AU615709B2

AU615709B2 - Method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface based on cholesterol affinant detecting agent and visualizing agent and application thereof

Info

Publication number: AU615709B2
Application number: AU28578/89A
Authority: AU
Inventors: Irina Pavlovna Adrianova; Lev Alexandrovich Andrianov; Dmitry-Borisovich Gudkov; Jury Mikhailovich Lopukhin; Alexandr Borisovich Rabovsky; Viktor Vikrotovich Zuevsky
Original assignee: NII FIZ KHIM MEDITSINY
Current assignee: NAUCHNO-ISSLEDOVATELSKY INSTITUT FIZIKO-KHIMICHESKOI MEDITSINY
Priority date: 1988-01-19
Filing date: 1989-01-18
Publication date: 1991-10-10
Anticipated expiration: 2009-01-18
Also published as: EP0338189A3; FI890266A; AU2857889A; FI890266A0; DE58909664D1; HUT56973A; ATE137336T1; SU1675770A1; CS274496B2; CN1048925A; EP0338189B1; DD289604A5; CA1335968C; EP0338189A2; JPH01289498A; SU1675769A1; PL277263A1; CS34889A2

Abstract

Used as detecting agent A are steroid glycosides, triterpene glycosides, hydrophobic proteins, proteinaceous toxins or polyene antibiotics which have an affinity for cholesterol, and as visualisation agent B are enzymes. To increase the sensitivity of the prepared compounds, the detecting agent A and the visualisation agent B are immobilised on a crosslinking agent C, for which low or high molecular weight compounds are used. <IMAGE>

Description

THE COMMISSIONER OF PATENTS OUR REF: 83522 S&F CODE: ,63320 5845/2 i Y. *iP w FORM 10 615709 SPRUSON FERGUSON COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int. Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: 0S 9*

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Name of Applicant: Address of Applicant: Address for Service: Nauchno-Issledovatelsky Institut Fiziko-Khimicheskoi Meditsiny Malaya Pirogovskaya ulitsa, la Moscow Union Soviet Soc. Republics Spruson Ferguson, Patent Attorneys, Level 33 St Martins Tower, 31 Market Street, Sydney, New South Wales, 2000, Australia *5S*

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*5 Complete Specification for the invention entitled: Method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface based on cholesterol afiinant detecting agent and visualizing agent and application thereof The following statement is a full description of this invention, including the best method of performing it known to me/us application made in a Convention country in respect of the invention the subject of the application.

Declared at MoSCow this 6 day of Decembe1988 Elvio Mustafovich Khalilov deputy direc r Signature of eclarant SPRUSON FERGUSON, SYDNEY.

The Commissioner of Patents,

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ABSTRACT

The invention deals with amethod for producing affino- -ensz.ymaic compounds for visual ndication of cholesterol on skin surface based on detecting gent A, which is an affinant of cholesterol, and visualizing agent B. Steroid glycosides, triterpene glycosides, bydrophobic proteins, protein 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 compounds are used.

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0 55 55 0 OS 0 a 0 @555 0 1A Method for producing aifino-enz.ymatic compounds for visual indication of cholesterol on skip-surface based on cholesterol affinant detecting agent and vizualizing agent and application thereof The proposed invention is in the field of bioorganic chemistry, namely, it deals with a method for producing affino-enzymatic compounds for visual indication of cholesterol on skin surface based on a detecting agent, which is an affinant of cholesterol, and a visualizing 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 phy- S" siological, histological and histochemical studies to detect S' high contents of cholesterol and its localization. Besides, the proposed, compounds enable to detect low contents of chole- S sterol 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 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 2.

of life style. The most difficult in atherosclerosis prevention is selection of high-risk groups by amount of cholesterol accumulated in the organism. The existing methods of atherosclerosis diagnostics are based. on quantization of general cholesterol content in the venous blood plasm. (Consensus Conference on Lowering Blood Cholesterol to Prevent Heart Disease, JAMA, 1985, 253, p.2080-2086; The Lipid Research Clinics 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 I cholesterol content over 260 mg is considered sufficient to include a patient into the risk group. A more precise diagnosis can be made by analyzing blood plasm lipoproteins and determining the atherogeneity index, which is a ratio of difference between total and high-density lipoprotein cholesterol to high-density lipoprotein cholesterol: S. Ix atber O C (total) Ch (h-d 1.) Ch (h-d 1.) A patied. 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, Medicine, 1975; Goldfourt

V.,

iHoltzman Neufeld H.N. Total and High Density Lipoproein Cholesterol in the Serum and Risk of Mortality. British Medical Journal, 1985, 290, p.I239-I2431).

Use of these methods necessitates blood taking, which may be traumatic for patients and, besides, is not altogether safe Aj 3.

for the reason of possible viral infections. Fractionation of plasm lipoproteins and cholesterol analysis still remain a complicated. and. expensive procedure. Moreover, in one case out of three quantization of total cholesterol and even a complete phenotyping do not correlate with severity of atherosclerosis (See: KNasnikov "Hypertensive disease", 1965, M., Medicine, p.300).

In addition, recent studies showed. that the blood plasm can not fully reflect processes of cholesterol accumulation, which are typical for the arterial wall and other bradytrophic tissues.

Solution of some fundamental problems related to atherosclerosis 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 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 atherosclerosis.

The above mentioed correlation between cholesterol content in the arterial wall and skin was found out through di- S. rect quantization of cholesterol in skin biopsy. Specimens were freezed in liquid ni~rogen and lyophilized; cholesterol was extracted. with Folch reagent and quantified using traditional chemical or biochemical methods. (See: Nikitin Gordienko Dolgov Filimonova T.A. "Cholesterol. content in the ,r Solera M.L. et al. Ann.Biol.Clin., 1982, vol.4 0 p.361-407).

due to its traumaticity and complicacy.

