Triglycerides Test
SUMMARY
Early methods used for determining triglycerides involved chemical hydrolysis of a solvent extract of the serum lipid. These methods required preliminary removal of interfering substances like phospholipids, carbohydrates and other difficulties, and produced unknown co-products so the tests were not readily available.
The enzymatic methods are based on the following advantages:
Two-step GPO-PAP method
Rapid colorimetric reaction within 10–15 minutes
Color reaction stable
Less susceptible to turbidity and pigments
Linearity up to 1000 mg/dL with hydrolytic concentration
Aim – Estimation of serum triglycerides by enzymatic method
PRINCIPLE
Glycerol dehydrogenase (GPO) catalyzes the specific oxidation of β-glycerol to glyceric acid and hydrogen peroxide (H₂O₂).
In peroxidase (POD) enzyme acts on hydrogen peroxide to liberate nascent oxygen (Nascent O₂).
Nascent oxygen couples with 4-aminoantipyrine and p-chlorophenol to form a red-colored quinoneimine dye.
Reaction:-
Triglyceride + H2O Lipoprotein
+ Lipase Glycerol
+ fatty acids
Glycerol + ATP Glycerol kinase Glycerol-3-phosphate + ADP
Glycerol-3-phosphate + 02 Glycerol
phosphate oxidase Dihydroxy acetone phosphate + H2O2
H2O2 + 4-amino-phenazone
+ p-chlorophenol Peroxidase Colored complex
REQUIREMENTS
Reagents:
-
Buffer/glycerol kinase/glycerol-3-phosphate oxidase/peroxidase – Enzyme reagent
-
Glycerol standard: 100 mg/dL
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Precipitating solution: 5%
-
Reagents contain 4-amino antipyrine in Tris buffer (pH 7.2 ± 0.02)
Preparation of Working Reagent
It is prepared fresh by mixing two parts of reagent 1 & one part of reagent 2.
PROCEDURE
Pipette in the tubes labeled as below:
| Contents | Blank | Standard | Test |
|---|---|---|---|
| Working reagent | 1000 µL | 1000 µL | 1000 µL |
| Distilled water | 10 µL | — | — |
| Glycerol standard | — | 10 µL | — |
| Sample | — | — | 10 µL |
Read absorbance of Test & Standard against Blank.
Calculations
Triglycerides (mg/dL)= Optical density of Test Optical density of Standard
×Concentration of Standard (100 mg/dL)
-
OD of Test = 0.23
-
OD of Standard = 0.22
-
Triglycerides= 0.22/0.23×100=104.54 mg/dl
Normal Values:
30 – 150 mg/dL
Clinical Significance
Triglycerides are esters of glycerol with three fatty acids and are the major naturally occurring lipids.
They are transported in plasma bound to lipoproteins.
Increased triglycerides may be observed in:
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Liver disease
-
Nephrotic syndrome
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Diabetes mellitus
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Endocrine disorders
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Alcoholism
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Acute pancreatitis
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Atherosclerosis & ischemic heart disease
Low triglycerides may be present in conditions like malabsorption.