SimpleAutomatedDeterminationof Serumor PlasmaGlucose
by a Hexokinase/Glucose-6-Phosphate
DehydrogenaseMethod
William E. Neeley
A simple, fast, efficient, and accurate automated
method is described for determining the quantity
of glucose in 50 l of serum or plasma. A hexokinase/gI ucose-6phosphate dehyd rogenase reac
tion requiring incubation at room temperature is
used. The method is specific for glucose, and is
not affected by increased concentrations
of
endogenous chemical substances in uremic sera
or by several common drugs that interfere with
other glucose methods. Results of parallel assays,
in which glucose oxidase and o-toluidine are used,
are compared.
Additional Keyphrases
dine
methods
AutoAnalyzer
.
globin, bilirubin,
drugs,
normal
incubation
range
#{149}
glucose
oxidase
glucose
by hemo-
.
lipemia,
reducing
at room
temperature
(6).
hexokinase
(ATP:
EC 2.7.1.1) and
From the Institute
of Pathology,
Case Western
versity School of Medicine, Cleveland, Ohio 44106.
Received Dec. 29, 1971; accepted Mar. 20, 1972.
+ ATP
Glucose-6-phosphate
+ ADP
hexokinaae> glucose-6-phosphate
+ NADP
gIucose-6-phoaphate
dehydrogenase
.......,..
NADPH + 6-phosphogluconate
(Absorbs at
340 nm)
Reserve
Materials and Methods
Instrumentation
sugars
Most clinical laboratories
determine
serum glucose by methods
based on its reducing properties
(1). These nonspecific
methods
also measure various reducing substances
(2). In contrast, enzymatic
procedures
are highly
specific.
Glucose
oxidase
(fl-n-glucose:
oxygen oxidoreductase,
EC 1.1.3.4)
has been coupled through
peroxidase
(donor: hydrogen-peroxide
oxidoreductase,
EC 1.11.1.7)
to
different
chromogenic
acceptors,
including
o-toluidine, o-dianisidine,
and potassium
ferrocyanide
(3-5). The first two acceptors
are carcinogens;
all three are subject
to numerous
interferences
In the proposed
method,
D-hexose
6-phosphotransferase,
Glucose
and o-tolui-
specificity for
comparative
interference
compared
glucose-6-phosphate
dehydrogenase
(D-glucose-6phosphate
: NADP
oxidoreductase,
EC 1.1.1.49)
are used, together with improved
instrumentation,
to provide a new and efficient system for glucose
analysis that is virtually
free from nonglucose
interference
from substances
in blood. The reaction
sequence is
Uni-
An AutoAnalyzer
Sampler
II, Proportioning
Pump II, and 6-inch dialyzer
(Technicon
Instruments Corp, Tarrytown,
N. Y. 10591) were used.’
Absorbance
was read by using a Model 300 N spectrophotometer
with a one-piece debubbler
flow cell
(Model 3019-A) made of Kel-F (all from Gilford
Instrument
Laboratories,
Oberlin,
Ohio 44074).
The output
was recorded
on a Model IR-18 M,
10 mV, 10-inch servo chart recorder
(Heath Company, Benton Harbor, Mich. 49022).
The spectrophometer’s
signal to the recorder
was dampened
with a low-pass filter by use of a
variable
resistor set at 8 k (“E-Z trim potentiometer,”
10 k,
0.5 W; Bourns
Co., Riverside, Calif. 92507) placed in series on the positive
line, followed by a 100-mF, 6-V capacitor
placed
in parallel to the recorder
inputs (Figure 1). The
1 The
6-inch U-groove dialyzer was obtained as subassemblies
No. 177-B008 and 177-0047 for the dialyzer bracket, and No.
157-0242 for the torque screws. Premounted
Cuprophane
membranes were obtained
as No. 170-0406-03.
