LIPIDS

LIPIDS

• Primary source of fuel, provide stability to cell membrane and allow for transmembrane transport

• Insoluble in water but soluble in organic solvents (chloroform, ether)

• Require special transport mechanisms known as lipoproteins for circulation in the blood.

• Increased plasma concentration of lipids contributes to the development of atherosclerosis and coronary heart disease.

• Major lipids: Phospholipids, Cholesterol, Triglycerides, Fatty Acid, Fat Soluble Vitamins

FATTY ACIDS

• Exists as short, medium, and long chains of molecules that are major constituents of triglycerides and phospholipids.

• Minimal amounts of fatty acids are bound to albumin and circulate free (unesterified) in plasma.

• Has two types:

• Saturated Fatty Acids

• Unsaturated Fatty Acids

• Major Function:  Building blocks for triglycerides & phospholipids; Sources of metabolic energy

PHOSPHOLIPIDS

• Most ABUNDANT LIPID from phosphatidic acid

• Formed from the conjugation of two fatty acids and phosphorylated glycerol = amphipathic lipid

• Sphingomyelin is an essential component of cell membranes of RBCs and nerve sheath

• RV: 150 – 380 mg/dL (serum)

• Forms of Phospholipids:

• Lecithin/Phosphatidyl choline 70%

• Sphingomyelin 20%

• Cephalin 10%

• Phosphatidyl ethanolamine

• Phosphatidyl serine

• Lysolecithin + Inositol phosphatide

• Premature babies are at risk of developing respiratory distress due to lack of surfactant. This is a mixture of phospholipids, including lecithin and sphingomyelin, which lowers the surface tension of the alveoli and facilitates the expansion and aeration of the fetal lungs at birth. The concentration of lecithin in amniotic fluid reflects production by fetal lungs. It increases rapidly after 32–34 weeks gestation, corresponding to increasing fetal lung maturity and decreasing risk of development of respiratory distress. Surfactant synthesis can be stimulated by giving corticosteroids to the mother, and this is now routine practice when elective premature delivery is planned for any reason. Natural and synthetic surfactants are available for use in the baby immediately after birth.

Functions:

• Phospholipids alter fluid surface tension (surfactant); it decreases surface tension within the alveolar space, thus allowing effective gas exchange and prevents alveolar collapse during expiration. (Fetal lung maturity test should be performed at less than 39 weeks or even at uncertain gestational age)

• Phospholipids participate in cellular metabolism and blood coagulation.

• They are also important substrates for a number of lipoproteins-metabolizing enzymes (e.g., lecithin cholesterol acyl transferase (LCAT) and lipoprotein lipase (LP)).

• Deficiency of surfactant leads to neonatal respiratory distress syndrome (RDS)

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METHODS OF PHOSPHOLIPID MEASUREMENT

Quantitative analysis of phospholipids is rare in laboratory medicine; it provides little added information in cases of dysbetalipoproteinemia.

Phospholipids can be measured in disorders characterized by altered phospholipids composition and lipoprotein distribution.

1. Estimation of Serum Lipid Phosphorus

• Each mole of phosphorus contributes to 4% to the total phospholipid mass, thus, phospholipid mass can be determined by multiplying the phospholipid phosphorus concentration (expressed in mg/dL) by 25.

2. Status of Fetal Lung Maturation – Lecithin/Sphingomyelin (L/S) Ratio

• The status of fetal lung maturation is estimated from the evaluation of pulmonary surfactant in amniotic fluid. The L/S and phosphatidylglycerol (PG) by chromatography or the microviscosity by fluorescence polarization are used.

• Thin layer chromatography followed by densitometric quantitation is the method for L/S ratio.

• Microviscosity of amniotic fluid can be measured by fluorescence polarization.

CHOLESTEROL

• Although cholesterol may be considered “bad” because of the previous association, it is actually a vital structural component of cell membranes and a precursor of steroid hormones and bile acids.

• Unsaturated steroid alcohol containing four rings with a single C-H side chain tail similar to fatty acid.

• Found on the surface of lipid layers: regulates fluidity of lipid bilayers.

