A Study of the Starch-Iodine
Modified
Colorimetric
of Amylase
Complex:
A
Micro Determination
in Biologic Fluids
N. R. Pimstone
The limitations, inaccuracies, and practical difficulties of saccharogenic methods are
discussed. A modified colorimetric microdetermination
of amylase is described in
which the digestion of starch is measured by the decrease in the starch-iodine color.
Experimental data show that there are two other serum factors that can also cause
a fall-off in color: (1) an immediate 10-15% depression of color, probably due to
serum proteins and countered by using serum in the control; (2) an acid-serum factor causing a progressive fall-off in color subsequent to the initial depression. Iodine
prevents this, and must be added as soon as the acid has been added to stop the enzyme activity. Results of 189 consecutive assays of human sera are presented.
Amylase activity of duodenal aspirate has been determined simultaneously by the
method described and the Lagerlof method. Results are compared. Changes in serum
amylase and lipase levels in artificially produced pancreatitis in dogs are presented.
Optimal conditions for amylase activity are reviewed, and in the light of these, different amyloclastic methods and their results compared. Achroic-point technics are
briefly evaluated.
F
OR PURPOSES
OF a study
dogs (3), it was necessary
logic fluids. In seeking the
that the many established
and a reassessment
of some
is increasing
evidence that
on experimentally
induced pancreatitis
in
to perform many assays
of amylase
in biomost suitable method, it soon became clear
procedures
were beset with shortcomings,
aspects of these became imperative.
There
amyloclastic
methods are far superior
to
From tile Department
of Chemical
Pathology,
Medical School, University
of Cape Town,
Cape Town, Republic of South Africa.
Thanks are due Prof. J. E. Kench, Dr. M. C. Berman, Dr. G. M. Potgieter,
Mr. E. J. Dun.
can, Mr. C. E. Edwards, and Miss P. Hendrikse.
Dr. E. J. Immelman,
of the Surgical
Research
Unit, and Dr. I. N. Marks, of the Gastrointestinal
Unit, gave permission
to publish results
of dog and human biological
fluid assays, respectively.
Miss C. Abraham
performed
many of
the sera and all the duplicate
pancreatic
fluid estimations.
Received for publication
Oct. 30, 1962.
891
892
PIMSTONE
Clinical Chemistry
the once-popular
saccharogenic
methods.
The latter measure
the reducing substances
formed
from starch by the action of amylase-a
collection
of large and small dextrins
and different
polymers
of maltose. As it is impossible
to compile a standard
for so hybrid a mixture,
one can never be certain whether
a particular
method is measuring
the
total reducing
power.
It is no wonder that such a diversity
of results
exists between the different
saccharogenic
methods.
These anomalies
are clearly reviewed
by Henry and Chiamori
(2)
and by Somogyi
(8),
who adds further
sources of error to be guarded
against.
Street
(11)
has demonstrated
serious limitations
of the Somogyi
method of determining the reducing
power by examining
“glucose
recovery”
in experiments
where known aliquots
of glucose were added to different
mixtures
which were then assayed.
He has shown that the method is
totally unreliable
when blood sugar is above 150 mg./100 ml. and serum amylase is normal.
This is due to interference
of starch, the effect
being greater
when more starch is present.
The possible physiochemical explanation
is discussed
in detail.
In addition
to these inaccuracies,
most saccharogenic
methods
(with
the notable exception
of the picric acid micro method of Marsters
et al.
(4).) use large quantities
of serum to assure a reasonable
gap between
the reading
and the blank.
Further,
these methods
are, in general,
more time-consuming
and laborious
than amyloclastic
technics.
Finally, as maltase
will add to the total reducing
power of the reaction
mixture, Lagerl#{246}f’ssaceharogenic
assay of amylase
in duodenal
aspirate
is not strictly specific (5).
The trend, therefore,
is towards
the amyloclastic principle,
where the breakdown
of a single substance,
starch, is
measured
by the decrease
in blue color of the starch-iodine
complex.
At first sight this appears
to be accurate,
specific, and easy, but the
work presented
in this paper demonstrates
two serum factors
other
than amylase
which may cause a fall-off
in color.
These “pseudoamylase”
effects have been studied.
Furthermore,
excellent
work by
Henry
and Chiamori
(2) has contributed
facts fundamental
to any
amylase
determination.
It is in the light of all these data that a method essentially
similar to that of Gomori (1), but with several important
modifications,
will be described.
