LIPIDS

Major Classes of Plasma Lipids

Free fatty acids (FFA / UFA / NEFA)

 Metabolic fuels; building blocks for other
lipids
Triglycerides (TAG)
 Major storage form of the body; metabolic
intermediates and regulation
Cholesterol
 Membrane stability and mother compound
of bile acids and steroid hormones
Phospholipids (PL)
 Membrane structure; membrane signal
transduction; pulmonary surfactant
Lipid Storage Diseases (Lipidoses)

a group of inherited disorders

characterized by the
accumulation of lipids in tissues
especially the brain
due to
deficiency in a particular
sphingolipid catabolic enzyme
Niemann-Pick disease
 Deficiency in sphingomyelinase and
accumulation of sphingomyelin

Gaucher’s disease
 Deficiency in -D-glucosidase and
accumulation of glucocerebroside
Krabbe’s disease
 Deficiency in -D-galactosidase and
accumulation of galactocerebsides

Fabry’s disease
 Deficiency in -D-galactosidase and
accumulation of ceramide trihexoside
Tay-Sach’s disease
 Deficiency in -D-hexaminidase A and
accumulation of ganglioside GM2

Metachromatic Leukodystrophy
 Deficiency in sulfatide sulfatase and
accumulation of -sulfogalactocerebroside
 Quick Quiz
 Bile acids that are synthesized in the liver
are derived from what substance?
a. bilirubin b. fatty acids
c. cholesterol d. triglycerides

 Which of the following is most associated

with nerve tissues?
a. cholesterol b. TAG
c. phospholipids d. sphingolipids
 Which of the following lipid tests is least
affected by the fasting status of the
patient?
a. cholesterol b. triglycerides
c. fatty acids d. lipoproteins

 Lipid storage disease are inherited disorders

due to mutations. What is the cause of these
diseases?
a. excessive ingestion of fat
b. excessive synthesis of chylomicrons
c. specific enzyme deficiency
d. inability of adipocytes to store fat
 Human Lipases
PANCREATIC LIPASE
 Found in pancreatic juice for the digestion of dietary
TAG

HORMONE-SENSITIVE LIPASE
 Found in adipocytes for fat mobilization

ACID LIPASE
 Found in lysosomes for intracellular catabolism of
lipoproteins

LIPOPROTEIN LIPASE (LPL)

 Found in capillaries for utilization of TAG in
lipoproteins

HEPATIC LIPASE
 Found in the liver for lipoprotein catabolism
 Esterification of Cholesterol

Mediated by Lecithin: Cholesterol

Acyltransferase (LCAT)
= Occurs in the plasma

Mediated by AcylCoA: Cholesterol

Acyltransferase (ACAT)
= Occurs in the peripheral tissues
including intestinal mucosa
 Lipoproteins
 The transport proteins of lipids having a
micellar structure composed of two parts as
follows:
Central core
 containsTAG and cholesteryl ester
 nonpolar moiety

Surface coat
 contains PL, free cholesterol, and
apolipoproteins
 polar moiety
 Quick Quiz
 Cholesteryl esters are formed via esterification
of the alcohol cholesterol with what substance?
a. protein b. triglyceride
c. fatty acids d. digitonin

 Which lipase is removed by allowing the blood

to clot so as to prevent interference in the
determination of serum TAG?
a. acid lipase b. hormone-sensitive lipase
c. hepatic lipase d. lipoprotein lipase
 Apolipoproteins
 according to Alaupovic nomenclature of 1971,
the apoproteins are:

Apo A: This is the major protein component

of HDL
 Apo A-I – major activator of the LCAT
 Apo A-II – activates hepatic lipase
 Apo A-IV – cofactor for LCAT
 Apo B: This is the major protein component
of LDL

 Apo B-100 – binds to cell receptor to

target LDL to LDL receptors or Apo B/E
receptors

 Apo B-48 – structural role in chylomicrons

 Apo C: This is the major protein component
of VLDL

 Apo C-I – may activate LCAT

 Apo C-II – potent activator of LPL
 Apo C-III – regulates rate of clearance of
TAG-rich lipoprotein remnants
(inhibit LPL)
Apo D: This is also called Apo A-III or ‘thin-
line apoprotein’. It activates LCAT
by serving as specific lysolecithin
carrier.

Apo E: This is rich in amino acid arginine.

