Lipids and Lipoproteins

Roger L. Bertholf, Ph.D.

Associate Professor of Pathology

Director of Clinical Chemistry & Toxicology

Classification of lipids

• Fatty acids (palmitic, linoleic, etc.)

• Glycerol esters (triglycerides)

• Sterols (cholesterol, hormones, vitamin D)

• Terpenes (vitamins A, E, K)

• Sphingosine derivatives (sphingomyelin)

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

OH

O

Lauric acid (C12, 12:0, n-dodecanoic acid)

Unsaturated fatty acids

• Double bonds in fatty acids are nearly always cis

OH

O

Palmitoleic acid (16:19, 9-hexadecanoic acid)

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

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)

• Triglycerides are the most abundant family of lipids in plant and animal cells, and are major components of the the human diet

H

CH OH

C

C

H OH

H

H OH

Glycerol

H

CH O

C

C

H O

H

H O

CO

CO

CO (CH2)n

(CH2)n

(CH2)n

CH3

CH3

CH3

Triglyceride

Measuring triglycerides (reference method)

• Triglycerides are extracted into chloroform prior to analysis

Triglycerides

KOHfatty acids + glycerol

Periodateformic acid + formaldehyde

chromotropic acidchromogen=570 nm

Measuring triglycerides (enzymatic method)

Triglycerides

Glycerol + FFAsLipase

Glycerophosphate + ADPGlycerokinase

ATP

Dihydroxyacetone + H2O2

Glycerophasphateoxidase

PeroxidaseQuinoneimine dye

max 500 nm

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

HO

12

34

56

7

89

1112

10

13

14 1516

17

A B

C D

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

Cholesterol biosynthesis

Acetyl-CoA 3-Hydroxy-3-methylglutaryl-CoA

Mevalonate

HMG-CoA reductase

Squalene

Cholesterol + Lecithin Cholesterol esterLCAT

“Statin” drugs inhibit this enzyme

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

HO

H2SO4/HOAc

HOO2S

Cholesterol Cholestahexaene sulfonic acid

max = 620 nm

L-B reagent

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

Cholesterylester

hydroxylase

Choles-4-en-3-one + H2O2

Cholesteroloxidase

Quinoneimine dye (max500 nm)

Phenol4-aminoantipyrinePeroxidase

Lipoproteins• In order to be transported in blood, lipids must

combine with water-soluble compounds, such as phospholipids and proteins.

Lipoprotein classes%TG %Chol LPE

Chylomicrons 86 3 Origin

VLDL 55 12 Pre-

IDL 23 29 Pre-/

LDL 6 42

HDL 3 15

Lp(a) (LDL) (LDL) Pre-

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

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-

- +

Chylomicrons LDL VLDLLp(a)

HDL

Migration

IDL

Fredrickson classificationType Refrig. LPE LPs

I Pos, clear Normal TG (chylos)

IIa Neg, clear band LDL

IIb Neg, cloudy , pre- LDL, VLDL

III Occ., cloudy pre- Chol, TG, VLDL

IV Neg, cloudy -2 VLDL

V Pos, cloudy -2 VLDL Chylos

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

• Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL 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 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

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

Apolipoproteins

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

Lipoprotein Apolipoprotein(s)

Chylomicron AI, B-48, CI, CII, CIII

VLDL B-100, CI, CII, CIII, E

IDL B-100, E

LDL B-100

HDL AI, AII

Lp(a) (a), B-100

Cholesterol metabolism (exogeneous)Dietary cholesterol,

triglycerides

C,TG

B

A

Chylomicron

Apo-C, E from HDL

Endotheliu

m

LPLC,TG

B

A

CE

C,TG

BE

Chylomicronremnant

Hepatocyte B/E receptors

Cholesterol metabolism (endogeneous)

C,TG

B

VLDL

Endotheliu

m

LPL

C,TG

BE

IDLLDL

EC

C

B

Hepatocyte B-100receptors

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

Dyslipoproteinemias

• Hyperchylomicronemia– LPL deficiency– Apo C-II deficiency

HyperchylomicronemiaDietary cholesterol,

triglycerides

C,TG

B

A

Chylomicron

Apo-C, E from HDL

Endotheliu

m

LPLC,TG

B

A

CE

C,TG

BE

Chylomicronremnant

Hepatocyte B/E receptors

ChylomicronsTriglyceridesHDLLDL

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

Hyperbetalipoproteinemia

C,TG

B

VLDL

Endotheliu

m

LPL

C,TG

BE

IDLLDL

EC

C

B

Hepatocyte B-100receptors

LDLNormal TG

Dyslipoproteinemias

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

Combined hyperlipoproteinemia

C,TG

B

VLDL

Endotheliu

m

LPL

C,TG

BE

IDLLDL

EC

C

B

Hepatocyte B-100receptors

LDLNormal TG

Dyslipoproteinemias

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

• Dysbetalipoproteinemia– Both cholesterol and triglyceride elevated– Mutant form of Apo E

DysbetalipoproteinemiaDietary cholesterol,

triglycerides

C,TG

B

A

Chylomicron

Apo-C, E from HDL

Endotheliu

m

LPLC,TG

B

A

CE

C,TG

BE

Chylomicronremnant

Hepatocyte B/E receptors

CholesterolTG

Dyslipoproteinemias

• Familial hypercholesterolemia– Defect in LDL receptor

• Absent

• Defective

– Incidence = 1:500

Familial hypercholesterolemia

C,TG

B

VLDL

Endotheliu

m

LPL

C,TG

BE

IDLLDL

EC

C

B

Hepatocyte B-100receptors

LDLor n TGHDL

Dyslipoproteinemias

• Familial hypercholesterolemia– Defect in LDL receptor

• Absent

• Defective

– Incidence = 1:500

• Familial defective Apolipoprotein B-100

Familial hypercholesterolemia

C,TG

B

VLDL

Endotheliu

m

LPL

C,TG

BE

IDLLDL

EC

C

B

Hepatocyte B-100receptors

or n LDL

High cholesterol, high LDL

• Diet/Lifestyle• 2° to hypothyroidism or nephrotic syndrome

(disruption of Apo-B metabolism)• Polygenic: (means we don’t know)• Familial hypercholesterolemia• Familial defective Apo-B• Rare disorders

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

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)

Low cholesterol, low/normal HDL

• Abetalipoproteinemia (ApoB degraded after synthesis causes fat malabsorption)

• Hypobetalipoproteinemia (genetically defective ApoB)

• Chylomicron retention disease (unknown cause)

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

High HDL

• Lifestyle (ethanol)

• 2° to phenytoin, phenobarbitol, rifampicin (p-450 inducers) and estrogens

• Cholesteryl Ester Transfer Protein defects