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Use of a non-carcinogenic reagent for the determination of blood glucose
475
CLINICACHIMICAACTA
BRIEF
NOTES
Use of a non-carcinogenic
reagent for the determination
of blood
glucose A method for determining glucose in blood, using only a single enzyme, glucose oxidase, and dispensing with the use of peroxidase has been applied to the precise determination of blood glucose levels in ketotic cattle and sheep’, where values down to IO mg/roo ml or lower are found. However, the chromogenic reagent, o-tolidine, used in this and many other methods is now widely regarded as carcinogenic, although less hazardous than benzidine and so its replacement by a less harmful reagent appeared desirable. Analogoues of o-tolidine, such as o-dianisidine and 2,7diaminofluorene were excluded from examination as these’also are reported to be carcinogeni@. Many reagents were tested in the glucose oxidase system and most were found to be unsatisfactory due either to lack of sensitivity or to their requiring pH conditions unfavourable for optimum enzyme activity. The best reagent for the purpose was found to be diethyl+phenylenediamine (DEPPD). This reagent gives a clear pink colour with free iodine which is an intermediate product in the method, and is only slightly less sensitive than o-tolidine. There are no reports of this compound having carcinogenic properties and indeed it is widely used as a component of photographic colourcoupling developers. The main problem in the routine application of this compound was to find a suitable medium for preparing its solution, that would be stable under working conditions, as it easily undergoes spontaneous oxidation. It was found that its preparation as a 5% solution in N-hydrochloric acid gives a reagent which keeps well, giving low blank values. This reaction is similar to the method for the determination of chlorine, where the liberated iodine reacts with dimethyl-p-phenylenediaminea. METHOD ,
The following method is suitable for glucose values np to about 200 mg/roo ml. Measure 0.2 ml blood, preserved with oxalate-fluoride mixture, into a centrifuge tube containing 3.0 ml 0.9% NaCl. Deproteinise by adding 0.4 ml of 5% &SO, solution and 0.4 ml of 0.3 N Ba(OH), solution, mixing gently after each addition. Centrifuge and pipette 1.0 ml of the supernatant liquid into test tubes (2.0 ml may be used in cases of low glucose levels). Standards: pipette 0.5 and 1.0 ml dilute glucose solution (= 25 and 50 ,ug) into other tubes. Blank: 1.0 ml water. Bring the volume in each tube to 3.0 ml with water. Add 0.5 ml of 0.5% K.I. solution, 0.5 ml of 1.0% ammonium molybdate solution and 2.0 ml buffer-enzymeDEPPD reagent, mixing after each addition. After 30 min at room temperature, measure the absorbance at 550 nm. (EEL Clin. C.hz.
Acta,
25
(1969) 475-477
476
BRIEF
NOTES
calorimeter with filter 624), in the same order in which the buffer-enzyme-DEPPD reagent was added. Calculation
Blood glucose (mg/roo ml) = (or: x
IOO
Test
Standard 25 when using the 50-pg standard)
x
50
Reagents
Z&O,
- 7 H,O in water.
(I)
5%
(2)
0.3 N Barium hydroxide. Standardise this against the ZnSO, solution using
phenolphthalein as indicator. Store free from CO, using a soda-lime guard-tube. (3) 0.9% Sodium chloride. (4) 0.5% Potassium iodide. (5) 1.0% Ammonium molybdate. (6) 5% Diethyl-$-phenylenediamine hydrochloride in N HCl. Keeps for several months when stored in a brown bottle at 4’. It should be discarded if the blank value rises above an acceptable level. Generally the absorbance of the blank is less than 0.05, using I-cm cells. (7) 0.5 M Acetate buffer (pH 5.0). Mix IOO ml N acetic acid with 70 ml N NaOH and dilute to 200 ml with water. Store cold, but do not use chloroform as a preservative. (8) Stock glucose standard (1.0 mg/ml); Dissolve IOO mg glucose in IOO ml saturated benzoic acid (about 0.3%). (9) Dilute glucose standard (50 /‘g/ml). Dilute the stock standard 5o-fold with water just before use. (IO) Buffer-enzyme-DEPPD reagent : Mix the following components in the ratio of 1.0 ml acetate buffer, 0.025 ml Fermcozyme*, 0.05 ml DEPPD solution and water to 2.0 ml. This may be conveniently done using a graduated cylinder, making only the volume required for the batch of tests on hand. Experimental
The same pH and concentration of acetate, iodide, molybdate and enzyme were found optimal as in the o-tolidine method’. DEPPD concentration. The method is not much influenced by a wide range of DEPPD concentration, except that using low levels, linearity starts to fall off at about IOO mg/roo ml blood glucose. Very high levels of DEPPD led to increase in blank value, and also tended to alter the pH of the system. Use of 0.05 ml of a 5% solution per test was found to give a linear relationship up to about 200 mg/roo ml (absorbance of 0.9 to 1.0). Recovery of added glucose. Glucose added to blood and carried through the complete procedure in amounts ranging from 12.5 to 125 mg/roo ml gave, for 35 instances a mean (f I S.D.) recovery of: 98.82 f 1.91%. Both the recovery of added glucose and precision of the method appeared to be limited only by accuracy of timing of measurement of the absorbance. In routine * Hughes C&a. Chim.
