WO2003069344A1 - Composition anti-glycolytique - Google Patents

Composition anti-glycolytique Download PDF

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Publication number
WO2003069344A1
WO2003069344A1 PCT/GB2003/000650 GB0300650W WO03069344A1 WO 2003069344 A1 WO2003069344 A1 WO 2003069344A1 GB 0300650 W GB0300650 W GB 0300650W WO 03069344 A1 WO03069344 A1 WO 03069344A1
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WO
WIPO (PCT)
Prior art keywords
tissue sample
glyceraldehyde
composition according
glucose
composition
Prior art date
Application number
PCT/GB2003/000650
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English (en)
Inventor
Carel Wynand Le Roux
Stephen P. Wilkinson
Bruce Ronald Muller
Jamshid Alaghband-Zadeh
Darrell Vincent Pavitt
Original Assignee
Ic Innovations Ltd
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 Ic Innovations Ltd filed Critical Ic Innovations Ltd
Priority to AU2003208411A priority Critical patent/AU2003208411A1/en
Publication of WO2003069344A1 publication Critical patent/WO2003069344A1/fr

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    • 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
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen

Definitions

  • the present invention relates to the field of glycolysis inhibition. More specifically, the invention relates to a composition for inhibiting glycolysis, especially in tissue samples, such as blood.
  • Glycolysis otherwise known as the Embden-Meyerhof pathway, refers to the series of biochemical reactions in which glucose is broken down to pyruvate with the release of usable energy in the form of ATP.
  • One molecule of glucose undergoes two phosphorylation reactions and is then split to form two triose phosphate molecules. Each of these is converted to pyruvate.
  • the net energy yield is two ATP molecules per glucose molecule.
  • pyruvate enters the Krebs cycle.
  • oxygen is in short supply or absent, the pyruvate is converted to various products by anaerobic respiration.
  • Other simple sugars such as fructose and galactose, and glycerol (from fats) enter the glycolysis pathway at intermediate stages.
  • the present invention seeks to provide new compositions that are capable of inhibiting glycolysis and which alleviate some of the problems associated with prior art glycolysis inhibitors.
  • the invention provides a composition comprising (i) glyceraldehyde, or a mimetic thereof; (ii) a glycolytic inhibitor; and (iii) an anti- coagulating agent. Said compositions are capable of exhibiting glycolytic inhibitory effects.
  • compositions are capable of exhibiting superior antiglycolytic effects compared to compositions known in the art.
  • the combination of glyceraldehyde, a glycolytic inhibitor and an anti-coagulating agent exhibits unexpectedly high anti-glycolytic activity compared to the prior art combination of sodium fluoride and potassium oxalate, and the use of glyceraldehyde alone. This finding is clearly illustrated by the experimental data provided in the accompanying
  • said glyceraldehyde is L/D-glyceraldehyde.
  • said glyceraldehyde is L-glyceraldehyde.
  • the composition of the invention comprises a mimetic of glyceraldehyde.
  • mimetic relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as glyceraldehyde.
  • Suitable glyceraldehyde mimetics include dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, pyruvate and glycerate dihydroxyacetate.
  • composition of the invention may also comprise one or more anti-coagulating agents.
  • the anti-coagulating agent is the salt of an oxalic acid.
  • compositions of the invention inhibit glycolysis equally well in the absence of an anti-coagulating agent. On the whole though, it is highly preferable that such a component is present.
  • the anti-coagulating agent is potassium oxalate or sodium oxalate.
  • EDTA ethylene diamine tetraacetic acid
  • said glycolytic inhibitor is selected from sodium fluoride and sodium iodoacetate.
  • the ratio of glyceraldehyde (or mimetic thereof): glycolytic inhibitor: anti-coagulating agent will vary depending on the exact choice of reagents.
  • the ratio of sodium fluoride: potassium oxalate: glyceraldehyde is typically 5 mg: 4 mg: lmg per ml of blood. Alternatively, this may be expressed as a molar ratio of 119 mMol: 22 mMol: 11 mMol.
  • the ratio of sodium iodoacetate: potassium oxalate: glyceraldehyde is typically 3.5 mg: 4 mg: 2 mg, or a molar ratio of 17 mMol: 22 mMol: 11 mMol.
  • the composition is an admixture.
  • the invention relates to the use of a composition according to the invention to inhibit glycolysis in a tissue sample.
  • the invention provides a method of preserving glucose levels in a tissue sample comprising:
  • the invention provides an additive for preserving glucose levels in a tissue sample, wherein said additive comprises a composition according to the invention.
  • the tissue sample is a fluid tissue, more preferably a blood sample.
  • the invention provides an assay for detecting the level of glucose in a tissue sample comprising: (a) preparing a tissue sample;
  • the invention provides a kit for preserving glucose levels in a tissue sample, said kit comprising (i) glyceraldehyde, or a mimetic thereof; (ii) a glycolytic inhibitor; and (iii) an anti-coagulating agent.
  • Example 2 The second experiment compared the combination of NaF + K 2 C 2 ⁇ and GA as anti- glycolytic agents.
  • One millilitre of venous blood from one individual was aliquoted into each tube.
  • One tube from each set was centrifuged at 3 minutes, 4 hours and 24 hours respectively. After separation all samples were stored at 4°C and then analysed in 3 consecutive runs on one analyser and the means calculated.
