Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

4. Add 0.05 ml serum in 5 ml of precipitating reagent, mix well and keep for 5 min,
   and centrifuge it at 3000 rpm for 5 min. Take 3 ml of supernatant and add to the
   tube labelled as T.


5. To each tube add 2 ml of conc. H 2 SO 4 through the sides of the tube. Mix well
   immediately. Incubate for 20 min at room temperature and read absorbance at
   550 nm.

Reagents Blank S 1 S 2 S 3 S 4 S 5 T

Cholesterol standard (μl) – 20 40 60 80 100 –

Cholesterol amount (μg) – 20 40 60 80 100 –

Precipitating reagent (ml) 3 2.98 2.96 2.94 2.92 2.90 –

Protein-freefiltrate (ml) –––––– 3

Conc. H 2 SO 4 (ml) 2 222222

Overlay and mixed thoroughly and keep at room temperature for 20 min

23.7 Calculations

Plot a graph by taking cholesterol amount at x-axis vs absorbance at y-axis, and
extrapolate cholesterol concentration of the given serum sample from the graph.The
amount of cholesterol extrapolated from graph will be in 0.03 ml of serum
sample since 0.05 ml of serum sample was mixed with precipitating reagent and
3 ml of this was used for cholesterol analysis. The cholesterol amount in serum
sample can also be calculated by using equation:

Cholesterol in serum¼

ODof testamount of standardðÞμg 100

ODof standardvolume of sample 0ðÞ:03ml 1000

¼xmg=dl

23.8 Clinical Significance...............................

Normal range of total cholesterol in blood ranges from 150 to 240 mg/dl. Serum
cholesterol rises with age. Hypercholesterolemia occurs in nephrotic syndrome,
diabetes mellitus, obstructive jaundice (due to obstruction of large ducts), coronary
thrombosis, angina pectoris, atherosclerosis, and other heart disease. In pregnancy,
the increase in cholesterol levels may reach 20–25% of the normal value. Hypercho-
lesterolemia occurs in hypothyroidism, liver damage, pernicious anemia, hemolytic
jaundice, abetalipoproteinemia, and cortisone administration.

23.8 Clinical Significance 95