Clinical Chemistry 1

1. Merlyn A. Baraclan,
RN, RMT

2.  Overview on Lipids
 Lipoproteins,
Apoliporoteins &
Related Proteins
 Lipid Transport &
Lipoprotein Metabolism
 Lipid & Lipoprotein
Measurement
 The NCEP Guidelines
 Lipids, Lipoproteins &
Disease
 References

3.  are biological
compounds
which are
soluble in
nonpolar organic
solvents but
relatively
insoluble in
polar solvents.

4. Triglycerides / TAG / TG
Cholesterol / C
Phospholipids
Glycolipids

5.  Primary source of fuel
 Components of cell membranes & many
cell structures
 Provide stability to cell membranes
 Means of transmembrane transport

6.  The function of lipoprotein particle is to
transport lipids around the body in the
blood.
Contain cholesterol in 2 forms:
_ free cholesterol
_ cholesterol ester
 Lipoproteins have a micellar structure.

7.  The transport proteins of lipids having a
micellar structure composed of two
parts as follows:
Central core
 contains TAG and cholesterol ester
 nonpolar moiety
Surface coat
 contains PL, free cholesterol, and
apolipoproteins

8. - Chylomicrons (CM)
- very low density lipoproteins
(VLDL)
- low density lipoproteins (LDL)
- high density lipoproteins (HDL)

9. CLASS DENSITY
(g/ml)
MEAN
DIAMETER
(mm)
source Principal
function
Electropho
retic
mobility
CM < 0.95 500 intestine Transport of
Exogenous
TAG
Remains at
Origin
VLDL 0.96 -1.006 43 liver Transport of
Endogenous TAG
Pre β
IDL 1.007-1.019 27 Catabolism of
VLDL
Precursor of LDL “broad β”
LDL 1.020-1.063 22 Catabolism of
VLDL via IDL
Cholesterol
Transport
β
HDL 1.064-
1.210
8 Liver,intestine,
catabolism of
CM & VLDL
“reverse
cholesterol
transport”
α

10. CLASS PROTEIN
%
TAG
%
CHOLESTEROL
%
PHOSPHO
LIPID
%
Chylomicron
/ CM
1-2 85 - 95 3 -5 5 -10
VLDL 10 60 -70 10 -15 10 – 15
LDL 15 -25 5 - 10 45 20 -30
HDL 50 Very
little
20 30

13.  MAJOR LIPOPROTEINS
1. CHYLOMICRONS / CM
- Produced by the intestine
- Transport lipids of dietary origin
- Poor in free cholesterol
- Has a “very high lipid/protein ratio
- “milky plasma”
- “Floating creamy layer”
- Removed by the liver

14.  2. VERY LOW DENSITY LIPOPROTEINS / VLDL
- Produced by the liver
- Supplies the body w/ TAG of endogenous
origin & also cholesterol
- “ turbid plasma”

15.  3. LOW DENSITY LIPOPROTEINS / LDL
- Produced by the metabolism of VLDL
- The particles do not scatter light
- Removed by the liver & macrophages

16.  4. HIGH DENSITY LIPOPROTEIN / HDL
- Consists mostly of proteins
- Produced by the liver
- “reverse cholesterol transport”
- Cardioprotective

17.  1. Lipoprotein (a) / Lp (a)
- Similar to LDL
- Synthesized in the liver
- “lipid staining pre β band”
- Has atherogenic properties
- 2. LPX Lipoprotein
- Seen in patients with obstructive biliary disease,
& with LCAT deficiency
- 3. β – VLDL
- “Floating β lipoprotein”

18. Food intestinal absorption
CM High TAG,Low Chol, Apo B48
TAG in adipose tissue
Muscle & FFA
chylomicron remnants taken by the liver

19. FFA TAG synthesis in liver, intestine VLDL
High TAG
Low Chol
FFA Apo B100
LPL Apo E
IDL
Apo E Binds onto hepatocytes through Apo E
LDL LDL binds to the receptors in liver (70%)
& other tissues (30%)

20. liver secretes Apo A1 + other Apos + PLs Nascent HDL
Cholesterol from tissues
HDL 3
Esterification of Cholestrol by LCAT
LDL
uptake by liver
Cholesterol transfer to VLDL
excretion into bile

21.  BIOLOGIC VARIATION
 FASTING
 POSTURE
 VENOUS VS. CAPILLARY
 PLASMA VS. SERUM
 STORAGE

22.  BIOLOGIC VARIATION
- Cholesterol level rises w/ age
- Women have lower levels than men
(except in childhood & after the early
50’s)
- Age related variation is the basis of
NCEP recommendation that
cholesterol screening be repeated
every 5 years.

