Note: Descriptions are shown in the official language in which they were submitted.
1335968
--1--
The proposed invention is in the field of bio
-organic
chemistry, namely, it deals with a method for prod
ucing affino-
enzymatic compounds for visual indication of chole
sterol on skin
surface based on a detecting agent, which is an ~f
fin~nt of
cholesterol, and a visll~li7.ing agent.
The compounds obtained can be most efficiently used in
diagnostics, including early diagnostics of atherosclerosis,
verification of the diagnosis in clinical and out-patient conditions and
for treatment control. They can also be used in physiological,
o histological and histochemical studies to detect high contents of
cholesterol and its localization. Besides, the proposed compounds
enable to detect low contents of cholesterol as well, typical for
patients with certain pathologies, in particular, of oncological origin.
Atherosclerosis and its complications such as infarctions,
apoplectic attacks and gangrenes, are one of the main mortality
causes in all countries of the world.
Studies which have been carried out for many years showed
that atherosclerosis is a disease whose detection and prevention is a
matter of top priority, and the leading risk factor of atherosclerosis
20 is accumulation of cholesterol in the organism. Prophylaxis of
atherosclerosis in population first of all presupposes identification of
the highest-risk group among the patients, followed by their
differentiated treatment and change of life style. The most difficult
in atherosclerosis prevention is selection of high-risk groups by
25 amount of cholesterol accumulated in the organism. The existing
methods of atherosclerosis diagnostics are based on quantification of
general cholesterol content in the venous blood plasma. (Consensus
Conference on Lowering Blood Cholesterol to Prevent Heart
Disease, JAMA, 1985, 253, p.2080; The Lipid Research Clinics
B ~
1335968
.
Population Studies Data Book, publication 80-152, Bethesda, Ma,
National Institute of Health, 1980, vol. I; Lipid Research Clinics
Program, JAMA, 1984, 251, p.351-364). In some cases, a
cholesterol content over 260 mg % is considered sufficient to include
a patient into the risk group. A morè precise diagnosis can be made
by analyzing blood plasma lipoproteins and determining the
atherogeneity index, which is a ratio of difference between total and
high-density lipoprotein cholesterol to high-density lipoprotein
cholesterol:
o I x ather = Ch (total) - Ch (h-d 1.)
Ch (h-d 1.)
A patient is considered to belong to the risk group when his
atherogeneity index is more than 3, and to the atherosclerosis group,
when said index is over 5.6. (Klimov A.N. "Phenotyping of
Lipoproteins", Methodological recommendations of the USSR
Ministry of (Health, M., Medicine, 1975; Goldfourt V., Holtsman E.,
Neufeld H.N. Total and High Density Lipoprotein Cholesterol in the
Serum and Risk of Mortality. British Medical Journal, 1985, 290,
p.l239-1243).
Use of these methods necessitates blood taking, which may be
traumatic for patients and, besides, is not altogether safe for the
reason of possible viral infections. Fractionation of plasma
lipoproteins and cholesterol analysis still remain a complicated and
expensive procedure. Moreover, in one case out of three
quantification of total cholesterol and even a complete phenotyping
do not correlate with severity of atherosclerosis (See: Myasnikov
A.L., "Hypertensive Disease", 1965, M., Medicine, p.300).
In addition, recent studies showed that the blood plasma can
not fully reflect processes of cholesterol accumulation, which are
typical for the arterial wall and other bradytrophic tissues.
,~
.~
3 13~5968
Solution of some fundamental problems related to
athero~clerosis pathogenesis made it possible to further improve
diagnostic methods. It has been shown that tissue cholesterol plays a
leading role in development of atherosclerotic disease. Tissues have
5 been identified which accumulate cholesterol in the same way as the
arterial wall.
, Recent studies have also shown a close correlation between
cholesterol content in the arterial wall and skin. This enabled to
develop fundamentally new diagnostic methods, in particular, for
10 atherosclerosis.
The above mentioned correlation between cholesterol content
in the arterial wall and skin was found out through direct
quantification of cholesterol in skin biopsy. Specimens were frozen
in liquid nitrogen and lyophilized; cholesterol was extracted with
Folch reagent and quantified using traditional chemical or
biochemical methods. (See: 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,
20 p.48-51; Bouisson H., De Graeve, Solera M.L. et al. Ann. Biol.
