Some
Applications
and
Enzymic,
Reducing
(Somogyi),
Methods
Frank
W. Fales,
Limitations
for
Estimating
Jane
A. Russell,
of the
and
Anthrone
Sugars
and
John
N. Fain*
The colorimetric
glucose-oxidase
and the Somogyi-Nelson
reducing-sugar
methods
yield the same blood-glucose
values when applied to carefully
prepared zinc
hydroxide filtrates of blood obtained from fasting individuals.
The anthrone method yields values only slightly higher than those obtained by the enzymic and the
reducing-sugar
methods. Use of the enzymic
method in conjunction
with the
reducing-sugar
or the carbohydrate
method allows the simultaneous
accurate
determination
of other carbohydrates added to the blood, with only microquantities
of blood being required for analysis.
Possible applications
include the galactose
and fructose
tolerance tests, the inulin clearance test, and the determination
of
the body water “space” of various carbohydrates.
To allow valid glucose
determinations
by the enzymic method, glutathione,
cysteine,
and amylase
substrates
such as glycogen
must be absent.
The anthrone reaction with glucose is enhanced by chloride, bromide, and iodide.
To eliminate interference
of chloride with estimation of blood glucose, the samples
must be diluted appropriately
before analysis.
for glucose,
the Somogyi
method for reducing
sugar,
and the anthrone
method
for carbohydrate,
used either
singly
or in combination,
offer a variety
of applications
because
of the differences
in specificity.
Because
of the specificity
of
the enzymic
method
for glucose
(1), one possible
application
is its
use for determining
blood-glucose
levels
in the presence
of other
carbohydrates.
The Somogyi
(2) or other
of the more
specific
reHE
QUANTITATIVE
ENZYMIC
METHOD
From
the Department
of Biochemistry,
Emory
University,
Atlanta,
Ga.
A portion
of the material
presented
in this article
was presented
before
the Annual
Meeting
of the American
Association
of Clinical
Chemists,
Cleveland,
Ohio, Aug. 28, 1959.
Received
for publication
Aug. 12, 1960.
‘Public
Health
Service
Predoetoral
Feliow,
National
Institute
of Arthritis
and Metabolic
Diseases.
289
290
FALES ET AL.
Clinical
Chemistry
ducing-sugar
methods,
used in conjunction
with the glucose-oxidase
method,
offer an alternative
for the determination
of reducing
sugars
other than glucose
added
to the blood, as in the galactose
or fructose
tolerance
tests.
Likewise,
the anthrone
method
used in conjunction
with the enzymic
method
offers
a means
for determining
carbohydrate
added
to blood,
but in this instance,
the carbohydrate
is not
restricted
to the reducing
sugars.
These
proposed
methods
offer
advantages
over previous
methods
that involve
the removal
of glucose by yeast
(3) or by glucose
oxidase
(4) prior
to the analysis
in that a precise
measure
of the glucose
level is also obtained
and
that the methods
are more
readily
amenable
to microanalytic
procedures.
Of equal relevance
to possible
applications
of the methods
are the
limitations
that were
uncovered
during
the study
reported
in this
article.
It appears
that too much
emphasis
may have been placed
upon
the specificity
of glucose
oxidase
in the colorimetric
enzymic
methods,
but too little
attention
paid to the many
substances
that
may interfere
with the method.
This has tended
to engender
the false
supposition
that the enzymic
methods
yield
true glucose
values
regardless
of the experimental
conditions.
These
methods
all employ
a
reagent
containing
a crude
glucose
oxidase
preparation,
peroxidase,
and a chromogenic
hydrogen
donor
for the peroxidase
reaction.
This
combined
reagent
is sensitive
to a number
of substances
other
than
glucose,
and some assurance
as to the absence
of these
interfering
materials
must be at hand before
the “true”
glucose
values
can be
considered
as valid.
Apart
from
the obvious
requirement
for the
exclusion
of activators
and inhibitors
of the two enzymes,
other less
obvious
materials
can cause
serious
errors.
The peroxides
that may
be released
in acid filtrates
of biologic
materials
cause
a positive
error,
whereas
materials
that compete
with the chromogen
as hydrogen donors,
e.g., uric acid, ascorbic
acid, bilirubin,
and the catechols,
cause
a negative
error.
It has been
found
that
glutathione
and
cysteine
also strongly
inhibit
color
formation,
and it is presumed
that these substances
also compete
as hydrogen
donors.
Also, in an
investigation
of the applicability
of the enzymic
method
for the
determination
of glucose
in tissue
extracts,
it was found
that there
was a large
positive
error
in the presence
of appreciable
concentrations of glycogen,
because
the glucose
oxidase
preparations
used in
the reagent
invariably
have high amylytic
activity.
This imposes
the
Vol. 7, No. 4, 1961
ESTIMATION
OF SUGARS
291
added
requirement
that amylase
substrates
be absent.
Thus,
there
is an imposing
list of materials
that may interfere
with the enzymic
method,
and hence,
despite
the specificity
of glucose
oxidase,
rigid
exclusion
of these materials
is required.
In conjunction
with these
studies,
the anthrone
method
was examined
for the determination
of blood sugar.
Since anthrone
does
not react
with
non-sugar-reducing
material
(“saceharoids”),
one
might
have expected
the reaction
to give true values
for total sugars
in blood.
However,
Roe (5) found
that with tungstic
acid filtrates,
which
are rich in saccharoids,
about
equal values
were obtained
by
the anthrone
and the reducing-sugar
methods.
For the most
part,
this positive
error
in the anthrone
reaction
appears
to be owing
to
the enhancement
of the anthrone
color
by chloride
ions
(6).
We
found
that the chloride
enhancement
can be overcome
by dilution,
the positive
error
of the anthrone
reaction
being
reduced
(by the
use of dilute
filtrates)
from
about
20 to 5 mg./100
ml., the residual
error
apparently
being
due to the presence
of small
amounts
of
polysaccharide.
Thus, under
properly
controlled
conditions,
the total
sugar
in the blood can be determined
with reasonable
precision.
By the judicious
combined
use of the Somogyi
and the enzymic,
and of the anthrone and enzymic
methods,
we have obtained
excellent
recoveries
of galactose
and/or
sucrose
added
to the blood.
It is suggested
that
this approach
may be of value
for the galactose
and
fructose
tolerance
tests
as well as for the determination
of the
clearance
and the body-water
“space”
of various
carbohydrates.
Methods
Preparation
and Materials
of Filtrates
Either
the Somogyi
(7) 0.5N sodium
hydroxide,
10% zinc sulfate
or the Somogyi
(8) 0.3N barium
hydroxide,
5% zinc sulfate
reagents
may be used with whole blood,
but only the latter
are suitable
with
serum
or plasma
(8, 9). We prefer
the former
reagents
with whole
blood because
a larger
volume
of filtrate
is obtained.
The concentration of the base and zinc sulfate
must
be carefully
adjusted
as directed.
The importance
of a careful
preparation
of the ifitrates
cannot be overemphasized,
since this process
accomplishes
the removal
of materials
that interfere
with the enzymic
as well as with the reducing-sugar
procedure.
Incomplete
removal
of these materials
can
cause large
discrepancies
between
the values
obtained
by the meth-
292
FALES ET AL.
Clinical
Chemistry
ods, since the materials
cause low values
with the enzymic
and high
values
with the reducing-sugar
method.
We prepared
the ifitrates
as follows:
The blood was laked with 17
or 22 volumes
of water.
One volume
of sodium
hydroxide
solution
was added,
and then,
after
thorough
mixing
but without
sufficient
delay to cause a significant
enolization
of the sugar,
1 volume
of zinc
sulfate
solution
was added
drop by drop, with mixing.
The solution
was then very thoroughly
mixed to avoid entrapment
of liquid within
the precipitate.
This mixing
may be accomplished
by shaking
in a
glassstoppered*
Erlenmeyer
flask or centrifuge
tube by vigorous
swirling
in an Erlenmeyer
flask, or by vigorous
and prolonged
stirring with a large
glass
rod in a centrifuge
tube.
In any event,
the
final mixture
should
be uniformly
opaque,
with no discernible
layers
or streaks
of liquid.
