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Sunday, December 13, 2009

Estimate blood serum cholesterol Lieberman Buchan reaction

To estimate blood (or serum) cholesterol by Lieberman-Buchan reaction. Acetic anhydride reacts with cholesterol in CHCI3 soln to produce a characteristic blue green co lour. The exact nature of chromospheres is not known but the reaction probably includes etherification of hydroxyl group in the 3 – posn as well as other in the mol.
Cholesterol is abs-from the diet (about 0.3 mgm/day) as well as is syn. In the body (1.5 – 2 mg/day) free cholesterol is equally distributed b/w the cells and the plasma while the etherification form occurs only in plasma. The normal serum cholesterol lies with in the rang 100-250 mgm/100ml. The average serum cholesterol is 200 mgs/100ml at the age of 25 ml in men and rises slowly with age reading a peak figure at age of 40-50 and then declining women in generation show a lower cholesterol level than men until monoplane. When the value rises above that found in men of the same age.


Note: - It is essential to use absolutely dry glassware for this estimation.

Requirements: - Std cholesterol soln (1 mg/ml). Chloroform, acetic anhydride, conc. H2SO4, blood or serum.

Procedure: - Prepare series of test tube, as given Add all the soln from burette mix the soln in each tube and plug with cork and keep in dark for 15 mins measure the co lour developed at 680 nm against blank.
Isolation of blood (or serum) cholesterol. Add 0.2 ml of serum or bld. to 10 ml. mix of acetone alcohol mix(1:1) in a centrifuge tube keep the tube in boiling water bath with intermittent shaking till the solvent begin to boil. Remove the tube the W.B.shake it for 10 min and centrifuge decant the supernatant liquid into a dry t.t.and evap. To dryness on a W.B. cool and dissolve in 2 ml chloroform. Use 1 ml for co lour development. Cholesterol level in blood is det to follow the treatment of diseases like hypo hyperthyroidism diabetes, coronary artery diseases and arthero scleroses.

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