Clin., 1982, vol.40, p.361-407), However, this method is not suitable
for population screening because it is tra~lm~tic and complicated.
US Patent No. 4,458,686 issued on July 10, 1984 describes a
method which enables to quantify glucose and ethanol, localized in
25 the blood, directly under the skin or on its surface, and points out
that cholesterol quantification using cholesterol oxidase is also
possible. The method is based on stoichiometric changes of oxygen
concentration when using redox enzymes, mostly oxidoreductases,
specific to the substrate in question. According to this method
30 quantification of oxygen concentration changes is made
electrochemically, for instance, polarography with the use of special
equipment and specially designed electrode.
.
-
- 4- 1335968
Such a sophisticated instrumentation necessitates the services of
high-skilled personnel for m~king diagnoses. All this inevitably
limits the possibility of using this method for population screening.
Canadian Patent No. 1,237,369, issued on May 31, 1988
proposes a detection-visll~liz~tion complex where the detecting and
visll~lizing elements are linked directly or through a binding agent.
! The complex is intended to detect low quantities of target molecules,
including lipids in biological tissues. however, this method of
detecting lipids can be used only in laboratory conditions and
o requires prior taking of biological fluid or tissue from a patient, i.e.
the method is traumatic, many-staged and complicated in
performance.
Correlation between cholesterol content in the skin and
severity of atherosclerotic vascular lesions is obtained on skin
specimens. Besides being traumatic, this method has several
disadvantages, because specimens 1 mm thick include various skin
layers: horny layer (average thickness 0.1 mm) and connective tissue
(derma proper) represented by two layers - papillary and retinal.
Both layers have a good blood supply and, consequently, specimens
include vessels and blood; besides, they contain perspiratory and
sebaceous glands and their secretions. The subcutaneous fat is located
directly under the derma and also can get into specimens, i.e.
heterogeneity of specimens may pervert the data on cholesterol
accumulated in the skin. From this point of view, a method which
enables to quantify the cholesterol content on skin surface in the
horny layer of the derma should be considered as the most accurate
one.
The purpose of the invention was to create a method for
producing affino-enzymatic compounds for visual indication of
cholesterol directly on patient's skin, in particular, in the epidermal
horny layer without taking blood or skin specimens from patients.
Any part of skin can be used for diagnostics, but the most suitable is
the surface of a palm because does not have sebaceous glands whose
~
13~5968
secretions, as well as the horny layer, contain cholesterol which may
affect diagnostic results.
The invention solves this problem by creating a method to
produce obtain affino-enzymatic compounds which are bi-functional
5 by their nature. The compounds are selectively bound to free
cholesterol of the skin, and then can be made visually detectable. At
least two components are necessary to obtain such a compound:
detecting agent A, selected from a group of substances capable of
discrimin~tely forming stable complexes with free cholesterol of the
o skin in order to make the whole bi-functional compound afferent to
cholesterol, and a visll~li7.ing agent B permitting visual detection of
the bi-functional compound bound to cholesterol.
The compounds produced according to this invention simplify
at most atherosclerosis diagnostics, are not traumatic and do not
require special instrumentation. Use of these compound in
diagnostics will make it possible to determine the belonging, of the
examined patients to one of the three groups: atherosclerotic, risk
group or normals.
The affino-enzymatic compounds for visual indication of
2 o cholesterol, obtained through the present invention, and the
diagnostic method based on their use, tentatively named as "three
point method", are most suitable for population screening. The
method is so simple in operation that it does not require specialized
personnel and even can be used in domestic conditions.
The compound according to the invention can only be obtained
either from detecting agent A, or visll~lizing agent B by chemically
activating functional groups of one of these agents. Then, after
adding to one agent, containing activated chemical groups, of the
other agent, they form under certain conditions a stable A-B bond
resulting in a high-molecular weight bi-functional compound for
visual indication of cholesterol on the skin of patients.
~ .
6 1335968
The following compound are used as cholesterol/detecting
agent A ~ffin~nts:
Steroid glycosides, containing as an aglycone a
cyclopentaneperhydrophenanthrene fragment of furostanole or
spirostanole series, and oligosaccharide fragment including 3 to 10
monosaccharide with linear or branched structures (Kintya P.K.
