ADAPTATION OF SHAFFER
TITRATION METHOD FOR BLOOD
SUGAR TO CLINICAL USE
By
HOWARD D. HASKINS, M.D.,
and
WILLIAM P. IIOLBROOK, B.S.,
Portland, Ore.
From the Department of  Biochemistry, University
of Oregon Medical School, Portland, Ore.
Reprinted from
THE JOURNAL OF LABORATORY
AND CLINICAL MEDICINE
St. Louis
Vol. VIII, No. 11, August, 1923
ADAPTATION OF SHAFFER TITRATION METHOD FOR BLOOD
SUGAR TO CLINICAL USE
BY HOWARD  D.  HASKINS, M.D., AND WILLIAM  P.  HOLBROOK, B.S.,
PORTLAND, ORE.
S
and Hartmann have recently published a complete system of
k--3  methods for estimation of sugar in urine, blood, milk, and other solu-
tions.In all of these methods a copper reagent is used which is similar to
Benedict. This is partly reduced by the sugar. The further treatment of
the mixture causes iodine to be set free. The iodine is titrated with thiosul-
phate, and from the amount of iodine estimated the copper equivalent of the
sugar can be calculated.
Our interest was attracted to the micro-blood-sugar method because it
enables physicians to make the estimations without the expense of investing
in a reliable colorimeter.
Folin method is  the  only colorimetrie method of estimating blood sugar
that we are willing to recommend. We find that the results secured by
Shaffer method are just as accurate as those by Folin method.
In addition to strongly recommending Shaffer method, we wish to pro-
pose a modification that removes the one difficulty which those who are not
expert chemists would encounter in using the method. The difficulty re-
ferred to is that of standardizing the thiosulphate. We have overcome this
satisfactorily by standardizing the thiosulphate against the copper reagent,
which is very easily done. We have proved that this modification does not
cause inaccuracy  in  estimation. Our experimental results will be reported
after the description of the method.
Preparing the Blood for Estimation.?Draw  into the syringe a strong
solution of neutral potassium oxalate (30 grams of oxalate dissolved in 100 c.c.
water), empty the syringe and expel the excess so that only a film of solu-
tion is left. Draw at least 3 c.c. of blood from  a vein and empty the syringe
into  a  dry tube. With an accurate pipette measure 2 c.c. of the blood into
a dry flask and add  14  c.c. of distilled water. When the blood is completely
laked add 2 c.c. of 10 per cent sodium tungstate solution and finally 2 c.c. of
2/3 normal sulphuric acid solution. The volume of the  ,liquid is now exactly
20 c.c. Cork the flask, shake well and let it stand 5 to 10 minutes.
If all the protein has been precipitated, the mixture will be brownish-red
rom the Department of Biochemistry, University of Oregon Medical School. Portland, Ore.
Received for publication, .Tune 18, 1923.
3
n color instead of bright red, and only a trace of foam will appear on shak-
ng. if the proper results have not been secured add 10 per cent sulphuric
teid a drop at a time until satisfactory. (If more than 2 drops are required,
he alkalinity of the stock solution of sodium tungstate must be reduced by
reatment with a definite quantity of sulphuric acid.) Use small size filter pa-
)er for filtration. If the filtrate is not clear and colorless, return all the liquid
and precipitate) to the flask, treat it with more acid, shake well, and after
few minutes filter again. This method of freeing the blood of protein is
!xactly in accordance with Folin 2 system of blood methods. What fol-
ows is Shaffer special technic.
Technic of the Estimation.easure  exactly 5 c.c. of filtrate into a large
est tube and mix with it exactly 5 c.c. of the special micro-reagent. Plug
he tube loosely with cotton and place it in a bath that is boiling actively.
kfter exactly 15 minutes remove the tube and cool it at once under the tap.
Then let it stand in a jar of cold water for 5 minutes. Add 5 c.c. of N/1 sul-
)hurie acid, mix and let it stand  1 minute.
Titrate the liberated iodine by adding N/200 sodium thiosulphate  as
st as drops can be counted (mixing well) until a distinct change is noticed,
hen add more slowly until the color becomes light yellow. Now add about
. c.c. of 2 per cent starch solution and continue the titration cautiously but
lot too slowly until the characteristic starch-blue color is lost and only a
)ale copper-blue remains. When near the end-point determine the effect of
ach drop added.
Calculation.n  the special table (devised by us to correspond to our
modification of Shaffer method) the per cent of glucose in the original blood
gill be found opposite the c.c. of thiosulphate used for titration.
If the sugar content is greater than the highest in the table, dilute some
if the blood filtrate with an equal volume of water and use 5 c.c. of this for
he estimation, multiplying the results by 2.
REAGENTS
(1)  Microcopper  Reagent.issolve each constituent separately.