Method
Reagents
Phosphate
(17.8
gm./L.)
buffer,
and
0.1 M (pH
K112P04
7.0)
Solutions
(1B.6 gm./L.)
are
of Na2H P04 .21120
prepared,
and 610 ml.
Vol. 10, No. 10. 1964
STARCH-IODINE
893
COMPLEX
Na2 HPO4. 21120 brought
to 1 L. by addition
of KH2
should be 7.0, but must be checked with a pH meter.
P04.
The
pH
Stock starch solution,
2% w/v
Ten grams
of soluble starch
are
suspended
in 250-300 ml. phosphate
buffer.
This is brought
slowly to
the boil in 10-15 mm.; the solution is stirred
continuously
with a glass
rod. The sides and base of the beaker must be stroked frequently
with
the rod to prevent insoluble
clumps from forming.
About 10 gm. NaCI
is added to the boiling solution,
which is then transferred
to a 500-ml.
volumetric
flask. The flask is filled to the stem with recently boiled dis.
tilled water-the
cap of water prevents
a skin from forming
on the sur.
face of the cooling starch-and
this is made up accurately
to the 500..
ml. mark when cool. There are many preservatives
in use currently,
such as propyl parasept
(propyl
parahydroxybenzoate)
and sodium
fluoride
(6, 8); benzoic
acid (1); and a combination
of methyl
and
propyl parasept
(4). Rice (7) claims that these are not adequate,
and
describes
a sorbic acid preparation
that keeps starch
stable for at
least 10 months.
However,
the addition
of preservatives
to an Unsterile
solution
of starch may fail to prevent
bacterial
degradation.
Reif and Nabseth
(6) have observed
as much as a 50% fall-off in starch
concentration
in 7-15 days, despite
preservation.
They have developed a fairly simple sterilizing
technic which, if used in conjunction
with preservatives,
keeps starch
stable for at least 35 days.
I have
found that sodium chloride,
a drop of toluene,
adequate
sterilization
and refrigeration
maintains
the stability
of starch satisfactorily.
Working
buffered
substrate
(starch
solution
0.6% w/v)
Thirty
milliliters
of starch solution
are made up to 100 ml. with phosphate
buffer in a volumetric
flask, and the solution is boiled gently for 1 mm.
One milliliter
of buffered
substrate
thus contains
6 mg. starch,
approximately
0.1 M NaC1, and 0.085-0.09
MI phosphate
buffer (pH 7.0).
The solution should be kept at 0 to 40
Sulphuric
acid, 5% v/v A. R.
Five milliliters
concentrated
H2S04,
S.G. 1.84, are added to 50 ml. distilled
1120. After cooling, the volume
is made up to 100 ml.
Stock
beaker
water.
rate of
at 0 to
iodine solution,
o.iN.
To 13 gm. iodine weighed out in a small
are added 30 gm. potassium
iodide and approximately
10 ml.
The high concentrations
of the iodine and iodide increase
the
solution.
This is made up to 1 L. with distilled
water and stored
4#{176}
in an amber bottle.
Working
iodine solution,
0.OiN
A 1 :10 dilution
of 0.O1N iodine
is
894
PIMSTONE
Clinical Chemistry
prepared
and stored at 0 to 4#{176}
in an amber
mains stable for a few weeks.
bottle.
This
solution
re-
Procedure
One milliliter
of buffered
starch is measured
accurately
by pipette
or burette
and added to each of two large test tubes marked
at 50 ml.
with a diamond
pencil.
One tube is labeled “test,”
the other, “control,”
and 1)0th are placed in a water bath at 37#{176}.
After about 5 mm.,
0.1 ml. serum is added to “test.”
The enzymatic
action is stopped
#{225}fter 30 miii. with 2 ml. 5% v/v 110504. Serum, 0.1 ml., is now added to
the control, followed immediately
by 2 ml. 112504.
After diluting both
to about 30 ml., 0.O1N iodine, 1 ml., is added to each tube and the mixture is made up to the 50-nil. mark with distilled
water.
After shaking
well, the tubes are left for 15-20 mm. so that the color can develop
fully. An iodine blank is prepared
with water, 0.1 ml. serum, iodine,
and sulfuric
acid. Tubes are read against
this blank using a mediumred filter (630-660
m)
in any standard
photoelectric
colorimeter.
For an obviously
jaundiced
serum, a separate
blank should be prepared.
