Method of determining skin tissue cholesterol

• US 6,365,363 B1
• Filed: 11/10/1999
• Issued: 04/02/2002
• Est. Priority Date: 02/20/1997
• Status: Expired due to Fees

• Current Assignee: IMI International Medical Innovations, Inc.
• Original Assignee: IMI International Medical Innovations, Inc.
• Inventors: Lopukhin, Yuri Michaelovich, Parfenov, Alexandr Sergeivich
• Primary Examiner(s): Gitomer, Ralph
• Assistant Examiner(s): CHAUDHRY, MAHREEN F
• Application Number:
  US09/367,724
• Time in Patent Office: 874 Days
• Field of Search: 435/11, 435/4, 435/25, 435/19
• US Class Current: 435/11
• CPC Class Codes: C12Q 1/60 involving cholesterol

Abstract

A method of determining skin tissue cholesterol includes preparing a section of skin surface, applying a dosed amount of an enzyme-containing mixture onto the skin, exposing, evaluating the concentration of cholesterol in a reaction solution and calculating the concentration of skin cholesterol, wherein the area of contact of the skin with the mixture is limited on the section of the skin surface by means of a sealing vessel without a bottom with a base fixed on the skin, a mixture comprising 2.0-2.5 units of cholesteroloxidase, 0.04-0.06% by weight of sodium dezoxycholate, 0.1-0.2.% by weight of 3-(dodecyl-dimethyl-ammonium)-propanesulfonate in a buffer solution with pH=6.8 is used as the mixture, evaluating the concentration of cholesterol in the reaction solution is carried out by measuring the level of hydrogen peroxide. Measurements of the level of hydrogen peroxide may be carried out by immersing an electrochemical sensor or a colorimetric indicator in the reaction solution.

Claims

1. A method of determining cholesterol present on skin surface, comprising the steps of:

a. providing an open-ended vessel having a base;

b. providing an enzyme solution comprising 2.0 to 2.5 units of cholesterol oxidase, 0.04 to 0.06 weight % of sodium dezoxycholate, and 0.1 to 0.2 weight % of 3-dodecyl-dimethyl-ammonium-propanesulfonate, buffered to a pH of 6.8 in water;

c. bringing said vessel into contac with the skin surface in a manner such that the base of said vessel sealingly engages the skin surface, thereby delimiting an area of said skin surface within said vessel;

d. adding a predetermined amount of said enzyme solution into said vessel so that said enzyme solution contacts the skin surface over the delimited area thereof;

e. allowing said enzyme solution to remain in contact with the skin surface over the delimited area thereof for a period of time sufficient for the cholesterol oxidase to catalyze oxidation of cholesterol and cause formation of hydrogen peroxide; and

,

f. measuring the amount of hydrogen peroxide formed in step (e), the amount of hydrogen peroxide measured being indicative of the concentration of skin cholesterol.

2. A method according to claim 1 wherein said enzyme solution further comprises 3 to 5 units of cholesterol esterase.

3. A method according to claim 2, wherein said 3 to 5 units of cholesterol esterase are added to said enzyme solution in said vessel between steps (d) and (e).

4. A method according to claim 1, wherein step (f) is carried out by spectrophotometry after the addition of peroxidase and a substrate to said enzyme solution in said vessel.

5. A method according to claim 1, wherein step (f) is carried out by means of an electrochemical sensor that is immersed into said enzyme solution.

6. A method according to claim 1, wherein step (f) is carried out by means of a colorimetric indicator immersed into said enzyme solution.