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Lipids and Lipoproteins Roger L. Bertholf, Ph.D. Associate Professor of Pathology Director of Clinical Chemistry & Toxicology.

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Presentation on theme: "Lipids and Lipoproteins Roger L. Bertholf, Ph.D. Associate Professor of Pathology Director of Clinical Chemistry & Toxicology."— Presentation transcript:

1 Lipids and Lipoproteins Roger L. Bertholf, Ph.D. Associate Professor of Pathology Director of Clinical Chemistry & Toxicology

2 Classification of lipids Fatty acids (palmitic, linoleic, etc.) Glycerol esters (triglycerides) Sterols (cholesterol, hormones, vitamin D) Terpenes (vitamins A, E, K) Sphingosine derivatives (sphingomyelin)

3 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 (C 12, 12:0, n-dodecanoic acid)

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

5 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

6 Clinical importance of fatty acids Fecal fatty acids are sometimes measured to detect malabsorptive and pancreatic disordersthe 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)

7 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 GlycerolTriglyceride

8 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

9 Measuring triglycerides (enzymatic method) Triglycerides Glycerol + FFAs Lipase Glycerophosphate + ADP Glycerokinase ATP Dihydroxyacetone + H 2 O 2 Glycerophasphate oxidase Peroxidase Quinoneimine dye max 500 nm

10 Sterols (cholesterol) Sterols are steroid backbones that have a hydroxyl group at position 3 and a branched aliphatic chain of 8 or more carbons at position 17

11 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

12 Cholesterol biosynthesis Acetyl-CoA3-Hydroxy-3-methylglutaryl-CoA Mevalonate HMG-CoA reductase Squalene Cholesterol + LecithinCholesterol ester LCAT Statin drugs inhibit this enzyme

13 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

14 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 Cholesterol Cholesteryl ester hydroxylase Choles-4-en-3-one + H 2 O 2 Cholesterol oxidase Quinoneimine dye ( max 500 nm) Phenol 4-aminoantipyrine Peroxidase

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

16 Lipoprotein classes %TG%CholLPE Chylomicrons863Origin VLDL5512 Pre- IDL2329 Pre- / LDL642 HDL315 Lp(a)(LDL) Pre-

17 Appearance of hyperlipidemia 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

18 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 Pre- -+ ChylomicronsLDLVLDL Lp(a) HDL Migration IDL

19 Fredrickson classification TypeRefrig.LPELPs IPos, clearNormal TG (chylos) IIaNeg, clear band LDL IIbNeg, cloudy, pre- LDL, VLDL IIIOcc., cloudy pre- Chol, TG, VLDL IVNeg, cloudy -2 VLDL VPos, cloudy -2 VLDL Chylos

20 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 HDL, IDL, LDL, VLDL HDL + (IDL, LDL, VLDL) Dextran sulfate Mg ++ Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol

21 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

22 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

23 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

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

25 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

26 Apolipoprotein content of LPs LipoproteinApolipoprotein(s) ChylomicronAI, B-48, CI, CII, CIII VLDLB-100, CI, CII, CIII, E IDLB-100, E LDLB-100 HDLAI, AII Lp(a)(a), B-100

27 Cholesterol metabolism (exogeneous) Dietary cholesterol, triglycerides C,TG B A Chylomicron Apo-C, E from HDL Endothelium LPL C,TG B A C E B E Chylomicron remnant Hepatocyte B/E receptors

28 Cholesterol metabolism (endogeneous) C,TG B VLDL Endothelium LPL C,TG B E IDLLDL E C C B Hepatocyte B-100 receptors

29 Dyslipoproteinemias Causes can be primary or secondary –Secondary causes include starvation, liver disease, renal failure, diabetes, hypothyroidism, lipodystrophies, drugs Primary causes of hyperlipidemia: –Increased production –Defective processing –Defective cellular uptake –Inadequate removal