Apo E recognizes cell receptors to
target chylomicrons and VLDL
remnants to the hepatic receptors.
 LIPOPROTEINS
Density Mean Electrophoretic Source Principal
LP (g/ml) diameter Mobility function
(mm)

<0.95 500 Remains at Intestine Transport of

CM exogenous
origin
TAG

VLDL 0.96 – 43 Pre- Liver Transport of

endogenous
1.006
TAG

IDL 1.007 – 27 ‘broad ’ Catabolism

of VLDL
Precursor of
LDL
1.019
LDL 1.020 – 22  Catabolism
of VLDL, via
Cholesterol
transport
1.063
IDL

HDL 1.064 – 8  Liver,

intestine;
Reverse
cholesterol
1.210
catabolism of transport
CM and VLDL
 Quick Quiz
 Exogenous TAG are transported in the
plasma in what form?
a. phospholipids b. cholesteryl esters
c. chylomicrons d. free fatty acids

 When lipoprotein agarose gel electrophoresis

is performed at pH 8.6, which fraction
migrates the fastest to the anode?
a. chylomicrons b. LDL
c. VLDL d. HDL
 The quantitation of HDL level is thought to be
significant in the risk assessment of what
disease?
a. pancreatitis b. cirrhosis
c. coronary heart disease d. hyperlipidemia

 The beta-lipoprotein consists primarily of what

lipid?
a. fatty acids b. cholesterol
c. triglycerides d. phospholipids
 Abnormal Lipoproteins

β – VLDL also known as floating beta- LP derived from

chylomicron and VLDL; rich in cholesterol thus a
serious risk factor for atherosclerosis

LP (a) is called sinking pre beta- LP; can cause coronary

heart disease when elevated

LP-X is seen in patients with obstructive jaundice; formed

within the bile canaliculi.

HDLc is also called HDL1; large HDL rich in cholesterol

and can cause atherosclerosis among animals
 Measurement of Lipids
Triglyceride Measurement
Nonenzymatic Methods
General Steps
1. Extraction
= to remove TAG from LPs
= accomplished by using MeOH, EtOH, isopropyl
alcohol, chloroform-ethanol (Folch’s rgt) or diethyl
ether
= removal of interferences by zeolite (Van Handel and
Zilversmit method)

2. Hydrolysis of TAG into FFA and Glycerol

= by saponification with alcoholic KOH at elevated
temperature or alkoxide transesterification

3. Measurement of Glycerol
Eegrine’s method,
Schryver’s method,
Pay’s method , and
Hantzsch method
 The glycerol liberated is oxidized by periodate to HCHO
(formaldehyde) and quantified using any of the following:
Eegrine reaction
= uses chromotropic acid and sulfuric acid to form a pink
product measured at 565 nm

Schryver’s reaction
= uses phenylhydrazine-ferriCN-HCl mixture to form a
product read at 540 nm

Pay’s reaction
= uses methylbenzothiazolinone and ferric chloride to
form a product measured at 620nm

Hantzsch reaction (method of choice)

= uses ammonium acetate and acetyl acetone to form
yellow diacetyl dihydrolutidine read at 410 nm.
 Enzymatic Methods of TAG Measurement
LPS
TAG ------------- 3 FFA + glycerol
glycerol kinase
Glycerol------------------ glycerol-3-PO4 + ADP
1.Baculo-David (NADH Consumption Method)
PK
ADP + PEP -------- ATP + pyruvate
LD
pyruvate + NADH + H+ --------- lactate + NAD+
2. Megraw (Formazan formation)
G-3-PD
glycerol-3-PO4 + NAD ---------- DHAP + NADH + H+
diaphorase
NADH + oxidized tetrazolium ------ reduced tetrazolium (formazan)
+ NAD +
 LPS
TAG ------------- 3 FFA + glycerol
glycerol kinase
Glycerol ----------- glycerol-3-PO4 + ADP
3. Winartasaputra method
G-3-PD
glycerol-3-PO4 + NAD ---------- DHAP + NADH + H+
diaphorase
NADH + resazurin ------------- reorufin (fluorescent) + NAD +

4. Nagele-Trinder method
G-3-POD
glycerol-3-PO4 + O2 ------------ DHAP + H2O2

H2O2 + 4-chlorophenol + peroxidase

4-aminophenazone -----------------------
+ pot. Hexacyanoferrate II 4-p-benzoquinonemonoiminophenazone
+ pot. Hexacyanoferrate III + HCl +
HOH
 Quick Quiz
 In the chemical methods of TAG measurement, the first
step is the
a. production of glycerol
b. hydrolysis of TAG into FFA and glycerol
c. hydrolysis of lipoproteins
d. hydrolysis of TAG with lipase

