Lipids and Lipoproteins

• organic substances that are insoluble in water but soluble in organic substances

• “like dissolves like”

• ether or chloroform > organic solvents

• lipids cannot be directly distributed > use lipoproteins to transport lipids

Lipids

• Lipids are transported in the blood by major lipoprotein which is (4)

• ULTRACENTRIFUGATION - categorized by density

• Chylomicrons

• VLDL

• LDL

• HDL

• Rich source of energy and efficient way to store excess calories > fats

• provide stability to the cell membrane > phospholipids

• also a precursor of steroid hormones > estrogen testosterone

Lipids

Forms of Lipids (5)

• arrange according to abundance

• phospholipid most abundant and so on

• Phospholipids

• Cholesterol

• Triglycerides

• Fatty acids

• Fat-Soluble Vitamins - A,D,E,K

PHOSPHOLIPIDS

• also known as -

conjugated lipids

PHOSPHOLIPIDS

• most abundant form of lipids that originates in the - and -

• liver

• intestine

PHOSPHOLIPIDS

• With similar structure as - except that two fatty acids and a phosphate group is attached to -

• triglycerides

• glycerol backbone

*triglycerides have 3 fatty acids and does not have phosphate

PHOSPHOLIPIDS

• reference value

150 to 380 mg/dL

PHOSPHOLIPIDS

• Functions of Phospholipids

→decreases surface tension within the alveolar space, allowing effective gas exchange and prevent alveolar collapse during expiration

Surfactant

PHOSPHOLIPIDS

• Functions of Phospholipids

→participate in - and -

• cellular metabolism and blood coagulation

PHOSPHOLIPIDS

• Functions of Phospholipids

→important substrates for lipoprotein-metabolizing enzymes like (2)

• LCAT > Lecithin cholesterol acyltransferase

• LPL > Lipoprotein lipase

PHOSPHOLIPIDS

• Functions of Phospholipids

→ Deficiency of surfactant lead to -

neonatal respiratory distress syndrome

PHOSPHOLIPIDS

• Forms of Phospholipids (3)

• Lecithin / Phosphatidylcholine - 70%

• Sphingomyelin - 20%

• Cephalin - 10%

PHOSPHOLIPIDS

• Forms of Phospholipids

→is the only phospholipid in membranes that is not derived from glycerol but from an amino alcohol

Sphingomyelin

PHOSPHOLIPIDS

• Forms of Phospholipids

→amino alcohol from which sphingosine is derived

sphingosine

PHOSPHOLIPIDS

• Forms of Phospholipids

→Sphingomyelin accumulates in the liver and spleen of patients with _ (lipid storage disorder)

• Niemann-Pick Disease

PHOSPHOLIPIDS

• Forms of Phospholipids

→Cephalin - 10% (3)

• Phosphatidylethanolamine

• Phosphatidylserine

• Lysolecithin + inositol phosphatide

PHOSPHOLIPID

• FETAL LUNG MATURATION

→ estimated from the evaluation of pulmonary surfactant in amniotic fluid

Lecithin/Sphingomyelin (L/S) Ratio

PHOSPHOLIPID

• FETAL LUNG MATURATION

→Method for estimating Lecithin/Sphingomyelin (L/S) Ratio

• Thin Layer Chromatography (TLC) followed by densitometric quantitation

PHOSPHOLIPID

• Phospholipids can be measured in disorders characterized by - and -

altered phospholipids cmposition and lipoprotein distribution

CHOLESTEROL

• also known as -

• 3-hydroxy-5,6-cholestene

CHOLESTEROL

• synthesized in the - and is found on the surface layer of -

• liver

• lipoproteins

CHOLESTEROL

• not catabolized by most cells, not a source of -

fuel

CHOLESTEROL

• transport and excretion is promoted by _

estrogen

CHOLESTEROL

• reference value

<200 mg/dL (desirable)

CHOLESTEROL

• Interpretation of Result

• 200 to 239 mg/dL (borderline)

• ≥ 240 mg/dL (high cholesterol)

CHOLESTEROL

• Functions of cholesterol

→ precursor of major steroids (4)

