THE NATURE OF BLOOD SUGAR. II.

BY MICHAEL SOMOGYI.
(From the Laboratory of the Jewish Hospital of St. Louis, St. Louis.)

(Received for publication, May 16, 1929.)

In a recent issue of this Journal (1) Folin criticized a paper by

Somogyi and Kramer (2) who controverted some findings of Folin
and Svedberg (3). The issue is this: Folin and Svedberg, having

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obtained lower values for fermentable sugar in blood by Folin’s
modified method (4) than by the Folin-Wu procedure, assumed
the presence in blood of a fermentable sugar other than glucose
“with so much lower reducing power than glucose that . . . it
escaped determination, in whole or in part, when the more weakly
alkaline copper reagent of Folin was used” (1). Somogyi and
Kramer, on the contrary, found practically identical values for
true sugar by several analytical methods. A justified exception
could have been taken, however, as to the comparability of their
results with those of Folin and Svedberg on the ground that the
reagents employed by Somogyi and Kramer were far more alkaline
than Folin’s new reagent. Yet, additional comparative sugar
determinations by four different procedures, including the two
employed by Folin and Svedberg, seem substantially to bear out
the previous findings of Somogyi and Kramer. Furthermore, we
are able to furnish evidence, we believe, to show that the substance
escaping oxidation in Folin’s method is not a fermentable sugar but
the complex of reducing non-sugars.

Analytical Procedure.
In the comparative determinations of true’ (fermentable) sugar
we made use of our zinc precipitation technique (5) which yields
blood filtrates containing no appreciable amounts of non-ferment-
able reducing substances. Thus by a single determination sugar
values are obtained representing at least as close an approach
to true sugar values as the fermentation technique might afford.
157
 Blood Sugar. II
TABLE I.
Comparative Sugar Determinations in Zinc Filtrates oj Human Blood by
Four Analytical Procedures.
-
Shaffer- 1 I I
E ‘~~~~n( Folin-Wu./ Folk. 1 Benedict.
SOU,C,. fied)

Mg. augrtr in 100 CD. blood.

J. Healthy man. 70 69 67 62
S. “ C‘ 91 93 93 94
G. “ ‘I 76 76 71 76

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Mixture of several sampIes. 96 97 95 96
“ “ “ “ 101 104 101 103
‘I “ “ “ 60 61 61 60
1011 Non-diabetic patient. 75 75 73 74
1032 “ “ 70 68 66 68
1047 ‘I “ 67 65 64 65
1063 “ “ 100 96 97 99
1068
‘I “ 82 83 83 83
1069
“ “ 91 91 89 95
1071 “ “ 75 75 75 75
1072 Fasting. 67 67 66 64
After 100 gm. glucose.
30 min. 126 126 125 124
1 hr. 145 148 142 143
2 hrs. 105 106 102 106
3 “ 96 94 91. 92
1008 Diabetic patient. 189 190 180 190
1014 “ I‘ 155 152 148 156
1019 “ ‘I 146 141 142 137
1027 “ “ 310 308 309 315
1028 ‘I “ 314 308 312 319
1026 “ “ 200 195 196 197
1033 ‘I ‘I 105 107 105 108
1041 “ “ 119 120 122 120
1042 “ “ 167 162 161 165
1050 “ “ 128 133 124 131
1053 “ “ 109 110 107 110
1057 “ “ 156 159 150 157
1058 ‘I “ 143 141 138 138
“ “
F1 266 262 263 266
F2 ‘I “ 234 239 220 244
Fs “ “ 178 174 178 174
1104
“ “ 187 183 181 182
 M. Somogyi 159

In the Folin and Folin-Wu methods we have followed the

authors’ directions with meticulous care, with the added precau-
tion that, in order to rule out errors arising from the lack of com-
plete proportionality in color development, the standard sugar
solutions were always so chosen as to agree with the sugar con-
centration of the blood filtrates within 2.5 mg. per cent (or 0.05 mg.
per 2 cc.). To make this possible, calorimetric work was always
preceded by titrimetric determinations.
In the Shaffer-Hartmann method an improved copper reagent

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was used, made up of 7.5 gm. of copper sulfate, 20 gm. of sodium
carbonate, 25 gm. of sodium bicarbonate, 25 gm. of Rochelle
salt, 5 gm. of potassium iodide, and 10 cc. of N potassium iodate
per liter. Details concerning this reagent will be given in another
paper.
Sugar Determinations.
Comparative sugar determinations were performed by three
calorimetric procedures: the Folin-Wu, the Folin, and the Bene-
dict (6) methods, and in addition by the modified Shaffer-Hart-
mann method. As stated above, true sugar values were ob-
tained by direct determinations in zinc filtrates. For the colori-
metric determinations, since the copper reagents involved possess
no buffer effect, the filtrates had to be neutralized by carbonate-
free sodium hydroxide, with phenolphthalein as indicator.
The results, recorded in Table I, show that the four analytical
procedures give identical sugar values within the rather narrow
limits of experimental errors. The new Folin reagent yields in
some cases slightly lower results than the other three, but the
difference is conspicuous in a single case (Case Fz). The close
agreement of the values of thirty-five determinations justifies the
conclusion that, on the whole, Folin’s new reagent gives the same
values for true (fermentable) sugar as the original Folin-Wu
method and the other two methods employed in this work, and, for
that matter, as any other adequate method.

Non-Fermentable Reducing Substances Escape Oxidation in

Folin Method.
In apparent contradiction to the foregoing, we can corroborate
the observation of Folin and Svedberg that Folin’s procedure
160 Blood Sugar. II

TABLE II.
Showing That in Folin’s Method Reducing Non-Sugars Escape Oxidation if
Su&ient Unoxidized Glucose Is Present.

Reduction values per 100 cc. blood, in terms of glucose.

i
Case No.
Time of
heating. Residual Added
T Total reduction.
reduction. glUCOSe. Error.
To be Found.
expected.

min. VW. ml. mg. ml. ml.

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la 10 12 75 87 80 -7
20 13 75 88 90 4-2
lb 10 12 200 212 198 -14
20 13 200 213 213 0

2 10 9 100 109 102 -7

20 11 100 111 114 $3

3a 10 10 50 60 61 $1
20 14 50 64 66 4-2
3b 10 10 200 210 189 -21
20 14 200 214 214 0

4a 10 75 83 80 -3
20 75 84 88 +4
4b 10 200 208 197 -11
15 200 209 2Q6 -3

5a 10 50 58 56 -2
20 50 59 59 0
5b 10 200 208 198 -10
20 200 209 206 -3

Folin-Wu method.

.z E 1 i ) :8” j 2i ) 2:: / 2;: ) +i

Shaffer-Hartmann method.
 M. Somogyi 161
yields lower values for fermentable blood sugar than the Folin-Wu
method (and, we may add, than the other two methods employed
in this work), when the determination is carried out in tungstic
acid jiltrates, by deducting the residual reduction from the total
reduction. Evidently some reducing substance does escape
oxidation in Folin’s procedure; some reducing substance which,
as we have seen, is obviously absent from zinc filtrates but is pres-
ent in tungstic acid filtrates. The assumption that it may be the
non-sugar reducing substances (residual reduction) that are in-

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volved in this process, is supported by Benedict’s interesting ob-
servation that “in the presence of glucose the residual reducing
material is without effect” upon the reduction values when his
new reagent is employed (7). This prompted an examination of
Folin’s method in this direction.
Tungstic acid filtrates were treated with washed yeast (8), and
the reduction values of the fermented filtrates were determined
before and after the addition of known amounts of pure glucose.
The results, presented in Table II, were obtained by a simple pro-
cedure, executed, for example, in Case la as follows: A substantial
sample of blood (about 20 cc.) was deproteinized by tungstic acid
at 1: 5 dilution. The filtrate was fermented, and after neutralrza-
tion 1 cc. portions of it were measured into two Folin-Wu tubes.
To one of the tubes, serving for the determination of the residual
reduction, 1 cc. of water, to the other 1 cc. of a 0.015 per cent
glucose solution were added; 1 cc. of thesame glucose solution plus
1 cc. of water furnished the standard for the latter, while a tube
with 2 cc. of a 0.001 per cent glucose solution was used as standard
for the residual reduction. After the addition of Folin’s reagent,
the entire set, supported by a rack as employed in the Shaffer-
Hartmann method, was placed in boiling water for 10 minutes.
Subsequently, an identical set was heated for 20 minutes.
From Table II the following information may be obtained: (1)
Folin’s method yields too low reduction values in tungstic acid
filtrates even when glucose is the sole fermentable substance
present. (2) T oo 1ow reduction values occur only if the duration
of heating does not exceed 10 minutes; upon more prolonged
heating (15 or 20 minutes) both the glucose and the non-ferment-
able reducing substances are fully oxidized. (3) Even within
10 minutes the non-fermentable reducing substances are nearly
 Blood Sugar. II
completely oxidized in the absence of sugar or if the sugar present
is not in excess of 50 to 60 mg. per cent of fermentable blood sugar.
With rising sugar concentrations a fraction of the reducing sub-
stances remains unoxidized, until at hyperglycemic sugar levels
this fraction is equal to the residual reduction.
These facts suggest the inference that glucose and non-ferment-
able reducing substances in tungstic acid filtrates are competing,
so to speak, for oxygen, glucose taking the precedence and thereby
inhibiting the oxidation of the non-sugars. In Folin’s procedure

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16 to 20 per cent of the glucose (or more correctly, its cleavage
products) are always left unoxidized after 10 minutes’ heating, and
if the initial sugar concentration has been high, as in diabetic blood,
the unoxidized fraction of it suffices completely to protect the non-
sugars from being oxidized in 10 minutes. At lower initial sugar
concentrations the protective effect is but partial and becomes
imperceptible at the lowest normal levels of blood sugar. The
oxidation of the non-fermentable reducing substances is not in-
hibited by sugar if the heating is prolonged to 20 minutes, since at
this stage but a very small fraction of the sugar is left unoxidized.
Two examples given at the end of Table II show that in the Folin-
Wu and the Shaffer-Hartmann methods the reducing non-sugars
are completely oxidized regardless of the amount of the sugar
present. This is in part due to the fact that in the Folin-Wu pro-
cedure only 6 to 8 per cent of the glucose is left unoxidized at the
end of 6 minutes heating, and almost none after 15 minutes in
the Shaffer-Hartmann method. But there is an additional factor
affecting the process; namely, the degree of alkalinity. If the
reaction is interrupted in the Folin-Wu procedure after less than
6 minutes heating, so that the oxidation is stopped about 20 per
cent short of completeness, no inhibition in the oxidation of the
non-sugars can be established. With the Shaffer-Hartmann re-
agent employed in this work, on the other hand, we were able to
demonstrate the protective effect of glucose if the heating was
interrupted after 6 minutes, at which stage 16 to 18 per cent of
the glucose is still unoxidized. A marked inhibition in the oxida-
tion of the non-sugars could be observed, however, only at a
sugar concentration about twice as high as suffices to produce the
like effect in Folin’s procedure. In other words, low alkalinity
favors the preferential oxidation of glucose.
 M. Somogyi

The original blood sugar shows the same behavior as the pure
giucose added to fermented bIood filtrates. Accordingly, in the
work of Folin and Svedberg the original Folin-Wu method gave
the more correct values for fermentable sugar, while in the new
Folin procedure, the deduction of the non-fermentable reducing
substances from the total reduction led to erroneous results. The
error is greater in hyperglycemic specimens, and this gave occa-
sion to Folin and Svedberg to point out the greater discrepancies
they had observed in diabetic than in non-diabetic cases.

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In conclusion we may add a few words in reply to Folin’s critical
remarks. (1) The first objection voiced by Folin is that Somogyi
and Kramer failed to take into account the autoreduction of the
reagent employed in their calorimetric work. This objection is
wholly without basis, since after a preliminary titrimetric deter-
mination, in subsequent calorimetric work standards were chosen
to approximate closely the sugar content of the blood filtrates.
It is obvious that in such a procedure the autoreductions in stand-
ard and blood filtrate cancel one another. (2) A second objec-
tion concerns the use of open test-tubes by Somogyi and Kramer.
It is unfortunate that Folin overlooked the following statement of
Somogyi and Kramer: “we employ ordinary test-tubes, covered
with glassbulbs.” As in the Shaffer-Hartmann method, the tubes
are held in place on copper racks while being heated in a water bath,
and it can be observed that condensed vapor soon forms an excel-
lent seal between the cover and the mouth of the tube, thus ex-
cluding convection currents of air before any appreciable reduction
had occurred. (3) Folin believes ‘(as a matter of fact” that our
results by the ferricyanide method are not “entirely reasonable”
because our figures ((are certainly not the kind of figures which
should be obtained by the Hagedorn-Jensen method.” They are
not, and cannot possibly be. Somogyi and Kramer made their
determinations on Folin-Wu (tungstic acid) filtrates, while Hage-
dorn and Jensen precipitated the blood proteins by heat coagula-
tion in the presence of zinc hydroxide. Just the observation of
the difference, to which Folin takes exception, led the writer to
recognize the fact that the Hagedorn-Jensen precipitation tech-
nique leaves only about half as much of non-fermentable reducing
substances in the filtrate as the tungstic acid technique.
 Blood Sugar. II

The author acknowledges the helpfulness in this work of much

valuable information he derived from his collaboration with Dr.
P. A. Shaffer in studies of chemical processes involved in sugar
determinations.

BIBLIOGRAPHY.

1. Folin, O., J. Biol. Chem., 81,377 (1929).

2. Somogyi, M., and Kramer, H. V., J. Biol. Chem., 80,733 (1928).
3. Folin, u., and Svedberg, A., J. Biol. Chem., 70,405 (1926).
4. Folin, O., J. Biol. Chem., 67,357 (1926).

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5. Somogyi, M., Proc. Sot. Exp. BioZ. and Med., 26,353 (1929).
6. Benedict, S. R., J. BioZ. Chem., 64,207 (1925).
7. Benedict, S. R., J. BioZ. Chem., 76,457 (1928).
8. Somogyi, M., J. BioZ. Chem., 78,117 (1928).
 THE NATURE OF BLOOD SUGAR. II
Michael Somogyi
J. Biol. Chem. 1929, 83:157-164.

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