Evaluation of Trinder's Glucose Oxidase Method ... - Clinical Chemistry

dIN. CHEM. 21/12, 1754-1760 (1975)
Evaluation of Trinder’s Glucose Oxidase Method
for Measuring Glucose in Serum and Urine
John A. Lott and Kathie Turner
Trinder’s method for glucose has nearly all the attributes
of an ideal automated colorimetric glucose oxidase
procedure. The chemicals used in the color reaction
with peroxidase are readily available, the solutions are
stable and can be prepared by the user, the method is
highly specific and largely free of interferences, the
sensitivity can. be adjusted by the user to cover a wide
range of glucose concentrations, and the reagents are
not hazardous. We found very good agreement between
results by this method and by the hexokinase and
Beckman Glucose Analyzer methods. The method has
been modified and adapted to the AutoAnalyzer I and
SMA 6/60 (Technicon) with manifolds that give very little
interaction between specimens. A study of the method
by the simplex technique revealed that the glucose
oxidase activity in the reagent is the most critical variable.
AddItIonal Keyphrases: continuous-flow analysis #{149}glucose
and reagent preservatives #{149} intermethod comparison
#{149} optimum analytical conditions #{149}normal (reference)
values #{149} sample stability
The glucose oxidase/peroxidase (EC 1.1.3.4/EC
1.11.1.7) method for glucose described by Trinder in
1969 is very attractive (1). The method is specific for
glucose and the reagents are all readily available. Solutions
of the reagents are stable. The Trinder reagents
are less of an occupational hazard than reagents
used in other methods-o-toluidine, o-dianisidine,
or N,N-dimethylaniline, which are all quite
toxic.
Here, we describe automated methods for serum
and urinary glucose with use of the AutoAnalyzer I or
SMA 6/60 (Technicon Instruments Corp., Tarrytown,
N. V. 10591) and of reagents that the user can
prepare. We also describe optimization of the method
with the simplex technique, method interferences,
normal values, and other data.
Materials and Methods
Reagents and Standards
Stock glucose oxidase, 106 U/liter, Type V, No.
G-6500, Sigma Chemical Co., St. Louis, Mo. 63178.
Division of Clinical Chemistry, Department of Pathology, Ohio
State University, 410 W. 10th Ave., Columbus, Ohio 43210.
Presented in part at the Ninth International Congress on Clinical
Chemistry, Toronto, Ont., 1975 (Clin. Chem. 21, 978 (1975),
abstract).
Received July 21, 1975; accepted Aug. 18, 1975.
1754 CLINICAL CHEMISTRY, Vol. 21, No. 12, 1975
Peroxidase, Type II, No. P-8250, Sigma. Note that
this peroxidase is as satisfactory as the much more
expensive Type VI, No. P-8375 from Sigma.
4-Aminoantipyrine, No. A-4382, Sigma (also
known as 4-aminophenazone).
Peroxidase/buffer reagent, pH 7.0. Dissolve 8.5 g
of anhydrous reagent-grade Na2HPO4 and 5.3 g of
anhydrous reagent-grade KH2PO4 in about 800 ml of
distilled water, and adjust the pH to 7.0 ± 0.1 with 1
mol/liter HC1 or NaOH if needed. Add 4 mg of peroxidase
and 300 mg of 4-aminoantipyrine and dissolve,
and dilute to 1 liter with distilled water. The final reagent
contains 0.1 mol of phosphate per liter, and is
stable for as long as four weeks when stored in
amber-colored glass bottles at 4 #{176}C.
Glucose oxidase/peroxidase reagent. Add 12 ml of
stock glucose oxidase (or 12 000 U) to 1 liter of the
peroxidase/buffer reagent. The complete reagent is
stable for 1 week at 4 #{176}C.
Phenol solution, 1.5 g/liter. Dissolve 1.5 g of phenol,
analytical-reagent grade, in enough distilled
water to make 1 liter of solution. Stable for at least
six months when stored in amber-colored glass bottles
at room temperature.
Saline, 9 g/liter. Dissolve 9 g of sodium chloride in
enough distilled water to make 1 liter of solution.
Add 1 ml of “Tween 20” surfactant (Technicon) per
liter just before use.
