Download Nature of Materials in Serum That Interfere inthe Glucose Oxidase

CLIN.CHEM.21/1, 119-124 (1975)
Nature of Materials in Serum That Interfere in the
Glucose Oxidase-Peroxidase--o- Dianisidine Method
for Glucose, and Their Mode of Action
Walter J. Blaedel and James M. Uhi
Separation
of blood serum on Sephadex
G- 100 reveals
three fractions that interfere with the glucose oxidaseperoxidase method for serum glucose when o-dianisidine is used as the chromogen. A low-molecular-weight
fraction containing primarily uric acid, a fraction containing protein with a molecular weight of about 40 000,
and a fraction of even higher molecular
weight (-.‘
500 000) each interfered with glucose recovery when
glucose was measured by this procedure. The uric acid
fraction and the isolated 40 000 molecular weight fraction interfere by competing with o-dianisidine for hydro-
gen peroxide in the peroxidase-catalyzed color-formation step. The high-molecular-weight fraction not only interferes with the peroxidase reaction, but also with the
glucose oxidase reaction itself. These agents cause
values to be low by as much as 20% in the manual determination of glucose in normal serum if thejr interference is not recognized.
AddItional
Keyphrases:
molecular-weight
materials
interference by uric acid and high#{149}
interfering material in urine
Keston (1) first conceived an enzymatic
method
for determining
glucose in biological fluids, in which
the glucose oxidase1 reaction is coupled to the peroxidase reaction in the presence of a chromogen.
Use of
o- dianisidine
in a quantitative
procedure
was reported by Teller (2) shortly thereafter.
Since then,
many glucose oxidase methods, both manual and au-
Department
Wis.
I
of Chemistry,
University
of Wisconsin,
Madison,
53706.
Terminology
reductase,
reductase,
1.7.3.3; and
Received
used:
glucose
oxidase,
l-D-glucose:oxygen
EC 1.1.3.4; peroxidase,
donor:hydrogen
peroxide
EC 1.11.1.7;
uricase,
urate:oxygen
oxidoreductase,
o- dianisidine,
3,3’-djmethoxybenzidine.
July 25, 1974; accepted
Oct. 21, 1974.
oxidooxidoEC
tomated, have been reported. About 20 references to
methods reported for clinical use appear in a paper
by Miskiewicz
et al. (3).
Attempts
to apply this method directly to plasma
or serum samples have not given highly precise results, owing to interferences
that are present in varying amounts. Plasma has been shown to contain inhibitors of glucose oxidase methods (4, 5), which are
generally removed by protein precipitation
(4, 6) or
extreme dilution
of serum (5, 7) in manual methods.
In automated
methods, serum is generally
dialyzed
against a buffer solution to remove the glucose from
the protein serum matrix (8-10), sq that interfering
material
of high-molecular-weight
is excluded.
Because automated
methods are most frequently
used
for routine analysis, the interferences
most often discussed are those caused by substances of low molecular weight, such as uric acid, ascorbic acid, and bilirubin. A recent study (3) showed that uric acid was the
only such low-molecular-weight
substance having a
significant
effect on determinations
of glucose in normal or slightly above-normal
physiological
concentration. Interference
by uric acid has been reported by
many workers and possible mechanisms
for it have
been proposed (8, 11, 12).
In early work on an electrochemical
method for tollowing the glucose oxidase reaction (13), a high-molecular-weight
serum fraction
separated with a column of Sephadex G-50 was found to inhibit glucose
oxidase. Separation
of serum by use of Sephade,
G100 has revealed two separate high-molecular-weight
fractions
and a low-molecular-weight
fraction,
each
of which interfered
with the glucose oxidase-peroxidase method. Preparations
of each of the fractions
CLINICAL
CHEMISTRY,
Vol. 21. No. 1, 1975
119
were tested for their effects on each step of the method. On the basis of the results obtained from these
tests, a mode of action of each of the interferences
is
proposed.
Materials and Methods
Apparatus
Visible absorbances at 460 nm were measured with
a Spectronic
88 spectrophotometer
(Bausch & Lomb,
Rochester, N. Y. 14602).
Ultraviolet absorbance studies of uric acid were
performed
with a Cary 14 double-beam
recording
spectrophotometer
(Applied
Physics Corp., Pasadena, Calif.).
The oxygen electrode used was a Clark-type
oxygen sensor, consisting
of a platinum
wire (20 am in
diameter),
heat-sealed
into a glass tube. The end of
the glass tube was ground off and polished, exposing
a 20-am cross-section
of the wire, which was covered
with a polypropylene
membrane
(0.001 inch thick).
The internal
reference
electrode
was silver-silver
chloride.
The platinum
microelectrode
was held at
-0.5 V with respect to the reference electrode. Currents were measured with a Model 414 S picoammeter (Keithley
Instruments,
Inc., Cleveland,
Ohio
44139) and recorded on an Omniscribe
chart recorder
(Houston Instruments,
Bellaire, Tex. 77401).
Rate measurements
were carried out in a 4.5-mi
cylindrical
cavity in a Plexiglas block equipped with
a small magnetic stirrer. Solutions
were added and
withdrawn
by means of hypodermic
syringes through
ports, which could be sealed to prevent access of air
to the reaction cavity.
Reagents
Glucose oxidase solutions of two different
concentrations (70 U/mI and 5 U/mI) were prepared from a
powder having a glucose oxidase (EC 1.1.3.4) activity
of 110 U/mg (Worthington
Biochemical
Corp., Freehold, N. J. 07728).
A reagent solution containing,
per liter, 100 mg of
o- dianisidine
dihydrochloride
and 3000 U of horseradish peroxidase
(EC 1.11.1.7; Worthington)
was prepared in 0.1 mol/liter
phosphate
buffer (12.814 g of
KH2PO4 and 1.02 g of K2HPO4 in 1 liter of solution,
adjusted to pH 6.0 with concentrated
KOH).
A 50 U/liter solution of uricase (urate oxidase; EC
1.7.3.3) was prepared by dissolving 0.0197 g of crude
uricase (17 U/g; Sigma Chemical Co., St. Louis, Mo.
63178) in 5 ml of 0.1 mol/liter
borate buffer (0.6184 g.
of H3B03 in 100 ml of solution, adjusted to pH 8.5
with concentrated
KOH).
Glucose standard solutions
(100 mg/100 ml) were
prepared by dissolving 0.1000 g of anhydrous glucose
in 100 ml of water. Two uric acid solutions,
7.0 mg/
100 ml and 4.2 mg/100 ml, were prepared by dissolving 0.0070 g and 0.0042 g of the solid, respectively,
in
100 ml of warmed water. A hydrogen peroxide solution having a concentration
of about 0.2 mmol/liter
120
CLINICAL
CHEMISTRY.
Vol. 21, No. 1, 1975
was prepared by two successive 250-fold dilutions
of
a 30% solution of hydrogen peroxide. All of the above
chemicals
were reagent grade.
Samples of pooled serum and “Q-Pak Automated
Chemistry Control Serum” (Hyland Div. Travenol
Laboratories,
Inc., Costa Mesa, Calif. 92626) were
used in the initial chromatographic
separations.
The
Hyland control serum was used in the preparative
separation of the two high-molecular-weight
frac-
tions, because we found it contained
greater
concentrations
of each interference
than did the pooled
serum.
For interference
studies, solutions of the two highmolecular-weight
fractions were used directly as collected
from
the preparative
separation
outlined
below.
Procedures
Serum was separated by gel-chromatography
on a
27.5 cm X 1.7 cm column of Sephadex G-100 (Pharmacia Fine Chemicals, Inc., Piscataway,
N. J. 08854).
To prove the presence of the various
interfering
agents, 3-ml samples of serum were separated
and
eluted at about 1 ml/min with phosphate buffer (0.1
mol/liter,
pH 6.0). After the first 20 ml was eluted,
5.0-ml fractions were collected. Each fraction was divided into two 2.0-ml aliquots. To each aliquot, 2.0
ml of peroxidase-odianisidine
reagent solution was
added. To one of the aliquots, 0.1 ml of standard glucose was added, to the other 0.1 ml of the phosphate
buffer. To initiate
the reaction, we added 0.1 ml of
glucose oxidase solution
(5 U/ml). The reaction was
allowed to proceed for 5.0 mm at 25 #{176}C,
and was then
quenched by adding 0.1 ml of KOH (400 g/liter). Absorbances were read within 10 mm at 460 nm vs. a reagent blank.
We calculated glucose recovery in each fraction by
taking the difference between the absorbances developed in the two aliquots and dividing by the absorbance developed in a reaction mixture containing
only
0.1 ml of glucose standard solution and the enzyme
reagents.
We made preparative
separations
of the two highmolecular-weight
fractions by adding 6.0-mi samples
of serum to the Sephadex G-100 column. The highmolecular-weight
fraction was collected between 20.5
and 27.5 ml of eluent, which corresponded
approximately to a 1.25-fold dilution
of the interfering
protein as compared
to its concentration
in the blood
serum. The 40 000 molecular-weight
fraction was collected between 30.0 and 44.0 ml, which approximately corresponded
to a 2.5-fold dilution
as compared to
its concentration
in the blood serum. A clean separation was obtained between the two fractions
by discarding some of the eluate between the two fractions.
The low-molecular-weight
fraction
was prepared
by dialyzing
3.0 ml of serum vs. 3.0 ml of the phosphate buffer for 2 h, by using a piece of Spectrapor
Type 2 dialysis membrane
(Spectrum
Medical Industries, Los Angeles, Calif. 90054). Presence of uric acid
in the dialysate
olet absorbance
was checked
at 290 nm.
We determined
by measuring
its ultravi-
the effect of interferences
on the
Table 1. Glucose Recovery Experiment’
Fraction
overall glucose
oxidase-peroxidase
system by reacting a series of solutions containing
a fixed amount of
glucose and various amounts of each interfering
fraction, with all other variables held constant. Each solution of the series consisted of 1.5 ml of chromogenperoxidase
reagent solution, 0.2 ml of standard glucose solution,
interfering
solution,
and sufficient
phosphate
buffer to make 4.0 ml (3.0 ml for the uric
1
2
3
4
Results and Discussion
Isolation of Interferences
Table 1 shows the glucose recovery data for the
fractions collected in a Sephadex G-100 separation of
pooled blood serum. The low recoveries in fractions
1-5 and 10-13 indicate the presence of two groups of
interferences.
Fractions
1 and 2 were cloudy and uncolored, distinctly
different
from fractions 3-5, which
were yellow. Glucose was eluted in fractions
8-11,
overlapping
the low-molecular-weight
interferences
in fractions 10-13.
,,
Glucose
recovery,
%
20-25
25-30
30-35
69
74
67
35-40
65
5
40-45
85
6
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
100
98
104
100
57
40
26
77
7
acid studies). The reactions were carried out and recoveries calculated as described above.
The effect of an interference
on the rate of the glucose oxidase reaction alone, independent
of the coupled peroxidasereaction, wasmeasured by noting the
rate of decrease in dissolved
oxygen concentration
with a Clark-type
oxygen electrode. For a rate measurement,
0.5 ml of standard glucose solution and a
measured quantity
of the interfering
fraction
to be
tested were added to the reaction chamber (see section on Apparatus),
which was then filled with the
phosphate buffer to give a total volume of 4.5 ml. To
start the reaction, we introduced
glucose oxidase (0.2
ml of 70 U/ml solution)
into the reaction chamber
with a syringe, and followed the fall-off
in current
caused by oxygen consumption.
Initial rates were calculated from the slope of the current-time
curve between 0 and 3 mm, divided
by the initial
current,
which gave the fraction of the oxygen originally
present that was consumed per minute. This procedure
corrected for run-to-run
variations
in electrode
sensitivity, and for oxygen originally
present.
The effect of an interference
on the peroxidase
step of the method alone, independent
of the glucose
oxidation
step, was evaluated
by measuring
the absorbance developed at 460 nm by 0.5 ml of hydrogen
peroxide
solution
when combined
with peroxidaseo- dianisidine
solution in the presence of the interference. Concentration
of interfering
material and chromogen concentration
were independently
varied in
separate
experiments,
the total
reaction
volume
being adjusted to 4.0 ml with the phosphate buffer. It
was not necessary to quench these reactions
with
KOH because, for the small quantities
of H202 involved, the reaction proceeded to completion
in less
than a minute.
Eluent
volume, ml
no.
8
9
10
11
12
13
3.0 ml of pooled
G-100, and fractions
Additional
serum
then
was chromatographed
used
in glucose
chromatographic
recovery
studies
on Sephadex
experiments.
with
Sepha-
dex G-100 and Sephadex G-200 showed that fractions 1-5 (Table 1) contain at least two different
interfering
substances. The molecular
weight of interference in fractions
3-5 was 40 000 to 45 000, as determined by the ratio of elution volume to void volume in several chromatographic
experiments.
The
molecular
weight of the cloudy fractions
is much
higher, greater than 500 000. We made no further at-
tempts to identify these two materials more specifically, although they presumably
are proteins.
The ultraviolet
absorption
spectrum
of a dialyzed
serum sample containing
the low-molecular-weight
interfering
material was very similar to that of a 0.1
mmol/liter
solution
of uric acid in the phosphate
buffer. In addition,
treatment
of another serum sample with uricase before dialysis removed ultraviolet
absorbance
in the dialysate entirely.
Also, after uncase treatment
of this dialysate,
interference
in the
glucose determination
was decreased to about a tenth
of that observed for serum dialysate not treated with
uricase. Because uric acid evidently
accounts for almost all of the low-molecular-weight
interference
observed, we studied the nature of its interference
by
using solutions of uric acid itself rather than dialysates.
Urine, when subjected to the same chromatographic procedure,
gave early fractions
(corresponding
to
high-molecular-weight
substances)
that did not interfere at all with glucose recovery.
On the other
hand, fractions corresponding
to 10-13 in Table 1 interfered severely and caused glucose recovery values
to be only about 10% of the true figure, an effect we
ascribed to high concentrations
of uric acid in the
urine.
Effects of Interferences
Figure
creasing
on the Overall Method
1 shows the effect of the presence of inamounts
of each interfering
substance on
CLINICAL
CHEMISTRY,
Vol. 21, No. 1, 1975
121
GLUCOSC+ 02
GLUCOSE OXOASE
C
GlUCONATE +
.!i
PEROXIDASE
U
0
H202
+0-DIANISIDINE
t
#{174}
uJ
0
2 I-lO+OXlDlZED
CHROMOPHORE
#{174}
Fig. 2. Possible sites of interference in the glucose oxidaseperoxidase-o-dianisidine
u-i
u-i
SItes of interference
system
are Indicated
by arabic numerals
0
Lu
Lu
U.)
0
Nature of Uric Acid Interference
0
2
ID
INTERFERENCE
ADDE
ML
Fig. 1. Relation between glucose recovery and increasing
amounts of each interfering fraction present in a reaction
mixture containing 0.1 ml of glucose solution (1 g/liter)
A, UricacId (4.2 mg/100 ml). 8, 40000 molecular weight chromatographic
traction. C. Higher molecular weight chromatographic fraction
the overall glucose oxidase-peroxidase
reaction. Uric
acid interferes quite strongly: 0.1 ml on the x- axis of
Figure l#{192}
is typical of the amounts of uric acid and
glucose found in 0.1 ml of normal serum, and this
amount causes recovery to be less than 80%, corresponding to a decrease in the apparent value of more
than 20%. The effect of the fraction of 40 000 molecular weight is shown in Figure 1B; 0.25 ml of added in-
terfering
solution,
typical
of the amount that would
be found in 0.1 ml of normal serum, causes a 77% recovery, again corresponding
to a decrease in the apparent value of more than 20%. The 500000 molecular weight fraction
gives a small but measurable
effect (Figure 1C), causing a relative error of about 3%
in normal serum.
Possible Modes of Interference
Five possible ways or modes in which interferences
could affect the glucose oxidase-peroxidase-o
-dianisidirie system are listed below and summarized
in
Figure 2.
1. Inhibition
of the glucose oxidase enzyme.
2. Nonenzymatic
destruction
of the H202 produced in the glucose oxidase reaction.
