoretic mobility | Major component | Apolipoproteins | Other comments | |:------------------|:---------|:-----------------------|:-----------------|:-----------------------------------------------------------------------|:---------------------------------------------------------------------------------------------------| | 1. Chylomicron | <0.93 | origin | TAG | ApoB-forty-eight, ApoC & ApoE | Transports lipids. Exogeneous/ dietary. | | 2. VLDL | 0.93-1.006 | pre-Beta | TAG | B-100, ApoC & ApoE | Transports lipids. Endogenous. | | 3. LDL | 1.019-1.063 | Beta | cholesterol | B-100 only | Transports lipids to peripheral tissues. | | 4. HDL | 1.063-1.21 | alpha | phospholipids | A-I, A-II, C & E | Reverse cholesterol transport. | ## Lipoprotein transport pathways - **1. Absorption pathway:** Dietary lipids are absorbed in the intestine and packaged into chylomicrons. - **2. Exogenous pathway:** Chylomicrons are transported from the intestine to tissues. - **3. Endogenous pathway:** VLDL is synthesized in the liver and transported to tissues. - **4. Reverse cholesterol transport pathway:** HDL picks up cholesterol from tissues and returns it to the liver. - **CETP (Cholesterol Ester Transfer Protein)**: Transfers cholesterol esters from HDL to VLDL and LDL. ## Gradient density centrifugation | Lipoprotein | Density (g/mL) | Description | |:--------------------|:--------------|:------------| | Chylomicrons | 0.95 | Floats above the surface because of its lower density than water. | | VLDL | 1.006 | Sinking nearly at the bottom because of its higher protein content. | | IDL | 1.019 | | | LDL | 1.063 | | | HDL | 1.090 | Density is based on both lipid and protein content. Higher lipid content, lower density. | ## Minor & Abnormal Lipoproteins ### IDL (Intermediate-density lipoprotein) - AKA: Floating Beta- lipoprotein. - Confusing because typical beta-LP is LDL. Does it float? - It has the same electrophoretic mobility with LDL but has low density (lower than the density of VLDL). - Differs in typical composition of VLDL: It has higher cholesterol content (Explains the fact of its lower density) - Associated with type III hyperlipoproteinemia/AKA dysbetalipoproteinemia. ### LP(a) - AKA: Sinking pre-B lipoprotein. - Typical pre-BLP is VLDL; does VLDL sink? It doesn't. - LDL like in structure. - Electrophoretic mobility: Similar to VLDL. - Ultracentrifugation sinks lower than in LDL. - Sinking to higher content of protein due to the addition of Apo(a)- Apo LP that has prothrombotic nature because it is homologous to plasminogen and inhibits fibrinolysis or it competes with fibronogen in binding site for clots that results to no fibrinolysis (lysis of clots) - **That in general will lead to cardiovascular disease (Brain clot: MI; arterial clot: stroke)** ### LPX - Associated with obstructive biliary disease and LCAT deficiency. - Mostly composed of NEC. ## Fredrickson Classification Of Hyperlipoproteinemia | Type | Particle | Representative disorder/s | |:----|:---------|:--------------------------------------------| | 1 | CM | Familial chylomicronemia or LPL deficiency | | 2a | LDL | Familial hypercholesterolemia | | 2b | LDL, VLDL | Familial combined hyperlipoproteinemia | | 3 | IDL, B-VLDL| Familial dysbetalipoproteinemia | | 4 | VLDL | Primary hypertriglyceridemia | | 5 | CM, VLDL | Mixed hyperlipidemia | | Type | Particle | Clinical Presentation | Lab Findings | |:----|:---------|:---------------------|:-----------------------------------------------| | 1 | CM | xanthomas | Increase in TAG | | 2a | LDL | xanthelasmas | Increase LDL & TC | | 2b | LDL, VLDL | xanthelasmas | Increase LDL, TAG & TC | | 3 | IDL, B-VLDL| xanthelasmas | Increase TC, TAG | | 4 | VLDL | xanthomas | Increase in TAG | | 5 | CM, VLDL | xanthomas | Increase in TAG | - **Xanthomas:** Increase TAG that deposits in the skin. - **High TAG:** High risk for pancreatitis; high TAG & low LDL in general, low for cardiac risk. - **Elevated LDL:** Increase total cholesterol; xanthelasmas deposits of high chole. - **Elevated TC:** High CHD (Coronary Heart Disease) ## Plasma Lipid Analysis ### 1. Analytical Methods #### A. Cholesterol - **Colorometric- spectrophotometry** - Utilizes chemical methods. - **Saponification step:** Hydrolyze CE using alcoholic KOH (break down of ester bond to release free cholesterol). - Reverse of LCAT catalyzed reaction. - **Liebermann-Burchardt:** Cholesterol + H2SO4 + (CH3CO)2O (acetic anhydride)-> Cholestadienyl monosulfonic acid (green) - **Salkowski:** Cholesterol + 2 H2SO4 + Fe³-> Cholestadienyl disulfonic acid (red) - **Steps** - **Saponification:** Hydrolysis of cholesterol esters - **Extraction:** Removal of protein interference - **Purification:** Precipitation of free cholesterol. - **Colorimetry:** Formation of a colored compound. - **Modified Abell-Kendall:** CDC reference method which involves saponification with alcoholic KOh, extraction with n-hexane or petroleum ether, and colorimetry using the Libermann-Burchardt rxn. - **Enzymatic method:** Peroxidase-catalyzed dye oxidation. - Cholesterol Ester + H2O -> Cholesterol + Fatty acid (cholesterol esterase) - Cholesterol + O2 -> Cholestenone+ H2O2 (cholesterol oxidase) - H2O2 + chromogen -> Oxidized chromogen +H2O (peroxidase) - **Reference values** - <200 mg/dL = desirable - 200-239 mg/dL = borderline high - ≥240 mg/dL = high - **Recommended cutoff points according to age:** | Age (years) | Moderate risk | High risk | |:-------------|:---------------|:-----------| | 2-19 | >170 | >185 | | 20-29 | >200 | >220 | | 30-39 | >220 | >260 | | 40 & above | >240 | >260 | #### B. Triglycerides - **Chemical methods:** - Triglyceride + H2O -> Glycerol + 3 Fatty acids (alcoholic KOH) - Glycerol + O2 -> Formaldehyde (HCHO) (periodic acid) - **Colorimetric:** HCHO + chromotropic acid -> colored compound - **Fluorometric:** HCHO + diacetyl acetone + NH3 -> diacetyl lutidine compound - **Enzymatic methods:** - Triglyceride + H2O -> Glycerol + 3 Fatty acids (Lipase) - Glycerol + ATP -> Glycerophosphate + ADP (Glycerokinase) - **Peroxidase-catalyzed dye oxidation (colorimetric):** Following oxidation of glycerophosphate: Glycerophosphate + O2 -> dihydroxyacetone + H2O2 (glycerophosphate oxidase) - H2O2 + dye -> colored product + H2O (peroxidase) - **Measurement of 1 absorbance at 340nm:** Following reaction of glycerophosphate with NAD: Glycerophosphate + NAD -> dihydroxyacetone phosphate + NADH (glycerophosphate dehydrogenase) - **Reference values:** - <200 mg/dL = - 150-199 mg/dL = - 200-499 mg/dL = - >/= 500 mg/dL = #### C. Lipoprotein Cholesterol - **1. HDL-C:** - **Ultracentrifugation:** Involves adjusting the sample to a density of ________ (potassium bromide), followed by centrifugation at high speed for 24 hours. - **Homogeneous (direct assay):** Immunologic assay involving blockage of non-HDL lipoproteins using an antibody to apo B-100. - **Precipitation:** Of apo B-containing lipoproteins by polyanion-divalent cations, followed by cholesterol determination on the supernatant using the PODcouple method. - **2. LDL-C:** - **B-Quantification:** Involves ultracentrifugation and precipitation. - **Homogeneous (direct assay):** Uses detergents or other chemicals to block or solubilize non-LDL lipoprotein classes to allow for quantitation of LDL. - **Calculation:** TC - (HDL + VLDL) - There are multiple formulas for calculating VLDL, including: - **Friedwald:** VLDL (mg/dL) = TG/__ VLDL (mmol/L) = TG/__ - **De Long:** VLDL (mg/dL) = TG/__ VLDL (mmol/L) = TG/__ - **Martin-Hopkins:** VLDL = TG/ adjustable factor. - **Reference values:** - <100 mg/dL = - 100-129 mg/dL = - 130-159 mg/dL = - 160-189 mg/dL = - >/= 190 mg/dL = ### 2. Clinical Significance #### A. Inulin clearance - Reference method; not routinely done because of the necessity for continuous IV infusion. - **Priming dose**: 25 mL of 10% inulin solution. - **Continuous infusion:** 500 mL of 1.5% inulin solution. - **Reference value:** - Male: 127 mL/min. - Female: 118 mL/min #### B. Creatinine clearance - Production and excretion is related directly to muscle mass (excretion is usually not affected by diet). - Excellent measurement of renal function - creatinine is freely filtered by the glomerulus but not reabsorbed. - **Reference value:** - Male: 85-125 mL. - Female: 75-112 mL. - **Increased creatinine clearance:** - High cardiac input. - Pregnancy. - Burns. - **Decreased creatinine clearance:** - Impaired kidney function. - Shock, dehydration. - Hemorrhage. - Congestive heart failure. #### C. Urea clearance - Does not give a reliable estimate of GFR. - Freely filtered by the glomerulus but variably reabsorbed by the tubules. - The faster the rate of urine flow, the less urea is reabsorbed, and vice versa. - It is about 50% of creatinine clearance in the presence of normal renal function with blood volume depletion. - It is significantly affected by protein diet and the enzymatic method is affected by smoking due to ammonia. - Can demonstrate progression of renal disease or response to therapy. #### D. Cystatin C - Freely filtered at the glomerulus, not secreted by the renal tubules but reabsorbed. - It is completely reabsorbed by the PCT hence its presence in the urine. - Tubule-serum level is an indirect measure of GFR. - It is a constant value, averaging body surface area of an adult individual. #### **I. Test for glomerular filtration rate** - **Glomerular filtration rate:** A measure of how well the kidneys filter blood. - It estimates the volume of filtrate produced by the kidneys’ glomeruli per minute, typically expressed in milliliters per minute (mL/min). - GFR is an important indicator of kidney function and helps assess the kidneys' ability to remove waste products and excess fluid from the bloodstream. - Decreases by 1.0 mL/min/yr after the age of 20-30 yrs. - **Clearance:** represents the volume of plasma that would contribute all the solute excreted. - The removal of the substance from plasma into urine over a fixed time. - Expressed: mL/min. - Plasma concentration and clearance is inversely proportional (as clearance of a substance decreases, its concentration in plasma increases). - **Formula:** Clearance (mL/min) = (U x Volume (mL) x 1.73) / (P x minutes) - **U:** concentration of the analyte in urine (mg/L). - **P:** concentration of the analyte in plasma (mg/L). - **V:** volume of urine in mL for 24 hours (L/min). - **Minutes:** time required to collect urine (1’440). - **1.73:** body surface area of the patient. #### **II. Test for renal blood flow** - **Blood urea nitrogen (BUN):** Major end product of protein and amino acid catabolism. - Used to obtain the concentration of urea from BUN = 2.14 x BUN = urea (mg%). - **Chemical (direct) method:** Used to measure the concentration of urea in blood. - **Enzymatic (indirect) method:** Uses an enzyme to convert urea to a measurable product. - **IDMS (isotope dilution mass spectrometry):** A reference method used to calibrate other urea assays. - **Creatinine:** End product of muscle metabolism derived from creatine. - It is also produced by 3 amino acid (methionine, arginine, and lysine). - It is most commonly used to monitor renal function and is an index of overall renal function. - **Reference value:** 0.5-1.5 mg/dL (44-133 umol/L) - Used to evaluate fetal kidney maturity. - **Patient and sample preparation:** - **Fasting is not required.