 The reaction needed before proceeding to the Hantzsch

reaction is the oxidative conversion by periodate of
glycerol to
a. hydrogen peroxide b. formaldehyde
c. ethanol d. glyceraldehyde
 Cholesterol Measurement
Nonenzymatic Methods
4 General Steps
1. Extraction
= using Bloor’s rgt (3:1 EtOH-ether) or zeolite extraction
2. Saponification
= using KOH
3. Purification
= precipitating interferents using digitonin
4. Color Development
Color Development Mixture
Glacial acetic acid
Acetic anhydride
Concentrated sulfuric acid
Leibermann-Burchardt reaction
= uses sulfuric acid and acetic anhydride
to produce unstable green cholestadienyl
monosulfonic acid; color stabilized by sodium
sulfate
Salkowski or Zak reaction
= uses sulfuric acid and ferric ions to
produce stable red to red-violet
cholestadienyl disulfonic acid
 CHOLESTEROL MEASUREMENT
One-step methods
Zlatkis-Zak-Boyle method
Ferro-Ham method
Pearson-Stern-MacGavak method
Wybenga et al method
Two-step method
Carr-Drekter method
Three-step method
Abell-Kendall method (standard reference
method)
Four-step methods
Schoenheimer-Sperry method
Parek-Jung method
Sperry-Webb method
 Enzymatic Method
CHOD-PAP method
Cholesteryl esterase
Cholesterol esters + HOH ----------- free cholesterol + FFA

CHOD
Cholesterol + oxygen ------- choles-4-ene-3-one + H2O2

Peroxidase
H2O2 + phenol + p-aminoantipyrine ----- quinone imine dye
+HOH
 Quick Quiz
 In the CHOD-PAP method, the cholesterol oxidase
reacts with
a. free cholesterol and cholesterol esters
b. free cholesterol and fatty acids
c. free cholesterol only
d. cholesterol esters only

 Which of the following tests would most likely be

included in a routine lipid profile?
a. TAG, FFA, chylomicrons
b. total cholesterol, TAG, phospholipids
c. HDL-chol, LDL-chol, chylomicrons
d. total cholesterol, TAG, HDL-chol
 Lipoprotein Measurement
1. HDL Measurement
Polyanion precipitation (Mn++, Mg++, or
dextran sulfate)
Electrophoresis – Spectrophotometric
determination
Ultracentrifugation (reference method)

2. Chylomicrons
Standing Plasma Test

3. Lipid Profile
use of the Friedewald equation
use of the De Long equation
 Quick Quiz
 A commonly used precipitating agent used to
separate HDL-cholesterol from other lipoprotein
fractions is
a. zinc sulfate b. trichloroacetic acid
c. heparin-manganese d. isopropanol

 Which of the following may be described as a

variant form of LDL associated with increased
risk of atherosclerotic cardiovascular disease?
a. Lp(a) b. HDL
c. Apo A-I d. Apo A-II
 FRIEDEWALD EQUATION
VLDL-Chol = TAG
(mg/dl) 5
VLDL-Chol = TAG
(mmol/L) 2.175

LDL-Chol = Total Chol - HDL-Chol - VLDL-Chol

 DE LONG EQUATION
VLDL-Chol = TAG
(mg/dl) 6.5
VLDL-Chol = TAG
(mmol/L) 2.825
LDL-Chol = Total Chol - HDL-Chol - VLDL-Chol
 Risk Factors of CHD
Positive
Age: Male 45 yrs and above, Females 55 yrs and
above or premature menopause without
estrogen therapy
Family history of premature CHD
Current cigarette smoking
Hypertension (equal or more than 140/90 mmHg or
on antihypertensive therapy)
Low HDL-Chol (<35 mg/dl)
Diabetes mellitus

Negative
High HDL-Cholesterol (equal to or above 60 mg/dl)
 Initial Classification of Adults based on TChol,
HDL-Chol, and LDL-Chol
mg/dl mmol/L
TChol desirable <200 <5.17
borderline 200-239 5.17-6.19
high ≥240 ≥6.20

HDL-Chol low <35 <0.91

LDL-Chol desirable <100 <2.59

borderline 130-159 3.36-4.11
high >160 >4.14

Conversion factor to convert mg/dl to mM is 0.02586

 Categories for TAG Levels
 By National Cholesterol Education Program (NCEP)
Normal <200 (mg/dl) <2.26 (mM)
Borderline High 200-400 2.26-4.52
High 400-1000 4.52-11.29
Very High >1000 >11.29

