Measurements of Cholesterol

Question 1

Functions of cholesterol (4)

Answer 1

Membrane component
Steroid synthesis
Bile acid/salt precursor
Vitamin D precursor

Question 2

What assays are available for cholesterol measurement?

Answer 2

Total serum cholesterol assay

Use of cholesterol dehydrogenase

Question 3

How does the assay for cholesterol work?

Answer 3

Total Serum Cholesterol Assay

1) Cholesterol ester —> cholesterol + fatty acid (cholesterol esterase)
2) Cholesterol –> Ketone (cholesterol oxidase) + H2O2

3) H2O2 + ADPH –> Resorufin
(horseradish peroxidase)

Resorufin = fluorescence

Question 4

How can cholesterol dehydrogenase be used to measure cholesterol?

Answer 4

Cholesterol undergoes oxidation

• NAD+ –> NADH
• absorbance of NADH can be measured (linear relationship)

Question 5

What constitutes as high risk cholesterol when measuring using the total serum cholesterol assay?

Answer 5

Around > 200mg/dL

Increased values as age increases

Question 6

How do cholesterol testing devices work?

Answer 6

Electrochemical assays (biosensor)

• cholesterol oxidase
• current is generated
• measured as a value

Question 7

What is specifically measured in total serum cholesterol assay?

Answer 7

VLDL + LDL + HDL

Hard to distinguish due to same size (difference in lipid proportion)

Question 8

How are HDLs calculated?

Answer 8

HDL does not contain apoprotein B
- VLDL, LDL and chyclomicrons do

Addition of reagent sulphated-a-cyclodextrin blocks apoprotein B, effectively excluding everything but HDL
- Only HDL will react with the cholesterol enzyme reagent thus allows estimate

Question 9

What is detected in pregnancy tests?

Answer 9

HCG - human chorionic gonadotropin (elevated levels)

Question 10

What type of assay is used in pregnancy testing?

Answer 10

Chromatographic immunoassay

Question 11

How do pregnancy tests work

Answer 11

LATERAL FLOW DEVICE

Sample of urine travels up absorbent element (capillary action)

• attach to mobilised antibodies labelled with dye (form antibody-antigen complex I)
• carried by sample fluid up the lateral flow wick
• attaches to secondary antibodies specific to HCG which are immobilised, thus producing a coloured line
• 1st coloured line shows positive result
• moves further to 3rd zone where reacts with anti-antibody: coloured line forms independent of HCG present (control zone to ensure test is working)