3. Peroxidase-catalyzed
chemical
reaction
with
H2O2, in competition
with o- dianisidine.
4. Inhibition
of the peroxidase enzyme.
5. Destruction
(bleaching)
of the oxidized form of
o- dianisidine.
In the following sections, experimental
evidence is
presented to elucidate and to test the nature of each
of the three previously
described interferences.
122
CLINICAL
CHEMISTRY,
Vol. 21, No. 1, 1975
The presence
of as much as 1.0 ml of uric acid solution (42 mg/liter)
in the reaction chamber had no effect on the rate of glucose oxidase reaction as measured with the oxygen electrode.
Because half this
amount of uric acid elicited only 30% recovery in the
full coupled method, Mode 1 (above) is ruled out. On
the other hand, uric acid had a marked effect on the
peroxidase reaction. Chromogen formation
in an
H2O2-peroxidase-o- dianisidine
system
decreased
with increasing amounts of added uric acid, the relationship between absorbance and uric acid concentration closely resembling the curve of Figure l#{192}.
Interference
Mode 2 was ruled out when it was
found that the order of adding H202 and peroxidase
to a uric acid buffer system made no difference in the
final absorbance
reached. If HO2
were destroyed
nonenzymatically
by uric acid, less absorbance would
be expected in those reactions
in which H202 and
uric acid were allowed to mix before addition
of per-
oxidase.
To test Mode 3, we increased the amount of peroxidase-o- dianisidine reagent in a series of solutions,
uric acid and H202 being held constant. The resulting absorbance increased correspondingly,
as shown
in the second column of Table 2, indicating
that uric
acid interferes
by Mode 3. The nonlinear
increase in
absorbance with increasing chromogen concentration
is expected for a system in which two reactants (chromogen and uric acid) compete for a constant amount
of H202 substrate.
Further evidence for Mode 3 was obtained by noting that the absorbance changes in Table 2 did not
occur in the absence of uric acid. We also found that
uric acid was consumed during the interfering
process, as indicated
by a decreased absorbance of the
reaction mixture at 290 nm (where o- dianisidine
does
not absorb) after the reaction. The decrease in uric
acid concentration
was accompanied
by a corresponding decrease in absorbance at 460 nm (where
the oxidized chromophore
absorbs). This could be explained by reaction of uric acid with H202, which in
turn causes decreased reaction of H202 with the odianisidine.
Inhibition
of peroxidase (Mode 4) could not be observed because the peroxidase reaction was so rapid.
For the glucose quantitation
procedure used here, the
peroxidase activity was made sufficiently
high to give
creased by increasing
Table 2. Relation Between Amount of Chromogen
and Final Absorbance in the Presence of Uric
Acid or 40 000 Molecular Weight Interfering
Material’
o.Dianisidine.
peroxidase
reagent, ml
Absorbance
with 0.2 ml Absorbance
of uric acid (7 mg/i00
ml) present
0.5
1.0
1.5
2.0
0.175
0.248
0.273
0.342
with 1.0 ml
of 40000 mel wt fraction
present
0.091
0.136
-
0.191
Absorbance
at 460 nm developed
by 0.5 ml of H,O, (0.2 mmol/
liter), the noted volumes of interfering
material,
and dianisidineperoxidase reagent, plus buffer to make a total volume of 4.0 ml.
Table 3. Effect of the High-Molecular-Weight
Inhibitor on the Rate of the Glucose Oxidase
Reaction’
Vol of
inhibitor
solution,
ml
0
0
0
1.0
2.0
Initial
slope,
pA/mm”
88
98
92
74
58
Initial
current,
pA’
486
536
511
507
519
Reaction
rate,
oxygen used
per mm
%
18.1
18.3
18.0
14.6
11.2
“Reaction
mixture consists of the indicated
volume of inhibitor
solution,
0.5 ml of glucose (1 mg/mI),
0.2 ml of glucose oxidase
(70 U/mI) and buffer to give a total volume of 4.5 ml.
From a current-time
curve obtained
with a Clark-type
oxygen
electrode
immersed
in the reaction
mixture.
Initial
currents
given are corrected
for background
currents
of 6-11 pA.
the concentration
of the 40 000
molecular
weight protein
present
in H202-peroxidase-o- dianisidine
systems.