CLINICAL CHEMISTRY, Vol. 18, No. 6, 1972 509
Reagents
MIMIN
Ta SAMPISI
WASH
Slut
IA
5*1151
Fig. 1. Manifold for glucose analyses. Coils A and B are
described in text
recorder
was set to a full-scale
deflection
of 1.0
absorbance.
Manifold.
Standard
Technicon
Tygon proportioning
pump tubing was used (Figure
1). Mixing and incubation
coils were made of polyethylene
tubing 0.062 inch i.d. (No. 21852; Bel-Art Products, Pequannock,
N.Y. 07440). Coil A was 125
cm long, with about 20 turns. Coil B was 245 cm
long, with 40 turns. Coils A and B together
provided a 7-mm incubation
and mixing period at
room temperature
before the samples reached the
spectrophotometer.
These coils were easily made
by wrapping
the tubing around a test tube 9/16
inch o.d., taping the ends securely with masking
tape, and then immersing
them in boiling water
for 2 mm, followed by a cold-water
rinse (7). A
245-cm coil of this type is unbreakable,
and costs
less than 10 cents. Alternatively,
glass coils may
be used, but must be long enough to allow the
7-mm incubation
period.
A 51-cm polyethylene
tube (0.034-inch
i.d.) was used to carry the enzyme
reagent to the manifold pump tube, to allow adequate time for the reagent to reach room temperature.
Technicon
connector
assembly No. 177-B004-02
was used to introduce
sample and diluent introduction,
and No. 177-B004-01
to introduce
enzyme. These connectors
produce a short and regular bubble pattern,
and do not require the use of
the “air bar” included
on Technicon
Proportioning Pump III.
The dialyzer
membrane
was changed
weekly.
Sample and recipient
streams should flow through
the dialyzer
at about the same speeds, to obtain
optimum peak heights and sample separation.
A PC-i glass pressure-chamber
(Technicon)
was
placed in the flow cell pull-through
line, to smooth
the sample peaks.
510 CLINICAL CHEMISTRY, Vol. 18, No. 6, 1972
All chemicals used were reagent grade, and only
demineralized
water was used. The enzyme
reagents are available commercially
in pre-mixed dry
mixtures
as “Glucose Stat-Pack,”
“Glucose
FastPack,” or “Glucose
500-Pack”
(Calbiochem,
San
Diego, Calif. 92112).
Sample
diluent. 9.0 g of NaCl was diluted to 1
liter with water, and 0.9 ml of “Triton
X-405”
(Technicon)
was added.
Reagent
diluent.
Triton
X-405,
0.24 ml, was
added to 250 ml of water.
Enzyme reagent. The NADP reagent was diluted
with the reagent
diluent listed above in the volume specified by the package instructions,
and was
added to the glucose reagent. It was mixed gently
(no shaking).
The reconstituted
reagent was kept
at 4#{176}C
in an ice bath.
Glucose standards.
Appropriate
amounts
of anhydrous
glucose were weighed out and diluted to
1 liter in a volumetric
flask with benzoic acid solution (2.5 mg/liter).
The standards
were prepared
in 50 mg/100 ml intervals
from 50 mg/100 ml to
350 mg/100
ml. The temperature
of the benzoic
acid solution
was adjusted
to the temperature
listed on the volumetric
flask.
The following
glucose methods
were chosen for
comparison
with the proposed method because they
represent
various commonly
used procedures,
each
of which is based on different
chemical
reactions
with glucose. The Sigma modification
of Hall and
Tucker’s
automated
glucose oxidase/peroxidase/
ferrocyanide
method
was used, because it represented a different
type of enzymatic
analysis and
because
it requires
no known carcinogenic
compounds
(5, 8). As a simple procedure,
the direct
o-toluidine
method was used as described
by the
AACC (9). Automatic
pipets (Eppendorf;
Brinkman Instruments,
Inc., Westbury,
N.Y. 11590)
and automatic
dilutors
(“Repipet”;
Labindustries, Berkeley,
Calif. 94710) were used with the
o-toluidine
method.