23.  FASTING
- 12 hours before venipuncture
- Chylomicrons are completely cleared w/in 6
– 9 hours
- NCEP Adult Treatment Panel III( ATP III), has
recommended that patients fast for at least
9 hours before blood specimens are taken for
lipid & lipoprotein analysis
 POSTURE
- Current NCEP guidelines recommend that
patients be seated for 5 minutes prior to
sampling.

24.  VENOUS VS. CAPILLARY SAMPLES
- Measurements in capillary blood samples are
lower than venous samples & tend to be
more variable.
 PLASMA VS. SERUM
- Either plasma or serum can be used when
only TAG, cholesterol & HDL are measured, &
LDL – C is calculated from these three
measurements.
- Plasma is preferred when lipoproteins are
measured by ultracentrifugal or
electrophoretic methods

25.  PLASMA VS. SERUM Cont.
- EDTA is the preferred anticoagulant - ♥
- Heparin – X
- Citrate – X
 STORAGE
- When serum or plasma must be stored for
long periods it should be maintained at a
temperature of -70⁰C.
- For short term storage, the samples can be
kept at – 20⁰C.

26.  A. TAG measurement
Chemical Nonenzymatic Methods
General Steps
1. Extraction
- To remove TAG from LP’s
- Accomplished by using MeOH, EtOH,
isopropyl alcohol, Folch’s rgt & diethyl
ether
- - removal of interferences by zeolite

27. 2. Hydrolysis of TAG into FFA & Glycerol
- By saponification w/ alcoholic KOH
3. Measurement of Glycerol
- The glycerol liberated is oxidized by
periodate to HCHO & quantified by using any
of the ff:
- Eegrine reaction
- Schryver’s reaction
- Pay’s reaction
- Hantzsch reaction (method of choice)

28.  Enzymatic Methods
- Based on the hydrolysis of TAG & the
measurement of glycerol that is released in
the reaction:
LPS
- TAG + 3H2O glycerol + fatty acid
- Methods:
- Boculo David
- Megraw
- Winartasaputra
- Nagele - Trinder

29.  Chemical Nonenzymatic Methods
4 General Steps
1. Extraction
- Using Bloor’s rgt (3:1 EtOH – ether) or
zeolite extraction
2. Saponification
- Using KOH
3. Purification
- Using digitonin
4. Color development
- May proceed w/ the Leibermann-Burchardt
reaction or Salkowski reaction

30.  Leibermann - Burchardt reaction
- Uses sulfuric acid & acetic anhydride to
produce an unstable green cholestadienyl
monosulfonic acid; color stabilized by sodium
sulfate
 Zak or Salkowski reaction
- Uses sulfuric acid & ferric ions to produce a
stable red to red – violet cholestadienyl
disulfonic acid

31.  1 Step Methods
- Zlatkis – Zak Boyle method
- Ferro – Ham method
- Pearson – Stern – MacGavack
- Wybenga et al
 2 Step Methods
- Carr – Drekter
 3 Step Methods
- Abell – Kendall method (Standard reference
method)
 4 Step Methods
- Schoenheimer – Sperry
- Parek – Jung
- Sperry - webb

32. cholesterol ester
Cholesterol ester + H2O cholesterol + FFA
hydrolase
cholesterol
Cholesterol + O2 cholest – 4 – en – 3 – one + H2O2
oxidase
peroxidase
H2O2 + phenol + 4 aminoantipyrine quinoneimine dye
+ 2 H2O

33.  1. HDL Measurement
- Polyanion precipitation
- Electrophoresis – spectrophotometric det’n
- Ultracentrifugation ( reference method)
 2. Chylomicrons / CM
- Standing Plasma Test
 3. Lipid Profile
- Use of the Friedewald equation

35.  Testing & Treatment
Cholesterol Goals:
ATP III recommends a complete lipoprotein
profile as the initial test for evaluating blood
cholesterol.
Testing should be performed on all adults aged
20 & older & should be repeated once every
5 years.
The need for a therapeutic lifestyle change &
drug therapy

