Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-04-14
2001-07-17
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S014000, C435S975000, C435S004000
Reexamination Certificate
active
06261796
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring mitochondrial activity. Moreover, the present invention relates to a diagnostic kit for measuring platelet mitochondrial activity.
As is known, Reactive Oxygen Species (ROS) play a fundamental role in pathological processes. Their chemical aggression towards all biological macromolecules causes a deterioration in cell structures which has been suggested to be the cause or pathogenic event leading to many degenerative diseases, cancer and ageing. In order to understand ROS biochemistry and pathology, research focuses chiefly on mitochondrions, since these are important ROS producers in the respiratory chain and are particularly vulnerable in the sophisticated mechanism of oxidative phosphorylation. The mitochondrial ageing theory is based on the idea that cells which are constantly exposed to ROS are gradually damaged, particularly through random errors in the mitochondrial DNA, in their energy functions, with a gradual loss of efficiency and consequent cell death.
In light of the above, ageing and age-related illnesses in humans must take into account mitochondrial activity. This activity can be directly investigated only in cells containing mitochondria, for example platelets. Blood platelets are a particularly suitable system, since the platelets contain mitochondria and are easily collected using a non-invasive technique.
As is known, platelets obtain the energy which allows them to function partly from glycolysis and partly from mitochondrial oxidative phosphorylation. The most important function is the aggregation process, part of the blood coagulation mechanism, and physiologically a result of stimulation and exocytosis of their secretion granules. The aggregation of platelets requires energy, conserved in the form of ATP generated by both the glycolysis and the oxidative phosphorylation. Inhibition of the respiratory chain partially inhibits platelet aggregation, indicating that the function is mainly, but not completely, provided with energy by the glycolytic ATP.
Therefore, a method is still required for measuring mitochondrial activity. In particular, a method is required for measuring the mitochondrial activity using platelets. More specifically, a method is required for measuring mitochondrial activity by measuring the amount of glycolytic and mitochondrial ATP in the platelets.
The use of platelets as markers for mitochondrial lesions is due to the fact that the alterations which accompany ageing and age-related illnesses are present in all cells and, therefore, platelets can indicate general bioenergetic changes.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a method for measuring mitochondrial activity.
Another aim of the present invention is to provide a method for measuring mitochondrial activity using platelets.
Another aim of the present invention is to provide a method for measuring mitochondrial activity by measuring the amount of glycolytic and mitochondrial ATP in the platelets.
Yet another aim of the present invention is to provide a bioenergetic marker (biomarker) for mitochondrial activity which is an individual and customised indicator of biological age.
Another aim of the present invention is to provide a biomarker for mitochondrial activity which is an indicator of a tendency towards those illnesses which accompany ageing through mitochondrial activity.
Another aim of the present invention is to provide a biomarker for mitochondrial activity which is a diagnostic and prognostic indicator of any pathology involving altered mitochondrial activity.
Another aim of the present invention is to provide an indicator for evaluating the preservation of bags containing blood for transfusions.
The final but no less important aim of the present invention is to provide a diagnostic kit (micromethod) for measuring platelet mitochondrial activity.
These aims and others are which are described in the detailed description which follows were achieved by the Applicant, who developed a method for measuring mitochondrial activity by measuring the amount of platelet &Dgr; lactate.
The present invention also relates to a diagnostic kit (micromethod) for measuring platelet mitochondrial activity by measuring the amount of platelet &Dgr; lactate.
The basic characteristics of the above-mentioned method and kit are defined in the main claims; some special preferred embodiments of the invention, which do not limit the scope of its application, are described in the secondary claims.
The method for measuring mitochondrial activity which is the subject matter of the present invention uses inhibition of the mitochondrial respiratory chain by means of inhibitor agents in such a way as to obtain a measurement of the amount of glycolytic and mitochondrial ATP through the so-called Pasteur effect.
The amount of mitochondrial ATP (mitATP) is equal to the &Dgr; lactate (lactate produced by the platelets inhibited with mitochondrial chain inhibitors minus the lactate produced by the uninhibited platelets (Ctl)).
The amount of glycolytic ATP (glycATP) is equal to the &Dgr; lactate (lactate produced by the uninhibited platelets (Ctl) minus the lactate produced by the platelets inhibited with the glycolysis inhibitors).
The effective oxidation of the glycolytic pyruvate through the Krebs cycle and respiratory chain leads to the formation of low or zero levels of lactate through the action of the cytosolic lactate dehydrogenase on the pyruvate. In contrast, a reduced or ceased mitochondrial activity leads to a reduction in the lactate pyruvate by the excess reducing force exerted by the NADH formed by the glycolysis; at the same time, glycolysis is stimulated with the aim of maintaining the ATP production constant over time. Since, in the absence of mitochondrial activity, glycolysis produces two ATP molecules and two lactate molecules for each glucose molecule broken down, the stimulation of lactate production when the mitochondria are inhibited, is equivalent to the mitochondrial ATP production when subjected to inhibition.
The Applicant has discovered a relationship between the &Dgr; lactate (more lactate is produced when the mitochondria are inhibited) and different categories of individuals. The Applicant found that there is less &Dgr; lactate in the platelets of elderly individuals than in those of young individuals, demonstrating that there is a change in the mitochondrial energy in the platelets of elderly subjects and that platelets are a useful material for investigation, on which to use a mitochondrial marker.
The &Dgr; lactate may be expressed as: &Dgr; lactate=(AA−Ctl) where AA=lactate produced by platelets in the presence of specific respiratory chain inhibitors; Ctl=lactate produced by platelets in the absence of specific inhibitors.
The Applicant found it useful to use particular specific inhibitors, such as antimycin A to inhibit platelet aggregation. The inhibited respiratory chain produces less ATP and, as a result, the reduced amount of ATP produced reduces platelet aggregation.
REFERENCES:
patent: 1046714 A2 (2000-10-01), None
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Holmsen; “Biochemistry of the Platelet: Energy Metabolism”; Lippincott, Philadelphia; 1987 pp. 631-643, month not available.*
Makler et al; “Measurement of the Lactate Dehydrogenase Activity of Plasmodium Falciparum as an assessment of Parasitemia”; American Journal of Tropical Medicine and Hygiene; vol. 48, No. 2 1993; pp. 205-210, month not available.*
Sajko et al; “Administration of Lactate Dehydrogenase From the Ox Heart to Determine the Content of Pyruvate in Milk”; Medycyna Weterynary JNA, vol. 41, No. 7, 1985 pp. 442-444, month not available.*
Homyk et al; “Steady-Stae Kinetics and the Inactivation by 2 3 Butanedione of the Energy Independent Transhydrogenase ofEscherichia-ColiCell Membranes”; Biochimica et Bioiphysica ACTA; vol. 571,
Bovina Carla
Catani Lucia
Lenaz Giorgio
Merlo Pich Milena
Tura Sante
Leary Louise N.
Nixon & Vanderhye PC
Universita′ Degki Studi di Bologna
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