Stock glucose standard, 10 g/liter. Dissolve 10.000
g of primary-standard grade dextrose in enough distilled
water that is saturated with benzoic acid (about
3 g/liter) to make 1 liter of solution.
Working glucose standards. Prepare dilutions of
the stock glucose standard with a saturated aqueous
benzoic acid solution to give standards containing
250, 500, 1000, 1500, 2000, 2500, 3000, and 4000 mg of
glucose per liter,
Procedure
The flow diagrams for the AutoAnalyzer and SMA
6/60 are shown in Figures 1 and 2. Freshly separated
serum or urine can be analyzed directly. In the case
of the AutoAnalyzer I, the standards are analyzed in
the order listed above and a calibration curve is prepared
on semilog paper. The glucose in patients’ samples
and in controls is estimated from the curve. The
standards should be run at the beginning of each

Fig. 1. Flow diagram for the AutoAnalyzer I
Added Founda
Recovery,
mg/liter %
SAIl results are means of three determinations.
Tube Flow,
id.in. rt/min
Table 1. Analytical Recovery of Glucose from
Bovine Albumin Solutions
500
1000
2000
3000
4000
510
1000
2000
3040
4020
102
100
100
101
101
third tray of 40 samples. At least two controls should
be analyzed on each tray.
The glucose concentration of the serum-based calibrating
material used to set the SMA 6/60 should be
established by analysis with the method proposed
here. The “insert” or “label” values must not be used
uncritically, because they may have been established
by a less-specific method. Standards and at least two
controls should be analyzed on every tray of 40 samples
on the SMA 6/60.
At the end of the day, 1 mol/liter NaOH is pumped
through all lines, including the dialyzer, for 15-30
mm, followed by a 30-mm wash with distilled water.
This washing effectively prevents shifting baselines,
drift, clogged tubing, etc. A sodium hypochlorite solution
must not be used, because it is difficult to
wash out completely, and the hypochlorite reacts to
form a color with the glucose oxidase/peroxidase re-
Month
6” Dialyzer
__________8-turn
I.
LU1709
2,9-1 ‘----‘ L
turn 37#{149}
coils Both
________ PhosingCoil
Color- Recorder
meter
505 nm ______
Fig. 2. Flow diagram for the SMA 6/60
Tube Flow,
id.in. mI/mm
Samole
[ Saline/Twn
.020
0l
0.16
10
I Air .035 0.42
Sdine/Tween .051
[ Air
.035
Gluc.Ox./Perox.
.035
0.42
0.42
Phenol .030 0.32
Flowcell Retum.05l 1.0
Sampler IV
agent. In the case of either the AutoAnalyzer I or the
SMA 6/60, replace the manifold tubing after no more
than 140 h of running time.
Resufts
Analytical Variables
Analytical recovery. Bovine albumin (No. 905-10,
Sigma) contained no glucose detectable by the AutoAnalyzer
I method described here or by the Calbiochem
hexokinase procedure (No. 869204; Calbiochem,
San Diego, Calif. 92112). Solutions were prepared
to contain, per liter, 70 g of albumin and 500,
1000, 2000, 3000, and 4000 mg of glucose, and they
were then assayed with the AutoAnalyzer I. Because
the analytical recoveries (Table 1) were all within
about 2% of the expected values, we concluded that
aqueous standards can be used to calibrate the AutoAnalyzer
I and that protein does not interfere in
the analysis for glucose.
Precision. The method described has been in routine
use here since October 1974. Between November
1, 1974 and March 31, 1975, we did about 44 000
serum glucose determinations. Blind controls (2)
from the same lot numbers were randomly distributed
between patients’ samples during that time, and
the results are listed in Table 2. An equal number of
blind controls were analyzed with the AutoAnalyzer I
and SMA 6/60. We think that the precision of the
method over this time span is satisfactory.