** - Hemolyzed and icteric samples should be avoided. - Both serum and urine creatinine have been used as indices of renal function. - 24-hour urine sample with < 0.8 g/day of creatinine indicates that some of the urine was probably discarded. ### **Methods** - **1. Enzymatic method** - Creatinine + H2O -> Creatinine (creatininase). - **Creatininase-CK coupled:** - Creatinine + ATP -> Creatine phosphate + ADP (CK). - ADP + Phosphoenolpyruvate -> Pyruvate + ATP (PK). - Pyruvate + NADH -> Lactate + NAD (LDH). - **Creatininase-POD coupled:** - Creatinine + H2O -> Sarcosine + urea (creatininase). - Sarcosine + O2 -> Glycine + H2O2 (sarcosine oxidase). - H2O2 + chromogen -> Oxidized chromogen + H2O (POD). - **2. Analytical methods:** - **Chemical (Jaffe method):** - Colorimetric, endpoint. - Not as specific, subject to a falsely increased result. - The original Jaffe method is nonspecific because there is an effect of various substances, both endogenous and exogenous, that are present in plasma. - These substances react with the picric acid reagent, contributing to the level of creatinine. - Techniques to improve the specificity of the Jaffe reaction: - **Kinetic:** A timed rate reaction. - **Use of adsorbents:** Used to remove non-creatinine chromogens and to increase specificity. - **Reference intervals:** - Jaffe: 0.9-1.3 mg/dL (M); 0.6-1.1 mg/dL (F). - Enzymatic: 0.6-1.1 mg/dL (M); 0.5-0.8 mg/dL (F). - **BUN/Creatinine ratio:** 10-20:1. - **Clinical significance of abnormal values:** - **BUN/Crea ratio >20:1 with Normal creatinine:** Pre-renal azotemia. - **BUN/Crea ratio >20:1 with elevated creatinine:** Renal post-renal azotemia. - **BUN/Crea ratio < 10:11:** Repeated dialysis, liver disease, low protein diet (low BUN). - **Decreased creatinine clearance:** Impairment in renal function or low GFR due to a decrease in cardiac output, blood volume, or renal blood flow. #### **Uric acid:** - Major product of purine (adenine and guanine) catabolism. - Final breakdown of nucleic acid catabolism in humans. - Formed from xanthine by the action of xanthine oxidase in the liver and intestine. - It is filtered, partially reabsorbed, and secreted in the renal tubules. - It is derived from three sources: 1. Catabolism of ingested nucleoproteins. 2. Catabolism of endogenous nucleoproteins. 3. Direct transformation of endogenous purine nucelotides. - About 1g of uric acid is excreted normally. - **Reference values:** - **Male:** 3.5-7.2 mg/dL (0.21-0.43 mmol/L). - **Female:** 2.6-6.0 mg/dL (0.16-0.36 mmol/L). ### Methods - **1. Chemical (Reduction-oxidation reaction).** - Uric acid + H3PW12O40 + O2 -> Allantoin + Tungsten + CO2. - **2. Enzymatic (Uricase) method:** - Uric acid + O2 + H2O -> Allantoin + CO2 + H2O2. - **Reference intervals:** - **Male:** 3.5-7.2 mg/dL - **Female:** 2.6-6.0 mg/dL - **Clinical significance of abnormal values:** - **1. Hyperuricemia (increased uric acid):** - **A. Gout:** - Pain and inflammation of the joints. - Presence of bifringent crystals in the synovial fluid. - Highly susceptible to nephrolithiasis. - **B. Increased nuclear metabolism:** Seen in leukemia (monitoring is important to avoid nephrotoxicity). - **C. Chronic renal disease:** - Due to decreased GFR and tubular secretion. - >10 mg/dL levels of plasma uric acid cause urinary tract calculi. - **D. Lesch-Nyhan syndrome:** Deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRT). - **2. Hypouricemia (decreased in uric acid):** - **A. Fanconi syndrome:** Renal-type aminoaciduria. - **B. Wilson's disease** - **C. Hodgkin's disease.** - **D. Purine inhibitors.** - **E. Allopurinol.** ## **Thank You**