Glucose
analyses
were performed
on (a) 20
hospitalized
patients
selected
at random,
(b) 10
patients
in uremia (pre-dialysis),
and (c) five different commercially
available quality-control
sera.
Bilirubin,
creatinine,
uric acid, and blood urea
nitrogen
(BUN)
were assayed,
in the case of the
uremic patients,
with an SMA-12/60
(Technicon).
Pharmaceutical
tablets and capsules were used
in the in vitro drug interference
tests, except for
L-dopa and ascorbic acid, which were obtained
in
pure form. Test solutions were prepared
by diluting an appropriate
amount of each substance
and
1.000 g of glucose to 1 liter in a volumetric
flask.
If the drug was insoluble in water, either 1 N sodium hydroxide
or 1 N hydrochloric
acid was used
to effect solution. The final pH was then adjusted
to about 7.0.
Dextran
solution was prepared
by diluting
170
ml of “Dextran-70”
(“Macrodex”;
Pharmacia
Laboratories,
Piscataway,
N.J. 08854), 60 g/liter,
in saline, along with 1 g of glucose to 1 liter. The
source of bilirubin was 5-ml quantities
of lyophilized bilirubin
standards
in albumin
(American
Monitor
Corp., Carmel,
Ind. 46032). The bilirubin standard
was dissolved in 2.5 ml of demineralized water. Exactly
2.00 ml of the bilirubin
solution was added to 2.00 ml of a solution containing
200 mg/100
ml of glucose, to give a final glucose
concentration
of 100 mg/100 ml. Hemoglobin-containing samples were prepared by washing erythrocytes 10 times in physiological
saline, followed by
lysis in demineralized
water. Exactly 1 g of glucose
was added to the hemoglobin
solution before dilution to 1 liter. The quantity
of hemoglobin
was
determined
by the cyanmethemoglobin
method
(10). All sera, test solutions,
and standards
were
analyzed four times by each of the three methods.
o-toluidine
method,
with no dialysis,
and their
manual
.o-toluidine
method
were slightly
lower
than values
obtained
by a manual
hexokinase
method. In their manual hexokinase
method essentially the same reagents
were used as in the proposed automated
hexokinase
method.
Regression
equations
correlating
the two methods
with the
proposed hexokinase
method are given in Table 2.
Yee et al. (12) correlated
their automated
o-toluidine method by using dialysis to a manual hexokinase method and found r = 0.997 with the regression
equation:
y (hexokinase)
=
1.03 x =
± 6.7,forn
=
30.
Table 1. Glucose Analyses for Sera
from 20 Randomly Selected Patients
Method
Glucose
Hexoklnss.
oxidase
mg/100 ml
84
Results and Discussion
86
156
153
131
353
255
83
205
186
Linearity
Beer’s law was obeyed for glucose concentrations up to at least 300 mg/100
ml (Figure
2).
Correlation
between
glucose
concentration
and
absorbance
was excellent (r = 0.9999) at a sample
rate of 60/h with a sample-to-wash
ratio of 1:1.
Accuracy
was determined
by comparing
results
of the proposed method to those of other accepted
methods
(Table 1). Statistical
comparisons
demonstrate excellent agreement
between the hexokinase
and glucose oxidase methods. The direct o-toluidine
procedure
yielded slightly lower results than either
enzymatic
procedure.
Sudduth
et al. (11) similarly
reported
that results
by both their automated
125
351
342
260
96
96
257
81
199
179
94
168
165
83
102
165
83
102
83
192
92
84
189
184
81
181
82
182
78
78
82
208
182
z
79
92
81
82
176
176
87
80
165
74
78
163
143
143
138
Mean deviation
Standard deviation
Paired
0.40
±2.2
Mean
Critical
value oft =
a = 0.05,D.F. = 19
.5
83
143
133
83
100
Accuracy
o-Toluldine
4.9
±4.8
4.5
2.09
0.8
2.09
0
(H,
w
U
z
.4
0
.2
Table 2. Regression Equations for Present
Method vs. Two Other Methods for Glucose
(I,
4
Glucose
oxidase
60/H
Fig.2.Actual tracing
Randomly
patients
Method
1:1
of 100,200,300,and 400 mg/100 ml
glucose standards. Sample carryover is negligible as
demonstrated by the sequence of low-high-low peaks.