Comparison studies. We compared our results for
this method to those obtained by the Calbiochem
Table 2. Summary of Quality-Control Data for Glucose in Samples Analyzed as Blinds
Versatola Versatol A0 Versatol A-AItarnate’
Pooled serum
n Mean CV n Mean CV n Mean CV n Mean CV
mg/liter % mg/liter % mg/liter % mg/liter %
Nov. ‘74 20 820 2.4 20 2020 2,9 30 2990 2.0 30 840 3.1
Dec. ‘74 21 820 3.0 21 1980 2.1 31 2980 1.8 31 840 2.3
Jan. ‘75 22 830 3.0 22 1990 1.7 31 2990 2,8 31 840 3.1
Feb. ‘75 20 820 4.3 20 1980 1.6 28 2980 2.2 28 810 2.7
Mar. ‘75 21 820 2.7 21 1980 2.6 31 2940 1.3 31 810 4.1
a General D iagnostics, Morris Plains, N.J. 07950. Lot numbers, left to right, were 2406103. 2262043, and 1176112.
CUNICAL CHEMISTRY, Vol. 21, No. 12, 1975 1755
I.0

Versatola
Table 3. Results by Three Methods for
Serum Glucose Compared
(Mean of Duplicate Results)
Auto-
Material Analyzer I SMA 6/60 Hexokinase
Versatol A
Versatol A Alternate
Calibratea
VersatoI’ Automated Lo
Serum Referenceb
Scale lb
Scale Ilb
Pool IC (lipemic)
Pool IIC
Pool IlIC
Pool lV’
Pool VC (lipemic)
Pool VIC
Pool VIIC
O General Diagnostics.
bTechnicon
C Freshly pooled human serum.
glucose, mg/liter
760 780 770
1840 1790 1840
2960 2820 2790
1830 1840 1810
770 800 840
2350 2310 2300
860 870 870
3710 3550 3590
470 470 320
560 550 540
850 850 850
940 940 910
1100 1050 840
1410 1380 1380
3000 2860 2930
hexokinase procedure for various lyophilized control
sera and pooled fresh sera. Two serum pools were
made up from lipemic samples. Agreement was good
(Table 3) except for the lipemic pools (pools I and V),
for which the hexokinase procedure gave somewhat
lower results.
We also assayed 54 freshly collected patients’ sera
containing 540 to 4760 mg of glucose per liter, with
the Glucose Analyzer (Beckman Instruments, Inc.,
Fullerton, Calif. 92634) and with the SMA 6/60. Results
obtained with the two instruments agreed well.
The means and standard deviations for the SMA and
Beckman were 1460 ± 930 and 1470 ± 910 mg of glucose
per liter, respectively, the correlation coefficient
was 0.9994, and the slope and intercept were: Beckman
= 0.972 (SMA) + 4.5 (3). To be certain that we
had no bias between the AutoAnalyzer I and SMA
6/60, we assayed 73 fresh patients’ sera with both instruments.
The range of values was 350 to 7890 mg/
liter, the means and SD were 1260 ± 970 (AutoAnalyzer
I), and 1260 ± 960 mg/liter (SMA 6/60). The
correlation coefficient was 0.9993, and the slope and
intercept were: SMA = 0.98.4(AutoAnalyzer I) + 2.51.
Interferences. Various anticoagulants, drugs, metabolites,
sugars and other compounds were tested
for potential interferences with the method (Table
4). For the first group the same glucose concentration
was observed when either saline or a solution of the
compound was added to pooled fresh serum. The
concentration of anticoagulants that we tested is
much higher than is commonly used, but none interfered.
The serum drug concentrations that we studied
are much higher than would be expected after a
therapeutic dose. It is significant that none of the
1758 CLINICAL CHEMISTRY, Vol. 21, No. 12, 1975
commonly used oral hypoglycemic agents interfered.
The concentrations of metabolites are far above the
normal range and generally exceed those seen for creatinine,
urea, and uric acid even in patients with severe
azotemia. For all of these, we observed no interference.
Uric acid was examined in more detail. Pooled
serum was diluted with saline or a stock uric acid solution
to give pools with 100, 250, and 500 mg of uric
acid per liter. The addition of uric acid did not
change the observed glucose concentration of 630
mg/liter as compared to the same pool diluted with
saline (Table 4). Likewise, when uric acid was added
to three other poois to give a concentration of 200 mg
of uric acid/liter, the glucose concentration of 510,
970, and 1940 mg/liter were the same as was observed
when saline was added to the poois.