Otherwise
one blank will suffice for a batch of tests.
Units are defined as the number of milligrams
of starch digested
by
10 ml. serum in 30 miii, at 37#{176}.
(This makes it numerically
similar to
the well-established
Somogyi
unit.)
As 1 ml. buffered
substrate
contains 6 mg. starch, the control reading,
divided by 6, gives (in terms of
absorbance)
the color-equivalent
of 1 mg. starch.
U./10 nil. serum = control
test X 100 X dilution factor =
eontrol/6
control
-
test
X 600 X dilution factor.
cntro1
The normal range for serum is 20-140 U./10 ml. serum.
The normal
urine values agree with those stated by Henry and Chiamori
(2), viz.,
random samples:
66-740 U./100 ml. urine; 6- or 24-hr. samples:
40-245
U./hr.
It is necessary
to point out several important
precautions
that have
resulted
from our experimental
work. The control should be incubated
with the test, as incubated
starch, being more soluble, forms a clear
blue color with iodine. Contrary
to observations
of Gomori (1), serum
does affect the color of the control-0.1
ml. serum can depress
it by as
much as 15% of the true value.
It follows that 0.1 ml. serum must be
added to all controls.
Further,
when acid is added to the serum-starch
Vol. 10, No. 10, 1964
STARCH-IODINE
895
COMPLEX
mixture,
an acid-serum
factor progressively
acts on the starch,
preventing
it from combining
with iodine to an extent equivalent
to 1-2
U./min.
Iodine stops this effect and should be added as soon as possible following
the 112504. If more than 75% of the starch has been degraded,
the test should be repeated
with serum diluted 1:5 (v/v), the
control now having 0.1 ml. of the diluted serum.
it is valid, where high
values are expected,
to read tubes after 15 mm. incubation
and multiply the answer by 2. This allows for an early answer in an emergency.
For urine, first dilute 1:2 (v/v) and for pancreatic
juice, 1 :200 (v/v),
setting
up appropriate
controls.
For reasons
to be discussed,
these
will have absorbances
greater
thaii those of the serum controls.
Results
Characteristics
of Starch-Iodine
Color
At room temperature,
0.6% \v/v starch exists as a hazy colloidal suspension which, with iodine, forms a coarse turbidity.
This results
in
inaccurately
low readings
on a photoelectric
colorimeter.
However,
after incubation
for 15-30 mm. at 37#{176},
starch becomes
“water-clear”
as the colloidal
suspension
goes into solution
and, especially
if first
diluted with distilled water, it now forms a clear blue color with iodine.
A fine sediment
sometimes
develops
on standing,
but this is readily
and accurately
reversible
on shaking.
When serum alone is added to an iodine solution,
the yellow color
pales, as indicated;
yet when serum and acid are added, the color is accentuated,
as measured
by a colorimeter
using a green 540 m filter.
Color
Composition
1 nil. 0.O1N iodine
solution,
1
+ 0.1 ml.
ml.
0.O1N
iodine
of solution
diluted
serum,
to 50 nil.
diluted
to 50 ml.
infex
Without
H2804
With
H2S04
32
32
26
45
This may account
for an odd phenomenon
relating
to tile starchiodine complex.
When there is an excess of iodine in the presence
of
acid and serum, starch and iodine form a dirty yellow, turbid solution.
If starch is now added in excess, a clear blue color is restored.
Gomori
(1) uses 1 ml. GramLugol’s
iodine (i.e., approximately
0.03N) as his
indicator.
In the presence
of acid and serum, this represents
an excess
of iodine, the yellow turbidity
reverting
to clear blueness
only when
more than 6 mg. starch are added to the mixture.
Iodine, 0.0IN, always produces
a clear blue color proportionate
to the amount of starch
_-
8%
PIMSTONE
Clinical Chemistry
used in the test. When the concentration
of potassium
iodide is raised
at constant
iodine concentration,
or when the pH is raised, the tendency for serum components
and hemoglobin
to cause precipitation
is
diminished
(6).
Acid accentuates
the blue color, whereas
alkali reduces
its intensity, the color disappearing
above a pH of about 9.5. Thus, acid must
be used to stop amylase
activity.
Heating
the solution
also reversibly
destroys
the color.
Table I presents
some further
interesting
and important
facts. Aliquots of starch are incubated
for 30 mm. To half of the specimens,
0.1
ml. serum is added, followed
immediately
by 112S04 and iodine.