Steady state for the 300mg/100 ml standard is shown
y=
S,,.,
r
=
o.Toluidine y=
=
selected
(n = 20)
Uremlc
(n
=
0.9995
0.950x+4.01
±1.53
0.9971
0.969x-0.515
1.Olx-4.86
0.997x+0.900
±2.16
8,,=
±4.18
±2.05
r
0.9982
0.9954
=
patients
10)
CLINICAL CHEMISTRY,
Vol. 18, No. 6, 1972 511
Table 3. Analyses for Glucose in 10 Uremic Sera, with Analyses for
Potential Interfering Substances
Glucose
Hexokinase
159
103
94
120
method
Glucose
oxldase
o-Toluidin.
BIlIrubin
mg/100 ml
C!fatinlne
0.5
0.4
0.3
13.9
19.2
96
158
97
91
117
112
0.3
15.9
18.0
14.5
12.7
17.7
154
103
15.0
87
84
113
113
83
109
0.4
0.2
106
82
104
105
0.2
81
0.3
126
116
125
114
80
123
112
0.3
22.2
9.4
111
109
107
0.3
16
Mean deviation
Standard deviation
Paired t
1.5
±2.0
2.4
3.6
±2.2
5.2
2.26
Mean
Critical value oft
a
BUN
=
=
=
2.26
0.3
Uric acid
BUNA
10.7
8.9
8.5
9.0
10.6
8.7
8.0
8.7
10.7
7.8
100
67
98
20
78
83
68
125
42
9.2
89
111
0.05,D.F. = 9
Blood urea nitrogen.
Numerous
authors
have reported
significant
differences in glucose values obtained by enzymatic
and chemical
methods
in uremic patients
(13).
Table 3 gives the data and Table 2 gives the regression
equations
obtained
on analysis
of predialysis uremic sera by the three different methods.
Results
of the hexokinase,
glucose oxidase, and
o-toluidine
methods
correlated
well. The o-toluidine results were slightly lower than the enzymatic
results, but this was consistent
with earlier findings on analyses of non-uremic
sera. These results
differed from the findings of Powell and Djuh (14),
who determined
glucose in uremic sera before and
after renal dialysis by a modification
of Sudduth’s
direct automated
o-toluidine
method (11) and by
an enzymatic
method
in which glucose oxidase/
peroxidase
coupled with the carcinogen,
o-dianisidine, is used (3). Their glucose values in predialysis uremic patients
were significantly
higher
by the o-toluidine
method than by their glucose
oxidase method.
Enzymatic
activity may be affected by the presence of numerous
chemical substances.
There was
close agreement
among results
of the two enzymatic and o-toluidine
methods in the analyses of
uremic sera. Moreover,
this agreement
was consistent with results of analyses of non-uremic
sera.
These findings indicate that no alterations
in enzymatic
activity
are produced
by constituents
found in uremic sera, which contain
abnormally
high concentrations
of many
dialyzable
substances. These substances
did not interfere
with
measurement
of NADPH
at 340 nm.
512 CLINICAL CHEMISTRY, Vol. 18, No. 6, 1972
Many authors
have validated
the accuracy
of
their methods
by comparing
their results
with
labeled values on commercial
quality-control
sera
(12, 15). I attempted
to establish
the accuracy
of the proposed
hexokinase
method
by similar
comparisons
with pre-assayed
commercial
quality
control sera (Table 4). There was excellent agreement between the two enzymatic
methods but poor
agreement
with the label values. Several commercial sera contained
abnormally
high amounts
of
bilirubin, which interfere with the direct o-toluidine
method.
