Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1994-06-06
1996-08-06
Kight, III, John
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 4, 435810, 435975, 435189, 435 28, 435 26, 436 63, 536 111, 560117, 540520, 540521, 514 1, 514 23, 514740, 514753, C12Q 126, C12Q 100, G01N 3348, C07H 100
Patent
active
055432980
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the assaying of superoxide dismutase (SOD) activity.
It relates more particularly to a new process for the assay of SOD activity, especially in biological samples, using auto-oxidizable compounds which are defined below, to kits for the implementation of this process and to new compounds which can be used in this process, and to their preparation.
The tissues of aerobic organisms, and those of the human organism in particular, are the site of continual production of superoxide ion O.sub.2..sup.-, which results from the process of cell respiration and from numerous essential metabolic pathways.
This production of superoxide ion increases considerably when these organisms are subjected to an "oxidizing stress" of toxicological origin (hyperoxia, irradiation, intoxication by xenobiotics which generate free radicals, in particular) or of physiopathological origin (inflammation, ischaemia, post-ischaemic reperfusion, in particular).
It is accepted by the entire scientific community that the superoxide ion is highly toxic, although the mechanisms underlying this toxicity remain the subject of controversy (reference 1).
In order to protect themselves against the harmful effects of the superoxide ion, aerobic organisms possess enzymatic systems, the superoxide dismutases (SODs), which catalyse the breakdown of the superoxide ion in accordance with the following dismutation reaction:
Because of the major protective role of this dismutation reaction with regard to the toxicity of the superoxide ion, the catalytic activity of intra- and extra-cellular SODs determines the survival of the tissues of an aerobic organism (references 2 to 5).
The assay of SOD activity is therefore of prime interest to biological researchers and to laboratories of clinical chemistry, on condition that it should be possible to carry it out using a method which is sensitive, specific, rapid and relatively simple.
Numerous methods for assaying SOD activity have been published and commented on (references 6 to 10), but it is generally accepted that there does not yet exist a satisfactory compromise between reliability and complexity in the existing methods, since the measurement of SOD activity poses two major problems which none of the methods published to date has been able to resolve in a simple fashion.
The first problem is that of the spontaneous decomposition of the sole substrate, the superoxide ion, which is very rapid at a pH of about 7 and which, although highly decelerated, remains significant at a pH of 9, the pH above which certain types of natural SODs are rapidly deactivated. To obtain a stationary concentration which is sufficiently stable therefore requires the presence of a dynamic source of superoxide ion in the assay solution.
Electrolytic sources require complex equipment and a prior extraction of the SOD catalyst, which is prohibitive in clinical chemistry.
Photochemical sources, such as riboflavin, lead to methods which cannot be standardized and which are complex to carry out.
Enzymatic sources, such as xanthine oxidase, require solutions to be prepared at the time of use. They are expensive, a source of artifacts, and are difficult to automate.
Purely chemical sources come down to auto-oxidizable compounds such as 6-hydroxydopamine, pyrogallol, hydroxylamine or the sulfite ion, whose autoxidation is generally inhibited by SOD.
These chemical reagents rarely give results which are precise and sensitive on crude biological extracts, and they require the extemporaneous preparation of an anaerobic solution of the reagent; their use is therefore difficult to automate.
The second problem is related to the fact that it is difficult to measure directly ("direct" assay) the disappearance of the superoxide substrate O.sub.2..sup.- as a function of time, or the appearance of its dismutation products, hydrogen peroxide H.sub.2 O.sub.2 and oxygen, because of the concentrations and the pH values which are accessible under the conditions of an assay of SOD activity.
The only "direct" spectrophotometri
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Chaudiere Jean R.
Moutet Marc E.
Xu Jinzhu
Yadan Jean-Claude Y.
Kight III John
Leary Louise N.
Oxis International S.A.
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