US Patent No.4458686 describes a method, which enables to skin antiy glucose and ethanol, localipid uotien in the serumblood, directly normals and in patients wi isurface, and points out that se", Cardiology, 1987, XXvII, No.IO, p.48-5I; Bouisson De Graeve, oler M.Lquantitaton usng BioCn.,loxidase is also40, p-40ossible.

However, this method is not suitable for population screening due to its traumaticity and complicac.

/US Patent No.4458686 describes a method, which enables to quantify glucose and ethanol, localized in the blood, directly under the skin or on its surface, and points out that cholesterol quantitation using cholesteroloxidase is also possible.

The method is based on stoichiometric changes of oxygen concentration when using radox enzymes, mostly oxidoreductases, specific to the substrate in question. According to this method quantitation of oxygen concentration changes is made electrochemically, for instance, polarography with the use of special fee S equipment and specially designed electrode.

Such a sophisticated instrumentation necessitates the services of high-skilled personnel for making diagnoses. All this inevitably limits the possibility of using this method for population screening.

i Application POT/US 84/00888 proposes a detection-visuali- S zation complex where the detecting and visualizing 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 S* 'lipids can be used only in laboratory conditions and 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 traumaticity, this method. has several disadvantages, because specimens I 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 suppl. and, consequently, specimens include vessels and blood; besides, they contain perspiratpry 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 epidermic 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 it does not have sebacous glands whose secretions, as well as the horny layer, contain cholesterol which may affect diagnostic results.

S* The invention solves this problem by creating a method to produce obtain affino-enzymatic compounds which are bi-functional 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 to discriminately form stable complexes with free cholesterol of the skin in order to make the whole bi-functional compound afferent to cholesterol, and visualizing ageivt 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 compounds in diagnostics will make it possible to determine whether the examined patients belong to one of the three groups: atherosclerotics, risk group or normals.

The affino-enzymatic compounds for visual indication of 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 can even be used in domestic conditions.

SUMMARY OF THE INVENTION 20 According to a first embodiment of this invention, there is provided a bifunctional compound for the visual detection of cholesterol on the surface of skin, comprising detecting agent A covalently linked to visualizing agent B, wherein: said detecting agent A is a moiety capable of discriminately 1' 25 forming a stable complex with said cholesterol: and said visualizitrg agent B is an enzyme moiety which converts a colourless substrate to a coloured product or a coloured substrate to a i. colourless product thereby indicating the presence of said cholesterol on said skin surface.

According to a second embodiment of this invention, there is provided a method for producing a bifunctional compound as defined in any one of claims 1 to 11, wherein detecting agent A is chemically activated in an aqueous medium at a molar ratio of agent A to activator of 1:1 1:10, at a concentration of agent A of 1-20 mg/ml, a temperature of 0-25 0 C and a pH of 4-11 for 0.1-24 hours, and to the solution obtained, visualizing agent B is added at a molar ratio of A:8 of 20:1 1:1 and the solution is Incubated at a temperature of 0-25 0 C for 1-I4 hours.

According to a third embodiment of this invention, there is .M L provlded a method for producing a bifunctional compound as defined in any LN 483 2 00rj 6A one of claims 1 to 11, wherein visualizing agent B is chemically activated in an aqueous medium at a molar ratio of B activator of 1:1 1:10 at a concentration of agent B of 1-20 mg/ml, a temperature of 0-25°C and a pH of 4-11 for 1-24 hours, and to the solution obtained, detecting agent A is added at a molar ratio of A:B of 20:1 1:1 and the solution is Incuibated at a temperature of 0-25°C for 1-24 hours.

According to a fourth embodiment of this invention, there is provided a method for producing a bifunctional compound as defined in any one of claims 12 to 14, wherein detecting agent A is dissolved in an aqueous salt solution at a pH of 5-9, at a concentration of A of 1-20 mg/ml, and a low-molecular asymmetric bifunctional binding agent C is added at a molar ratio of A:C of 1.0.5 1:10, then visualizing agent B is added at a molar ratio of A:B of 20:1 1:1 and the solution is incubated at a temperature of 0-20 0 C for 1-48 hours.

According to a fifth embodiment of this invention, there is provided a method for producing a bifunctional compound as defined in any *0 one of claims 12 to 14, wherein visualizing agent B is dissolved in an aqueous salt solution at a pH of 5-9 and a concentration of 1-20 mg/ml, a Slow-molecular asymmetric bifunctional binding agent C is added at a molar 20 ratio of B:C of 1:0.5 1:1 and the resulting solution is incubated at a temperature of 0-20 0 C for 1-20 hours, detecting agent A is added at a molar ratio of A:B of 20:1 1:1 and the solution is incubated at a temperature of 0-20°C for 1-48 hours.

According to a sixth embodiment of this invention, there is S 25 provided a method for producing i bifunctional compound as defined in any one of claims 15 to 18, wherein: detecting agent A and visulizing agent B are dissolved at a molar ratio of A:B of 20:1 1:1 in an aqueous buffer solution at a pH of 5-9, at a concfntration of each of agents A and B of 0.1-20 mg/ml, and a high-molecular weight polymer binding agent C is added to the resulting solution it a molar ratio of of 1:0.5 1:10, and the obtained solution is incubated at a temperature of 0-20°C for 1-20 hours.

According to a seventh eabodiment of this invention, there is provided a method for produciIg a bifunctional compound as defined in any one of claims 15 to 18, wherein detecting agent A is preliminarily dissolved in an aprotonic polar solvent and high-molecular weight polymer binding agent C is added thereto at a concentration of each of agents A and C of 1-20 mg/ml and a ratio of A:C of 1:1 1:10, and the solution is Incubated at a temperAure of 20-120°C for 0.5-6 hours, thereafter the SiM/483Z -I U 6B resulting product (A C) Is precipitated from the solvent and held in vacuum over phosphorus pentoxide at 80-120 0 C for 4-10 hours, the dried product at a concentration of 2-20 mg/ml is added to an aqueous salt solution having a pH of 5-9 containing dissolved visualizing agent B at a concentration of 1-10 mg/ml, and the solution is incubated at a temperature of 4-8 0 C for 2-.12 hours.

According to a eighth embodiment of this invention, there is provided a method of assessing the cholesterol level in the skin of an individual, which method comprises applying to an area of said skin a bifunctional compound as defined in any one of claims 1 to 18 or washing unbound compound from said area of skin, and applying a substrate for said visualizing agent B.

DETAILED DESCRIPTION OF THE INVENTION The compound according to the invention can only be obtained either from detecting agent A, or visualizing agent B by chemically activating functional groups of one of these agents, Then, after adding to one agent containing activated chemical groups, the other agent, there is formed under certain conditions a stable A-B bond resulting in a 20 high-molecular bi-functlonal compound for visual Indication of S. cholesterol on the skin of patients.

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The following oompound are used, as cholesterol/detecting agent A affinants: Steroid glycoside, containing as an aglicone a c.vclopentanojporhydrophenetrene fragment of furostanole or spirostanole series, and-oligosaoharide fragment including 3 to monosaocharides with linear or branched structures (KintyaP.K.

"Structure and biological activity of steroid glycosides of spirostan and furostan series", Kishinev, Stinza, 1987, P.142), or Triterpene P lycosides, containing an aglioxoe of alphaor beta-amwvril, lupane, gopane, dammarane, linostan or holostan series, and oligosacoharide of 2-B residues of branched or linear structures (Dekanosidze Chirva Sergienko Uvarova N.I. "Study on triterpene glbyoosides", Tbilisi, Mezniereba, 1982), or gyropho ic p oteins, capable to discriminately form a complex compound with cholesterol (Klimov Titova GV., Kozhevnikov Biochemistry, 1982, vol.47, No.2, p.226-232); Klimov Kozhevnikov Klvueva N.N. et al. Voprosy Med.

Khiniii, 1984, vol.3 0 No.5, p.86-90; Titova Klyueva N.N., Kozhevnikov et al. Biochemistry, 1980, vol.45, No.1, f P.51-55), or **elkProtein toxdins, capable to discriminateLy form complex compounds with cholesterol. They are obtained from bacteria, marine microorganisms, insects or snakes (flalin Fish N.G.

*9 "Protein toxins of microorganisms", Moscow, Medicine, 1980), or Pql.eneAntibiotica, capable to discriminately form complex

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compounds with cholesterol (I.J.Katzenstein, A.M.Spielvogel, A.W.Norman, J.Antibiot., 27, 12, 1974, p-943-95I; Jong Shang-Shyng, Wang Hsi-Hua. Clin.J.Microbiol., 1976, 9, p.19-50; Readig Josephine D. et al. Biochim.Bioph.vs.Acta, 1982, 685 p.219-224), or High-affinit.y enzymes, whose substrate is cholesterol, and which have a high affinity to it.

As a visualizing agent B, the following enzymes are used: acet.lcholinesterase, tyrosinase, glucose-6-phosphatedeh.ydrogenase, glucooxidase, glucoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin or pyrophosphatase.

The problem is solved, because the affinant-detecting agent selected from a group of steroid gl.cosides containing as an aglicone a cyclopentanoperhydrophenanthrene fragment of furostanols or spirostanole series, and oligosaccharide fragment including 3 to IO monosaccharides with linear or branched structures, or from a group of triterpene glycosides, containing an aglicone of alpha- or beta-amyrane, lupane, gonane, dammarane, lanostane or holostene series, and oligosaccharides of 2-8 resides with branched or linear structures, or from a group of hydrophobic proteins capable to discriminately form complex compounds with cholesterol, or from a group of protein toxins capable to discriminately form complex compounds with cholesterol, and obtained from bacteria, marine microorganisms, insects or snakes, or from a group of pol.yene antibiotics capable to discriminately form complex compounds with cholesterol, or S9.

from enzymes having a high affinity to cholesterol, is chemically activated in an aqueous medium with detecting agent A /activator molar ratio I:I 1:I1, detecting agent A concentration 1-20 mg/ml, temperature 0-250C, pH 4-II, during 0.1-24 hrs.

To the solution obtained, a visualizing agent B is added, selected among the following enzymes: acetylcholinesterase, t.yrosinase, glucose-6-phosphatedehydrogenase, glucosooxidase, glucosoamilase, galactosidase, peroxidase, alkaline or acid phosphatase, alpha-oh .Ymotrypsin or pyrophospbatase in A/B molar ratio 20:1 I:1. Then the solution is incubated at 0-250C during 1-24 hrs until the final product is obtained.

As mentioned above, functional groups of visualizing agent B can also be subjected to chemical activation. In this case, agent B is activated in an aqueous medium with B/activator

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molar ratio I:I 1:I0, agent B concentration 1-20 mg/ml, at temperature 0-250°C, pH 4-II, during 0.1-24 hrs. Then detecting agent A, which is one of the above-listed substances, is added to the solution, with A/B molar ratio 20:I I:1, and S; the solution is incubated at 0-25 0 C for I- 24 hrs until the final product is obtained.

Under the present invention, detecting agent A or visualizing agent B are activated using the well-known azide, carbodiimide and succinimide methods, or the method of periodate oxidation.

In cases when visualizing agent B is a high-molecular cornpound such as enzyme, and detecting agent A is a low-molecular compound, for instance, glycosis, a molecule of the final ;Li SI0.

product can contain only one molecule of visualizing agent B.