• Synthesized in the liver; Almost exclusively synthesized by animals.

• Not catabolized by most cells – not a source of energy

• Cholesterol can, however, be converted in the liver to primary bile acids, such as cholic acid and chenodeoxycholic acid, which promote fat absorption in the intestine by acting as detergents. A small amount of cholesterol can also be converted by some tissues, such as the adrenal gland, testis, and ovary, to steroid hormones, such as glucocorticoids, mineralocorticoids, and estrogens. Finally, a small amount of cholesterol, after first being converted to 7-dehydrocholesterol, can also be transformed to vitamin D3 in the skin by irradiation from sunlight.

• Should be measured in all adults 20 years and older at least once every 5 years

• Important in the assembly of cell membranes, bile acids and steroid hormones

• Transport and excretion are promoted by estrogen

• Evaluates risk for atherosclerosis, myocardial infarction and coronary arterial occlusions

TWO FORMS OF CHOLESTEROL

1. Cholesterol esters (CE) – 70%

• Cholesterol bound to fatty acids and is found in plasma and serum

• Neutral lipid; found in the center of lipid drops and lipoproteins (along with TG)

• Esterified by LCAT (Lecithin-Cholesterol Acyl Transferase)

• Excess cholesterol is re-esterified by the microsomal enzyme acyl cholesterol acyl transferase (ACAT). LCAT activated by Apo-A1 and enables HDL to accumulate cholesterol as cholesterol esters by promoting transfer of fatty acids from lecithin to cholesterol = lysolecithin and cholesterol ester.

2. Free Cholesterol (FC) - 30% 

• Polar non-esterified alcohol found in plasma, serum and RBCs

• Produced by lysosomal hydrolysis and becomes available for membrane, hormone, and bile acid synthesis

LABORATORY METHODS

Specimen: FASTING 12 – 14 hours OR NON-FASTING PLASMA OR SERUM

• Use tube with gel separator to avoid exchange of cholesterol with RBC membranes if not refrigerated at 4°C

• Two weeks prior to testing: Patient should be in their usual diet and neither losing nor gaining weight.

CHEMICAL METHODS TO MEASURE CHOLESTEROL

PRINCIPLE: Dehydration and oxidation of cholesterol to form a colored compound (COLORIMETRY)

1. Salkowski Reaction End Product: Cholestadienyl Disulfonic Acid (RED)

2. Liebermann-Burchardt End Product: Cholestadienyl Monosulfonic Acid (GREEN)

Color Developer:

1. Glacial Acetic Acid

2. Acetic Anhydride

3. Concentrated Sulfuric Acid

GENERAL METHODS TO MEASURE CHOLESTEROL

1. ONE STEP: Pearson, Stern & Mac Gavack 

COLORIMETRY

2. TWO STEP METHOD: Bloors

EXTRACTION + COLORIMETRY

Cholesterol is extracted using an alcohol ether mixture

Measured using Liebermann-Burchardt

3. THREE STEP METHOD: Abell – Kendal

SAPONIFICATION + EXTRACTION + COLORIMETRY

Cholesterol saponified/hydrolyzed with alcoholic KOH

Extracted with petroleum jelly

Measured with Liebermann-Burchardt

4. FOUR STEP METHOD: Shoenheimer Sperry, Parekh and Jung

SAPONIFICATION + EXTRACTION + COLORIMETRY + PRECIPITATION

Precautions:

• Avoid hemolyzed blood – false increase total cholesterol

• Avoid ecteric specimens – 5 mg% to 6 mg% increase in cholesterol/mg of bilirubin above normal; bilirubin can interfere with total cholesterol measurement because of its own spectral properties; bilirubin absorbs light at 500nm.

• Avoid water contamination

• Precise and accurate timing for color development must be observed.