The third column of Table 2 shows that the absonbance developed in a solution containing
a fixed
amount of the 40 000 molecular
weight protein
increases with the amount of peroxidase-odianisidine
reagent. No such absorbance increases were observed
in the absence of the interfering
material.
These results indicate that the way in which this fraction interferes is the same as that in which uric acid interferes (Mode 3). Similarly,
neither nonenzymatic
destruction
of H202 nor bleaching
was observed, and
any inhibition
of peroxidase was not great enough to
be significant.
Nature of the Interference by the Fraction
Containing Protein of 500 000 Molecular Weight
When
the
glucose
oxidase reaction
was followed
increasing amounts of interference
by this high-molecular-weight
fraction
caused the rate of the reaction to decrease markedly.
Table 3 summarizes data for three rate measurements
made
in the absence of this high-molecularweight inhibitor
(to check the reproducibility
of the
measurement
procedure)
and in the presence of two
different
concentrations
of the inhibitor.
Table 3
with the oxygen electrode,
(last column) shows a linearly decreasing reaction
rate with increasing
concentration
of the interference, indicating
that the high-molecular-weight
material is a glucose oxidase inhibitor operating through
Mode 1.
The above experiment
does not rule out the possi-
bility that catalase, as an impurity
immediate chromogen oxidation
by the H202 produced by glucose oxidation.
Thus, uric acid inhibition
of peroxidase is not completely
ruled out by the preceding experiments-its
inhibition
could be masked
by the large excess of peroxidase used.
Chinh (12) observed that uric acid caused bleaching (Mode 5) when 2,2’-azine-diethylbenzthiazoline
sulfonic acid was used instead of o- dianisidine.
When
uric acid was added to the H202-peroxidase-odianisidine system after color development
was complete,
no such bleaching
was observed,
indicating
that
Mode 5 was inoperative.
Nature of the Interference by the Fraction
Containing Protein of 40 000 Molecular Weight
Experiments
similar
to those for uric acid were
performed
by using the chromatographically
separated fraction containing
the protein of 40 000 molecular weight, concentrated
by as much as eight-fold
as
compared
with its concentration
in normal serum.
Results were similar to those observed for uric acid.
We saw no effect on the rate of the glucose oxidase
reaction as followed with the oxygen electrode. Interference with the peroxidase
reaction alone was in-
in
the glucose oxi-
dase preparation,
is the target of the interfering
agent. Activation
of catalase by the high-molecularweight material would appear to cause an inhibition
of glucose oxidase. Because cyanide is known to inactivate
catalase
but not glucose oxidase,
we repeated
the experiment
in the presence of 1 mmol of NaCN
per liter.
We obtained the same results as before,
confirming
that the interfering
fraction
actually inhibits glucose oxidase, and not artifactually
through
an impurity
in the catalase.
Some additional
work was done that indicated that
the high-molecular-weight
fraction
also interfered
with the peroxidase
reaction. Two milliliters
of the
fraction
(corresponding
to a concentration
about 15fold greater than that encountered
in serum), was
added to H202-peroxidase-.-odianisidine
mixture,
and the resulting
absorbance
was 84% of that observed in the absence of the interference.
This degree
of interference,
when combined with that operating
on the glucose oxidase reaction alone, accounts for all
of this interference
by the high-molecular-weight
fraction, as measured directly on the overall reaction.
The mechanism
of interference
with the peroxidase
reaction was not resolved further, mainly because the
turbidity
of the fraction made quantitative
measurements difficult.
CLINICAL
CHEMISTRY,
Vol. 21, No. 1, 1975
123
This work was supported
in part by the National
Science Foundation (Grant GP-40694
X). During
1973, J. M. U. was the recipient of a Du Pont Summer
Fellowship.
Special thanks go to Ronald
Laessig, Director,
Wisconsin
State Laboratory
of Hygiene,
for providing the serum samples,
and to Professor
Stuart Updike, University of Wisconsin
School of Medicine,
for helpful discussions
and
for the use of the oxygen electrode.
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A. S., Colorimetric
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reagents
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of
serum
or plasma
glucose.
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American
Chemical
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