and Hughes,
Ltd.,
Iza High Street, Brentwood,
Acta, 25 (1969) 475-477
Essex.
BRIEF
477
NOTES
use this is not a serious obstacle, as a difference of I min in the time taken for colour development between test and standard gives an error of f 1.0% in the final answer. As an example of precision found under routine conditions, blood samples were analysed in replicate. The following mean (* I SD.) values were found, the samples being analysed twenty or ten times, respectively. (i) 26.7 _I 0.84 mg/roo ml (20 instances) (ii) 99.0 f 3.8 mg/roo ml (IO instances) Veterinary Research Laboratories, ~~~~~styyof Agricultwe, S~o~rno~~, Be~ast~ BT4, 3SLl ~~re~an~~ I R. H.THOMPSON, Cliff. Chim. Acta, 13(IQ66) 133. 2 Carcinogenic Aromatic Amines. The Chester Beatty Research Institute, 3 G. A. TEPLOUKHOVA, Zav. Lab., 33 (1967) 565. Quoted by Anal. Abstv.,
Received
R. H.
THOMPSON
1966. 15 (1968) 4676.
April 22, 1969 Clin. Chim.
Acta,
25 (1969) 475-477
CLINICACHIMICAACTA
BRIEF
NOTES
Use of a non-carcinogenic
reagent for the determination
of blood
glucose A method for determining glucose in blood, using only a single enzyme, glucose oxidase, and dispensing with the use of peroxidase has been applied to the precise determination of blood glucose levels in ketotic cattle and sheep’, where values down to IO mg/roo ml or lower are found. However, the chromogenic reagent, o-tolidine, used in this and many other methods is now widely regarded as carcinogenic, although less hazardous than benzidine and so its replacement by a less harmful reagent appeared desirable. Analogoues of o-tolidine, such as o-dianisidine and 2,7diaminofluorene were excluded from examination as these’also are reported to be carcinogeni@. Many reagents were tested in the glucose oxidase system and most were found to be unsatisfactory due either to lack of sensitivity or to their requiring pH conditions unfavourable for optimum enzyme activity. The best reagent for the purpose was found to be diethyl+phenylenediamine (DEPPD). This reagent gives a clear pink colour with free iodine which is an intermediate product in the method, and is only slightly less sensitive than o-tolidine. There are no reports of this compound having carcinogenic properties and indeed it is widely used as a component of photographic colourcoupling developers. The main problem in the routine application of this compound was to find a suitable medium for preparing its solution, that would be stable under working conditions, as it easily undergoes spontaneous oxidation. It was found that its preparation as a 5% solution in N-hydrochloric acid gives a reagent which keeps well, giving low blank values. This reaction is similar to the method for the determination of chlorine, where the liberated iodine reacts with dimethyl-p-phenylenediaminea. METHOD ,
The following method is suitable for glucose values np to about 200 mg/roo ml. Measure 0.2 ml blood, preserved with oxalate-fluoride mixture, into a centrifuge tube containing 3.0 ml 0.9% NaCl. Deproteinise by adding 0.4 ml of 5% &SO, solution and 0.4 ml of 0.3 N Ba(OH), solution, mixing gently after each addition. Centrifuge and pipette 1.0 ml of the supernatant liquid into test tubes (2.0 ml may be used in cases of low glucose levels). Standards: pipette 0.5 and 1.0 ml dilute glucose solution (= 25 and 50 ,ug) into other tubes. Blank: 1.0 ml water. Bring the volume in each tube to 3.0 ml with water. Add 0.5 ml of 0.5% K.I. solution, 0.5 ml of 1.0% ammonium molybdate solution and 2.0 ml buffer-enzymeDEPPD reagent, mixing after each addition. After 30 min at room temperature, measure the absorbance at 550 nm. (EEL Clin. C.hz.