The
mixture
is then diluted
to the 50-ml. mark on the test tube. The table
records
the direct colorimetric
readings,
using a red 660 rn/A filter.
Color reaches a maximum
in 15-20 mm. and is stable for another
2 hr.
It is proportional
to the amount
of starch present,
whether
there is
serum or not. Serum depresses
color by about 15%.
Effects of Serum on Starch-Iodine
Color
Figure 1 and Tables 2 and 3 illustrate
the effects of 0.1 ml. serum on
the blue starch-iodine
complex.
In the experiments
tabulated
in Table
2, 6 mg. starch are incubated
under various
circumstances.
At intervals, iodine is added and colorimetric
readings
of the starch-iodine
color made,
It is apparent
that there are two serum factors
that have a “pseudoamylase”
effect by producing
(1) an initial immediate
depression
in
Table
1.
RATE
OF
DEVELOPMENT
AND
STABILITY
Color v&uea
Starch
in substrate
(mg.)
6
6
6
6
COMPLEX
(mm.)
10
15
20
4.5
60
150
270
271
283
283
288
285
288
285
288
285
0.1 ml. serum
0.1 ml. serum
230
229
238
237
242
238
240
239
240
237
288
285
240
238
285
284
242
238
+ 0.1 ml.
0.1 ml. serum
serum
335
340
290
287
336
347
297
290
340
350
301
290
345
352
300
291
347
352
302
290
347
350
299
290
347
352
299
290
+
+
0.1 ml. serum
0.1 ml. serum
403
414
350
350
410
420
352
351
417
420
359
355
420
424
360
354
419
425
360
355
420
424
360
355
420
424
360
355
+
+
5
5
5
5
STAacH-IoDnE
5
4
4
4
4
OF
rea d at intervals
+
Vol. 10, No. 10, 1964
STARCH-IODINE
897
COMPLEX
4S
A
__________
CONTROL
400
Fig. 1. Effect
of serum
SERLR4
on
starch-iodine
color.
Graphic representation
of
Table
2 shows effect
of
amy]ase
(C and
of two nonspecific
amylase
factors
D).
350
I
SERUM-5AL_c
‘ZZ
-I----
‘
-
VALUE
‘
C.
____________
B) and
pseudo(B and
300
a
.(
#{149}SALIVA
CON1.0
is.
w
a0o
.4
I-
w
-j
I1
S.
E.
is
Jo
TIME
Table 2. EFcr
OF SERUM
ON
(IN
Control
(6 mg. starch
+
Miws)
CoLoR
5rARH-IODINE
Color
acid)
Serum control
(6 mg. starch + 0.1 ml. serum + acid)
Test-low
value
(6 mg. starch + 0.1 ml. serum of low amylase
con
tent incubated;
acid added just before iodine)
Test-high
value
(6 mg. starch + 0.1 ml. serum + 0.1 ml. saliva;
acid and iodine added at intervals)
Serum-saliva
control
(6 mg. starch + 0.1 ml. serum + 0.1 ml. saliva +
acid)
Eo
values
read
at intervals
(nun.)
0
15
30
45
60
412
415
410
415
414
360
339
320
310
305
354
350
347
340
310
309
Decolorized
364
340
315
898
Table
PIMSTONE
3.
EXPERIMENTS
ON THE
DEPRESSANT
Clinical
EFFECT
OF SERUM
ON
TIlE
Chemistry
STARCH-IODINE
COMPLEX
Color
index
Experiment
(6 mg. starch
+
A
Control
B
0.1 ml. serum
C
Protein-free
supernatant
(m 0.1 ml. serum)
added immediately
after acid.
+
+
starch
acid.
+
added
0.1 ml. serum
then added.
E
Protein-free
supernatant
bated
at 37#{176}
for 30 mm.
F
0.1 ml. serum
G
0.1 ml. serum,
then added.
II
0.1 ml. human albumin
(7% w/v)
+ starch
(a) iodine added immediately
after acid
(b)
I
color
(Fig.
acid,
410
Iodine
D
+ iodine
starch
acid)
+
incubated
immediately
+
after
+
starch
acid.
acid.
355
Iodine
415
at 370
for 30 mm.
Iodine
297
+
acid,
+
0.1 ml. serum)
Iodine then added.
(m
incubated
+
starch
acid,
incU-
415
at 37#{176}
for 30
miii.
Incubated
starch
then added.
incubated
incubated
360
at 37#{176}
for 30 mm.
