Determination of triglyceride

in serum

Dept.of Biochemistry

Blood lipid

• Concept: All the lipids contained in plasma, including fat, phosphalipids, cholesterol, cholesterol ester and fatty acid.

• Blood lipid exist and transport in the form of lipoprotein.

blood lipids

freeTG

cholesterol

phospholipidslecithinsphingolipidscephalin

ester

FFA

Triglyceride (TG) or triacylglycerol (TAG)

Glycerol

Triacylglycerols,TG

CH2

CH

O C R1

O

CH2

O

O

C

C

R2

O

O

R3

Ultra centrifugation method ：

high density lipoprotein (HDL) high

low density lipoprotein ( LDL)

very low density lipoprotein ( VLDL)

CM (chylomicron ) low

Classification of plasma lipoproteins

electron microscope

Structure of lipoprotein

Composition of lipoprotein

CM VLDL LDL HDL

Density(g/ml) <1.0060.95-1.006

1.006-1.063

1.063-1.210

Protein 2 10 23 55

Phospholipids 9 18 20 24

Cholesterol 1 7 8 2

Cholesteryl esters 3 12 37 15

TG 85 50 10 4

The function of lipoproteins

• Chylomicron transport triglycerides (fat) from the intestinal mucosa to the liver.

• VLDL carry newly synthesised triacylglycerol from the liver to adipose tissue.

• In the blood, VLDL release triglycerides and some cholesterol and become LDL.

• LDL then carries fat and cholesterol to the body’s cells.

• HDL carry fat and cholesterol back to the liver for excretion.

Lipoprotein Profile

Includes:Total CholesterolLDL CholesterolHDL CholesterolTriglycerides

CLINICAL SIGNIFICANCE

• Triglyceride measurements are used in the diagnosis and treatment of diseases involving lipid metabolism (hyperlipoproteinemia), various endocrine disorders (diabetes mellitus, nephrosis) and liver obstruction (extrahepatic biliary obstruction).

• Obesity, excessive consumption of simple sugars and saturated fats, inactivity, alcohol consumption and insulin resistance, renal failure are commonly associated with hypertriglyceridaemia.

• Triglyceridaemia determined by levels of plasma lipids after an overnight fast.

Hyperlipidemia

• Hyperlipidemia, hyperlipoproteinemia, or dyslipidemia is the presence of raised or abnormal levels of lipids and/or lipoproteins in the blood.

• Lipid and lipoprotein abnormalities are extremely common in the general population, and are regarded as a highly modifiable risk factor for cardiovascular disease, atherosclerosis.

“ Normal” valuesTriglycerides < 1.7 mmol/L Total cholesterol < 5.5 mmol/LHDL – C > 1.1 mmol/LLDL – C < 3,4 mmol/L

< 150 mg/dl < 200 mg/dl> 35 mg/dl< 130 mg/dl

xanthoma

Classification of Hyperlipidemias

ClassificationIncreased lipoprote

in Blood lipids

Serum appearance

Ⅰ (rare) CM TG↑ ↑ ↑ CH↑ Creamy top layer

Ⅱa LDL CH↑ ↑ Clear

Ⅱb LDL, VLDL CH↑ ↑ TG↑ ↑ Clear

Ⅲ (rare) IDL CH↑ ↑ TG↑ ↑ Turbid

Ⅳ VLDL TG↑ ↑ Turbid

Ⅴ (rare) VLDL, CM TG↑ ↑ ↑ CH↑Creamy top layer & turbid bottom

Normal rare common common rare common rare

Normal CM LDL LDL+VLDL IDL VLDL CM+VLDL

Principle (GPO method)

• Triglycerides in the sample are hydrolyzed by lipase to glycerol and fatty acids.

• The glycerol is then phosphorylated by ATP to glycerol-3-phosphate (G3P) and ADP in a reaction catalyzed by glycerol kinase (GK).

• G3P is then converted to dihydroxyacetone phosphate (DAP) and hydrogen peroxide(H2O2) by glycerophosphate oxidase (GPO).

• H2O2 then reacts with 4-aminoantipyrine (4-AAP) and 4-chlorophenol in a reaction catalyzed by peroxidase(POD) to yield a red colored quinoneimine dye.

• The intensity of the color produced is directly proportional to the concentration of triglycerides in the sample when measured at 510nm.

Triglycerides + H2Olipases

glycerol + fatty acids

Glycerol + ATPGK glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2

GPOdihydroxyacetone phosphate + H2O2

2H2O2 + 4-aminoantipyrine + 4 chlorophenolperoxidae

quinoneimine + HCl +4 H2O

Trinder reaction

Specimens & Materials

• Specimen: serum （血清） • Reagent:

– R1: Tris-HCl buffer (pH 7.0) ， chlorophenol– R2: 4-AAP, lipase, GK, GPO, POD, ATP

• Working reagent: – R1 : R2 =4:1

• Standard: CS=2.26mmol/L • Water bath, Test tubes, Pipettes• Spectrophotometer

Method

B S T

Working reagent 1.0ml 1.0ml 1.0ml

dH2O 10 l - -

Standard - 10 l -

Serum - - 10 l

Mix well, incubate for 10mins at 37CdH2O 0.5ml 0.5ml 0.5ml

•Mix well, measure the absorbance of T and S setting zero with B, λ=510nm.

Calculation

• CT (mmol/L)=AT /AS x CS

• Normal value: 0.4~1.7 mmol/L

Next experiment

• Urea （ p84-86 ）