(a) Dissolve 40 gm. C.P. anhydrous sodium carbonate (or 47 gm. pure
)holographic monohydrated carbonate) in 400 c.c. warm distilled water.
(b) Dissolve 5 gm. C. P. copper sulphate (crystals that have not efflor-
.sced) in about 100 c.c. water.
(c) Dissolve 7.5 gm. pure tartaric acid in 100 c.c. of water.
(d) Dissolve exactly 0.7 gm. pure potassium iodate in 100 c.c. of water.
(e) Dissolve 10 gm. pure potassium iodide in 100 c.c. of water.
(f) Dissolve 18.4 gm. pure neutral potassium oxalate in 100 c.c. of water.
When each is dissolved, mix (c) with (b), and pour the mixture slowly
with stirring) into (a). Combine (d), (e) and (f) and pour this at once into
he carbonate-copper mixture. Transfer to a measuring flask, Rinse all the
lissolving beakers with small portions of water. When cooled to room tern-
rerature fill to the liter mark and mix thoroughly. Keep the reagent in a
The Shaw Supply Co. (of Portland, Seattle, and Tacoma) will supply any or all of the
!hemicals used for the method, including ready prepared solutions.
4
tightly corked bottle. A little sediment will be deposited, use the clear top
liquid for estimations.
If the chemicals are pure, the microreagent run as a control will give
the same titration whether heated in a bath 15 minutes or not heated. With
each batch of reagent run one heated control and compare it with the unheated.
(2) Standard Thiosu.lphate.-Very  dilute thiosulphate does not keep well.
Prepare a stock solution of sodium thiosulphate a little stronger than deci-
normal (dissolve about 26 gm. in about 1 liter of distilled water) and let it
stand 2 (lays.
Dilute exactly 5 c.c. to 100 c.c. in a measuring flask. Mix and use this
to titrate 5 c.c. of the copper reagent (after adding 5 c.c. water and 5 c.c. of
N/1 sulphur:c acid) following the directions given above for the rate of titra-
TABLE I
PER. CENT GLUCOSE IN BLOOD CORRESPONDING TO C.C. THIOSULPIIATE USED FOR TITRATION
C.C. PER CENT C.C. PER CENT C.C. PER CENT C.C. PER CENT
18.6 .036 16.3 .101 14.0 .162 11.4 .232
18.5 .039 16.2 .104 13.9 .165 n.2 .237
18.4 .042 16.1 .106 13.8 .167 11.0 .242
18.3 .045 16.0 .109 13.7 .169 10.8 .217
18.2 .048 15.9 .111 13.6 .172 10.6 .252
18.1 .052 15.8 .114 13.5 .175 10.4 .257
18.0 .055 15.7 .116 13.4 .177 10.2 .263
17.9 .058 15.6 .119 13.3 .180 10.0 .268
17.8 .061 15.5 .122 13.2 .183 9.8 .273
17.7 .064 15.4 .124 13.1 .185 9.6 .279
17.6 .066 15.3 .127 13.0 .188 9.4 .285
17.5 .069 15.2 .129 12.9 .191 9.2 .289
17.4 .071 15,1 .132 12.8 .194 9..0 .294
17.3 .074 15.0 .134 12.7 .197 8.8 .298
17.2 .076 14.9 .137 12.6 .200 8.6 .303
17.1 .079 14.8 .140 12.5 .202 8.4 .307
17.0 .082 14.7 .143 12.4 .205 8.2 .312
16.9 .084 14.6 .146 12.3 .207 8.0 .316
16.8 .087 14.5 .149 12.2 .210 7.6 .325
16.7 .089 14.4 .151 12.1 .212 7.2 .335
16.6 .092 14.3 .154 12.0 .215 6.8 .345
16.5 .095 14.2 .157 11.8 .220 6.4 .357
16.4 .098 1.4.1 .159 11.6 .226 6.0 .368
tion. The titration will be less than 19.5 c.c. When duplicate titrations agree
within 0.1 c.c. prepare a dilution of the thiosulphate such that exactly 19.5 c.c.
will be required for the titration. For example, if the titration is 19 c.c.
it will be necessary to dilute 5 c.c. of the stock solution to 102.6 c.c. (i.e.,
19.5 x 100). First dilute to 100 c.c. in a measuring flask then add the 2.6 c.c.
19.0
with a reliable pipette, and mix well. This ratio of dilution should be deter-
mined by similar titration of a control once in two weeks. Prepare the
dilute solution each day that estimations are made. This dilute solution is
approximately N/200, and is the one referred to in the technic. Keep the
stock solution in a brown bottle well corked, set away where it will not get
warm, under these conditions it does not deteriorate.
(3) Starch Solution.-This  should be made once a week. We prefer
5
"soluble starch" but undoubtedly common corn starch could be used. Mix
about 2 grams of starch with about 10 c.c. of water and pour it into about
90 c.c. of boiling water, mix and boil one minute. A few drops of toluol may
be added as a preservative.
(4) Standard Sulphuric Acid.-The  normal solution may be purchased
if one is not used to checking standard solutions. For the % normal solu-
tion dilute 100 c.c. of the normal sulphuric acid with 50 c.c. of distilled water.
(5) Sodium Tungstate Solution.-C.P  tungstate should be used. The
solution must be testedfor excess of carbonate as follows. Measure with
a pipette 5 c.c. of the solution, add a drop of methyl orange solution and
some distilled water, then titrate with the % normal acid until the yellow
color changes to a slightly reddish yellow. Between 3 and 3.3 c.c. should be
required. If the titration is greater add twice normal sulphuric acid (made
by diluting about 11.5 c.c. C.P. acid. to 200 c.c.) to the whole stock of 10
per cent sodium tungstate solution to bring its alkalinity down to the
proper limits. Mix and titrate again.
TABLE  II
COMPARISON OF ESTIMATIONS BY ''OLIN'S AND SHA FFER 'S METHODS
Blood 1. 2 3 4 5 6 7
Folin .070 .109 .117 .124 .130 .167 .222
Reagent  I .069 .106 .118 .124 .132 .165 .222
Reagent TI .072 .109 .123 .126 .134 .169 .220
Reagent III .069 JOS .117 .122 .132 .167 .219
Note:  Control titrations of Shaffer reagents were as follows:
1-19.55 c.c., 11-19.3 c.c., 111-19.7 c.c.
TABLE III
COMPARISON OF BLOOD SUGAR ESTIMATIONS BY SHAFFER 'S ORIGINAL METHOD AND BY THE
PROPOSED MODIFIED METHOD
Blood 1 2 3 4 5 6 7 8 9
Shaffer .047 .072 .099 .102 .120 .129 .229 .338 .433
Reagent V .048 .071 .100 .102 .122 .129 .230 .342 .436
Reagent  -VI .045 .069 .101 .103 .123 .130 .231 .341 .431
Variation .002 .003 .002 .001 .003 .001 .002 .004 .003
Note:  Reagent IV used for the original method gave a titration of 19.6 c.c. The control
titration of reagent V used for the modified method was 19.9 c.c. and of reagent
VI-19.0 c.c.
DISCUSSION
Different samples of copper reagent do not always give exactly the same
titration value when accurate N/200 thiosulphate is used. We find that
this value is generally close to 19.5 c.c., therefore, we have taken this figure
as the average titration value. Varying the amount of iodate in the reagent
changes the titration value.
Estimations by Shaffer method using accurate reagents are practically
identical with those by Folin method (see Table II). Also when we used
copper reagents that had a slightly different titration value (using exact
N/200 thiosulphate the control estimation on reagent 2 is 19.3 c.c., and on
6
reagent 3 it is 19.7 c.c.) and made the thiosulphate correspond to the re-
agent in each case in accordance with our modification of the method, the
results agreed very closely with the estimations both by Folin method and
by Shaffer (with accurate reagents).
By changing the iodate content we secured copper reagents having a
titration value differing more widely (19.0 c.c. and 19.9 c.c.). With these
less accurate reagents we obtained very satisfactory results by using our
modification of the method (see Table III). In most cases the variations
from the accurate estimations by the original method were only 1 to 3 mg.,
and the greatest variation was 6 mg. Most of the variations were of the
same order as were secured by running duplicate or triplicate estimations
by any one of the methods. The Folin duplicates which we ran, differed by
1. to 2.3 mg. Shaffer triplicates varied by 0.5 to 4.5 mg. These results
convince us that our modification does not perceptibly affect the accuracy
of the Shaffer method.
The explanation for such good results with slightly inaccurate reagents
is, undoubtedly, that the variation of the thiosulphate from a true N/200
solution compensates for the deviation of titration value of the copper re-
agent from the average value. For example, if the reagent is a little weak,
then the thiosulphate solution prepared for use with it will be more dilute
than N/200, thus avoiding the undertitration that would occur in a sugar
estimation if an exact N/200 solution were used.
SUMMARY
An easy method of standardizing the thiosulphate is proposed as a
modification of the Shaffer method, the thiosulphate being checked against
the copper reagent.
Because of this adjustment of the solution the control estimation of the
reagent is always 19.5 c.c. of dilute thiosulphate.
This constant titration value has enabled the authors to construct a
table from which the per cent of glucose in the blood can be read without
calculation.
REFERENCES
haffer,  P.  A., and Hartmann, A. F.: Journal of Biol. Chem., 1921, xlv, 365.
2Folin, 0. and Wu, H.: Jour. of Biol Chem., 1919, xxxviii, 81.
3Pueher, G. W.: Jour. of Biol. Chem., 1922, lii, 333.
7