Estimation of serum Triglycerides by GPO-Trinder method.

Principle

Triglycerides in the sample are hydrolyzed to yield glycerol and free fatty acids by
Lipoprotein Lipase (LPL) enzyme. Glycerol is then phosphorylated to Glycerol-3-phosphate
by Glycerol kinase (GK). Glycerol-3-phosphate is then oxidized by the enzyme
Glycerophosphate oxidase (GPO) to yield Dihydroxyacetone phosphate(DHAP) and H 2O2.
Hydrogen peroxide then reacts with 4-Aminoantipyrine and Trinders reagent to generate a
Quinoneimine dye this reaction is catalyzed by peroxidase.

LPL
Triglycerides + H2O Glycerol + Free fatty acids

Glycerol kinase
Glycerol + ATP Glycerol-3-phosphate + ADP
Mg 2+

GPO
Glycerol-3-phosphate + O2 Dihydroxyacetone phosphate + H 2O2

Peroxidase
H2O2 + 4-Aminoantipyrine + Trinder reagent Quinoneimine Dye + 2H 2O

The Quinoneimine dye generated shows the absorbance at 540 nm, and its intensity is
directly proportional to the concentration of Triglycerides in the sample.

Requirements
Reagent : Buffer (pH 7) 40 mmol/L
4-Aminoantipyrine 0.4 mmol/L
ATP 2 mmol/L
Mg2+ 2.5 mmol/L
TOOS (Tinders reagent) 0.2 mmol/L
Glycerol kinase 1500 IU/L
Glycerol-3-phospate oxidase 4000 IU/L
Peroxidase 2200 IU/L
Lipoprotein lipase 4000 IU/L
Triglycerides standard ( 200 mg/dl)
colorimeter, test tubes, micro-pipettes.

Procedure
Pipette into tubes marked Blank Standard Test
Reagent 1000 μL 1000 μL 1000 μL
Distilled water 10 μL -- --
Standard -- 10 μL --
Test Sample -- -- 10 μL
 • Take three sterile test tubes and Blank, Standard and Test.
• To the Blank test tube pipette out 1000 μL of reagent and to that add 10 μL of
distilled water.
• To standard test tube 1000 μL of reagent and 10 μL of standard.
• To the Test add 1000 μL of the reagent and 10 μL of the sample solution.
• Mix well and incubate at 37°C for 10 minutes.
• Read the absorbance at 540 nm.

Calculation

The concentration of the Triglycerides in the serum is given by the following formula,

Triglycerides (mg/dl) = OD of Test x concentration of standard (mg/dl)

OD of standard
Report

The concentration of Triglycerides in the given serum sample is _______ mg/dl.

Clinical significance

Normal range : 50 – 150 mg/dl.

Triglycerides are simple lipids, formed in the liver by glycerol and fatty acids. They are
transported by VLDL, LDL and constitute about 95% of fat, stored as source of energy in
the tissues. Increased levels are a risk factors for atherosclerotic coronary artery disease,
peripheral vascular diseases and acute pancreatitis.

• Serum Triglycerides are increased in,

◦ Familial hypertriglyceridemia.
◦ Hypothyroidism.
◦ Nephrotic syndrome and Chronic kidney disease.
◦ Diabetes mellitus.
◦ Obesity.
◦ Chronic alcoholism and smoking.

• Serum Triglycerides are decreased in,

◦ Hyperthyroidism.
◦ Malabsorption syndrome.
◦ Malnutrition.