For more accurate
comparisons,
analyses were also performed
by an indirect o-toluidine
procedure,
with use of a protein-precipitation
step
with trichloroacetic
acid (9). In this case, there
was poor agreement
between results of the indirect
o-toluidine
method
and label values.
The large
difference
between
values obtained
by the direct
o-toluidine
method on the Monitrol
serum was not
consistent
with previous results on normal sera by
the enzymatic
methods.
The manufacturer
found
their direct o-toluidine
values to be 8 mg/100 ml
higher than by their own Nelson-Somogyi
determinatibn.
This finding is difficult to explain, because bilirubin
or hemoglobin
values
were not
high and because
Cooper and McDaniel
(9) recently reported that they found no significant
differences between analyses
by the o-toluidin#{232}
and
Nelson-Somogyi
methods.
Precision
Precision was assessed both by repeatability
and
reproducibility.
Repeatability
of the hexokinase,
Table 4. Analyses of Commercial Quality Control Sera
Method
Label
Reference
Mfr’s method(s)
sera
for glucose
o-Toluidine
(direct)
Nelson-Somogyi
Folin-Wu
o-Toluidine and
ferricyanide
Glucose oxidase
(Hyland)
“Monitrol”
(Dade)
“Q-Pak”
(Hyland)
“Versatol-A”
(General
Diagnostics)
“Ledertrol”
(Lederle)
...
SMA.6/60 and
o-toluidine
SMA.12
Reference
(Techn icon)
...
glucose
value
88
Hexokinase
Glucose
Other
o-Toluidine
oxides.
(direct)
o-Toluidine
(indirect)
mg/100 ml
Bill.
label values
rubln
Creatlnine
Uric
acid
BUN”
68
70
87
70
0.8
1.0
4.6
13.4
178
178
176
170
7.2
5.1
10.1
51
201
192
193
195
190
5.1
4.1
8.3
30
230
215
216
223
214
7.2
4.7
15.2
52
235
214
213
206
204
2.3
5.3
7.4
65
80
93
187
165
Mean deviation
Standard deviation
Paired t
Critical
value oft =
a = 0.05,D.F. = 4
±5.8
5.7
2.78
0.9957
a
BUN
=
±5.9
±10.8
±7.4
5.4
2.2
5.6
2.78
2.78
2.78
0.9951
0.9875
0.9922
Blood urea nitrogen.
glucose oxidase, and o-toluidine
methods
was determined
by 15 replicate
analyses on sera containing three different glucose concentrations,
and was
excellent for all methods (Table 5).
Reproducibility
of the hexokinase
method
was
determined
by daily analyses of pooled and frozen
quality-control
serum for eight days. The mean
was 158 mg/100 ml, (± 1.9, SD), with a coefficient
of variation
of 1.3%.
Interferences
Effects of reducing sugars. The specificity of each
method for glucose in the presence of other sugars
is given in Table 6. Only the enzymatic
methods
were acceptably
specific for glucose under these
conditions.
The o-toluidine
method was nonspecific
and other sugars markedly interfered.
Effects
of reducing
substances.
Ascorbic
acid,
creatinine,
glutathione,
and uric acid are often
present
in abnormal
amounts.
The combined
effects of some of these substances
were observed in
the analyses of uremic sera. To determine
the individual effects, I analyzed
glucose solutions
containing a single other reducing substance
(Table 6).
Ascorbic acid interfered
with the glucose oxidase
and o-toluidine
methods.
Creatinine
and uric
acid interfered
with none of the three methods.
Glutathione
depressed
values in the glucose oxidase method.