However, the molar quantity of detecting agent A, responsible for binding skin cholesterol in the final product, can exceed the content of visualizing agent B only by several times, according to the number of functional groups in the enzyme molecule capable to bind detecting agent A without significant loss of enzymatic activity. In such compounds, the content of visualizing 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.

To expand technical performance of affino-enzymatic compounds for visual indication of cholesterol on the skin, and in view of increasing their sensitivity, it is proposed to bind the above detecting agent A and visualizing agent B using binding agent C, taken from a series of low-molecular asymmetric Sbi-functional compounds, such as bromocan, trichlortriazine or S 2-amino-4,6-dichlor-3-triazino. In this case, compounds can be obtained in two ways: either detecting agent A is first linked to binding agent C and then visualizing agent B is added to the intermediate product to obtain the final product, or the intermediate product is B+C to which detecting agent A is added.

In the first option, detecting agent A is dissolved in a usual 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 0, selected among the above-mentioned substances, is added to the solution, with A/C molar ratio ,0 i r tnereot The following statement is a full description of this invention, including the best method of performing it known to me/us

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1-20 hrs; then visualizing agent B, selected from the above substances, is added with A/B molar ratio 20:I I:I, and the solution is incubated at 0-2000C for 1-48 hrs to obtain the final product.

To obtain an affino-enzymatic compound through the intermediate product B+0, visualizing agent B is dissolved in a usual 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 1/C molar ratio I:0.5 I:10. The solution obtained is incubated. at 0-20 0 for 1-20 brs; then detecting agent A is added with A/B molar ratio 20:I I:I, and the solution is incubated at 0-20 0 C for 1-48 brs to obtain the final product.

SHowever, the best sensitivity of affino-enz.matic compounds for visual indication of cholesterol on the skin are obtained when using as a binding agent C some high-molecular pol.yfunctional compounds, such as polysaccharides, proteins or synthetic polymers, i.e. any high-molecular compounds containing any of functional groups selected among the following: ol primary amines, carboxyl, hydrox.yl, aldehide, haloid anh.ydride, mixed anhy.drides, iminoesters, azide, hydrazide, maleimide, isocyanate or epoxide. Moreover, both detecting agent A, being S one of the above compounds or their mixture, and visualizing S agent B, also one of the above, are independently immobilized by anyone of these compounds. Such an affino-enzymatic compound enables most widely vary A/B ratios, depending on the extent of selected agent A affinity to skin cholesterol and, i priaryamies, arbxyl hydoxy, adehie, alod, ahydide I2.

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.

Such affino-enzymatic compounds are obtained as follows: 4detecting 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 I:1 and dissolved in an aqueous salt solution with pH 5-9, and in concentration of each of the agents 0.1-20 mg/mi. High-molecular pol.functional binding agent 0 is added, to the solution obtained; it is selected from polysacbcharides, proteins or s.ynthetic polymers according to the above. A+B/C molar ratio 0 96: 1:0.5 1:10. Then the solution is incubated at 0-200C for I-20 hrs to obtain the final product.

Among steroid glycosides, used as detecting agent A and containing as an aglicone a cyclopentanoperhbydrophenanthrene fragment of furostanole and spirostanole series, and oligosaccharide fragment including 3 to 10 monosaccharides with linear or branched structures, the most preferable are: agavosides A,B,C,D,F,H and G, trilin, funcoside I, dioscine, gracllin, ptotodosoine, kikubaseponine, juncosides E and H, lanotigonine, desgly.oodigitonin, digitonin, gitonin, rockosides C,f and E, funcoside BE, alliumosid.e C, polygonatonine, tigogenin tetraoside, togigenin hexaoside, kapsycoside, ammumosides B,C,D and E, desglycodesramnoparillin, desgl.Yooparillin, pa- S rillin, sarmaparilloside, asparagosides C,D,G and H, juocoside B, protojuocoside H, lanotigoside, monoside, bioside, trioside, presupposeS identification of the highest-risk group among the patients, followed, b' their differentiated treatment and change ii: 13.

purpureagitoside, tomatonine, alliogenine liquotetraoside, karatavioside A, cyclosiversioside H, acanthophiloside 0, alliofusoside A, alliospiroside A, cyclosiversioside F, tea saponin, acanthophiloside B, tigonine (total), glycoside from Calba Pol.vpatala or cyclosiversioside G.

Among the above steroid glycosides more preferable are funcosides C,D,E,F,G or I, dioscine, rockoside, lanotigonine, digitonine and tomatonine. The most preferable are digitonine and tomatonine.

Among triterpene glycosides used as detecting agent A and containing an aglicone of alpha- or beta-am.yril, lupane, gopane, dammarane, lanostane or holostane series, and oligosaccharide s of Z-8 residues with branched or linear structures, the most preferable are: escine, avenacine, tea saponin, alpha-'aderine, kauloside C, stichinoside A, cyclamine, ohinovine, saponins S from sugarbeet, hbypsoside and triterpene gl.ycosides obtained CP i: from Fatsia Japonica.

Among hydrophobic proteins used as detecting agent A and capable to discriminately form complex compounds with obolesterol, the most preferable are: Folch meilin protein, l.ysosomic proteins, proteins of m.ytochondria, fibrinogen, immuno- S globulins, cerebrone, myoglobin, tripsin, cytochrome C, cytochabrome 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: streptolgysine 0, pneumolisine, listerio lysin, 0-toxin C.I obtained from Perfrigens of A and IIj Use of these methods ,necessitates blood taking, which may be traumatic for patients and., besides, is not altogether safe S4.

C t.pes, 6 -toxin C.I. novyi of A and C types, Hemblysine C.I histol.ticum, hemolysine C.I boulinum of C and D types, tetano- 1.sine, cerebrolysine, alveol.sine, turingeol.ysine or c.ytotoxin from Metridium senile.