ENZYMATIC METHOD TO MEASURE CHOLESTEROL

• Most common method of quantifying the cholesterol oxidase reaction is to measure the amount of hydrogen peroxide produced

• If the cholesteryl ester hydrolase step is omitted, it can be used to measure unesterified cholesterol

Cholesterol ester + H2O –cholesterol esterase-- 🡪 Cholesterol + Fatty Acids

Cholesterol + O2 –cholesterol oxidase-- 🡪 cholest-4-en-3-one + H2O2

H2O2 + phenol + 4-aminoantipyrine –peroxidase-- 🡪 Quinoneimine dye

REFERENCE METHOD TO MEASURE CHOLESTEROL

NCEP Guideline Recommendation for Adults in terms of Risk for CHD:

DESIRABLE: <200 mg/dL (<5.8 mmol/L)

BORDERLINE HIGH: 200 – 239 mg/dL (5.18 – 6.19 mmol/L)

HIGH CHOLESTEROL: >240 mg/dL (>6.72 mmol/L)

ASSESSMENT OF RISK ACCORDING TO AGE GROUP

INCREASED CHOLESTEROL

1. Hyperlipoproteinemia types II, III, V

2. Biliary cirrhosis

3. Nephrotic syndrome

4. Poorly controlled diabetes mellitus

5. Alcoholism

6. Primary hypothyroidism

DECREASED CHOLESTEROL

1. Severe hepatocellular disease

2. Malnutrition

3. Severe burns

4. Hyperthyroidism

5. Malabsorption syndrome

TRIGLYCERIDES / TRIACYLGLYCEROL (Neutral Fat)

• Main storage lipid in man

• Resynthesized in the intestinal epithelial cells after absorption then combines with cholesterol and Apo-B48 to form chylomicrons. 

• Function: When Triglycerides are metabolized, their fatty are released into cells and converted into energy – provides excellent insulation.

• Contains 3 molecules of fatty acids and one molecule of glycerol.

• The breakdown is facilitated by lipoprotein lipase (LP), epinephrine and cortisol

• Fasting TAG ≥ 200 mg/dL are at risk for coronary artery disease because of atherogenic VLDL remnants

• TAG and Cholesterol are the most important lipids in the management of CAD

• Laboratory measurement: Based on hydrolysis of fatty acids to produce glycerol

• Specimen: FASTING for 12 – 14 hours, plasma or serum

• Interference: Ascorbic acid, Bilirubin and Hemolysis

<150 mg/dL TG: Normal/ Desirable/ Optimal

<200 mg/dL TG: Clear serum

>300 mg/dL TG: Turbid serum

>400 mg/dL TG: Lactescent serum

>600 mg/dL TG: Opaque or milky serum

CDC TRIGLYCERIDE LEVELS:

NORMAL: <150 mg/dL

BORDERLINE HIGE 150-199 mg/dL

HIGH 200-499 mg/dL

VERY HIGH ≥500 mg/dL

CHEMICAL METHODS IN MEASURING TRIGLYCERIDES

ENZYMATIC METHOD TO MEASURE TRIGLYCERIDES

• Specific, rapid and easy to use

• Major interference: Glycerol (corrected by using blank assay – without addition of lipase)

REFERENCE METHOD TO MEASURE TRIGLYCERIDES

MODIFIED VAN HANDEL & ZILVERSMITH METHOD (COLORIMETRIC)

• Time consuming manual method

• Involves alkaline hydrolysis 9saponification) with alcohol KOH, solvent extraction with chloroform and treatment with silicic acid (chromatography) to remove phospholipids and isolate Triglycerides

Glycerol + Sodium periodate 🡪 Formaldehyde (HCHO) + Formic Acid

HCHO + Chromotropic Acid 🡪 (+) PINK colored compound

NIST Method for Triglyceride: GC-IDMS

CURRENT REFERENCE METHOD: GC-MS

ELEVATED TRIGLYCERIDES LEVEL

• Fredrickson Type I, IIb, IV, V

• Hyperlipoproteinemias

• Pancreatitis

• Alcholism

• Obesity

• Hypothyroidism

• Nephrotic Syndrome

• Storage Disease (Gaucher, Niemann-Pick)

DECREASED TRIGLYCERIDES LEVEL

• Malabsorption Syndrome

• Hyperthyroidism

• Malnutrition burns

• Brain infarction

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LIPOPROTEINS

• Spherical lipid and protein complex, liquid core (TG and CE) and an outer shell of phospholipids, protein, and free cholesterol

