Abstract:
Skin cholesterol is measured by applying an adhesive tape onto a selected area of the skin to adhere the tape to the selected skin area and stripping the tape off the selected skin area to obtain a sample representative of the outer stratum corneum layer of the skin, the sample adhering to the tape so as to have exposed skin constituents. The sample is assayed using a detector reagent that specifically binds to cholesterol and in addition has an indicator component that allows quantitation of the amount of cholesterol present in the exposed skin constituents.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     The present invention relates to a method of measuring skin cholesterol. More particularly, the invention pertains to a method for the direct assay of cholesterol in skin samples removed by tape stripping, with a view to identifying individuals at risk of having atherosclerosis as well as those at risk of developing atherosclerosis and similar diseases associated with and attributable to high cholesterol levels.  
         [0002]     Numerous studies have shown that atherosclerosis and its complications, such as heart attacks and strokes, are major causes of morbidity and mortality in almost all countries of the world.  
         [0003]     Cost effective prevention of atherosclerosis requires the identification of individuals at risk, thereby allowing their medical treatment and change of life style. A desired goal is identifying those individuals belonging to the high-risk group but there are difficulties in selecting optimum methods for discriminating individuals at risk.  
         [0004]     A widely used method for identifying individuals at risk of having atherosclerosis is based on the measurement of total cholesterol levels in venous blood plasma (Consensus Conference on Lowering Blood Cholesterol to Prevent Heart Disease, JAMA, 1985, 253, pg. 2080). Patients are considered to be at high-risk if their cholesterol level is over 240 mg/dL and there have been recent moves to lower this threshold level to lower values.  
         [0005]     However, total cholesterol levels alone do not accurately predict a patient&#39;s risk level. A better prediction can be made by analyzing blood plasma lipoproteins; in particular, measurement of low density and high-density lipoprotein (HDL) cholesterol levels is advantageous (Total and High Density Lipoprotein Cholesterol in the Serum and Risk of Mortality, British Medical Journal, 1985, 290, pg. 1239-1243).  
         [0006]     Despite their advantage, use of the above methods requires blood sampling after a period of fasting. Additionally, the sampling is uncomfortable, poses a risk of infection and the required analysis of plasma lipoproteins and cholesterol is complicated and expensive. Moreover, studies have shown that blood plasma analysis may not entirely reflect the process of cholesterol accumulation in the arterial wall and other tissues. In many cases, neither plasma cholesterol levels nor even complete lipid profiles correlate with the severity of atherosclerosis.  
         [0007]     Significant levels of cholesterol occur in tissue as well as in plasma and it has been shown that tissue cholesterol plays a leading role in development of atherosclerosis. Tissues, including skin, have been identified which accumulate cholesterol in the same way as the arterial wall and studies have demonstrated a close correlation between cholesterol content in the arterial wall and the skin. For example, cholesterol was extracted from lyophilized skin samples and measured using traditional chemical and biochemical techniques. (Nikitin Y. P., Gordienko I. A., Dolgov A. V., Filimonova T. A. “Cholesterol content in the skin and its correlation with lipid quotient in the serum in normals and in patients with ischemic cardiac disease”, Cardiology, 1987, II, No. 10, P. 48-51). While useful, this method is too complicated and painful to be employed for large scale population screening.  
         [0008]     U.S. Pat. No. 4,458,686 describes a method of quantifying various compounds in the blood directly under the skin or on its surface. The method is based on measuring oxygen concentration changes electrochemically, for instance, via polarography. In the case of non-volatile substances that do not diffuse through the skin, it is necessary to implant enzymes under the skin to effect oxygen changes at the skin surface. This patent also discloses the potential of using such methods to quantify the amount of cholesterol using cholesterol oxidase. The complex instrumentation and procedures needed require the services of highly skilled personnel for making measurements, thus limiting the usefulness of the method for screening large numbers of people.  
         [0009]     Determination of the cholesterol content in skin gives a measure of the extent of atherosclerosis and can be obtained through standard laboratory analysis of skin biopsy specimens. However, there is considerable pain involved in taking a skin sample and a risk of infection at the sampling site. In addition, this method has other disadvantages because the thick skin specimens incorporate several skin layers, including the outermost horny layer (stratum corneum), epidermis and dermis. Since the dermal layer is highly vascularized, skin biopsy samples contain blood vessels and blood elements. They may also contain sweat and sebaceous glands and the secretions contained therein. Additionally, subcutaneous fat is located directly under the derma and may also contaminate specimens. Therefore, skin biopsy specimens are heterogeneous and their analysis may give false data on cholesterol content in the skin.  