• glucocorticoids

• mineralocorticoids

• androgens

• estrogens

CHOLESTEROL

• Functions of cholesterol

→important constituent in the assembly of - and -

• cell membranes

• bile acids

CHOLESTEROL

• Functions of cholesterol

→cholesterol after being converted to - can also be transformed to - in the skin by irradiation from sunlight

• 7-dehydrocholesterol

• vitamin D3

CHOLESTEROL

• Diagnostic significance

→evaluates the risk of (3)

• atherosclerosis

• myocardial infarction

• coronary arterial occlusions

CHOLESTEROL

• Diagnostic significance

→used as (3) function tests and for - studies

• thyroid, liver, renal function tests

• DM studies

CHOLESTEROL

• Forms of Cholesterol (2)

• Cholesterol Ester (70%)

• Unesterified/Free Cholesterol (30%)

CHOLESTEROL

• Forms of Cholesterol

→found in plasma and serum

→bound to fatty acid

→inactive form with protective property and stored in cells

Cholesterol Ester (70%)

CHOLESTEROL

• Forms of Cholesterol

→found in plasma, serum, and RBCs

→active form of cholesterol with cytotoxic property

Unsterified/Free Cholesterol (30%)

CHOLESTEROL

• is a detoxification step to reduce the accumulation of free cholesterol

Esterification

CHOLESTEROL

• Esterification is a detoxification step to reduce the accumulation of free cholesterol through _

acyltransferases

CHOLESTEROL

• METHODS OF CHOLESTEROL DETERMINATION

→_ is measured rather that its forms

Total cholesterol

CHOLESTEROL

• METHODS OF CHOLESTEROL DETERMINATION

→TC can be assayed using _ samples

non-fasting

CHOLESTEROL

• METHODS OF CHOLESTEROL DETERMINATION

→It increases with - , with women having lower values than men before age -

• age

• 45

CHOLESTEROL

• METHODS OF CHOLESTEROL DETERMINATION

→Serum TC increases at - between 45 to 65 years ols

2 mg/dL/year

CHOLESTEROL

• METHODS OF CHOLESTEROL DETERMINATION

→Decreased levels of _ among menopausal women contribute to increase of serum cholesterol

estrogen

CHOLESTEROL

• METHODS OF CHOLESTEROL DETERMINATION

→specimen:

plasma/serum

CHOLESTEROL

• METHODS OF CHOLESTEROL DETERMINATION (2)

• chemical method

• enzymatic method

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→ Principle:

Dehydration and oxidation of cholesterol to form a colored compound

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→General methods (4)

• one-step

• two-step

• three-step

• four-step

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→General methods > ONE-STEP

• colorimetry > Pearson, Stern, & Mac Gavack

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→General methods > TWO-STEP

• extract + colorimetry > Bloors

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→General methods > THREE-STEP

• saponification + extraction + colorimetry > Abell-Kendall

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→General methods > FOUR-STEP

• saponification + extraction + colorimetry + precipitation > Schoenheimer, Sperry, Parekh, and Jung

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→Reagents for the steps: (4)

• Liebermann-Burchardt > colorimetry

• Petroleum ether > saponification

• Alcoholic potassium hydroxide > saponification

• Digitonin > precipitation

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→Color Developer Mixture (3)

• Glacial acetic acid

• acetic anhydride

• Concentrated sulfuric acid

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→Precautions

1. Avoid hemolysis > may falsely - total cholesterol

2. Avoid icteric specimen > causes - to - mg increase in cholesterol/ mg bilirubin above normal

3. Avoid - contamination

4. Precise and accurate timing for - must be observed

1. Avoid hemolysis > may falsely INCREASE total cholesterol

2. Avoid icteric specimen > causes 5 to 6 mg increase in cholesterol/ mg bilirubin above normal

3. Avoid WATER contamination

4. Precise and accurate timing for COLOR DEVELOPMENT must be observed

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→ ABELL-KENDALL (3 steps)