• Transports lipids throughout the body

• Attached with apolipoproteins

Three main functions of apolipoproteins:

• Activate enzymes to aid in lipid metabolism

• Maintain structural integrity of lipoprotein molecule

• Enhance cellular uptake of lipoproteins

• Used in the assessment of atherosclerosis and CAD

• Lipoprotein metabolism/lipolytic enzymes

1. Lipoprotein Lipase - hydrolyzes TG to glycerol, monoglycerol, and FA which are rapidly removed by the liver

2. Hepatic lipase - hydrolyzes TAG and phospholipids from HDL, and lipids in VLDL and IDL 

3. Lecithin cholesterol acyltransferase (LCAT) - converts free cholesterol to CE

4. Endothelial lipase - hydrolyzes HDL for the release of TG and phospholipids

5. ATP-binding cassette protein A1 (ABCA1) - for efflux of cholesterol from peripheral tissues into HDL

LIPID METABOLISM:

• Dietary or exogenous pathway of lipid transport:

• Absorption of cholesterol and triglycerides through the intestine with the formation of chylomicrons (CM) into the lymph (chyle) and into the blood by way of the thoracic duct

• LPL liberates fatty acids from TAG, reducing the size of CM to become CM remnants which are taken up by the liver

• Free fatty acids are taken up by the muscles and adipose tissue

• Endogenous pathway

• Production of TAG from free fatty acids by the liver with the synthesis of VLDL 

• VLDL particles are converted to IDL that can either be removed by the liver by apo-E or converted to cholesterol-rich LDL

• LDL can be taken up by the liver or into other tissues for steroid or cell membrane synthesis

• Reverse Cholesterol Transport pathway 

• HDL particles mobilize Ch from tissues and reintroduces it into the circulation for exchange with VLDL 

REMEMBER: DENSITY DICTATES THE NAME OF THESE LIPOPROTEINS

Summary of Major Lipoprotein Comparison 

Comparison of Major Lipoproteins Based on Content

Abbreviations: HDL, high-density lipoprotein: IDL, intermediate-density lipoprotein; LCAT, lecithin-cholesterol acyltransferase: LDL, low-density lipoprotein; Lp(a), lipoprotein A: LPL, lipoprotein lipase; VLDL: very low-density lipoprotein.

• Apo A-1 is the major protein found in HDL. It activates lecithin-cholesterol acyltransferase (LCAT) and removes free cholesterol from extrahepatic tissues. Thus, it is considered antiatherogenic.

• Apo B-100 is the major protein found in LDL. It is associated with increased risk of coronary artery disease.

•  Lp(a) is an independent risk factor associated with impaired plasminogen activation and thus decreased fibrinolysis. A high level suggests increased risk for coronary heart disease and stroke.

Test Methodology

Apo-A, Apo-B, and Lp(a) are measured by Immunochemical methods such as Immunoturbidimetric and immunonephelometric.

LABORATORY METHODS FOR LIPOPROTEINS: 

Specimen: EDTA PLASMA (TRADITIONAL SPECIMEN OF CHOICE

PREFERRED SPECIMEN: Serum collected in a serum separator vacuum tube with clotting enhancer

12-14 hrs fasting

1. ULTRACENTRIFUGATION

• Sample is adjusted to density of 1.063 with Potassium Bromide and centrifuged at high speed for 24 hours

• Sample separates based on density

• REFERENCE METHOD

• Based on the protein and TG content of lipoproteins

Order from most to least dense: HDL, LDL, VLDL, Chylomicrons

2. ELECTROPHORESIS

• Separation based on size and charge

• Lipid stains: Oil red O, Sudan black B and Sudan III, Sudan IV, Fat Red B

Order from fastest/most anodic: Chylomicrons, LDL, VLDL, HDL

STANDING PLASMA TEST

• An aliquot of plasma (2 mL) is placed into a 10 x 75-mm test tube and allowed to stand in the refrigerator at 4° C undisturbed overnight. Chylomicrons accumulate as a floating "cream" layer and can be detected visually. The presence of chylomicrons in fasting plasma is considered to be abnormal. A plasma sample that remains turbid after standing overnight contains excessive amounts of VLDL; if a floating "cream" layer also forms, chylomicrons are present as well. (Henry's)

