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An Enzymatic Method For Glucose Determination in Body Fluids
An Enzymatic Method For Glucose Determination in Body Fluids
An Enzymatic Method For Glucose Determination in Body Fluids
462
Vol. 4, No. 6, 1958 ENZYMATIC METHOD FOR GLUCOSE DETERMINATION 463
urine and in blood filtrates. The enzymatic method will not respond
to any other physiologic constituent except glucose.
Until recently the few published accounts of this new system for
glucose determination came from sources concerned with its com-
mercial development (10, 11, 12) and were lacking, therefore, in de-
tail required for practical laboratory application. Experience in de-
velopment of the method in our own laboratory and successful appli-
cation to a large number of urine specimens led to the belief that a
presentation of the details of this analytical procedure would be of
value. More recently papers by Huggett and Nixon (14) and by
Middleton and Griffiths (15) have described fully the enzymatic
method for blood sugar. Development of the procedure in our own
hands has been somewhat different, inasmuch as it has been applied
both qualitatively and quantitatively to urine as well as to blood
filtrates. The practical handling of this system of glucose determina-
tion is the subject of the present report, together with a comparison
of the results obtained thereby with those yielded by other common
methods. The procedures should prove useful in any well-equipped
laboratory in which “true glucose” estimation is required.
METHODS
GLUCOSE QUALITATIVE TEST FOR URINE SCREENING
buffer at pH 7.0, add 250 mg. glucose oxidase. Stir a little to dissolve
the enzyme and then filter. Just before use add 1 per cent orthoani-
sidine at the rate of 2 drops for every 3 ml. of enzyme-buffer mixture.
6. Peroxidase solution. Use horseradish root extract as described
previously under the qualitative test. This solution must be glucose
free and can be tested by running a blank determination as described
below.
7. Standard glucose solution. 0.005 per cent glucose in saturated
benzoic acid.
Procedure:
The method here described is useful on urines containing 0.5 per
cent glucose or less. Urines under examination that exceed this level
will show too intense and rapid color development and must be
diluted further to fall into the correct range of sugar concentration.
Place 1 ml. of urine in a 125-ml. Erlenmeyer flask and add 19 ml. of
water. Add 50 mg. decolorizing charcoal and 1.5 Gm. Lloyds reagent.
It is convenient and sufficiently accurate to use metal measuring
spoons calibrated to deliver the right weight of each adsorbent. Mix
with the diluted urine specimen by shaking the flasks frequently, and
after 6 minutes remove the suspended material by filtration (What-
man No. 40 ifiter paper is suitable). Pipet 1 ml. of this filtrate into a
spectrophotometer tube and add 1 ml. of horseradish root peroxidase
extract. Prepare similarly a blank tube containing 1 ml. of water
and a standard tube containing 1 ml. of 0.005 per cent glucose solution
in lieu of urine filtrate. Because the reaction must be timed carefully
for reading later in the photoelectric colorimeter, the next addition
must be made at measured intervals. To each tube in the series
(standard, unknowns, and blank) add at the proper time 3 ml. of
glucose oxidase-indicator solution and mix thoroughly. All solutions
must be at room temperature. The indicator changes from colorless
to pink as peroxide is liberated from the glucose. The photoelectric
colorimeter is now set to measure at wave length 440 m and the
blank is set so that it gives 100 per cent transmittance.* Then exactly
20 minutes after adding glucose oxidase each tube is read in the in-
strument to determine optical density. To calculate the glucose con-
eThe blank should be tested against water set at 100 per cent transmittance, once near
the time reagent is added and then 30 or more minutes later. Any substantial tendency for
transmittance of the blank to decrease with time suggests that not all glucose has been
removed from the peroxidase solution by dialysis. Such extracts are unsatisfactory for use
without further purification.
Vol. 4, No. 6 958 ENZYMATIC METHOD FOR GLUCOSE DETERMINATION 467
tent of the unknown urine (before dilution with 19 ml. of water) USQ
the formula:
O.D. unknown
X 0.1 = per cent glucose in urine.
O.D. standard
Reagents:
1. 0.3 N barium hydroxide solution.
2. 5 per cent zinc sulfate solution.
3. 1 per cent orthoanisidine in 95 per cent alcohol.
4. Peroxidase solution. Horseradish root extract as described for
urine methods.
5. Glucose oxidase-indicator solution. Identical with that de-
scribed for urine quantitative method.
6. Glucose standard. 0.005 per cent glucose in saturated benzoic
acid (5 mg. glucose per 100 ml).
Procedure:
The method may be applied to serum or whole blood only after re-
moval of protein. Tungstic acid filtrates cannot be used, but the
Somogyi zinc filtrates made by using barium hydroxide as the alkali
are free of metallic ions and therefore satisfactory. Blood samples
in which 10 mg. sodium fluoride and 1 mg. mercuric chloride per ml.
have been used as preservative and antocoagulant are suitable.
In a 15-ml. centrifuge tube place 4.5 ml. of water and mix with this
0.3 ml. of blood. Add 0.6 ml. of 0.3 N barium hydroxide solution, mix
and let stand a minute or two until it turns brown. Then add 0.6 ml.
of 5 per cent zinc sulfate with mixing. After 5 minutes, centrifuge and
use 1 ml. of the clear supernatant for analysis in the identical manner
described above for diluted urine except that the step of treating
with charcoal and Lloyds reagent is omitted. The following formula
is used to calculate the glucose concentration in the original blood:
O.D. unknown
x 100 = mg. per cent glucose in blood.