Table 5. Repeatability of Results for Glucose in
Sera in Three Concentrations (n = 15)
Glucose concentration,
52
100
Method
Coefficient
Hexokinase
Glucose oxidase
o-Toluidine
1.0
1.2
2.0
mg/100 ml
300
of variation,
%
0.9
1.1
1.7
0.6
0.8
1.2
Table 6. In Vitro Effects of Other Reducing
Substances in Solutions Containing
100 mg of Glucose per Milliliter
Amount
present
Method and apparent glucose
concentration
HexoGlucose o-Toluidifle
kinase
oxidase
(direct)
mg/100 ml
Sugars
Fructose
Man nose
Xylose
Other reducing
100
100
100
100
100
100
100
100
100
127
200
131
10
20
60
10
100
100
99
100
96
100
90
100
105
100
102
100
sub8tances
Ascorbic acid
Creatinine
Glutathione
Uric acid
CLINICAL CHEMISTRY, Vol.18,No.6,1972 513
Effects of anticoagulants
and preservatives.
The
hexokinase
and glucose oxidase methods were unaffected by the use of anticoagulants
and preservatives (Table 7). There was a slight but definite
elevation
produced
by potassium
oxalate
and
EDTA in results
by the o-toluidine
method.
In the
o-toluidine
reaction EDTA has been reported
to enhance color (9).
Effects of drugs. Some commonly used drugs, administered
in 500 mg doses or greater, were tested
to determine
the degree of interference
with the
three methods
(Table 7). I used higher concentrations than usually found in sera, to aid in identifying potential
interfering
substances.
Ampidillin
produced
only a slight elevation
in the o-toluidine
method.
r-Dopa
depressed
values by the glucose
oxidase method. Dextran caused marked turbidity
in the o-toluidine
method.
Tetracycline
interfered with all methods
except the glucose oxidase method.
The pharmaceutical
manufacturer
included
in their
tetracycline
capsules
small
amounts
of lactose, which elevated the o-toluidine
values. Because the lactose is diluted in a large
blood volume its effect would not be detected
in
actual serum analyses.
In the hexokinase
method,
interference
was produced
by direct absorption
of
the tetracycline
at 340 nm. This effect was confirmed by on-line sampling
of the tetracycline
solution
(10 mg/100
ml) while using water in
place of the enzyme reagent. Since the therapeutic
concentrations
of tetracycline
are 0.1-0.3 mg/100
ml instead of the 10 mg/100 ml used in the test, its
interference
with the hexokinase
method
is negligible (16).
Sharp et al. (17) recently
reported
that two
sulphonylureas,
tolzamide
and tolbutamide,
interfere with determination
of blood glucose by two
separate
glucose methods.
In the first enzymatic
method,
2-2 ‘-azino-diethylbenzothiazoline-6-sulfonic acid is used as the oxygen acceptor,
and in
the second o-dianisidine
is used as the oxygen acceptor. They found that in both methods
tolazamide
depressed
glucose
values,
whereas
tolbutamide
produced
a false elevation
in glucose
values. The data in Table 7 show that tolazamide
lowered
glucose
values
in the glucose oxidase/
peroxidase/ferrocyanide
method
but did not effect the hexokinase
or o-toluidine
methods.
Tolbutamide
did not interfere
with any of the three
methods.
Effects
of bilirubin,
hemoglobin,
and tipemia.
Wright et al. (15) recently evaluated
a colorimetric
hexokinase
method for glucose analysis and found
that abnormally
high concentrations
of biirubin
or hemoglobin
interfere.
The use of a dialysis step
in the hexokinase
and glucose oxidase methods
eliminates
such interference
(Table 7). There was
significant
interference
by biirubin
or hemoglobin
with the o-toluidine
method.