Starch
+
acid
+
iodine
357
+
acid
340
for 30 mm. at 37#{176}
and iodine
0.1 ml. human albumin
(25% w/v) + starch
(a) iodine added immediately
after acid
(b) incubated
for 30 miii. at 37#{176}
and iodine
then added
+
340
acid
then added
180
173
(Fig. 1, A-B), and (2) a subsequent
progressive
fall-off in color
1, B-D).
This latter factor acts only in the presence
of acid.
Immediate Effect
if 0.1 ml. serum is added to starch amid this is followed immediately
by the addition
of iodine, the color obtained
is only about 85% of its
true value in the presence
of acid, the 15% depression
increasing
more
than fourfold
if acid is withheld.
A 7% human albumin
preparation
to the amount of 0.1 ml. acts similarly,
the effect being enhanced
by a
more concelitrated
protein
solution.
Furthermore,
as a protein-free
supernatant
of serum
(equivalent
to 0.1 ml.) has no effect on color
whatsoever,
the initial
depression
described
is probably
a physical
phenomenon
dependent
upon the serum-protein
level. It follows that
most sera exhibit this effect to a similar
degree.
We have already
shown that, at a low pH, serum intensifies
the yellow color of iodine.
Serum
proteins,
therefore,
probably
depress
the blue starch-iodine
color by acting on the starch, the latter possibly
being adsorbed
on to
receptor
surfaces
on the protein molecule.
in view of this supposition,
other large organic anions, e.g., caffeine-sodium
benzoate,
were added
in an attempt
to compete with the starch for these receptor
surfaces.
Vol. 10. No. 10, 1964
STARCH-IODINE
COMPLEX
899
However,
these were without
effect. Hemoglobin
also interferes
with
starch-iodine
color in a similar way (6), and allowance
for this must
be made in assay of hemolysed
serum by using a separate
control.
The
action is probably
similar to that of serum proteins.
ProgressiveFaIl.off (Due to Acid-Serum Factor)
In our early experimental
work,
assays
of amylase
frequently
yielded erroneous,
and on occasions,
even negative
results. Subsequent
events showed that this was related
to an interesting
and unexpected
phenomenon:
the fault lay in the control,
which was then constituted
by incubating
starch, serum, and acid at the same time as the test, with
iodine added only after the 30-mm. incubation.
Table 2 and Fig. 1 illustrate
clearly where we went wrong.
If one
incubates
acid, serum, and starch together-even
though there is no
amylase
activity
at this p11 (approximately
1)-there
is some other
factor that causes a progressive
fall-off in color roughly
proportional,
over 30 mm., to the time-interval
before iodine is added.
Thus if iodine
follows immediately
upon the addition
of serum and acid to starch
(Fig. 1 B and Table 2), values are significantly
higher than if iodine is
added after 30 mm. (Fig. ID and Table 2). The results of Tests B-D
(Fig. 1) therefore
show a pseudoamylase
effect to I)e avoided for valid
amylase
assay.
This phenomenon
obviously
called for further
study,
and although
we still do not know what this factor is, we have made
some interesting
discoveries
as to the nature of its action. For this action, the acid-serum
factor
must be in contact
with the incubating
starch; the effect is not reproduced
if acid and the serum are incubated
together
for 30mm. and the starch iii en added (Table 3, Exp. F). The
fall-off in color is roughly
proportional
to the time of contact before
the iodine is added, more rapid in the first few minutes,
then trailing
off after 30 mm. Like the factor responsible
for the immediate
15%
depression,
this one is also a protein.
(A protein-free
filtrate of serum
does not affect color in any way, according
to Table 3, Exp. C and E.)
It differs
in that it acts only in the presence
of acid; 7% w/v human
albumin
cannot reproduce
the phenomenon
(Table 3, Exp. H and I);
and different
sera vary considerably
in their effect, the variation
being in no way related to the initial depression
they produce
or to their
amylase activity
(Table 2 and Fig. 1 D).
Fortunately,
the addition
of iodine to the mixture
prevents
further
fall-off, the final color being stable for hours.
The importance
of adding iodine as soon as possible after the acid has already
been stressed.
Figure
1 illustrates
clearly the need for Control
B, in which starch is
PIMSTONE
900
Clinical Chemistry
incubated
alone for 30 mm. and serum, acid, and iodine are then added
one after the other.
