Triglyceride (TG) Content Assay Kit (Acetylacetone, Colorimetric Method)

Cat. No.: T1509111

GRADE & PURITY BioReagent

Overview

Triglycerides (TG), also known as triacylglycerol or triolein, are fat molecules formed from three long-chain fatty acids and one glycerol molecule. They are the most abundant lipids in the human body. Most tissues can utilize the breakdown products of triglycerides for energy, while organs such as the liver and adipose tissue can also synthesize triglycerides. Currently, common methods for detecting triglycerides include enzymatic and chemical methods. The enzymatic method is simple, rapid, requires minimal sample volume, and offers stable reagents, making it suitable for both manual and automated assays. Chemical methods involve extracting triglycerides from serum using organic solvents such as isopropanol, removing interfering factors like phospholipids through layering or adsorption, followed by saponification, oxidation, and colorimetric determination. The most commonly used chemical method is the acetylacetone colorimetric method, which includes adsorption extraction and solvent extraction techniques.

Detection Principle

Triglycerides in tissue homogenates or serum are treated with TG extraction solution, retaining the triglycerides in the upper layer. They are then saponified to produce glycerol, which is oxidized by an oxidizing agent to form formaldehyde. In the presence of ammonium ions, formaldehyde condenses with acetylacetone (Hantzsch reaction) to form a yellow fluorescent compound, 3,5-diacetyl-1,4-dihydrodimethylpyridine. The absorbance is measured at 420 nm using a spectrophotometer, and the content is calculated by comparison with a similarly treated standard. The reagents used in this method can be stored at room temperature for over six months, demonstrating good stability. The assay shows a good linear relationship within the range of 0.25–11.44 mg/ml (0.28–12.93 mmol/L) and has a wide detection range. This product is intended for scientific research purposes only and is not suitable for clinical diagnosis or other uses.

Applicable Samples: Human or animal serum, plasma, cerebrospinal fluid, etc.

Reagents, consumables and Equipments not provided

• Spectrophotometer (capable of measuring absorbance at 420 nm)
• Water bath or incubator, centrifuge, balance
• Centrifuge tubes or small test tubes, cuvettes
• Physiological saline
  

Operating Steps

1. Sample Preparation

1.1 Serum Samples

Use directly for detection after collection.

1.2 Tissue Samples

Accurately weigh an appropriate amount of tissue sample (mass = m), add physiological saline at a mass (g) to volume (ml) ratio of 1:4, homogenize manually or mechanically under ice bath conditions to obtain a homogenate (total volume = V_T). Take 0.1 ml of the homogenate for use.

2. TG Sample Addition

Set up blank, standard, and test tubes according to the table below. Add solutions in the specified order, taking care to avoid bubbles. If the TG content in the sample is too high, reduce the sample volume or dilute appropriately before measurement.

Substance Added (ml)	Blank Tube	Standard Tube	Test Tube
Distilled Water	0.1	—	—
TG Standard (2 mg/ml)	—	0.1	—
Sample	—	—	0.1
Extraction Solution	1.25	1.25	1.25
Acidic Buffer Solution	0.25	0.25	0.25

Mix while adding, allow to stand for layering, then take the supernatant.

Supernatant	0.15	0.15	0.15
Saponification Agent	0.5	0.5	0.5

Mix well, incubate in a 56°C water bath for 5 min.

Oxidizing Agent	0.5	0.5	0.5
Chromogenic Agent	0.5	0.5	0.5

Mix well, incubate in a 56°C water bath for 25 min.  

3. TG Measurement

Remove the tubes and cool under running water. Zero the spectrophotometer with the blank tube, using a 1 cm light path cuvette, measure the absorbance of the standard and test tubes at 420 nm (A_Standard, A_Test).

4. Result Calculation

Triglyceride content in 100 ml of serum:

TG (mg/100 ml) = (A_Test / A_Standard) × 2 × 100 / 0.1 = (A_Test / A_Standard) × 2000

Triglyceride content in 100 g of tissue:

TG (mg/100 g) = (A_Test / A_Standard) × 2 × V_T / 0.1 × 100 / m = (A_Test / A_Standard) × 2000 × V_T / m

Triglyceride concentration in the sample:

TG (mg/ml) = (A_Test / A_Standard) × 2

Parameter Explanation:

A_Test: Absorbance of the test tube

A_Standard: Absorbance of the standard tube

V_T: Total volume of the homogenate from a given mass of tissue (ml)

m: Actual mass of tissue used (g)

TG concentration conversion: 1 mg/mL = 1.13 mmol/L

5. Reference Range

Normal serum TG range: 0.55–1.70 mmol/L; Critical value: 2.30 mmol/L; Risk value: 4.50 mmol/L.

Precautions

1. This method can be directly used to detect TG levels in cerebrospinal fluid and urine.

2. Blood samples should be separated promptly to avoid hydrolysis of red blood cell membrane phospholipids by phospholipase, which produces free glycerol, or dilution of plasma TG values due to water release from red blood cells in the presence of anticoagulants.

3. Before plasma separation, samples should be placed in ice water and processed as soon as possible.

4. The linear range of this method is 0.3–12 mg/mL. Results are more accurate above 0.6 mg/mL, with slight deviations possible above 11 mg/mL. Samples exceeding this range should be processed accordingly.

5. Absorbance after color development changes over time, so measurements should be taken promptly. If there are many samples, they can be stored in the refrigerator and measured one by one.

6. Saponification, oxidation, color development time, and temperature can affect color development and final absorbance. Therefore, standard controls should be included in each batch of tests.

7. For plasma samples, 1 mg/mL EDTA·2K is typically used as the anticoagulant.

8. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.

9. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.

Contents & Storage

T1509111	Component	50T	Storage
T1509111A	TG Standard (2 mg/mL)	1 mL	2-8℃. Store in the dark.
T1509111B	TG Extraction Solution	70 mL	RT. Store in the dark.
T1509111C	Acidic Buffer Solution	6 mL	RT.
T1509111D	Saponification Agent	30 mL	RT.
T1509111E	TG Oxidizing Agent	30 mL	2-8℃. Store in the dark.
T1509111F	TG Chromogenic Agent	30 mL	2-8℃. Store in the dark.