That the sugars listed in Table 4 do not interfere
reflects the specificity of the method, maltose being
an exception. The interference from maltose was due
to the presence of maltase in the glucose oxidase. Hemoglobin
did not interfere, as was also reported by
Gochman et al. (4). Ascorbic acid produced dramatic
decreases in the observed glucose value, in contrast to
the findings of others (5) who observed no effect on
results by Trinder’s method (1) of ascorbic acid, 1000
mg/liter.
In vivo concentrations of ascorbic acid are too low
to significantly interfere. In a study by Schrauzer and
Rhead, the maximum ascorbic acid concentration in
plasma or erythrocytes never exceeded 27.5 ± 6.5
(SD) and 15.1 ± 3.6 mg/liter, respectively, in 17 volunteers
who had taken 2 g of the drug daily for nine
days (6). A large fraction of ingested ascorbic acid is
excreted unchanged in the urine (7), hence a potential
interference exists in cases of renal failure.
Gentisic acid, a metabolite of salicylic acid, interferes
with the method. However, only a “small fraction
[of salicylic acid] is converted to this metabolite”
(8), so that, in vivo, gentisic acid is probably not a
source of interference.
Reduced glutathione is present in whole blood at a
concentration of 280-340 mg/liter (9). When we
added reduced glutathione to serum, we observed
falsely low glucose values with the method. But when
whole blood was intentionally hemolyzed to produce
plasma with 20 g of hemoglobin per liter, we observed
no interference. The intentional hemolysis may have
destroyed the reduced glutathione so it remains a potential
interferant in hemolyzed blood.
Levodopa (L-DOPA) in the concentrations indicated
in Table 4 seriously interferes. These concentrations
of L-DOPA are much higher than have been observed
in vivo. Muenter and Tyce (10) observed peak
concentrations of 0.40 to 7.3 mg/liter of plasma after
0.25- to 2-g doses of L-DOPA, in a study involving 26
patients. In another study with 15 patients (11), peak
concentrations of 1.0 to 4.0 mg/liter of plasma were
observed after doses of 0.25 to 1.9 g. Whether the metabolites
of L-DOPA interfere is an open question; it

Glucose concn
Glucose concn
Concns in pooled serum, in poo1, Concns in pooled serum, in pool,
Substance mg/liter or as stated#{176} mg/liter Substance mg/liter or as stated0 mg/liter
Compounds that do not change the obser,.ed glucose Compounds that interferea
Anticoagulants Ascorbic acid 0
Heparin 75000and 150000 1080
50
units/liter
100
Sodium citrate 5000, 10 000, and
1020
200
20 000
500
Sodium
Sodium
fluoride
oxalate
10 000, 20 000,
40000
2000, 4000, 8000
1070
990
Gentisic acid 0
100
200
Drugs 400
Acetohexamideb 50, 100, 200 1010
800
ChlorpropamideC 50, 100, 200
1080
Phenformind 20, 40, 80 1010
Glutathione 0
Tolbutamidee 50, 100, 200, 400 1100
(reduced) 250
Tolazamidee 50, 100, 200 1080
500
Sodium
salicylate
100, 200, 500, 1000 1070
‘woo
‘Metabo/ites L4)QPAI 0
1110
Bilirubin 50, 100, 200
1160
100
820
Creatinine 500, 1000, 2000
1060
200
620
Hemoglobin
standard
5000, 10 000, 20 000 440
400
530
Urea 500, 1000, 2000 1090
Maltose
0
1070
Uric acid 100, 250, 500
630
1000
1230
Uric acid 200
510, 970, 1940
2000
1380
Sugars
5000
1830
Fructose
Galactose
Lactose
1000 2000
1000, 2000,
1000, 2000,
5000
5000
5000
1030
1020
1010
a Either saline or the indicated compound was added to a serum
pool. For any compound, the actual concentration of glucose in the
particular pool was constant. For the uric acid study, see text.
bEli Lilly and Co., Indianapolis, md. 46206.
Mannose
Sucrose
1000,2000,
1000, 2000,
5000
5000
1030
1070
C Pfizer Inc., New York, N.Y. 10017.
dGeigy Pharmaceuticals, Ardsley, N.Y. 10502.
e The Upjohn Co., Kalamazoo, Mich. 49001.
d-Xylose 1000, 2000, 5000 1080 f Eaton Laboratories, Norwich, N.Y. 13815.
is metabolized by at least three major pathways (12).