1. Saponification

2. Extraction

3. Colorimetry

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→ABELL-KENDALL

Step 1: Saponification

Reagent

Purpose

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→ABELL-KENDALL

Step 1: Saponification

Reagent > Alcoholic KOH

Purpose > Convert cholesterol esters into free cholesterol and fatty acids

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→ABELL-KENDALL

Step 1: Extraction

Reagent

Purpose

Reagent > Petroleum Ether

Purpose > Separate cholesterol from protein carriers

CHOLESTEROL

• Methods of cholesterol determination - CHEMICAL METHOD

→ABELL-KENDALL

Step 1: Colorimetry

Reagent

Purpose

Reagent > Acetic anhydride

Purpose > Liebermann-Burchardt reaction - colorimetric step, meausres GREEN COLOR absorbance at 410 nm

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→it measures total cholesterol in -

serum/plasma

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→rapid, uses - quantities of sample

→does not require -

• microliter quantities

• preliminary extraction

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→elevated levels of - can lead to low Total Cholesterol

• ascorbic acid

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→Chromogen:

Phenol + 4-aminoantipyrine

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→ Hemoglobin has a - activity that can diminish the hydrogen peroxide produced in the reaction

pseudo-peroxidase

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→Bilirubin exceeding 5 mg/dL decreases total cholesterol by -

5% to 15%

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→3 steps

1. Cholesterol esterase

2. Cholesterol oxidase

3. Peroxidase

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→Step 1: Cholesterol esterase

cholesterol esters →free cholesterol + fatty acids

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→Step 2: Cholesterol oxidase

→reacts only to free cholesterol

→free cholesterol + O2 → cholest-4-en-3-one + H2O2

CHOLESTEROL

• Methods of cholesterol determination - ENZYMATIC METHOD

→Step 3: Peroxidase

H2)2 + phenol + 4-aminoantipyrine → quinoneimine dye + H2O

CHOLESTEROL

• CDC REFERENCE METHOD (2)

• Abell, Levy, and Brodie Method

• Isotope Dilution Mass Spectrometry (IDMS)

CHOLESTEROL

• CDC REFERENCE METHOD - ABELL, LEVY, and BRODIE METHOD

→uses hexane extraction after hydrolysis with alcoholic - followed by reaction with - color reagent

→end color:

→uses hexane extraction after hydrolysis with alcoholic KOH followed by reaction with LIEBERMANN-BURCHARDT color reagent

→end color: GREEN

CHOLESTEROL

• CDC REFERENCE METHOD - ISOTOPE DILUTION MASS SPECTROMETRY (IDMS)

→- order method for serum cholesterol developed by NIST

→- method for cholesterol

• highest

• gold standard

CHOLESTEROL

• INCREASED (6)

• Hyperlipoproteinemia (Types II, III, V)

• Biliary cirrhosis

• Nephrotic syndrome

• Poorly controlled DM

• Alcoholism

• Primary hypothyroidism

CHOLESTEROL

• DECREASED (5)

• Severe hepatocellular disease

• Malnutrition

• Severe burns

• Hyperthyroidism

• Malabsorption syndrome

TRIGLYCERIDES

• also known as -

triacylglycerol / neutral fats

TRIGLYCERIDES

• formed from one - molecule with three - attached via - bonds

• glycerol

• fatty acids

• ester bond

TRIGLYCERIDES

• main storage form of - in man > adipose tissue

• constitutes - % of stored fat

• lipids

• 95%

TRIGLYCERIDES

• when they are metabolized, their _ are released to the cells and converted into energy

fatty acids

TRIGLYCERIDES

• their breakdown is facilitated by (4)

• lipase

• lipoprotein lipase

• epinephrine

• cortisol

TRIGLYCERIDES

• an average person ingests, absorbs, resynthesizes and transports - grams of fat daily

60 to 130 grams

TRIGLYCERIDES

• FASTING:

10 to 12 hours

TRIGLYCERIDES

• Reference Range:

<150 mg/dL

TRIGLYCERIDES

• Interpretation (3)

• 150 to 199 mg/dL > Borderline high

• 200 to 499 mg/dL > High TAG

• >500 mg/dL > Very high TAG

TRIGLYCERIDES

• Diagnostic significance

→evaluates suspected - and measures the body's bility to -

• atherosclerosis

• metabolize fat

TRIGLYCERIDES

• Diagnostic significance

→FASTING TAG of _ > at risk for coronary artery disease

≥ 200 mg/dL

TRIGLYCERIDES

• Diagnostic significance

→ are the most important lipids for the managemnet of CAD (2)

