WO2003100418A2 - Methode et trousse de mesure de l'activite mitochondriale dans des cellules isolees - Google Patents

Methode et trousse de mesure de l'activite mitochondriale dans des cellules isolees Download PDF

Info

Publication number
WO2003100418A2
WO2003100418A2 PCT/IT2003/000238 IT0300238W WO03100418A2 WO 2003100418 A2 WO2003100418 A2 WO 2003100418A2 IT 0300238 W IT0300238 W IT 0300238W WO 03100418 A2 WO03100418 A2 WO 03100418A2
Authority
WO
WIPO (PCT)
Prior art keywords
cells
lactate
isolated cells
isolated
incubation
Prior art date
Application number
PCT/IT2003/000238
Other languages
English (en)
Other versions
WO2003100418A3 (fr
Inventor
Giorgio Lenaz
Carla Bovina
Milena Merlo Pich
Original Assignee
Universita' Degli Studi Di Bologna
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universita' Degli Studi Di Bologna filed Critical Universita' Degli Studi Di Bologna
Priority to AU2003230208A priority Critical patent/AU2003230208A1/en
Publication of WO2003100418A2 publication Critical patent/WO2003100418A2/fr
Publication of WO2003100418A3 publication Critical patent/WO2003100418A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • 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

Definitions

  • the present invention relates to a method for measuring mitochondrial activity in cells, in particular in isolated cells, immortalized cells, transformed cells and tissue-isolated cells.
  • the present invention further relates to a diagnostic kit for measuring mitochondrial activity in isolated cells.
  • ROS reactive oxygen species
  • Isolated cells are an excellent model to be used for many research purposes in which the metabolic characterization of said cells would be very important.
  • immortalized cells present on the market 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.
  • 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 isolated 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 mitochondrial ATP in isolated cells.
  • an aim of the present invention is to provide a diagnostic kit (micromethod) for measuring mitochondrial activity in isolated cells.
  • the method for measuring mitochondrial activity according to the present invention exploits the inhibition of mitochondrial respiratory chain by using inhibitors so as to obtain the quantitative measure of glycolytic and mitochondrial ATP through the so-called Pasteur effect.
  • mitochondrial ATP The quantitative measure of mitochondrial ATP (mit ATP) corresponds to ⁇ -lactate (lactate produced by isolated cells inhibited with inhibitors of the mitochondrial respiratory chain (AA) minus the lactate produced by non-inhibited isolated cells (Ctl) ) .
  • glycolytic ATP The quantitative measure of glycolytic ATP (glyc ATP) corresponds to the lactate produced by non-inhibited isolated cells (CT1) ..
  • 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.
  • ⁇ -lactate (AA- Ctl)
  • AA lactate produced by isolated cells in the presence of specific inhibitors of the respiratory chain
  • Ctl lactate produced by isolated cells without specific inhibitors.
  • Figure 1 shows the titration curve of antimycin A as a function of ⁇ -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 lxlO 6 HL60 cells.
  • Figure 4 is a table showing the time course referring to lactate production with different concentrations of HL60 cells, 1x10 s , 2xl0 6 and 3xl0 6 .
  • Figure 5 is a graph showing lactate production comparing different cell types: HL60; H9C2; JURKATT; ECV; and U937.
  • Figure 6 is a table showing lactate production comparing different cell types (HL60, U937, JURKATT, H9C2 and ECV 304) and the percentage of ATP produced in mitochondria.
  • 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 .
  • 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 instance fibroblasts, keratinocytes, cardiomyocytes, stem cells, spermatozoa.
  • 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% C0 2 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 metabolic alterations involving cell suffering.
  • incubation systems according to the type of re- suspension buffer used it is possible to use simple test tubes with plug, which can be incubated in a thermo-controlled bath or multiwell plates to be placed in a thermo-controlled incubator.
  • the isolated cells that are present in the culture medium, in suspension with the latter can be chosen among: immortalize cells, transformed cells and tissue-isolated cells.
  • 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 10 6 cells/ml and divided into several tubes (1 x 10 6 cells/aliquot) , each representing a different experimental condition.
  • the cells that are present in the culture medium adhering to the growth support can be chosen among immortalized cells, transformed 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 suspension. 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 10 5 cells pro well, so as to obtain a semi-confluent growth after around 2 days for instance.
  • the cells undergo an incubation with and without inhibitor AA (step b and step c) .
  • the isolated cells 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 temperature of 37°C.
  • 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 lactate concentration.
  • RT room temperature
  • 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 instance, protein measurement.
  • the inhibitors used by the Applicant are specific inhibiting agents.
  • Anti ycin A has been used to inhibit the respiratory chain at a common stage at the entrance of all physiologic substrates.
  • the samples of cells incubated with and without inhibitors have undergone a second separation (step d) .
  • the suspension of cells has been centri- fuged at 1500 rpm for a period between 5 and 20 minutes at a temperature between 18 and 25°C.
  • the centrifuged sample comprises a fraction in the upper 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.