Acta,
25
(1969) 475-477
476
BRIEF
NOTES
calorimeter with filter 624), in the same order in which the buffer-enzyme-DEPPD reagent was added. Calculation
Blood glucose (mg/roo ml) = (or: x
IOO
Test
Standard 25 when using the 50-pg standard)
x
50
Reagents
Z&O,
- 7 H,O in water.
(I)
5%
(2)
0.3 N Barium hydroxide. Standardise this against the ZnSO, solution using
phenolphthalein as indicator. Store free from CO, using a soda-lime guard-tube. (3) 0.9% Sodium chloride. (4) 0.5% Potassium iodide. (5) 1.0% Ammonium molybdate. (6) 5% Diethyl-$-phenylenediamine hydrochloride in N HCl. Keeps for several months when stored in a brown bottle at 4’. It should be discarded if the blank value rises above an acceptable level. Generally the absorbance of the blank is less than 0.05, using I-cm cells. (7) 0.5 M Acetate buffer (pH 5.0). Mix IOO ml N acetic acid with 70 ml N NaOH and dilute to 200 ml with water. Store cold, but do not use chloroform as a preservative. (8) Stock glucose standard (1.0 mg/ml); Dissolve IOO mg glucose in IOO ml saturated benzoic acid (about 0.3%). (9) Dilute glucose standard (50 /‘g/ml). Dilute the stock standard 5o-fold with water just before use. (IO) Buffer-enzyme-DEPPD reagent : Mix the following components in the ratio of 1.0 ml acetate buffer, 0.025 ml Fermcozyme*, 0.05 ml DEPPD solution and water to 2.0 ml. This may be conveniently done using a graduated cylinder, making only the volume required for the batch of tests on hand. Experimental
The same pH and concentration of acetate, iodide, molybdate and enzyme were found optimal as in the o-tolidine method’. DEPPD concentration. The method is not much influenced by a wide range of DEPPD concentration, except that using low levels, linearity starts to fall off at about IOO mg/roo ml blood glucose. Very high levels of DEPPD led to increase in blank value, and also tended to alter the pH of the system. Use of 0.05 ml of a 5% solution per test was found to give a linear relationship up to about 200 mg/roo ml (absorbance of 0.9 to 1.0). Recovery of added glucose. Glucose added to blood and carried through the complete procedure in amounts ranging from 12.5 to 125 mg/roo ml gave, for 35 instances a mean (f I S.D.) recovery of: 98.82 f 1.91%. Both the recovery of added glucose and precision of the method appeared to be limited only by accuracy of timing of measurement of the absorbance. In routine * Hughes C&a. Chim.
and Hughes,
Ltd.,
Iza High Street, Brentwood,
Acta, 25 (1969) 475-477
Essex.
BRIEF
477
NOTES
use this is not a serious obstacle, as a difference of I min in the time taken for colour development between test and standard gives an error of f 1.0% in the final answer. As an example of precision found under routine conditions, blood samples were analysed in replicate. The following mean (* I SD.) values were found, the samples being analysed twenty or ten times, respectively. (i) 26.7 _I 0.84 mg/roo ml (20 instances) (ii) 99.0 f 3.8 mg/roo ml (IO instances) Veterinary Research Laboratories, ~~~~~styyof Agricultwe, S~o~rno~~, Be~ast~ BT4, 3SLl ~~re~an~~ I R. H.THOMPSON, Cliff. Chim. Acta, 13(IQ66) 133. 2 Carcinogenic Aromatic Amines. The Chester Beatty Research Institute, 3 G. A. TEPLOUKHOVA, Zav. Lab., 33 (1967) 565. Quoted by Anal. Abstv.,
Received
R. H.
THOMPSON
1966. 15 (1968) 4676.
April 22, 1969 Clin. Chim.
Acta,
25 (1969) 475-477