The results of amylase activity
(C and E) can also
be seen. If starch, serum, and acid were to be incubated
as a control
(Fig. 1 D), sera of low activity
would give negative
results
(Fig. 1 C).
That incubation
of starch and acid together
produces
no alteration
in
color (Fig. 1 A) confirms the evidence that a serum factor is responsible for this subsequent,
progressive
fall-off in color.
Color as Guide to Quantity of Starch
Figure
2 represents
two standard
curves.
Different
aliquots
of
starch were incubated
for 30 mm. in 50-ml. volumetric
flasks and then
0.1 ml. each of serum, acid, and iodine were added, in that order.
The
volume was made up to 50 ml. and the color read after 15 mm. Each
point is the mean of four readings,
the individual
values not differing
by more than 2%. This has been termed the serum-control
curve.
A
control
curve
omitting
serum
was similarly
prepared.
Both graphs
are nearly straight
lines passing
through
zero, the slope of the serum
control
graph being approximately
13% less than the control.
It is
clear from the graph that the difference
between a serum control and
test reading
(Fig. 1, B-C) is an extremely
accurate
measure
of the
starch breakdown.
02
‘a
w
zo
OW
-II.-
Il
U)
Mt STARCH
Fig. 2. Standard
and iodine;
“serum
curves.
control”
“Control” curve represents
shows
colors
obtained
with
color of increments
addition
of starch,, acid,
of 0.1 ml. serum.
Vol. 10, No. 10, 1964
STARCH-IODINE
901
COMPLEX
Applications
Our experiments
using dog sera show that amylase
values are inversely
proportional
to the serum dilutions.
Two assays
are performed,
one at a dilution
of 1:10 and the other at 1:50. The values
shown are already corrected
for their respective
dilutions.
Dilutions
No.
Dog
Time serum taken
1:10
1:50
43
53
58
4 hr. postop.
Postop.
postop.
58
1 day postop.
3,100
2,200
85
85
85
Preop.
45 mm. postop.
833
2,200
1,200
4,360
4,375
85
1 day
1,500
1,430
1 hr.
41/2
hr. postop.
postop.
4,400
5,000
1,830
1,900
1,500
1,400
2,200
The values also increase
in a roughly linear fashion with the time of
incubation,
and the agreement
of duplicate
readings
provide
further
evidence of the accuracy
of the method.
The method has been used extensively
for assay of amylase
in various biologic fluids. At the time of writing,
189 consecutive
duplicate
tests on human sera have been performed.
An arbitary
norm of 20-140
U./10 ml. serum was adopted
from findings of other workers,
and Fig.
3 shows that the majority
of our results fall within this range.
Results
of duodenal
aspirate
assay by the Lagerl#{246}fand new method
are
set out graphically
(Fig. 4), and there is an obvious
close correlation between
the two methods.*
The straight
line passes
through
0.4 Lagerl#{246}f(5) units, an interesting
finding probably
related
to the
fact that the saccharogenic
Lagerlof
method measures
both amylase
and maltase
of the duodenal
aspirate.
Experiments
on dogs with artificially produced
pancreatitis
(3) have necessitated
serial amylase
and
lipase
(naphtholic
method)
estimations
on blood, chyle, peritoneal
fluid, and pancreatic
fluid. The results
of one such experiment
can be
seen in Fig. 5. This typifies the observations
made in the experimental
animal.
Discussion
The
ready
practical
been
*1sults
of 5.6 X 10
points
discussed.
of 450 assays
U./nl.
(-2
arising
this
experimental
It will suffice to reiterate
on duodenal
S.D.)
from
aspirate
in normals
by the new method
tested.
that
indicate
work
have
al-
an amyloclastic
a mean
lower
limit
902
PIMSTONE
Clinical Chemistry
method is accurate
if one has serum in the control and adds iodine immediately
after the amylase reaction
has been stopped by the acid.
At this point it is pertinent
to mention some of the excellent and fundamental
data arising
from the paper of Henry
and Chiamori
(2).
Jo
if
LIMITS
OF
NORUAL
/....
a
UNITS
Fig. 3.
of 20-140
Distribution
curve,
OF
189 consecutive
AMYLASE
ACTIVITY
tests.
values
Most
fall
within
arbitrary
norms
U.
They have pointed
out that -amy1ases
of serum,
urine,
saliva,
and
pancreatic
juice digest starch and larger dextrins
at a linear maximal
rate to smaller reducing
dextrins.