• TAG

• Cholesterol

TRIGLYCERIDES

• METHODS

→ Specimen:

• Plasma or serum

TRIGLYCERIDES

• METHODS

→Interferences (3)

• ascorbic acid

• bilirubin

• hemolysis

TRIGLYCERIDES

• METHODS

→TAG level increases to - between 45 and 65 years old

2 mg/dL/year

TRIGLYCERIDES

• METHODS

→if processing will be delayed and lipemic serum will be frozeb, the specimen should be - and - before test

• warm thoroughly and mixed

TRIGLYCERIDES

• METHODS (2)

• Chemical Methods

1. Colorimetric > Van Handel & Silversmith

2. Fluorometric > Hantzsch Condensation Method

• Enzymatic Method

1. Glycerol Kinase Method

TRIGLYCERIDES

• CHEMICAL METHOD - Van Handel & Silversmith

>4 Steps

1. Extraction Phase

2. Saponification Phase

3. Oxidation Pahse

4. Colorimetry

TRIGLYCERIDES

• CHEMICAL METHOD - Van Handel & Silversmith

→ Step 1: Extraction Phase

Reagent:

Purpose:

TRIGLYCERIDES

• CHEMICAL METHOD - Van Handel & Silversmith

→ Step 1: Extraction Phase

Reagent: CHLOROFORM

Purpose: Separates TAG from protein carrier

TRIGLYCERIDES

• CHEMICAL METHOD - Van Handel & Silversmith

→ Step 2: Saponification Phase

Reagent:

Purpose:

TRIGLYCERIDES

• CHEMICAL METHOD - Van Handel & Silversmith

→ Step 2: Saponification Phase

Reagent: Alcoholic KOH

Purpose: Hydrolysis; divides TAG to fatty acids and glycerol

TRIGLYCERIDES

• CHEMICAL METHOD - Van Handel & Silversmith

→ Step 3: Oxidation Phase

Reagent:

Purpose:

TRIGLYCERIDES

• CHEMICAL METHOD - Van Handel & Silversmith

→ Step 3: Oxidation Phase

Reagent: Sodium Periodate

Purpose: Converts glycerol to something that can be measured

TRIGLYCERIDES

• CHEMICAL METHOD - Van Handel & Silversmith

→ Step 4: Colorimetry

Reagent:

Purpose:

TRIGLYCERIDES

• CHEMICAL METHOD - Van Handel & Silversmith

→ Step 4: Colorimetry

Reagent: Color reagent

Purpose: Formation of pink color at 410 nm

TRIGLYCERIDES

• ENZYMATIC METHOD

→ ALL 3 reactions has initial reaction that uses - and -

lipase & glycerokinase

• TAG is converted by lipase into glycerol + fatty acids

• Glycerol + ATP is converted by glycerokinase into glycerophosphate + ADP

TRIGLYCERIDES

• ENZYMATIC METHOD (Reaction 1)

→ Initial reaction +

• Glycerophosphate + O2 is converted by GLYCERPHOSPHATE OXIDASE into dihydroxyacetone + H2O2

• Two H2O2 + Phenol + 4-aminoantipyrine is converted by PEROXIDASE into quinoneimine dye + four H2O

TRIGLYCERIDES

• ENZYMATIC METHOD (Reaction 2)

→Initial reaction +

• Glycerophosphate + NAD is converted by GLYCEROPHOSPHATE DEHYDROGENASE into dihydroxyacetone phosphate + NADH + H4

• NADH + tetrazolium dye is converted by DIAPHORASE into formazan + NAD

TRIGLYCERIDES

• ENZYMATIC METHOD (Reaction 3)

→Initial reaction + -

• ADP + Phosphoenolpyruvate is converted by PYRUVATE KINASE into ATP and pyruvate

• Pyruvate +NADH + H is converetd by LACTATE DEHUYDROGENASE into Lactate = NAD+

TRIGLYCERIDES

• Clinical Significance

→Decreased (5)

• Malabsorption syndrome

• Hyperthyroidism

• Malnutrition

• Burns

• Brain Infarction

FATTY ACIDS

• major constituents of - and -

• triglycerides

• phospholipids

FATTY ACIDS

• mainly derived from hydrolysis of - in adipose tissues

TAG