WO2003069344A1

WO2003069344A1 - Anti-glycolytic composition

Info

Publication number: WO2003069344A1
Application number: PCT/GB2003/000650
Authority: WO
Inventors: Carel Wynand Le Roux; Stephen P. Wilkinson; Bruce Ronald Muller; Jamshid Alaghband-Zadeh; Darrell Vincent Pavitt
Original assignee: Ic Innovations Ltd
Priority date: 2002-02-12
Filing date: 2003-02-12
Publication date: 2003-08-21
Also published as: AU2003208411A1; GB0203280D0

Abstract

The present invention provides a composition comprising (i) glyceraldehyde, or a mimetic thereof; (ii) a glycolytic inhibitor; and (iii) an anti-coagulating agent. The invention also relates to the use of said composition to inhibit glycolysis in a tissue sample. Further aspects of the invention relate to an assay for detecting the level of glucose in a tissue sample, and a kit for preserving glucose levels in a tissue sample.

Description

ANTI-GLYCOLYTIC COMPOSITION

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. In aerobic respiration, pyruvate then enters the Krebs cycle. Alternatively, when 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 measurement of glucose levels in tissue samples is an important diagnostic tool in modern medicine. However, the World Health Organisation states that glucose concentrations should not be determined in plasma unless red blood cells are immediately removed, otherwise glycolysis will result in an unpredictable underestimation of the true . concentration. It is emphasised that glucose preservatives to date, do not totally prevent glycolysis. The WHO advises that if whole blood is obtained, the sample should be kept at 0-4 °C or centrifuged immediately, or assayed immediately.

It is known in the art that glycolysis can be partially inhibited by the use of sodium fluoride in combination with potassium oxalate. However, it is acknowledged that glycolysis continues with an accompanying loss in measurable glucose in the presence of this combination of reagents. As a result, this leads to patients being misdiagnosed (Chan A Y W, Cockram C S, Swaminathan R., Ann. Clin. Biochem, 1990; 27: 73-74). Thus, the issue of incorrect glucose measurements must receive more prominence, especially now more emphasis is being placed on the diagnosis of impaired fasting glucose and diabetes mellitus. This issue has been recognised for several years, but has not been successfully addressed yet in view of the practical problems it poses.

It is of course impractical to analyse all glucose samples immediately. Generally, it is accepted that a routine sample may take several hours to reach the laboratory, hence the importance of glycolysis inhibition (Meites S, Saniel-Banrey K., Clin. Chem, 1979; 25:531-534). Most general chemical pathology laboratories will deal with vast numbers of routine glucose specimens per annum and very few will be able to cope with analysing all of the samples urgently. Many of these samples will also come from general practitioners who are not located close to the laboratory. Samples are often transported over considerable distances and logistically this would be both difficult and expensive if there had to be a strict adherence to the WHO guidelines.

The prior art has demonstrated that sodium iodoacetate is capable of inhibiting glycolysis, but only for up to 2 hours when a sample is left uncentrifuged at room temperature (Kaplan LA, Gau N, Stein E., Clin Chem 1980;26: 175-176). Likewise, sodium fluoride in combination with potassium oxalate slows, but does not eliminate glycolysis or the production of lactate (Astles R, Williams CP, Sedor F, Clin Chem. 1994 Jul; 40: 1327-1330). It has also been shown that glucose values may fall by as much as 0.5 mmol/L after a 2-4 hour period (Chan A Y W et al, ibid). More recently, it has been reported that glyceraldehyde is also capable of acting as a glycolytic inhibitor (Landt M, Clin. Chem, 2000; 46: 1144-1149), although again, glycolysis is not completely eliminated.

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. Thus, in a first aspect, 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.

The present compositions are capable of exhibiting superior antiglycolytic effects compared to compositions known in the art. Surprisingly, 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

Examples.

In a preferred embodiment, said glyceraldehyde is L/D-glyceraldehyde.

In a more preferred embodiment, said glyceraldehyde is L-glyceraldehyde.

In one preferred embodiment, the composition of the invention comprises a mimetic of glyceraldehyde. As used herein, the term "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.

The composition of the invention may also comprise one or more anti-coagulating agents. Preferably, the anti-coagulating agent is the salt of an oxalic acid.

It is noteworthy that in certain specific embodiments of the invention, the 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.

In a particularly preferred embodiment, the anti-coagulating agent is potassium oxalate or sodium oxalate.

Other preferred anti-coagulating agents include lithium heparin, sodium citrate and ethylene diamine tetraacetic acid (EDTA).

Preferably, said glycolytic inhibitor is selected from sodium fluoride and sodium iodoacetate.

It will be apparent to the skilled person that the ratio of glyceraldehyde (or mimetic thereof): glycolytic inhibitor: anti-coagulating agent will vary depending on the exact choice of reagents. By way of example, 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. Likewise, 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.

In one preferred embodiment of the invention, the composition is an admixture.

In a second aspect, the invention relates to the use of a composition according to the invention to inhibit glycolysis in a tissue sample.

In a third aspect, the invention provides a method of preserving glucose levels in a tissue sample comprising:

(a) preparing a tissue sample;

(b) contacting said tissue sample with a composition according to the invention. In a fourth aspect, the invention provides an additive for preserving glucose levels in a tissue sample, wherein said additive comprises a composition according to the invention.

In said second, third and fourth aspects, preferably the tissue sample is a fluid tissue, more preferably a blood sample.

In a fifth aspect, the invention provides an assay for detecting the level of glucose in a tissue sample comprising: (a) preparing a tissue sample;

(b) contacting said tissue sample with a composition according to the invention; and

(c) measuring the level of glucose in said tissue sample.

In a sixth aspect, 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.

The invention will now be further described by way of example.

EXAMPLES

All glucose measurements were performed in the laboratory on a single Olympus AU600 automatic chemical analyser, using a hexokinase method. The coefficient of variation (CV) of glucose controls, with a target concentration of 3.48 mmol/L measured over a 3 month period was 3%. All additives were freeze dried to negate dilutional effects.

The following abbreviations are used: NaF (sodium fluoride), K₂C₂O₄ (potassium oxalate), GA (glyceraldehyde). Example 1

In the first experiment three types of vacutainers (no additives, lithium heparin and

NaF + K₂C₂θ₄) were used. Each set had 4 tubes containing increasing amounts of

GA. 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.

Example 2 The second experiment compared the combination of NaF + K₂C₂θ 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.

Example 3

The third experiment compared NaF + K2C2O₄, and the combination of NaF + ₂C₂O₄ + 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 K₂C₂O-1, while the concentration of GA was kept constant. This was achieved by adding increasing amounts (11 μmol, 22 μmol, 33 μmol, 44 μmol) of GA to a constant mass of NaF and K₂C₂O₄. 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 K₂C₂O . 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 + K₂C₂θ , GA, GA + NaF, NaF + K₂C₂0₄+ 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. One millilitre of venous blood from one individual was aliquoted into each tube (n=60). The two tubes from each set, were centrifuged at 3 minutes, 1 hour, 2 hours, 4 hours, 8 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 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.

RESULTS Example 1 demonstrated that the combination of NaF + K₂C₂θ₄ + 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.

The possibility that all the glucose measurements were within experimental error was evaluated. However, the observed differences in the tubes with GA alone and in combination with lithium heparin were found not to be attributable to instrumental imprecision.

Example 2 compares GA with NaF + K₂C₂θ₄. 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, K₂C₂O and GA is used. This combination is also markedly better than the current state of the art, a combination of NaF and K₂C₂θ₄ (Tables 3 and 4).

Example 4 demonstrates that provided the concentration of GA remains at 11 mmol/L, reducing the concentrations of NaF and K₂C O₄ does not seem to influence the anti-glycolytic effect of the combination (Table 5).

Table 5: Mean glucose (mmol/L) at different concentrations of component B

Example 5 demonstrates that the combination of NaF, GA and an anticoagulant gives the best anti-glycolytic results (Tables 6 and 7).

Table 6

Table 7

Glycolysis is not completely inhibited by the conventional glycolysis inhibitors, such as NaF and K₂C₂θ₄. 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. However, the combination of NaF + GA and an anticoagulant such as lithium heparin or K₂C₂θ 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.

It is interesting to confirm previous work using NaF and K₂C₂O₄ showing that 50% of the loss in measurable glucose that takes place over 24 hours occurs within the first 2 hours. Many samples will not have reached the laboratory or have been centrifuged before the expiry of 2 hours and therefore it becomes clear why so many misdiagnoses are possible. Again the combination of NaF, GA and an anticoagulant is far superior to NaF + K₂C₂O₄ (p < 0.001). The absolute values obtained from the samples centrifuged after 3 minutes, 24 and 48 hours are also the same.

Example 4 (Table 5) demonstrates that by keeping the concentration of GA constant and reducing the NaF and K₂C₂O₄ 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.

Table 8

Table 9

By way of summary, the above results demonstrate that the combination of NaF, GA and an anticoagulant is far superior to the conventional combination of NaF and K₂C₂O₄. Indeed, 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. In addition, the new anti- glycolytic composition will also be welcomed in routine laboratories as it will increase the reliability of plasma glucose analysis. Various modifications and variations of the described methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

Claims

1. A composition comprising (i) glyceraldehyde, or a mimetic thereof; (ii) a glycolytic inhibitor; and (iii) an anti-coagulating agent.

2. A composition according to claim 1 wherein the glyceraldehyde is L/D- glyceraldehyde.

3. A composition according to claim 1 wherein the glyceraldehyde is L- glyceraldehyde.

4. A composition according to claim 1 wherein the glyceraldehyde mimetic is selected from dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3- bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenol- pyruvate, pyruvate and glycerate dihydroxyacetate.

5. A composition according to any preceding claim wherein said anti- coagulating agent is the salt of an oxalic acid.

6. A composition according to any preceding claim wherein said anti- coagulating agent is potassium oxalate or sodium oxalate.

7. A composition according to any one of claims 1 to 4 wherein said anti- coagulating agent is selected from sodium citrate, EDTA and lithium heparin.

8. A composition according to any preceding claim wherein said glycolytic inhibitor is selected from sodium fluoride and sodium iodoacetate.

9. A composition according to any preceding claim wherein said composition is an admixture.

10. Use of the composition according to any one of claims 1 to 9 to inhibit glycolysis in a tissue sample.

11. Use according to claim 10 wherein said tissue sample is a fluid tissue.

12. Use according to claim 10 or claim 11 wherein said tissue sample is a blood sample.

13. A method of preserving glucose levels in a tissue sample comprising:

(a) preparing a tissue sample;

(b) contacting said tissue sample with a composition according to any one of claims 1 to 9.

14. A method according to claim 13 wherein said tissue sample is a fluid tissue.

15. A method according to claim 14 wherein said tissue sample is blood.

16. An additive for preserving glucose levels in a tissue sample, wherein said additive comprises a composition according to any one of claims 1 to 9.

17. An additive according to claim 16 wherein said tissue sample is a fluid tissue.

18. An additive according to claim 16 or 17 wherein said tissue sample is blood.

19. An assay for detecting the level of glucose in a tissue sample comprising:

(a) preparing a tissue sample;

(b) contacting said tissue sample with a composition according to any one of claims 1 to 9; and

(d)- measuring the level of glucose in said tissue sample.

20. 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.