When the latter begin to form a significant part of the substrate,
the reaction
rate slows and becomes nonlinear (Fig. 6). The initial rapid phase is curvilinear
when the initial
concentration
of starch in the incubating
mixture
is less than 375 mg./
100 ml. They have also shown
that the point of deviation
from the
rapid linear to the slower nonlinear
phase is proportional
to the initial
starch-serum
ratio.
Where
30, 75, and 130 mg. starch/ml.
serum were
employed,
deviation
occurred
at mean values of 325, 600, and 1,400
Somogyi
units respectively.
Figure 6 demonstrates
that this is never
a problem
in amyloclastic
methods,
as all the starch is broken
down
while the reaction
rate is still maximal.
Further,
the fall-off in color is
linear until about 75% of the starch
is degraded
(2, 6).
There are
some interesting
figures arising from Henry and Chiamori’s
work (2)
STARCH-IODINE
Vol. 10, No. 10, 1964
concerning
is 6.9-7.2,
amylase
buffers
optimal
conditions
for
COMPLEX
amylase
with a distinct
optimum
at
maximally
above a concentration
are employed,
a final concentration
903
activity.
The optimum
pH
6.9. Chloride
ion activates
of 0.01 M. Where phosphate
of 0.0875 M phosphate
ion
.
.
‘S
Fig. 4. Assay
lase
in duodenal
Simultaneous
new method
method.
and
of amy.
aspirate.
assay
by
Lagerl#{246}f
Qs
‘C
z
4
$
LERI&
UNITS1
I
is without effect on amylase and satisfactorily
regulates
pH in assay of
fresh and aged sera, urine, and pancreatic
fluid. The optimal temperature is 50 to 550, but
and 40#{176}
are standard
laboratory
temperatures
-the
reaction
rate being 13.5% faster at the latter temperature.
Gomori’s
method
(1) has been modified
in the light of all the data
just presented.
The present
procedure
is simple, quick, requires
only
0.2 ml. serum and measures
amylase activity
under optimal conditions.
The unit is logical and easy to understand,
as it records
directly
the
number of milligrams
of substrate
broken down. It is fortuitous
that
the number of milligrams
of starch broken down by 10 ml. serum in 30
370
PIMSTONE
904
Clinical
Chemistry
mm. is
similar,
numerically,
to the number
of milligrams
of reducing
substances
(expressed
as glucose)
formed by 100 ml. serum in 30 mm.
(the latter comprising
the well-known
Somogyi
unit).
Comparison
of the normal range of different
amyloclastic
methods,
4000
N0
hO
Fig.
5.
R000
SI
experiment
1100
IC
artificially
produced
7000
TI
000
60
I!
t
Bile,
No.
Results
of
an
on a dog with
(bile.induced)
pancreatitis.
10 cc., used on Dog
85 (wi.
25 lb.).
I
2
50
U,
1
4o
3000
I1
U)
10
-
30
<aloe
SOH.
JO
PRE-OP---1
I HOUR
POST-Op
SERUM AMYLA$
SERUM LIPASE
6
____J
AFTER
g
TIME
BILE
9
(Nouns)
INJECTION
when time of incubation
and the amount of serum have been taken into
account is interesting.
Gomori
(1) has understandably
higher
values
than those in our series, as he used no serum in his controls.
The Huggins and Russell unit, as one would expect, is equivalent
to a fifth of the
Somogyi
unit, being expressed
as milliliters
of serum per hour.
Despite the use of a very dilute (0.1%)
starch solution,
Rice (7) published similar
results.
Street and Close (9, 10) have together
intensively investigated
the amyloclastic
method.
They claim that amylose
is a better substrate,
as its purity allows for more accurate
work. However, Henry and Chiamori
(2), who have compared
various
substrates,
have demonstrated
that amylase
hydrolyzes
amylopectin
at a significantly faster rate than it does amylose.
Further,
although
starch does
vary in the amylose :amylopectin
ratio, the variation
between different
brands of starch is less than 8%, and is thus of little practical
importance. Somogyi
(8)
severely criticizes
the use of soluble starch, a find-
Vol.
10,
STARCH-IODINE
No. 10. 1964
905
COMPLEX
ing confirmed
by Reif and Nabseth
(6) but not borne out by Henry and
Chiamori
(2)
in their study of saccharogenic
methods.