Among polyene antibiotics, used as detecting agent A and capable to form complex compounds with cholesterol, the most preferable are amphotericine B, philipine or nistanine.

Among enzymes with a high affinity to cholesterol, used as detecting agent A, the most preferable are cholesteroloxidases, cholesteroldeh.ydrogenases or cholesterolesterases.

Among all the above-listed affinants used as detecting agent A, namely: glyoosides, h.drophobic proteins, protein toxins, polyene antibiotics and high-affinity enzymes, more preferable are gl.cosides, 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 molecular weight of glycosides makes it possible to obtain a oompound with a high molar content of detecting agent, thus providing multiple points of interaction between the compound and skin cholesterol.

he most preferable among gl.ycosides are steroid gl oosides which are more efficient in making complexes with cholesterol, compared to triterpene glycosides.

SHydrophobi proteins, protein toxins, pol.ene antibiotics and high-affinity enzymes are also successfully used for obtaining diagnostic tools, however, the.y introduce certain limitations in the metbod of produ'ng affino-enzymatic compounds, due to their low chemical stablity and inactivation I, ad caabl towithtan durng yntesisbot orgnicsol neinical or plocnemicai menoas. 'jOee: likitin (rordienko IA., Dolgov Filimonova T.A. "Cholesterol content in the tendencies. Moreover, a high molecular weight of these detecting agents somewhat reduces sensibilit. 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 visualizing 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: acetvlcholinesterase, tyrosinase, gl.Yooso-B-phosphatdeb.ydrogenase, glycosooxidase, gl.ycosoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, alphach.motr.ypsin or p.yrophosphatase.

All these enzymes can be used with any of the above-listed detecting agent A, however, steroid glycosides should be pre- S" ferred.

Use of binding agent C expands technical performance of the method to obtain the compound in question, while preserv- I ing at most the functional activity of agents A and B.

The most preferable are compounds using as an affinant-de- S" tecting agent A steroid gly.cosides which contain as an aglicone a cyclopentanoperhbrdrophenanthrene fragment of furostanole or spirostanole series, and oligosacoharide fragment including 3 to 10 monosaccharides with linear or branched structures.

Such steroid glycosides possess a high chemical stability and can be immobilized onto a binding polymer in hard conditions, i.e. high temperatures and dissolution in organic sol- I) I 16.

vents 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 visualizing agent B onto the product containing both A and C, in soft conditions for preserving agent B enz.matic activity.

As binding agent C the following asymmetric low-molecular bi-functional compounds can be used: bromcyan, trichlortriazine or 2-amino-4,6-dichlor-5-triazine.

Use as a binding agent C of high-molecular polyfunctional compounds enables to widely vary proportions of agent A and B in the final product, for instance, with the purpose to get S an optimal ratio between the number of points binding the product with skin surface (detecting agent A) and quantity of visualizing agent B.

As a high-molecular polyfunctional binding agent C it is possible to use various polysaccharides, proteins or synthetic polymers, i.e. any high-molecular compounds containing any of the following functional groups: primary amine, oarboxyl, Hydrooxyl, aldehyde, baloidenhydride, mixed anhydride, iminoester, azide, hydrazide, maleimide, isocyanate or epoxide.

As to high-molecular polyfunctional binding agent C, copol.vmers of acrylic acid or maleic anhydride as to be preferred.

The most preferable is copolymer of N-vin.lpyrrolidone and maleio anhydride. The abovp agents C can be used with any detecting agent A and visualiiing agent B. The final A-C-B tional by their nature. The compounds are selectively bound. to 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 agents A and B into reactable state are used as activators of these agents. Such groups as primary amine, carboxy1, alpha- -glycol can be activated using the well-known methods (azide, carbodiimide, succinimide, periodate etc.).

The most preferable are the periodate and carbodiimide methods, because they ensure maximum mild synthesis conditins and preserve activity of the immobilized enzyme.

Aqueous-salt 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, 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 as steroid glycosides, triterpene glycosides and pol.yene antibiotics, inadequately or incompletely dissolve in water and therefore require organic solvents. For this purpose, polar aprotonic solvents can be used, for instance, dimethylsulfoxide, dimethyl formamide, hexamethan. ol, 2:1 dimethylformamide/toluol mixture, or 2:I dimethylsee formamide/hexane mixture.

Conditions to obtain compounds for cholesterol indication (temperature, incubation time) dep >i on the choice of A, B and C components. Use of enzmes as visualizing agent B 2imits the 1 1 *1 j I IIIuU ctL ct tcemperature Of 0-250C for 1- .4 hours.- According to a third embodiment of this invention, there is .RA1provided a melthod for producing a bifunctional compound as defined in any ~L L'483Z N reaction temperature to 200C. Preferablyv, the reaction should be conducted at 400 in buffer solution with pH 5-9. Use of synthetic pol.Vpners for binding agent C, and glycosides for detecting agent, A enables to perform, the reaction in organic solvents even at high temperatures (u fp to T2000 and choose a reaction timq~e sufficient to get the maximum possible yield of final product.

Molar ratios of the components are fixed in accordance with molecular weights of 8elected A or B agents, affinitN to skin cholesterol and activity of the enzyme used.

Below, the invention is illustrated by several examples and drawings: Fig.I symbols Fig.2 compounds obtained and their interaction with cholesterol on a patient's skin surface according S. To Ex. 1,2.

Fig.3 compounds obtained and thelz interarntion 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

*S

Example I 100 mng of tornatonine are placed in 10 ml of water, mng of sodium periodate are added, and the mixture is incubated at 2000 for 4 hrs. I ml) of alpha-hyNmotrypsin solution (10 mg/nil) in 0.2 M phosphate buffer with PH= 7.5 is added to the solution obtained, nnd the mixture is incubated 1C arir- Ir ~IU- W~-L^iLL--__~IC~-_ICIICIXC_~~ I* 19.

for 12 hrs at 40C. The result ,i aqueous solution of the final product.

