High ascorbic acid in a sample gives a falsely low cholesterol by an enzymatic method that ends in a peroxidase (Trinder) reaction. Why?

It uses up the peroxide before the dye forms

It inhibits the cholesterol esterase

It precipitates the LDL in the sample

It adds color at the read wavelength

A 45-year-old man's fasting sample gives total cholesterol 310 mg/dL (8.02 mmol/L), HDL cholesterol 34 mg/dL (0.88 mmol/L) and triglyceride 980 mg/dL (11.1 mmol/L). Which approach gives the most reliable LDL cholesterol?

Ultracentrifugation (beta-quantification)

Friedewald after a 1:2 dilution

A visibly clear serum has a triglyceride of 720 mg/dL (8.13 mmol/L) by a standard enzymatic method, but 95 mg/dL (1.07 mmol/L) by a glycerol-blanked method. What best explains the difference?

High free glycerol in the sample

Chylomicrons from a recent meal

Hemoglobin from in vitro hemolysis

Lipids: test procedures and interferences

8 ASCP MLS practice questions on Lipids: test procedures and interferences, part of Chemistry. The first 8 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Hard
A 50-year-old man's first lipid panel is drawn 3 hours after a large, high-fat breakfast: total cholesterol 204 mg/dL [5.28 mmol/L], HDL cholesterol 45 mg/dL [1.16 mmol/L], triglyceride 330 mg/dL [3.73 mmol/L]. His clinician asks for a repeat after a 12-hour fast one week later, with no change in diet or drugs: total cholesterol 207 mg/dL [5.35 mmol/L], HDL cholesterol 46 mg/dL [1.19 mmol/L], triglyceride 145 mg/dL [1.64 mmol/L]. The laboratory calculates LDL cholesterol with the Friedewald equation. Which value from the first, non-fasting sample agrees best with the fasting result?
1. AFriedewald LDL cholesterol
2. BTriglyceride
3. CNon-HDL cholesterol
4. DCalculated VLDL cholesterol
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Answer: C · Non-HDL cholesterol
Non-HDL cholesterol is total minus HDL cholesterol: 159 mg/dL non-fasting and 161 mg/dL fasting, almost identical. Friedewald LDL (total - HDL - TG/5) is 204 - 45 - 66 = 93 mg/dL non-fasting but 207 - 46 - 29 = 132 mg/dL fasting. After a fatty meal, chylomicrons and their remnants add triglyceride that carries relatively little cholesterol, so TG/5 overstates VLDL cholesterol and the calculated LDL comes out too low. Total and HDL cholesterol change little with eating, which is why non-HDL cholesterol is reliable on non-fasting samples. When a calculated LDL is needed, a fasting sample is preferred.
- A: Calculated LDL is 93 mg/dL non-fasting but 132 mg/dL fasting, because TG/5 overstates VLDL cholesterol after a meal.
- B: Triglyceride fell by 185 mg/dL once he fasted.
- D: TG/5 gives 66 versus 29 mg/dL; it rises with meal triglyceride.
Question 2 · Medium
High ascorbic acid in a sample gives a falsely low cholesterol by an enzymatic method that ends in a peroxidase (Trinder) reaction. Why?
1. AIt inhibits the cholesterol esterase
2. BIt precipitates the LDL in the sample
3. CIt uses up the peroxide before the dye forms
4. DIt adds color at the read wavelength
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Answer: C · It uses up the peroxide before the dye forms
In the Trinder step, peroxidase uses hydrogen peroxide to oxidize a chromogen into a colored dye. Ascorbic acid is a reducing agent that consumes the peroxide or reduces the dye, so less color forms and the result reads low. High bilirubin causes a similar negative interference. Some reagents contain ascorbate oxidase to reduce this effect.
- A: Ascorbic acid does not inhibit the esterase step.
- B: Ascorbic acid does not precipitate lipoproteins.
- D: Added color would raise the result, and ascorbic acid is colorless.
Question 3 · Easy
In the enzymatic method for total cholesterol, the enzyme that acts first on the sample is:
1. ALipoprotein lipase
2. BCholesterol esterase
3. CCholesterol oxidase
4. DPeroxidase
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Answer: B · Cholesterol esterase
About two thirds of plasma cholesterol is esterified, so cholesterol esterase first frees the cholesterol from its fatty acids. Cholesterol oxidase then oxidizes all the free cholesterol, producing hydrogen peroxide. Peroxidase uses the peroxide to form a colored dye (Trinder reaction) that is read by photometry. If the esterase step fails, results are falsely low because only free cholesterol is measured.
- A: Lipoprotein lipase or a microbial lipase is used in triglyceride methods.
- C: Cholesterol oxidase acts second, on free cholesterol.