  • the third experiment compared NaF + K2C2O 4 , and the combination of NaF + 2 C 2 O 4 + GA as anti-glycolytic agents over 11 time points covering a 48 hour period. A millilitre of blood from one individual was used and at each time point the relevant sample was centrifuged after which all the plasma was stored at 4 °C and then analysed in 3 consecutive runs on one analyser and the means calculated. This experiment was then repeated at a higher level of plasma glucose.
  • Example 4 The fourth experiment compared different concentrations of NaF and K 2 C 2 O-1, while the concentration of GA was kept constant. This was achieved by adding increasing amounts (11 ⁇ mol, 22 ⁇ mol, 33 ⁇ mol, 44 ⁇ mol) of GA to a constant mass of NaF and K 2 C 2 O 4 . Increasing volumes of blood (lmL, 2mL, 3mL, 4mL) were added. This was done to keep GA constant at 1 lmmol/L, while diluting the NaF and K 2 C 2 O . One tube from each set was centrifuged at 3 minutes, 4 hours and 24 hours respectively. After separation all samples were stored at 4°C and then analysed in 3 consecutive runs on one analyser and the means calculated.
  • Example 5 The fifth experiment compared whole blood with no additives, NaF + K 2 C 2 ⁇ , GA, GA + NaF, NaF + K 2 C 2 0 4 + GA, NaF + lithium heparin + GA.
  • the experiment was planned to demonstrate glucose values at time points 0, 1, 2, 4, 8 and 24 hours. At each time point duplicate samples were obtained.
  • Example 6 The sixth experiment compares the effect of whole blood with NaF + sodium oxalate, GA + NaF, GA + NaF + lithium heparin, GA + citrate, GA + citrate + NaF, GA + EDTA, GA + NaF + EDTA.
  • Example 7 The seventh experiment compares the effect of whole blood with NaF + sodium oxalate, NaF + GA, sodium iodide + GA, sodium iodide + GA + EDTA, sodium iodide + GA + citrate.
  • the reagents in Examples 6 and 7 were used in the following amounts: GA: 35 ml of 310 mmol/L (11 mmol); NaF: 5 mg, sodium iodoacetate: 350 mg; sodium citrate: 4.5 ml; EDTA: 3 ml; lithium heparin, sodium oxalate.
  • the sodium citrate and EDTA were sub-optimally freeze-dried.
  • Example 1 demonstrated that the combination of NaF + K 2 C 2 ⁇ 4 + GA gives the best anti-glycolytic results (Table 1).
  • Table 1 Mean glucose (mmol/L) at different concentrations of GA alone or in combination with antiglycolytic agents.
  • Example 2 compares GA with NaF + K 2 C 2 ⁇ 4 . It demonstrates that neither inhibits glycolysis completely. (Table 2)
  • Table 2 Mean glucose (mmol/L) for different anti-glycolytic agents.
  • Example 3 demonstrates the remarkable preservation of the initial glucose level when the combination of NaF, K 2 C 2 O and GA is used. This combination is also markedly better than the current state of the art, a combination of NaF and K 2 C 2 ⁇ 4 (Tables 3 and 4).
  • Example 4 demonstrates that provided the concentration of GA remains at 11 mmol/L, reducing the concentrations of NaF and K 2 C O 4 does not seem to influence the anti-glycolytic effect of the combination (Table 5).
  • Example 5 demonstrates that the combination of NaF, GA and an anticoagulant gives the best anti-glycolytic results (Tables 6 and 7).
  • Glycolysis is not completely inhibited by the conventional glycolysis inhibitors, such as NaF and K 2 C 2 ⁇ 4 .
  • the loss of measurable glucose is significant as it impairs the ability of a clinician to exclude the diagnosis of impaired fasting glucose or even diabetes mellitus with confidence.
  • the combination of NaF + GA and an anticoagulant such as lithium heparin or K 2 C 2 ⁇ inhibits glycolysis to such an extent that the glucose value obtained from the sample left at room temperature and only centrifuged after 24 hours is no different from the values from the samples that were centrifuged 3 minutes and 4 hours after venepuncture.
  • the glucose value obtained from this combination is also the same as that from the sample with no additives that was centrifuged at 3 minutes.
  • Example 4 (Table 5) demonstrates that by keeping the concentration of GA constant and reducing the NaF and K 2 C 2 O 4 concentration by as much as 4-fold, no difference in the anti-glycolytic effect can be detected.
  • the results from Examples 6 and 7 (Tables 8 and 9 respectively) illustrate that anticoagulants other than oxalate salts (for example, EDTA, lithium heparin and sodium citrate), are also effective in the combination of the invention.
  • the above results demonstrate that the combination of NaF, GA and an anticoagulant is far superior to the conventional combination of NaF and K 2 C 2 O 4 .
  • the combination of NaF, GA and an anticoagulant does not allow any glycolysis to take place; thus, a sample analysed at 48 hours gives the same results as a sample analysed within 3 minutes.
  • the new anti-glycolytic composition should therefore be ideal for epidemiological studies especially in places where immediate centrifugation or analysis is not an option.
  • the new anti- glycolytic composition will also be welcomed in routine laboratories as it will increase the reliability of plasma glucose analysis.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Neurosurgery (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention a trait à une composition contenant (i) un glycéraldéhyde, ou un mimétique de ce dernier ; (ii) un inhibiteur glycolytique ; et (iii) un agent anti-coagulant. L'invention se rapporte également à l'utilisation de ladite composition pour inhiber la glycolyse dans un échantillon tissulaire. Dans d'autres aspects, l'invention concerne un dosage visant à détecter le niveau de glucose présent dans un échantillon tissulaire, et une trousse permettant de maintenir les niveaux de glucose dans un échantillon tissulaire.
PCT/GB2003/000650 2002-02-12 2003-02-12 Composition anti-glycolytique WO2003069344A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003208411A AU2003208411A1 (en) 2002-02-12 2003-02-12 Anti-glycolytic composition

Applications Claiming Priority (2)

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GB0203280A GB0203280D0 (en) 2002-02-12 2002-02-12 Anti-glycolytic composition
GB0203280.3 2002-02-12

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WO2003069344A1 true WO2003069344A1 (fr) 2003-08-21

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GB (1) GB0203280D0 (fr)
WO (1) WO2003069344A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7390663B2 (en) 2005-02-23 2008-06-24 Streck, Inc. Process, composition and kit for providing a stable whole blood calibrator/control
US20110111410A1 (en) * 2009-11-09 2011-05-12 Streck, Inc. Stabilization of rna in intact cells within a blood sample
US20130034860A1 (en) * 2009-02-18 2013-02-07 Streck, Inc. Preservation of cell-free rna in blood samples
US9956281B2 (en) 2011-05-04 2018-05-01 Streck, Inc. Inactivated virus compositions and methods of preparing such compositions
US10091984B2 (en) 2013-07-24 2018-10-09 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US10568317B2 (en) 2015-12-08 2020-02-25 Biomatrica, Inc. Reduction of erythrocyte sedimentation rate
US10772319B2 (en) 2014-06-10 2020-09-15 Biomatrica, Inc. Stabilization of thrombocytes at ambient temperatures
US10966421B2 (en) 2002-10-16 2021-04-06 Streck, Inc. Method and device for collecting and preserving cells for analysis
US11168351B2 (en) 2015-03-05 2021-11-09 Streck, Inc. Stabilization of nucleic acids in urine
US11299764B2 (en) 2015-11-20 2022-04-12 Streck, Inc. Single spin process for blood plasma separation and plasma composition including preservative
US11506655B2 (en) 2016-07-29 2022-11-22 Streck, Inc. Suspension composition for hematology analysis control
US11634747B2 (en) 2009-01-21 2023-04-25 Streck Llc Preservation of fetal nucleic acids in maternal plasma

Citations (3)

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Publication number Priority date Publication date Assignee Title
US3847738A (en) * 1971-11-01 1974-11-12 American Hospital Supply Corp Blood collection and preservation unit
JPS63173966A (ja) * 1987-01-14 1988-07-18 Sekisui Chem Co Ltd 血糖測定用添加剤
EP0696643A1 (fr) * 1994-08-09 1996-02-14 Becton, Dickinson and Company Appareil pour inhiber le glycolyse dans des échantillons de sang

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3847738A (en) * 1971-11-01 1974-11-12 American Hospital Supply Corp Blood collection and preservation unit
JPS63173966A (ja) * 1987-01-14 1988-07-18 Sekisui Chem Co Ltd 血糖測定用添加剤
EP0696643A1 (fr) * 1994-08-09 1996-02-14 Becton, Dickinson and Company Appareil pour inhiber le glycolyse dans des échantillons de sang

Non-Patent Citations (3)

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Title
LANDT MICHAEL: "Glyceraldehyde preserves glucose concentrations in whole blood specimens.", CLINICAL CHEMISTRY, vol. 46, no. 8 Part 1, August 2000 (2000-08-01), pages 1144 - 1149, XP002242605, ISSN: 0009-9147 *
PATENT ABSTRACTS OF JAPAN vol. 012, no. 451 (P - 791) 28 November 1988 (1988-11-28) *
STAHL M ET AL: "Optimization of preanalytical conditions and analysis of plasma glucose. 1. Impact of the new WHO and ADA recommendations on diagnosis of diabetes mellitus.", SCANDINAVIAN JOURNAL OF CLINICAL AND LABORATORY INVESTIGATION, vol. 61, no. 3, May 2001 (2001-05-01), pages 169 - 180, XP009011240, ISSN: 0036-5513 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11647743B2 (en) 2002-10-16 2023-05-16 Streck Llc Method and device for collecting and preserving cells for analysis
US10966421B2 (en) 2002-10-16 2021-04-06 Streck, Inc. Method and device for collecting and preserving cells for analysis
US7390663B2 (en) 2005-02-23 2008-06-24 Streck, Inc. Process, composition and kit for providing a stable whole blood calibrator/control
US11634747B2 (en) 2009-01-21 2023-04-25 Streck Llc Preservation of fetal nucleic acids in maternal plasma
US11761025B2 (en) 2009-02-18 2023-09-19 Streck Llc Preservation of cell-free nucleic acids
US8586306B2 (en) * 2009-02-18 2013-11-19 Streck, Inc. Preservation of cell-free RNA in blood samples
US20180216165A1 (en) 2009-02-18 2018-08-02 Streck, Inc. Preservation of cell-free nucleic acids
US20130034860A1 (en) * 2009-02-18 2013-02-07 Streck, Inc. Preservation of cell-free rna in blood samples
US10144955B2 (en) 2009-02-18 2018-12-04 Streck, Inc. Methods for preservation of cell-free nucleic acids
US10294513B2 (en) 2009-02-18 2019-05-21 Streck, Inc. Preservation of cell-free nucleic acids
US9926590B2 (en) 2009-02-18 2018-03-27 Streck, Inc. Devices and compositions for preservation of cell-free nucleic acids
US10689686B2 (en) 2009-02-18 2020-06-23 Streck, Inc. Preservation of cell-free nucleic acids
US20110111410A1 (en) * 2009-11-09 2011-05-12 Streck, Inc. Stabilization of rna in intact cells within a blood sample
US9956281B2 (en) 2011-05-04 2018-05-01 Streck, Inc. Inactivated virus compositions and methods of preparing such compositions
US10674721B2 (en) 2013-07-24 2020-06-09 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US10091984B2 (en) 2013-07-24 2018-10-09 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US11547111B2 (en) 2013-07-24 2023-01-10 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
US10772319B2 (en) 2014-06-10 2020-09-15 Biomatrica, Inc. Stabilization of thrombocytes at ambient temperatures
US11168351B2 (en) 2015-03-05 2021-11-09 Streck, Inc. Stabilization of nucleic acids in urine
US11299764B2 (en) 2015-11-20 2022-04-12 Streck, Inc. Single spin process for blood plasma separation and plasma composition including preservative
US10568317B2 (en) 2015-12-08 2020-02-25 Biomatrica, Inc. Reduction of erythrocyte sedimentation rate
US11116205B2 (en) 2015-12-08 2021-09-14 Biomatrica, Inc. Reduction of erythrocyte sedimentation rate
US11506655B2 (en) 2016-07-29 2022-11-22 Streck, Inc. Suspension composition for hematology analysis control

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AU2003208411A1 (en) 2003-09-04

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