Example 2 3 mg peroxid)se from horse-radish is dissolved in I ml of water, 0.5 mg of sodium periodate are added, and the mixture is incubated for 8 hrs at 400C. 3 mg of cholesteroloxidase (A) in I 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 400.

The result is aqueous solution of the final product.

Example 3 IO mg of polyacrylic acid are dissolved in 10 ml of acetate buffer with pH 4.8, and 15 mg of P-c.volohexyl-2- (4-mofoline) eth.yl-carbodiimide-meto-p-toluolsulfonate are added. The mixture is incubated for 1.5 hrs at OOC. 5 ml of buffer containing 5 mg of O-streptolysine and 5 mg of peroxidase from horse-radish are added to the solution obtained. The mixture is incubated for 2 brs at 200C. The o result is final product.

i o Example 4 Agavoside (100 mg) are placed in 5 ml of water, and 100 mg of bromc.,\4 are added; pH II is maintained by 9'9" adding IM of Na0H solution. The mixture is incubated for mii at 400, and pH is reduced to 8.5 by adding phosphoric acid., 3 ml of 0.M phosphate buffer with pH 8.5 containing 6 mg of alkaline phosphatase are added to I ml of the solution obtained. The mixtiue is incubated for 12 hrs at I A r

'A

The result is solution of the final product.

Example Nistatijne (500 mg) and copol.ymer of ethxlene with maleic anhbydride (250 mg) are dissolved in 5 ml of dimethylformamide and incubated in argon current at 500C for 3 hrs.

The product obtained is precipitated in 20 ml of ester, and dried in vacuum at 200C. 6 ml of the product obtained are added to I ml of beta-galactosidase solution (2 mg/ml) in 0.2 M phosphate buffer with pH 7.5, and the mixture is incubated, at 400 for 12 brs. The result is solution of the final product.

Example 6 Escine (50 mg) and copol.ymer of N-vin.ylpirrolydone with maleie anhydride (100 mg) are dissolved, in I ml of dimethlsuJlfoxide and incubated for 2 hrs at OOC10000. The pro- S duct obtained is precipitated. in 3 ml of actone, dried in vacuum above phosphorus pentoxide for 4 bre at IOOOC10000. 8 mg of the product obtained are added to I ml of alpha-ch.ymotr.ypsin solution (I mg/ml) in 0.2 M phosphate buffer with pH 7.5, and the mixture is incubated at 600 for 15 brsa. 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 binding agent 0, permits to preserve at most the enzYmatic activit.7 of visualizing agent B due to soft conditions of the enusme immobilization.

S 21.

Use of the compounds for visual indication of cholesterol on a patient's skin Diagnostics using the compounds produced according to the present invention is based not on an accurate quantitation of cholesterol, but on a possibility to determine the belonging of a patient to one of the three groups: atherosolerotics, risk group and normals. In order to determine the belonging of a patient to one of these groups it is necessary to evaluate the cholesterol content in the epidermal horny lay.er typical for each of the above groups.

Accordingly, for every compound, produced according to the present invention, three concentrations are selected and used in diagnostics. Such three different concentrations of an affino-enzymatic compound are applied as three points on S a patient's skin, preferably on the palm. The maximum conoentration leads to development of stain in all subjects, O' including normals in whom the skin cholesterol coitent is the least. This stain is used as a kind of control for the choles- Sterol effect. 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 o. compound solution with intermediate concentration develops a stain both in atherosclerotic patients, and in subjects usually relating to the risk 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, *9a nately form complex compounds with cholesterol, or 22.

with two to the risk group, and in case of three stains to clinical-stage 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 tha 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 I min. The redundant amount of the compound, not bound to skin cholesterol, is removed by washing. Then,a substrate of the corresponding enzyme, used as visualizing agent B in production of the present compound, is applied. 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 colorless solution is stained red, yellow, blue, green, violet or other, or the stained solution is decolorized.

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: 1 Solution I: ABTC (2.2'azinobis-(3-ethyl-benzthioazoline-6- S sulfonic acid)' ImM, 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 Solution 3: 5.5'-tetramethylenebenzidine 0.4 mM, hydrogen peroxide 0.004% in acetate buffer with pH 6.

or other peroxidase substrates widely used in immuno-enzymatic analysis.

ml 23.

For instance, if alkaline phosphatase is used as visualizing agent B, th6 'following solution can be used as a substrate of' this enz".rne: p-nitrophenol phosphate 2.5 mM, magnesium chloride 0.5 mM in diethbylainine bufftor with PH or any other alkaline phosphatase substrate used in immuno- -enzymatic analysis.

200 patients with verified diagnosis have been examined, and the results shown in the table give a high correlation between the proposed diagnostic method and a level of atherosclerotic vascular lesion.

I.

a. 0 eS *5 *r

S

S S *5 S S S 55 5 S S 555 S.* i r S S* S S S S S S S S* 5.5 5 5 S

A/

(ij T ABLE 24.

Family Ischemic Hypertension Ischemic Ischemia Ischemic hyperlipi- heart heart of lower heart Normals demia disease disease+ extremi- disease+ hvyper- ties bypert entension sion ischemia of lower extrem No. of patients 9 7 44 30 8 66 Females 5 2 13 12 Males 4 5 31 18 8 66 Age (middle) F-45, I-40 F-55, M-47 F-50, M-52 F-54, 48 54 F-24, M-52 M-26 Plasm cholesterol Atherogeneity index Skin assay 3 stains 3- stains-3 3 stains-I0O 3 stains- 3 stains-6 3 stains I stain according to in all 2 stains-4 2 stains -3 24 2 stains-2 in all in all invention 2 stains- 6

Claims

1. A bifunctional compound for the visual detection of cholesterol on the surface of skin, comprising detecting agent A covalently linked to visualizing agent B, wherein: said detecting agent A is a moiety capable of discriminately forming a stable complex with said cholesterol; and said visualizing agent B is an enzyme moiety which converts a colourless substrate to a coloured product or a coloured substrate to a colourless product thereby indicating the presence of said cholesterol on said skin surface.