After
5 mm., the mixture
was filtered
or centrifuged.
We prefer
the above
sequence
for the addition
of the reagents
because
the base insures
the complete
hemolysis
of the red
blood cells, which is essential
for a valid estimate
of the whole blood
glucose
concentration.
Reducing-Sugar
Determination
The improved
copper
reagent
and the method
of Somogyi
(2) were
used in conjunction
with the Nelson
(10) arsenomolybdic
acid reagent.
The procedure
is readily
adaptable
for microquantities
and
so is well suited
for the proposed
microprocedure
for the galactose
tolerance
test.
The following
procedure,
which
closely
follows
the
recommendations
of Somogyi
(2), was found
satisfactory.
To 1.8
ml. of water
in a colorimeter
tube was added
0.2 ml. of 1:20 dilution
filtrate
and 2 ml. of copper
reagent.
Duplicate
tests,
standards,
and
a blank
were capped
with glass marbles
to prevent
evaporation
and
were simultaneously
placed
in a boiling-water
bath.
After
20 mm.,
the tubes were simultaneously
removed
a few minutes,
2 ml. of arsenomolybdic
each tube, the contents
of the tubes were
several
minutes
the colorimetric
readings
Total
Carbohydrate
Determination
Total
carbohydrate
Fales
(11).
Versene
‘A
rubber
formation
Biochemical
to a cold-water
bath.
After
acid reagent
was added
to
thoroughly
mixed,
and after
were made.
was determined
by the anthrone
or potassium
oxalate
(no fluoride)
stopper
should
not be used because
derivatives
in the enzymic
procedure.
This
information
was
Corporation
with their
reagent.
method
was used
from
the rubber
provided
by the
of
as
inhibit
color
Worthington
Vol. 7, No. 4. 1961
ESTIMATION
OF SUGARS
293
the anticoagulant.
The use of wood applicator
sticks,
ifiter
paper,
and cloth or paper
pipet
swipes
was avoided
throughout
the procedure, as they can cause a serious
positive
error.
Enhancement
of the
anthrone
color by chloride
(see below)
was avoided
by dilution.
Glucose
Determination
The prepared
reagent
Glucostat*
was used.
The reagent
was prepared
as directed
for the microprocedure
except
that 0.05M
phosphate
buffer,
pH 7, was used as the diluent
rather
than water.
Two
milliliters
of reagent
were added
to 0.2 ml. of blood filtrate.
The reaction
was stopped
after
40 mm. by the addition
of 1 drop
of 4N
hydrochloric
acid.
The readings
were made
after
5 mm. at a wavelength
of 400 m.
It was found desirable
to determine
frequently
the
range
of glucose
concentrations
over which
a linear
response
was
obtained,
since there
seemed
to be a variability
of response
with
different
batches
of reagent.
Also, the pH of the final reagent
was
checked
and adjusted
to pH 7.0 if necessary.
The pH given
by the
buffer
incorporated
into the reagent
by the manufacturer
was at considerable
variance
from
pH 7.0 in many
batches.
The more
convenient
reagent
and method
described
by Washko
(12) were used in
some of the later experiments.
Results
Limitations
of the Enzymic
Method
It was found
that the low blood-glucose
values
obtained
by the
enzymic
method
with
tungstic
acid filtrates
result
almost
entirely
from
the glutathione
contained
in the filtrates.
The inhibition
of
color formation is not brought about by residual tungstic acid in the
filtrates,
as suggested
by Beach
and Turner
(13),
since equivalent
amounts
of tungstic
acid added
to glucose
standards
have no measurable
effect.
Also, the concentrations
of uric acid and ascorbic
acid
in the filtrates
were insufficient
to cause
a measurable
effect.
However, glutathione
strongly
inhibited
color formation,
and the glutathione
content
of tungstic
acid filtrates
is sufficient
to account
for the
low values.
Glutathione
evidently
competes
with the chromogen
as a hydrogen
donor
for the peroxidase
reaction.
When
excess
glutathione
is added
during
the course
of the reaction,
color formation
is arrested
but no
‘Worthington
Biochemical
Corp.
FALES ET AL.
