Purification of lipoprotein cholesterol for use as a cholesterol reference material
AssigneeMiles Laboratories, Inc.
InventorsMakram M. Girgis, David E. Jackson, Frank J. Mannuzza
Life of this patent
Estimating the term…
Abstract
A method for the purification of lipoprotein cholesterol. The method involves the steps of adsorbing lipoprotein material on a silica adsorbant, eluting the adsorbed lipoprotein with water at a pH of about 10 to 11.5, adjusting the salt concentration to about less than 0.05M, heating the lipoprotein to about 50.degree. to 100.degree. C. for a period of about 5 minutes to 24 hours, adding an alkaline carbonate and alkaline earth salt to form a precipitate and removing the precipitate, adjusting the pH from about 6.5 to 9.0 and recovering purified lipoprotein cholesterol. Also a method for using the cholesterol as a cholesterol reference material.
Claims11
In a method for purifying lipoprotein cholesterol which involves the steps of adsorbing a solution of a lipoprotein-containing substance onto a silica adsorbant, separating the adsorbed lipoprotein from any excess solution present, freezing and thawing the adsorbed lipoprotein, eluting the adsorbed lipoprotein, concentrating the lipoprotein to a desired concentration and recovering purified lipoprotein cholesterol therefrom, the improvement comprising the steps of eluting the adsorbed lipoprotein at a pH from about 10 to 11.5, adjusting the salt concentration to about less than 0.05 M, heating the eluted lipoprotein to a temperature of from about 50.degree. to 100.degree. C. for a period of time sufficient to increase the storage stability of the purified lipoprotein cholesterol, adding an alkaline carbonate and an alkaline earth salt;
- adjusting the pH from about 6.5 to 9.0;
- and recovering therefrom said lipoprotein cholesterol.
A method for purifying lipoprotein cholesterol which comprises the steps of:
- (a) adsorbing a solution of lipoprotein-containing substance onto a silica adsorbant;
- (b) separating the adsorbed lipoprotein from any excess solution present;
- (c) freezing and thawing the adsorbed lipoprotein;
- (d) eluting the adsorbed lipoprotein at a pH of from about 10 to 11.5;
- (e) concentrating the lipoprotein obtained from step (d) to a desired concentration of cholesterol;
- (f) adjusting the pH of the lipoprotein from about 7.0 to 10.0;
- (g) adjusting the salt concentration to about less than 0.05 M;
- (h) heating the lipoprotein to a temperature of from about 50.degree. to 100.degree. C. for a period of about 5 minutes to 24 hours;
- (i) adding an alkaline carbonate and an alkaline earth salt;
- (j) adjusting the pH of the lipoprotein from about 6.5 to 9.0;
- and
- (k) recovering therefrom purified lipoprotein cholesterol.
A purified lipoprotein cholesterol prepared by the steps of:
- (a) adsorbing a solution of lipoprotein-containing substance onto a silica adsorbant;
- (b) separating the adsorbed lipoprotein from any excess solution present;
- (c) freezing and thawing the adsorbed lipoprotein;
- (d) eluting the adsorbed lipoprotein at a pH of from about 10 to 11.5;
- (e) concentrating the lipoprotein obtained from step (d) to a desired concentration of cholesterol;
- (f) adjusting the pH of the cholesterol from about 7.0 to 10.0;
- (g) adjusting the salt concentration to about less than 0.05 M;
- (h) heating the cholesterol to a temperature of from about 50.degree. to 100.degree. C. for a period of about 5 minutes to 24 hours;
- (i) adding an alkaline carbonate and an alkaline earth salt;
- (j) adjusting the pH of the cholesterol from about 6.5 to 9.0;
- and
- (k) recovering therefrom purified lipoprotein cholesterol.
A method of determining the cholesterol content of a test sample which includes the step of using as a cholesterol reference material a composition consisting essentially of purified lipoprotein cholesterol prepared by the steps of:
- (a) adsorbing a solution of lipoprotein-containing substance onto a silica adsorbant;
- (b) separating the adsorbed lipoprotein from any excess solution present;
- (c) freezing and thawing the adsorbed lipoprotein;
- (d) eluting the adsorbed lipoprotein at a pH of from about 10 to 11.5;
- (e) concentrating the lipoprotein obtained from step (d) to a desired concentration of cholesterol;
- (f) adjusting the pH of the cholesterol from about 7.0 to 10.0;
- (g) adjusting the salt concentration to about less than 0.05 M;
- (h) heating the cholesterol to a temperature of from about 50.degree. to 100.degree. C. for a period of about 5 minutes to 24 hours;
- (i) adding an alkaline carbonate and an alkaline earth salt to form a precipitate and removing the precipitate;
- (j) adjusting the pH of the cholesterol from about 6.5 to 9.0;
- and
- (k) recovering therefrom purified lipoprotein cholesterol.
Dependencies are inferred from the claim text.