"Structure and biological activity of steroid glycosides of spirostan
, and furostan series", Kishinev, Stinza, 1987, p. 142),
Triterpene glycosides, containing an aglycone of alpha or beta-
amyryl, lupane, hopane, ~l~mm~rane, lanostane or holostane series,
and oligosaccharide of 2-8 residues of branched or linear structure
(Dekanosidze G.E., Chirva V.Y., Sergienko T.V., Uvarova N.I.
"Study on triterpene glycosides", Tbilisi, Mezniereba, 1982), or
Hydrophobic proteins, capable to discriminately form a
complex compound with cholesterol (Klimov A.N., Titova G.V.,
Kozhevnikov K.A., Biochemistry, 1982, vol. 47, No. 2, p. 226-232);
Klimov A.N., Kozhevnikov K.A., Klyueva N.N. et al. Voprosy
Med., Khimii, 1984, vol. 30, No. 3, p. 86-90; Titova G.V., Klyueva
N.N., Kozhevnikov K.A., et al. Biochemistry, 1980, vol. 45, No. 1,
p. 51-55), or
Protein toxins, capable to discriminately form complex
compounds with cholesterol. They are obtained from bacteria,
marine microorganisms, insects or snakes (Dalin M.V., Fish N.G.
"Protein toxins of microorganisms", Moscow, Medicine, 1980), or
Polyene antibiotics, capable to discriminately form complex
compounds with cholesterol (I.J. Katzenstein, A.M.Spielvogel, A.W.
Norman, J. Antibiot., 27, 12, 1974, P-943-951; Jong Shang-Shyng,
Wang Hsi-Hua. Clin. J. Microbiol., 1976, 9, (1-2), p.19-30; Readig
Josephine D. et al. Biochim. Biophys. Acta, 1982, 685 (2), p.219-
224), or
High-affinity enzymes. whose- substrate is cholesterol, and
which have a high affinity to it.
As a visualizing agent B, the following enzyme, are used:
acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase,
glucose oxidase, glucoamylase, galactosidase, peroxidase, ~lk~line or
acid phosphatase, alpha-chymotrypsin or pyrophosphatase.
~7~ 1335968
The problem is solved, because the ~ffin~nt-detecting agent
(A) selected from a group of steroid glycosides containing as an
aglycone a cyclopentaneperhydrophenanthrene fragment of
furostanole or spirostanole series, and oligosaccharide fragment
5including 3 to 10 monosaccharide with linear or branched structures,
or from a group of triterpene glycosides, containing an aglycone of
alpha- or beta-amyryl, lupane, hopane, d~mm~rane, lanostane or
holostane series, and oligosaccharides of 2-8 resides with branched
or linear structures, or
0from a group of hydrophobic proteins capable to
discrimin~tely form complex compounds with cholesterol, or
from a group of protein toxins capable to discriminately form
complex compounds with cholesterol, and obtained from bacteria,
m~rine microorganisms, insects or snakes, or
15from a group of polyene antibiotics capable to discriminately
form complex compounds with cholesterol, or
from enzymes having a high affinity to cholesterol, is
chemically activated in an aqueous medium with detecting
agent A /activator molar ratio = 1:1 - 1:10, detecting agent A
20concentration 1-20 mg/ml, temperature 0-25C, pH = 4-11, during
0.1-24 hrs.
To the solution obtained, a visualizing agent B is added,
selected among the following enzymes: acetylcholinesterase,
tyrosinase, glucose-6-phosphate dehydrogenase, glucose-oxidase,
2 5glucose-amylase, galactosidase, peroxidase, alkaline or acid
phosphatase, alpha-chymotrypsin or pyrophosphatase in A/B molar
ratio = 20:1 - 1:1. Then the solution is incubated at 0-25C during 1-
24 hrs until the final product is obtained.
As mentioned above, functional groups of visll~li7ing Agent B
30can also be subjected to chemical activation. In this case, agent B is
activated in an aqueous medium with B/activator molar ratio = 1:1 -
1:10, agent B concentration 1-20 mg/ml, at temperature 0-25C, pH
= 4-11, during 0.1-24 hrs. Then detecting agent A, which is one of
f~
- 1~35968
--3 -
the above-listed substances, is added to tie solution, with A/B molar
ratio = 20:1 - 1:1, and the solution is incubated at 0-25C for 1-24
hrs until the final product is obtained.
Under the present invention, detecting agent A or visll~lizing
agent B are activated using the well-known azide, carbodiimide and
succinamide methods, or the method of periodate oxidation.
In cases when visll~li7.ing agent B is a high-molecular weight
compound such as enzyme, and detecting agent A is a low-molecular
compound, for instance, glycosides, a molecule of the final product
o can contain only one molecule of visu~li7.ing agent B. However, the
molar quantity of detecting agent A, responsible for binding skin
cholesterol in the final product, can exceed the content of visuali7ing
agent B only by several times, according to the number of functional
groups in the enzyme molecule capable of binding with detecting
agent A without significant loss of enzymatic activity. In such
compounds, the content of visll~lizing agent B, as which an enzyme
was used, is limited. The result is a relatively low sensitivity of the
compounds thus obtained, and the need to increase exposure time in
diagnostic procedures.
2 o To expand technical performance of affino-enzymatic
compounds for visual indication of cholesterol on the skin, and view
of increasing their sensitivity, it is proposed to bind above detecting
agent A and visu~li7.ing agent B using binding agent C, taken from a
series of low-molecular asymmetric functional compounds, such as
bromcyan, trichlorotriazine or 2-amino-4,6-dichloro-3-triazine. In
this case, compounds can be obtained in two ways: either detecting
agent A is first linked binding agent C and then visll~li7ing agent B is
added to tie intermediate product (A+C) to obtain the final product,
or tie intermediate product is B+C to which detecting agent A is
3 0 added.
In the first option, detecting agent A is dissolved in a usual
manner in an aqueous-salt buffer with pH = S-9, in concentration 1-
B
.
9 13~5968
20 mg/ml, then a low-molecular asymmetric bi-functional binding
agent C, selected among the above-mentioned substances, is added to
the solution, with A/C molar ratio = 1:0.5 - 1:10. The solution
obtained is incubated at 0 - 20 for 1 - 20 hours; then visu~li7.ing
5 agent B, selected from the above substances, is added with A/B molar
ratio = 20:1 - 1:1, and the solution is incubated at 0 - 20 for 1 - 48
hours to obtain the final product.
To obtain an affino-enzymatic compound through the
intermediate product B+C, visll~li7.ing agent B is dissolved in a usual
lO manner in an aqueous-salt buffer with pH 5-9, in concentration 1-20
mg/ml. Then a low-molecular asymmetric bi-functional binding
agent C, selected among the above substances, is added with B/C
molar ration = 1:0.5 - 1:10. The solution obtained is incubated at 0 -
20C for 1 - 20 hours; then detecting agent A is added with A/B
molar ratio 20:1 - 1:1, and the solution is incubated at 0 - 20C for 1
- 48 hours to obtain the final product.
However, the best sensitivity of affino-enzymatic compounds
for visual indication of cholesterol on the skin are obtained when
using as a binding agent C some high-molecular weight poly-
20 functional compounds, such as polysaccharides, proteins or syntheticpolymers, i.e. any high-molecular weight compounds containing any
of functional groups selected among the following: primary amines,
carboxyl, hydroxyl, aldehyde, haloid anhydride, mixed anhydrides,
iminoesters, azide, hydrazide, maleimide, isocyanate or epoxide.
2 5 Moreover, both detecting agent A, being one of the above
compounds or their mixture, and visll~li7.ing agent B, also one of the
above, are independently immobilized by any one of these
compounds. Such an affino-enzymatic compound enables most
widely vary A/B ratios, depending on the extent of selected agent A
30 affinity to skin cholesterol and, accordingly, activity of enzyme B
used. For instance, when using a detecting agent A with lower
affinity to cholesterol, it is possible to significantly increase its molar
content in the final product.
B
. ~,~
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- lo
Such affino-enzymatic compounds are obtained as follows:
detecting agent A, which is one of the above substances or their
mixture, and visualizing agent B, which is also one of the above, are
taken in A/B molar ratio = 20:1 - 1:1 and dissolved in an aqueous
salt solution with pH = 5-9, and in concentration of each of the
agents 0.1 - 20 mg/ml. High-molecular weight poly-functional
binding agent C is added to the solution obtained; it is selected from
polysaccharides, proteins or synthetic polymers according to the
above. A+B/C molar ratio = 1:0.5 - 1:10. Then the solution is
incubated at 0 - 20C for 1 - 20 hours to obtain the final product.
Among steroid glycosides, used as detecting agent A and
containing as an aglycone a cyclopentaneperhydrophenanthrene
fragment of furostanole and spirostanole series, and oligosaccharide
fragment including 3 to 10 monosaccharide with linear or branched
structures, the most preferable are: agavosides A, B, C, d, F, H and
G, trillin, funcoside I, dioscin, gracillin, protodioscin,
kikubasoponine, juccosides E and H, lanotigonin, desglycodigitonin,
digitonin, gitonin, rocosides C, D and E, funcoside E. alliumosides
C, polygonatin, tigogenin tetraoside, tigogenin hexaoside,
kapsycoside, alliumosides B, C, D and E, desglycodesramnoparillin,
desglycoparillin, parillin, sarmaparilloside, asparagosides C, D, G
and H, juccoside B, protojuccoside H, lanotigoside, monoside,
bioside, trioside, purpureagitoside, tomatine, alliogenin
liquotetraoside, karatavioside A, cyclosiversioside H.
acanthophylloside C, alliofuroside A, alliospiroside A,
cyclosiversioside F, tea saponin, acanthophylloside B, tigonin (total),
glycoside from Calha Polypatala or cyclosiversioside G.
Among the above steroid glycosides more preferable are
funcoside C, D, E, F, G, or I, dioscin, rocoside, lanotigonin,
digitonin and tomatine. The most preferable are digitonin and
tomatine.
Among triterpene glycosides used as detecting agent A and
containing an aglycone of alpha- or beta-amyryl, lupane, hopane,
1335968
(l~mm~rane, lanostane or holostane series, and oligosaccharide of 2-8
residues with branched or linear structures, the most preferable are:
escin, avenacin, tea saponin, alpha-gederine, kauloside C, stichinoside
A, cyclamine, chinovine, saponins from sugar beet, hypsoside and
triterpene glycosides obtained from Fatsia Japonica.
Among hydrophobic proteins used as detecting agent A and
capable of discriminately forming complex compounds with
cholesterol, the most preferable are: Folch meilin protein, lysosomal
proteins, proteins of mitochondria, fibrinogen, immunoglobulins,
o cerebrone, myoglobin, typsin, cytochrome C, cytochrome P-450 or
apo-proteins of lipoproteins.
Among protein toxins, used as detecting agent A and capable
to form complex compounds with cholesterol, which are obtained
from bacteria, marine microorganisms, insects or snakes, the most
preferable are: streptolysins o, pneumolysins, listerio lysine, O-
toxin C.l obtained from Perfringens of A and C types, a-toxin C.l.
novvi of A and C types, hemolysine C.l histolyticum, hemolysine
C. 1 botulinum of C and D types, tetanolysine, cerebrolysins,
alveolysine, turingeolysine or cytotoxin from Metridium senile.
Among polyene antibiotics, used as detecting agent A and
capable to form complex compounds with cholesterol, the most
preferable are amphotericin B, filipin or nystatin.
Among enzymes with a high affinity to cholesterol, used as
detecting agent A, the most preferable are cholesterol oxidases,
2 5 cholesterol dehydrogenases or cholesterol esterases.
Among all the above-listed ~ffin~nts used as detecting agent A,
namely: glycosides, hydrophobic proteins, protein toxins, polyene
antibiotics and high-affinity enzymes, more preferable are
glycosides, as being most chemically stable and capable to withstand
during synthesis both organic solvents and high temperatures while
retaining the ability to form complexes with skin cholesterol. A low
r~
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-12- 1335968
molecular weight of glycosides makes it possible to obtain a
compound with high molar content of detecting agent, thus
providing multiple points of interaction between the compound and
skin cholesterol.
'rhe most preferable among glycosides are steroid glycosides
which are more efficient in making complexes with cholesterol,
compared to triterpene glycosides.
Hydrophobic proteins, protein toxins, polyene antibiotics and
high-affinity enzymes are also successfully used for obtaining
o diagnostic tools, however they introduce certain lirnitation in the
method of producing affino-enzymatic compounds, due to their low
chemical stability and inactivation tendencies. Moreover, a high
molecular weight of these detecting agents somewhat reduces
sensibility of the final product, and that necessitates higher
concentrations of the compound and longer exposures to get
cholesterol indication.
Naturally, it would be more advantageous to perform
reactions in mild conditions, i.e. neutral pH, low ionic force, low
temperatures and short reaction time.
Enzymes used as visll~li7ing agent B, which as a result of
reacting with detecting agent A produce affino-enzymatic compounds
for visual indication of cholesterol on the skin, are selected from the
following list: acetylcholinesterase, tyrosinase, glucose-6-phosphate
dehydrogenase, glucose-oxidase, glucose-amylase, galactosidase,
peroxidase, ~lk~,line or acid phosphatase, alpha-chymotrypsin or
pyrophosphatase.
All these enzymes can be used with any of the above-listed
detecting agent A, however, steroid glycosides should be preferred.
B
~,. ., . ~
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-13 -
Use of binding agent C expands technical performance of the
method to obtain the compound in question, while preserving at most
the functional activity of agents A and B.
The most preferable are compounds using as an affinant-
5 detecting agent A steroid glycosides which contain as an aglycone acyclopentaneperhydrophenanthrene fragment of furostanole or
spirostanole series, and oligosaccharide fragment including 3 to 10
monosaccharides with linear or branched structures.
Such steroid glycosides possess a high chemical stability and
o can be immobilized onto a binding polymer in hard conditions, i.e.
high temperatures and dissolution in organic solvents when required,
thus ensuring their high content in the final product. Compounds for
visual indication of cholesterol on the skin are obtained through
consecutive immobilization of detecting agent A onto binding agent
C in hard conditions, and then immobilization of visu~li7ing agent B
onto the product containing both A and C, in soft conditions for
preserving agent B enzymatic activity.
As binding agent C the following asymmetric low-molecular
bi-functional compounds can b used: bromcyan, trichlorotriazine or
2 o 2-amino-4,6-dichloro-3-triazine.
Use as a binding agent C of high-molecular weight poly-
functional compounds enables to widely vary proportions of agent A
and B in the final product, for instance, with the purpose to get an
optimal ratio between the number of points binding the product with
25 skin surface (detecting agent A) and quantity of visu~li7ing agent B.
As a high-molecular weight poly-functional binding agent C it
is possible to use various polysaccharides, proteins or synthetic
polymers, i.e. any high-molecular weight compounds containing any
of the following functional groups: primary amine, carboxyl,
30 hydroxyl, aldehyde, haloid anhydride, mixed anhydride, iminoester,
azide, hydrazide, maleimide, isocyanate or epoxide.
~ ., . . ~ .
1~5968
- 14 -
As to high-molecular weight poly functional binding agent C,
copolymers of acrylic acid or maleic anhydride as to be preferred.
The most preferable is copolymer of N-vinylpyrrolidone and
maleic anhydride. The above agents C can be used with any
5 detecting agent A and visu~li7ing agent B. The final A-C-B product
is always obtained.
The most preferable are compounds where steroid glycosides
are used as detecting agent A.
Compounds capable to transform functional groups of agent A
o and B into reactable state are used as activators of these agents. Such
groups as primary amine, carboxyl, alpha-glycol can be activated
using the well-known methods (azide, carbodiimide, succinamide,
periodate etc.).
The most preferable are the periodate and carbodiimide
methods, because they ensure maximum mild synthesis conditions
and preserve activity of the immobilized enzyme.
Aqueous-sale buffers are solutions of compounds possessing
buffer action with pH = 5-9, and not provoking inactivation or
inhibition of enzymes. These may be, for instance, borate, citrate,
20 phosphate and other buffer solutions. As a rule, aqueous-salt
solutions are used where salt concentration is sufficient to ensure a
stable pH during immobilization, but not too high to cause
denaturation of the enzyme.
Some of detecting agent A, such a steroid glycosides,
2 5 triterpene glycosides and polyene antibiotics, inadequately or
incompletely dissolve in water and therefore require organic
solvents. For this purpose, polar aprotonic solvents can be used, for
instance, dimethysulfoxide, N,N-dimethylformamide, hexamethanol,
.,
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- 15 -
2:1 N,N-dimethylformamide/toluene mixture, or 2:1
dimethylformamide/hexane mixture.
Conditions to obtain compounds for cholesterol indication
(temperature, incubation time) depend on the choice of A, B and C
compounds. Use of enzymes as visualizing agent B limits the
reaction temperature to 20C. Preferably, the reaction should be
conducted at 4C in buffer solution with pH = 5-9. Use of synthetic
polymers for binding agent C, and glycosides for detecting agent A
enables to perform the reaction in organic solvents even at high
o temperatures (up to 120C) and choose a reaction time sufficient to
get the maximum possible yield of final product.
Molar ratios of the components are fixed in accordance with
molecular weights of selected A or B agents, affinity to skin
cholesterol and activity of the enzyme used.
Below, the invention is illustrated by several examples and
drawings:
Fig. 1 - symbols
Fig. 2 - compounds obtained and their interaction with
cholesterol on a patient's skin surface according to Ex. 1, 2
20 Fig. 3 - compounds obtained and their interaction with
cholesterol on a patient's skin surface according to Ex. 3, 4.
Fig. 4 - compounds obtained and their interaction with
cholesterol on a patient's skin surface according to Ex. S, 6.
Example 1
100 mg of tomatine (A) are placed in 10 ml of water, 40 mg
of sodium periodate are added, and the mixture is incubated at 20C
for 4 hrs. 1 ml of alpha-chymotrypsin solution (B) (10 mg/ml) in 0.2
M phosphate buffer with pH = 7.5 is added to the solution obtained,
and the mixture is incubated for 12 hrs a 4C. The result is aqueous
solution of the final product.
1335968
- 16-
Example 2
3 mg peroxidase (B) from horse-radish is dissolved in 1 ml of
water, 0.5 mg of sodium periodate are added, and the mixture is
incubated for 8 hrs. at 4C. 3 mg of cholesterol oxidase (A) in 1 ml
of 0.2 M phosphate buffer with pH = 7.5 are added to the solution
obtained, and the mixture is incubated for 12 hrs. at 4C.
.
The result is aqueous solution of the final product.
Example 3
10 mg of polyacrylic acid (C) are dissolved in 10 ml of acetate
buffer with pH = 4. 8, and 15 mg of p-cyclohexyl-2-(4-
mofoline)ethyl-carbodiimide-meta-p-toluenesulfonate are added.
The mixture is incubated for l.S hrs. at 0C. S ml of buffer
containing S mg of 0-streptolysins (A) and S mg of peroxidase (B)
from horse-radish are added to the solution obtained. The mixture is
incubated for 2 hrs. at 20C. The result is final product.
Example 4
Agavoside G (100 mg) (A) are placed in S ml of water, and
100 mg of bromcyan (C) are added; pH = 11 is maintained by adding
lM of NaOH solution. The mixture is incubated for 30 nun. at 4C
and pH is reduced to 8.5 by adding phosphoric acid. 3 ml of 0.5M
phosphate buffer with pH = 8.5 containing 6 mg of ~lk~line
phosphatase (B) are added to 1 ml of the solution obtained. The
mixture is incubated for 12 hrs. at 4C.
The result is solution of the ~lnal product.
Example S
Nystatin (A) (500 mg) and copolymer of ethylene with maleic
anhydride (C) (250 mg) are dissolved in 5 ml of dimethylformamide
and incubated in argon current at 50C for 3 hrs. The product
obtained is precipitated in 20 ml of ester, and dried in vacuum at
20C. 6 ml of the product obtained are added to 1 ml of beta-
D
L~
. ._
1335968
- 17 -
galactosidase (B) solution (2 mg/ml) in 0.2 M phosphate buffer with
pH = 7.5, and the mixture is incubated at 4C for 12 hrs. The result
is solution of the final product.
Example 6
Escin (A) (50 mg) and copolymer of N-vinylpyrrolidone with
maleic anhydride (C) (100 mg) are dissolved in 1 ml of
dimethysulfoxide and incubated for 2 hrs. at 100C. The product
obtained is precipitated in 3 ml of acetone7 dried in vacuum over
phosphorous pentoxide for 4 hrs. at 100C. 8 mg of the product
o obtained are added to 1 ml of alpha-chymotrypsin solution (B) (1
mg/ml) in 0.2 M phosphate buffer with pH = 7.5, and the mixture is
incubated at 6C for 15 hrs. The result is solution of the final
product.
In the last example the product is characterized by a high
molar content of detecting agent A, and the stage of obtaining the
intermediate product, containing agent A and high-molecular weight
binding agent C, helps to preserve the activity of the enzyme serving
as visll~li7.ing agent B because of the gentle conditions under which
the enzyme is immobilized.
20 Use of the compounds for visual indication of cholesterol on a
patient's skin
Diagnosis using the compounds produced according to the
present invention is not based on an accurate quantification of
cholesterol, but on the ability to determine which of the three groups
25 a patient belongs to; these groups are: atherosclerotic, risk group and
normals. In order to determine which group a patient belongs to, it
is necessary to evaluate the cholesterol content in the epidermal
horny layer typical for each of the above groups.
Accordingly, for every compound, produced according to the
30 present invention, three concentrations are selected and used for the
diagnosis. These three different concentrations of an affino-
enzymatic compound are applied to three spots on a patient's skin,
B
1335968
- 18-
preferably on the palm. The maximum concentration leads to
development of colour or stain in all subjects, including normals in
whom the skin cholesterol content is the least. This stain acts as a
kind of control for the cholesterol effect by showing that the
reagents are working. The minimum concentration of the compound
stains only patients having the clinical form of atherosclerotic
disease, in whom the cholesterol content is the greatest. A compound
solution with intermediate concentration develops a stain both in
atherosclerotic patients, and in subjects usually relating to the risk
o group (high cholesterol content), but fails to stain normals.
Thus, three different concentrations of compounds produced
according to the invention are applied on a patient's skin. With one
stain, a patient is considered to belong to normals, with two - to the
risk group, and in case of three stains - to clinical-stage
lS atherosclerotic patients.
Several sq. cm of skin area, selected on any part of the body,
are sufficient for a diagnostic procedure. The most preferable is the
palm, which is easily accessible and, besides, has no sebaceous
glands.
10-20 mcl of a compound obtained are applied in three
different concentrations on skin surface and incubated for 1 min.
The compound which does not bind to skin cholesterol, is removed
by washing. Then, a substrate of the corresponding enzyme, used as
visll~li7ing agent B in production of the present compound, is applied
2s on the same skin area. A reaction between the enzyme and the
compound results in a stained product.
Depending on the selected enzymatic substrate, a colour-less
solution may be stained red, yellow, blue, green, violet or some
other colour; or a coloured solution may become a colour-less
3 o solution.
B
-l9_ 13359~8
For instance, if horse-radish peroxidase is used as visualizing
agent B, any of the following solutions can be used as a substrate of
this enzyme immediately after preparation:
Solution 1: ABTC (2.2' azinobis-(3-ethyl-benzthioazoline-6-sulfonic
acid) lmM. hydrogen peroxide - 0.002% in phosphate-citrate buffer
with pH = 4.3.
Solution 2: c-phenylenediamine 4 mM, hydrogen peroxide 0.004% in
phosphate-citrate buffer with pH = 5.
Solution 3: 3.3', 5.5'-tetramethylenebenzidine 0.4 mM, hydrogen
o peroxide 0.004% in acetate buffer with pH = 6 .
or other peroxidase substrates widely used in immuno-enzymatic
analysis.
For instance, if ~lk~line phosphatase is used as visu~1i7ing
agent B, the following solution can be used as a substrate of this
enzyme: p-nitrophenol phosphate 2.5 mM, magnesium chloride 0.5
mM in diethyl amine buffer with pH = 9.5. or any other ~lk~line
phosphatase substrate used in immuno-enzymatic analysis.
200 patients with verified diagnosis have been examined, and
the results shown in the table given a high correlation between the
proposed diagnostic method and a level of atherosclerotic vascular
lesion.
-20- 13~59~8
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