NZ510471A - Methods of determining fructosamine oxidase activity in blood plasma of patients predisposed to diabetes - Google Patents

Abstract

Methods whereby, by reference to fructosamine oxidase activity in blood plasma of a patient or patients, the risk of diabetes associated vascular complications can be assessed. Candidate fructosamine oxidase inhibitors and/or antagonists can be identified or tested and the inhibition and/or antagonism of the fructosamine oxidase inhibition and/or antagonism of a patient are assessed. The graph depicts fructosamine oxidase activity that was measured in non-diabetic sera and the results were compared with serum fructosamine concentrations.

Description

510471 WO 00/18891 PCT/NZ99/00160 FRUCTOSAMINE OXIDASE ASSAY: METHODS AND MATERIALS THE CURRENT INVENTION The present invention relates to methods and materials for the assay of 5 fructosamine oxidase enzyme in patients and particularly but not solely those predisposed to or with diabetes mellitus.

Diabetes mellitus is a common disease characterised by serious long-term vascular complications. Diabetic individuals have a 25-fold increase in the risk of blindness, a 20-fold increase in te risk of renal failure, a 20-fold increase in the risk of 10 amputation as a result of gangrene, and a 2- to 6-fold increased risk of coronary artery disease and ischemic brain damage. See, Klein R, Klein B, Davis M, DeMets D. Diabetes Care 8:311-5 (1985). Almost half those diagnosed as diabetic before the age of 31 years, die before they reach 50 years largely as a result of cardiovascular or renal complications, often with many years of crippling and debilitating disease beforehand. 15 See, Deckert T, Poulsen J, Larsen M. Diabetologia 14:363-70 (1978).

Elevated blood glucose levels are now regarded as causative of diabetic complications based on results of the Diabetes Complications and Control Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS). See, The Diabetes Control and Complications Trial Research Group. NEngJMed. 379:977-85 (1993) and 20 Lancet 352:837-53 (1998). The DCCT and the UKPDS have both demonstrated that the development of complications of diabetes are related with degree of hyperglycaemia and that long-term outcome may be ameliorated by rigorous treatment. After controlling for current HbAlc levels, the development of micro vascular complications in DCCT patients was strongly correlated with the degree ofnonenzymatic glycation of structural 25 proteins such of skin collagen, but not with advanced glycation end product (AGE) markers such as pentosidine, carboxymethylysine, and tissue fluorescence (V Monnier -personal communication). These findings imply that the nonenzymatic glycation of tissue proteins has greater pathophysiological importance than AGE formation.

Many of the features of diabetic vascular disease may also be attributed to 30 oxidative stress, defined as an increase in the steady-state level of reactive oxygen or oxygen radicals in abiological system See, Baynes JW. Diabetes 40;405-12 (1991). For WO 00/18891 PCT/NZ99/00160 example, superoxide anions increase intracellular calcium which modulates the activity of nitric oxide synthase in the endothelium. Nitric oxide is a potent vasodilator and it has been implicated in the vascular dysfunction of early diabetes See, Ido Y, Kilo C, Williamson JR. Nephrol Dial Transplant 11 Suppl 5:72-5 (1996). Reactive oxygen species precipitate a drastic dose-dependent decrease in de novo synthesis of heparan sulphate proteoglycans leading to a reduction in anionic sites on the basement membrane and to an increased permeability to positively charged proteins such as albumin See, Kashira N, Watanabe Y, Makin H, Wallner EI, & Kanwar YS. Proc Natl Acad Sci USA 89:6309-13 (1992). Such leaky capillaries manifest clinically as background retinopathy and microalbuminuria. Microalbuminuria, in turn, is a recognised risk factor both for diabetic nephropathy in IDDM and for coronary artery disease and sudden death in elderly NIDDM See, Mogensen CE, Christensen CK. N Eng J Med 311;89-93 (1984) & Mogensen CE, Damsgaard EM, Froland A, et al Clin Nephrol 38 (suppl l);s28-39 (1992).

Once natural anti-oxidant defences are exceeded, there is the potential for hydroxyl radical generation from superoxide via a copper catalysed Haber-Weiss reaction See, Halliwell B & Gutteridge JMC "Free radicals in Biology and Medicine" Clarendon Press, Oxford (pp. 136-76 1989). Hydroxyl radicals are extremely reactive species that cause serious site-specific damage.

Oxygen radicals have also been implicated in the oxidative modification of low density lipoprotein (LDL) See, Witztum JL. Br Heart J 69; S12-S18 (1993). Oxidised LDL is a specific risk factor for atherosclerosis, binding with a scavenger receptor on tissue macrophages leading to the formation of foam cells and to cholesteryl ester accumulation in the intimal fatty streak, a feature of atheromatous plaque formation.

To date, the source of the oxidative stress in diabetes has not been identified. I have isolated a novel extracellular enzyme which catalyses the elimination of fructosamines from glycated protein. The existence of this enzyme has not previously been recognised in the world literature. The reaction is important because fructosamine is the precursor of all the Maillard products. Based on its high specificity for glycated protein substrates and its use of oxygen as acceptor, the enzyme may be classified as fructosamine oxidase 1.5.3 See, Enzyme nomenclature, Recommendations of the WO 00/18891 PCT/NZ99/00160 Nomenclature Committee of the International Union of Biochemistry, Academic Press, London pp. 19-22, (1979). Fructosamine oxidase is a metalloenzyme with copper and quinone cofactors. Reaction products are free unglycated protein, a-dicarbonyl sugar, and superoxide (Figure 1).

SUMMARY OF THE INVENTION The existence of the fructosamine oxidase enzyme has not previously been recognised in the world literature. This is a novel enzyme. The present invention relates to methods of monitoring fructosamine oxidase inhibition and/or antagonism of 10 patients, methods for testing or identifying fhictosamine oxidase inhibitors, methods of screening patients to determine patients at risk to vascular (particularly microvascular) damage and methods of identifying those individuals who will benefit by treatment with fhictosamine oxidase inhibitors and/or antagonists, methods of determining fructosamine oxidase levels in a mammal, methods of detennining blood plasma 15 fructosamine oxidase levels in a diabetic individual or a suspected individual, methods of assaying blood serum or blood plasma in vitro for fructosamine oxidase and to related methods and procedures.

In one aspect the invention consists in a method of detennining fructosamine oxidase activity in blood plasma of mammalian patients or a mammalian patient to 20 determine patients or a patient at risk to vascular damage, which method comprises determining the levels of fructosamine oxidase and/or the superoxide reaction product of fructosamine oxidase and/or any other oxygen free radical product of fructosamine oxidase in the population of patients andmaking the determination dependant upon such levels.

Preferably the patients are humans suffering from or predisposed to diabetes.

Preferably said fructosamine oxidase activity is measured in blood taken from each patient.

Preferably the measurement conducted in vitro is of the superoxide reaction product or any other oxygen free radical product of fructosamine oxidase. 30 Preferably at risk patients are or an at risk patient is then treated inter alia to inhibit and/or to antagonise fructosamine oxidase.

WO 00/18891 PCT/NZ99/00160 Accordingly, in another aspect the present invention consists in a method of screening mammalian patients (preferably humans suffering from or predisposed to diabetes) to determine patients at risk to vascular (particularly microvascular) damage, which method comprises determining the levels of fructosamine oxidase and/or the superoxide reaction product (or any other oxygen free radical product) of fructosamine oxidase in the population of patients and making the determination dependant upon such levels.

Preferably said screening is of blood taken from each patient.

Preferably the measurement conducted in vitro is of the superoxide reaction product (or any other oxygen free radical product) of fructosamine oxidase.

Preferably at risk patients are then treated inter alia to inhibit and/or to antagonise the fructosamine oxidase.

Preferably the procedure is substantially as hereinafter described.

In still a further aspect, the present invention consists in a method of identifying those individuals who will benefit by treatment with fructosamine oxidase inhibitors and/or antagonists, which method comprises testing an individual or a group of individuals for fructosamine oxidase in their blood directly or by reference to the superoxide reaction product (or any other oxygen free radical product) of fructosamine oxidase.

Preferably at risk patients are then treated inter alia to inhibit and/or to antagonise the fructosamine oxidase.

Preferably the procedure is substantially as hereinafter described.

In still a further aspect, the present invention consists in a method of monitoring fructosamine oxidase inhibition and/or antagonism of a patient which comprises or includes testing (directly or indirectly) the fructosamine oxidase level of such patient.

Preferably such testing is by reference to the superoxide reaction product (or any other oxygen free radical product of fructosamine oxidase) in the blood of the patient.

Preferably each of the methods involves a determination of a particular level attributed to fructosamine oxidase and/or the reaction products referred to in comparison to such level or levels of a patient or patients (as the case may be) who is or are not at risk to such vascular damage, or will not benefit by treatment with fructosamine oxidase inhibitors and/or antagonists or have no need for fructosamine oxidase inhibition and/or antagonism.

In another aspect the invention consists in a method of testing and/or identifying fructosamine oxidase inhibitors or a fructosamine oxidase inhibitor which comprises measuring the effect a candidate inhibitors) has on one or more of the quinone co-factor, or the copper co-factor of fructosamine oxidase.

In yet another aspect the invention consists in a method of identifying a 10 candidate fructosamine oxidase inhibitors) for trial for the amelioration of diabetes induced vascular damage in a mammal which comprises testing such a substance for fructosamine oxidase ^ inhibition and/or antagonism and choosing to trial the substance where (i) it has a specifity for such an enzyme or its co-factors 15 and (ii) it has an effectiveness for such inhibition and/or antagonism at dosage levels not known to be toxic or contraindicated in such a mammal.

In still a further aspect the present invention consists in the measurement in vitro of the superoxide reaction product (and/or any other oxygen free radical product) of 20 fructosamine oxidase in the blood of a mammal by exploiting its reductant properties or its oxidant properties or by enzymatic means.

In one preferred form said measurement procedure involves (preferably at a pH 7 to 8 (most preferably at pH greater than 7.5)) the disabling of the superoxide scavenging mechanism (such as superoxide dismutase) (SOD) [ using potassium 25 cyanide or (more preferably) by pretreatment with antihuman CuZn SOD antisera] and then exposure [ by addition] to a suitable fructosamine oxidase substrate [ e,g glycated bovine serum albumin modified to eliminate copper chelating activity which might disable the fructosamine oxidase].

Preferably the measurement following from the preferred procedure described 30 involves a consideration [ measurement] of an absorbance change, chemiluminescent change, or some other characterising change in an indicator of the modified sample.

In still a'further aspect the present invention consists in a method of determining the fructosamine oxjdase levels in a mammal (human or non-human) which at least 'nisWcsfral Prcpsrty Ovrsc? of MZ *> / 'I "O */ tTisjf'U o"* C&lV&f- PCTINZ99/00160 includes procedures as previously set forth.

In still a further aspect the present invention consists in a method of determining blood plasma fructosamine oxidase levels in a diabetic individual or a suspected diabetic individual which comprises at least steps of a method as previously set forth.

In still a further aspect the present invention consists in a method of assaying blood serum or blood plasma in vitro (directly and/or indirectly) for fructosamine oxidase which involves at least one or more of the steps or procedures hereinbefore described and/or hereinafter described.

Preferably said fructosamine oxidase substrate is bovine serum albumin that has been modified to eliminate copper chelating activity which might disable fructosamine oxidase.

In still a further aspect the present invention consists in the use of a sample in accordance with the present invention for the purpose of any of the methods previously set forth.

The attention of the reader is drawn to my simultaneously filed PCI Application (claiming New Zealand priorities from NZ 332084, NZ 332079 and NZ 334471) in which there are disclosed a variety of procedures, methods, pharmaceutical compositions, dosage units etc. involving the use of fructosamine oxidase inhibition and/or antagonism in order to reduce vascular (preferably microvascular) damage to patients (particularly although not solely diabetic or suspected diabetic patients). Preferably any such inhibitor or antagonist is selected from the groups (i) copper chelating agents (eg: triethylenetetramine dihydrochioride, penicillamine, sar, diamsar, ethylenediamine tetraacetic acid, o phenanthroline and histidine) (iii) substrate analogues (eg: N-acetylcysteine, captropril and enalapril). (iii) hydraziiie compounds (eg: diaminoguanidine, hydralazine and WO 00/18891 PCT/NZ99/00160 carbidopa).

As used herein including the appended claims the term "and/or" means "and" or "or".

The full content of the simultaneously filed PCT International patent 5 specification is hereby included by way of cross reference.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a detailed reaction mechanism for the formation of fhictosamine and Maillard products from glucose and protein. Fructosamine oxidase degrades 10 fructosamine by a two-step reaction with initial release of an a-dicarbonyl sugar and subsequent oxidation of the enzyme/protein complex to release free unglycated protein. The reduced copper cofactor is oxidised in vivo by molecular oxygen and the oxidation product is superoxide.

Figure 2 shows the relationship between fructosamine oxidase measurements and 15 plasma fhictosamine. Linear regression equation (y = 0.0349x - 5.9589; r2 = 0.7455).

Figure 3 shows the effect of fructosamine oxidase inhibitors on enzyme activity in human blood plasma. The three inhibitors are selected, merely by way of example, from the three classes of compounds which inhibit the enzyme (i.e. captopril is a substrate analogue, carbidopa is a hydrazine compound, and potassium cyanide is a 20 copper chelator).

Detailed Description of the Invention (i) Assay principle Fructosamine oxidase catalyses the degradation of fructosamine(s) with 25 concurrent reduction of molecular oxygen yielding a superoxide reaction product (FIGURE 1). Superoxide is unstable in aqueous solution with spontaneous dismutation to hydrogen peroxide and oxygen. The dismutation reaction is strongly pH dependent with maximal reactivity in acidic solutions and reducing reactivity in alkaline solution. Therefore, enzyme activity is best determined atpH values 7.0-8.0 and preferably about 30 pH 7.5 where superoxide is more stable using one of the assay compounds listed in Table 1. -8-TABLE1 Assay compound Assay pH Type of reaction Reference Femcytochrome c 7.8 Reduction McCord J & Fridovich 1.

J Biol Chem 244;6087-93 (1969) Nitroblue tetrazolium 7.8 Reduction Hailiwell B FEBS Lett 72;8 (1976) Dichlorphenol indophenol 7.0 Reduction Greenstock CL & Ruddock GW.

Int J Radial Phys Chem 8;367 (1976) Epinephrine 7.8 Oxidation Misra HP & Fridovich I J Biol Chem 247;3170-5 (1972) Hydroxyiamine 7.8 Oxidation Elstner EF, Heupel A.

Anal Biochem 70:616-20 (1976) Peroxidase 7.8 Enzymatic Misra HP, Fridovich I Anal Biochem 79;553-60 (1977) NADH...LDH 7.0 Enzymatic Chan PC & Bielski BHJ.

J Biol Chem 249;1317-9 (1974) NADH...GDH 7.2 Enzymatic Chan PC & Bielski BHJ.

J Biol Chem 255:874-6 (1980) (ii) Interference Because superoxide is potentially a noxious substance, superoxide degrading enzyme, superoxide dismutase (SOD), is elaborated in plasma as a physiological response to increasing superoxide concentrations. Compared with healthy non-diabetic individuals, SOD levels are significantly elevated in the plasma of patients with diabetes mellitus and particularly amongst those patients with microvascular disease such as diabetic nephropathy and diabetic retinopathy. See, Mizobuchi N, Nakata H, Horimi T, Takahashi I. Rinsho 5yor/41;673-8 (1993). The major SOD isoenzyme in extracellular fluids like plasma is extracellular SOD which is a tetrameric glycoprotein that contains four copper atoms and four zinc atoms. See, Karlsson K & Marklund SL Biochem J 242;55-9 (1987). Unless it is disabled, such SOD activity will cause significant interference in any blood plasma assay based on the detection systems listed in Table 1.

Almost all of the SOD activity of human plasma is sensitive to inhibition with millimolar concentrations of potassium cyanide, sodium azide or sodium fluoride. Alternatively, SOD activity ofplasma may be eliminated by pre-treatment of the plasma sample with anti-human CuZn SOD antisera. See, Marklund SL, Holme E, Hellner L Clin ChimActa 126;41-51 (1982). (iii) Procedure Fructosamine oxidase activity may be measured using the redox-active colour reagent, ferricytochrome c, which is readily reduced by superoxide to form ferrocytochrome c with a characteristic increase in absorbance at 550nM (eJS0 = 22.1 5 mM'1 .cm'1). The reagent is 50mM TES buffer pH 7.4 containing 10^M cytochrome c (Sigma), and 50fiM fructosamine as glycated bovine serum albumin. The parameters for performance of die assay in a Cobas Bio (Roche) automated analyser are as shown in Table 2.

TABLE 2 PARAMETER LISTING 1 UNITS U/L 2 CALCULATION FACTOR 473.9 3 STANDARD 1 CONCENTRATION 0 4 STANDARD 2 CONCENTRATION 0 STANDARD 3 CONCENTRATION 0 6 LIMIT 0 7 TEMPERATURE [DEG.C] .0 8 TYPE OF ANALYSIS 6 9 WAVELENGTH [NM] 550 SAMPLE VOLUME [UL] 11 DILUENT VOLUME [UL] 45 12 REAGENT VOLUME [UL] 200 13 INCUBATION TIME [SEC] 300 14 START REAGENT VOLUME [UL] TIME OF FIRST READING [SEC] 0.5 16 TIME INTERVAL [SEC] 300 17 NUMBER OF READINGS 2 18 BLANKING MODE 1 19 PRINTOUT MODE 1 One unit of enzyme was defined as the amount which reduced 1 fimol/minute of cytochrome c in solution under the above assay conditions. The calculation factor is determined from the molar absorptivity for ferrocytochrome c (eSSOmn) according to the formula: U/L (nmol.min'.L"') = TV x 103 / e550 x SV where TV = total reaction volume SV = sample volume (iv) Materials _10_ Glycated bovine serum albumin substrate was prepared as follows: (a) Bovine serum albumin (BSA) (Sigma) was reduced with sodium borohydride to eliminate protein hydroperoxides. BSA (60g/L) was dissolved in 0.145M NaCl, pH was adjusted to 9.0 with molar NaOH, sodium borohydride (200mmol/L) was added, and the solution was stirred gently at room temperature for 24 hours. Excess sodium borohydride was discharged with glacial acetic acid and the solution was dialysed exhaustively against 0.145M NaCl at 4 °C. (b) Borohydride-reduced BSA was glycated by mixing protein solution with an equal volume 0.4M Na2P04 buffer pH 7.4 containing 50mM glucose and 0.02% sodium azide and incubating at 37 °C for 7 days. Excess glucose was removed by exhaustive dialysis against 0.145M NaCl. (c) Glycated BSA (gBSA) was acetylated by adding 0.2M iodoacetic acid, adjusting pH to 6.8, and incubating at room temperature for 24 hours. Excess iodoacetate was removed by exhaustive dialysis against 0.14SM NaCl. (d) Remaining copper binding sites on gBSA were saturated by dialysing against 0.145M NaCl containing lOOfiM copper sulphate. Excess copper was removed by exhaustive dialysis against 0.145M NaCl. (e) Degree of glycation of gBSA substrate was determined by fructosamine assay (Hoffmann La-Roche). (v) Substrate specificity The specificity of the assay for reactive oxygen species was tested by measuring the degree of inhibition of ferricytochrome c reduction after adding the following oxygen free radical scavengers to the reaction mixture: (a) Superoxide dismutase to selectively remove superoxide; (b) catalase to selectively remove hydrogen peroxide; & (iii) mannitol to scavenge hydroxyl radicals. Results are shown in Table 3.

TABLE 3 Free radical scavenger Enzyme activity* Significance (U/L) (P) Control 15.34 ±0.16 superoxide dismutase (20kU/L) 9.99 ± 0.03 <0.0001 catalase (1000kU/L) 12.23 ± 0.03 <0.0001 superoxide dismutase + catalase 6.7810.12 <0.0001 mannitol (50mmol/L) 14.96 ± 0.19 0.0421 • determined with free Tadical scavenger added to the reagent Results imply that the assay reaction is 10 measuring both superoxide and hydroxyl radicals formed from the reaction of superoxide with hydrogen peroxide. (v) Specificity Cytochrome c is a non-specific reductant and other reducing substances in sera or 15 anticoagulants added to the blood sample at specimen collection may interfer in the assay as shown in Table 4.

TABLE4 Additive* Activity compared with control (%) Control 100 Heparin (1000U/L) 24.4 EDTA(100mM) 26.3 • Human fructosamine oxidase analysed with and without (control) additive in the reagent (vi) Comparison with fructosamine concentrations Fructosamine oxidase activity was measured in non-diabetic sera and results were compared with serum fructosamine concentrations Figure 2. (vii) Identifying fructosamine oxidase inhibitors An important application of the current activity assay is as a means to identify potential fructosamine oxidase antagonists and inhibitors . Fructosamine oxidase inhibitors may be hydrazine compounds which bind and block the quinone co-factor, copper chelators which bind and block the copper co-factor, or substrate analogues which mimic the normal substrate of the enzyme. Micromolar amounts of candidate WO 00/18891 PCT/NZ99/00160 substance are added to the reaction mixture, and the decrease in fructosamine oxidase activity of a human plasma sample is measured. The inhibitory potential of carbidopa (hydrazine compound), potassium cyanide (copper chelator), and captopril (substrate analogue) are demonstrated in FIGURE 3.

The effectiveness of an enzyme inhibitor is usually expressed by a velocity constant (K) which determines the fraction of the enzyme inhibited in a given period of time by a certain concentration of inhibitor. The specificity of the inhibitor for the . active centre of the enzyme is indicated by the concentration of inhibitor causing 50% inactivation of the enzyme (IC50). Results of this in vitro assay would suggest that, at 10 lp.M inhibitor concentration, the most effective enzyme inhibitor is carbidopa (K = 15% per minute) followed by captopril (K = 2.6% per minute) followed potassium cyanide (K = 1.2% per minute). Carbidopa also shows the greatest specificity for the active centre offructosamine oxidase (IC50= 0.50|*M) compared with captopril (IC50 = 0.83 fiM) and potassium cyanide (ICS0 = 6.36jiM).

WO 00/18891 PCT/NZ99/00160

Claims (23)

WHAT WE CLAIM IS:

1. A method of determining fructosamine oxidase activity in blood plasma of mammalian patients or a mammalian patient to determine patients or a patient at risk to vascular damage, which method comprises determining the levels of fructosamine 5 oxidase and/or the superoxide reaction product of fructosamine oxidase and/or any other oxygen free radical product of fructosamine oxidase in the population of patients and making the determination dependant upon such levels.

2. A method of claim 1 wherein the patients are humans suffering from or predisposed to diabetes. 10

3. A method of claim 1 or 2 wherein said fructosamine oxidase activity is measured in blood taken from each patient.

4. A method of claim 3 wherein the measurement conducted in vitro is of the superoxide reaction product or any other oxygen free radical product of fructosamine oxidase. 15

5. A method of any one of the preceding claims wherein at risk patients are or an at risk patient is then treated inter alia to inhibit and/or to antagonise fructosamine oxidase.

6. A method of identifying those individuals who will benefit by treatment with fructosamine oxidase inhibitors and/or antagonists, which method comprises testing an 20 individual or a group of individuals for fructosamine oxidase in their blood directly or by reference to the superoxide reaction product of fructosamine oxidase or any other oxygen free radical product of fructosamine oxidase.

7. A method of claim 6 wherein at risk patients are then treated inter alia to inhibit and/or to antagonise the fructosamine oxidase. 25

8. A method of monitoring fructosamine oxidase inhibition and/or antagonism of a patient which comprises or includes testing directly or indirectly the fructosamine oxidase level of such patient.

9. A method of claim 8 wherein such testing is by reference to the superoxide reaction product of fructosamine oxidase any other oxygen free radical product of 30 fructosamine oxidase in the blood of the patient.

10. A method of any one of the preceding claims which involves a determination of a WO 00/13891 - 14 - PCTINZ99/00160 particular level attributed to fructosamine oxidase and/or the reaction products referred to in comparison to such level or levels of a patient or patients (as the case may be) 5 who is or are not at risk to such vascular damage, or will not benefit by treatment with fructosamine oxidase inhibitors and/or antagonists or have no need for fructosamine oxidase inhibition and/or antagonism.

11. A method of testing and/or identifying fructosamine oxidase inhibitors or a fructosamine oxidase inhibitor which comprises measuring the effect a candidate 10 inhibitors) has on one or more of the quinone co-factor, the copper co-factor, or at least one substrate analogue of fructosamine oxidase.

12. A method of identifying a candidate fructosamine oxidase inhibitors) for trial for the amelioration of diabetes induced vascular damage in a mammal which comprises testing such a substance for fructosamine oxidase inhibition and/or antagonism and choosing to trial the substance 15 where (ii) it has a specifity for such an enzyme, or its co-factors, and (iii) it has an effectiveness for such inhibition and/or antagonism at dosage levels not known to be toxic or contraindicated in such a mammal. 20

13. The measurement in vitro of the superoxide reaction product (and/or any other oxygen free radical product) of fmctosamine oxidase in the blood of a mammal by exploiting its reductant properties or its oxidant properties or by enzymatic means.

14. The measurement of claim 13 wherein the procedure involves the disabling of the superoxide scavenging mechanism (such as superoxide dismutase) (SOD) and then 25 exposure to a suitable fructosamine oxidase substrate.

15. The measurement of claim 14 wherein it is made at a pH of 7 to 8.

16. The measurement of claim 15 wherein the pH is at greater than 7.5.

17. The measurement of claim 16 wherein the disabling is by pre-treatment of the plasma sample with anti-human CuZn SOD antisera. 30

18. The measurement of claim 16 wherein the fructosamine oxidase substrate is bovine serum albumin that has been modified to eliminate copper chelating activity which might diasable the fructosamine oxidase.

19. The measurement of claim 17 which involves measurement of an absorbance Fii.. -?v J . wilt H . i*"- /Tt — <T IN • —7 W(|TKjO Ci" f- ■ 3 £03 WO 00/18891 -15- PCTINZ99/00160 change, chemiluminescent change, or some other characterising change in an indicator of the modified sample.

20. A method of determining the fhictosamine oxidase levels in a mammal which involves the measurement as claimed in claim 19.

21. A method of determining the blood plasma fructosamine oxidase levels in a diabetic individual or a suspected diabetic individual which involves the measurement procedure of claim 19.

22. A method of assaying blood serum or blood plasma in vitro directly and/or indirectly for fructosamine oxidase which involves the measurement procedure of claim 19.

23. A method of claim 11 or 12 which involves a method of any one of claims 13 to 22. END OF CLAIMS