         [0010]     U.S. Pat. No. 5,489,510 describes a non-invasive method for the visual identification of cholesterol on skin using a reagent having a specific cholesterol binding component in combination with a reagent having an indicator component to provide a visual color change corresponding to the presence of the component bound to cholesterol of the skin. The method overcomes many of the objections of earlier procedures and meets many of the desired goals required for a simple mass screening to identify individuals at risk of having atherosclerosis. The procedure is done directly on the palmar skin and, while it is quick and simple, it requires all individuals to be tested to be present at a doctor&#39;s office or clinic where the test is conducted. This of course limits effective large scale screening.  
         [0011]     Molar ratios of the lipids, including cholesterol, in stratum corneum have been determined on samples obtained by direct, solvent extraction of skin (Norlen L., et al. J. Invest. Dermatology 72-77, 112, 1999). High performance liquid chromatography (HPLC) and gas liquid chromatography in conjunction with mass spectrometry were used to separate and analyze the lipids. The analytical methods are complex, but more importantly, the use of corrosive and irritant organic solvent systems to extract human skin for routine determinations is not practical.  
         [0012]     The lipid profile of the stratum corneum layer of skin has been determined using a tape stripping method as described by A. Weerheim and M. Ponec (Arch. Dermatol. Res., 191-199, 293, 2001). In this study, lipids, including cholesterol, were solvent extracted from stratum corneum after tape stripping of skin. The resultant lipid extract was separated by high performance thin-layer chromatography. This method is very laborious. It requires three consecutive solvent systems to effect the separation of the lipids, a staining and charring method to visualize the components and a densitometry step to determine the relative amounts of the lipids. The method does not lend itself to the simple and rapid determination of cholesterol levels in large numbers of samples.  
       SUMMARY OF THE INVENTION  
       [0013]     It is therefore an object of the present invention to overcome the above drawbacks and to provide a simple and non-invasive method of measuring skin cholesterol, which allows for effective large scale screening.  
         [0014]     According to a first aspect of the invention, there is provided a method of measuring skin cholesterol, which comprises the steps of: 
        a) providing a tape comprising a backing member coated on at least one side thereof with a medical adhesive;     b) applying the tape onto a selected area of skin to adhere the tape to the selected skin area;     c) stripping the tape off the selected skin area to obtain a sample representative of an outer stratum corneum layer of the skin, the sample adhering to the tape so as to have exposed skin constituents;     d) providing a source of an affinity-enzymatic compound of formula A-C-B, wherein A is a detecting agent having affinity for cholesterol, B is an enzymatic visualizing agent and C is a binding agent linking the detecting agent and the visualizing agent to one another;     e) applying a predetermined amount of the affinity-enzymatic compound onto a predetermined surface area of the sample and allowing the compound to remain in contact therewith for a period of time sufficient to cause binding of the detecting agent to cholesterol present in the exposed skin constituents; and     f) applying a predetermined amount of a color developing agent onto the predetermined surface area of the sample, whereby the color developing agent reacts with the enzymatic visualizing agent to form a colored product having a color indicative of cholesterol level.        
 
         [0021]     According to a second aspect of the invention, there is provided a method of measuring skin cholesterol, which comprises the steps of: 
        a) providing a tape comprising a backing member coated on at least one side thereof with a medical adhesive;     b) applying the tape onto a selected area of skin to adhere the tape to the selected skin area;     c) stripping the tape off the selected skin area to obtain a sample representative of an outer stratum corneum layer of the skin, the sample adhering to the tape so as to have exposed skin constituents;     d) providing a source of an affinity signal-generating compound of formula A-C-B′, wherein A is a detecting agent having affinity for cholesterol, B′ is a signal-generating indicator agent and C is binding agent linking the detecting agent and the indicator agent to one another;     e) applying a predetermined amount of the affinity signal-generating compound onto a predetermined surface area of the sample and allowing the compound to remain in contact therewith for a period of time sufficient to cause binding of the detecting agent to cholesterol present in the exposed skin constituents; and     f) measuring the signal generated by the indicator agent to provide a value indicative of cholesterol level.        
 
         [0028]     According to a third aspect of the invention, there is provided a method of measuring skin cholesterol, which comprises the steps of: 
        a) providing a tape comprising a backing member coated on at least one side thereof with a medical adhesive;     b) applying the tape onto a selected area of skin to adhere the tape to the selected skin area;     c) stripping the tape off the selected skin area to obtain a sample representative of an outer stratum corneum layer of the skin, the sample adhering to the tape so as to have exposed skin constituents;     d) providing a source of cholesterol oxidase as a detecting agent having affinity for cholesterol;     e) applying a predetermined amount of cholesterol oxidase onto a predetermined surface area of the sample and allowing the cholesterol oxidase to remain in contact therewith for a period of time sufficient to cause oxidation of cholesterol and 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 cholesterol level.        
 
         [0035]     The present invention also provides, in a fourth aspect thereof, a kit for use in carrying out a method according to the first aspect. The kit comprises: 
        the aforesaid tape;     the aforesaid source of affinity-enzymatic compound of formula A-C-B, wherein A, B and C are as defined above; and     a source of the aforesaid color developing agent.        
 
         [0039]     The invention further provides, in a fifth aspect thereof, a kit for use in carrying out a method according to the second aspect. The kit comprises: 
        the aforesaid tape; and     the aforesaid source of affinity signal-generating compound of formula A-C-B′, wherein A, B′ and C are as defined above.        
 
         [0042]     The invention additionally provides, in a sixth aspect thereof, a kit for use in carrying out a method according to the third aspect. The kit comprises: 
        the aforesaid tape; and     the aforesaid source of cholesterol oxidase.        
 
         [0045]     Applicant has found quite surprisingly that the measurement of skin cholesterol can be carried out directly on the skin sample adhering to the aforementioned tape. The procurement of skin samples removed by tape stripping from donor individuals allows assays to be conducted at distant and centralized sites and also allows assays from many individuals to be run concurrently. Thus, the method according to the invention is suitable for large scale screening of individuals for assessing their risk of cardiovascular disease.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0046]     Use is preferably made of a tape comprising a backing member formed of polyester. The tape is coated on at least one side thereof with a medical adhesive. The term “medical adhesive” as used herein refers to an adhesive which is hypoallergic and safe for application to the skin. Such an adhesive is preferably a pressure-sensitive adhesive, for example, an adhesive comprising an elastomer formed of block polymers of styrene-isoprene-styrene or styrene-butadiene-styrene.  
         [0047]     A particularly preferred tape for use in the method of the invention is a double-coated pressure-sensitive medical grade tape sold by 3M under Product #9877, or by Adhesive Research, Inc. under Product #8570.  
         [0048]     Double-coated pressure-sensitive tapes are generally available with an easily removable protective liner. The liner protects the tape from adhering until it is removed and keeps the adhesive from becoming contaminated. Liners may be placed on either side of the double-coated tape or the tape may have a single liner and be wound onto itself, thereby protecting both surfaces.  
         [0049]     Liners with differential release properties may be used so that a first side of adhesive may be exposed while protecting the second adhesive surface. A double-coated tape with differential liners is particularly advantageous for skin sampling. Removal of the first liner allows the tape to be stuck onto the backing support of a sampling device and leaves the skin-sampling side covered with the second liner. This second liner protects the skin sampling adhesive area from sticking and from contamination until it is to be used. When required for skin sampling, the second liner is removed.  
         [0050]     The tape can be applied onto any part of skin, but the most suitable part is the surface of a palm because the palm does not have sebaceous glands whose secretions contain cholesterol which may affect diagnostic results. Additionally, the skin on the palm is readily accessible for sampling.  
         [0051]     It is desirable to obtain uniform amounts of skin samples for analysis. Application of the adhesive tape for sampling is typically and routinely done using a single application of the tape to the skin. Additional amounts of stratum corneum material can be obtained by additional applications of the tape to the skin. Each subsequent application of the tape to the skin results in additional skin adhering to the tape. This process continues until the tape becomes saturated with skin material after which it is no longer sticky. The number of applications required to saturate a tape depends on the type of adhesive used, but for most commonly used adhesive tapes, saturation is achieved with less than ten applications. Applying tape to a fresh area of skin for each subsequent stripping results in better and faster saturation of the tape. Therefore, for consistent and good sampling, it is convenient to make ten applications of a tape to the skin, using new areas of skin for each application.  
         [0052]     After skin sampling, the sampling device is closed and shipped to a central laboratory for assay of cholesterol.  
