Factors influenc,ing the Reduction of Alkaline Copper

Reagents by Glucose '.

BY
W. H. Forbes and A. Andreen-Svedberg.
(From the Fatigue Laboratory, Morgan Hall, Harvard University,
and the Clinical Laboratory of the Board of Health of Stockholm.)
(With 6 flew in the text.)

In a previous paper (1) one of us compared the values of blood

sugar found in Somogyi filtrates (2) with those in F o l i n unlaked
blood atrates (3) made from the same sample of blood, using the
revised Folin-Wu method (4) for the sugar determinations. Out of
32 samples of fasting mammalian blood (man, normal and diabetic,
pig, calf, rabbit, but not dog), identical values were obtained from both
filkates seven times. Fourteen times the Somogyi filtrate gave a
higher value (1 to 5 mg.), and eleven times the unlaked blood filtrate
was higher (1 to 4 mg.). The average post-fermentation reduction was
the same for both filtrates, (2.4 mg.). (The figures are milligrams
per 100 C.C. of blood.) It wi19 therefore concluded that both filtrates
contain the same amount of fermentable reducing substance.
The validity of these results was questioned, and it was suggested
that the coincidence between the values obtained might be apparent
rather than real. The bases of this criticism lay in the differences
in pE between the two filtrates (Somogyi 7-0-7.6; F o l i n 3.6-4.0),
and in the fact that the reduction of copper by glucose is a function
of the pH of the solution in which the reduction takes place. The
investigation which was started in order to elucidate this question soon
revealed that the pH of the filtrate is a factor of no importance. In
the course of the work, however, certain similar questions arose, as
for example the effect of the salt content of filtrates, and the study
of these led in turn to a brief study of a few general characteristics of the
reduction of copper solutions by glucose. The many factors involved in
Der Redaktion am 17. Juli 1934 eugegangen.
 FACTORS
INFLUENCING THE REDUCTION,
&c. 169
the determination of glucose in blood filtrates by alkaline copper
reagents have always meant a danger of experimental errors. This
danger is greatly increased when a method is used under different
conditions than those for which it was designed, e. g. the determination
of glycogen by hydrolysis to glucose followed by the determination
of the glucose by a method designed for use on blood filtrates.
In order to be able to discuss the general question of the reduction
of copper by glucose three methods were studied: the revised Folin-Wu (4),
the S h a f f e r - H a r t m a n n as revised by Shaffer and Somogyi (5)
(hereafter referred to as the Sh a ff e r - S o m o g j i), and the B en e d i ct
1931 (6). The large majority of the work was done on the first of
these, which was the method involved in the original problem, and
which is fast and reliable and has a simple copper reagent. This last
characteristic makes it peculiarly suitable for studying the fundamental
features of the reduction. Studies of the other two methods are included
later in the paper and show that the second behaves approximately
as could be predicted from the study of the revised F o l i n - W u reagent,
while the Benedict 1931, though having a very different composition,
shows some of the same phenomena.
The literature of this subject contains many piecemeal studies and
observations of the effect of one factor upon one reagent, but there are
few papers in which any of the several factors are correlated. A recent
and comprehensive investigation is that by S haffer and Somogyi (5),
dealing largely with the effects of changing the composition of the
reagent and the time of heating in the S h a f f e r - H a r t m a n n method
- a paper of especial interest to those who would design a new method
or who are interested in distinguishing between the various sugars.
They deal principally with changes in the method. The present paper
on the other hand deals primarily with the effect of the presence of
various substances in the solution whose sugar content is to be deter-
mined. The point of view adopted throughout is that of the user of
the method who is interested in knowing under what conditions a given
method will give good results and under what conditions it will not;
consequently the inquiry was restricted for the most part to those factors
which may vary in the ordinary use of the method.
Solutions whose glucose content one wishes to determine may
differ from each other in five respects which a prim' seem likely to
be of significance in this inquiry. These are: pR, titratable acidity, salt
content (including kind of salt and concentration), concentration of sugar,
and presence of organic substances other than glucose. This paper is
concerned mostlj- with the first three, the fourth is touched upon, the
fifth is omitted.
170 W. H. FORBES
AND A. ANDREEN-SVEDBERG:

The first may be summarily disposed of. The pEr of the unknown
solution whose glucose content is to be measured does not determine
the pH of the mixture of unknown and reagent during the time of
heating. This is determined by the titratable acidity of the unknown.
If other factors are held constant, it is the pIr of the mixture during
the heating period which determines the amount of copper reduced
b\- 1 milligram of glucose, and therefore it is the titratable acidity of
the unknown, and not its pH, which is significant. Furthermore it is
not the titratable acidity to pR 7, but to the pH of the mixture which
is important. This pn is not known and can be found only bF ex-
periment, or calculated from the titration curves of the unknown and
the reagent. However the reduction takes place very slowly on the
acid side of pR 9.0 and therefore the pH of the mixture must be at
least as high as that. As a reasonable approximation one mag take 9 5, -
(the blue color of thymol blue), and say that it is the titratable acidity
of the unknown or filtrate to pH 9.5 which should be considered. Most
modern copper reagents have pH values lying between 9.1 and 10.3
and are so well buffered that unless the titratable acidity of the
unknown is quite large the pH of the mixture will be close to that
of the reagent.
-
Experiments with the revised F o 1i n W n method.
The effect of varying the titratable acidity of the unknown was
investigated as follows. Two C.C. of a solution containing 0 . 1 mg. of
glucose per C.C. (the “100 mg. per cent sugar standard”) were put into
each of a number of sugar tubes. Then small amounts of strong acid
or strong base, usually 4 W H,SO, or 1N NaOH, were added. Then
2 C.C. of the freshly made up revised Folin-Wu reagent (4) were added,
and the tubes put in boiling water for 8 minutes, then cooled, molyb-
date added, and the depth of color resulting measured in a colorimeter.
See (4)for a full description of the method. H,SO, was chosen because
it is a common ingredient of filtrates and because, being a strong acid,
its titratable acidity to pH 9.5 is so close to its titratable acidity to pH 7 . 0
that they may be taken as the same, and equal to the amount of acid
added. The acid (or alkali) added must be fairly concentrated because
the addition of fluid lowers the amount of copper reduced by a little
less than 1 per cent for each 0.1 C.C. added. This effect seems to be
about the same at different sugar concentrations and is not easy to
explain. It means that sugar tubes should be dry when used, or if
not dry, that they should contain less than 2 drops of water. In these
experiments, with the exception of a few of the extreme points, the amount
of fluid added did not exceed 0.12 C.C. The same experiment was
 FACTORS
INFLUENCING THE REDUCTION,
&c. 171
performed at different sugar concentrations ranging from 0.0 1 to