294
Clinical
Chemistry
significant
bleaching
is observed.
Therefore,
the action
of the glutathione
is not the reduction
of the colored
product.
The reagent
is
affected
in a similar
manner
by cysteine.
Oxidized
glutathione
or
cystine
have no effect.
A study
of the course
of the color formation
with a tungstic
acid filtrate
indicated
that the inhibition
was exerted
early
in the course
of the reaction.
After
all the glutathione
was
oxidized
(10 mm., in this case),
the color formation
proceeded
at a
rate identical
to that observed
with a zinc hydroxide
filtrate
of the
same blood.
These
data are shown
in Table
1. Thus,
a simple
test
Table
1.
COTJRSE
or
Cot.oa
DEVELOPMENT
WITH
TUNOSTIC
ACID
AND
ZINC
HYDROXIDE
FILTRATES
Incubation
time
Apparent
(tam.)
Tungatic
5
10
‘Values
blood
filtrates
concentrations
(mg./IOO
Zinc
34
40
20
30
glucose
acid
30
minus
10’
based
on the
and
of
changes
the
in absorbancy
ml.)
hydroxide
64
67
47
69
53
69
66
69
from
the
10.
to the
30-mm.
readings
of
the
standard.
for the presence
of glutathione
or other interfering
hydrogen
donors
is suggested.
If higher
values
are obtained
with increasing
incubation times, the presence
of a competing
hydrogen
donor
is suggested.
Consistently
low or decreasing
values
would be indicative
of enzyme
inhibition.
A comparison
of the blood glucose
values
obtained
with the use of
tungstic
acid and zinc hydroxide
filtrates
was carried
out with nine
blood samples.
Under
the conditions
used, the average
underestimation with tungstic
acid ifitrates
was 13 mg., with a range
of 2-24
mg./100
ml. There
seemed
to be no correlation
between
the underestimation
and the glucose
concentration.
The use of tungstic
acid
filtrates
in the determination
of serum
or plasma
glucose
levels
by
the enzymic
method
may be admissible
because
the blood glutathione
resides
almost
wholly
within
the red blood cells.
The possible
use of the enzymic
method
for the determination
of
glucose
content
of tissue
extracts
was investigated.
Upon testing
the
effect of various
constituents
of the tissue
extracts,
it became
evident
that glycogen
strongly
interfered
with the determination.
When
the
ESTIMATION
Vol. 7, No. 4, 1961
295
OF SUGARS
reagent
was added
to glycogen
solutions,
a considerable
color formation was observed.
The course
of the color development
with various
concentrations
of glycogen
and with a glucose
standard
is shown
in
Fig. 1. The autocatalytic
reaction
curves
with the glycogen
solutions
Fig.
time
1.
by
Increase
the
in
glucose
absorbancy
with
oxidase
method
of glycogen
and
with various
amounts
with glucose:
A, 25 pg. of glycogen;
B, 100 pg. of glycogen;
C, 400 pg. of
glyeogen;
D, 5 mg. of glycogen;
and
E,
50
pg.
of
glucose.
20
TIME.
30
40
MINUTES
suggest
an a-amylase
contaminant
in the reagent.
The endoamylase
catalyzes
the hydrolysis
of the polysaccharide
into small
fragments
that are further
split into maltose
units,
but a molecule
of glucose
is
derived
from fragments
having
an odd number
of glucose
residues.
Although
the relative
yield
of glucose
from
glycogen
is not large,
very much more glycogen
than free glucose
is likely to be present
in
most tissues,
and so the results
obtained
when crude
oxidase
is applied
to aqueous
protein-free
extracts
are entirely
unreliable.
Preliminary
observations
indicate
that
the amylase
can be removed
from a solution
of the crude glucose
oxidase
by passing
it through
a
starch
column
under
properly
controlled
conditions
of pH and ionic
strength.
However,
even with such a preparation,
it would
still be
necessary
to remove
glutathione
and other
reducing
materials
from
the tissue
extracts
before
the method
can be applied
to them
with
confidence.
Limitations
of the Anthrone
Enhancement
observed
some
Method
of the reaction
by chloride,
mentioned
time ago by one of us (J. A. R.) and was
above,
briefly
was
men-
296
Clinical
FALES ET AL.
Ch.misfry
tioned
by Scott
and Melvin
(6).
However,
no extensive
report
of
this potential
error
has appeared,
and Mokrasch
(14) erroneously
reported
that chloride
at a concentration
of 1M did not interfere.
The
enhancement
of the anthrone
reaction
with
glucose
by the
I-.
z
taJ
FIg.
U
2.
absorbancy
the halides.
w
a.
expressed
z
Enhancement
of
anthrone
with glucose
(40 pg.) by
Halide
concentrations
are
in terms
of the final sam-
pie-reagent
mixture.
Fluoride
values
are approximate,
owing
to some
etch-
0
I-
ing
of
the
glass.
I-
z
L,J
I,
D
-6
-5
LOG.(M)
-4
HALIDE
-3
-2
CONCENTRATION
-I
halides
is shown
in Fig. 2. Included
with the chloride
data are two
experiments
by the method
of Morris
(15), in which reagent
is added
to the sample
in a ratio of 10:5, and two experiments
by the method
of Fales,
in which
the ratio
is 10:1.
It is evident
that the enhancement is dependent
upon the final concentration
of halide,
regardless
of the ratio of sample
to reagent.
Investigations
at higher
concentrations were not feasible
because
of the etching
of glass with fluoride,
the fuming
of hydrogen
halides
with chloride
and bromide,
and the
formation
of iodine and other colored
products
with iodide.
The data
indicate
that the maximal
halide
concentrations
free from
interference are 0.04, 4 X 10,
1.8 X 10,
and 4.5 X 10
molar,
respectively, for fluoride,
chloride,
bromide,
and iodide.
With
the assumption
of a whole-blood
chloride
concentration
of 85 mEq./L.,
a 65-fold
dilution of the blood by the Morris
method
and a 20-fold
dilution
by the
Fales
method
would
be required
to remove
the chloride
effect.
The
data
also point
to the danger
of interference
in specimens
from
patients
on bromide
and iodide
therapy.
Although
fluoride
at the
Vol. 7, No. 4, 1961
ESTIMATION
297
OF SUGARS
concentration
used as a preservative
does not interfere,
it appears
to
lower
the permissible
chloride
concentration.
Also,
troublesome
bubbles
often adhere
to the sides of the tubes when fluoride
is present, and there
is danger
of etching
the colorimeter
tubes.
Another
difficulty
in the use of the anthrone
method
for the determination
of blood
sugar
is some lack of specificity.
Both
zinc hydroxide
and tungstic
acid filtrates
contain
small
amounts
of nonfermentable
carbohydrate.
The nonfermentable
carbohydrate
found
in blood filtrates
by the anthrone
method
is shown
in Table
2. The
fermentations
were performed
by the method
of Somogyi
(16), except that double
centrifugations
were carried
out to insure
the complete removal
of the yeast
and that two water
blanks
were included
with
each run to correct
for the small
quantity
of carbohydrate
exuded
from
the yeast.
The nonfermentable
carbohydrate
content
of the blood ifitrates
appeared
to be in no way related
to the bloodsugar
levels
or to the method
of protein
precipitation.
It appears
that
the nonglucose
carbohydrate
is not accounted
for by sugar
phosphates,
as suggested
by Roe (15),
since identical
results
were
obtained
when anthrone
was used with ifitrates
prepared
either
with
zinc sulfate
and sodium
hydroxide
or with zinc sulfate
and barium
hydroxide.
The barium
would
induce
the precipitation
of the sugar
phosphates.
This suggests
that sugar
phosphates
in the erythrocytes
Table
2.
TOTAL
AND
FERMENTABLE
CARBOHYDRATE
IN
Bwon
FILTRATES
DETERMINED
WITh
ANTHEONE
Carbohydrate
Sample
No.
Fermentable
92
5
87
2
99
7
92
3
4
147
78
136
4f
77
3
81
81
7
97
104
7
8
6
6
6
3
4
4
7f
105
5
100
6
90
Of
‘Values
based
concentration
an
f Same
than
ml.)
1
Sf
give
(mg./100
Nonfsrmentable*
Total
1:20
on
glucose
standard.
of nonfermentable
absorbancy
blood
of
value
as previous
dilution
zinc
These
carbohydrate,
with
anthrone
sample
hydroxide
quite
but
with
filtrate,
do
because
figures
not
the
70
71
75
75
87
93
100
necessarily
represent
carbohydrate
different
from
that
given
1:20
dilution
of tungstic
as was the case in the other
the
in question
by glucose.
acid filtrate
samples.
actual
may
rather
298
FALES ET AL.
Table
3.
Bwon
3
CoMPARrIvE
BY
Sample
No.
GLUCOSE
vALuES
INDEPENDENT
Glucose
METHODS
Oxidas.
Clinical
(Mo.J100
ML.)
OF
ANALYSIS
OF
FASTING
Somogyi-Nelson
Chemistry
INDIvIDUALS
Ant hron,
1
2
50
56
48
56
55
3
63
60
4
5
6
65
66
66
66
70
71
73
72
7
67
68
71
8
68
69
77
9
68
70
10
11
12
13
14
70
72
73
72
67
75
75
75
15
16
76
80
75
75
17
74
80
82
82
18
86
87
83
19
84
20
86
86
86
21
86
85
22
23
94
112
98
113
24
117
123
25
120
123
26
130
132
are in concentrations
that are insignificant
as compared
to the blood
glucose.
The identity
of the nonfermentable
carbohydrate
found
in
the filtrates
is unknown,
but the fact that it is nonreducing
in character suggests
that it may be polysaccharide.
The error
in blood-sugar
determinations
with anthrone
attributable
to this factor
is not serious, averaging
about
5 mg./100
ml., but it must be considered
in the
comparison
of the anthrone
and the enzymic
blood-glucose
values.
Applications
In
Table
individuals*
‘The
use
and
galactose
milk
products.
3 are
with
shown
three
of blood from
transiently
fasting
appear
the
blood-sugar
independent
values
methods
individuals
was indicated
in the blood
following
obtained
of analysis.
on fasting
These
data
for
this study
because
fructose
a meal
containing
sucrose
and
Vol. 7, No. 4, 1961
ESTIMATION
299
OF SUGARS
indicate
that glucose
was the only reducing
substance
present
in the
filtrates
at an appreciable
concentration.
The values
obtained
by the
three methods
are in quite good agreement,
except
that the anthrone
values
tend to be slightly
higher
than the enzymic
or the reducingsugar
values.
If 5 mg./100
ml., the average
overestimation
of glucose
deduced
from the fermentation
studies
(Table
2), is subtracted
from
the anthrone values, good agreement
between the three methods
is
obtained.
The recovery
of galactose
added
to blood,
determined
by the proposed
procedure
for the galactose
tolerance
test, is shown
in Table
4. The total reducing
value was determined
by the Somogyi-Nelson
method,
and the glucose
by the enzymic
method.
Both
glucose
and
galactose
standards
were included
with the reducing-sugar
determinations
because
glucose
has a higher
reducing
power
than
does
galactose.
Since
the same standard
glucose
solution
and the same
blood
filtrate
were used in both methods,
the portion
of the total
Somogyi-Nelson
absorbancy
resulting
from
the oxidation
of the
blood glucose
was calculated
directly
from the colorimetric
measurements
as follows:
AG =
AR/AE
X Au
where
A0
is the
absorbancy
Table
4.
due
RECOVERY
to glucose;
OF
Milligrams
SUGARS
sugar
A5,
ADDED
per
100
ml.
I’O
of
that
of the
glucose
BLooD
whole
blood
Glucose*
found
Gahx.ctose
added
Galactossf
1
1
1
1
1
2
2
131
127
131
129
131
72
73
0
40
80
120
160
0
0
1
42
81
123
163
2
72
0
80
89
2
72
0
120
129
160
164
80
82
lood
sample
2
71
2
74
by the
by the
‘Determined
$Determined
oxidase
0
40
0
80
glucose
oxidase
combined
use
Sucrose
found
78
5
42
method.
of the
somogyi-Nelson
reducing-sugar
and
the
glucose
methods.
tDetermined
last
Sucroce
added
found
figure
in
by the combined
use of the anthrone
and
the column
was determined
by the combined
average
overestimation
recovery
values
are
(5
obtained
mg./100
(see
ml.)
Table
by
2).
the
anthrone
the glucose
use of all
method
oxidase
methods.
three
methods.
is subtracted,
The
If the
improved
FALES fT AL.
300
Clinical
Chemistry
standard
with the reducing
sugar
method;
A5, that
of the glucose
standard
with the enzymic
method;
and A, that of the blood ifitrate
with the enzymic
method.
The absorbancy
due to galactose
was calculated
by difference,
and the concentration
of galactose
was determined
in the usual way with the use of the absorbancy
of the galactose standard.
The results
of a recovery
study
with added
sucrose
are also shown
in Table
4. In this instance
the sucrose
was determined by the combined
use of the anthrone
and the enzymic
methods.
In one case, both sucrose
and galactose
were added,
and the total
carbohydrate
was determined
by the anthrone
method,
the total reducing
sugar
by the Somogyi-Nelson
method,
and the glucose
by the
enzymic
method.
Glucose,
sucrose,
and galactose
standards
were
included
with the anthrone
determinations
since they give different
absorbancy
values
per unit
concentration,
that
for sucrose
being
somewhat
higher
and that
for galactose
being
considerably
lower
than that of glucose.
These
data point
to the general
utility
of the
proposed
method
of parallel
analyses.
In addition
to its use for the
galactose
tolerance
test,
it offers
an alternative
method
for the
fructose
tolerance
test.
Also, it can be applied
to the measurement
of the distribution
of various
carbohydrates
in the body water
and
to the measurement
of the clearance
of various
carbohydrates
from
the blood.
In Fig. 3 are shown
the results
of a galactose
tolerance
test carried
out by the proposed
method
on a galactosemic
infant.
The normal
infant
has a very high tolerance
for galactose,
the blood
galactose
level declining
after
1 hour and falling
close to zero after
3 hours.
In addition
to the information
gained
from
the galactose
Fig.
3.
galactosemic
combined
Somogyi-Nelson
0
2
I
TIME
Hours
3
Galactose
tolerance
infant
use
of
methods.
test
on
carried
out
by
enzymic
and
Vol. 7, No. 4, 1961
ESTIMATION
OF SUGARS
301
levels,
the precise
measurements
of the changes
in the blood-glucose
level
may be of some
value
in conjunction
with
the intravenous
galactose
tolerance
test carried
out as a measure
of liver function.
In this instance,
both the capacity
of the liver to remove
galactose
and its capacity
to restore
the blood-glucose
level would be measured.
Discussion
In the present
study,
we obtained
excellent
agreement
between
the
Somogyi-Nelson
and the enzymic
methods
for whole
blood-glucose
from fasting
individuals.
Beach
and Turner
(13) likewise
found
no
significant
differences
between
the values
obtained
by the two methods.
Also,
Middleton
and Griffiths
(17) found
agreement
with the
enzymic
method
when they used the Asatoor
and King
(18) modification
of the Harding
and Downs
(19) procedure.
The copper
reagents
used in these
procedures
are modifications
of the ShafferSomogyi
Reagent
50 (20),
as is the Somogyi-Nelson
reagent,
and
each is a low-alkaline
reagent
having
a bicarbonate
:carbonic
acid
ratio
of maximal
sensitivity
for glucose
relative
to “saccharoids.”
Recently,
Kingsley
and Getchell
(21) obtained
excellent
agreement
between
the enzymic
and Somogyi-Nelson
methods
for plasma
glucose levels.
However,
in contradiction
to these four reports
of agreement,
Saifer
and Gerstenfeld
(22) found
consistently
lower
plasma
glucose
levels
with the enzymic
than with the Somogyi-Nelson
procedure.
The discrepancy
was quite
closely
proportional
to the glucose level and ranged
from
several
milligrams
with plasma
of low
glucose
content
to over 45 mg./100
ml. with plasma
having
a high
glucose
content
of some diabetic
patients.
Despite
the preponderance of evidence
for agreement
between
the enzymic
and the more
specific
copper
reduction
methods,
this
single
exception
has generated
some
confusion.
Because
of. the specificity
of the glucose
oxidase,
‘Saifer
and Gerstenfeld
contend
that the disagreement
must
be due to reducing
substances
other
than glucose
(“saccharoids”).
This would
not necessarily
follow
since, as we have shown,
there
is
little
to choose
between
the two methods
on the basis
of interfering
materials.
On the other hand,
there
are those who would
insist
that
if the blood-sugar
values
obtained
by the enzymic
method
are to be
considered
valid,
they
must
agree
with
those
obtained
by the
Somogyi
methods,
because
of the large
body of evidence
supporting
the validity
of the latter
methods.
302
FALES fT AL.
Clinical
Chemistry
Because
of the confusion
concerning
this point,
this evidence
will
be reviewed.
Using
the Shaffer-Somogyi
Reagent
50 and a modification thereof,
Somogyi
(7, 9) found
that little or no reducing
material
remained
in the zinc hydroxide
filtrates
after
the removal
of the
sugar
with yeast.
This finding
was confirmed
by MacKay
(23).
We
have
repeatedly
verified
this finding,
using
the Somogyi
reagents
modified
for colorimetry
by Nelson
(10) and by Somogyi
(2) in the
performance
of galactose
tolerance
tests by the fermentation
method
in the clinical
laboratory.
Of the materials
that have been suggested
by various
authors
(5, 24-27)
to account
for the high values
obtained
with
tungstic
acid filtrates
(glutathione,
ergothioneine,
glucuronic
acid, uric acid, creatinine,
creatine,
ascorbic
acid, and sugar
phosphates),
only the sugar
phosphates
are acted
upon
by yeast,
and
they are fermented
at such a slow rate-less
than one-twentieth
the
rate of glucose
fermentation
(28)-that
they would
remain
largely
unfermented
after
the 10-mm.
fermentation
period
employed
by
Somogyi
(16).
Thus,
the absence
of nonfermentable
reducing
material
in the zinc hydroxide
filtrates,
as measured
by the more specific
copper
reagents,
constitutes
strong
evidence
in support
of the specificity
of these
methods
for blood
sugar.
This conclusion
is in no
way vitiated
by the several
reports
of the presence
of nonfermentable reducing
material
in zinc hydroxide
filtrates
measured
by less
specific
reagents.
In this category
are the reports
of Benedict
(29)
and of Fujita
and Iwatake
(26), who used a copper
reagent
of high
alkalinity
and an alkaline
ferricyanide
reagent,
respectively.
The
latter
authors
found
non-sugar-reducing
material
equivalent
to 5-10
mg. of glucose
per 100 ml. in zinc hydroxide
ifitrates
when measured
with the ferricyanide
reagent,
but with these same filtrates
they fully
confirmed
the validity
of the blood-sugar
values
obtained
with the
Somogyi
low-alkali
copper
reagent.
Further
support
for the validity
of the Somogyi-Nelson
procedure
is its agreement
with chemical
methods
not of identical
specificity.
The relative
agreement
presented
in this paper
with the fermentable
sugar
determined
with
anthrone,
a reagent
specific
for carbohydrates,
may be cited, as well as the excellent
agreement
obtained
by
Anthanail
and Cabaud
(30) with the o-aminodiphenyl
reagent.
The
latter
is a modified
Tauber
reaction
with the requirement
of a sugar
having
a potentially
free or easily accessible
aldehyde
group
(31, 32),
the green-colored
product
measured
by Anthanail
and Cabaud
being
Vol. 7, No. 4, 1961
ESTIMATION
303
OF SUGARS
quite specific
for the aldoses
(32).
Unlike
the anthrone
reaction,
no
colored
product
is obtained
with polysaccharides
because
the acetic
acid contained
in the reagent
is not of sufficient
strength
to cause
their
hydrolysis
(33).
Because
of the wholly
different
specificities
of these
chemical
methods,
their
agreement
strongly
supports
the
validity
of the Somogyi-Nelson
procedure.
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