The major components excreted in urine are the unchanged
drug, dopamine, and homovanillic acid (13).
Serum Normal Values
Serum from 72 presumed healthy adult volunteers
who had fasted for at least 12 h was examined for glucose
by this method (Table 5). Our median and mean
were both 840 mg of glucose per liter and the histogram
was reasonably gaussian. For comparison, we
have also listed normal values for several methods in
which glucose oxidase but different chromophores
were used. In his review, Free lists normal (reference)
values for glucose in serum as measured by glucose
oxidase methods published before 1962 (14).
Glucose Estimation in Urine
Trinder’s method is also suitable for quantitating
glucose in urine. We added glucose to four different
glucose-free urines to give glucose concentrations of
Table 4. Interferences Study
1300
1220
1110
790
0
1030
830
760
630
570
1240
1100
1000
840
2500, 5000, and 10000 mg/liter. The analytical recovery
was 98-104% (average, 100%). The urinary glucose
estimations were done with the AutoAnalyzer I.
They can be done with the SMA 6/60, but then aqueous
glucose solutions must be used to calibrate the
instrument.
As much as 1.6 g of uric acid in per liter of urine, or
boric acid at concentrations of 0.4, 0.8, and 1.6 g/liter,
or a saturated aqueous solution of thymol do not interfere
with the method.
Stability of glucose in urine. Four patients’ urine
samples were chosen for study on the basis that they
were free of glucose and they contained more than
100 000 organisms per milliliter. Glucose was added
to each urine to prepare samples of each to contain
about 5000 and 10000 mg of glucose per liter. One
gram of boric acid, or about 200 mg of thymol, or
nothing was added to three 100-ml aliquots of each
sample. These 24 samples were analyzed for glucose
with the AutoAnalyzer I immediately after prepara-
CUNICAL CHEMSTRY, Vol. 21, No. 12, 1975 1757

Table 5. Normal (Fasting) Values for Serum
Glucose, by Various Glucose Oxidase Methods
Mean Mean ± 2S0
mg/liter No. Comment
830 750-1080 32 AutoAnalyzer I,
MBTH-D MAO
880 700-1070 32 AutoAnalyzer II,
M BTH-D MA0
920 720-1160 151,age SMA12/60,
20-49 MBTH-DMA0
(fasting?)
1110 840-1280 185,age SMA12/60,
over 50 MBTH-DMA0
(fasting?)
890 670-1120 58 AutoAnalyzer II,
glucose oxidase/
neocuproine
880 660-1110 58 Glucose oxidase/
dianisidine
920 700-1150 58 Glucoseoxidase/
M BTH-D MA0
840 670-1010 72b This method,
AutoAnalyzer I
a Chromophore linked to peroxidase described in ref. 18.
b 61 women, 11 men; age range 20-57 yr (mean, 29).
tion and again after 1, 2, 3, 7, and 14 days of storage
at room temperature. Boric acid is somewhat superior
to thymol as a preservative, although neither is
ideal. The maximum loss in glucose after 1, 2, 3, 7,
and 14 days was 8, 14, 26, 27, and 34%, respectively,
for boric acid and 14, 28, 28, 36, and 54%, respectively,
for thymol. Unpreserved samples lost as much as
40% of their glucose in one day. From these limited
data, we concluded that analysis for glucose in urine
is invalid after the urine has stood for more than 1
day at room temperature, even when thymol or boric
acid is present.
Simplex Optimization of Analytical Conditions
There are many variables in this method: pH, type
of buffer, incubation temperature, concentrations of
the reagents and sample in the final reaction mixture,
sample-to-wash ratio, etc. The simplex method for
optimizing analytical conditions has been described
elsewhere (15-17). We chose to use a 0.1 mol/liter
phosphate buffer because it has been used successfully
by others (18), but our decision was really arbitrary.
A pH of 7.0 was chosen because it is close to
both the pK2 of phosphoric acid (7.13) and the reported
(19) optimal range for glucose oxidase (pH 4.0
to 6.5). The variables we investigated by using the
simplex method were the glucose oxidase and peroxidase
activity and the concentrations of 4-aminoantipyrine
and phenol in the final reaction mixture.