 By National Institutes of Health (NIH)

Normal <250 <2.82
Borderline 250-500 2.82-5.65
High >500 >5.65
Conversion factor to convert mg/dl to mM is 0.011
 Quick Quiz
 A patient’s total cholesterol/HDL-cholesterol ratio
is 10.0. What level of risk for coronary heart
disease does this result indicate?
a. no risk b. half average risk
c. average risk d. twice average risk

 Which of the following techniques can be used

to quantify apolipoproteins?
a. GC b. ISE
c. ELISA d. Refractometry
 Lipoprotein Phenotypes by
Fredrickson
Phenotype Frequency Chylomicrons Approx. Approx. Approx. Total Total Refrigerated
VLDL LDL HDL Cholesterol TAGS Serum or
Plasma
I Very rare     N or   “Cream”/
clear or
turbid
II a Common N N  N  N Clear
II b Common N   N   + or ++
turbid
III Uncommon N or  These two bands N    or +++ turbid
merge 
IV Very N  N or  N or  N or    or ++ turbid
common 
V Rare   N or  N or    “Cream”/ ++
turbid

The Familial Hyperlipoproteinemias

 Clinicopathologic Significance of Lipoprotein
Phenotypes

Phenotype Familial Syndrome May Occur Secondary Remarks

to
I Abdominal pain Insulin-dependent DM LPL is deficient
Eruptive xanthomas LE
Lipemia retinalis Dysglobulinemias
Early vascular dse absent Pancreatitis
II Early, severe vascular disease High cholesterol diet Familial trait is
Prominent xanthomas Nephrotic syndrome autosomal dominant;
Porphyria homozygotes are esp.
severely affected
Hypothyroidism
Dysglobulinemias
Obstructive liver dses
III Accelerated vascular dse, Hypothyroidism Diet, lipid-lowering
onset in adulthood Dysglobulinemias drugs very effective
Xanthomas, palmar yellowing Uncontrolled diabetes
Abnormal glucose tolerance
Hyperuricemia
IV Acclerated vascular dse, onset Obesity Weight loss lowers
in adulthoo High alcohol intake VLDL
Abnormal glucose tolerance Oral contraceptives High-fat diet may
Hyperuricemia Diabetes convert to type V
Nephrotic syndrome
Glycogen storage dse
V Abdominal pain High alcohol intake Weight loss does not
Pancreatitis Diabetes lower VLDL
Eruptive xanthomas Nephrotic syndrome
Abnormal glucose tolerance Pancreatitis
Vascular disease not Hypercalcemia
associated
Exogenous (Dietary) Lipid Pathway
Food Intestinal absorption

Chylomicrons (CM)

CM High TAG

TAG in Low Chol

adipose Apo B48
tissue

Muscle
and FFA
adipose
tissue

Chylomicron remnants
taken by the liver
Endogenous Lipid Pathway (Synthesis)
FFA TAG synthesis in VLDL
liver, intestine

High TAG VLDL

Low Chol
Apo B100,
Apo E
FFA LPL

IDL

Binds onto
Apo E hepatocytes
through apo E

LDL

LDL binds to receptors in

liver (70%) & other
tissues (30%)
 HDL Pathway
Liver secretes
Apo AI + other Nascent
apos + PLs HDL

Chol from
tissues

HDL3

Esterification of
Chol by LCAT

LDL
Uptake by liver
Chol transfer to VLDL

Excretion into bile

 Lipoprotein Composition

TAG Cholesterol Phospholipid Protein Electrophoretic

% % % % Mobility
Chylomicrons 85 – 95 3–5 5 – 10 1–2 Remain at
origin
VLDL 60 – 70 10 – 15 10 – 15 10 2-lipoprotein,
Pre-
lipoprotein
LDL 5 – 10 45 20 – 30 15 – 25 -lipoprotein
HDL Very little 20 30 50 1-lipoprotein
 Quick Quiz
 The surfactant / albumin ratio by fluorescence
polarization is performed to assess what physiological
state?
a. hyperlipidemia
b. coronary heart disease
c. hemolytic disease of the newborn
d. fetal lung maturity

 A patient’s total cholesterol is 300 mg/dl, HDL-

cholesterol is 50 mg/dl and his TAG is 200 mg/dl. What
is his calculated LDL-cholesterol?
a. 200 b. 210
c. 290 d. 350