Analyses
of several
514 CLINICAL CHEMISTRY,
Vol. 18, No. 6, 1972
Table 7. In Vitro Effects of Anticoagulants and
Preservatives, of Some Drugs, and of
Hemoglobin and Bilirubin in Solutions
Containing 100 mg of Glucose per 100 Milliliters
Method
ahd apparent glucose
concentration
Amount
present
Hexo-
klnase
Glucose
oxides.
mg/100
o-Toluldlne
(direct)
ml
An&oagulants
Sodium fluoride
Potassium
oxalate
EDTA, disodium
salt
200
200
100
100
100
100
100
200
100
100
103
10
100
101
103
1020
100
100
Turbid
10
101
74
102
10
102
100
104
10
100
93
99
10
100
100
100
18
708
100
100
100
100
105
115
102
Drugs
Ampicillin
(Pen britin)
Dextran-70
(“Macrodex”)
L-Dopa
Tetracycline
(“Achromycin”)
Tolazamide
(“Tolinase”)
Tolbutamide
(“Orinase”)
Hemoglobin
and bilirubin
Bilirubin
Hemoglobin
lipemic samples before and after protein precipitation by each of the three methods revealed that no
significant differences were produced by the turbidity.
Enzyme
Stability
The main disadvantage
in the past with hexokinase methods
was the lack of reagent
stability
after reconstitution
at room temperature.
This
disadvantage
was easily overcome
by keeping the
mixed reagent in an ice bath at 4#{176}C,
where it remains stable for at least five days. The stability of
the reconstituted
reagent at 4#{176}C
was measured
by
adherence
of the standards
to Beer’s law. Reagent
decomposition
was characterized
by a gradual decrease in absorbance
of the 300 mg/100 ml standard
in relationship
to the lower standards.
On the 6th
day after reconstitution
the absorbance
of the 300
mg/100 ml standard
fell to 98% of the value required to follow Beer’s law and progressively
declined to 93% on the 9th day. The total absorbance
of the 100 mg/100
ml and 200 mg/100
ml standards remained
constant
for the nine-day
period,
with only minor day-to-day
fluctuations
owing to
the usual variations
in the pump tubing.
Normal
Range
Table 8. Kinetic Parame ter s of
Proposed Method
The fasting
normal
range for the hexokinase
method
averaged
66-100 mg/100
ml for serum
specimens
from 30 fasting adults. This is consistent with the reports of others (8, 15).
Proposed method (hexokinase)
4.5
5.0
10
21
System
SMA.12/30(neocuproine)
SMA-6/60(neocuproine)
6.0
5.0
Efficiency
The combined
use of the Technicon
connector
assemblies
(which gives the regular
bubble pattern),
the 6-inch dialyzer,
the small-bore
polyethylene
coils, and the one-piece
debubbler
flow
cell results in an efficient miniaturized
manifold.
The enzyme reagent consumption
and liquid flow
rate to the debubbler
flow cell is only 0.6 ml/min.
The system efficiency
was measured
in terms of
percent carryover,
percent steady state, and by the
kinetic parameters
of half-wash
time (W 1/2) and
lag phase (L) (18). At 60 samples per hour with a
1: 1 sample-to-wash
ratio, the percent
carryover
was determined
by running
a 100 mg/100
ml
standard followed by a 300 mg/ 100 ml standard,
and
another
100 mg/100 ml standard
(Figure 2). The
difference in absorbance
between the two 100 mg/
100 ml standards
divided by the absorbance
of the
300 mg/100 ml standard
multiplied
by 100 gave
0.5% carryover.
The absorbance
values were approximately
93% of steady state. Table 8 gives the
kinetic parameters
with a comparison
to the values
obtained
with the neocuproine
method
on the
SMA-12/30
and SMA-6/60
(18, 19). The lower
values obtained
indicate the high efficiency of the
proposed system.
System efficiency was improved
by use of polyethylene tubing, in agreement
with the findings of
Reid and Wise (20). Furthermore,
the system does
not require a high-temperature
heating bath as do
most other glucose methods
(o-toluidine,
ferricyanide, and neocuproine),
a requirement
that adds
to the cost of the system,
and decreases
system
efficiency.
The present
method
is simple, fast, efficient,
accurate,
and requires
only 50 ul of serum or
plasma. Of the three methods
examined,
only the
hexokinase
method
was not significantly
affected
by any of the interfering
substances
tested.