One milliliter each of solutions of glucose oxidase,
peroxidase, and 4-aminoantipyrine were mixed and
6.5 ml of a phenol solution was added. The mixture
was incubated at 37 #{176}C for 10 mm, and the absorb-
1758 CLINICAL CHEMISTRY, Vol. 21, No. 12. ‘75
ance was measured at 505 nm in a Model 2000 spectrophotometer
(Gilford Instrument Laboratories,
Inc., Oberlin, Ohio 44074) vs. a water blank. The op-
Ref. timum sought was the maximum color intensity.
The concentrations of each of the reagents used in
the final reaction volume (9.6 ml) is given in Table 6
18 along with the progress of the simplex. The simplex
has four dimensions and five vertices and was there-
22 fore treated by Long’s calculation technique (15).
The starting concentrations (vertex 1) were deliberately
set far from the presumed optimum, and a step
2 size of 80% of the starting concentrations was used.
2 The simplex study was stopped at 18 experiments,
even though we had not found the optimum. We did
find that glucose oxidase activity is a primary deter-
23 minant of the final color intensity. Glucose oxidase
activity plotted vs. absorbance gives a straight line
with some scatter of the points (correlation coeffi-
23 cient, r = 0.96). The final color intensity is less sensitive
to changes in the concentration of 4-aminoan-
23 tipyrine (r = 0.43) and still less sensitive to changes
in the peroxidase activity (r = 0.27) or the concentration
of phenol (r = 0.14).
The concentrations of the reagents in the solution
entering the 37 #{176}C bath (see Figures 1 and 2) of the
AutoAnalyzer I and SMA 6/60 are also listed in Table
6. The peroxidase activity and the concentrations of
4-aminoantipyrine and phenol are somewhat in excess
of what is needed. The solutions with concentrations
described at vertices 13, 14, and 15 (Table 6)
give nearly the same absorbancies and were obtained
by using about the same amount of glucose oxidase.
A large variation in the peroxidase activity (vertex 13
vs. 15), the concentration of 4-aminoantipyrine (vertex
13 vs. 15) or of phenol (vertex 13 vs. 14) had practically
no effect on the absorbance,
In another experiment, the glucose oxidase activity
was varied, and the concentrations of the other reagents
were the same as described under Materials
and Methods. We found that the sensitivity of the
1.2
1.0
0.8
0
.0 . 0.6
0
.0
0.4
0.2
0
0000
Sc
0o
‘; .!
0
o
0
.
/ 1
,
o = ,/“mg/liter
0
0 / 2000
‘ N0 0 0 -w
.// $000
,/,, .-,
,-
- -
:-
4 5
Log Glucose Oxidose Activity, U/liter
Fig. 3. Absorbance of the 1000, 2000, and 4000 mg per liter
glucose standards after reaction with glucose oxidase/peroxidase
reagent containing increasing amounts of glucose oxidase

Vertex no.
Table 6. Data for Simplex Optimization of Glucose Oxidase Reaction.
