CA1335968C - Method for producing affinity-enzymatic compounds for visual indication of cholesterol on skin surface - Google Patents
Method for producing affinity-enzymatic compounds for visual indication of cholesterol on skin surfaceInfo
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- CA1335968C CA1335968C CA000588652A CA588652A CA1335968C CA 1335968 C CA1335968 C CA 1335968C CA 000588652 A CA000588652 A CA 000588652A CA 588652 A CA588652 A CA 588652A CA 1335968 C CA1335968 C CA 1335968C
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
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Abstract
The invention deals with a method for producing affino-enzymatic compounds for visual indication of cholesterol on the skin surface based on detecting agent A, which is an affinant of cholesterol, and visualizing agent B. Steroid glycosides, triterpene glycosides, hydrophobic proteins, proteins toxins or polyene antibiotics which are affinants of cholesterol are used as detecting agent A, and enzymes - as visualizing agent B. Agents A and B are immobilized on binding agent C to increase sensitivity of compounds obtained. As agent C, low-molecular or high-molecular weight compounds are used.
Description
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 ~
.
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.
,~
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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.
.
-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.
~ .
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 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
. ~,~
- 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, (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~
~. ~
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
~,. ., . ~
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 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, ., 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.
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~
. ._ 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
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 ~ ~ o ~, ~o U~ U~ ~ +l+l ,c rC ~ C
t_ ~ _ O 'L
~ 1~ - oo I ~'~ +l +
C ~_ ~ ~ IC C
~ _ _ Lr> O t~ ~
,_ ~
C`~ ~ O
$ ~ ~ o ~~ ~ o ,, _ ` o +1 5 5 :~ ~, ~ ~o ~ ~
:~ a ~ ~ ~ ~ +l +I r o 'L _~ ~L ._ ~3 Z ~ ~ ~ ~ u s .5 a~ . . ~
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 ~
.
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.
.
-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.
~ .
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 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
. ~,~
- 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, (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~
~. ~
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
~,. ., . ~
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 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, ., 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.
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~
. ._ 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
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 ~ ~ o ~, ~o U~ U~ ~ +l+l ,c rC ~ C
t_ ~ _ O 'L
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C ~_ ~ ~ IC C
~ _ _ Lr> O t~ ~
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Claims (20)
1. Method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface, comprising the steps of:
a) chemically activating a detecting agent in an aqueous medium containing an activator in a detecting agent/activator molar ratio ranging from about 1:1 to about 1:10, at a temperature of about 0°
to about 25°C, a pH of about 4 to about 11 and for a period of about 0.1 to about 24 hours, said detecting agent being present in said aqueous medium in a concentration ranging from about 1 to about 20 mg/ml, said detecting agent having affinity for cholesterol and being selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol;
b) adding to the solution obtained in step (a) a visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase, in a detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1; and c) incubating the solution obtained in step (b) at a temperature of about 0° to about 25°C for a period of about 1 to about 24 hours.
a) chemically activating a detecting agent in an aqueous medium containing an activator in a detecting agent/activator molar ratio ranging from about 1:1 to about 1:10, at a temperature of about 0°
to about 25°C, a pH of about 4 to about 11 and for a period of about 0.1 to about 24 hours, said detecting agent being present in said aqueous medium in a concentration ranging from about 1 to about 20 mg/ml, said detecting agent having affinity for cholesterol and being selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol;
b) adding to the solution obtained in step (a) a visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase, in a detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1; and c) incubating the solution obtained in step (b) at a temperature of about 0° to about 25°C for a period of about 1 to about 24 hours.