0.40 mg. of glucose per c. c. In each experiment there was one tube
which had neither acid nor alkali added and this was used as a standard.
The results are shown in fig. 1. The ordinates are percentages
of the standards. There are three scales for t,he abscissae. One shows the
amounts of acid or alkali added to the tube expressed as C.C. of normal
acid or alkali; although to avoid dilution errors, as explained above, one-
fourth as much 4 normal acid was generally used. The second scale, which
is numerically equal to half of the first within the limits of experimental
error, is the titratable acidity of 1 C.C. of the sugar solution. The
third scale is the pH of the mixture of the reagent and the unknown
just before the heating in boiling water. (It is the same after heating
except in the more acid solutions which lose a little CO,.) The pH
was obtained from the titration curve of the reagent diluted half and
half with water (fig. 2), and was checked by direct measurement. The
measurements were made with a glass electrode, but cannot claim the
usual accuracy of that instrument because, below pH 9.2, CO, is given
off when the acid is added, and above 9.8 the glass electrode becomes
less reliable. The figures on the curves show the strengths of the
sugar solutions, “100 mg.” signifying the 100 mg. per cent. standard,
containing 0.1 mg. glucose per c. c. and so forth.
Rough titration curves were done colorimetrically on several fil-
trates, (fig. 3). These show that for these common filtrates, (7) (2), (81,
(3), (9), the largest titratable acidity to pH 9 . 5 for 1 C.C. of filtrate is
under 0.01 normal. The titratable acidities to pa 9.0 for a number of
Somogyi whole blood filtrates were 0.0034, 0.0075, 0.0080, 0.0060,
0.0059, 0.0060, 0.006, 0.006, 0.0058, 0.0050, average 0.0060.
Corresponding values for Folin unlaked blood filtrates were 0 - 007,
0.006, 0.006, 0.0073, 0.0075, 0.0063, 0.0072, 0.0058, average
0.067. These values are for 1 C.C. of filtrate. The blood was taken
from fasting and non-fasting individuals, some normal and some diabetic.
The Folin filtrates are very slow in reaching their end-points at room
temperature and should be heated.
An analysis of the curves in fig. 1 shows that above pH 9 . 5 the
percentage error introduced by a given titratable acidity in the filtrate
varies inyersely as the logarithm of the strength of the sugar solution,
but the absolute error in milligrams per 100 C.C. increases with the
strength of the sugar solution. A comparison of figs. 1 and 3 shows
that the buffer values of these ordinary filtrates are so low that the
error they introduce is not significant, running from 3 per cent at 10 mg.
to 0 . 5 per cent at 400 mg. This is slightly less than the probable
error of the method as a whole. (N.B. The abcissae in figs. 1, 2, 4
172 W. H. FORBES
AND A. ANDRENN-SVEDBERG:

and 5 are in C.C. of normal acid or alkali, but in fig. 3 in C.C. of

hundredth normal.) One might say as an approximate rule for the
revised F o l i n - W u method that in order to produce a significant error
(2 per cent) in a comparison between two solutions the solutions must
differ in titratable acidity by about 0.018 N acid per c. c. More exact
figures can be got from fig. 1. As the largest titratable acidity found
for any filtrate was 0.0085 per C.C. the error thus introduced is
almost always less than 2 per cent and in most cases less than
0.7 per cent. Since all filtrates are more acid than pH 9 . 5 the differences
between the titratable acidities of filtrates made by different methods
must be smaller than the titratable acidities themselves and there-
fore negligible. Perhaps it should be mentioned that the revised
F o l i n - W u method is relatively stable in this respect, (with the
F o l i n 1926 method (4) these differences are not negligible); and that,
even with the revised Folin-Wu, a change in the manner of making
the filtrate (using N/2H,S04 for example instead of N/3) maj- intro-
duce an error of 4 per cent.
The effect of the concentration of sugar is discussed by Folin (4)
who gives tables showing the deviation from proportionality as the con-
centration is changed. We found the same lack of proportionality,
though it does not shorn in fig. 1, as in each experiment the standard
had the same amount of sugar as the other tubes. It is interesting
to note, however, that if one takes the values for the reduction at
the maxima of the curves in fig. 1 the proportionality is good over large
-
ranges, i. e. a solution containing 0 8 mg. in 2 c. c. at pH 9.9 has
eight times the color of one containing 0 . 1 mg. at pII 9.6 within the
experimental error. If, instead of taking the values at the maxima,
which fall at different pH values, one takes the values at a constant pn,
the proportionality is still good provided the pH chosen lies between
9.3 and 9.8. F o l i n has observed this in connection with the .Folin
reagent (p, about 9.2) which, he says, gives better proportionality than
the revised Folin-Wu (p, 10.3). (These figures are for the reagents
diluted half and half with water or glucose solution. Before dilution
the values are slightly lower.) There are indications that the pro-
portionality may not be really exact in either of the foregoing cases
if the difference between the solutions exceeds 400 per cent.
In the experiments with salts in the higher concentrations the salt
was added to the glucose solution as crystals and the tube shaken
gently until it dissolved. For convenience in the case of the lower
concentrations'the salt was added in solution, the volume of solution
added being kept down to a fraction of 1 C.C. The effects of salts
proved complex. Ther have been investigated bj- E v e r e t t , Shoemaker
 FACTORS
INFLUENCING THE REDUCTION,
&c. 173
and S h e p p a r d (lo), by Somogyi (ll),by E v e r e t t (12), by Shaffer
and Somogyi (5), by Levy and Doisy (13), by L u n d i n (14), and by
others, but no studies have been reported of their effects on the revised
F o l i n - W u method. Salts ma?- affect the reduction by changing the
ionic strength, by changing the pH, by reacting with the sugar or
copper, by acting as positive or negative catalysts, or by any combina-
tion of these four ways. This applies to all methods depending on
the reduction of copper (or of other metals) by sugars in alkaline
solutions. In colorimetric methods such as the revised F o l i n - W u
there is an additional way in which salts can influence the results,
namelj- by influencing the production of color or its fading.
The revised Folin-Wu method is relatively insensitive to the salts
commonly found in filtrates, if the reagent is freshly mixed each day
and if the determinations are carried through without undue delays.
These qualifications are important if the salt content is high. On
general principles it is well to have the salt content of the standard
approximate that of the unknown, but the following data show that
this precaution is often unnecessary. A solution of glucose and water
(0.1 mg. glucose per c.c.) was used as the standard and compared
with various salt solutions all containing the same amount of glucose.
The results are shown in table 1. The values are in per cent of the
standard.
From this table it appears that there is no large error
introduced even by the presence of considerable quantities of NaCl or
Na&!O,. The results with NaCl are in contrast to the findings of
E v e r e t t , Shoemaker and Sheppard (10) working with the original
Folin-Wu reagent. They found such rapid fading of the blue color
when the filtrate contained 1 - 6 per cent NaCl that there was an error
of 29 per cent in 10 minutes. There is slight fading with the revised
Folin-Wu but even in a 10 per cent NaCl solution it is not significant
for about 20 minutes after the addition of the molybdate and there-
after proceeds at a rate of about 10 per cent an hour. With lower
salt content the fading is slower. If, at3 is generally the case in the
original F o l i n - W u method, the reagent has stood mixed for some days
and if the dilution to 25 C.C. is made with water instead of with
dilute molyhdate, the fading is increased, the latter factor being the
more important; but we did not find any fading approaching that
reported by Everett, Shoemaker and Sheppard. Na,SO, seems
to have little effect. ZnSO, on the other hand has a large one. This
is due largely to the change of pH it produces, and it has no appreciable
effect on the Folin-Wu method in the small concentrations in which it
occurs in Somogyi whole blood filtrates. Salts containing the SO, ion
174 MT.H. FORBES
AND A. ANDREEK-SVEDBERG:

or the CK- ion have large effects and must be avoided in filtrates.
The cuprous and ferrous ions of course must be avoided also.

Glucose solution 0-2 mg. per c. c. All others 0.1 mg.

Experiments with the Shaffer-Bomogyi method.

