285 PHL

$H:\beautiful%20Nature%20photos.jpg$

Determination of Triglyceride in Serum or Plasma

Outline of Today’s lectureOutline of Today’s lecture

1. Introduction of Triglyceride1. Introduction of Triglyceride

2. Clinical significance of Triglyceride2. Clinical significance of Triglyceride

3. Principle of Triglyceride estimation3. Principle of Triglyceride estimation

ObjectivesObjectives

To determine serum Triglyceride levelTo determine serum Triglyceride level

Master how to determine the conc. ofTriglyceride in serum.Master how to determine the conc. ofTriglyceride in serum.

Understand the clinical significance ofTriglycerideUnderstand the clinical significance ofTriglyceride

What are triglycerides?What are triglycerides?

•A•A

t t

i i

a a

e e

f f

g g

t t

f f

a a

g g

T T

a a

m m

c c

o o

n n

f f

a a

o o

http://www.familyhealthavenue.com/wp-content/uploads/2009/04/triglyceride-structure.gif

The major lipids present in the plasma are fattyacids, triglycerides, cholesterol andphospholipids.The major lipids present in the plasma are fattyacids, triglycerides, cholesterol andphospholipids.

Triglycerides consist of glycerol esterified withthree long-chain fatty acids, such as stearicacid (18 carbon atoms) or palmitic (16 carbonatoms) acids.Triglycerides consist of glycerol esterified withthree long-chain fatty acids, such as stearicacid (18 carbon atoms) or palmitic (16 carbonatoms) acids.

Triglyceride is present in dietary fat, and can besynthesized in the liver and adipose tissue toprovide a source of energy, which can bemobilized when required, for example, duringstarvation.Triglyceride is present in dietary fat, and can besynthesized in the liver and adipose tissue toprovide a source of energy, which can bemobilized when required, for example, duringstarvation.

c c

b b

s s

u u

f f

a a

w w

a a

c c

o o

c c

m m

a a

t t

m m

c c

o o

v v

e e

g g

e e

t t

a a

b b

l l

e e

o o

i i

l l

a a

n n

i i

m m

a a

l l

f f

T T

a a

c c

o o

h h

u u

m m

a a

n n

s s

k k

i i

n n

o o

i i

l l

s s

Lipoprotein Structure and Composition

Roles of TriglyceridesRoles of Triglycerides

Fat storesFat stores

–Energy–Energy

–Protection- fat layer protect organs–Protection- fat layer protect organs

–Insulation- fat does not conduct heat–Insulation- fat does not conduct heat

Causes of High TriglyceridesCauses of High Triglycerides

High carbohydrate dietsHigh carbohydrate diets

Several diseases (type 2 diabetes, chronicrenal failure, nephrotic syndrome)Several diseases (type 2 diabetes, chronicrenal failure, nephrotic syndrome)

Certain drugs (corticosteroids, estrogens,retinoids, higher doses of beta-blockers)Certain drugs (corticosteroids, estrogens,retinoids, higher doses of beta-blockers)

Various genetic dyslipidemiasVarious genetic dyslipidemias

Physical inactivityPhysical inactivity

Cigarette smokingCigarette smoking

Increased Triglycerides Occur With TheFollowing Conditions:Increased Triglycerides Occur With TheFollowing Conditions:

Hyperlipoproteinemia type II, IIb, III, IV,and I.Hyperlipoproteinemia type II, IIb, III, IV,and I.

Liver disease, alcoholism. Liver disease, alcoholism.

Nephritic syndrome, renal disease.Nephritic syndrome, renal disease.

Myocardial infarction.Myocardial infarction.

Hypothyroidism.Hypothyroidism.

T T

L L

O O

T T

F F

C C

α α

ββ

w w

l l

o o

l l

d d

How can you low high triglycerides?How can you low high triglycerides?

c c

m m

d d

i i

e e

t t

l l

c c

t t

l l

y y

l l

a a

h h

e e

a a

l l

t t

h h

y y

w w

e e

i i

g g

h h

t t

f f

a a

s s

i i

y y

d d

m m

a a

s s

m m

o o

k k

i i

n n

g g

Determination of serum TriglycerideDetermination of serum Triglyceride

Types of Samples:Types of Samples:

PlasmaPlasma

Water + solids (e.g. glucose, urea, albumin, fibrinogen)Water + solids (e.g. glucose, urea, albumin, fibrinogen)

No cellsNo cells

SerumSerum

Serum = plasma – clotting factorsSerum = plasma – clotting factors

http://loudoun.nvcc.edu/vetonline/vet131/plasma&serum.JPG

RequirementsRequirements

Requirements:Requirements:

Automatic pipettesAutomatic pipettes

TipsTips

CuvettesCuvettes

 Spectrophotometer Spectrophotometer

 Reagents Reagents

Specimen: Specimen:

Serum or plasma Serum or plasma

Preparation of CuvettesPreparation of Cuvettes

Procedure Procedure

Sample

(Reagent+Serum)

Standard

(Reagent+ St. sample)

Blank

(only Reagent)

Triglyceride Lipase Glycerol + Fatty acidsTriglyceride Lipase Glycerol + Fatty acids

Glycerol + ATP Gk Glycerol-3-phosphate + ADPGlycerol + ATP Gk Glycerol-3-phosphate + ADP

Glycerol-3-Phosphate + O2 GPOGlycerol-3-Phosphate + O2 GPO

Dihydroxyacetone-phosphate + H2O2 Dihydroxyacetone-phosphate + H2O2

2H2O2 + 4-aminoantipyrine + 4-chlorophenol POD 2H2O2 + 4-aminoantipyrine + 4-chlorophenol POD

Quinoneimine + HCL + 4H2O Quinoneimine + HCL + 4H2O

ProcedureProcedure

Kit MethodKit Method

ReagentsReagents

BlankBlank

SampleSample

StandardStandard

Sample (Sample (l)

10 10

Standard (Standard (l)

1010

Working reagent (ml)Working reagent (ml)

ProcedureProcedure

Add the reagents and sample as directedAdd the reagents and sample as directed

Mix and let stand at room temperature for 10 min.Mix and let stand at room temperature for 10 min.

Read the absorbance at 505 nm (495-550).Read the absorbance at 505 nm (495-550).

Zero the spectrophotometer with the blank reagent.Zero the spectrophotometer with the blank reagent.

Read the absorbance's of the standard and sampleswithin 1 hrRead the absorbance's of the standard and sampleswithin 1 hr

Enter the absorbance's readings and calculate theconcentration of the sample.Enter the absorbance's readings and calculate theconcentration of the sample.