REDUCING NON-SUGARS AND TRUE SUGAR IN

HUMAN BLOOD.

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

(Received for publication, June 24, 1927.)

It is known that the commonly used methods for blood sugar

determination yield higher values than the sugar actually present.
The new alkaline copper reagent of Benedict (1) and Folin’s
modified reagent (2), which are more specific to sugars than the
older reagents, yield values still too high. In using mercury salts
for the precipitation of the blood proteins, simultaneously a

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considerable portion of the non-sugar reducing substances is also
removed, but this effect is only partial and inconsistent. As
Harned (3) has shown, sugar values obtained by the Folin-Wu
method in mercury filtrates are not lower than those determined
by Benedict’s new method in tungstic acid filtrates. Lapa (4)
recently recommended precipitation by mercuric acetate as a
means of obtaining values close to true sugar. His determina-
tions were carried out only on plasma, and show 3 to 6 mg. per
cent reducing non-sugars. Since the tungstic acid filtrate of
plasma contains, as we shall show, not much more of these sub-
stances, Lapa’s method offers no material advantage.
The older attempts to obtain true sugar values were based upon
Otto’s (5) original precedure of determining the “residual reduc-
tion” after alcoholic fermentation of blood, and to subtract this
from the “apparent sugar.” But this seemingly simple method
led only to a maze of conflicting results, various authors having
obtained residual reduction values ranging from zero up to 60 mg.
per 100 cc. of blood, in terms of glucose. One cause of discrepan-
cies, pointed out in the work of Frank and Brettschneider (6)
and of Ege (7), is the disregard by earlier workers of the fact that
various reagents, used in different reduction methods, yield differ-
ent values with identical reducing substances. The main source
33
34 Sugar in Human Blood

of error, however, was recognized by Salkowski (S), Neuberg (9),

and Mayer (lo), who noted that variable amounts of reducing,
and also optically active, substances are given up by yeast in the
course of fermentation, even if pure glucose solutions and pure
cultures of yeast were used in the experiments.
Van Slyke and his collaborators (11) have overcome this diffi-
culty by a drastic abbreviation of the fermentation period. They,
too, found that in the course of prolonged fermentation reducing
substances are produced in the reaction mixture, but that this
does not occur within short periods which are at the same time
sufficient completely to ferment the sugar in the blood. They

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chose incubation for 20 minutes at body temperature as a suitable
procedure. For the reduction derived from impurities intro-
duced with the yeast they made correction.
Ege (12), taking exception to the brevity of this fermentation
period, asserts on the basis of his previous experiments (7) that
at least 24 hours are required for the complete fermentation of
blood sugar. We have found, however, that in the course of such
long fermentation periods chemical, and occasionally bacterial,
changes set in, which frequently lead to an almost complete disap-
pearance of all the non-fermentable reducing substances (13).
Subsequently Folin and Svedberg (14) showed that even 8 minutes
incubation with yeast suffice completely to dispose of the blood
sugar.
In a preliminary note, concerning reactions between yeast and
various sugars, we reported (15) the extremely rapid disappearance
of glucose from solutions under certain conditions. In the course
of the experiments, still in progress in this laboratory, we attempted
the practical application of our finding in the determinaton of
true sugar in blood (and other biological fluids) by the separation
and determination of the reducing non-sugars or residual reduc-
tion. We arrived at the conclusion that it is unnecessary to
incubate the blood with yeast even for the brief periods recom-
mended by Van Slyke or by Folin and Svedberg, as the sugar is
completely removed at room temperature in the course of the
Folin-Wu precipitation of the blood proteins, if the water for
dilution and laking is replaced by a 10 per cent (moist weight)
yeast suspension.
 M. Somogyi 35

The procedure is as follows: Into 7 volumes’ of a 10 per cent

yeast suspension introduce 1 volume of blood, thoroughly mix
and agitate for a few seconds, then add 1 volume of 10 per cent
sodium tungstate, mix, and finally add 1 volume of 0.66 N sul-
furic acid; shake well, allow to stand for 5 minutes, and filter.
Centrifugation may be inserted before filtration if small amounts
of blood are used.
Preparation of Yeast.-Commercial yeast is always contami-
nated with adhering particles of wort which contain unferment-
able reducing substances. Since the determination of these,
for the purpose of correcting the reducing non-sugar values, led

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to inconsistent results, we decided to wash the yeast entirely
free from reducing substances. This is performed as follows:
A weighed amount of fresh commercial yeast (Fleischmann’s)
is suspended in 5 to 10 parts of water, centrifuged, and the water
decanted. This operation is repeated until the supernatant liquid
is practically clear and colorless, and the last washing gives a
zero reduction with the copper reagent. The yeast is then sus-
pended in 10 parts of water and is ready for use. In this condi-
tion it keeps well in the cold, especially if at intervals the water is
centrifuged off and replaced by fresh water.

Apparent sugar, mg. per cent.. . . . . . . . . . . . . . . . . . . . 104 116 314

Reducing non-sugars, mg. per cent.
1. Precipitation performed immediately after
mixing yeast and blood.. . . . . . . . . . . . . . . . . . . . 31 27 22
2. Precipitation 2 min. after mixing.. . . . . . . . . . . 31 25 24
3. “ 5 “ “ “ .... ... .. ... 31 26 22
4. “ 20 “ “ ‘I . . . . . . . . . . . . . 31 23
5. “ 45 “ “ “ .... ... .. ... 26 22

With the technique outlined we obtained consistent results in

over 100 experiments with human blood. The temperature of
our reaction mixtures was always that of the laboratory, 20-23°C.
in most of the cases, the maximum temperature observed being
28°C. In over twenty caseswe ascertained that the removal of

1 We use 7) instead of 7 volumes, allowing 4 volume for the cell volume

of the yeast; but the error from disregarding this correction is practically
negligible.
 Sugar in Human Blood

the blood sugar is complete without allowing any extra time for
fermentation preceding the precipitation of proteins. A few
examples-tabulated on the preceding page-will illustrate this,
the rest of the experiments showing identical results.
Numerous experiments, performed in work in this laboratory
along other lines, show a rapid disappearance of glucose from
solutions of considerably higher concentrations than occurred in
our blood samples. In the experiment given in Table I solutions
containing 160, 400, and 800 mg. per cent glucose were subjected
to the same procedure, including dilution and precipitation, as
blood; to furnish the protein, the yeast was suspended in a

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neutral solution of pure casein. According to the titration
figures in Table I, at a concentration of 400 mg. per cent the re-

TABLE I.

Showing the Rapid Disappearance of Glucose from Solutions in

Mixture with Yeast.

Mg. per cent glucose.

Time from mixing of

mgar and yeast till 160 400 800
precipitation. I I

Cc. of 0.005 N sodium thiosulfste used in titration.

min.

<1 22.21 22.23 21.58

2 22.22 22.22 22.02
5 22.20 22.22 22.20
20 22.21 22.19 22.23

sult is still the same whether the precipitation is performed im-

mediately after the glucose and yeast were united, or 20 minutes
later. At the extreme concentration of 800 mg. per cent, how-
ever, 5 minutes must be allowed before precipitation in order to
reach a constant titration figure. The filtrate in blank experi-
ments, in which the glucose was omitted, gave a titration figure
of 22.23 cc. at the beginning of the experiment, and 22.21 cc.
after the yeast suspension had been standing for 3 hours at 27.5”C.
Thus the constant titration figures in Table I indicate the com-
plete disappearance of glucose.
 M. Somogyi 37
TABLE II.
Reducing Non-Sugars in Normal and Pathologic Human Blood.
Normal individuals. IIospital patients

Apparent Redwing Apparent Reducing

NO. blood sugar. non-sugar. NO. blood sugar. non-sugar.

mg. pm cent mg. per cent mg. per cent mg. per cent
1 loo 31 34 108 31
2 76 28 72 416 29
3 108 27 401 211 29
4 94 27 404 144 24

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5 102 29 405 113 26
6 99 28 538 100 23
7 120 28 544 87 25
8 107 27 545 101 25
9 108 26 548 109 29
10 110 31 549 175 27
11 120 25 550 103 26
12 113 28 551 226 28
13 136 25 556 234 2a
14 124 28 559 90 25
15 94 30 561 175 23
16 147 25 010 108 27
17 100 27 018 174 25
18 112 25 019 82 23
19 96 25 020 113 28
20 97 23 021 215 30
21 163 28 029 214 22
22 116 26 030 89 27
23 150 29 031 133 24
24 137 29 034 224 29
25 123 27 035 244 -30
26 110 24 084 99 26

Lowest. ... .. . . . . . . . . 23 23
Highest ... . . . . 31 31
Average.. ... . . .. ... 27 20

Reducing Non-Sugars in Health and Disease.

We have determined the reducing non-sugars in the blood of

twenty-six healthy individuals and in a large number of samples
from hospital patients. Of these twenty-six are given in Table
38 Sugar in Human Blood

II, picked in the chronological order as they were examined, with

the exclusion only of samples showing high nitrogen retention.
The values in this work were obtained by the Shaffer-Hartmann
method, with the reagent modified by the writer (16). An in-
teresting fact disclosed in this table is the remarkable uniformity
of the amount of reducing non-sugars: both in health and in
disease it is 23 to 31, an average of 27 mg. in 100 cc. of blood,
expressed in terms of glucose. The only exceptions to this were
cases with high nitrogen retention, in most of which-as shown
in Table III-the amount of reducing non-sugars rises above the
normal level. This is in line with the findings of Hiller, Linder,

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and Van Slyke (11) who reported residual reduction values of 40

TABLE III.
Reducing Non-Sugars in Nitrogen Retention.

Reducing Non-protein
Apparent sugar. non-sugar. nitrogen.
Case No.

Mg. per 100 co.

517 109 36 88
555 312 31 82
298 28 68
303 41 185
387 123 43 160
403 119 38 60
404 144 24 56
405 113 26 44
418 297 36 71

to 48 mg. per cent (Folin-Wu method) in the fermented blood of

patients with glomerulonephritis. There is, however, no direct
relation between non-protein nitrogen and reducing non-sugars,
and even normal values for the former are compatible with high
non-protein nitrogen.
The other important characteristic of the reducing non-sugars,
as shown in Table II, is their independence of the blood sugar
level. Previous workers found greatly increased residual reduc-
tion in various cases of hyperglycemia, such as diabetic and hemor-
rhagic hyperglycemia (5, 17). Lund and Wolf (18) recently de-
termined in diabetic blood as much as 36 to 91 mg. per cent
 M. Somogyi 39
of unfermentable reducing substances. Among the specimens in
our experiments some were drawn from fasting individuals, some
in the state of alimentary hyperglycemia, and a few after the sub-
ject had given 500 to 600 cc. of blood for transfusion; but the
reducing non-sugars are unaffected by these factors. The samples
from hospital patients comprise a number of diabetic bloods which
exhibit, without exception, a normal amount of reducing non-
sugars. The lack of parallelism between variations of the sugar
level and the reducing non-sugars-as also pointed out by Folin
and Svedberg (14)-is still more accentuated in a few experiments

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TABLE IV.
Relation between Reducing Non-Sugars and Changes in Blood Sugar Level.

Case 523. I Case 023. I Case 048.

Time after
ingestion of
glucose.

Mg. per 100 cc.

min.
0 (Fasting.) 109 28 272 27
30 355 33 375
60 183 30 414 28 410
120 142 30 433 29 459 23
180 77 28 447 26 465 23
-

given in Table IV, in which the amount of reducing non-sugars

was followed up in sugar tolerance tests, after the ingestion of 100
gm. of glucose. While in none of the three cases are the reducing
non-sugars increased with rising blood sugar, in the severe diabetic
cases, Nos. 023 and 048, there is a distinct decline as the high
sugar level persists. Van Slyke and his associates (11) found that
the residual reduction is likewise unaffected by insulin hypogly-
cemia in rabbits even though the blood sugar be reduced to zero
in insulin convulsions.

True Xugar Values.

Before concluding the difference between apparent sugar and
reducing non-sugars to be true sugar, we had to consider another
possible source of error. According to Holden (19) amino acids,
 Sugar in Human Blood

as glycine, cystine, glutamic acid, giving alone little or no reduc-

tion with alkaline copper solutions, appreciably increase the re-
duction values of added glucose. We had to ascertain, therefore,
whether or not similar induced or coupled reactions occur in the
determination of apparent sugar; in other words, whether the
apparent sugar represents the actual sum of true sugar and re-
ducing non-sugars or perhaps some higher value. To this end
we prepared sugar-free blood filtrates and determined their re-
duction values with and without the addition of known amounts
of glucose. The result invariably was the sum of the reduction
exerted by the non-sugars plus the reduction calculated for the

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added glucose. For example:
With 5 cc. of copper reagent were heated for 15 minutes in a water bath
3 cc. of sugar-free filtrate of corpuscles + 2 cc. of HZO; reduction = 23 mg.
per cent in terms of glucose.
3 cc. of Hz0 + 2 cc. of glucose solution; reduction = 96 mg. per cent
glucose.
3 co. of sugar-free filtrate of corpuscles + 2 cc. of glucose solution; re-
duction = 118 mg. per cent in terms of glucose.
23+96 = 119.
Thus the difference between apparent sugar and reducing non-
sugars is the true sugar. In most cases it is not necessary to
determine the exact amount of the reducing non-sugars; because
of the uniformity of this value, it is sufficiently accurate for most
purposes to use 27 mg. per cent as a correction. By subtracting
this amount from the apparent sugar, the true sugar is obtained
with a maximum error of f 4 mg. per cent. The probable
error is even less, as reducing non-sugar values in excessof 29 mg.
per cent and below 24 mg. per cent are relatively infrequent.
Similar corrections may be readily established for any other
method for blood sugar determination, the above figure applying
only to the Shaffer-Hartmann method.

Distribution and Chemical Nature of Reducing Non-Sugars.

In previous, unpublished experiments (carried out in the De-
partment of Biochemistry, Washington University School of
Medicine) we have found that Folin-Wu filtrates and mercury
filtrates of beef plasma yield practically the same sugar values,
whereas from corpuscles, 50 to 70 mg. per cent lower values are
 M. Somogyi 41
obtained in mercury filtrates than in tungstic acid filtrates. This
finding prompted an examination of the distribution of reducing
non-sugars in corpuscles and plasma of human blood. Twenty
specimens, obtained from healthy persons, were analyzed with
the following result.
Reducing Non-Sugars, Mg. per 100 Cc.
In In cm-
‘n,l:2’e plasma. puscles.
Lowest........................................23~ 7 41
Highest.......................................31 13 51
Average.......................................27 10 47

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These figures show that corpuscles contain, on an average,
about 5 times as much reducing non-sugars as plasma. Rock-
wood (20) too, found that a substance, oxidized by the Folin-Wu
sugar reagent but unaffected by Benedict’s new reagent, is chiefly
confined to the corpuscles.
We concur in the opinion of Rockwood and of Sjollema (21)
that only a small fraction of the reducing non-sugars is represented
by those constituents of Folin-Wu filtrates which are known to
reduce alkaline copper solutions. Van Slyke (11) has demon-
strated that in normal blood the sum of the reduction values of
uric acid and creatinine does not exceed the equivalent of 2 to
3 mg. per cent of glucose, and only in casesof high nitrogen re-
tention doesit rise to 10 to 15 mg. per cent. This increase closely
corresponds to the excess over the normal values in the speci-
mens from nephritic patients given in our Table III. Thus it is
safe to assume that some other substance or substances, as yet
unidentified, are responsible for the greater part of the reduction
derived from non-sugars.
The fact that the distribution of the reducing non-sugar sub-
stances in corpuscles and plasma coincides so conspicuously with
that of Benedict’s (22) thioneine or the substance X of Hunter
and Eagles (23), renders it highly probable that this interesting
sulfur compound-shown to be identical with ergothioneine
(2426)-has a part in the reduction exerted by tungstic acid
titrates. By the courtesy of Dr. Eagles we obtained a sample of
ergothioneine and performed the following experiments :
With 5 cc. of copper reagent were heated for 15 minutes in a water bath
3 cc. of 10 mg. per cent ergothioneine + 2 cc. of HZO; reduction value = 23
mg. per cent in terms of glucose.
 Sugar in Human Blood
3 cc. of Hz0 + 2 cc. of glucose solution; reduction value = 95 mg. per cent
glucose.
3 cc. of 10 mg. per cent ergothioneine + 2 cc. of glucose solution; reduc-
tion value = 116 mg. per cent in terms of glucose.
95+23 = 118.

Results of the same nature were obtained with other concen-

trations of ergothioneine and of glucose. As can be seen, ergoth-
ioneine in a concentration at least as high as the maximum found
in corpuscles, yields only a reduction equivalent to 23 mg. per
cent of glucose so that an additional non-sugar must account for
the high reducing non-sugar content of corpuscles. Thompson

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and Voegtlin (27) found that glutathione is present only in the
corpuscles, furthermore that it is largeiy in the reduced form.
Hunter and Eagles (28) estimate the amount of glutathione in
human and animal corpuscles as 100 mg. per 100 cc. and concur
in the opinion that it is, at least for the most part, in the reduced
form. Thus, we believe that glutathione is responsible for a
substantial fraction of the residual reduction in tungstic acid
filtrates of corpuscles.
This contention is apparently in contradiction to the fact that
thioneine does not reduce alkaline copper solutions (22), and that
probably the same holds true for glutathione, at least in its
oxidized form. But it must be borne in mind that glutathione
as well as thioneine consumesiodine even in the cold so that in
our method, and at least in all of those which conclude in iodo-
metric titration, their presence increases the reduction values
irrespective of their reducing effect upon the copper reagent. In
fact, tungstic acid filtrates of blood consume iodine at room
temperature in acid medium, although somewhat less than the
equivalent of the reducing non-sugars. This iodine consumption
is the same whether or not the sugar is removed from the filtrate.

SUMMARY.

A method is presented for the determination of reducing non-

sugars (residual reduction) and thereby for the estimation of true
sugar in blood.
The amount of reducing non-sugars in htiman blood is found to
be very uniform, averaging 27 mg. per 100 cc. of blood in terms of
glucose, as determined by the Shaffer-Hartmann method with
 M. Somogyi

the modified reagent. It is independent of the blood sugar level,

and rises above the normal only in cases of high nitrogen retention.
The distribution of reducing non-sugars in corpuscles and plasma
is unequal; the average value for corpuscles is 47 mg. per 100 cc.,
for plasma 10 mg. per 100 cc.
In human blood the subtraction of 27 mg. per cent from the
apparent sugar, as determined with the modified Shaffer-Hart-
mann reagent, gives the true sugar with a maximum error of f 4
mg. per cent.
BIBLIOGRAPHY.

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1. Benedict, S. R., J. Biol. C&m., 1925, Ixiv, 207.
2. Folin, O., J. Biol. Chem., 1926, lxvii, 357.
3. Harned, B. K., J. Biol. Chem., 1925, lxv, 555.
4. Lapa, V., Bull. Sot. chim. biol., 1927, ix, 310.
5. Otto, J. J., Arch. ges. Physiol., 1885, xxxv, 467.
6. Frank, E., and Brettschneider, A., 2. physiol. Chem., 1911, lxxi, 157.
7. Ege, R., Biochem. Z., 1920, cvii, 229.
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10. Mayer, P., Biochem. Z., 1913,1,362.
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12. Ege, R., J. Biol. Chem., 1926, lxviii, 317.
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14. Folin, O., and Svedberg, A., J. Biol. Chem., 1926, lxx, 405.
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20. Rockwood, R., .I. Biol. Chem., 1926, Ixix, 187.
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Ixvii, 267.
23. Hunter, G., and Eagles, B. A., J. Biol. Chem., 1925, Ixv, 623.
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REDUCING NON-SUGARS AND TRUE
SUGAR IN HUMAN BLOOD
Michael Somogyi
J. Biol. Chem. 1927, 75:33-43.

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