HDL MEASUREMENT

1. Polyanion precipitation (3-step chemical precipitation)

• Apo B containing lipoproteins (CM, VLDL, LDL, IDL) are precipitated out using a polyanion (heparin sulfate, dextran sulfate, or phosphotungstate) and divalent cation (Mg, Ca, or Mn) solutions

• Reagent: Dextran sulfate-magnesium chloride or heparin sulfate-manganese chloride

• HDL is then quantitated in the supernatant by cholesterol oxidase and cholesterol esterase

• Cannot be automated

2. Homogenous assay

• Uses an antibody to Apo B-100 to bind LDL and VLDL so that they will not react

• HDL is then measured enzymatically 

• Principle used in automated measurements

LDL MEASUREMENT

NOTE:

• Errors in LDLc become NOTICEABLE at TAG levels >200 mg/dL or 2.26 mmol/L

• LDLc becomes UNACCEPTABLY LARGE at TAG levels >400 mg/dL or 4.52 mmol/L

1. Friedewald equation:

• LDLc = Total cholesterol - (HDLC + VLDL)

• VLDL = TG ÷ 5 (mg/dL)

• VLDL = TG ÷ 2.175 (mmol/L

• Not reliable when TG >400 mg/dL or for patients with B-VLDL

2. DeLong Equation

• More accurate than Friedewald when TG >400 mg/dL

• LDLC = TC - (HDLC + VLDL)

• VLDL = TG ÷ 6.5 (mg/dL)

• VLDL = TG ÷ 2. 825 (mmol/L)

3. Beta Quantification – Reference method

• Uses ultracentrifugation (at least 18 hours at 105 K x g) to separate VLDL and CM

• Remaining solution is measured for cholesterol

• LDL is precipitated out and the solution is again measured for cholesterol. 

• The difference between the 2 measurements is the concentration of LDL

4. Homogenous Direct LDL-c Method

• Useful when TG is elevated (>600 mg/dL)

• Use a combination of two reagents.

• First reagent: selectively removes non-LDL and/or inhibits it from reacting with enzymes

• Second reagent: releases cholesterol from LDL to be measured enzymatically

5. Gel chromatography or Affinity chromatography

6. Immunochemical methods

7. Immunoassay or Immunonephelometry

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DISEASES ASSOCIATED WITH LIPIDS AND LIPOPROTEINS

NCEP

• National Cholesterol Education Program (USA)

• Provides evidence-based guidelines for cholesterol testing and management, and provides detailed information on other topics, including the classification of lipids and lipoprotein particles, CHD risk assessment, lifestyle intervention, drug treatment, specific dyslipidemias, and treatment adherence issues.

ATP (Adult treatment panel) Ill

• Recognizes additional positive risk factors for CHD, including elevations in Lp(a). remnant lipoproteins, small LDL particles, fibrinogen, homocysteine, high-sensitivity C-reactive protein (hs-CRP), impaired fasting plasma glucose (110-125 mg/dL) and preexisting subclinical atherosclerosis (as evidenced by myocardial ischemia on exercise testing, carotid intimal-medial thickening, and/or coronary artery calcium deposition). (Henry's)

DYSLIPIDEMIAS

• Can be subdivided into two major categories: 

• HYPERLIPOPROTEINEMIAS, which are diseases associated with elevated lipoprotein levels, and;

• HYPOLIPOPROTEINEMIAS, which are associated with decreased lipoprotein levels.

HYPERLIPOPROTEINEMIAS

• Have been classified using the Fredrickson-Levy classification system, which is not commonly used today. 

• The hyperlipoproteinemias can be subdivided into hypercholesterolemia, hypertriglyceridemia, and combined hyperlipidemia, with elevations of both cholesterol and triglycerides.