30 Dyslipoproteinemias Hyperchylomicronemia –LPL deficiency –Apo C-II deficiency

31 Hyperchylomicronemia Dietary cholesterol, triglycerides C,TG B A Chylomicron Apo-C, E from HDL Endothelium LPL C,TG B A C E B E Chylomicron remnant Hepatocyte B/E receptors Chylomicrons Triglycerides HDL LDL

32 Dyslipoproteinemias Hyperchylomicronemia –LPL deficiency –Apo C-II deficiency Hyperbetalipoproteinemia –Overproduction of VLDL –Enhanced conversion of VLDL to LDL –LDL enriched with cholesteryl esters –Defective LDL structure –Decreased LDL receptors

33 Hyperbetalipoproteinemia C,TG B VLDL Endothelium LPL C,TG B E IDLLDL E C C B Hepatocyte B-100 receptors LDL Normal TG

34 Dyslipoproteinemias Combined hyperlipoproteinemia –Normal LDL receptors –Overproduction of VLDL and Apo B-100

35 Combined hyperlipoproteinemia C,TG B VLDL Endothelium LPL C,TG B E IDLLDL E C C B Hepatocyte B-100 receptors LDL Normal TG

36 Dyslipoproteinemias Combined hyperlipoproteinemia –Normal LDL receptors –Overproduction of VLDL and Apo B-100 Dysbetalipoproteinemia –Both cholesterol and triglyceride elevated –Mutant form of Apo E

37 Dysbetalipoproteinemia Dietary cholesterol, triglycerides C,TG B A Chylomicron Apo-C, E from HDL Endothelium LPL C,TG B A C E B E Chylomicron remnant Hepatocyte B/E receptors Cholesterol TG

38 Dyslipoproteinemias Familial hypercholesterolemia –Defect in LDL receptor Absent Defective –Incidence = 1:500

39 Familial hypercholesterolemia C,TG B VLDL Endothelium LPL C,TG B E IDLLDL E C C B Hepatocyte B-100 receptors LDL or n TG HDL

40 Dyslipoproteinemias Familial hypercholesterolemia –Defect in LDL receptor Absent Defective –Incidence = 1:500 Familial defective Apolipoprotein B-100

41 Familial hypercholesterolemia C,TG B VLDL Endothelium LPL C,TG B E IDLLDL E C C B Hepatocyte B-100 receptors or n LDL

42 High cholesterol, high LDL Diet/Lifestyle 2° to hypothyroidism or nephrotic syndrome (disruption of Apo-B metabolism) Polygenic: (means we dont know) Familial hypercholesterolemia Familial defective Apo-B Rare disorders

43 High TG, normal cholesterol Diet/Lifestyle 2° to diabetes, thiazide diuretics, Cs, beta- blockers, CRF/Nephrotic syndrome Familial hypertriglyceridemia (etiology unknown) ApoC-III excess (interferes with LPL) LPL deficiency ApoC-II deficiency

44 High cholesterol, TG Obesity 2° to steroids, Cs, hypothyroidism, CRF Familial combined hyperlipidemia (multifactorial) Peroxisome proliferator-activator receptor Dysbetalipoproteinemia (Type III) Hepatic lipase deficiency (rare)

45 Low cholesterol, low/normal HDL Abetalipoproteinemia (ApoB degraded after synthesis causes fat malabsorption) Hypobetalipoproteinemia (genetically defective ApoB) Chylomicron retention disease (unknown cause)

46 Low HDL Lifestyle 2° to steroids, beta-blockers, progestogens Familial hypoalphalipoproteinemia (ApoA-I, C-III, or A-IV defects) ApoA-I variants Tangier disease (enhanced HDL degredation) LCAT deficiency

47 High HDL Lifestyle (ethanol) 2° to phenytoin, phenobarbitol, rifampicin (p-450 inducers) and estrogens Cholesteryl Ester Transfer Protein defects


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