CA2025316A1 - Process for the determination by means of free radicals, of the anti-oxydant properties of a living organism or a potentially agressive agent - Google Patents
Process for the determination by means of free radicals, of the anti-oxydant properties of a living organism or a potentially agressive agentInfo
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- CA2025316A1 CA2025316A1 CA002025316A CA2025316A CA2025316A1 CA 2025316 A1 CA2025316 A1 CA 2025316A1 CA 002025316 A CA002025316 A CA 002025316A CA 2025316 A CA2025316 A CA 2025316A CA 2025316 A1 CA2025316 A1 CA 2025316A1
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- cell material
- free radicals
- free radical
- radical generator
- cell
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
The present invention relates to a novel method of assaying or evaluating the antioxidizing activities of a living organism or a potentially aggressive agent, said method, which comprises using free radicals as a means of inducing cell lysis. being characterized in that 1°) a free radical generator is brought into contact. in an appropriate liquid biological medium, with a cell material selected from the group consisting of (a) human, animal and plant cells, (b) fragments of said cells, and (c) synthetic walls and fragments thereof con-taining liposomes, said cell material having first been contaminated with a potentially aggressive agent;
2°) the release of free radicals from said free radical generator is induced; and 3°) the lysis of the cell material by the free radicals is evaluated by comparison with a control con-taining said cell material which has not been contami-nated.
The present invention relates to a novel method of assaying or evaluating the antioxidizing activities of a living organism or a potentially aggressive agent, said method, which comprises using free radicals as a means of inducing cell lysis. being characterized in that 1°) a free radical generator is brought into contact. in an appropriate liquid biological medium, with a cell material selected from the group consisting of (a) human, animal and plant cells, (b) fragments of said cells, and (c) synthetic walls and fragments thereof con-taining liposomes, said cell material having first been contaminated with a potentially aggressive agent;
2°) the release of free radicals from said free radical generator is induced; and 3°) the lysis of the cell material by the free radicals is evaluated by comparison with a control con-taining said cell material which has not been contami-nated.
Description
-;`` 2~2~3~
PROCESS FOR THE DETE~MINATION ~Y MEA~S OF FREE RADICALS, OF THE ANTI-OXYDANT PROPERTIES OF A-LIVING ORGANISM OR A
POTENTIALLY AGRESSIVE AGENT.
05 FI~LD OF THE INV~TION
The precent invention relates to a novel method of as~ayin~ or determinin~ the antioxidlzing prop~rtie~
of a living organi~m or a potentially aggre~q~ive agent by mean~ of free radical~. ~
It relateq in particular to the method of e~alua-tin~ on the one hand the antioxidative ~tate of cells of a li~ing organi~m, and on the other hand the oxidative or antioxidizing activities of a potentiall~ aggre~qive chemical or phy3ical agent~ i.e. a chemical or physical agent capable either of increa~ing or accelerating or of inhibiting or retarding the cell lY~iY induced by free radicals.
PRIOR ART
It is known that free radicals generally have an adverse effect on the organism and in particular on the cells of thi~ organiqm. Free radicalq attack the cell ; walI at a rate which depend~ on the cell re~i~tance imparted by the enzymatic and mole~cular equipment of 3aid cells. When the cell wall has been degraded7 perforated ~25 ~or opened by free radical~, the contents of the cell ~
spread outcide the wall. See the followin~ documen~ in particular: Ghemical Ab~tractq lnl, 213235v, Chemical ~AbstractAq lQ~ 232454~, Chemical Ab~tract~ lQQ, 99345j.
iological Ab~tracts l2 (n 9)~, page 5914, abotract nJ
57169, (1981) and Biological Abstract~ 1~ tn 12~, page 8817, ab~tract n 84420, (1982).
The abstract CA 107, 213235v, which refer~ to an article by M. MIKI et al., Arch. Biochem. Biophy~.l 2 ~n 2), pages 373-380 (1987), indicate~ that alpha-tocopherol protects rat erythrocyte~ from the ly8i9 :. : : - ., :: ,:~ ` . , ~ .
: .... d , ~2~3~
induced by free radicals.
According to the abstract CA lQQ~ ~9345j cited above, which refers to an article by E.B. SPEKTOR et al., Lab. Delo (n 1), pages 26-28 (1984), the total anti-05 oxidizing acti~ity of a sample (blood plasma or spinalfluid) is determined by absorption at 532 nm after the induction of free radicals in a cell material (erythro-cyte membranes in this particular case) by means of a UV
lamp.
According to the invention, a novel technical solution is recommended whereby (i) the free radicals are not generated in said cell material, but originate from a free radical initiator added to said cell material, and (ii) the cell material has first been contaminated with a potentially aggressive agent.
SUEJECT OF THE INV~NTION
According to the invention, a novel method of assaying or evaluating the antioxidizing activities of a living organism or a potentially aggressive agent is recommended, said method, which comprises using free ~ radicals as a means of in~ucing cell lysis, being ; characterized in that 1) a free radical generator is brought into contact, in an appropriate liquid biological medium, with a cell~material (I) selected from the group consi~ting of (a) human, animal and plant cells, (b? fragments~of said cell~, and (c) synthetic TRalls and fragments thereo~ con-taining liposomes, 30 ~ said cell material having first been contaminated with a potentially aggre3sive agent (II)~
PROCESS FOR THE DETE~MINATION ~Y MEA~S OF FREE RADICALS, OF THE ANTI-OXYDANT PROPERTIES OF A-LIVING ORGANISM OR A
POTENTIALLY AGRESSIVE AGENT.
05 FI~LD OF THE INV~TION
The precent invention relates to a novel method of as~ayin~ or determinin~ the antioxidlzing prop~rtie~
of a living organi~m or a potentially aggre~q~ive agent by mean~ of free radical~. ~
It relateq in particular to the method of e~alua-tin~ on the one hand the antioxidative ~tate of cells of a li~ing organi~m, and on the other hand the oxidative or antioxidizing activities of a potentiall~ aggre~qive chemical or phy3ical agent~ i.e. a chemical or physical agent capable either of increa~ing or accelerating or of inhibiting or retarding the cell lY~iY induced by free radicals.
PRIOR ART
It is known that free radicals generally have an adverse effect on the organism and in particular on the cells of thi~ organiqm. Free radicalq attack the cell ; walI at a rate which depend~ on the cell re~i~tance imparted by the enzymatic and mole~cular equipment of 3aid cells. When the cell wall has been degraded7 perforated ~25 ~or opened by free radical~, the contents of the cell ~
spread outcide the wall. See the followin~ documen~ in particular: Ghemical Ab~tractq lnl, 213235v, Chemical ~AbstractAq lQ~ 232454~, Chemical Ab~tract~ lQQ, 99345j.
iological Ab~tracts l2 (n 9)~, page 5914, abotract nJ
57169, (1981) and Biological Abstract~ 1~ tn 12~, page 8817, ab~tract n 84420, (1982).
The abstract CA 107, 213235v, which refer~ to an article by M. MIKI et al., Arch. Biochem. Biophy~.l 2 ~n 2), pages 373-380 (1987), indicate~ that alpha-tocopherol protects rat erythrocyte~ from the ly8i9 :. : : - ., :: ,:~ ` . , ~ .
: .... d , ~2~3~
induced by free radicals.
According to the abstract CA lQQ~ ~9345j cited above, which refers to an article by E.B. SPEKTOR et al., Lab. Delo (n 1), pages 26-28 (1984), the total anti-05 oxidizing acti~ity of a sample (blood plasma or spinalfluid) is determined by absorption at 532 nm after the induction of free radicals in a cell material (erythro-cyte membranes in this particular case) by means of a UV
lamp.
According to the invention, a novel technical solution is recommended whereby (i) the free radicals are not generated in said cell material, but originate from a free radical initiator added to said cell material, and (ii) the cell material has first been contaminated with a potentially aggressive agent.
SUEJECT OF THE INV~NTION
According to the invention, a novel method of assaying or evaluating the antioxidizing activities of a living organism or a potentially aggressive agent is recommended, said method, which comprises using free ~ radicals as a means of in~ucing cell lysis, being ; characterized in that 1) a free radical generator is brought into contact, in an appropriate liquid biological medium, with a cell~material (I) selected from the group consi~ting of (a) human, animal and plant cells, (b? fragments~of said cell~, and (c) synthetic TRalls and fragments thereo~ con-taining liposomes, 30 ~ said cell material having first been contaminated with a potentially aggre3sive agent (II)~
2) the release of free radicals from ~aid free radical generator is inducedi and 3) the ly~is of the cell material by the free radicals is evaluated by comparison with a control con-:,~ ' : ~ , .
