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.

Triglyceride (TG) or triacylglycerol (TAG) Glycerol

Triacylglycerols,TG

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 CMVLDLLDLHDL Density(g/ml)< Protein Phospholipids Cholesterol1782 Cholesteryl esters TG

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 Cholesterol LDL Cholesterol HDL Cholesterol Triglycerides

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” values Triglycerides < 1.7 mmol/L Total cholesterol < 5.5 mmol/L HDL – C > 1.1 mmol/L LDL – C < 3,4 mmol/L < 150 mg/dl < 200 mg/dl > 35 mg/dl < 130 mg/dl

xanthoma

Classification of Hyperlipidemias Classification Increased lipoprotein Blood lipids Serum appearance Ⅰ (rare) CMTG↑ ↑ ↑ CH↑Creamy top layer ⅡaⅡa LDLCH↑ ↑Clear ⅡbⅡb LDL, VLDLCH↑ ↑ TG↑ ↑Clear Ⅲ (rare) IDLCH↑ ↑ TG↑ ↑Turbid Ⅳ VLDLTG↑ ↑Turbid Ⅴ (rare) VLDL, CMTG↑ ↑ ↑ 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(H 2 O 2 ) by glycerophosphate oxidase (GPO). H 2 O 2 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. Trinder reaction

Specimens & Materials Specimen: serum （血清） Reagent: –R 1 : Tris-HCl buffer (pH 7.0) ， chlorophenol –R 2 : 4-AAP, lipase, GK, GPO, POD, ATP Working reagent: –R 1 : R 2 =4:1 Standard: C S =2.26mmol/L Water bath, Test tubes, Pipettes Spectrophotometer

Method BST Working reagent1.0ml dH 2 O 10  l -- Standard- 10  l - Serum-- 10  l Mix well, incubate for 10mins at 37  C dH 2 O0.5ml Mix well, measure the absorbance of T and S setting zero with B, λ=510nm.

Calculation C T (mmol/L)=A T /A S x C S Normal value: 0.4~1.7 mmol/L

Next experiment Urea （ p84-86 ）