Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1990-09-17
1992-08-04
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
4352401, 436 63, C12Q 102
Patent
active
051358506
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a novel method of assaying or determining the antioxidizing properties of a living organism or a potentially aggressive agent by means of free radicals.
It relates in particular to the method of evaluating on the one hand the antioxidative state of cells of a living organism, and on the other hand the oxidative or antioxidizing activities of a potentially aggressive chemical or physical agent, i.e. a chemical or physical agent capable either of increasing or accelerating or of inhibiting or retarding the cell lysis 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 this organism. Free radicals attack the cell wall at a rate which depends on the cell resistance imparted by the enzymatic and molecular equipment of said cells. When the cell wall has been degraded, perforated or opened by free radicals, the contents of the cell spread outside the wall. See the following documents in particular: Chemical Abstracts 107, 213235v, Chemical Abstracts 107, 232454g, Chemical Abstracts 100, 99345j, Biological Abstracts 72 (n.degree. 9), page 5914, abstract n.degree. 57169, (1981) and Biological Abstracts 73 (n.degree. 12), page 8817, abstract n.degree. 84420, (1982).
The abstract CA 107, 213235v, which refers to an article by M. MIKI et al., Arch. Biochem. Biophys., 258 (n.degree. 2), pages 373-380 (1987), indicates that alphatocopherol protects rat erythrocytes from the lysis induced by free radicals.
According to the abstract CA 100,99345j cited above, which refers to an article by E. B. SPEKTOR et al., Lab. Delo (n.degree. 1), pages 26-28 (1984), the total antioxidizing activity of a sample (blood plasma or spinal fluid) is determined by absorption at 532 nm after the induction of free radicals in a cell material (erythrocyte 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.
SUBJECT OF THE INVENTION
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 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 (I) selected from the group consisting of material having first been contaminated with a potentially aggressive agent (II);
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 containing said cell material which has not been contaminated.
In this method, the oxidative state of the cell material I or the influence of the agent II on said 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 samples (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 measurements 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 evaluation, the resistance to free radicals of the cell material I is expressed as the time which corresponds to lysis of 50% of said cell material.
In the case of the dose-response evaluation, the resistance to free radicals of the cell material is expressed as the concentration of free radical generator which in
REFERENCES:
Miki Chem. Abstract #107:213235v 1987.
Oberley Cancer Research 39 pp. 1141-1149 Apr. 1, 1979.
M. Miki et al., Archives of Biochemistry and Biophysics 258(2):373-380 (1987).
V. N. Ushkalova et al., "Spectrophotometry, fluorometry, and kinetic methods used for analysis of blood lipid free radicals", Chemical Abstracts, vol. 107, No. 25, Dec. 21, 1987.
E. B. Spektor et al., "Determination of the total antioxidizing activity of the blood plasma and spinal fluid", Chemical Abstracts, vol. 100, No. 13, Mar. 26, 1984.
R. A. Lovstad, "The protective action of ceruloplasmin on iron (II) stimulated lysis of rat erythrocytes", Biological Abstracts, vol. 72, No. 9, 1981.
B. N. Ames et al., "Uric acid provides an antioxidant defense in humans against oxidant-caused and radical-caused aging and cancer: A hypothesis", Biological Abstracts, vol. 73, No. 12, 1982.
Robinson Douglas W.
Spiral Recherche et Developpment
Williams Jane A.
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