; ' ' , " , . ' ' taining said cell material which has not been contami-nated In this methodl the oxidative state of the cell material I or the influence of the agent II on said 05 material I is assayed.
In particular, evaluation of the lysis of the cell material can be followed '`kinetically" [by making measurements at regular time intervals on ~amples (of a constant volume) of liquid test medium containing the free radical generator, the cell material and, if appropriate, the agent II to be tested] or "non-kinetically" ~by making measurement~ of the dose-response type on samples of the liquid test medium containing the cell material associated, if appropriate, with the agent II to be tested, and increasing aliquots of the free radical generator].
In the case of the kinetic eYaluation, the resistance to free radicals of the cell material I is expressed as the time which corre~ponds to lysis of 50%
of said cell material.
In the case of the dose-response evaluation, the resi~tance to free radicals of the cell material is expressed as the concentration of free radical ~enerator which~induces 1 y9i~ of ~0% of said cell material.
~DETAlLED D~SCRIPTION OF THE INVENTION
Products which release frae radicals and which are commonly~used in~the fièld of polymerization to produce macromolecules may be indicated in particular ~ among the free radical generators which are suitable 30 according to the invention. The following may be men-tioned in particular among these~products: oxidizing free radical generators such as benzoyl peroxide, C1-Cs and preferably C3-C4 alkyl perbenzoates (especially n-butyl, t-butyl, i-propyl and n-propyl perbenzoates), dialkyl peroxydicarbonates in which the alkyl groups contain from ; , - ' :, .
, I
~- :
r~
l to 8 carbon atoms and preferably from 3 to 4 carbon atoms (especially diisopropyl peroxydicarbonate~, cumene hydroxyperoxide, azo-bis(isobutyronitrile), 2,2'-azo-bis(2,4-dimethylvaleronitrile), 2,2'-azo-bis(2-amidino-0~ propane) and~ if appropriate, their addition salts suchas the hydrochlorides, and analo~ues thereof.
The preferred oxidizing free radical generators are those which have a zero order kinetics or reaction rate (the release of free radicals is constant with time~
: lO or, preferably 7 a 1st order kinetics or reaction rate : (the release of free radicals is linear with time). The preferred oxidizing free radical generator~ according to the invention are on the one hand 2,2'-azo-bi~(2-amidino-propane) dihydrochloride, which give~ a 1st order : 15 kinetics in an aqueous medium, and on the other hand ,2'-azo-bis(2,4-dimethylvaleronitrile), which gives a 1st order kinetics in an oily or organic liquid medium.
The release of free radicals from a free radical genera-tor:is effected according to a method known per se, for : 20 example by means of heat~ light (e~specially light of the visible spectrum or UV light~g protons, electrons or X-: : rays; this release will preferably b~ initiated by : photons or heat. For example, heating an a~ueous solu-: ' tion of 2,2'-azo-bis(2-amidinopropane) dihydrochloride to ~:: 25 ~a temperature of 37C is sufficient to trigger the release of oxidizing free radic~als according to the : reaction :
Cl-H-N~ CH, : C~ ~NH~ a-~N ' c~
~ ~ ~ I ~: : ~ I
C--~-N=*--G--C --~2 C--C ~ N2 ~N Ctb ~b ~}6 ' : ~N CH, The cell material I according to the invention includes cells of human, animal, vegetable or synthetic origin or fragments of said cells~ such as the walls.
It will be advanta~eous to use a material I
containing a coloured or fluorescent marker which can be released under the action of free radicals.
05 Among the cell materials I which are suitable according to the invention, those which will be used in particular are pigmented living cells of human, animal or ve~etable origin, i.e. living cells containing a pigment or colourant~ such as haemoglobin, chlorophyll, xantho-I0 phyll, carotene and anthocyanins? whose release at the moment o~ cell lysis is detectable especially by mea ur-ing the variation in optical density by means of a spec-trophotometer. The recommended li~ing cells are eryth-rocytes of animal origin and preferably erythrocytes taken from warm-blooded animals such as mammals, in particular man. The choice of erythrocyte~ i~ based on the following factors:
- blood cells such as erythrocytes have a rela-tively short half-life tao days for erythrocytes of human origin);
- erythrocytes posses~ all the molecular and enzymatic equipment for protecting a~ain~t free radicals, so they are considered as representative of the other cells of the organism~ and - the aocessibility and wide availability of erythrocyte~, especially by way of a simple blood sample with a volume of a few millilitre3.
~ When erythrocyte~ isolated from their plasma are subjeoted to oxidative-type a~gression ~ith free radi-cals. they apply all their enzymatic and molecular eguip-ment to resist this aggression until the cell membrane or wall is modified thereby to the point of allowing the cell contents to escape~ In the case of erythrocyte~, this release of said content3 can easily be determined by ~pectrophotometry by measuring the amount o~ haemo810bin ', . ' ' .
~ 3 ~ ~
which passes into the biological medium. The reæistance of the erythrocyte population which is being tested is expressed either as the time taken to release 50% w/w of the haemoglobin contained in the erythrocytes (kinetic 05 evaluation) or as the concentration of free radical generator (CH60~) which causes 50% haemolysis (dose-response evaluation).
The cell material can also consist of walls or wall fragments of cells, preferably of pigmented cells.
The walls which are most suitable are tho~e containing a sufficient amount of pigment or colourant which can be released at the moment of lysis by free radicals.
The cell material can al~o consi3t of a synthetic cell wall. Preferred synthetic walls are substances con-taining liposomes in which a coloured marker, which canbe released at the moment of lysis by free radicals, is coated, fixed or immobilized. Membrane substances containing liposomes and such coloured markers are particularly advanta~eous in that they make it pos~ible 20~ to avoid looking for healthy human, animal or plant subjects for the control tests, thereby ensuring a better standardization~of the assay method according to the inventlon.
According to the invention, the cell material to be used will consist preferably o:E erythrocytes and most pre~erably of 1iposomes masking, coating, fixing or immobilizing a coloured marker which can be relea~ed during lysis by free~radical~. In this ca~e, the resis-tancs to free radicals which is te~tsd is sxpressed either as the 50% lysis time, i.e. the time taken to release 60% w/w of the coloured marker (kinetic measure-ment~, or as the concentration of free radical generator which induce3 50% lysis ~dose-respon3e measurement~.
The expression "cell material contaminated with a ~5 potentially aggressive agent II" i~ understood as meaning .: . . , ~ ., , ,~ . '- I
, : ' ' : ~' ': ,;:. ' ,. . ~ "
, ' a cell material I. as defined above, which has been brought into contact with said agent II at least 0.5 h before the method is carried out, or which has been subiected to the action of said agent II at least 0.5 h 0~ before the method of the invention is carried out.
The process of bringing said cell material I into contact with said agent II can comprise introducing the agent II into the living organism and then recovering pigmented cells contaminated with said agent II for carrying out the method of the invention. It can also comprise incorporating the agent II into cells of the material I according to a techni~ue known per se, either by injection (according to a technology related to that of in vitro fertilization) or by osmosi~.
The potentially aggressive agent II can be of a physical nature (irradiation with X-rays, beta rays, protons, etc.) or of a chemical nature (test substances or reference substance~ metabolites, etc.) or even of a physicochemical nature (tobacco smoke, involving especi-ally the release of free radicals by pyrolysi~).
The biological test medium, which is an aqueous liquld medium or an organic liquid medium, comprises the cel~l material I contaminated with the agent II before-~hand, a free radical generator and, if appropriate, one 25~ or more additives conventionally used in the field of cell cultures and biological a~says, in particular a preservative. The agent II forming part of said ~ uid test medium is introduced either as such into ~aid bio-logical medium containing the cell material and the free radical generator at least 0.5 h before the release of free radicals is triggered, or already associatsd with said cell material after contamination. Contamination can re~ult from the (deliberate or accidental) admini~-tration of said agent II (or one of its precursors) to 3~ the organism rom which the cells are taken, or ~rom the :
, 3 ~ ~
in vitro adsorption of said agent II by the cell mem-brane.
In the case of the contamination of living cells by administration to the organism, the cells carry said 05 contaminating substance, or at least one of it~ metabo-lites, in their contents and/or on their membranes In the case of the contamination of livin~ or synthetic cells by adsorption, the bulk of the contaminating sub-stance fixed is to be found in and/or on the cell wall.
Cont~amination by adsorption is advantageously applied to lipophilic contaminatlng substanc0s. Adsorption on the cell wall can be effected by incubating the cell material and the contaminating substance in an appropriate medium and at an appropriate temperature, said contaminating substance having been diluted in a selective solvent beforehand; depending on the inc~lbation time and the concentration of the cell matPrlal used, all or (more frequently) only part of the contaminating substance in the incubation medium is fixed by adsorption to the natural or synthetic cell wall.
If the means II is a chemical substance, this can be any test substance. especially an oxidizing sub3tance, ~an antioxidizing substance, a composition or association of products or else a metabolike. Among the substance~
which can be tested andfor assayed, it is al80 possible ; to ~indicate piements (especially flavonoids), proteins, enzymes,~ peptides,~amino~acids,~antibodies and antigens and, in general, any products from which antibodies can be generated. Products or means which act on the organism and~or the cells ma~ be mentioned in particular among these substances capable of being tested and/or assayed.
To carry out the assay method according to the invention, the isolated and washed cell material is SU9-pended in a li~uid biological medium which i3 preferably , ~, . . " ~ .
2 ~ i3J
isotonic. Incubation is carried out at a temperaturewithin the ran~e from 10 to 6QC, preferably from 15 to 40C and most preferably at a temperature of 37C, in the presence of a free radical generator at a rate of 50 to 05 200 mmol/l of free radical generator for a concentration of 10 to 20% w/v of cell material.
The best mode of carrying out the method of the invention consistæ in suspending either a cell material consisting of erythrocytes, or synthetic wall fragments containing liposomes and a relea~able coloured marker, in an isotonic physiolo~ical serum which may be buffered, and in incubating the suspension at 37C in the presence of 2,2 -azo-bis(2-amidinopropane) dihydrochloride at a concentration of 100 mM for a final volume of 2 ml of (preferably) aqueous li~uid biological medium, or in the ; presence of 2,2 -azo-bis(2,4-dimethylvaleronitrile~ in the same proportions for an organic biological li~uid medium. The release of free radicals from the free radical generator is preferably initiated by photons and most preferably by a thermal shock (in this particular case, heating to 37C). Samples ~0.02 ml) are taken at regular time intervals (for examp:le every 20 minute~) until the cell residue has disappeared (this period generally 'oeing of the order of 150 to 600 minute~ and 25 egpecia11y 200-300 minut-s); each sample i9 diluted with 1 ml of physiological serum and centrifuged [for examp~le for 10-60 seconds at 3000-9000 g, preferably at 3000-6000 g (excessively severe centrifugation, e~pecially above 9000 g, can upset the measurement b* inducing mechanical lysis)~; an aliquot (0 2 ml) of the re3ulting supernatant is then transferred to the wells of a micro-plate or of a set of microcells for reading of the optical densitY by spectrophotometry (especially at 350-600 nm, irrespective of the origin of the pigmented cell~
35 or synthetic walls, and in particular at 405-410 and 540 - , : ~ , ,: : :
3 ~ ~
nm when the cell material consists of erythrocytes).
In practice, the results of the variations in optical density are expressed as a percentage relative to maximum lysis of the cell material (100%) A theoretical 05 curve adjusted at the experimental points makes it pog-sible in particular to obtain the time corresponding to ~0% lysis (this time is the longer, the better the resis-tance of the erythrocytes or synthetic wall to the in ~i~Q oxidative stress applied under the abovementioned operating conditions)j the slope of the sigmoid curve of the~peak and the latency time (determined by the tangent to the point of inflexion of said sigmoid curve).
The kinetic evaIuation described above can be replaced with an evaluation of the dose-respon~e type.
The a~say method according to the invention is v0ry simple ~o carry out and can be marketed in the form of an assay kit, either for diagnostic purposes or for screening studies on various molecules and their metabo-lites, especially on the one hand molecules having an oxidizing or antioxidizing activity and on the other hand molecules having a long-term action. The assay method can also be carried out on human or animal plasma for the :~ :
purpose of assessing the influence of said molecules on said plasma, which then constitutes said agent II in ~uch an assay.
The method of the invention is particularly valu-able for (i) assaying~antimalaria agents, thece generally being substances which reduce ~he re~istance o erythro-cytes and liposome~ to free radicals, (ii~ diagnosing diabetes, since this disease produces a hypooxidative effect in the organism, and (iii) a~sessing the influence of foods or food additives, especially colourants, on the organism.
This assay method has the advantage of being carried out over very short period~ of time, which are preferably less than or equal to 5 h.
If liposomes are to be used as the cell material according to the invention, the following liposome pre-parations are recommended:
05 P~EPARATION A:
Particles containing an association of liposomes, a coloured or fluorescent marker which can be relea~ed under the action of free radicals, and a binding means ensuring the cohesion of each particle.
PREPARATION B:
A liposome matrix consisting of an as~ociation of liposomes, a coloured or fluore~cent marker which can be released under the action of free radicals, and a binding means ensuring the cohesion of the matrix.
PREPARATION C:
An inert support to which a liposome layer con-~isting of the matrix according to Preparation B above is adhesively bound.
~REPA~ATIQN D:
~0 An inert support having, on at least one of its faces, at least one zone coated with a coloured or fluorescent marker which can be r~leased under the action of free radicals. said face and said zone being coated with a layer con~isting of a mixture of liposomes and a ~5 binder ensuring the cohesion of ~aid layer, here the liposome layer ma~k~ the zone coated with the coloured or ~fluorescent mar~er.
PREPARA:TION E:
A porous inert support containing a coloured or fluore~cent marker which can be relea~ed under the action of free radical and which ha been introduced by impreg-nation, said support being coated with a liposome layer analogous to that of Preparation D, masking said marker.
PREPA~ATION F:
3~ A liposome matrix according to Preparation D, ,.. : ~ : ,' , :' , , :, - ::
, :
sensitized on the surface by a chromo~enic marker of a type known in the field of chromogenic peptide sub~trates (see especially patent document EP-A-0 280 61~`), under the action of free radicals, fragments or debri~ of lipo-05 somes containing said marker are separated from thematrix, these fragments or debris are isolated from the matrix and collected by filtration or centrifugation and said fragments or debris are then resuspended in a bio-logical medium appropriate for developin~ the colouration according to a known technique (see patent document EP-A-0 280 610 cited abo~e).
In Preparations ~-E above, the coloured or fluo-rescent marker which can be released under the action of free radicals is physically masked by the liposome matrix, the thickness of which must be relatively small so as to allow easy access to said marker under the action of free radicals. In Preparations A-C7 it is possible to have either purely~physical maskin~ of the coloured or fluorescent marker which can be released under the action of free radical~, or a more elaborate association of said marker with the liposome~, such as flxation, immobilization or seque~tration.
Finally, an assay kit is recommended which com-prises (i) the cell material according to the invention ; ~ ~5 and (ii) if appropriate, the~free radical generator and/
or the liquid;biological diluting media.
Further advantages a~d characteri~tics of the invention will be understood more clearly from the following description of Examples. Taken as a whole, .30 this information in no way implie~ a limitatio~ but i~
~iven by way of illustration. Example~ 1-9 relate to kinetic assays and Examples 10-15 to assays of the dose-response type.
To quantify the method according to the in~en-:, ,: . , ~ ~ - , : :
: , , .
~,'. ' ' , ' 1 tion, the influence of the concentration of 2,2'-azo-bis(2-amidinopropane) dihydrochloride on rat erythrocytes was studied.
After removal and washing, the rat erythrocytes 05 are suspended (haematocrit 15% w/v) in isotonic physio-logical serum. These erythrocytes are brought into contact with 2,2'-azo-bis(2-amidinopropane) dihydro-choride at doses of 0 mM (controi), 25 mM, 50 mM and 100 mM, the final volume of the aqueous liquid biological medium being 2 ml. The release of free radicals is initiated by heating the reaction medium to 37~C. 0.0 ml samples are taken evéry 20 minuteY for 200-240 minutes, each sample being diluted in 1 ml of phy~io-lo~ical serum and centrifuged (15 second3; 4000 g). An ~;~ 15 aliquot (0.2 ml) of the supernatant is then tran~ferred to the wells of a microplate for reading of the optical density (especially at 540 nm) by spectrophotometry.
The results collated in Figure 1 are expre~ed as the time (in minutes) which corre~3ponds to a haemoly~is rate of 50% (t~ox). Curve (a) re:Lates to the 100 mM
concentration of 2,~2'-azo-bi~(2-a~idinopropane) dihydro-chloride and gives a t60~ value of 158 minute~; curve (b~
relates to the 50 mM concentration of 2,2'-azo-bi~2-amidinopropane) dihydrochloride and gives a t~ox value of 176 minutes; curve (c) relate~ to the 25 mM concentrakion of~2,2'-azo-bis(2-amidinopropane) dihydrochloride and gives a t6c% value of 214 minutes; curve ~d) relateF to the control test, i.e. the test perfo~rmed in the absence of 2,2'-azo-bis(2-amidinopropane) dihydrochloride.
The results ~ show the relation~hip between do~e and dependence of the efect on cell ly~i~
with respect to the free radical generating molecule.
The present Example concern~ the effect of an antioxidant, which in this particular case i8 ascorbic ~ ;
~ ~2~3~
acid.
The operating procedures described in Example 1 above are followed using an aqueous liquid biological medium (physiological serum) containing rat erythrocytes, 05 100 mmol/1 of 2.2'-azo-bis(2-amidinopropane) dihydro-chloride and 0 (absence of antioxidant) or 0 1 mmol/1 of ascorbic acid, the release of free radicals being initiated 0.5 h after the ascorbic acid has been brought into contact with the erythrocytes and the free radical I0 generator.
The results collated i ~ show that in the absence of ascorbic acid ~curve (a)], the t60% value is equal to 158 minutes and that in the presence of 0 1 mmol/l of ascorbic acid [curve (b)], the t~ox value is equal to 244 minutes. In other words, the presence of an antioxidant such as ascorbic acid slows down the cell Iysis caused by -free radicals.
EgAMPLE 3 The present Example concerns the effect of an antioxidant incorporated into the membrane of rat eryth-rocytes, namely butylhydroxytoluene [abbreviated to BHT;
systematic nomenclature: 2,6-di(1~1-dimethylethyl)-4-methylphenol].
The operating procedures described in Exampl0 1 25 are followed using healthy rat erythrocytes (control) and erythrocytes which have been preincubated beforehand at 37~C for 0.& h in the presence of BHT and its solvent, namely ethanol (for adsorption of the BHT on the cell membrane of the erythrocytes), and then resuspended for performing the assay. The erythrocytes treated in this way are placed in the phy~iological serum of Example 1 and brought into contact with 100 mmol/l of 272'-azo-bis(2-amidinopropane) dihydrochloride, the resulting reaction medium then being heated to 37C.
The re~ults are collated in Figure 3. Control .: , ,,, . , , : ", .; :;, , , .; . , ,;, 3~ ~
curve ( a ) gives a t60~ of 163 minutes. Curve (b), which relates to the healthy rat erythrocytes which have been preincubated with the solvent for BHT, namely ethanol at a concentration of 0.5% w/v, for 0.5 h at 37C~ gives a 0~ t60~ value of 157 minutes. Curve (c), which relates to the same erythrocytes as curve (b), except that they have been preincubated under the same conditions with BHT and ethanol (the test medium containing 0.03 mmol/l of BHT
and 0.5% w/v of ethanol), gives a t60x value of 193 minutes. Comparison of curve (c) with curves (a) and (b) shows the protection which BHT provides again~t cell lysis. In other words, the antioxidant used here 310ws down the cell lysis caused by *ree radicals.
EXoMPLE 4 The present Example concerns the comparison of erythrocytes originating from two different subjects.
After separation and washing, these erythrocytes are re-suspended at the same concentration and subjected to the action of free radicals originating from a free radical generator, which in this particular case is 100 mmol~l of 2,2 -azo-bis(2-amidinopropane) dihydrochloride.
The results are collated in Figure 4. Curve (a) shows the percentage haemolysis ~ C~ for an adult smoker and gives a t60~ value of 84.4 minutes.
Curve~(b) shows the percentage haemolysis for an adult non-smoker and gi~es a t~ox value of 93.3 minutes. Com-pari~on of curves (a) and (b) show~ that, in smokers, the normal resistance to aggression by free radicals is lower than in subjects who are non-s~okers.
EXAMPLC ~
The present Example concerns the use of plant cells or as~essing the influence of irradiation on the oxidative state.
Camomile cell~ are divided up into two batches, one of the two batches is subjected to irradiation with a , "
. . .
radioactive source of 3.7 x 10~ B~ (100 microcuries) and the irradiated batch and non-irradiated batch are then stored under an inert atmosphere (nitrogen or argon) for 7 months. The cel]s of both batche3 are then su~pended 0~ in an aqueous liquid biological medium and brought into contact with a free radical generator, namely 2,2 -azo-bis(2-amidinopropane) dihydrochloride, at 15C for 0.5 h.
The release of free radicals i~ initiated by heating to 37-40C and ~amples are then taken and analyzed according to the procedures of Example 1. The kinetic study of the lysis of the cell material shows that the cells which were irradiated beforehand have a lower resistance to free radicals than the non-irradiated cell~. The corresponding curves are similar to those of Figure 3 (the kinetics of lysis of the irradiated cells and that of lysis of the non-irradiated cells having approximately the shape of curves a and, re3pectively, b or c of said Fi~ure 3) A batch of camomile seeds is irradiated with a radioactive source of 3.7 x 10~ Bq and this batch i9 stored, together with a non-irradiated batch, for 8 months under an inert atmosphere. The irradiated and non-irradiated seeds are then ground and the resultin~
ground products are brought into contact with a su3pen-sion of rat erythrocytes in a phy3iological serum for 1 h at 15C. The free radical generator, namely 2,2 -azo-bis(~-amidinopropane) dihydrochloride, i~ then introduced and the procedure indicated in Example 1 is folIowed.
The ~inetic study of the lysi3 of the erythrocytes in the presence of ground irradiated and non-irradiated cam~mile seeds give3 curves approximately similar to curve~ a and, respectively, b or c of Figure 3. This demonstrate~ that the enzymatic molecular equipment of the ~eeds ha3 been modified and, in this particular case, partially de~-, ,.. .. . ,, . , , ~
;.; ~ !
. ` ~ .
$
- i7 -troyed by irradiation The present Example 6 and Example 5 above illus-trate that the method of the invention makes it pos~ible to assess the quality of foods of animal and/or vegetable 05 ori~in in order to determine whether or not the food to be tested has be0n degraded to a greater or lesser extent before consumption~
E~AMPL~ 7 The procedure indicated in Example 3 is followed, the BHT being replaced with phenothiazine. It is ound that phenothiazine has an unexpected antioxidizing effect `~ ~ in the sense that it slows down the ly~is of er~throcytes which is induced by free radical~.
XAMPL~ 8 15The procedure indicated in Example 7 i~ followed?
the erythrocytes being replaced with a synthetic cell material comprisin~ liposomes accordin~ to Preparation C
above. The antioxidizing effect of phenothiazine i~
observed in the same way as in Example 7.
A tea lyophilizate i9 prepared by extraction of dried leave~ with water which ha~ first been brought to the boil, and lyophilization of the re~ulting filtrate, one batch being subjeeted to irradiation with ionizing radiation (~0 kGyj before lyophilization and the control - batch~not being irradiated. Each batch is then stored under vacuum for~9 months.
The procedure indicated in Example 3 is then followed, the BHT bein~ replaced with the irradiated batch of tea or~the non-lrradiated control batch. It i9 found that the irradiated batch has a lower resistance to free radical~ than the control batch.
Example~ 10-15 below illu trate the measurement of the anti-free radical activity under a~ay condition~
.. ~ .
." ., ,. -,-", of the dose-response type~
Increasing aliquots (20 to 300 mmol) of free radical generator C2,2'-azo-bis(2-amidinopropane) di-hydrochloride] dissolved in an aqueous medium (water or 05 physiological serum) are placed in tubes and then lyo-philized After resolubilization of said aliquot~ of free radical generator in a constant volume of test physiological serum which may or may not contain a sub-stance to be studied, a constant amount of the cell material according to the invention is added.
The corresponding test media are incubated at ; 37~C for a defined period o* time (2.5 h3. The effect of ; free radicals is then assessed by lysis of the cell material Example 10 was carried out with healthy rat erythrocytes and it was found that the concentration of free radical generator which induced the lysis of 50% of the erythrocytes (CH60x) was 108 0 ~ 20 mmol/l.
Example 11 was carried out with the same healthy rat erythrocytes as those used in~Example 10, the te3t medium additionall~ containing 10 mmol/l of mannitol. It is found that the CHsox is eq~al to 155.6 ~ 6.1 mmol~l, whlch~confirms the antioxidizing effect of mannitol by compari~on with the CH60~ of Example I0 ~ Example 12 was carried out with the same healthy rat erythrocytes as those used in Example 10, the test medium additionally containing an oxidizing mean~, namely ~azodicarboxylic acid bis(dimethylamide) ~a compound which oxidizes thiols], at~a dose of~250 mM. It is found that said peroxidizing means makes the erythrocytes more~sen-sitive to the effect of frèe radicals as regards deple-tion of the glutathione in the erythrocyte~7 the CH~ox being reduced (compared with that of Example 10) to a value of 60.8 ~ 8 1 mmol/l.
In Examples 13-15, similar results to those of , ~.,, ' :, ' Examples 10-12 were obtained on replacing the erythro-cytes with a 3ynthetic cell material containing lipo30mes according to Preparation C above.
::
~, , ~
~ 15 ~
: : : :
, 20 :
:: ~ : : : : :
:
' ' '' ', " ' ~ , .
; ' ' , " , . ' ' taining said cell material which has not been contami-nated In this methodl the oxidative state of the cell material I or the influence of the agent II on said 05 material I is assayed.
In particular, evaluation of the lysis of the cell material can be followed '`kinetically" [by making measurements at regular time intervals on ~amples (of a constant volume) of liquid test medium containing the free radical generator, the cell material and, if appropriate, the agent II to be tested] or "non-kinetically" ~by making measurement~ of the dose-response type on samples of the liquid test medium containing the cell material associated, if appropriate, with the agent II to be tested, and increasing aliquots of the free radical generator].
In the case of the kinetic eYaluation, the resistance to free radicals of the cell material I is expressed as the time which corre~ponds to lysis of 50%
of said cell material.
In the case of the dose-response evaluation, the resi~tance to free radicals of the cell material is expressed as the concentration of free radical ~enerator which~induces 1 y9i~ of ~0% of said cell material.
~DETAlLED D~SCRIPTION OF THE INVENTION
Products which release frae radicals and which are commonly~used in~the fièld of polymerization to produce macromolecules may be indicated in particular ~ among the free radical generators which are suitable 30 according to the invention. The following may be men-tioned in particular among these~products: oxidizing free radical generators such as benzoyl peroxide, C1-Cs and preferably C3-C4 alkyl perbenzoates (especially n-butyl, t-butyl, i-propyl and n-propyl perbenzoates), dialkyl peroxydicarbonates in which the alkyl groups contain from ; , - ' :, .
, I
~- :
r~
l to 8 carbon atoms and preferably from 3 to 4 carbon atoms (especially diisopropyl peroxydicarbonate~, cumene hydroxyperoxide, azo-bis(isobutyronitrile), 2,2'-azo-bis(2,4-dimethylvaleronitrile), 2,2'-azo-bis(2-amidino-0~ propane) and~ if appropriate, their addition salts suchas the hydrochlorides, and analo~ues thereof.
The preferred oxidizing free radical generators are those which have a zero order kinetics or reaction rate (the release of free radicals is constant with time~
: lO or, preferably 7 a 1st order kinetics or reaction rate : (the release of free radicals is linear with time). The preferred oxidizing free radical generator~ according to the invention are on the one hand 2,2'-azo-bi~(2-amidino-propane) dihydrochloride, which give~ a 1st order : 15 kinetics in an aqueous medium, and on the other hand ,2'-azo-bis(2,4-dimethylvaleronitrile), which gives a 1st order kinetics in an oily or organic liquid medium.
The release of free radicals from a free radical genera-tor:is effected according to a method known per se, for : 20 example by means of heat~ light (e~specially light of the visible spectrum or UV light~g protons, electrons or X-: : rays; this release will preferably b~ initiated by : photons or heat. For example, heating an a~ueous solu-: ' tion of 2,2'-azo-bis(2-amidinopropane) dihydrochloride to ~:: 25 ~a temperature of 37C is sufficient to trigger the release of oxidizing free radic~als according to the : reaction :
Cl-H-N~ CH, : C~ ~NH~ a-~N ' c~
~ ~ ~ I ~: : ~ I
C--~-N=*--G--C --~2 C--C ~ N2 ~N Ctb ~b ~}6 ' : ~N CH, The cell material I according to the invention includes cells of human, animal, vegetable or synthetic origin or fragments of said cells~ such as the walls.
It will be advanta~eous to use a material I
containing a coloured or fluorescent marker which can be released under the action of free radicals.
05 Among the cell materials I which are suitable according to the invention, those which will be used in particular are pigmented living cells of human, animal or ve~etable origin, i.e. living cells containing a pigment or colourant~ such as haemoglobin, chlorophyll, xantho-I0 phyll, carotene and anthocyanins? whose release at the moment o~ cell lysis is detectable especially by mea ur-ing the variation in optical density by means of a spec-trophotometer. The recommended li~ing cells are eryth-rocytes of animal origin and preferably erythrocytes taken from warm-blooded animals such as mammals, in particular man. The choice of erythrocyte~ i~ based on the following factors:
- blood cells such as erythrocytes have a rela-tively short half-life tao days for erythrocytes of human origin);
- erythrocytes posses~ all the molecular and enzymatic equipment for protecting a~ain~t free radicals, so they are considered as representative of the other cells of the organism~ and - the aocessibility and wide availability of erythrocyte~, especially by way of a simple blood sample with a volume of a few millilitre3.
~ When erythrocyte~ isolated from their plasma are subjeoted to oxidative-type a~gression ~ith free radi-cals. they apply all their enzymatic and molecular eguip-ment to resist this aggression until the cell membrane or wall is modified thereby to the point of allowing the cell contents to escape~ In the case of erythrocyte~, this release of said content3 can easily be determined by ~pectrophotometry by measuring the amount o~ haemo810bin ', . ' ' .
~ 3 ~ ~
which passes into the biological medium. The reæistance of the erythrocyte population which is being tested is expressed either as the time taken to release 50% w/w of the haemoglobin contained in the erythrocytes (kinetic 05 evaluation) or as the concentration of free radical generator (CH60~) which causes 50% haemolysis (dose-response evaluation).
The cell material can also consist of walls or wall fragments of cells, preferably of pigmented cells.
The walls which are most suitable are tho~e containing a sufficient amount of pigment or colourant which can be released at the moment of lysis by free radicals.
The cell material can al~o consi3t of a synthetic cell wall. Preferred synthetic walls are substances con-taining liposomes in which a coloured marker, which canbe released at the moment of lysis by free radicals, is coated, fixed or immobilized. Membrane substances containing liposomes and such coloured markers are particularly advanta~eous in that they make it pos~ible 20~ to avoid looking for healthy human, animal or plant subjects for the control tests, thereby ensuring a better standardization~of the assay method according to the inventlon.
According to the invention, the cell material to be used will consist preferably o:E erythrocytes and most pre~erably of 1iposomes masking, coating, fixing or immobilizing a coloured marker which can be relea~ed during lysis by free~radical~. In this ca~e, the resis-tancs to free radicals which is te~tsd is sxpressed either as the 50% lysis time, i.e. the time taken to release 60% w/w of the coloured marker (kinetic measure-ment~, or as the concentration of free radical generator which induce3 50% lysis ~dose-respon3e measurement~.
The expression "cell material contaminated with a ~5 potentially aggressive agent II" i~ understood as meaning .: . . , ~ ., , ,~ . '- I
, : ' ' : ~' ': ,;:. ' ,. . ~ "
, ' a cell material I. as defined above, which has been brought into contact with said agent II at least 0.5 h before the method is carried out, or which has been subiected to the action of said agent II at least 0.5 h 0~ before the method of the invention is carried out.
The process of bringing said cell material I into contact with said agent II can comprise introducing the agent II into the living organism and then recovering pigmented cells contaminated with said agent II for carrying out the method of the invention. It can also comprise incorporating the agent II into cells of the material I according to a techni~ue known per se, either by injection (according to a technology related to that of in vitro fertilization) or by osmosi~.
The potentially aggressive agent II can be of a physical nature (irradiation with X-rays, beta rays, protons, etc.) or of a chemical nature (test substances or reference substance~ metabolites, etc.) or even of a physicochemical nature (tobacco smoke, involving especi-ally the release of free radicals by pyrolysi~).
The biological test medium, which is an aqueous liquld medium or an organic liquid medium, comprises the cel~l material I contaminated with the agent II before-~hand, a free radical generator and, if appropriate, one 25~ or more additives conventionally used in the field of cell cultures and biological a~says, in particular a preservative. The agent II forming part of said ~ uid test medium is introduced either as such into ~aid bio-logical medium containing the cell material and the free radical generator at least 0.5 h before the release of free radicals is triggered, or already associatsd with said cell material after contamination. Contamination can re~ult from the (deliberate or accidental) admini~-tration of said agent II (or one of its precursors) to 3~ the organism rom which the cells are taken, or ~rom the :
, 3 ~ ~
in vitro adsorption of said agent II by the cell mem-brane.
In the case of the contamination of living cells by administration to the organism, the cells carry said 05 contaminating substance, or at least one of it~ metabo-lites, in their contents and/or on their membranes In the case of the contamination of livin~ or synthetic cells by adsorption, the bulk of the contaminating sub-stance fixed is to be found in and/or on the cell wall.
Cont~amination by adsorption is advantageously applied to lipophilic contaminatlng substanc0s. Adsorption on the cell wall can be effected by incubating the cell material and the contaminating substance in an appropriate medium and at an appropriate temperature, said contaminating substance having been diluted in a selective solvent beforehand; depending on the inc~lbation time and the concentration of the cell matPrlal used, all or (more frequently) only part of the contaminating substance in the incubation medium is fixed by adsorption to the natural or synthetic cell wall.
If the means II is a chemical substance, this can be any test substance. especially an oxidizing sub3tance, ~an antioxidizing substance, a composition or association of products or else a metabolike. Among the substance~
which can be tested andfor assayed, it is al80 possible ; to ~indicate piements (especially flavonoids), proteins, enzymes,~ peptides,~amino~acids,~antibodies and antigens and, in general, any products from which antibodies can be generated. Products or means which act on the organism and~or the cells ma~ be mentioned in particular among these substances capable of being tested and/or assayed.
To carry out the assay method according to the invention, the isolated and washed cell material is SU9-pended in a li~uid biological medium which i3 preferably , ~, . . " ~ .
2 ~ i3J
isotonic. Incubation is carried out at a temperaturewithin the ran~e from 10 to 6QC, preferably from 15 to 40C and most preferably at a temperature of 37C, in the presence of a free radical generator at a rate of 50 to 05 200 mmol/l of free radical generator for a concentration of 10 to 20% w/v of cell material.
The best mode of carrying out the method of the invention consistæ in suspending either a cell material consisting of erythrocytes, or synthetic wall fragments containing liposomes and a relea~able coloured marker, in an isotonic physiolo~ical serum which may be buffered, and in incubating the suspension at 37C in the presence of 2,2 -azo-bis(2-amidinopropane) dihydrochloride at a concentration of 100 mM for a final volume of 2 ml of (preferably) aqueous li~uid biological medium, or in the ; presence of 2,2 -azo-bis(2,4-dimethylvaleronitrile~ in the same proportions for an organic biological li~uid medium. The release of free radicals from the free radical generator is preferably initiated by photons and most preferably by a thermal shock (in this particular case, heating to 37C). Samples ~0.02 ml) are taken at regular time intervals (for examp:le every 20 minute~) until the cell residue has disappeared (this period generally 'oeing of the order of 150 to 600 minute~ and 25 egpecia11y 200-300 minut-s); each sample i9 diluted with 1 ml of physiological serum and centrifuged [for examp~le for 10-60 seconds at 3000-9000 g, preferably at 3000-6000 g (excessively severe centrifugation, e~pecially above 9000 g, can upset the measurement b* inducing mechanical lysis)~; an aliquot (0 2 ml) of the re3ulting supernatant is then transferred to the wells of a micro-plate or of a set of microcells for reading of the optical densitY by spectrophotometry (especially at 350-600 nm, irrespective of the origin of the pigmented cell~
35 or synthetic walls, and in particular at 405-410 and 540 - , : ~ , ,: : :
3 ~ ~
nm when the cell material consists of erythrocytes).
In practice, the results of the variations in optical density are expressed as a percentage relative to maximum lysis of the cell material (100%) A theoretical 05 curve adjusted at the experimental points makes it pog-sible in particular to obtain the time corresponding to ~0% lysis (this time is the longer, the better the resis-tance of the erythrocytes or synthetic wall to the in ~i~Q oxidative stress applied under the abovementioned operating conditions)j the slope of the sigmoid curve of the~peak and the latency time (determined by the tangent to the point of inflexion of said sigmoid curve).
The kinetic evaIuation described above can be replaced with an evaluation of the dose-respon~e type.
The a~say method according to the invention is v0ry simple ~o carry out and can be marketed in the form of an assay kit, either for diagnostic purposes or for screening studies on various molecules and their metabo-lites, especially on the one hand molecules having an oxidizing or antioxidizing activity and on the other hand molecules having a long-term action. The assay method can also be carried out on human or animal plasma for the :~ :
purpose of assessing the influence of said molecules on said plasma, which then constitutes said agent II in ~uch an assay.
The method of the invention is particularly valu-able for (i) assaying~antimalaria agents, thece generally being substances which reduce ~he re~istance o erythro-cytes and liposome~ to free radicals, (ii~ diagnosing diabetes, since this disease produces a hypooxidative effect in the organism, and (iii) a~sessing the influence of foods or food additives, especially colourants, on the organism.
This assay method has the advantage of being carried out over very short period~ of time, which are preferably less than or equal to 5 h.
If liposomes are to be used as the cell material according to the invention, the following liposome pre-parations are recommended:
05 P~EPARATION A:
Particles containing an association of liposomes, a coloured or fluorescent marker which can be relea~ed under the action of free radicals, and a binding means ensuring the cohesion of each particle.
PREPARATION B:
A liposome matrix consisting of an as~ociation of liposomes, a coloured or fluore~cent marker which can be released under the action of free radicals, and a binding means ensuring the cohesion of the matrix.
PREPARATION C:
An inert support to which a liposome layer con-~isting of the matrix according to Preparation B above is adhesively bound.
~REPA~ATIQN D:
~0 An inert support having, on at least one of its faces, at least one zone coated with a coloured or fluorescent marker which can be r~leased under the action of free radicals. said face and said zone being coated with a layer con~isting of a mixture of liposomes and a ~5 binder ensuring the cohesion of ~aid layer, here the liposome layer ma~k~ the zone coated with the coloured or ~fluorescent mar~er.
PREPARA:TION E:
A porous inert support containing a coloured or fluore~cent marker which can be relea~ed under the action of free radical and which ha been introduced by impreg-nation, said support being coated with a liposome layer analogous to that of Preparation D, masking said marker.
PREPA~ATION F:
3~ A liposome matrix according to Preparation D, ,.. : ~ : ,' , :' , , :, - ::
, :
sensitized on the surface by a chromo~enic marker of a type known in the field of chromogenic peptide sub~trates (see especially patent document EP-A-0 280 61~`), under the action of free radicals, fragments or debri~ of lipo-05 somes containing said marker are separated from thematrix, these fragments or debris are isolated from the matrix and collected by filtration or centrifugation and said fragments or debris are then resuspended in a bio-logical medium appropriate for developin~ the colouration according to a known technique (see patent document EP-A-0 280 610 cited abo~e).
In Preparations ~-E above, the coloured or fluo-rescent marker which can be released under the action of free radicals is physically masked by the liposome matrix, the thickness of which must be relatively small so as to allow easy access to said marker under the action of free radicals. In Preparations A-C7 it is possible to have either purely~physical maskin~ of the coloured or fluorescent marker which can be released under the action of free radical~, or a more elaborate association of said marker with the liposome~, such as flxation, immobilization or seque~tration.
Finally, an assay kit is recommended which com-prises (i) the cell material according to the invention ; ~ ~5 and (ii) if appropriate, the~free radical generator and/
or the liquid;biological diluting media.
Further advantages a~d characteri~tics of the invention will be understood more clearly from the following description of Examples. Taken as a whole, .30 this information in no way implie~ a limitatio~ but i~
~iven by way of illustration. Example~ 1-9 relate to kinetic assays and Examples 10-15 to assays of the dose-response type.
To quantify the method according to the in~en-:, ,: . , ~ ~ - , : :
: , , .
~,'. ' ' , ' 1 tion, the influence of the concentration of 2,2'-azo-bis(2-amidinopropane) dihydrochloride on rat erythrocytes was studied.
After removal and washing, the rat erythrocytes 05 are suspended (haematocrit 15% w/v) in isotonic physio-logical serum. These erythrocytes are brought into contact with 2,2'-azo-bis(2-amidinopropane) dihydro-choride at doses of 0 mM (controi), 25 mM, 50 mM and 100 mM, the final volume of the aqueous liquid biological medium being 2 ml. The release of free radicals is initiated by heating the reaction medium to 37~C. 0.0 ml samples are taken evéry 20 minuteY for 200-240 minutes, each sample being diluted in 1 ml of phy~io-lo~ical serum and centrifuged (15 second3; 4000 g). An ~;~ 15 aliquot (0.2 ml) of the supernatant is then tran~ferred to the wells of a microplate for reading of the optical density (especially at 540 nm) by spectrophotometry.
The results collated in Figure 1 are expre~ed as the time (in minutes) which corre~3ponds to a haemoly~is rate of 50% (t~ox). Curve (a) re:Lates to the 100 mM
concentration of 2,~2'-azo-bi~(2-a~idinopropane) dihydro-chloride and gives a t60~ value of 158 minute~; curve (b~
relates to the 50 mM concentration of 2,2'-azo-bi~2-amidinopropane) dihydrochloride and gives a t~ox value of 176 minutes; curve (c) relate~ to the 25 mM concentrakion of~2,2'-azo-bis(2-amidinopropane) dihydrochloride and gives a t6c% value of 214 minutes; curve ~d) relateF to the control test, i.e. the test perfo~rmed in the absence of 2,2'-azo-bis(2-amidinopropane) dihydrochloride.
The results ~ show the relation~hip between do~e and dependence of the efect on cell ly~i~
with respect to the free radical generating molecule.
The present Example concern~ the effect of an antioxidant, which in this particular case i8 ascorbic ~ ;
~ ~2~3~
acid.
The operating procedures described in Example 1 above are followed using an aqueous liquid biological medium (physiological serum) containing rat erythrocytes, 05 100 mmol/1 of 2.2'-azo-bis(2-amidinopropane) dihydro-chloride and 0 (absence of antioxidant) or 0 1 mmol/1 of ascorbic acid, the release of free radicals being initiated 0.5 h after the ascorbic acid has been brought into contact with the erythrocytes and the free radical I0 generator.
The results collated i ~ show that in the absence of ascorbic acid ~curve (a)], the t60% value is equal to 158 minutes and that in the presence of 0 1 mmol/l of ascorbic acid [curve (b)], the t~ox value is equal to 244 minutes. In other words, the presence of an antioxidant such as ascorbic acid slows down the cell Iysis caused by -free radicals.
EgAMPLE 3 The present Example concerns the effect of an antioxidant incorporated into the membrane of rat eryth-rocytes, namely butylhydroxytoluene [abbreviated to BHT;
systematic nomenclature: 2,6-di(1~1-dimethylethyl)-4-methylphenol].
The operating procedures described in Exampl0 1 25 are followed using healthy rat erythrocytes (control) and erythrocytes which have been preincubated beforehand at 37~C for 0.& h in the presence of BHT and its solvent, namely ethanol (for adsorption of the BHT on the cell membrane of the erythrocytes), and then resuspended for performing the assay. The erythrocytes treated in this way are placed in the phy~iological serum of Example 1 and brought into contact with 100 mmol/l of 272'-azo-bis(2-amidinopropane) dihydrochloride, the resulting reaction medium then being heated to 37C.
The re~ults are collated in Figure 3. Control .: , ,,, . , , : ", .; :;, , , .; . , ,;, 3~ ~
curve ( a ) gives a t60~ of 163 minutes. Curve (b), which relates to the healthy rat erythrocytes which have been preincubated with the solvent for BHT, namely ethanol at a concentration of 0.5% w/v, for 0.5 h at 37C~ gives a 0~ t60~ value of 157 minutes. Curve (c), which relates to the same erythrocytes as curve (b), except that they have been preincubated under the same conditions with BHT and ethanol (the test medium containing 0.03 mmol/l of BHT
and 0.5% w/v of ethanol), gives a t60x value of 193 minutes. Comparison of curve (c) with curves (a) and (b) shows the protection which BHT provides again~t cell lysis. In other words, the antioxidant used here 310ws down the cell lysis caused by *ree radicals.
EXoMPLE 4 The present Example concerns the comparison of erythrocytes originating from two different subjects.
After separation and washing, these erythrocytes are re-suspended at the same concentration and subjected to the action of free radicals originating from a free radical generator, which in this particular case is 100 mmol~l of 2,2 -azo-bis(2-amidinopropane) dihydrochloride.
The results are collated in Figure 4. Curve (a) shows the percentage haemolysis ~ C~ for an adult smoker and gives a t60~ value of 84.4 minutes.
Curve~(b) shows the percentage haemolysis for an adult non-smoker and gi~es a t~ox value of 93.3 minutes. Com-pari~on of curves (a) and (b) show~ that, in smokers, the normal resistance to aggression by free radicals is lower than in subjects who are non-s~okers.
EXAMPLC ~
The present Example concerns the use of plant cells or as~essing the influence of irradiation on the oxidative state.
Camomile cell~ are divided up into two batches, one of the two batches is subjected to irradiation with a , "
. . .
radioactive source of 3.7 x 10~ B~ (100 microcuries) and the irradiated batch and non-irradiated batch are then stored under an inert atmosphere (nitrogen or argon) for 7 months. The cel]s of both batche3 are then su~pended 0~ in an aqueous liquid biological medium and brought into contact with a free radical generator, namely 2,2 -azo-bis(2-amidinopropane) dihydrochloride, at 15C for 0.5 h.
The release of free radicals i~ initiated by heating to 37-40C and ~amples are then taken and analyzed according to the procedures of Example 1. The kinetic study of the lysis of the cell material shows that the cells which were irradiated beforehand have a lower resistance to free radicals than the non-irradiated cell~. The corresponding curves are similar to those of Figure 3 (the kinetics of lysis of the irradiated cells and that of lysis of the non-irradiated cells having approximately the shape of curves a and, re3pectively, b or c of said Fi~ure 3) A batch of camomile seeds is irradiated with a radioactive source of 3.7 x 10~ Bq and this batch i9 stored, together with a non-irradiated batch, for 8 months under an inert atmosphere. The irradiated and non-irradiated seeds are then ground and the resultin~
ground products are brought into contact with a su3pen-sion of rat erythrocytes in a phy3iological serum for 1 h at 15C. The free radical generator, namely 2,2 -azo-bis(~-amidinopropane) dihydrochloride, i~ then introduced and the procedure indicated in Example 1 is folIowed.
The ~inetic study of the lysi3 of the erythrocytes in the presence of ground irradiated and non-irradiated cam~mile seeds give3 curves approximately similar to curve~ a and, respectively, b or c of Figure 3. This demonstrate~ that the enzymatic molecular equipment of the ~eeds ha3 been modified and, in this particular case, partially de~-, ,.. .. . ,, . , , ~
;.; ~ !
. ` ~ .
$
- i7 -troyed by irradiation The present Example 6 and Example 5 above illus-trate that the method of the invention makes it pos~ible to assess the quality of foods of animal and/or vegetable 05 ori~in in order to determine whether or not the food to be tested has be0n degraded to a greater or lesser extent before consumption~
E~AMPL~ 7 The procedure indicated in Example 3 is followed, the BHT being replaced with phenothiazine. It is ound that phenothiazine has an unexpected antioxidizing effect `~ ~ in the sense that it slows down the ly~is of er~throcytes which is induced by free radical~.
XAMPL~ 8 15The procedure indicated in Example 7 i~ followed?
the erythrocytes being replaced with a synthetic cell material comprisin~ liposomes accordin~ to Preparation C
above. The antioxidizing effect of phenothiazine i~
observed in the same way as in Example 7.
A tea lyophilizate i9 prepared by extraction of dried leave~ with water which ha~ first been brought to the boil, and lyophilization of the re~ulting filtrate, one batch being subjeeted to irradiation with ionizing radiation (~0 kGyj before lyophilization and the control - batch~not being irradiated. Each batch is then stored under vacuum for~9 months.
The procedure indicated in Example 3 is then followed, the BHT bein~ replaced with the irradiated batch of tea or~the non-lrradiated control batch. It i9 found that the irradiated batch has a lower resistance to free radical~ than the control batch.
Example~ 10-15 below illu trate the measurement of the anti-free radical activity under a~ay condition~
.. ~ .
." ., ,. -,-", of the dose-response type~
Increasing aliquots (20 to 300 mmol) of free radical generator C2,2'-azo-bis(2-amidinopropane) di-hydrochloride] dissolved in an aqueous medium (water or 05 physiological serum) are placed in tubes and then lyo-philized After resolubilization of said aliquot~ of free radical generator in a constant volume of test physiological serum which may or may not contain a sub-stance to be studied, a constant amount of the cell material according to the invention is added.
The corresponding test media are incubated at ; 37~C for a defined period o* time (2.5 h3. The effect of ; free radicals is then assessed by lysis of the cell material Example 10 was carried out with healthy rat erythrocytes and it was found that the concentration of free radical generator which induced the lysis of 50% of the erythrocytes (CH60x) was 108 0 ~ 20 mmol/l.
Example 11 was carried out with the same healthy rat erythrocytes as those used in~Example 10, the te3t medium additionall~ containing 10 mmol/l of mannitol. It is found that the CHsox is eq~al to 155.6 ~ 6.1 mmol~l, whlch~confirms the antioxidizing effect of mannitol by compari~on with the CH60~ of Example I0 ~ Example 12 was carried out with the same healthy rat erythrocytes as those used in Example 10, the test medium additionally containing an oxidizing mean~, namely ~azodicarboxylic acid bis(dimethylamide) ~a compound which oxidizes thiols], at~a dose of~250 mM. It is found that said peroxidizing means makes the erythrocytes more~sen-sitive to the effect of frèe radicals as regards deple-tion of the glutathione in the erythrocyte~7 the CH~ox being reduced (compared with that of Example 10) to a value of 60.8 ~ 8 1 mmol/l.
In Examples 13-15, similar results to those of , ~.,, ' :, ' Examples 10-12 were obtained on replacing the erythro-cytes with a 3ynthetic cell material containing lipo30mes according to Preparation C above.
::
~, , ~
~ 15 ~
: : : :
, 20 :
:: ~ : : : : :
:
' ' '' ', " ' ~ , .
Claims (11)
1. A method of assaying or evaluating the antioxidizing activities of a living organism or a potentially aggres-sive agent, said method, which comprises using free radicals as a means of inducing cell lysis, being charac-terized in that 1°) a free radical generator is brought into contact, in an appropriate liquid biological medium, with a cell material selected from the group consisting of (a) human, animal and plant cells, (b) fragments of said cells, and (c) synthetic walls and fragments thereof con-taining liposomes, said cell material having first been contaminated with a potentially aggressive agent;
2°) the release of free radicals from said free radical generator is induced; and
3°) the lysis of the cell material by the free radicals is evaluated by comparison with a control con-taining said cell material which has not been contami-nated.
2. A method according to Claim 1, characterized in that the cell material is selected from the group consisting of pigmented cells of human, animal or vegetable origin.
3. A method according to Claim 2, characterized in that said pigmented cells are erythrocytes of human or animal origin.
2. A method according to Claim 1, characterized in that the cell material is selected from the group consisting of pigmented cells of human, animal or vegetable origin.
3. A method according to Claim 2, characterized in that said pigmented cells are erythrocytes of human or animal origin.
4. A method according to Claim 1, characterized in that the cell material is a synthetic wall fragment.
5. A method according to Claim 4, characterized in that the cell material is a liposome material.
6. A method according to Claim 1, characterized in that said free radical generator is selected from the group consisting of products which release free radicals according to a 0 or 1st order kinetics.
7. A method according to Claim 1, characterized in that said free radical generator is selected from the group consisting of products which release oxidizing free radicals.
8. A method according to any one of Claims 1, 6 and 7, characterized in that said free radical generator is 2,2'-azo-bis(2-amidinopropane) dihydrochloride.
9. A method according to Claim 1, characterized in that 50 to 200 mmol/l of free radical generator are brought into contact with a cell material at a concentration of
10 to 20% w/v in an aqueous biological medium.
10. A method according to Claim 1 or Claim 9, charac-terized in that the free radicals are generated by in-cubation at 37°C.
10. A method according to Claim 1 or Claim 9, charac-terized in that the free radicals are generated by in-cubation at 37°C.
11. An assay kit, characterized in that it comprises (i) the cell material according to Claim 1 and, if approp-riate, (ii) the free radical generator and/or the liquid biological diluting media.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8900999A FR2642526B1 (en) | 1989-01-27 | 1989-01-27 | USE OF FREE RADICAL GENERATOR IN THE FIELD OF BIOLOGICAL ASSAYS |
| FR8900999 | 1989-01-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2025316A1 true CA2025316A1 (en) | 1990-07-28 |
Family
ID=9378160
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002025316A Abandoned CA2025316A1 (en) | 1989-01-27 | 1990-01-26 | Process for the determination by means of free radicals, of the anti-oxydant properties of a living organism or a potentially agressive agent |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US5135850A (en) |
| EP (1) | EP0418335B1 (en) |
| JP (1) | JPH03505257A (en) |
| KR (1) | KR910700456A (en) |
| CN (1) | CN1057675A (en) |
| AT (1) | ATE153762T1 (en) |
| AU (1) | AU629611B2 (en) |
| CA (1) | CA2025316A1 (en) |
| DE (1) | DE69030797D1 (en) |
| FR (1) | FR2642526B1 (en) |
| HU (1) | HU209588B (en) |
| WO (1) | WO1990008955A1 (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3842699A (en) * | 1998-06-04 | 1999-12-20 | Procter & Gamble Company, The | Method for detecting potential cellular toxicity of compounds |
| KR20010073833A (en) * | 2000-01-21 | 2001-08-03 | 복성해 | Method for high speed measurement of free radical of cell unsing 2',7'-dichlorofluorescin |
| DE10049732C2 (en) * | 2000-09-30 | 2003-06-26 | Stiftung A Wegener Inst Polar | Method for testing substances with lytic activity, arrangement for carrying out and using the method |
| US6923939B1 (en) * | 2001-07-05 | 2005-08-02 | Uop Llc | Heat activated membrane introduction apparatus and method for screening materials |
| FR2861463B1 (en) * | 2003-10-22 | 2006-02-10 | Michel Prost | METHOD FOR DETERMINING ANTIRADICAL DEFENSE POTENTIAL AND USE IN PARTICULAR IN HUMAN AND VETERINARY PREVENTIVE THERAPEUTICS. |
| US7241622B2 (en) * | 2004-04-22 | 2007-07-10 | Kemin Industries, Inc. | Method for high throughput screening of antioxidants at near ambient temperatures |
| KR100561873B1 (en) * | 2004-11-30 | 2006-03-17 | 삼성전자주식회사 | Cell Lysis Method Using Free Radicals |
| EP2108692A1 (en) | 2008-04-11 | 2009-10-14 | Brasseries Kronenbourg | Method of obtaining concentrated extracts of polyphenols resulting from the brewing process |
| FR2931361B1 (en) * | 2008-05-20 | 2012-08-31 | Menvielle Bourg Fabienne Joanny | MAGNESIUM-BASED SYSTEM AND ITS USE IN COSMETICS |
| FR2931359B1 (en) * | 2008-05-20 | 2012-12-21 | Menvielle Bourg Fabienne Joanny | USE OF MATRIX FOR EXTENDED RELEASE MAGNESIUM ORAL DELIVERY, AND COMPOSITION CONTAINING SAME |
| FR2953687B1 (en) | 2009-12-15 | 2012-10-05 | Labo Concept Nature | SYSTEM AND METHOD FOR TREATING A PLANT OR ANIMAL ORGANISM PRODUCT |
| RU2563838C1 (en) * | 2014-03-24 | 2015-09-20 | Федеральное государственное бюджетное учреждение "Всероссийский научно-исследовательский институт по проблемам гражданской обороны и чрезвычайных ситуаций МЧС России" (федеральный центр науки и высоких технологий) | Method of express-detection of aggressive chemical substances |
-
1989
- 1989-01-27 FR FR8900999A patent/FR2642526B1/en not_active Expired - Fee Related
-
1990
- 1990-01-26 AT AT90902287T patent/ATE153762T1/en not_active IP Right Cessation
- 1990-01-26 US US07/576,460 patent/US5135850A/en not_active Expired - Lifetime
- 1990-01-26 EP EP90902287A patent/EP0418335B1/en not_active Expired - Lifetime
- 1990-01-26 JP JP2502653A patent/JPH03505257A/en active Pending
- 1990-01-26 KR KR1019900702164A patent/KR910700456A/en not_active Withdrawn
- 1990-01-26 HU HU901439A patent/HU209588B/en not_active IP Right Cessation
- 1990-01-26 WO PCT/FR1990/000061 patent/WO1990008955A1/en active IP Right Grant
- 1990-01-26 AU AU50271/90A patent/AU629611B2/en not_active Ceased
- 1990-01-26 CA CA002025316A patent/CA2025316A1/en not_active Abandoned
- 1990-01-26 DE DE69030797T patent/DE69030797D1/en not_active Expired - Lifetime
- 1990-06-25 CN CN90103117A patent/CN1057675A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0418335A1 (en) | 1991-03-27 |
| HU901439D0 (en) | 1991-06-28 |
| CN1057675A (en) | 1992-01-08 |
| AU5027190A (en) | 1990-08-24 |
| ATE153762T1 (en) | 1997-06-15 |
| DE69030797D1 (en) | 1997-07-03 |
| KR910700456A (en) | 1991-03-15 |
| HU209588B (en) | 1994-08-29 |
| FR2642526B1 (en) | 1992-11-13 |
| AU629611B2 (en) | 1992-10-08 |
| WO1990008955A1 (en) | 1990-08-09 |
| FR2642526A1 (en) | 1990-08-03 |
| EP0418335B1 (en) | 1997-05-28 |
| JPH03505257A (en) | 1991-11-14 |
| HUT56970A (en) | 1991-10-28 |
| US5135850A (en) | 1992-08-04 |
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