Wi,,
Lag
Seconds
2. Somogyi, M., Reducing non-sugars and true sugar in human
blood. J. Biol. Chem. 75, 33 (1927).
3. Robin, M., and Saifer, A., Determination
of glucose in biologic
fluids with an automated
enzymatic procedure. CLIN. CHEM. 11,
840 (1965).
4. Fales, F. W., Russell, J. A., and Fain, J. N., Some applications
and limitations
of enzymatic,
reducing
(Somogyi), and anthrone
methods for estimating sugars. CLIN. CREM. 7, 289 (1961).
5. Hall, J. W., and Tucker, D. M., Automated
determination
of
glucose using glucose
oxidase
and potassium
ferrocyanide.
Anal. Biochem. 26, 12 (1968).
6. Precautions
for Laboratory Workers Who Handle Carcinogenic
Aromatic
Amines, Chester Beatty
Research
Institute,
Royal
Cancer Hospital, London, 1966, p 3.
7. Amador, E., Polyethylene
coils for continuous flow analyzers.
CLIN. CHEM. 18, 164 (1972).
8. The automated
determination
of glucose, Sigma Chemical
Co., St. Louis, Mo. 63178, Tech. Bull. No. 970, July 1970.
9. Cooper, G. R., and McDaniel, V., The determination
of glucose
by the ortho-toluidine
method. Stand. Methods Clin. Chem. 6,
159 (1970).
10. Hainline, A., Hemoglobin.
Stand. Methods Clin. Chem. 2, 49
(1958).
11. Sudduth,
of glucose
N., Widish,
measurement
J. R., and Moore,
using
ortho-toluidine
J. L., Automation
reagent.
Amer.
1.
Clin. Pat hot. 53, 181 (1970).
12. Yee, H. Y., Jenest, E. S., and Bowles, F. R., Modified
manual or automated
o-toluidine system for determining
glucose in
serum, with an improved aqueous reagent. CLIN. CHEM. 17, 103
(1971).
13. Fingerhut,
B., Automated
serum glucose levels in uremia.
Amer. J. Clin. Pathol. 51, 157 (1969).
14. Powell, J. B., and Djuh, Y. Y., A comparison of automated
methods for glucose analysis in patients with uremia before and
after dialysis. Amer. J. Clin. Pat hot. 56, 8 (1971).
15. Wright, W. R., Rainwater,
J. C., and Tolle, L. D., Glucose
assay systems: Evaluation
of a colorimetric
hexokinase
procedure. CLIN. CHEM. 17, 1010 (1971).
16. Weinstein, L., In The Pharmacological
Basis of Therapeutics,
4th ed., L. S. Goodman, and A. Gilman, Eds. Macmillan
Company, New York, N. Y., 1970, p 1256.
the
17. Sharp, P., Riley, C., Cook, J. G. H., and Fink, P. J. F.,
Effect of two sulfonylureas
on glucose determinations
by enzymatic methods. Clin. Chim. Ada 36, 93 (1972).
18. Thiers, R. B., Cole, R. R., and Kirsch, W. J., Kinetic parameters of continuous flow analysis. CLIN. CIIEM. 13, 451 (1967).
1. Gilbert, R. K., Analysis of results of the 1969 comprehensive
chemistry survey of the College of American Pathologists.
Amer.
J. Clin. Pat hot. 54, 463 (1970).
19. Brown, M. E., Ultra-micro
sugar determinations
using 2,9dimethyl-1 ,10-phenanthroline
hydrochloride
(neocuproine).
Diesbetes 10,60 (1961).
20. Reid, R. H., and Wise, L., A study of analyses done under
non-steady
state conditions. In Automation in Analytical
Chemistry,
Technicon Symposia
1967, 2, N. B. Scova et al., Eds.
Mediad, White Plains, N. Y., 1968, p 159.
Supported
by Training
NIH, Department
Grant
5-T01-GM
01784-05 from
of HEW.
References
CLINICAL CHEMISTRY,
Vol. 18, No. 6, 1972 515