FREDRICKSON CLASSIFICATION OF HYPERLIPIDEMIAS

Type I hyperlipoproteinemia: Elevated chylomicrons

1. Serum appearance: Creamy layer of chylomicrons over clear serum

2. Total cholesterol: Normal to moderately elevated

3. Triglyceride: Extremely elevated

4. ApoB-48 Increased, ApoA-IV increased

Type lla hyperlipoproteinemia: Increased LDL

1. Serum appearance: Clear

2. Total cholesterol: Generally elevated

3. Triglyceride: Normal

4. Apo-B 100: increased

Type lIb hyperlipoproteinemia: Increased LDL and VLDL

1. Serum appearance: Clear or slightly turbid

2. Total cholesterol: Elevated

3. Triglyceride: Elevated

4. Apo B-100 increased

Type III hyperlipoproteinemia: Increased IDL

1. Serum appearance: Creamy layer sometimes present over a turbid layer

2. Total cholesterol: Elevated

3. Triglyceride: Elevated

4. Apo E-II increased, Apo E-Ill decreased, and Apo E-IV decreased

Type IV hyperlipoproteinemia: Increased VLDL

1. Serum appearance: Turbid

2. Total cholesterol: Normal to slightly elevated

3. Triglyceride: Moderately la severely elevated

4. ApoC-ll either increased or decreased, and ApoB-100 increased

Type V hyperlipoproteinemia: Increased VLDL with increased chylomicrons

1. Serum appearance, Turbid with creamy layer

2. Total cholesterol Slightly to moderately elevated

3. Triglyceride: Severely elevated

4. Apo C-ll increased or decreased, Apo B-48 increased, and Apo B-100 increased

The most common familial form is familial combined hyperlipidemia (FCHL). 

FCHL is characterized by increased plasma levels of total and LDL cholesterol (type lla), or triglyceride (type IV), or a combination of both (type IIb). Also, apo B-100 is increased. The level of HDL cholesterol may be decreased.

Hyperapobetalipoproteinemia is associated with VLDL and apo B-100 overproduction in the liver. It is characterized by normal or moderate elevation of LDL cholesterol with an elevated apo B-100. Total cholesterol and triglyceride are generally elevated but may be normal. HDL cholesterol and apo A-l levels are decreased.

Secondary lipoproteinemia: Many conditions cause lipoproteins to be abnormally metabolized. Some of those conditions include diabetes mellitus, hypothyroidism, obesity, pregnancy, nephrotic syndrome, pancreatitis, alcoholism. and myxedema.

HYPOLIPOPROTEINEMIAS

1. Abetallpoproteinemia: (also known as Bassen-Kornzwelg syndrome) Total cholesterol level very low, triglyceride level nearly undetectable, LDL and Apo B-100 absent

2. Hypobetalipoproteinemia: Unable to synthesize apo B-100 and apo B-48, low total cholesterol level and normal to low triglyceride level

3. Hypoalphalipoproteinemia: Severely elevated triglyceride level and low HDL level

4. Tangier disease: HDL absent, apo A-1 and apo A-ll very low levels, LDL low, total cholesterol level low, triglyceride level normal to slightly increased. Due to a mutation in the ABCA1 gene on chromosome 9.

Arteriosclerosis - hardening and narrowing of arteries

Atherosclerosis - narrowing of arterial due to plaque build-up on arterial walls due to deposition of cholesterol and TG

• Coronary Artery Disease → heart

• Peripheral vascular disease → arms/legs

• Cerebrovascular disease→ brain (stroke)

Sitosterolemia - is an extremely rare autosomal recessive disorder wherein phytosterols (plant sterols) are absorbed and accumulate in plasma and peripheral tissues.

CETP deficiency - is an autosomal recessive disorder in which the transfer of cholesterol esters is inhibited. As a result, HDL particles are large and laden with cholesterol ester, and apoA-I is increased, as is HDL-C (typically >100 mg/dL).

Chylomicron retention disease (Anderson's disease) - presents in childhood with fat malabsorption and low levels of plasma lipids

METABOLIC SYNDROME:

Definition

• Group of risk factors that seem to promote development of atherosclerotic cardiovascular disease & type 2 diabetes mellitus.

Risk factors                                      

 ↓ HDL-C

 ↑ LDL-C

 ↑ Triglycerides

 ↑ blood pressure

 ↑ blood glucose