  • the upper portions of supernatant liquor 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 lactate 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 alternative 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 + .
  • the method for measuring lactate according to the present invention is based on the following reaction:
  • the method for measuring lactate suggested by the Applicant 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.
  • the method according to the present invention is recommended for measuring at low ' concentrations lactate, since in the field of low concentrations 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 according to the present invention sets up a relation between the measure of ⁇ -lactate and mitochondrial activity. 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 ⁇ -lactate in isolated cells that are present 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 mitochondrial respiration, and therefore the maximum cell production of lactate (expressed as ⁇ -lactate) , is in a range between final 1 and 4 ⁇ M of AA; preferably between 1.5 and 3 ⁇ M; advantageously at final 2 ⁇ M of AA. For concentrations of inhibitor above 7 ⁇ M there is a citotoxic effect.
  • the method for .pleasuring mitochondrial activity through the measure of ⁇ -lactate in cells according to the present invention is reproducible and has no methodological obstacles.
  • the Applicant suggests the method for measuring mitochondrial activity in isolated cells:
  • tissue-isolated cells obtained from human biopsies, for instance fibroblasts, keratinocytes, cardiomyo- cytes, stem cells and spermatozoa
  • biomarker of mitochondrial function of said cell type in the organism 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 alterations present in the various tissues. Therefore, the method according to the present invention allows to test a mitochondrial lesion on isolated cells that are easy to be handled, and offers an excellent individual biomarker of mitochondrial activity.
  • the dosage suggested by the Applicant does not depend on the alteration spot in the oxidative phosphorylation 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.
  • the Applicant designs to suggest the use of this biomarker in pathologies in which a mitochondrial alteration has been assessed or supposed, for instance in mitochondrial genetic illnesses.
  • this dosage it is possible to suppose an assessment of the bioenergetic metabolism of isolated cells, as a useful functional index in the field of scientific research.
  • the method has been carried out using some cell types as disclosed below.
  • Example of lactate measurement in HL60 isolated cells The measurement has been carried out on a cell suspension.
  • 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:
  • 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.
  • 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 order to prevent contaminations disposable sterile containers and lab equipment have been used for all operations.
  • Isolated cells have been prepared and washed as described above. Aliquots of the cell suspensions (0.5xl0 6 /500 ⁇ l and lxl0 6 /500 ⁇ l and 1.5xl0 6 /500 ⁇ l) have been incubated in a thermo-controlled bain-marie at 37 °C or in a thermo-controlled incubator with controlled atmosphere without (control) and with Antimy- cin A (2-20 ⁇ g/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 according to the invention is spectrophotometric and is based on the following reaction:
  • Lactate concentration has been calculated using two standard curves: 0-15 ⁇ g of lactate/ml and 15-50 ⁇ g of lactate/ml.
  • FIG 4 shows the time development of lactate production with the three concentrations of HL60 cells used (lxlO 6 cells/ml; 2xl0 6 cells/ml and 3xl0 6 cells/ml without (Ctrl) and with Antimycin A (AA) 2 ⁇ M.
  • the development 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 ( ⁇ -lactate) are reproducible. It can thus be inferred that the value of ⁇ -lactate represents mitochondrial activity.
  • 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.
  • a package of the kit (micromethod) of mitochondrial 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:
  • the pH of the solution should be adjusted at 7.4.
  • the hydrated buffer can be kept at 4°C for 3 days.
  • 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 C0 2 (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.
  • 1 sealed dark glass bottle of Antimycin A in powder 2.67 mg, to be re-suspended in 5 ml of absolute ethanol (1 mM solution, from which 1 ⁇ l is taken to be adjusted up to a final volume of 0.5 ml of cell suspension, so as to achieve a final concentration of 2 ⁇ M) .
  • lactic acid 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 H 2 0; 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 H 2 0 (non-preservable buffer) .
  • the kit should be kept at a temperature of 4°C.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode destinée à mesurer l'activité mitochondriale par mesure du Δ-lactate de cellules isolées. La présente invention concerne également une trousse (microméthode) destinée à mesurer le Δ-lactate dans un échantillon de cellules isolées.
PCT/IT2003/000238 2002-05-28 2003-04-15 Methode et trousse de mesure de l'activite mitochondriale dans des cellules isolees WO2003100418A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003230208A AU2003230208A1 (en) 2002-05-28 2003-04-15 Method and kit for measuring mitochondrial activity in isolated cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITMI2002A001148 2002-05-28
IT2002MI001148A ITMI20021148A1 (it) 2002-05-28 2002-05-28 Metodo per la determinazione della funzionalita' mitocondriale in cellule isolate e relativo kit

Publications (2)

Publication Number Publication Date
WO2003100418A2 true WO2003100418A2 (fr) 2003-12-04
WO2003100418A3 WO2003100418A3 (fr) 2004-01-29

Family

ID=11449988

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IT2003/000238 WO2003100418A2 (fr) 2002-05-28 2003-04-15 Methode et trousse de mesure de l'activite mitochondriale dans des cellules isolees