2. Method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface, comprising the steps of:
a) chemically activating a visualizing agent in an aqueous medium containing an activator in a visualizing agent/activator molar ratio ranging from about 1:1 to about 1:10, at a temperature of about 0°
to about 25°C, a pH of about 4 to about 11 and for a period of about 1 to about 24 hours, said visualizing agent being present in said aqueous medium in a concentration ranging from about 1 to about 20 mg/ml, said visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase;
b) adding to the solution obtained in step (a) a detecting agent which has affinity for cholesterol and is selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol, in a detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1; and c) incubating the solution obtained in step (b) at a temperature of about 0° to about 25°C for a period of about 1 to about 24 hours.
a) chemically activating a visualizing agent in an aqueous medium containing an activator in a visualizing agent/activator molar ratio ranging from about 1:1 to about 1:10, at a temperature of about 0°
to about 25°C, a pH of about 4 to about 11 and for a period of about 1 to about 24 hours, said visualizing agent being present in said aqueous medium in a concentration ranging from about 1 to about 20 mg/ml, said visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase;
b) adding to the solution obtained in step (a) a detecting agent which has affinity for cholesterol and is selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol, in a detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1; and c) incubating the solution obtained in step (b) at a temperature of about 0° to about 25°C for a period of about 1 to about 24 hours.
3. Method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface, comprising the steps of:
a) dissolving a detecting agent in an aqueous salt buffer solution at a pH of about 5 to about 9, said detecting agent being present in said aqueous salt buffer solution in a concentration ranging from about 1 to about 20 mg/ml, said detecting agent having affinity for cholesterol and being selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures;
- triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol, b) adding to the solution obtained in step (a) an asymmetric bifunctional binding agent of low molecular weight selected from the group consisting of bromcyan, trichlorotriazine and 2-amino-4,6-dichloro-5-triazine, in a detecting agent/binding agent molar ratio ranging from about 1:0.5 to about 1:10;
c) incubating the solution obtained in step (b) at a temperature of about 0° to about 20°C for a period of about 1 to about 20 hours;
d) adding to the incubated solution of step (c) a visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphatedehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase, in detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1; and e) incubating the solution obtained in step (d) at a temperature of about 0° to about 20°C for a period of about 1 to about 48 hours.
a) dissolving a detecting agent in an aqueous salt buffer solution at a pH of about 5 to about 9, said detecting agent being present in said aqueous salt buffer solution in a concentration ranging from about 1 to about 20 mg/ml, said detecting agent having affinity for cholesterol and being selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures;
- triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol, b) adding to the solution obtained in step (a) an asymmetric bifunctional binding agent of low molecular weight selected from the group consisting of bromcyan, trichlorotriazine and 2-amino-4,6-dichloro-5-triazine, in a detecting agent/binding agent molar ratio ranging from about 1:0.5 to about 1:10;
c) incubating the solution obtained in step (b) at a temperature of about 0° to about 20°C for a period of about 1 to about 20 hours;
d) adding to the incubated solution of step (c) a visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphatedehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase, in detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1; and e) incubating the solution obtained in step (d) at a temperature of about 0° to about 20°C for a period of about 1 to about 48 hours.
4. Method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface, comprising the steps of:
a) dissolving a visualizing agent in an aqueous salt buffer solution at a pH of about 5 to about 9, said visualizing agent being present in said aqueous salt buffer solution in a concentration ranging from about 1 to about 20 mg/ml, said visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline and acid phosphatase, alpha-chymotrypsin and pyrophosphatase;
b) adding to the solution obtained in step (a) an asymmetric bifunctional binding agent of low molecular weight selected from a group consisting of bromcyan, trichlorotriazine and 2-amino-4,6-dichloro-
a) dissolving a visualizing agent in an aqueous salt buffer solution at a pH of about 5 to about 9, said visualizing agent being present in said aqueous salt buffer solution in a concentration ranging from about 1 to about 20 mg/ml, said visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline and acid phosphatase, alpha-chymotrypsin and pyrophosphatase;
b) adding to the solution obtained in step (a) an asymmetric bifunctional binding agent of low molecular weight selected from a group consisting of bromcyan, trichlorotriazine and 2-amino-4,6-dichloro-
5-triazine, in a visualizing agent/binding agent molar ratio ranging from about 1:0.5 to about 1:10;
c) incubating the solution obtained in step (b) at a temperature of about 0° to about 20°C for a period of about 1 to about 20 hours;
d) adding to the incubated solution of step (c) a detecting agent which has affinity for cholesterol and is selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol, in detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1, e) incubating the solution obtained in step (d) at a temperature of about 0° to about 20°C for a period of about 1 to about 48 hours.
5. Method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface, comprising the steps of:
a) dissolving a detecting agent and a visualizing agent in an aqueous salt buffer solution at a pH of about 5 to about 9, said detecting agent and said visualizing agent being each present in said aqueous buffer solution in a concentration ranging from about 0.1 to about 20 mg/ml and in a detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1, said detecting agent having affinity for cholesterol and being selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol, said visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline and acid phosphatase, alpha-chymotrypsin and pyrophosphatase;
b) adding to the solution obtained in step (a) a polyfunctional binding agent of high molecular weight selected from the group consisting of polysaccharides, proteins and synthetic polymers containing at least one functional group selected from the group consisting of primary amine, carboxyl, hydroxyl, aldehyde, haloid-anhydride, mixed anhydride, azide, hydrazide, maleimide, isocyanate and epoxide, in a detecting agent and visualizing agent/binding agent molar ratio ranging from about 1:0.5 to about 1:10; and c) incubating the obtained solution in step (b) at a temperature of about 0° to about 20°C for a period of about 1 to about 20 hours.
c) incubating the solution obtained in step (b) at a temperature of about 0° to about 20°C for a period of about 1 to about 20 hours;
d) adding to the incubated solution of step (c) a detecting agent which has affinity for cholesterol and is selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol, in detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1, e) incubating the solution obtained in step (d) at a temperature of about 0° to about 20°C for a period of about 1 to about 48 hours.
5. Method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface, comprising the steps of:
a) dissolving a detecting agent and a visualizing agent in an aqueous salt buffer solution at a pH of about 5 to about 9, said detecting agent and said visualizing agent being each present in said aqueous buffer solution in a concentration ranging from about 0.1 to about 20 mg/ml and in a detecting agent/visualizing agent molar ratio ranging from about 20:1 to about 1:1, said detecting agent having affinity for cholesterol and being selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - hydrophobic proteins capable of discriminately forming complex compounds with cholesterol, - protein toxins capable of discriminately forming complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - enzymes having affinity for cholesterol, said visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline and acid phosphatase, alpha-chymotrypsin and pyrophosphatase;
b) adding to the solution obtained in step (a) a polyfunctional binding agent of high molecular weight selected from the group consisting of polysaccharides, proteins and synthetic polymers containing at least one functional group selected from the group consisting of primary amine, carboxyl, hydroxyl, aldehyde, haloid-anhydride, mixed anhydride, azide, hydrazide, maleimide, isocyanate and epoxide, in a detecting agent and visualizing agent/binding agent molar ratio ranging from about 1:0.5 to about 1:10; and c) incubating the obtained solution in step (b) at a temperature of about 0° to about 20°C for a period of about 1 to about 20 hours.
6. Method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface, comprising the steps of:
a) dissolving a detecting agent in an aprotic polar solvent, said detecting agent having affinity for cholesterol and being selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - mixtures of the above;
b) adding to the solution obtained in step (a) a polyfunctional binding agent of high molecular weight selected from the group consisting of polysaccharides, proteins and synthetic polymers containing at least one functional group selected from the group consisting of primary amine, carboxyl, hydroxyl, aldehyde, haloid-anhydride, mixed anhydride, imino-ester, azide, hydrazide, maleimide, isocyanate and epoxide, said detecting agent and said binding agent being each present in said solution in a concentration ranging from about 1 to about 20 mg/ml and in a detecting agent/binding agent molar ratio ranging from about 1:1 to about 1:10;
c) incubating the solution obtained in step (b) at a temperature of about 20° to about 120°C for a period of about 0.5 to about 6 hours;
d) precipitating a product from the incubated solution of step (c);
e) drying the precipitated product by holding same in vacuum over phosphorus pentoxide at a temperature of about 80° to about 120°C for a period of about 4 to about 10 hours;
f) adding the dried product of step (e) to an aqueous salt buffer solution containing a visualizing agent dissolved therein, said visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase, said product and said visualizing agent being present in said aqueous salt buffer solution in a concentration of 2-20 mg/ml and about 1-10 mg/ml, respectively, and g) incubating the solution obtained in step (f) at a temperature of about 4° to about 8°C for a period of about 2 to about 12 hours.
a) dissolving a detecting agent in an aprotic polar solvent, said detecting agent having affinity for cholesterol and being selected from the group consisting of:
- steroid glycosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanole or spirostanole series, and an oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, - triterpene glycosides containing an aglicone of alpha- or beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues with branched or linear structures, - polyene antibiotics capable of discriminately forming complex compounds with cholesterol, and - mixtures of the above;
b) adding to the solution obtained in step (a) a polyfunctional binding agent of high molecular weight selected from the group consisting of polysaccharides, proteins and synthetic polymers containing at least one functional group selected from the group consisting of primary amine, carboxyl, hydroxyl, aldehyde, haloid-anhydride, mixed anhydride, imino-ester, azide, hydrazide, maleimide, isocyanate and epoxide, said detecting agent and said binding agent being each present in said solution in a concentration ranging from about 1 to about 20 mg/ml and in a detecting agent/binding agent molar ratio ranging from about 1:1 to about 1:10;
c) incubating the solution obtained in step (b) at a temperature of about 20° to about 120°C for a period of about 0.5 to about 6 hours;
d) precipitating a product from the incubated solution of step (c);
e) drying the precipitated product by holding same in vacuum over phosphorus pentoxide at a temperature of about 80° to about 120°C for a period of about 4 to about 10 hours;
f) adding the dried product of step (e) to an aqueous salt buffer solution containing a visualizing agent dissolved therein, said visualizing agent comprising an enzyme selected from the group consisting of acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin and pyrophosphatase, said product and said visualizing agent being present in said aqueous salt buffer solution in a concentration of 2-20 mg/ml and about 1-10 mg/ml, respectively, and g) incubating the solution obtained in step (f) at a temperature of about 4° to about 8°C for a period of about 2 to about 12 hours.
7. Method according to claim 1, 2, 3, 4, 5 or 6, wherein said detecting agent is a steroid glycoside selected from the group consisting of agavosides A, B, C, D, F, H and G, trillin, funcosides C, D, F, G
and I, dioscin, gracillin, protodioscin, kikubasoponin, juccosides E and H, lanotigonin, desglycodigitonin, digitonin, gitonin, rocosides C, D
and E, funcoside E, alliumoside C, polygonatonin, tigogenin tetraoside, tigogenin hexaoside, kapsycoside, ammumosides B, C, D and E, desglycodesramnoparillin, desglycoparillin, parillin, sarmaparilloside, asparagosides C, D, G and H, protojuccoside H, juccoside B, lanotigoside, monoside, bioside, trioside, purpureagitoside, gecogenin, rocogenin, diogenin, tigogenin, protopolyagamatoside, tomatine, laxogenin, liquotetraoside, alliogenin, lactotetraoside, karatavioside A, cyclosiversioside H, acanthophylloside C, alliofuroside A, alliospiroside A, cyclosiversioside D, tea saponin, acanthophylloside B, tigonin (total), glycoside from Calha Polypatala and cyclosiversioside G.
and I, dioscin, gracillin, protodioscin, kikubasoponin, juccosides E and H, lanotigonin, desglycodigitonin, digitonin, gitonin, rocosides C, D
and E, funcoside E, alliumoside C, polygonatonin, tigogenin tetraoside, tigogenin hexaoside, kapsycoside, ammumosides B, C, D and E, desglycodesramnoparillin, desglycoparillin, parillin, sarmaparilloside, asparagosides C, D, G and H, protojuccoside H, juccoside B, lanotigoside, monoside, bioside, trioside, purpureagitoside, gecogenin, rocogenin, diogenin, tigogenin, protopolyagamatoside, tomatine, laxogenin, liquotetraoside, alliogenin, lactotetraoside, karatavioside A, cyclosiversioside H, acanthophylloside C, alliofuroside A, alliospiroside A, cyclosiversioside D, tea saponin, acanthophylloside B, tigonin (total), glycoside from Calha Polypatala and cyclosiversioside G.