         [0053]     When using a compound of formula A-C-B or A-C-B′ for the analysis of cholesterol in the skin samples, the detecting agent A can be for example a steroid glycoside, a triterpene glycoside, a hydrophobic protein, a polyene antibiotic or an anti-cholesterol antibody. Use is preferably made of a steroid glycoside, such as digitonin. The binding agent C, on the other hand, is preferably a copolymer of maleic anhydride and N-vinylpyrrolidone.  
         [0054]     In the case where use is made of a compound of formula A-C-B, the enzymatic visualizing agent B is preferably an enzyme selected from the group consisting of peroxidase, alkaline phosphatase, urease, galactosidase, glucose oxidase and acetylcholinesterase. Peroxidase such as horseradish peroxidase is preferred. In this particular case, after step (e), the peroxidase is activated with hydrogen peroxide to form an activated peroxidase, and the color developing agent used in step (f) reacts with the activated peroxidase to form the aforesaid colored product. To this end, a predetermined amount of an aqueous solution containing hydrogen peroxide and the color developing agent is applied in step (f) onto the predetermined surface area of the sample. Examples of suitable color developing agents which can be used in step (f) include 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) and 3,3′,5,5′-tetramethyl benzidine. 3,3′5,5′-Tetramethyl benzidine is preferred.  
         [0055]     In the case where use is made of a compound of formula A-C-B′, the indicator agent B′ can be for example a dye, a fluorophore, a radioisotope, a metal sol compound or a chemiluminescent compound. When the indicator agent is a dye, step (f) can be carried out by spectrophotometry, such as colorimetry. When the indicator agent is a fluorophore, step (f) can be carried out by fluorometry. When the indicator agent is a radioisotope, step (f) can be carried out by means of a radioactivity sensor. When the indicator agent is a metal-sol compound, step (f) can be carried out by colorimetry. When the indicator agent is a chemiluminescent compound, step (f) can be carried out by luminometry.  
         [0056]     In the case where use is made of cholesterol oxidase as a detecting agent having affinity for cholesterol, step (f) is preferably carried out by means of an electrochemical sensor, for instance, amperometrically using an electrode. Step (f) can also be carried out by spectrophotometry after addition of peroxidase and a colorimetric indicator. The peroxidase used is preferably horseradish peroxidase. Examples of suitable colorimetric indicators which can be used include 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) and 3,3′,5,5′-tetramethyl benzidine. A colorimetric indicator consisting of a multicomponent oxidative coupling reagent of Trinder or Ngo-Lenhoff type can also be used. When use is made of peroxidase and a colorimetric indicator, the aforementioned kit for carrying out the method according to the third aspect of the invention further comprises a source of peroxidase and a source of the colorimetric indicator.  
         [0057]     The method according to the invention enables to achieve a simple, high-throughput skin cholesterol assay. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0058]     The following non-limiting examples illustrate the invention, reference being made to the accompanying drawings, in which:  
         [0059]      FIG. 1  is a top view of a sampling device as used in Example 2; and  
         [0060]      FIG. 2  is a fragmentary view of the sampling device illustrated in  FIG. 1 , showing details of the sampling member thereof. 
     
    
     EXAMPLE 1  
       [0061]     A double-coated pressure-sensitive medical grade tape having a protective release liner on an upper sampling side and sold by Adhesive Research, Inc. was used. A piece of tape  1  inch by 1 inch was cut. The piece of tape was stuck, using the exposed, lower adhesive surface to one end of a 1 inch by 3 inch thin plastic (white polystyrene) member, leaving a 1 inch by 2 inch piece of uncovered plastic as a handle for applying the tape to the skin and for labeling the sample.  
         [0062]     To obtain a skin sample, the protective liner was removed and the exposed adhesive area applied to a clean dry section of skin. Pressure was applied to the back of the plastic member over the adhesive area to effect good contact of the adhesive with the skin. The plastic member with the attached tape and stratum corneum sample was then peeled from the skin.  