Country Status (3)

Country Link
AU (1) AU2003230208A1 (fr)
IT (1) ITMI20021148A1 (fr)
WO (1) WO2003100418A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110517776A (zh) * 2019-08-16 2019-11-29 四川大学华西医院 一种老年人健康风险评估方法及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4273869A (en) * 1979-04-16 1981-06-16 The Regents Of The University Of Minnesota Cystic fibrosis detection method
EP1046714A2 (fr) * 1999-04-20 2000-10-25 Universita' degli studi di Bologna Une méthode et trousse pour mesurer l'activité mitochondriale
US6232060B1 (en) * 1996-01-19 2001-05-15 Galileo Laboratories, Inc. Assay system for anti-stress agents

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4273869A (en) * 1979-04-16 1981-06-16 The Regents Of The University Of Minnesota Cystic fibrosis detection method
US6232060B1 (en) * 1996-01-19 2001-05-15 Galileo Laboratories, Inc. Assay system for anti-stress agents
EP1046714A2 (fr) * 1999-04-20 2000-10-25 Universita' degli studi di Bologna Une méthode et trousse pour mesurer l'activité mitochondriale

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CUI LIXIN ET AL: "Cellular and Molecular Events Leading to Mitochondrial Toxicity of 1-(2-Deoxy-2-Fluoro-1-beta-D-Arabinofurano syl)-5-Iodouracil in Human Liver Cells" JOURNAL OF CLINICAL INVESTIGATION, vol. 95, no. 2, 1995, pages 555-563, XP008024304 ISSN: 0021-9738 *
D'AURELIO MARILENA ET AL: "Decreased Pasteur effect in platelets of aged individuals" MECHANISMS OF AGEING AND DEVELOPMENT, vol. 122, no. 8, June 2001 (2001-06), pages 823-833, XP001172496 ISSN: 0047-6374 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110517776A (zh) * 2019-08-16 2019-11-29 四川大学华西医院 一种老年人健康风险评估方法及其应用
CN110517776B (zh) * 2019-08-16 2023-06-16 四川大学华西医院 一种老年人健康风险评估方法及其应用

Also Published As

Publication number Publication date
AU2003230208A8 (en) 2003-12-12
ITMI20021148A0 (it) 2002-05-28
AU2003230208A1 (en) 2003-12-12
WO2003100418A3 (fr) 2004-01-29
ITMI20021148A1 (it) 2003-11-28

Similar Documents

Publication Publication Date Title
Anderson et al. A specific enzymatic assay for the diagnosis of congenital galactosemia: I. The consumption test
Barrientos et al. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays
Roth et al. The quantitative determination of galactose—an enzymic method using galactose oxidase, with applications to blood and other biological fluids
Lanza et al. Functional assessment of isolated mitochondria in vitro
Vigue et al. Isozyme variability in species of the genus Drosophila. VI. Frequency-property-environment relationships of allelic alcohol dehydrogenases in D. melanogaster
Zhang et al. A method for determination of pyridine nucleotides using a single extract
Tulpule et al. Primary cultures of astrocytes and neurons as model systems to study the metabolism and metabolite export from brain cells
Carrieri et al. Renewable hydrogen production by cyanobacteria: nickel requirements for optimal hydrogenase activity
Lippi et al. A new colorimetric ultramicromethod for serum glutamicoxalacetic and glutamic-pyruvic transaminase determination
Blake et al. A colorimetric assay for the measurement of D-glucose consumption by cultured cells
US20010006794A1 (en) Screening for modulators of biomolecules
Outlaw Jr Critical examination of the quantitative evidence for and against photosynthetic CO2 fixation by guard cells
Carnicer et al. Quantitative metabolomics of the thermophilic methylotroph Bacillus methanolicus
Van Bergen et al. Oxidative phosphorylation measurement in cell lines and tissues
Byer et al. Mechanistic studies of radical SAM enzymes: pyruvate Formate-Lyase activating enzyme and lysine 2, 3-Aminomutase case studies
Ke et al. A new spectrophotometric assay for measuring pyruvate dehydrogenase complex activity: a comparative evaluation
Seigneurin-Berny et al. Percoll-purified and photosynthetically active chloroplasts from Arabidopsis thaliana leaves
Heijnen et al. Metabolic flux control analysis of branch points: an improved approach to obtain flux control coefficients from large perturbation data
Takahashi et al. γ-glutamyl transpeptidase and glutathione in aging IMR-90 fibroblasts and in differentiating 3T3 L1 preadipocytes
Kepka‐Lenhart et al. Determination of mammalian arginase activity
Singh et al. Evaluation of biomass
Merlo-Pich et al. Methods to detect mitochondrial function
Kouzuma et al. An enzymatic cycling method for the measurement of myo-inositol in biological samples
WO2003100418A2 (fr) Methode et trousse de mesure de l'activite mitochondriale dans des cellules isolees
Dewey et al. Evidence for the absence of the urea cycle in Tetrahymena

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP