A modified automated enzymatic glucose determination

CLINICA CHIMICA ACTA

325

A MODIFIED AUTOMATED ENZYMATIC GLUCOSE DETER~IINATION W. VAN D E R SLIK, A. L. KOEVOET, B. R. VAN N EER B O S, G. M. A L K E M A D E Atop P. VAN D E R H A R S T

Department of Clinical Chemistry, .State University, Leiden and Laboratory for Clinical Chemistry, Municipal Pharmacy, The Hague (The Netherlands) (Received September 9. 1969)

SUMMARY

A modified method is described for tlle enzymatic glucose determination in blood using the Auto-Analyzer. Optimal conditions were obtained by investigating all the reactions separately. The variation coefficient, measured over 35 estimations, was 3.6%. The influence of other compounds was also examined.

Using the method described by Kawerau ~ we were not always successful in obtaining good, reproducible values. The preparation of the membranes by means of dialysis under pressure did not give more sensitive results in our case. The values were especially likely to vary at lower concentrations. After examination of the different reactions of the system, we altered the flow diagram so that the reactions could proceed under optimal conditions. The sensitivity of the analysis became much better and special preparation of the membranes was therefore no longer necessary. METHODS AND RESULTS

Reagents r. Acetate buffer of 0.5 31, pH 5.0. 30 rnl acetic acid 8oo ml distilled water 35 ml sodium hydroxide (4o%) to adjust the solution to pH 5.0 Fill up to xooo ml with distilled water. Io drops of Bry 35 (Technicon) are added.

z. xo% Sulphuric acid. 3. Peroxidase. 5o mg peroxidase, R.Z.x.o grade, activity 9o units (Hughes and Hughes Ltd.) are dissolved in 250 ml o.9% sodium chloride solution. 4. Enzyme buffer. 5o ml peroxidase solution (see. 3), 5- I ° ml glucose oxidase (Fermcozym 653 AM, about 75° U per ml, Hughes and Hughes Ltd.). The amount Clin. Chim. Acta. 27 (t97 o) 325-33 °

326

SLIK a al.

is dependent on the activity of the charge glucose oxydase. Fill up to iooo ml with acetate buffer (see x). 5. 2 % o-Tolidine. 2.5 g o-tolidine (3,3'-dimethyl benzidine, Fa. Schuchardt, Mt~nchen) are dissolved in ~5o ml absolute ethanol. 6. o-Tolidine-enzyme buffer. IO ml 1% o-tolidine (see 5) are filled up to xooo ml with enzyme buffer (see 4). 7. Sodium chloride solution, 9 % . 9o g sodium chloride, p.a. Merck IOO mg N-ethylmaleinimide 5 ml glucose solution, 2ooo mg per xoo ml (see Io) fill up to xooo ml with distilled water. 8. Sodium chloride solution, 0.9O/o. Fill zoo ml sodium chloride solution (see 7) up to xooo ml with distilled water. Add xo drops of Bry 35 (Technicon). 9. Preservation solution. 2 ml heparin, 5000 E per ml xoo nag N-ethylmaleinimide (Merck, Schuchardt) fill up to lOOO ml with distilled water. to. Glucose stock solution, 2000 mg per zoo rot. 2o g glucose p.a. are dissolved in iooo ml of a saturated benzoic acid solution (about 8 g benzoic acid per 1). This solution must be preserved 24 h before diluting with preservation solution (see 9) to make standard solutions. All reagents must be kept at 4 °, except the acetate buffer and sulphuric acid. Procedure Blood and standards are diluted IO times by adding o.I ml sample to 0. 9 nd of preservation solution. The flow diagram of the Auto-Analyzer system is shown in Fig. I. The system is pre-washed before use for IO man with a x% haemosol solution and for 15 man with distilled water. The sampler I I is run at 4 ° samples an hour with o.9% sodium chloride as a washing solution and a 1:2 sample to wash ratio cam.

Q

wa see

t

i

.

i

dialyzer 37°C •

., I

tube size ~ preperttoning linches) |

,

.o I

~,~p

.o3o

~®

.o.

°'1/

~® X@

Dl .

waste ~ waste

PSM.C.

o, heating

~ ~

lSmm tubular f/c L/.Onrn titters

~ sa,,,~t. sa..

.~.~ ,o. ~®

Sarnpter 11 rate: t.OI)er ho~r Sampte : wash ritlo 1 : |

,,r

:oss

..,,.r

°.

A,r

.o30

.,so,

I

|

r,eorder

Fig. I. Auto-Analyzer flow diagram for the determination of glucose in capillary blood. Clan. Chem. dcta, 27 (I97 o) 325-33 °

327

AUTOMATED GLUCOSE DETERMINATION TABLE I

I N F L U E N C E OF T H E I N C U B A T I O N T I M E ON T H E C O L O U R Y I E L D , B E F O R E A D D I T I O N O F O - T O L I D I N E

o- Tolidine added after

A bsorbance

5min to ,,

o.752 0.333

15

,,

0-242

20

,,

o.188

2.0 1.6 o( J

c

1.2

.o

I..,

o

tit

,o

O.e

0.4

V

..__

,

i

i

I

!

.5

10

15

20

,, !

25 rain

incubation time Fig. 2. Influence of t h e i n c u b a t i o n time, with addition of o-tolidine, on the colour yield.

T A B L E 11 COLOUR Y I E L D A F T E R I N C U B A T I O N AT T W O D I F F E R E N T T E M P E R A T U R E S

Glucose solution rag/too ml

A bsorbance at a5 °

A bsorbance at 37 ~

o 7 I4 21 28 35

0.026 o. x85 0.335 0.493 0.694 0.898

o.oi 7 0.083 0.134 °.2°4 o.258 o.3z4 .

.

.

.

.

.

.

A single dialyser with Cuprophan membrane and a colorimeter with a x5-mm tubular flow cell and interference filters of wave length 44 ° nm are used. The concentration of glucose oxidase in reagent 4 is so adjusted, that the highest glucose standard (60o rag/too ml) alter diluting x :to gives a read-out on the recorder between scale units to and x2. A one-day charge of the o-tolidine enzyme buffer must be brought up to room temperature before use. The remainder should be kept at 4 ° .

Discussion of the procedure Several steps o! the estimation were investigated in order to construct an optimal scheme for the Auto-Analyzer. Clin. Chim. Acta, 27 (197 o) 325-33 o

328

VAN DER SLIK et ~ll.

I. o-Tolidine was added to a solution of glucose in enzyme buffer (reagent 4) after increasing reaction times and the reaction was stopped 8 min later with sulphuric acid. The colour intensity was measured in a colorimeter at 44 ° n m . Table I shows, that o-tolidine must be present as soon as the enzyme reaction begins. 2. To measure the enzyme activity with increasing incubation times, after increasing periods of time sulphuric acid was added to a solution of glucose, enzyme buffer and o-tolidine. The colour intensity must then be measured immediately since the compomxd formed di~colours at about I % per min at 25 °. The optimal incubation time is xo min, see Fig. 2. 3. The influence of the temperature on tile enzyme reaction was investigated at two different temperatures, 25 ° and 37 °. Table II shows that 25 ° is preferable and that higher temperatures decrease the sensitivity. These experiments resulted in the flow diagram for the Auto-Analyzer in Fig. i. The sample was not dialysed against the enzyme buffer since a solution of enzyme buffer and o-tolidine was used. The o-tolidine would soil the membrane and therefore we had to dialyse against the acetate buffer and add the enzyme and otolidine simultaneously to the dialysate. The enzyme- and oxidation reactions proceeded at 25 °, after which the sulphuric acid was added to stop the reactions and stabilize the colour of the oxidized o-tolidine. ~.U. Abs.

~ /

20

30 /.0 50 60

?0 80 90

:

i

50

v

100

'

'

i

150

'"

v

200

.

.

!

.

250

.

I

300

!

350

i

tOO

i

1.50

'

-'

m g glucose 500 per 100 m t i

Fig. 3. Relationship between the recorder scale units and concentration of glucose standard solutions. RESULTS

The standard curve is linear over the whole distance (Fig. 3). The variation coefficient of the dilution of the serum and the estimation of glucose, calculated from 35 samples of a serum containing 176 mg per IOO ml glucose, is 3.6~/o. Fig. 4 shows that there is a negligible carry-over at a sample wash rate of I :z and no cross-contamination of the samples. Clin. Chim. Acta, 27 (x97 o)

325-3 o3

329

AUTOMATED GLUCOSE DETERMINATION

..i I . i- • _~:. ::.": 1

i

~ :

,--

x

T

:

T

' - ~

"

;'

, -i

•

:

:

,

:"~ ~

....

t.

i - - - . !

.

:

]~

.

.!.

!

i .~

:

..j

i

• }"•••i :

!

:

,

1

;

•

t

;

1

.

itl

4

i

u- ,- ,

i

•

|1;

5

: !i!

i

:

Ill iiii;

F i g . 4, C a r r y - o v e r e x p e r i m e n t s glucose.

!

].

I I.

"i

~

i

i|

;I t i

TABLE

•

f

i

!

i

b e t w e e n t h e s t a n d a r d s o l u t i o n s o f 30 m g a n d 5 0 0 m g per 1 o o m l

I11

R E C O V E R I E S FROM G L U C O S E S T A N D A R D S O L U T I O N S A D D E D TO S E R U M

Glucose mg/loo ml

Calculated rag/too ml

Found mg/loo ml

Recovery %

Found after 3 days mglzoo ml

Recto ery %

o 5° too 15o 200

97 122 I47 t72

72 Io4 ]28 X5o t74

Io7.3 io4.9 !o2.o ]ot.z

76 io2 128 15o 172

Io5.5 io5.2 1o4.9 1o2.o IOO.O

25o

~97

3oo 35 °

222 247

2oz

~o2.5

zoo

~oI.5

216 242

97.3 98.0

218 252

98.2 102.0

Recoveries were made by diluting a serum t : t with solutions containing from o to 350 mg per too ml glucose, and were determined at once, and again after keeping for 3 days at 4 ° in the preservation solution, see Table III. Some biological compounds which can interfere with the glucose estimation were also studied. The results are given in Table IV. It can be said, in general, that oxidizing agents may give too high values for the glucose concentration and reducing agents too low values. DISCUSSION

On comparing the methods of Kawerau t and Cramp z with the method recommended by us, the differences are obvious. Both investigators use sodium fluoride as preservation solution. Kawerau t and Saifer a did not find that the sodium Clin. Chim. Acta. 27 ( ] 9 7 o) : $ 2 5 - 3 3 0

330

vA~ DER SLIK et al.

T AB L E IV INFI.13F~C'E n g O T H E R r~IOLOGICAL C O M P O U N D S ON T H E ]gSI"IMATION OF G L U C O S E

Inhibitor rag/too rnl

Glucose concentration --Se-ru--m l Serum II rag/too ml rag/zoo ml

Vitamin C: o 5 1o

176 172 152

372 36o 326

x76 x76 17z

38x 38o 386

x76 t8o 176

383 394 386

176 176 I7 z

380 372 372

Glutathione o z5 50

Cystein. HC! o io zo

Uric acid o 5 Io

fluoride had any influence on the enzyme reactions. Cramp, however, removed the sodium fluoride as the insoluble magnesium salt before dialysing. Cross-experiments with sodium fluoride and N-ethyl maleinimide as preservation solution and potassium oxalate and heparin as anti-coagulants were made. There was no difference in sensiti-Aty between the glucose concentrations estimated in these solutions, but the solution of N-ethyl maleinimide with heparin became more homogeneous with added blood than other solutions. Furthermore, our experiments showed that o-tolidine has to be reacted at once with the hydrogen peroxide formed, to keep the loss of the latter as low as possible. Cramp 2 also used o-tolidine and the enzymes in a single solution, but lead the reagent through the dialyser. Our experience is that in this case, the membranes soon become soiled because of the o-tolidine. Another disadvantage of Cramp's method is that the reactions proceed in the dialysis bath at 37 °, while our experiments indicate z5 ° as a much better reaction temperature. The method of Kawerau~ does not specify the temperature, but allows the reactions to proceed at room temperature. In this method, competition may arise between the o-tolidine and other oxidizable compounds present in blood with the peroxide formed, since the o-tolidine is added somewhat later to the system. REFERENCES x E. KAWERAU, Z. Klin. Chem., 4 (I966) 224 2 D. G. C R A M P , J. Clin. Pathol.o 20 (i967) 970. 3 A. SMFER ANt> S. GERSXENFBLt), J. Lab. Clin. Med., 5I (I958) 448.

Clin. Chim. Acta, 27 (x97 o) 325-330