8. Method according to claim 1, 2, 3, 4, 5 or 6, wherein said detecting agent is a steroid glycoside selected from the group consisting of funcosides C, D, E, F, G or I, dioscin, rocoside, lanotigonin, digitonin and tomatine.
9. Method according to claim 1, 2, 3, 4, 5 or 6, wherein said detecting agent is a triterpene glycoside selected from the group consisting of ascine, avenacine, tea saponin, alpha-gederine, kauloside C, stichionoside A, cyclamine, chinovine, saponins from sugar-beet, hypsoside and triterpene glycosides obtained from Fatsia Japonica.
10. Method according to claim 1, 2, 3, 4 or 5, wherein said detecting agent is a hydrophobic protein selected from the group consisting of Folch meiline protein, lysosomal and mitochondrial proteins, fibrinogen, immunoglobulins, cerebrone, myoglobin, tripsin, cytochrome C, cytochrome P-450 and apo-proteins of lipoproteins.
11. Method according to claim 1, 2, 3, 4 or 5, wherein said detecting agent is a protein toxin selected from the group consisting of streptolysine O, pneumolysine, listeriolysine toxin C.I obtained from Perfringens of A and C types, .delta.-toxin C.I novyi of A and C types, hemolysine C.I, histolyticum, homolysine C.I botulinum of C and D types, tetanolysine, cereolysine, alveolysine, turingeolysine and cytotoxin from Metridium senile.
12. Method according to claim 1, 2, 3, 4, 5 or 6, wherein said detecting agent is a polyene antibiotic selected from the group consisting of amphotericin B, filipin and nystatin.
13. Method according to claim 1, 2, 3 or 5, wherein said detecting agent is a high-affinity enzyme selected from the group consisting of cholesterol oxidase, cholesterol dehydrogenase and cholesterol esterase.
14. Method according to claim 5, wherein said binding agent is a copolymer of acrylic acid or maleic anhydride.
15. Method according to claim 14, wherein said binding agent is a copolymer of N-vinylpyrrolidone with maleic anhydride.
16. Method according to claim 1 or 2, wherein said activator is a carbodiimide, succinimide, azide or periodate compound.
17. Method according to claim 6, wherein said aprotic polar solvent is selected from the group consisting of dimethyl-sulfoxide, dimethylformamide, hexamethanol, 2:1 dimethylformamide/toluol mixture and 2:1 dimethylformamide/hexanol mixture.
18. Method according to claim 3, 4, 5, 6 or 17, wherein said aqueous salt buffer solution is a borate, citrate or phosphate solution ensuring a buffer action within a pH range of about 5 to about 9.
19. An affino-enzymatic compound produced by a method according to claim 1, 2, 3, 4, 5 or 6.
20. Use of an affino-enzymatic compound produced by a method according to claim 1, 2, 3, 4, 5 or 6, for providing a visual indication of cholesterol on skin surface.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SU884357046A SU1675769A1 (en) | 1988-01-19 | 1988-01-19 | Method for preparation of agent, used for visual detection of cholesterol on the skin surface |
| RU4357046 | 1988-01-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1335968C true CA1335968C (en) | 1995-06-20 |
Family
ID=21347013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000588652A Expired - Lifetime CA1335968C (en) | 1988-01-19 | 1989-01-19 | Method for producing affinity-enzymatic compounds for visual indication of cholesterol on skin surface |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0338189B1 (en) |
| JP (1) | JPH01289498A (en) |
| CN (1) | CN1048925A (en) |
| AT (1) | ATE137336T1 (en) |
| AU (1) | AU615709B2 (en) |
| CA (1) | CA1335968C (en) |
| CS (1) | CS274496B2 (en) |
| DD (1) | DD289604A5 (en) |
| DE (1) | DE58909664D1 (en) |
| FI (1) | FI890266A (en) |
| HU (1) | HUT56973A (en) |
| PL (1) | PL277263A1 (en) |
| SU (2) | SU1675770A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5489510A (en) * | 1988-01-19 | 1996-02-06 | 2860601 Canada Inc. | Method for visual indication of cholesterol on skin surface agents used therefor and methods for producing such agents |
| RU94044169A (en) * | 1994-12-16 | 1996-10-20 | И.А. Кочетов | Multiple analytical member |
| LT4121B (en) | 1995-06-01 | 1997-03-25 | Akcine Bendrove Biofa | Kit for the cholesterol vizualization on the skin and process for preparing conjugate for use in cholesterol determination |
| RU2130189C1 (en) * | 1997-02-20 | 1999-05-10 | Александр Сергеевич Парфенов | Method for determining tissular cholesterol in skin |
| CA2465427A1 (en) * | 2004-04-28 | 2005-10-28 | Imi International Medical Innovations Inc. | Direct assay of cholesterol in skin samples removed by tape stripping |
| CN100564539C (en) * | 2004-12-31 | 2009-12-02 | 浙江伊利康生物技术有限公司 | The mensuration reagent and the preparation method of cholesterol in the high-density lipoprotein (HDL) |
| CN104181312B (en) * | 2014-08-08 | 2016-06-01 | 安徽易康达光电科技有限公司 | A kind of skin free cholesterol noninvasive detection device for atherosclerosis risk assessment |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4071020A (en) * | 1976-06-03 | 1978-01-31 | Xienta, Inc. | Apparatus and methods for performing in-vivo measurements of enzyme activity |
| US4401122A (en) * | 1979-08-02 | 1983-08-30 | Children's Hospital Medical Center | Cutaneous methods of measuring body substances |
| US4458686A (en) * | 1979-08-02 | 1984-07-10 | Children's Hospital Medical Center | Cutaneous methods of measuring body substances |
| EP0118543A1 (en) * | 1982-09-01 | 1984-09-19 | University Of Southern California | Substituted n-benzenesulfonyloxyphthalimides |
-
1988
- 1988-01-19 SU SU4357046K patent/SU1675770A1/en active
- 1988-01-19 SU SU884357046A patent/SU1675769A1/en active
-
1989
- 1989-01-18 DD DD89325117A patent/DD289604A5/en not_active IP Right Cessation
- 1989-01-18 CS CS34889A patent/CS274496B2/en unknown
- 1989-01-18 FI FI890266A patent/FI890266A/en not_active IP Right Cessation
- 1989-01-18 EP EP89100828A patent/EP0338189B1/en not_active Expired - Lifetime
- 1989-01-18 DE DE58909664T patent/DE58909664D1/en not_active Expired - Lifetime
- 1989-01-18 AT AT89100828T patent/ATE137336T1/en not_active IP Right Cessation
- 1989-01-18 AU AU28578/89A patent/AU615709B2/en not_active Expired - Fee Related
- 1989-01-19 JP JP1008737A patent/JPH01289498A/en active Pending
- 1989-01-19 HU HU89212A patent/HUT56973A/en unknown
- 1989-01-19 CA CA000588652A patent/CA1335968C/en not_active Expired - Lifetime
- 1989-01-19 PL PL27726389A patent/PL277263A1/en unknown
- 1989-07-15 CN CN89106704.3A patent/CN1048925A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0338189A3 (en) | 1990-05-02 |
| FI890266A (en) | 1989-07-20 |
| AU2857889A (en) | 1989-07-20 |
| FI890266A0 (en) | 1989-01-18 |
| DE58909664D1 (en) | 1996-05-30 |
| HUT56973A (en) | 1991-10-28 |
| ATE137336T1 (en) | 1996-05-15 |
| SU1675770A1 (en) | 1991-09-07 |
| CS274496B2 (en) | 1991-04-11 |
| CN1048925A (en) | 1991-01-30 |
| EP0338189B1 (en) | 1996-04-24 |
| AU615709B2 (en) | 1991-10-10 |
| DD289604A5 (en) | 1991-05-02 |
| EP0338189A2 (en) | 1989-10-25 |
| JPH01289498A (en) | 1989-11-21 |
| SU1675769A1 (en) | 1991-09-07 |
| PL277263A1 (en) | 1989-09-04 |
| CS34889A2 (en) | 1990-08-14 |
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