Method of determining skin tissue cholesterol

Details Method of determining skin tissue cholesterol Method of determining skin tissue cholesterol

1999-11-10

2002-04-02

Gitomer, Ralph (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving cholesterol

C435S004000, C435S019000, C435S025000

Reexamination Certificate

active

06365363

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the field of medicine, more exactly to a method of examining the skin with biochemical methods. The invention may also be used in cosmetology, pharmacology, criminalistics, etc., to determine the content of cholesterol, both free and total, in the skin.
BACKGROUND ART
In clinical conditions the determination of cholesterol in the skin makes it possible in a noninvasive manner, i.e. without taking blood samples or biopsy material, to evaluate the level of tissue cholesterol.
A method is known for determining the presence of different chemical substances on the surface of the skin, these substances being in the blood channel of a human or animal (glucose, alcohol, lactate, hypoxanthine). The method includes the subcutaneous administration of an enzyme immobilized on a silicon surface, heating a section of the skin to 38-44° C. and registering a local change in the concentration of oxygen or hydrogen peroxide on the skin by means of an electrochemical sensor (U.S. Pat. No. 4,458,686, A 61 B 5/00, published July 1984).
A drawback of the known method is the complexity of subcutaneous administration of an immobilized enzyme, the formation of hydrogen peroxide directly in tissues, which may result in undesirable consequences (the transition of hemoglobin into methemoglobin)—the development of hypoxia.
A method is known for determining skin cholesterol by taking samples of tissue with subsequent extraction of cholesterol. The method makes it possible to determine not only the amount of total cholesterol, but separately free and esterified (H. Bouissou, M. Th. Pieraggi, M. Julian, Identifying arteriosclerosis and aortic ateromathosis by skin biopsy. Atherosclerosis, v. 19, pp. 449-458).
A development of this method is an extraction method, in accordance with which extraction of cholesterol is carried out without taking a tissue sample directly on a section of live skin. A mixture of ethyl alcohol and ether is used as the extractant. A test tube with the extractant is held against the surface of the skin for several minutes. The lipids which are in the surface layers of the skin dissolve and pass into the extractant solution, after which evaporation of the solvent is carried out and the substances passing into a liquid phase are determined (Yu. M. Lopukhin. The skin and atherosclerosis [a three-drop test]. 1992, Gordon and Breach Science Publishers S.A. UK).
The content of cholesterol in the epidermis reflects the accumulation of cholesterol in the skin and correlates with the content of the latter in the aorta, and also with the area of damage of the aorta in the case of atherosclerosis (Yu. M. Lopykhin. The skin and atherosclerosis [a three-drop test], 1992, Gordon and Breach Science Publishers S.A. UK). Thus, determination of the content of cholesterol in the skin makes it possible to obtain unique information on the state of the tissue pool of cholesterol, and this is extremely important from both the point of view of early diagnosis of atherosclerosis (preclinical state) and for the purpose of monitoring the treatment of patients with atherosclerosis.
In pharmacology, the method of determining cholesterol in the skin makes it possible to carry out evaluation of the effectiveness of drugs which affect both the synthesis of cholesterol in tissues (a group of statins), and its removal from the organism by means of different sorbents.
A drawback of the known method is the necessity of carrying out a biopsy, contact of skin with the solution of extractants, and also the lengthy time necessary to carry out the analysis.
The method most similar to the proposed method is the method of determining skin cholesterol including preparing a section of skin surface, applying onto the skin (or another cholesterol-containing surface) a dosed amount of a solution that is an affino-enzymatic conjugate of a general structure A—C—B, wherein A is a compound capable of linking with cholesterol (affinate), C is a linking bridge or polymer, B is an enzyme providing a color product as a result of conversion of an according substrate. The amount of cholesterol is determined according to the intensity of the transition of the dye to a dyed form (U.S. Pat. No. 5,489,510, A 61 B 10/00, published February 1996—prototype).
Drawbacks of this method are the limited field of use, i.e. the impossibility of determining esterified cholesterol, the low specificity of the analysis, in particular interaction of the conjugate not only with cholesterol, but also with other lipids.
DISCLOSURE OF THE INVENTION
The object to the attainment of which the instant invention is directed is to increase the specificity, simplify and expand the field of utilization of the method, increase the accuracy of determination of cholesterol in the skin of a human and other condensed mediums.
The stated object is attained in a method of determining skin cholesterol, including preparing a section of a surface, applying onto the skin surface a dosed amount of an aqueous solution of an enzyme with addition of a surfactant, exposing, evaluating the concentration of cholesterol in a reaction solution and calculating the concentration of skin cholesterol, in that in accordance with the proposal, after preparing the section of the surface, an area of contact of the skin with the enzyme solution and the surfactant is limited by means of an appliance, the enzyme solution with the surfactant for determination of free tissue cholesterol has the following composition (% by weight):
cholesteroloxidase
2.0-2.5 units
sodium dezoxycholate
0.04-0.06
3-(dodecyl-dimethyl-ammonium)-
0.1-0.2
-propanesulfonate
phosphate buffer pH 6.8
balance to 100%,
while in order to determine the total cholesterol, the composition has the following makeup (% by weight);
cholesteroloxidase
2.0-2.5 units
cholesterolesterase
3-5 units
sodium dezoxycholate
0.04-0.06
3-(dodecyl-dimethyl-ammonium)-
0.1-0.2
-propanesulfonate
phosphate buffer pH 6.8
balance to 100%,
exposing the solution on the skin is carried out for 2 minutes to determine free cholesterol and for 10 minutes to determine total cholesterol, while evaluation of the concentration of cholesterol is carried out by direct measurement of the concentration of hydrogen peroxide in the reaction solution. Limitation of the area of contact of the skin with the surfactant solution is carried out using a sealed cuvette without a bottom with a base fixed on the skin.
Cholesteroloxidase, obtained from different sources (Brevibacterium sp., Nocardia erythropolis), is used. The hydrogen peroxide generated during interaction of cholesterol with cholesteroloxidase is determined by means of an electrochemical sensor or by using spectrophotometric equipment.
The cholesteroloxidase catalyzes oxidation of cholesterol with the participation of molecular oxygen and the formation of hydrogen peroxide:
cholesterol+O
2
=4-cholestine-3-OH+H
2
,O
2
When a mixture of a surfactant—ionic detergent (sodium dezoxycholate [DOC])—in a concentration of 0.04-0.06% and a zwitterion detergent - 3-(dodecyl-dimethyl-ammonium)-propanesulfonate (DAPS) in a concentration of 0.1-0.2% is used, optimum speed and completeness of oxidation of cholesterol are detected. This reaction system makes it possible to enhance the effectiveness of action of cholesteroloxidase as a result of reduction of the inhibiting action of the surfactant and to reduce the consumption of expensive detergents. Such a composition of detergents ensures access to free cholesterol and a high speed of its oxidation. This makes it possible to use relatively low concentrations of cholesteroloxidase in the determination of free cholesterol. When a kinematic variant of measurement is used, only 4-8 nmols per ml of reaction mixture, and during determination according to the final point to carry out complete oxidation of cholesterol in 10 minutes, 20 nmols of enzyme per ml of reaction mixture are sufficient.
Photometric methods make it possible to achieve the lower limit of detecting oxidase substrates of about

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