Triglycerides

Introduction

1. Triglycerides are esters derived from glycerol and three fatty acids, serving as the primary storage form of fat in humans and animals.
2. They are transported in the bloodstream as part of lipoprotein particles, including very low-density lipoproteins (VLDL) and chylomicrons.
3. Accurate measurement of serum triglyceride levels is crucial for assessing an individual’s lipid profile and risk for cardiovascular and metabolic diseases.
4. Biochemical determination of serum triglycerides involves enzymatic and colorimetric methods, which provide reliable and reproducible results.
5. Factors such as fasting status, metabolic rate, and diet influence triglyceride levels, making it vital to standardize testing conditions for accuracy.
6. This article explores the methods, principles, sample handling, reagents, procedures, calculations, normal reference ranges, and clinical significance of triglyceride determination.

 

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Method

Colorimetric enzymatic test using glycerol-3-phosphate-oxidase (GPO PAP)

Principle

 Triglycerides are determined after enzymatic hydrolysis with lipases. The quinonemine indicator is formed from hydrogen peroxide, 4- aminophenazone, and 4-chlorophenol under the catalytic influence of peroxidase.

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Reagent 

1. Reagent: Triglycerides reagent 
2. Triglycerides Standard: 200 mg/dl (2.25 mmol/L)

Sample Collection and Handling

1. Sample Type:
   • Serum or plasma is collected in a plain or heparinized tube.
   • EDTA or fluoride anticoagulants are not recommended as they may interfere with enzymatic reactions.
2. Patient Preparation:
   • A 12-hour fasting period is required to minimize postprandial lipemia.
   • Water is allowed during fasting, but no other drinks or food should be consumed.
3. Storage:
   • Serum samples should be analyzed promptly.
   • If delayed, samples can be stored at 2–8°C for up to 48 hours or frozen at -20°C for longer storage.

 

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Procedure

	Test	Standard	Blank
Reagent, ml	1 ml	1 ml	1 ml
Sample, ml	0.010 ml	–	–
Standard, ml	–	0.010 ml	–
Distilled water	–	–	0.010ml

 

Mix well and incubate for 5 mins. at 37°C (or 10 mins. at 20 – 25° C). Measure the absorbance of Sample (AT) and Standard (AS) against reagent blank at 505 nm

Sample Dilutions

This method is linear up to a concentration of 1300 mg/dL.  Dilute samples above this concentration 1:1 with 0.9% saline  Repeat assay. Multiply the result by 2.

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Calculation:

• Calculate triglyceride concentration using the formula:

Triglyceride Concentration (mg/dL) = Absorbance of Sample − Absorbance of Blank/Absorbance of Standard − Absorbance of Blank×Concentration of Standard (mg/dl)

Normal Range

The reference range for serum triglycerides varies slightly depending on the laboratory and population studied. Typical adult reference ranges are:

• Normal: <150 mg/dL (1.7 mmol/L)
• Borderline High: 150–199 mg/dL (1.7–2.2 mmol/L)
• High: 200–499 mg/dL (2.3–5.6 mmol/L)
• Very High: ≥500 mg/dL (≥5.6 mmol/L)

 

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Clinical Significance

1. Hypertriglyceridemia:
   • Common causes include obesity, diabetes mellitus, hypothyroidism, excessive alcohol consumption, and genetic disorders like familial hypertriglyceridemia.
   • Associated with increased risk of atherosclerosis, cardiovascular disease, and acute pancreatitis when levels exceed 1000 mg/dL.
2. Hypotriglyceridemia:
   • Rare and associated with malabsorption syndromes, malnutrition, hyperthyroidism, and certain metabolic disorders.
3. Monitoring and Risk Assessment:
   • Triglyceride measurement is part of a lipid panel, alongside total cholesterol, HDL, and LDL cholesterol, to assess cardiovascular risk.
   • Used to monitor response to interventions such as lifestyle changes, dietary modifications, and lipid-lowering medications.