         [0063]     The sample was cut into four equal pieces each measuring ½ inch by ½ inch. One piece was placed in a well of a 12 well tissue culture plate, or similar container, with the skin sampling side facing up. An aliquot of reagent of the type A-C-B was then applied onto a predetermined surface area of the skin sample. The A-C-B reagent used was a conjugate of digitonin (A) linked to horseradish peroxidase (B) through a maleic anhydride-N-vinylpyrrolidone copolymer (C). The reagent was left in contact with the skin sample for 15 minutes at room temperature, after which it is removed by aspiration. Thereafter, the sample was washed with three separate aliquots of a wash solution to remove non-specifically bound reagent. The piece was then placed in a new, clean well of a 12 well tissue culture plate, or similar container, with the skin sampling side facing up. An aliquot of substrate solution was applied to the sample and left in contact with the skin sample for  15  minutes at room temperature. The substrate solution used was Enhanced K-Blue reagent available from Neogen Corp.(Lexington, Ky., USA) and containing hydrogen peroxide and tetramethyl benzidine as color developing agent. An aliquot of the developed substrate solution was removed from the well and added to an aliquot of 1 N sulfuric acid in a well of a 96 well microwell plate. The optical density of the resulting solution, which is a measure of the amount of cholesterol in the skin sample, was read at 450 nm on a plate reading spectrophotometer.  
       EXAMPLE 2  
       [0064]     Use was made of a sampling device as shown in  FIG. 1 . The sampling device which is generally designated by reference numeral  10  is formed of plastic (polypropylene) and comprises a sampling member  12  connected to a closure member  14  by an integral hinge  16 . The closure member  14  has a peripheral rim  18  and four pins  20 , adapted to lock into, respectively, a peripheral groove  22  and four holes  24  formed in the sampling member  12 . Folding the hinge  16  causes engagement of the rim  18  with the groove  22  and of the pins  20  with the holes  24 , thereby ensuring that the two halves of the device  10  remain closed and sealed to prevent dust and contamination of the interior surfaces. The outer surface (not shown) of the closure member  14  has a flat area for receiving a label and barcode strip, for sample identification. The sampling member  12  and closure member  14  are respectively provided with finger-tabs  26  and  28  for opening the device  10 .  
         [0065]     A double-coated pressure-sensitive medical grade tape  30  having a protective Kraft paper release liner  32  and sold by 3M under Product #9877 was adhered to the central area of the sampling member  12 . The release liner  32  is wider than the adhesive tape  30 , thereby defining a strip  32 ′ along one edge with no attached tape. This strip  32 ′ of liner overhangs the edge of the device to form a tab for easy removal of the liner. Immediately before use, the liner  32  is removed using the overhanging tab  32 ′ and this exposes the adhesive of the tape  30  for skin sampling.  
         [0066]     The palmar skin area for sampling was cleaned and dried. The tape  30  with the exposed adhesive was applied onto the palm. The tape  30  was pressed against the skin by applying pressure to the back of the sampling member  12  above the adhesive area, thereby causing adherence of the stratum corneum layer. The device  10  was peeled away, reapplied to a new area of the palm and again pressed to the skin. The device is peeled away and applied to the palmar skin in this way for a total of 10 applications.  
         [0067]     Two small dipsticks 4 mm in width were cut from the device  10  as follows. An end portion of the sampling member  12  was removed by cutting along the portion of groove  22  which is adjacent to the tab  26 . Three cuts were then made along guide lines  36  (shown in  FIG. 2 ) molded into the sampling member  12 , to delineate the 4 mm sticks, cutting from the edge to just past the centre line. The two 4 mm wide sticks were released from the sampling member  12  by making a third cut across the center of the member  12 , using guide line  38  molded into the member  12 . These sticks had an upper portion devoid of tape and a lower portion with tape having the skin sample adhered thereto.  
         [0068]     The sticks were each placed into 100 uL solution of an A-C-B reagent in wells of a 96 well microwell plate. The reagent was a conjugate of digitonin (A) linked to horseradish peroxidase (B) through a maleic anhydride-N-vinylpyrrolidone copolymer (C) and was used at a concentration of approximately 1 μg/mL. The sticks were left in the solution for 15 minutes at room temperature, after which they were removed and placed into new wells of a microwell plate containing 200 μL of wash solution. The microwell plate was agitated to effect washing and after 1 min the sticks were removed to new wells containing 200 μL of fresh wash solution and again agitated for 1 min. Washing with agitation was done a third time, after which the sticks were removed and placed in 100 uL of a substrate solution (Enhanced K-Blue reagent). The sticks were incubated with the substrate solution, in the dark, for 15 minutes at room temperature, and then removed. One hundred (100) μL of 1 N sulfuric acid were added to the wells with the substrate solution to stop further reaction and the optical density of the resulting solution was read at 450 nm on a plate reading spectrophotometer, to provide a measure of the amount of cholesterol in the skin sample.