- D: Peroxidase catalyzes the final color-forming step.
Question 4 · Hard
A 45-year-old man's fasting sample gives total cholesterol 310 mg/dL (8.02 mmol/L), HDL cholesterol 34 mg/dL (0.88 mmol/L) and triglyceride 980 mg/dL (11.1 mmol/L). Which approach gives the most reliable LDL cholesterol?
1. ALipoprotein electrophoresis
2. BThe Friedewald equation
3. CUltracentrifugation (beta-quantification)
4. DFriedewald after a 1:2 dilution
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Answer: C · Ultracentrifugation (beta-quantification)
The Friedewald equation is invalid above a triglyceride of 400 mg/dL (4.5 mmol/L), because the TG/5 estimate of VLDL cholesterol no longer holds. Beta-quantification, the reference method, ultracentrifuges plasma at density 1.006 g/mL to float off VLDL and chylomicrons. LDL cholesterol is the cholesterol of the bottom fraction minus its HDL cholesterol. Diluting the sample does not help, because the VLDL composition is still abnormal.
- A: Electrophoresis shows lipoprotein patterns but does not give a reliable LDL value.
- B: The Friedewald equation is invalid when triglyceride is above 400 mg/dL.
- D: Dilution lowers all values equally, so the TG/5 error remains.
Question 5 · Hard
A visibly clear serum has a triglyceride of 720 mg/dL (8.13 mmol/L) by a standard enzymatic method, but 95 mg/dL (1.07 mmol/L) by a glycerol-blanked method. What best explains the difference?
1. AA very high HDL cholesterol
2. BHigh free glycerol in the sample
3. CChylomicrons from a recent meal
4. DHemoglobin from in vitro hemolysis
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Answer: B · High free glycerol in the sample
Enzymatic triglyceride methods use lipase to release glycerol and then measure total glycerol. Free glycerol already in the sample is counted as triglyceride unless it is measured first and subtracted (glycerol blanking). A true triglyceride of 720 mg/dL would make serum turbid, so a clear sample is a clue. Free glycerol is high in glycerol kinase deficiency and after IV infusions that contain glycerol.
- A: HDL carries little triglyceride and does not affect the glycerol blank.
- C: Chylomicrons would make the serum turbid and would raise both methods.
- D: The serum is clear, and hemolysis causes only a small bias, not a sevenfold difference.
Question 6 · Medium
In precipitation methods for HDL cholesterol, a reagent such as dextran sulfate with magnesium is added and the tube is centrifuged. The pellet contains the lipoproteins that:
1. ACarry apo B
2. BMigrate as alpha bands
3. CCarry apo A-I
4. DAre denser than HDL
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Question 7 · Easy
A fasting lipid profile is ordered. For how long should the patient go without food beforehand, with water allowed?
1. A9 to 12 hours
2. B2 to 4 hours
3. C24 to 36 hours
4. D4 to 6 hours
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Question 8 · Medium
A request for lipoprotein electrophoresis does not record whether the patient fasted, and his triglyceride is 260 mg/dL [2.94 mmol/L]. Refer to the gel shown. The most appropriate action is:
Figure: MedExams
1. ARecollect the sample after a 12-hour fast
2. BReport a raised lipoprotein(a)
3. CReport type I hyperlipoproteinemia
4. DReport type V hyperlipoproteinemia
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Lipids: test procedures and interferences

High ascorbic acid in a sample gives a falsely low cholesterol by an enzymatic method that ends in a peroxidase (Trinder) reaction. Why?

In the enzymatic method for total cholesterol, the enzyme that acts first on the sample is:

A 45-year-old man's fasting sample gives total cholesterol 310 mg/dL (8.02 mmol/L), HDL cholesterol 34 mg/dL (0.88 mmol/L) and triglyceride 980 mg/dL (11.1 mmol/L). Which approach gives the most reliable LDL cholesterol?

A visibly clear serum has a triglyceride of 720 mg/dL (8.13 mmol/L) by a standard enzymatic method, but 95 mg/dL (1.07 mmol/L) by a glycerol-blanked method. What best explains the difference?

In precipitation methods for HDL cholesterol, a reagent such as dextran sulfate with magnesium is added and the tube is centrifuged. The pellet contains the lipoproteins that:

A fasting lipid profile is ordered. For how long should the patient go without food beforehand, with water allowed?

A request for lipoprotein electrophoresis does not record whether the patient fasted, and his triglyceride is 260 mg/dL [2.94 mmol/L]. Refer to the gel shown. The most appropriate action is: