Interference in glucose oxidase-peroxidase blood glucose methods

CLINICA CHIMICA ACTA

115

CCA 5117

INTERFERENCE

IN GLUCOSE OXIDASE-PEROXIDASE

BLOOD GLUCOSE

METHODS

P. SHARP

Defiartment ofBiochemistry, (Received

Royal Sussex County Hospital, Brighton, BNz

gBE (U.K.)

March 20, 1972)

SUMMARY

Interference by certain compounds with the glucose oxidase-peroxidase system used in blood glucose estimations appears to be a function of the oxygen affinity of the oxygen acceptor chromogen or the oxidative coupling system. It has been shown1 that tolazamide interferes seriously with the Boehringer GOD-PERID blood glucose method. By utilising other compounds with similar chemical groups, it was found possibleto identify the group in the tolazamide molecule that caused interference and consequently to predict the effects of other compounds on such blood glucose methods. A comparison of three chromogenic systems was also performed. These were guaiacum2, phenol-4-aminophenazone3 and the Boehringer z’2-diazo-di(3-ethyl benzthiazoline-6-sulphonic acid) (ABTS) S ome of the advantages and disadvantages of these three systems are discussed.

INTRODUCTION

Tolazamide has been shown to act as a competitive oxygen acceptorl. In the following study the chemical group which effects this interference has been identified by observing the interference caused by compounds containing similar groups. In doing this other drugs frequently used therapeutically were identified as potential sources of interference. By comparing the relative interferences produced by these compounds it was possible to identify other chemical groups which enhanced or decreased the ability of the molecule to bind oxygen. To provide a standard for comparison ascorbic acid, which is known to interfere with GOD-PERID method9, was examined under similar conditions. MATERIALS

AND METHODS

All drugs used were prepared from the pharmaceutical tablet and dissolved in either alcohol or water depending on solubility. The pH was adjusted to 7.0 in all cases before further dilution to the appropriate concentrations. Details of all drugs investigated except trypan blue are given by Clarke5. Information concerning trypan Cl&. Chim. Acta, 40 (1972) IIS-IZO

116

SHARP

blue is given by Busch and Lane6. Solutions graded drug concentrations were prepared by glucose standards. These solutions were used the estimation of glucose by the Boehringer (Part I).

containing IOO mg/Ioo ml glucose and diluting the solutions of each drug with to determine the effect of each drug on * GOD-PERID autoanalyser method

Peroxide substrate

This was prepared by diluting I volume of 30% w/v (IOO ~01s) hydrogen peroxide (A.R.) in IOOO volumes water. This is equivalent to about 160 mg/roo ml glucose assuming full conversion of glucose. Boehringer GOD-PERID

method

The auto-analyser method was used for part I of the investigation and utilised two reagents : (a) Glucose oxidase-peroxidase-ABTS reagent : prepared as recommended by the manufacturers and stored at 4 ‘. (b) Saline diluent : 9.0 g sodium chloride (A.R.) was dissolved in I 1 of deionised water and 0.5 ml Brij 35 added. Protein precipitation was only carried out in the manual method which was used during the reaction rate studies when comparing the three glucose oxidaseperoxidase methods (Part 2). Protein $recipitating

reagents:

(a) Copper sulphate-sodium sulphate reagent: 18 ml 7% w/v copper (CuSO, .5 H,O) was mixed with 192 ml 3% w/v sodium sulphate (Na,SO,. (b) Tungstate reagent: 10.0 g sodium tungstate (Na,WO, .z H,O) was in IOO ml deionised water. 0.1 ml sample was added to 1.7 ml copper sulphate-sodium sulphate 0.2 ml of tungstate was then added and mixed. After centrifugation 0.5 natant was used for analysis.

sulphate IO H,O). dissolved reagent. ml super-

Guaiacum method of Morley et aL2

(a) Acetate buffer pH 5.0, 0.105 M: 14.28 g sodium acetate trihydrate was dissolved in 800 ml deionised water. About 2.5 ml glacial acetic acid (A.R.) was added until the pH was 5.0. This was made up to I 1 with distilled water. (b) Stock peroxidase solution: peroxidase R.Z. = 1.0 (Hughes and Hughes)** 20 mg was dissolved in IOO ml of acetate buffer. This solution was stored at 4’. (c) Fermcozyme: Fermcozyme (653 AM) (Hughes and Hughes)“* 0.75 ml was diluted to IOO ml with acetate buffer and stored at 4’. (d) 1.5 g/Ioo ml Ethanolic guaiacum solution : the required weight of guaiacum powder, approximately 1.8 g calculated from the proportions of non-volatile and ethanol-insoluble components in the sample was shaken mechanically for I h with IOO ml ethanol (A.R.). This mixture was then filtered and the clear brown filtrate stored in a dark bottle. (e) Solution A: 7.5 ml of ethanolic guaiacum was mixed with 67.5 ml acetone * Boehringer Mannheim, GmbH,

Biochemical

Department,

W. Germany.

** Hughes and Hughes (Enzymes) Ltd., rza High Street, Brentford, Clin. Chim. Acta,

40 (1972) I IS-120

Essex.

INTERFERENCE

OF COMPOUNDS

11:

IN BLOOD GLUCOSE ESTIMATION

(A.R.) and 7.5 ml stock peroxidase. This mixture was diluted to IOO ml with acetate buffer. This solution was prepared well in advance because the initial intense blue colouration may take up to 24 h to fade. Immediately before use 2 parts (by volume) of solution A was mixed with I part (by volume) of Fermcozyme (solution C). Fermcozyme is slowly deactivated in an acetone-water solvent, therefore this solution was prepared fresh daily. 0.1 ml of test substrate was diluted with 4.9 ml water and mixed. 0.5 ml of this solution was then incubated at 37” for exactly 15 min with 3.0 ml of working colour reagent. At 15 min the absorbance was read at 600 nm. Phenol-4-aminophenazone method of Trinder3 (a) Protein precipitation reagents: 10.0 g sodium tungstate (Na,WO,.z H,O), 10.0 g disodium hydrogen phosphate (N%HPO,) and 9.0 g sodium chloride were dissolved in 800 ml deionised water. About 125 ml N hydrochloric acid was added to bring the pH to 3.0 (narrow range paper) then 1.0 g phenol was added and the volume made up to I 1 with deionised water. Colour reagent: 75 ml 4% w/v disodium hydrogen phosphate (Na,HPO,) was mixed with 215 ml deionised water and 5 ml Fermcozyme (635 A.M.) (Hughes and Hughes). To this was added 3 ml 0.1% w/v peroxidase (R.Z. = I.o), 300 mg sodium azide and IOO mg 4-aminophenazone. 0.1 ml of test substrate was added to 2.9 ml of protein precipitation reagent and mixed. 1.0 ml of this mixture was then incubated at 37’ for IO min with 3.0 ml colour reagent. The absorbance was read at 515 nm. RESULTS AND DISCUSSION

Part I The nature of the chemical group in the tolazamide molecule (Fig. I) which interferes with the Boehringer GOD-PERID method was investigated. A series of other sulphonylureas; tolbutamide, glibenclimide, chlorpropamide and acetohexamide were studied and found to be without significant effect on this blood glucose method.

P

S-O-N-C-N-H

Fig. I. Tolazamide

(N-hexahydroazepin-I-yl-N’-toluene-P-sulphonylurea).

As the N-hexahydro-azepin-r-y1 group is present in tolazamide and not in the other sulphonylureas examined, four other compounds containing this group or a similar group were investigated. These were nitrazepam, opipramol, nortriptyline and oxazepam. Nitrazepam appeared to interfere slightly but none of the other compounds interfered. These results were surprising as tertiary amines have a reducing potential Clin. Chim.

Acta,

40 (1972)

IIS--120

SHARP

118

and opipramol contains two such groups. Tolazamide also contains a tertiary amine group which was suspected as the probable source of interference. To eliminate the possibility that interference was the property of the sulphonamide moiety, five sulphonamides were studied. The five chosen covered as wide a range of sulphonamide structures as possible. These included: sulphasalazine, sulphadiazine, sulphafurazole, sulphaguanidine and sulphamethizole. None of these produced the slightest interference. The only remaining possibility was the -N-N-group where the sulphonylurea moiety proper couples with the N-hexahydroazepin-I-y1 group. Several drugs and compounds containing this -N-N-group in various chemical combinations were found and studied. These included : hydrallazine, isoniazid, iproniazid, isocarboxazid, guanoclor, trypan blue and other compounds containing a hydrazine group. All depressed the blood glucose result by varying amounts depending on the molecular site and chemical combination of the double nitrogen linkage and the adjacent chemical groups. It appeared that the oxygen affinity of these compounds was enhanced by the presence of a benzene nucleus in close proximity to the active group. The oxygen affinities were lowered by the presence of strong electronegative side groups. These observations were confirmed using a series of hydrazines and comparing their effects on the GODPERID ABTS method (Fig. 2). The relative effects of the drugs investigated were also plotted and compared with tolazamide (Fig. 3). Ascorbic acid depressed the results to a similar degree as did isoniazid. These compounds interfere by being oxidisedin preference to the chromogen and several mechanisms of oxidation may exist depending on the chemical nature of the compound being studied. Hydrazine and phenyl hydrazine probably undergo oxidative decomposition producing nitrogen and water plus benzene and perhaps a little biphenyl in the latter. a) isocarboxazid b) guanoclor _x_x_

p-nitrophenyl

-+-+-

dinitrophenyl

-O-O-

hydrazine

-A-A-

phenylhydrazine

c) hydrallazine

hydrazine

d) iproniazid

hydrazinc

e) isoniazid

sulphate

f) ascorbic

hydrochloride

acid

g) tolazamide 10

3 ki ~~

s

%

-==+=e +=-A\’

-x-_

60’

&

k 60. ‘s 540. z s ; 0.

20.

Fig. 2. Effect of hydrazine compounds

on the Boehringer GOD-PERID

Fig. 3. A comparison of the interferences GOD-PERID ABTS method. Clin. Chinz.

Acta,

40 (1972)

rrg-120

produced

ABTS method.

by certain compounds

on the Boehringer

INTERFERENCE

OF COMPOUNDS IN BLOOD GLUCOSE ESTIMATION

119

The nitrophenyl hydrazines are less likely to be oxidised owing to the presence of the strong electro-negative nitro groups. However, they do have a small reducing potential which could be the result of an oxidative decomposition mechanism similar to phenyl hydrazine or be due to a nitrogen atom in the hydrazine moiety forming a co-ordinate link with nascent oxygen. The formation of a co-ordinate link with nascent oxygen is probably the most common mechanism of interference in the compounds studied. The more potent interfering compounds may form two co-ordinate linkages on the same molecule. This reaction is illustrated using azobenzene as the oxygen acceptor which on oxidation produces azoxybenzene’.

Q-“=N~+

[o-J-

(=$=,a

Part 2. Possible

interference with other chromogenic oxygen acceptors Of all the compounds studied tolazamide had the greatest oxygen affinity. Therefore this drug was used to determine whether the guaiacum2 and phenol-4aminophenazone3 methods were also susceptible to interference. Using these two methods it was found that tolazamide did not interfere significantly even at I g/roe ml levels. Thus, the oxygen affinities of these chromogens are much stronger than ABTS. This was demonstrated using the peroxide substrate for the reaction instead of glucose. Reaction rates were measured using the Unicam SP-1800 (Fig. 4). Using the guaiacum a) guoiacum b) ABTS c) 4-aminophenazone 1.0

t

b

c 0.2.

h

secon4dOs

Fig. 4. Reaction rates with three chromogenic oxygen acceptor systems

or the 4-aminophenazone chromogens the peroxide-oxygen-chromogen reaction is calorimetrically complete in less than IO sec. After IO set however, the peroxideoxygen-ABTS reaction is only 69% complete calorimetrically. The delay in oxygen consumption must be attributed to the ABTS and would explain why it is vulnerable to competitors for oxygen. This experiment also illustrated the extremely fast action of peroxidase indicating that it is the glucose oxidase reaction that is rate limiting. In our hands the guaiacum method2 presented other problems. A straight line standard calibration curve could not be obtained. Below 50 mg/roo ml there was slight deviation whilst above 400 mg/roo ml the plot tended towards its asymptote. Clin. Chim. Acta, 40 (1972) 115-120

SHARP

I20

This appears to be due to the fact that the blue colour by oxidation of the guaiacum fades, the initial rate of colour loss being related to the intensity of colour produced. Above 400 mg/Ioo ml the intense colour fades rapidly. This colour instability means that the incubation of each test must be accurately timed otherwise there may be serious loss of precision in the hyperglycaemic range. The loss of precision would be negligible at lower blood glucose levels. The absorbance of the colour developed in the 4-aminophenazone method is only about half that obtained with the guaiacum method but this does not constitute a serious disadvantage. A standard glucose solution containing 25 mg/roo ml glucose will produce an absorbance of about 0.060 thus retaining adequate sensitivity well into the range of hypoglycaemia. This method also produces a straight line standard calibration curve from o to at least 500 mg/roo ml. The final colour is stable for at least 30 min. The preparation of reagents is more complex and time consuming in the guaiacum method so that phenol-4-aminophenazone appears to be the chromogen of choice. The increased specificity afforded by the high oxygen affinities of guaiacum and 4-aminophenazone makes it possible to interchange the manual protein precipitation procedure with the automated technique using dialysis. This cannot be done reliably with the ABTS chromogen. It is important when monitoring blood glucose levels that the same method of analysis be used for emergency night-duty specimens as for emergency and routine daily specimens. The 4-aminophenazone method can be used on the auto-analyser or manually with the same ease and precision. The guaiacum method, whilst a useful method when automated, is not so suitable as an emergency manual procedure. REFERENCES I P. SHARP, C. RILEY, J. G. H. COOK AND P. J. F. PINK, Clin. Chim. Acta, 36 (1972) 93. G. MORLEY, A. DAWSON AND V. MARKS, Proc. Assoc. Clin. Biochem., 5 (1968) 42. 3 P. TRINDER, Ann. CLin. Biochem., 6 (1969) 24. 4 J. I. PETERSON AND D. S. YOUNG, Anal. Biochem., 23 (1967) 301. 5 E. G. C. CLARKE, Isolation and Identi&ation of Drugs, Pharmaceutical Press, London, 1969. 6 H. BUSCH AND M. LANE, Chemotherapy, Year Book Medical Publishers Inc., Chicago, 1967, p. 20. 7 I. L. FINAR, Organic Chemistry, Longmans, London, 1954, p. 484. 2

Clin. Chim. Acta, 40 (1972)

1rg-120