PRIOR ART It is known that reactive oxygen species (ROS) are deeply involved in pathologic processes. Their chemi- cal aggressiveness towards all biological macromole- cules results in a deterioration of cell structures, which is thought to be the causal or pathogenic event of many degenerative illnesses, of cancer and of age- ing. In order to understand the biochemistry and the pathology of ROS, research has strongly-focused on mi- tochondria since these are important producers of ROS through the respiratory chain and are particularly vulnerable within the sophisticated mechanism of oxi- dative phosphorylation. The mitochondrial theory ap- plied to ageing is based on the idea that cells that are continuously exposed to ROS are progressively dam- aged, in particular through casual errors in mitochon- drial DNA, in their energetic functions, with progres- sive loss of efficiency and subsequent cell death.

From the above description human ageing and age- related illnesses should take into consideration the mitochondrial function. Said function can be investi- gated directly only in cells containing mitochondria.

It is known that isolated cells get energy for their functions partly from glycolysis and partly from mito- chondrial oxidative phosphorylation. Isolated cells proliferate using in this process energy preserved as ATP generated both by glycolysis and by oxidative phosphorylation.

Isolated cells are an excellent model to be used for many research purposes in which the metabolic charac- terization of said cells would be very important. As a matter of fact, immortalized cells present on the mar- ket can be used, such as line cells, cells transformed in laboratory, cells deriving from biopsies such as for instance fibroblasts, keratinocytes, cardiomyo- cytes, stem cells, spermatozoa.

Therefore, there is the need for a method for measur- ing bioenergetic mitochondrial activity, which di- rectly investigates isolates cells. In particular, there is the need for a method for measuring mitochon- drial activity using immortalized cells, transformed cells and tissue-isolated cells. More particularly, there is the need for a method for measuring mitochon- drial activity through the quantitative measurement of glycolytic and mitochondrial ATP in isolated cells.

DESCRIPTION OF THE INVENTION An aim of the present invention is to provide a method for measuring mitochondrial activity in isolated cells.

Another aim of the present invention is to provide a method for measuring mitochondrial activity using iso- lated cells such as immortalized cells, transformed cells and tissue-isolated cells.

A further aim of the present invention is to provide a method for measuring mitochondrial activity through the quantitative measurement of glycolytic and mito- chondrial ATP in isolated cells.

Last but not least, an aim of the present invention is to provide a diagnostic kit (micromethod) for measur- ing mitochondrial activity in isolated cells.

These and other aims that will be evident in the course of the following detailed description have been achieved by the Applicant, who has developed a method for measuring mitochondrial activity through the quan- titative measure of A-lactate in isolated cells.

A further object of the present invention is a diag- nostic kit (micromethod) for measuring mitochondrial activity in isolated cells through the quantitative measurement of A-lactate.

The essential characteristics of the aforesaid method and kit referred to above are defined in the appended main claims.

Some particular preferred though non-limiting embodi- ments of the present invention are defined in the ap- pended dependent claims.

The method for measuring mitochondrial activity ac- cording to the present invention exploits the inhibi- tion of mitochondrial respiratory chain by using in- hibitors so as to obtain the quantitative measure of glycolytic and mitochondrial ATP through the so-called Pasteur effect.

The quantitative measure of mitochondrial ATP (mit ATP) corresponds to A-lactate (lactate produced by isolated cells inhibited with inhibitors of the mito- chondrial respiratory chain (AA) minus the lactate produced by non-inhibited isolated cells (Ctl)).

The quantitative measure of glycolytic ATP (glyc ATP) corresponds to the lactate produced by non-inhibited isolated cells (CT1)..

The oxidation of glycolytic pyruvate and of other aerobic substrates such as glutamine through the Krebs cycle and the respiratory chain keeps lactate levels relatively low by means of cytosolic dehydrogenase lactate acting on the pyruvate.

Conversely, a reduced or ceased mitochondrial function results in the reduction of pyruvate to lactate through the excess of reducing power exerted by NADH generated by glycolysis; simultaneously, the glycoly- sis is stimulated in, order to keep the production of ATP constant in time. Since glycolysis without mito- chondrial function produces two molecules of ATP and two molecules of lactate pro molecule of glucose that has been destroyed, as a consequence the stimulation of lactate production-when mitochondria are inhib- ited-corresponds quantitatively to the mitochondrial production of ATP undergoing inhibition.

A-lactate can be expressed as: A-lactate = (AA-Ctl) where: AA = lactate produced by isolated cells in the pres- ence of specific inhibitors of the respiratory chain; - Ctl = lactate produced by isolated cells without spe- cific inhibitors.

The description of some embodiments of the present in- vention shall make reference to the appended figures, in which: Figure 1 shows the titration curve of antimycin A as a function of A-lactate in HL60 cells (incubation period 120 minutes).

Figure 2 is a table showing lactate production of HL60 cells with different concentrations of AA (incubation period 120 minutes).

Figure 3 shows the time course referring to lactate production with 1x106 HL60 cells.

Figure 4 is a table showing the time course referring to lactate production with different concentrations of HL60 cells, lx106, 106 and 3x106.

Figure 5 is a graph showing lactate production compar- ing different cell types: HL60; H9C2 ;. JURKATT ; ECV; and U937.

Figure 6 is a table showing lactate production compar- ing different cell types (HL60, U937, JURKATT, H9C2 and ECV 304) and the percentage of ATP produced in mi- tochondria.

The description of the following experimental part should be regarded as a mere and therefore non- limiting example of the present invention.

The method according to the present invention has been applied to some cell types commonly used in research labs.

Advantageously, the method suggested by the Applicant applies to immortalized cells present on the market such as cell lines, to cells transformed in laboratory and to cells deriving from biopsies such as for in- stance fibroblasts, keratinocytes, cardiomyocytes, stem cells, spermatozoa.

METHOD FOR PREPARING ISOLATED CELLS Material required for preparing cells: Washing buffer, pH 7.4 containing: - NaCl, 142 mM - KCl, 2 mM - K2HPO4, 1.2 mM - MgS04, 1 mM - Hepes, 10 mM - CaCl2, 1.3 mM - Glucose, 10 mM -Glutamine, 1% pH 7.4 'Krebs-Ringer buffer NaCl 115 mM KC1 3 mM K2HP04 2 mM MgS04 1 mM Glucose 10 mM CaCl2 1 mM NaHCO3 25 mM Glutamine 1% pH 7.4 Advantageously, every cell type taken into considera- tion should be first characterized in order to set up the following parameters: 1. re-suspension buffer: some cell types cannot use artificial buffer systems (for instance Hepes buffer), so that it is preferable to use buffers such as Krebs-Ringer buffer, which requires a balancing step in a controlled 5% C02 atmosphere and at a temperature of 37°C (thermo-controlled incubators). Moreover, it is necessary to carry out the vitality analysis at the beginning and at the end of incubation in order to exclude meta- bolic alterations involving cell suffering.

2. incubation systems: according to the type of re- suspension buffer used it is possible to use sim- ple test tubes with plug, which can be incubated in a thermo-controlled bath or multiwell plates to be placed in a thermo-controlled incubator.

3. time course: tests on lactate production as a function of incubation time should be carried out in order to characterize the amount of lactate produced under the different experimental condi- tions.

4. sensibility to inhibitor Antimycin A: titration curves of the inhibitor should be carried out in order to find maximum lactate production under conditions of total inhibition of mitochondrial activity.

In a preferred embodiment the isolated cells that are present in the culture medium, in suspension with the latter, can be chosen among: immortalize cells, trans- formed cells and tissue-isolated cells.

The isolated cells that are present in the culture me- dium, in suspension with the latter, have been centri- fuged at 200 rpm for 10 minutes at room temperature.

The supernatant liquor has been removed since it in- terferes in the spectrophotometric measurement exe- cuted later.

The pellet of cells has been re-suspended with washing buffer. Then a further washing has been carried out.

The cells have been diluted in the same buffer at a concentration of about 2 x 106 cells/ml and divided into several tubes (1 x 106 cells/aliquot), each rep- resenting a different experimental condition.

In another preferred embodiment the cells that are present in the culture medium adhering to the growth support can be chosen among immortalized cells, trans- formed cells and tissue-isolated cells.

The isolated cells that are present in the culture me- dium adhering to the growth support have undergone trypsinization so as to obtain a homogenous cell sus- pension. Concerning this, it should be pointed out that every cell type used requires specific treatments and care.

The cells are then counted and seeded in multiwell plates with 6 wells (diameter 2 cm), using 2 x 105 cells pro well, so as to obtain a semi-confluent growth after around 2 days for instance.

At the end of the semi-confluent growth the cells un- dergo an incubation with and without inhibitor AA (step b and step c).

In both embodiments referred to above, the isolated cells (cells suspended in the culture medium or cells adhering to the growth support) have undergone a first incubation both with and without inhibitors.

The duration of said first incubation is preferably between 1 and 3 hours, for instance in a thermo- controlled bain-marie or in a thermostat at a tempera- ture of 37°C.

At the end of the incubation the supernatant liquor has been taken from every well and centrifuged at 1500 rpm for 10 minutes at room temperature (RT) so as to eliminate cell contaminations in order to measure lac- tate concentration.

In the case of adhering cells, after the incubation with and without inhibitor, in order to determine the precise number of cells that have undergone incubation producing lactate, the adhering cells undergo a second trypsinization, are counted again and then settled at 1500 rpm for 10 minutes so as to carry out, for in- stance, protein measurement.

The inhibitors used by the Applicant are specific in- hibiting agents.

Preferably, Antimycin A (AA) has been used to inhibit the respiratory chain at a common stage at the en- trance of all physiologic substrates.

At the end of said first incubation the samples of cells incubated with and without inhibitors have un- dergone a second separation (step d).

Preferably, the suspension of cells has been centri- fuged at 1500 rpm for a period between 5 and 20 min- utes at a temperature between 18 and 25°C.

The centrifuged sample comprises a fraction in the up- per portion, known as (supernatant liquor), and a fraction in the lower portion comprising the cells.

The supernatant liquor contains the lactate produced by the cells during said first incubation.

Said second separation separates the lactate produced by the cells during said first incubation from the cell suspension.

The lactate obtained from said second separation has been measured.

Alternatively, the upper portions of supernatant liq- uor can be taken directly to lactate dosage or they can be kept at a temperature of about minus 20°C. If needed, the upper portions are defrosted and the lac- tate is measured. Lactate dosage can be carried out with any method known on the market.

The Applicant has found a method for measuring lactate that can successfully replace or be used as an alter- native to methods for measuring lactate present on the market.

A further object of the present invention is a spec- trophotometric method for measuring lactate using as chromogen 3-acetyl-pyridine-NAD+. 3-acetyl-pyridine- NAD+ has a molar extinction coefficient s = 9.1 mM cm.

The method for measuring lactate according to the pre- sent invention is based on the following reaction: LDH Lactate+3-acetyl-pyridine-NAD+ Pyruvate+3-acetyl- pyridine-NADH+H+ LDH enzyme can be chosen among enzymes known on the market, preferably LDH enzyme is from ox's heart.

3-acetyl-pyridine-NADH has a maximum absorbance at 363 nm.

The method for measuring lactate suggested by the Ap- plicant differs from methods known on the market in that it has a higher sensibility in the whole field of lactate concentrations having a diagnostic relevance.

In particular, the method according to the present in- vention is recommended for measuring at low concentra- tions lactate, since in the field of low concentra- tions said method has a higher sensibility than known methods.

The production of low concentrations of lactate by isolated cells can be obtained both by reducing the periods of said first incubation of isolated cells though their number remains the same, or by reducing the number of incubated grown cells though the period of said first incubation remains the same.

The high sensibility of the method suggested by the Applicant for measuring lactate enables to reduce the time needed for measuring lactate and/or enables to reduce the number of grown cells to be incubated.

The method for measuring mitochondrial activity ac- cording to the present invention sets up a relation between the measure of A-lactate and mitochondrial ac- tivity.

The aforesaid method is validly used to measure accu- rately the low levels of lactate that are produced during the incubation period.

The Applicant has carried out a second study aiming at determining A-lactate in isolated cells that are pre- sent in scientific labs, washed and incubated without and with Antimycin A.

Figure 1 shows the titration curve of AA in HL60. It can be observed that the maximum inhibition of mito- chondrial respiration, and therefore the maximum cell production of lactate (expressed as A-lactate), is in a range between final 1 and 4 pM of AA; preferably be- tween 1.5 and 3 AMi advantageously at final 2 jeu, M of . AA. For concentrations of inhibitor above 7 pM there is a citotoxic effect.

From the data shown in Figure 2 it can be observed that in HL60 cells lactate production (see glycolytic ATP, [AA 2 pM = 47.98, pmoles/ml] is stimulated about 1.5 times as much after incubation with inhibitor (71.57/47. 98 = 1. 49). From these parameters the dif- ference between basic glycolytic lactate and lactate after inhibition (A-lactate = about 24) and the ratio A-lactate/basal lactate can be calculated: the latter represents the ratio of mitochondrial ATP/glycolytic ATP = 0.49.

Figure 2 contains the values referring to lactate pro- duction expressed both as Fg of lactate/ml and as moles of lactate/minute/1011 cells.

From Figure 2 it is possible to infer the data in Fig- ure 6 concerning the percentage of mitochondrial ATP, which is quite low (33%) in HL60 cells.

However, the Applicant has found out that percentages change in the different cell types that have been studied.

As a matter of fact, in the case of H92C cells (see Figure 6) the ratio of mitochondrial ATP/glycolytic ATP is 8.49 and the percentage of ATP produced by mi- tochondria is 89%.

The method for measuring mitochondrial activity through the measure of A-lactate in cells according to the present invention is reproducible and has no meth- odological obstacles.

Advantageously, the Applicant suggests the method for measuring mitochondrial activity in isolated cells: - immortalized cells ; - transformed cells; and - tissue-isolated cells obtained from human biopsies, for instance fibroblasts, keratinocytes, cardiomyo- cytes, stem cells and spermatozoa) as biomarker of mi- tochondrial function of said cell type in the organ- ism, based on the assumption that the bioenergetics in isolated cells suitably represents the bioenergetics of all analogue cells. It can thus be supposed that the alteration found out by the Applicant in the cells isolated from biopsies can be due to the analogous al- terations present in the various tissues.

Therefore, the method according to the present inven- tion allows to test a mitochondrial lesion on isolated cells that are easy to be handled, and offers an ex- cellent individual biomarker of mitochondrial activ- ity.

The dosage suggested by the Applicant does not depend on the alteration spot in the oxidative phosphoryla- tion system, since it is based on the assessment of the total energetic yield of mitochondrial ATP. That is why the present method is versatile and can detect mitochondrial alterations of various origin giving rise to a reduced synthesis of ATP.

Advantageously, the Applicant designs to suggest the use of this biomarker in pathologies in which a mito- chondrial alteration has been assessed or supposed, for instance in mitochondrial genetic illnesses.

First of all, with this dosage it is possible to sup- pose an assessment of the bioenergetic metabolism of isolated cells, as a useful functional index in the field of scientific research.

Merely by way of example, the method has been carried out using some cell types as disclosed below.

Example of lactate measurement in HE60 isolated cells The measurement has been carried out on a cell suspen- sion.

The cells, separated and washed as described above, have been incubated without (Ctrl) and with inhibitor Antimycin A (AA), measuring lactate production at various time intervals (60-120 up to 180 min).

The tests have been carried out using: - 1 million cells for each test ; data are shown in Figure 4.

- 2 millions cells for each test ; data are shown in Figure 4.

- 3 millions cells for each test ; data are shown in Figure 4.

The test proves the reproducibility of the results: a) using half and 1/3 grown cells, b) using an incubation period below 3 hours, according to usual needs of analysis labs.

In order to meet needs involving a higher sensibility, due to the low lactate concentrations produced in short times by a relatively small number of grown cells, the method for measuring lactate according to the present invention has been advantageously used.

All steps involving the isolation and washing of grown cells have been carried out at 10-37°C so as to avoid freezing-related ultra-structural cell changes. In or- der to prevent contaminations disposable sterile con- tainers and lab equipment have been used for all op- erations.

Preparation of cell material Isolated cells have been prepared and washed as de- scribed above. Aliquots of the cell suspensions (0. 5x106/500 pl and lxl06/500 p1 and 1. 5x106/500 pl) have been incubated in a thermo-controlled bain-marie at 37°C or in a thermo-controlled incubator with con- trolled atmosphere without (control) and with Antimy- cin A (2-20 Ag/sample). The incubation has been blocked in the various assays after 0,60, 120 or 180 minutes by centrifugation at 1500 rpm for 10 min.

The supernatant, containing the lactate produced by the cultured cells, were taken at-20°C till the time of dosage.

Lactate dosage The method for quantitatively measuring lactate ac- cording to the invention is spectrophotometric and is based on the following reaction: LDH <BR> <BR> <BR> Lactate+3-acetyl-pyridine-NAD+ Pyruvate+3-acetyl- pyridine-NADH+HA The LDH enzyme used is from ox's heart.

For lactate dosage absorbance variations at 363 nm are followed, at which wave length 3-acetyl-pyridine-NADH has s = 9.1.

For the dosage, which is carried out. directly in the cuvette of the spectrophotometer, 0.8 ml of borate buffer 0.01 M pH 9.20 are added with 0.1 ml of 3- acetyl-pyridine NAD+ (20 mg/ml), 1 ml of supernatant liquor, 0.01 ml of lactate dehydrogenase (LDH 5 mg/ml).

After 5 minutes the absorbance has been read at 363 nm against a blank without the LDH enzyme solution.

Lactate concentration has been calculated using two standard curves: 0-15 p, g of lactate/ml and 15-50 Fg of lactate/ml.

Both curves have been suitably used according to the concentration of lactate in the samples.

Figure 4 shows the time development of lactate produc- tion with the three concentrations of HL60 cells used (lux106 cells/ml; 2x106 cells/ml and 3x106 cells/ml without (Ctrl) and with Antimycin A (AA) 2 J. M. The de- velopment of lactate production is linear in all cases taken into consideration ; in particular (Figure 3), there is a relation between the line representing the development of controls and the line corresponding to samples treated with Antimycin A also with the other concentrations tested, so that differential values at every time interval (A-lactate) are reproducible.

It can thus be inferred that the value of A-lactate represents mitochondrial activity.

Moreover, considering the linearity of the develop- ments and the high sensibility of the present method it can be thought to block incubation after only 60 minutes, thus reducing to a half the time necessary for carrying out this analytical step.

On the other hand, since a high number of cells can be used in a small volume, incubation time can be further reduced.

It should be considered that with concentrations above 3x106 cells/ml after 120 minutes there is a decrease of lactate production and a simultaneous increase of cell death, probably due to cell density and to the competition for the substrate.

The method carried out provides means for measuring lactate in a reproducible way at two ranges of low concentrations (0-15 pg/ml and 15-50 pg/ml) using two setting curves. The methods and kits commonly avail- able on the market are hardly reproducible at low con- centrations.

A further object of the present invention is a kit (micromethod) of mitochondrial activity. The aforesaid kit consists of a package comprising two containers.

The first container A contains the reagents and the material for washing and incubating the cells.

The second container B contains the reagents and the material for the quantitative measuring (dosage) of lactate.

For instance a package of the kit (micromethod) of mi- tochondrial activity for 10 experiments comprises: Washing and incubation of cells (container A): a) 10 sealed sterile bottles containing the powders to be hydrated up to a final volume of 20 ml, which shall be the washing buffer for the isolated cells having the following composition: - NaCl, 142 mM - KCl, 2 mM -K2HP04, 1.2 mM - MgS04, 1 mM -Hepes, 10 mM -CaCl2, 1.3 mM -Glucose, 10 mM -Glutamine, 1% The pH of the solution should be adjusted at 7.4. The hydrated buffer can be kept at 4°C for 3 days. b) 10 sealed sterile bottles containing the powders to be hydrated up to a final volume of 20 ml, which shall be the Krebs-Ringer washing buffer for the isolated cells having the following composition: -NaCl 115 mM - KC1 3 mM -KH2 PO4 2 mM - MgS04 1 mM - Glucose 10 mM - CaCl2 1 mM - NaHC03 25 mM - Glutamine, 1% - pH 7.4 The pH of the solution should be adjusted at 7.4. The solution shall be balanced at least for 30 minutes in an incubator at 37°C and with a controlled C02 (5%) atmosphere before being used with the cells, and kept in said incubator (under the same conditions) for the whole experiment. The buffer cannot be kept. c) 1 sealed dark glass bottle of Antimycin A in powder 2.67 mg, to be re-suspended in 5 ml of absolute etha- nol (1 mM solution, from which 1 1 is taken to be ad- justed up to a final volume of 0.5 ml of cell suspen- sion, so as to achieve a final concentration of 2 fJJM).

Dosage of lactic acid (container B) a) 10 plastic 1 ml cuvettes for spectrophotometer ; b) 4 sealed bottles each containing 50 mg of 3-acetyl- pyridine NAD+ to be re-suspended in 2.5 ml of bi- distilled H20 ; c) 10 bottles containing 1 ml of LDH enzyme (250 U) from ox's heart in ammonium sulfate (3.2 M) 5 mg/ml ; d) 1 bottle of 0.01 M sodium-borate buffer pH 9.2, to be adjusted up to a volume of 50 ml with bi-distilled H20 (non-preservable buffer).

The kit should be kept at a temperature of 4°C.