Note: Descriptions are shown in the official language in which they were submitted.
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DIRECT ASSAY OF CHOLESTEROL IN SKIN
SAMPLES REMOVED BY TAPE STRIPPING
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.
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.
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.
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.
However, total cholesterol levels alone do not accurately predict a
patient'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
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Lipoprotein Cholesterol in the Serum and Risk of Mortality, British Medical
Journal, 1985, 290, pg. 1239-1243).
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.
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.
U.S. Patent 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
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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.
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
I 5 and their analysis may give false data on cholesterol content in the skin.
US Patent 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's office or clinic where the test is conducted. This of
course limits effective large scale screening.
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, I 12, 1999). High performance
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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
S determinations is not practical.
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
1 S the simple and rapid determination of cholesterol levels in large numbers
of
samples.
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.
According to a first aspect of the invention, there is provided a method
of measuring skin cholesterol, which comprises the steps o~
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
2S the selected skin area;
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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.
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;
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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.
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
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fJ measuring the amount of hydrogen peroxide formed in step (e),
the amount of hydrogen peroxide measured being indicative of cholesterol
level.
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.
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.
1 S 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.
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
CA 02465427 2004-04-28
concurrently. Thus, the method according to the invention is suitable for
large
scale screening of individuals for assessing their risk of cardiovascular
disease.
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.
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.
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.
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.
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
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whose secretions contain cholesterol which may affect diagnostic results.
Additionally, the skin on the palm is readily accessible for sampling.
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.
After skin sampling, the sampling device is closed and shipped to a
central laboratory for assay of cholesterol.
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 malefic anhydride and N-vinylpyrrolidone.
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
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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
S (f) onto the predetermined surface area of the sample. Examples of suitable
color developing agents which can be used in step (fj include 2,2'-azino-di-(3-
ethylbenzthiazoline-6-sulfonic acid) and 3,3',S,S'-tetramethyl benzidine.
3,3'S,S'-
Tetramethyl benzidine is preferred.
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 (fj 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
1 S carried out by means of a radioactivity sensor. When the indicator agent
is a
metal-sol compound, step (fj can be carried out by colorimetry. When the
indicator agent is a chemiluminescent compound, step (f) can be carried out by
luminometry.
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
2S include 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) and 3,3',S,S'-
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
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the invention fi~rther comprises a source of peroxidase and a source of the
colorimetric indicator.
The method according to the invention enables to achieve a simple,
high-throughput skin cholesterol assay.
The following non-limiting examples illustrate the invention, reference
being made to the accompanying drawings, in which:
Figure 1 is a top view of a sampling device as used in~Example 2; and
Figure 2 is a fragmentary view of the sampling device illustrated in
Figure 1, showing details of the sampling member thereof.
EXAMPLE 1
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.
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.
The sample was cut into four equal pieces each measuring %2 inch by '/2
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
CA 02465427 2004-04-28
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sample. The A-C-B reagent used was a conjugate of digitonin (A) linked to
horseradish peroxidase (B) through a malefic 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 1 S 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
Use was made of a sampling device as shown in Figure 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 I4 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
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sampling member 12 and closure member 14 are respectively provided with
finger-tabs 26 and 28 for opening the device 10.
A double-coated pressure-sensitive medical grade tape 30 having a
protective Kraft paper release liner 32 and sold by 3M under Product #9877
S 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.
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.
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. Tbree 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.
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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 malefic anhydride-N-
vinylpyrrolidone copolymer (C) and was used at a concentration of
approximately 1 ~g/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 ~L of wash solution. The microwell plate was
agitated to effect washing and after 1 min the sticks were removed to new
wells
containing 200 ~.L 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
minutes at room temperature, and then removed. One hundred ( 100) ~,L of 1
N sulfuric acid were added to the wells with the substrate solution to stop
15 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.