The starting
concentration
of amylose in the Street and Close micro and ultramicro
method is far below the 375 mg./100 ml. mentioned
above. Yet the two
REDUCING SUBSTANCES
Fig. 6. Change
of concentrations
of reducing
AS GLUCOSE
FOUl-Wu
BY
I
.4’
.ugJ.*t
I
substances
and of starch
(Folin.Wu)
with time.
(Reproduced
from
Henry
I
(Z),
by per-
I
and Chiamori
mission.)
STARCH CONC.
BY I COLOUR
,ug/ut
60
MINUTES
methods
agree fairly well, probably
indicating
little practical
deviation from linearity
in the amylase-reaction
rate.
There are some criticisms
of the Street and Close method.
Enzymatic action is stopped
by adding distilled
water and iodine, which is
certainly
not adequate
in sera with high activities.
This we have shown
quite clearly in our assays of pancreatic
juice and dog’s sera. Further,
Street and Close have not used serum in their controls,
thereby adding
another
source of error.
They have, in addition,
chosen a rather
complicated
unit based on
the formula
(Rs-Rt)
.
100
B
/100
ml. serum/15
mm.
incubation
where ils is the color index of 2 mg. amylose
(in the micro method)
and Rt the reading of the serum under assay.
In their micro method, 0.1 ml. serum is incubated
with amylose
for
15 mm. As the amount
of amylose
degradation
is multiplied
by 100,
the units should be expressed
per 10 ml. serum, not per 100 ml., as
above.
One would therefore
expect that, when corrected,
the normal
906
PIMSTONE
Clinical Chemistry
range would be numerically
similar to that of Somogyi.
Here, the falloff iii color in 13 miii. is divided by the reading
for 2 mg. amylose.
If
this was divided by 1 mg. and expressed
Ier 30 mm. incubation,
the
value would be four times greater
than the Street and Close unit and
should compare with the Somogyi unit. This is indeed so, as the range
of normal in Street and Close units is 6-33. In their ultramicro
method, 1/10 of the above amount of serum is used, but the values are identical as the standard
has 0.2 mg. amylose.
Other “achroic-point”
technics
exist where the time taken for full
digestion
of starch to the reddish-brown-erythrodextrins
is measured.
From Fig. 6, it is clear that results will be lower than Somogyi
unitsthe curve flattens out before the achroic point. The Wohlgemuth
method, by measuring
the least amount of serum to fully digest the starch,
expresses
the units as milligrams
of starch broken down per milliliter
of serum.
As expected,
the unit is slightly
less than 1/10 of the
Somogyi
unit.
While accurate,
the method
is laborious;
it involves
many
tubes,
many accurate
nieasurements,
and 1.5-2 ml. serum (3-6 ml. may be
necessary
where sera with high activity
are assayed).
The units rise
in stepwise fashion,
the “jumps”
becoming
disconcertingly
large when
higher values are reached.
The amyloclastic
method of Somogyi
(8)
measures
the time needed
for 1 ml. serum to completely
digest 3 mg. starch at 40#{176}.
The technic,
although
fairly simple, requires
many tubes; 1 ml. clear, nonlipaemic,
nonjaundiced
serum; and the full attention
of the technician.
A constant, 1800, worked out by simultaneous
saccharogenic
procedures,
is
divided by the time for the achroic point to be reached,
and the activity
is expressed
as Somogyi
units per 100 ml. This conversion
thus involves the inaccuracies
of two methods
and makes it obligatory
to accept the Somogyi
reducing
method and the results he has obtained.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Gomori, G., Am. J. Chin. PaIhol. 27, 714 (1957).
Henry,
R. J., and Chiamori, N., ChinS. Cheim. 6, 434 (1960).
Immelman,
B. .1., Unpublished
data, Surgical
Research
Unit, Medical
School,
of Cape Town.
Marsters,
R. w., Kinney, T. D., and Lin, K. Y., Chin. Chem. 6, 130 (1960).
Lagerl#{246}f,H. 0., Ada med. scandinav.
Suppl. 128 (1942).
Reif, A. E., and Nabseth,
D. C., Chin. Chem. 8, 113 (1962).
Rice, E., Chin. Chem. 5, 592 (1959).
Somogyi,
M., Chin. Cheni. 6, 23 (1960).
Street,
H., and Close, J., Chin. Chini. Acta 1, 256 (1956).
Street, H., and Close, ,J., Chin. Chin,. Acta 3, 476 (1958).
Street,
H., Chin. Chin,. Acta 3, 501 (1958).
University