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2.17: Triglycerides

  • Page ID
    38645
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    RELATED READING: Chapter 33. See Methods in CD-ROM for Triglycerides.

    OBJECTIVES

    Upon completion of this exercise, appropriate discussions, and related readings, the student will be able to:

    1. Determine the triglyceride value of serum with 90% accuracy using an endpoint, coupled enzyme reaction.
    2. Explain what steps need to be taken to deal with specimens with unusual characteristics.
    3. Describe the appearance of samples with markedly elevated triglycerides.
    4. List secondary causes of elevated triglycerides.

    PRINCIPLES

    Triglycerides are completely hydrolyzed to free glycerol and free fatty acids by the enzyme lipase. The liberated free glycerol content is then determined enzymatically as shown by the following coupled sequenced reactions.

    \[\text{Triglycerides} \xrightarrow{\text{Lipase}} \text{Glycerol} + \text{Free Fatty Acids}\]

    \[\text{Glycerol} + \text{ATP} \xrightarrow{\text{Glycerol}} \text{Glycerol-1-Phosphate} + \text{ADP Kinase}\]

    \[\text{ADP} + \text{Phosphoenolpyruvate} \xrightarrow{\text{Pyruvate}} \text{ATP} + \text{Pyruvate Kinase}\]

    \[\text{Pyruvate} + NADH + H^{+} \xrightarrow{\text{LD}} \text{Lactate} + NAD^{+}\]

    The resulting decrease in absorbance at 340nm (decrease in NADH) is a stoichiometric measure of the glycerol present and is directly related to the triglyceride content of the sample.

    MATERIALS

    • Triglyceride reagent
    • Patient samples
    • Standard
    • Control serum
    • UV Spectrophotometer
    • Cuvettes
    • Pipets 37°C incubator

    PROCEDURE

    1. For each standard, control, and sample to be tested, pipet 3.0 mL of reagent into a clean labeled cuvette.
    2. Incubate each cuvette for 3 minutes at 37°C.
    3. Adjust a spectrophotometer to 340 nm and set zero absorbance (100% T) with distilled water.
    4. Add 50 \(\mu\)L of standard to the warmed reagent.
    5. Mix gently by inversion and read the absorbance immediately (within 15 seconds) and record.
    6. Start a stopwatch and continue incubation.
    7. Repeat steps 4 & 5 for each control serum to be tested; you will find it useful to space each timing at 30 second or 1 minute intervals from the standard.
    8. After exactly 10 minutes of incubation take a second absorbance reading or each mixture and record your results on the data sheet.
    9. Calculate the total \(Delta\)A for each standard, control, and serum sample and determine the triglyceride value using the following formula: $$\frac{\Delta A_{Unknown}}{\Delta A_{Standard}} \times \text{conc. std} = \text{conc. mg/L}$$

    Note

    This procedure is linear to 4000 mg/L. Samples with higher values should be diluted with normal saline and retested.

    OPTIONAL EXERCISE

    1. Your instructor will provide a series of plasma samples that have been placed in a refrigerator (4-8 °C) overnight.
    2. Observe these samples and indicate whether they are cloudy (turbid) or clear, exhibit a cream layer on the top, or some combination of these.
    3. Record your observations on the data sheet.
    4. From Chapter 33, what type, if any, of hyperlipidemia may be associated with each sample?
    DATA SHEET, EXERCISE #17

    NAME: ___________

    DATE: ___________

    RESULTS

      Initial Absorbance
    (10 Minutes)
    \(\Delta\)A
    (Total)
    Triglycerides
    (mg/L)
    Standard       (Target Value)
    Control        
    Serum #        
    Serum #        

    OPTIONAL EXERCISE

    Check one or more of these observations for each sample

    Sample # Clear Slightly Cloudy Very Cloudy Creamy Layer Type of Hyperlipidemia
    1          
    2          
    3          
    4          
    5          
    6          

    Discussion Questions

    1. What is the expected appearance of a serum sample containing elevated triglycerides?
    2. What type (classification) of lipid abnormality will show marked elevation of triglycerides and the presence of chylomicrons?
    3. What disease or condition can be a secondary cause of elevated triglycerides?

    2.17: Triglycerides is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by LibreTexts.

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