2. The compound according to claim 1, characterized in that said detecting agent A is: a steroid glycoside containing as an aglicone a cyclopentanoperhydro- phenanthrene fragment of furostanole or spirostanole series and an oligosaccharide fragment including 3 to monosaccharides having linear or branched structure; a triterpene glycoside containing an aglicone of alpha- or i beta-amyral, lupane, gopane, dammarane, lanostane or holostane series, and an oligosaccharide of 2-8 residues having branched or linear 20 structure; a hydrophobic protein; a protein toxin produced by bacteria, marine microorganisms, Insects or snakes; S..a polyene antibiotic; or 25 an enzyme having high affinity for cholesterol.

3. The compound according to claim 2, characterized in that said steroid glycoside is agavoside A, B, C, D, F, H, or G, trillin, funcoside SC, D. E, F, G or I, dioscine, gracillin, protodiscine, kikubasanine, juncosides E or H, lanotigonine, desglycodigitonine, digitonine, gltonine, rockosides C, D and E, alliumoside C, polyganatonine, tigogenine hexaoside, kapsycoside, ammumosides B, C, D or E, desglucodesramnoparillin, desglucoparillin, parillin, sarsaparilloside, asparagosides C, D, G or H, protojuccoside H, juccoside B, lanotigoside, monoside, bioside, trioside, purpureagitoside, gecogenine, rockogenine, diogenine, tigogenine, protopolygamatoside, tomatonine, laxogenine liquotetraoside, alliogenine lactotetraoslde, karataloslde A, cyclosiversioside H, acanthophiloside C, alliofusoside A, alliospiroside RAZ/X A, cyclosiversioside F, tea saponin, acanthophiloside b, tigonine (total), glycoside from Calha Polypatala or cyclosiversloside G. LMM/483Z listerio lysin, 0-toxin C.I obtained from Perfrigens of A and I 1 M 26

4. The compound according to claim 2 or claim 3, characterized in that said steroid glycoside is funcoside C, D, E, F, G or I, dioscine, rockoside, lanotigonine, digitonine or tomatonine. The compound according to any one of claims 2 to 4, characterized in that said steroid glycoside is digitonine or tomatonine.

6. The compound according to claim 2, characterized in that said tritarpene glycoside is escine, avenacine, ted saponin, alpha-gederine, kauloside C, stichionoside A, cyclamine, chinovine, sugar-beet Japonins, hypsoslde or a triterpene glycoside produced by Fatsia Japonica.

7. The compound according to claim 2, characterized in that said hydrophobic protein is Folch meiline protein, lysosomal or mitochondrical proteins, fibrinogen, an immunoglobulin, cerebrone, myoglobin, trypsin, cytochrome C, cytochrome R-450, or an apo-protein of a lipoprotein.

8. The compound according to claim 2, characterized in that said protein toxin is streptolysine 0, pneumolysine, listeriolysine, C.I. -toxin produced from type A or C Perfringens, C.I 5-toxin, type A or C novvi, Histolyticum C.I. hemolysine, C and D type Botulinum C.I. hemolysine, tetanolysine, cereolysine, alveolysine, turingeolysine, or Metridium senile actinium cytotoxin. 20 9. The compound according to claim 2, characterized in that said polyene antibiotic is amphotericine B, philipine or nistatine. The compound according to claim 2, characterized in that said Shigh-affinity enzyme is cholesteroloxidase, cholesteroldehydrogenase or cholesterolesterase. 25 11. The compound according to any one of claims 1 to characterized in that said visualizing agent B is acetylcholinesterase, tyrosinase, glucose-6-phosphate dehydrogenase, glucosooxidase, glucosoamylase, galactosidase, peroxidase, alkaline or acid phosphatase, alpha-chymotrypsin or pyrophosphatase. 12, The compound according to any one of claims 1 to 11, characterized in that said covalent linkage of A and B is via binding agent C.

13. The compound according to claim 12. characterized in that said binding agent C Is a low-molecular weight asymmetric bifunctional compound.

14. The compound according to claim 13, characterized In that said low-molecular weight asymmetric bifunctional compound is bromocyan, trichlorotriazlne or 2-amino-4,6-dichloro-S-triazine. S'LMM/483 t V I c- .L.Luua .U±u .1L1 uLnc utaiuUu uiL J)ru puciong a.illno-enz.matlc com- pounds, due to their low chemical stailit.y and inactivation M4 1> .1 i 27 The compound according to claim 12, characterized in that said binding agent C is a high-moleular weight polymer containing functional groups selected from p imary amine, carboxyl, hydroxyl, aldehyde, haloid anhydride, mixed anhydride, azide, hydrazide, maleimide, isocyanate or epoxide.

16. The compound according to claim 15, characterized in that said high-molecular weight polymer is a polysaccharide, a protein or a synthetic polymer.

17. The compound according to claim 15 or 16, characterized in that said high-molecular weight polymer is a copolymer of acrylic acid or maleic anhydride.

18. The compound according to claim 15 or 16, characterized in that said high-molecular weight polymer is a copolymer of N-vinylpyrrolidone and a maleic anhydride. 15 19. A method for producing a bifunctional compound as defined in any one of claims 1 to 11, wherein detecting agent A is chemically activated in an aqueous medium at a molar ratio of agent A to activator of 1:1 1:10, at a concentration of agent A of 1-20 mg/ml, a temperature of 0,?5°C and a pH of 4-11 for 0,1-24 hours, and to the solution 20 obtained, visualizing agent B is added at a molar ratio of A:B of 20:1 1:1 and the solution is incubatd at a temperature of 0-25 0 C for 1-24 hours. C.* p. p. p k 4 A method for producing a bifunctional compound as defined in any one of claims 1 to 11, wherein visualizing agent B is chemically 25 activated in an aqueous medium at a molar ratio of B activator of 1:1 1:10 at a concentration of agent B of 1-20 mg/ml, a temperature of 0-25°C and a pH of 4-11 for 1-94 hours, and to the solution obtained, detecting agent A is added at a molar ratio of A:B of 20:1 1:1 and the solution is incubated at a temperature of 0-25°C for 1-24 hours.

21. The method according to claim 19 or 20, characterized in that said chemical activation of detecting agent A or visualizing agent B Is effected by carbodlimide, succinlmide, azide or periodate oxidation.

22. The method according to claim 21, characterized in that said chemical activation of detecting agent A or visualizing agent B is effected by a carbodiimlde or periodate oxidation.

23. A method for producing a bifunctional compound as defined in any one of claims 12 to 14, wherein detecting agent A Is dissolved in an aqueous salt solution at a pH of 5-9, at a concentration of A of 1-20 mg/ml, and a low-molecular asymmetric bifunctional binding agent C is 1 'LMM/483Z and. can be immobilized onto a binding polymer in hard. condi- tions, i.e. high temperatures and dissolution in organic sol- I I iii_ i 0* *r S S. 'T 28 added at a molar ratio of A:C of 1:0.5 1:10, then visualizing agent B is added at a molar ratio of A:B of 20:1 1:1 and the solution is Incubated at a temperature of 0-20 0 C for 1-48 hours.

24. A method for producing a bifunctional compound as defined in any one of claims 12 to 14, wherein visualizing agent B is dissolved in an aqueous salt solution at a pH of 5-9 and a concentration of 1-20 mg/ml, a low-molecular asymmetric bifunctional binding agent C is added at a molar ratio of B:C of 1:0.5 1:1 and the resulting solution is incubated at a temperature of 0-20°C for 1-20 hours, detecting agent A is added at a molar ratio of A:B of 20:1 1:1 and the solution is incubated at a temperature of 0-20 0 C for 1-48 hours. A method for producing a bifunctional compound as defined In any one of claims 15 to 18, wherein: detecting agent A and visualizing agent B are dissolved at a molar 15 ratio of A:B of 20:1 1:1 in an aqueous buffer solution at a pH of 5-9, at a concentration of each of agents A and B of 0.1-20 mg/ml, and a high-molecular weight polymer binding agent C is added to the resulting solution at a molar ratio of of 1:0.5 1:10, and the obtained solution is incubated at a temperature of 0-20°C for 1-20 hours. 20 26. A method for producing a bifunctional compound as defined in any one of claims 15 to 18, wherein detecting agent A is preliminarily dissolved in an aprotonic polar solvent and high-molecular weight polymer binding agent C is added thereto at a concentration of each of agents A and C of 1-20 mg/ml and a ratio of A:C of 1:1 1:10, and the solution Is 25 incubated at a temperature of 20-120*C for 0.5-6 hours, thereafter the resulting product (A C) is precipitated from the solvent and held in vacuum over phosphorus pentoxide at 80-1200C for 4-10 hours, the dried product at a concentration of 2-20 mg/ml is added to an aqueous salt solution having a pH of 5-9 containing dissolved visualizing agent B at a concentration of 1-10 mg/ml, and the solution Is incubated at a temperature of 4-80C for 2-12 hours.

27. The method according to claim 26, characterized in that detecting agent A Is a steroid glycoside, a triterpene glycoside or a polyene antibiotic, or a mixture of said compounds.

28. The method according to claim 26 or claim 27, characterized in that said aprotonic polar solvent Is dimethylsulfoxide, dimethylformamide, hexamethanol, a mixture of dimethylformamide and toluene at a ratio of 2:1 or a mixture of dimethylformamide and hexanol at a ratio of 2:1. Mo: I LMIY/483Z and, maleic anbhdride. The above agents C can be used with an.y detecting agent A and, risuali ing agent B. The final A-C-B 29 The method according to any one of claims 23 to 27, characterized in that said aqueous salt solution is a borate, citrate or phosp.hte solution providing buffer action within the pH range of 5-9. The bifunctional compound product when produced by the method according to any one of claims 19 to 29.

31. A method of assessing the cholesterol level in the skin of an individual, which method comprises applying to an area of said skin a bifunctional compound as defined in any one of claims 1 to 18 or washing unbound compound from said area of skin, and applying a substrate for said visualizing agent B.

32. The method of claim 31, when used for the diagnosis of an abnormal disease state.

33. The method of claim 32, wherein said abnormal disease state is athersclerosis.

34. A bifunctional compound as defined in claim 1, which compound is substantially as hereinbefore described with reference to Example 1 or Example 2. A method for producing a bifunctional compound as defined in claim 1, which method is substantially as hereinbefore described with 20 reference to Example 1 or Example 2.

36. A bifunctional compound as defined in claim 12, which compound is substantially as hereinbefore described with reference to any one of Examples 3 to 6. a 37. A method for producing a bifunctional compound as defined in claim 12, which method is substantially as hereinbefore described with reference to any one of Examples 3 to 6.

38. A method of assessing the cholesterol level in the skin of an individual, which method comprises applying to an area of said skin the Sbifunctional compound of claim 34 or claim 36, washing unbound compound from said area of skin, and applying a substrate for said visualizing agent B. DATED this TWENTY-FIFTH day of JULY 1991 Nauchno-2ssledovatelsky Institut Fiziko-Khimicheskol Meditsiny A i tPatent Attorneys for the Applicant SPRUSON FERGUSON LMM483