O.D. standard
A NOTE ON THE ASSESSMENT OF PEROXIDASE ACTIVITY IN HORSERADISH
ROOT EXTRACTS
The validity of the enzymatic spot test for use in screening urine
samples for glucose has been assessed by comparison with 2 other
methods.
The first, used for many years by life insurance laboratories, was
devised by Benedict (17). Based upon the ability of glucose in alka-
line solution to reduce picrate to orange-colored picramate, it is non-
specific and responds to numerous other reducing substances that are
normally present in urine in variable amounts. The color due to
creatinine is suppressed during the test by adding a few drops of
acetone. The test provides for comparison against 5 permanent
visual color standard tubes ranging from 0.1 to 0.5 per cent glucose.
Higher glucose levels are assessed by dilution to proper range. Long
Vol. 4, No. 6. 1958 ENZYMATIC METHOD FOR GLUCOSE DETERMINATION 469
experience with the procedure has proven that when the 0.3 per cent
level is indicated, “true glucose” is extremely likely to be present in
the urine. However, at this or even higher readings glucose may be
absent. Comparison of this procedure with the enzymatic test on a
selected group of 1,600 urines is presented in Table 1. These urines
were chosen because they fell in the “doubtful” range of the picric
acid method, where reduction is substantial but the presence of true
glucose is uncertain. The results show that as the picrate reduction
level rises the percentage of specimens responding to the enzymatic
test rises and the intensity of those giving a positive reaction also in-
creases. At the 0.25 per cent level indicated by the picrate method
only 14.4 per cent of the samples react to the enzyme test, but at the
0.50 per cent picrate level 84.0 per cent are enzyme positive. This dis-
tribution is consistent with the fact that the enzymatic test is specific
and precise.
The second method used for comparison with the enzyme spot test
was the qualitative copper reduction test of Benedict (18). Table 2
presents a summary of findings in 500 random urine specimens.
Agreement between the 2 tests is very good. In a few instances the
enzymatic reagent gave a slight positive reaction with the Benedict
Table 1. COMPARISON OF’ ENZYMATIC Spor TEST WITH PIcarE REDUCTION METHOD FOR
GLUCOSE DETECTION
Pereentag.
Glucose by Reaction to Enzyme Test Total
Picrate Method Negative Slight Moderate Marked Cases
(Percent age) (Number)
Table 2. COMPARISON 01’ ENZYMATIC Sor TEST WITH BsmnICTs COPPER REDUCTION TEST
FOR GLUCOSE DETECTION
Picrate Reaction
Number % Glucose by Decreased Lntt of Sensitivity to Enzyme Test
Oases Picrate Test by Fermentation .OOS..014%* .015-.O3O%*
(Nmnber of Cases)
17 0.35 1 8 9
19 0.40 1 8 11
8 0.45 1 1 7
6 0.50 1 2 4
1 0.70 0 0 1
1 0.80 1 0 1
1 1.25 0 1 0
53 5 20 33
* Percentage of glucose added to the urine that is just detectible by the enzymatic reagent.
Vol. 4. No. 6, 1958 ENZYMATIC METHOD FOR GLUCOSE DETERMINATION 471
the enzymatic test as here described would miss detecting any signifi-
cant level of urinary glucose.
THE QUANTITATIVE METHOD FOR URINE GLUCOSE
The quantitative procedure was studied in regard to the relation-
ship between glucose concentration and optical density measured by
spectrophotometer. Figure 1 shows that color intensity obtained for
various times of reaction up to 60 minutes falls on a series of straight
lines when plotted against glucose concentration.
As will be seen, the color intensity changes with time as the en-
zymatic reaction proceeds. It may be desirable to stop the reaction
and stabilize the color at a specified time. This can be done by adding
to each tube, at the correct moment, 3 drops of 6 N hydrochloric acid.
This shifts the pH to an unfavorable one, stops the reaction, and at
the same time changes the color to much yellower, due to the fact that
the colored product from orthoanisidine is itself an acid base indica-
tor. Thus the optical density at 440 m is decreased by acidification.
This is ifiustrated in Fig. 2, which shows, however, that Beer’s Law is
still obeyed and demonstrates that the acidified solutions are valid for
determining glucose concentration.
20 30 40 30 6070
MO Glucos.
Fig. 1. Relation of color intensity to glucose concentration at various reaction times.
Carried out at room temperature, 22.5#{176}.
Spectrophotometer readings at 440 nit.
472 BEACH & TURNER Clinical Chemistry
30 40 50 60
g Glucos.
Fig. 2. Relation of color intensity to glucose concentration at 60 minute reaction time.
Upper curve at pH 7.0; Lower curve, 3 drops 6 N HCI added to terminate reaction at 60
minutes. Spectrophotometer readings at 440 n1c.
*0.050% Glucose was the known concentration of the solutions used for these recovery
experiments.
*Glucose was added to the urine to make 1 per cent concentration; this was diluted further
with the glucose-free to yield the series.
Table 6. BLOOD SUGAR BY THE ENZYMATIC METHOD COMPARED WITH VALUES BY OTHER
PRoCEDURES
1 47 50 62
2 63 60 74
3 64 59 71
4 64 62 74
5 68 62 77
6 68 69 83
7 72 70 71
8 76 69 82
9 83 86
10 85 81 91
11 86 87 94
12 89 80 94
13 92 80 100
14 97 94 100
15 100 87 112
16 100 94 100
17 101 100 115
18 107 106 118
19 113 129
20 118 123 118
21 128 123 120
22 130 143
23 132 119 125
24 137 141 158
25 165 150 153
26 169 147 157
27 182 187 188
28 207 175 197
29 232 225 221
30 239 214
31 242 242 229
3! 261 236
33 266 260 229
34 285 274 250
35 460 466 400