36. For Cholesterol: conversion factor to convert mg/dL to mM is 0.02586
For triglyceride : conversion factor to convert mg / dL to mM is 0.011
LDL Cholesterol HDL
Cholester
ol
Total
Cholesterol
Triglyceride
<100 optimal < 40 low < 200 desirable < 150 normal
100-129 Near
optimal /
above
optimal
≥ 90 high 200-
239
Borderline
high
150 -
199
Borderline
high
130-159 Borderline
high
≥ 240 high 200 -
499
high
160 -
189
High ≥ 500 Very high
≥190 Very high

37. Drug Class Mechanism of Action Example
Statins Lowers LDL cholesterol Lovastatin , simvastatin,
Pravastatin, fluvastatin
Fibric acid
derivatives
Lowers TAG Gemfibrosil, Fenofibrate
Bile acid
resins
Lowers LDL cholesterol Colestipol, cholestyramine
Niacin
(nicotinic
acid)
Lowers TAG niacin

38.  CHD
(coronary heart
disease)
 Atherosclerosis

39. 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)

40.  hyperbetalipoproteine-
mia
 Elevated LDL – C
 Normal TAG
 High cardiac risk
 Commonly encountered
 SPECIFIC DISEASES
- Polygenic (Nonfamilial)
Hypercholesterolemia
- Familial
hypercholesterolemia/FH
- Familial Defective Apo B
- Sitosterolemia

41.  Due to the elevation
of TAG rich particles
 Hyperprebetalipo –
proteinemia
 secondary to excess
alcohol & high
carbohydrate intake
 SPECIFIC DISEASES
- Diabetic dyslipidemia
- Familial
hypertriglyceridemia
- Lipoprotein lipase
deficiency
- Apo C II deficiency
- Apo C III excess

42. A group of inherited disorders
characterized by the accumulation of
lipids in tissues especially the “brain”
due to a deficiency in a particular
sphingolipid catabolic enzyme

43.  Niemann – Pick disease
- Deficiency in sphingomyelinase & accumulation
of sphingomyelin
 Gaucher’s disease
- Deficiency in β – D glucosidase & the
accumulation of glucocerebroside
 Krabbe’s disease
- Deficiency in β – D galactosidase & the
accumulation of galactocerebrosides
 Fabry’s disease
- Deficiency in α – D galactosidase & the
accumulation of ceramide trihexoside
 Tay – Sach’s disease
- Deficiency in β – D hexaminidase & the
accumulation of β - sulfogalactocerebroside

44.  Ultracentrifugation & electrophoretic techniques are
of historical significance, MOST useful lipid &
lipoprotein testing are now enzymatic.
 LDL – cholesterol can be measured directly , but is
usually calculated using the Friedewald formula.
 LDL – C is now considered the MOST important
value in assessing cardiac risk & directing therapy.
 The profile for initial adult screening aged 20 &
above, includes TC, TAG, HDL – C & LDL – C &
should be repeated every 5 years.

45.  INTERNET SOURCES
 Bhagavan NV (2002). Medical Biochemistry.
San Diego: Harcourt/Academic Press.
ISBN 0-12-095440-0.
http://books.google.com/?id=vT9YttFTPi0C
&printsec=frontcover.
 http://biology.clc.uc.edu/courses/bio
104/lipids.htm
 BOOK SOURCES
 Clinical Diagnosis and Management
by Laboratory Methods / John
Bernard Henry. 20th ed. 224 – 248
Henry’s Clinical Diagnosis and
Management by Laboratory Methods
/ Richard McPherson & Mathhew
Pincus. 21st ed. 200 – 219
 Clinical Chemistry:
Principles, Procedures &
Correlations / Michael Bishop, Janet
Engelkirk & Edward Fody. 4th ed. 232
– 259
 Southwestern University College of
Medical Technology Clinical
Internship Manual / 2005 ed.
 Southwestern University College of
Medical Technology: Lecture Handbook
in Routine Clinical Chemistry/ Julius
Mario. 2008 ed. 44 -54
 Danny Donor ♥

46. learn to love
the things you
hate
You're on the road to success when
you realize that failure is only a
detour.