Concentrations in Final Reaction Mixture
Glucose oxidase
U/liter
Peroxidase 4-aminoantipyrine Phenol
mg/liter
Absorbance
observed
Vertices retained from
previous simplex
1 417 0.596 26.0 169.2 0.092 -
2 750 0.596 26.0 169.2 0.138 -
3 583 1.009 26.0 169.2 0.113 -
4 583 0.734 43.0 169.2 0.104 -
5 583 0.734 30.2 276.0 0.106 -
6 833 0.941 36.6 223.0 0.139 2, 3,4, 5
7 792 0.906 16.4 250.0 0.129 2, 3, 5, 6
8 896 0.992 22.3 129.7 0.138 2, 3, 6, 7
9 1052 0.709 24.7 216.8 0.148 2, 6, 7, 8
10 973 0.713 38.4 119.4 0.151 2,6,8,9
20 750 0.596 26.0 169.2 0.130
6’ 833 0.941 6.6 223.0 0.145 -
11 1125 1.082 35.0 175.3 0.179 6a,8,9,10
12 1097 0.731 45.1 237.6 0.187 6a, 9, 10, 11
13 1291 0.677 35.0 151.6 0.179 9,10,11,12
14 1308 0.887 31.5 271,3 0.192 9, 11, 12, 13
90 1052 0.709 24.7 216.8 0.155 -
15 1358 0.980 48.6 201.1 0.185 11, 12, 13, 14
Auto- 3200 1.07 80 267
Analyzer
1a
SMA 6/600 2900 0.966 72.4 276
0 Concentrations in final reaction mixtures by proposed method.
method could be altered by simply changing the glucose
oxidase activity, as is illustrated in Figure 3. At a
glucose oxidase activity of 48000 U/liter, the curve
begins to flatten. For the 2000 mg/liter glucose standard,
the absorbance increases by 0.142 when the glucose
oxidase activity is doubled (from 12 000 to
24 000 U/liter). When the glucose oxidase activity is
doubled again (from 24 000 to 48 000 U/liter), the absorbance
increases by only 0.067 for the same standard.
We chose to use 12000 U of glucose oxidase per
liter of reagent because this gave us a linear curve to
4000 mg of glucose per liter and good sensitivity in
the 0-1500 mg/liter region with a 0.2 ml serum sample.
Limits of the Method
Glucose concentration and absorbance are linearly
related to at least 4000 mg/liter on the AutoAnalyzer
I and to 5000 mg/liter of the SMA 6/60. The AutoAnalyzer
I method can be altered to permit analysis of
samples containing as much as 5000 mg of glucose
per liter by reducing the sample line one size, to 0.10
ml/min, but some precision is lost in the low concentration
range results.
With either instrument the method is suitable for
analysis of samples with glucose concentrations of

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SteodVStole Stondords Interoction test ltgh control Low Control
Fig. 4. Recorder tracing from the AutoAnalyzer I
Steady-State tracing obtained by constant sampling of the 4000 mg/liter
standard for 5 mu; Standards are the eight aqueous glucose standards dosctlbed
In the text; Interaction test, tracings for the 1000, 4000, 1000, and
1000 mg/liter standards; b ConVol and Low ConVol replIcate analyses of
two commercial control set-a
dase reagent should be added through the middle
connection
interaction.
of the GO cactus (Figure 1), to minimize
Reagent stability. We examined several different
preservatives for the peroxidase/buffer reagent listed
earlier. This reagent developed a fine sediment after
one to two weeks at room temperature, and after
three to four weeks, sensitivity declined. The reagent
is stable for at least four weeks at 4 #{176}C. We have
found the stability of phosphate buffers to be quite
capricious. Some appear to be stable for months at
room temperature, while mold is growing in other
lots soon after preparation. Apparently the stability
of the buffer is determined by what spores, dust, etc.,
fall into the solution at the time of preparation.
Trig (hydroxymethyl)aminomethane buffers also
showed mold growth.
We investigated three preservatives in some detail.
Sodium azide (4 g/liter) was unsatisfactory; the peroxidase/buffer
reagent became yellow after one week,
and linearity and sensitivity deteriorated after two
weeks. According to Bergmeyer et al. (21), azide inhibits
peroxidase. Thimerosal (Merthiolate), 20 mg/
liter, is unsatisfactory. Results for glucose with the
SMA 6/60 were lower when thimerosal was present in
the final reagent (glucose oxidase/peroxidase reagent)
vs. the reagent without thimerosal. This was
not true of the AutoAnalyzer I; identical sera assayed
with and without thimerosal in the reagent gave the
same results.
Cacodylic acid (dimethylarsinic acid) looked promising
as a preservative because the peroxidase/buffer
reagent containing only 10 mmol of cacodylate per
liter was stable for five months at room temperature.
Unfortunately, reagent with cacodylate gave consistently
higher results on the AutoAnalyzer I with fresh
patients’
late.
sera than did the reagent without cacody-
The 1.5 g/liter phenol solution was colorless and
1780 CLINICAL CHEMISTRY, Vol. 21, No. 12 ‘75
free of sediment after six months of storage at room
temperature in amber-colored glass bottles, and
could not be distinguished from a freshly prepared
phenol solution when used in conjunction with the
other reagents for glucose.
We thank Drs. H.-D. Gruemer and G. F. Grannis for helpful
comments on the manuscript, and Rita Beal, B. W. Durham, Joan
Mercier, Kathy Rieger, and Tim Walters for technical assistance.
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