TRIGLYCERTRIGLYCERIDES MR (Ready To Use) Biochemistry Reagent

PRINCIPLE:

The method^1,2 is based on the enzymatic hydrolysis of serum or plasma triglyceride to glycerol and free fatty acids (FFA) by lipoprotein lipase (LPL). The glycerol is phosphorylated by adenosin triphosphate (ATP) in the presence of glycerolkinase (GK) to form glycerol-3-phosphate (G-3-P) and adenosine diphosphate (ADP). G-3-P is oxidized by glycerophosphate oxidase (GPO) to form dihydroxyacetone phosphate (DHAP) and hydrogen peroxide.

A red chromogen is produced by the peroxidase (POD) catalyzed coupling of 4-aminoantipyrine (4-AA) and phenol with hydrogen peroxide (H2O2), proportional to the concentration of triglyceride in the sample.

REAGENT COMPOSITION:

Monoreagent. PIPES buffer 50 mmol/L pH 6.8, LPL³ 12 KU/L, GK ³ 1 KU/L, GPO ³ 10 KU/L, ATP 2.0 mmol/L,

Mg²⁺ 40 mmol/L, POD ³ 2.5 KU/L, 4-AA 0.5 mmol/L, phenol 3 mmol/L, non-ionic tensioactives 2 g/L (w/v). Biocides.

Triglycerides standard. Glycerol 2.26 mmol/L, equivalent to 200 mg/dL of glycerol trioleate. Secondary standard

STORAGE AND STABILITY:  Store at 2-8ºC.

The Monoreagent and Standard are stable until the expiry date stated on the label.

Discard the reagent if presents an absorbance above 0.150 at 500 nm against distilled water or if it fails to recover the declared values of control sera.

REAGENT PREPARATION:

 The Monoreagent and the Standard are ready-to-use.

SAMPLES:

Serum or EDTA plasma obtained by the patient after an overnight fast. Remove from cells within 2 hours of venipuncture. Analyze samples immediately or refrigerate. Stable for 1 week at 4-8ºC

INTERFERENCES:

 Bilirubin levels up to 14 mg/dL interfere. Hemolysis (hemoglobin > 0.5 g/dL) does not interfere. Glycerol in rubber stoppers or in contaminated glassware will give higher triglycerides values.3

MATERIALS REQUIRED:

1.Photometer or colorimeter capable of measuring absorbance at 500 ± 20 nm.

2.Constant temperature incubator set at 37ºC.

3.Pipettes to measure reagent and samples

PROCEDURE:

1. Bring reagents and samples to room
2. Pipette into labelled tubes

3. TUBES	Blank	Sample	Standard
   Monoreagent	1.0 mL	1.0 mL	1.0 mL
   Sample Standars	– –	10 mL –	– 10 mL

1. Mix and let stand the tubes 15 minutes at room temperature (16-25ºC) or 5 minutes at 37ºC.
2. Read the absorbance (A) of the samples and the standard at 500 nm against the reagent

The color is stable for at least 1 hour protected from light.

CALCULATIONS:

Samples with concentrations higher than 600 mg/dL should be diluted 1:2 with saline and assayed again. Multiply the results by 2.

If results are to be expressed as SI units apply: mg/dL x 0.0113 = mmol/L

REFERENCE VALUES :

 Updated clinical values of triglycerides used to classify risk groups.

It is recommended that each laboratory establishes its own reference range.

QUALITY CONTROL:

The use of a standard to calculate results allows to obtain an accuracy independent of the system or instrument used.

To ensure adequate quality control (QC), each run should include a set of controls (normal and abnormal) with assayed values handled as unknowns.

CLINICAL SIGNIFICANCE:

 The plasma level of lipids (triglycerides and cholesterol) and lipid derivates, especially lipoproteins (HDL and LDL), aids in the diagnosis of many metabolic disorders. An imbalance in the level of lipoproteins in plasma leads to hyperlipoproteinemia, a group of disorders that affects lipid and lipoproteins levels in plasma, causing coronary heart disease (CHD) and atherosclerosis. Each type of hyperlipoproteinemia is associated with an abnormal elevation of triglycerides, cholesterol or lipoprotein subfraction.

Prospective studies⁴ indicate that elevated triglycerides are also an independent risk for coronary heart disease. The finding that elevated triglycerides are an independent CHD risk factor suggest that some triglyceride-rich lipoproteins are atherogenic. The latter are partially degraded VLDL, commonly called remnant lipoproteins. In clinical practice, VLDL colesterol is the most readily available measure of atherogenic remnant lipoproteins, and as such can be a target of colesterol-lowering therapy.

ANALYTICAL PERFORMANCE

 Linearity:Up to 800 mg/dL

• Precision 

Replicates: 10 for each level.         Replicates: 10 for each level Instrument: UVIKON 930       for 8 days.

• Sensitivity. Using a 1:100 sample/reagent at 505 nm, 1mg o triglyceride will produce a net absorbance of approximately 003.
• Correlation. This assay (y) was compared with a similar commercial method (x). The results were:

N = 30      r = 0.996      y = 1.116 + 0.439

REFERENCES:

1. Buccolo G and David, Clin. Chem. 19 : 476 (1973).
2. Fossati, R. and Prencipe, Clin. Chem. 28: 2077 (1982).
3. Young, S. Effects of Drugs on Clinical Laboratory Tests. 4^th Edition. AACC Press (1995).
4. SPECIAL Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 285 : 2486 (2001).