A number of the experiments which were made with the revised
Folin-Wu method were repeated with the Shaffer-Somogyi. The
reagent used waa that designated “No. 50” (5), containing 25 gms.
Na,CO,, 20 gms. NaHCO,, 20 gms. Rochelle salt, 7 - 5 gms. CuSO,5 H,O,
5 gms. KI, and 250 C.C. of KIO, N/10 it8 to &, per liter. As in the
previous experiments a small volume of acid was added to the tubes
 FACTORS
INFLUENCING THE REDUCTION,&c. 175
before the sugar solution; otherwise the procedure of Shaffer-Somogyi
was closely adhered to. Three strengths of sugar solution were used:
0.02, 0.12, and 0.40 mg. per C.C. The results are shown in fig. 4.
The abscissae are the same in terms of titratable acidity per C.C. of
solution as in fig. 1 and therefore the curves are directly comparable
with those in fig. 1. The curves can not be followed any further to
the acid side as the acid will then liberate the iodine giving results
which have no relation to the sugar content of the solution.
The experiments with salts were made in the same way as corre-
sponding experiments with the revised Folin-Wu method. The results
are given in table 1. A few other experiments mostly on time of
heating will be mentioned in the discussion.
Experiment with the B e n e d i c t 1931 method.
Experiments on titratable acidity were done at two sugar con-
centrations: 0.02 and 0.10 mg. per C.C. Benedict’s method was
followed throughout except that Folin’s acid molybdate reagent was
used instead of the one suggested by Benedict. The results are
shown in fig. 5. The scales are the same as those in figs. 1 and 4.
This reagent differs considerably from the other two in its behavior,
on account of the presence of alanine and NaHSO,. In order to bring
out the resemblance to the other reagents the results for two different
times of heating are shown. The longer heating period gives cur-ies
which are closer to those in figs. 1 and 4. By shortening the heating
period with either of the other reagents the curves they give can be
made to resemble those in fig. 5 in that they give lower values than
the standard with acid solutions and higher with alkaline.
PH determinations.
Determinations of pH were made on four reagents and on these
reagents mixed with an equal volume of glucose solution. The latter
is the condition under which the reagents are used and is therefore the
more important. The measurements were made with a glass electrode.
The pH’sfound were as follows:
Revised F o l i n - W u . . . . . . . . . . 10-1
Revised F o l i n - W u diluted to half strength. 10.3
Shaffer-Somogyi
‘IReagent 50” . . . . . . . . . . . 9.6
“Reagent 50” diluted to half strength . . 9.3
Benedict 1931 . . . . . . . . . . . 10.3
Benedict 1931 diluted to half strength . . 10-4
F o l i n 1926 . . . . . . . . . . . . 9.1
F o l i n 1926 diluted to half strength. . . . 9.2
176 W. H. FORBES
AND A. ANDREEN-SVEDBERG:

Discussion.
The findings with respect to the four variables in filtrates considered
in this investigation may now be summarized. The pH of filtrates has
been shown to be immaterial. The titratable acidity of the several
filtrates studied was so small as to be without significant effect upon
the results obtained with the revised Folin- Wu, the Shaffer-
Somogyi, and the Benedict 1931 methods. See figs. 1, 3, 4, 5. The
two remaining rariables, salt content and concentration of sugar, require
further discussion.
It has been shown that the sensitivity to NaCl of the original F o l i n -
W u method described by E v e r e t t , Shoemaker, and Sheppard (10)
is not exhibited by the revised Folin-Wu method. That these two
methods so similar in reagents and technique should be so different
in sensitivity to NaCl serves as an indication that salt content is a
factor capable of causing unexpected deviations of significant proportions.
Table 1 shows however, that for the two methods studied, NaCl and
N%SO, have little or no effect at concentrations under 1 per cent and
that for the higher concentrations the revised Folin-Wu method is
less affected than the Shaffer-Somogyi. With zinc sulphate however
the former shows a distinct rise below 1 per cent and fall above
1 per cent as might be expected from the acidity of the salt, but with
the latter method distinctly lowered values are obtained in the presence
of this salt even when its concentration is too low to have a significant
effect upon the titratable acidity. This was the reason that Somogyi (9)
suggested the use of copper instead of zinc in the deproteinization of
plasma. It is of interest in this connection that Mac Kay (15) working
with the Sh a f f e r- S om o g y i reagent found a high corpuscle plasma
ratio for sugar. He used zinc sulphate in making not only the whole
blood but also the plasma filtrates. Whole blood filtrates do not contain
noticeable amounts of zinc mlphate according t o Som o g y i.
NaHSO, and NaCN have very large effects on both methods for
two reasons; they influence the reduction by the sugar, and they have
large blanks. The higher values for NaHSO, in table 1 are chiefly
blanks; those for NaCN almost entirely blanks. A brief investigation
indicated that NaHSO, in low concentrations increased the reduction
by the sugar while in higher concentrations it depressed it. NaCN
always depressed it. Levy and Doisy (13), and L u n d i n (14) have
observed the effect of borates and the latter has shown that a glucose
solution containing 5 per cent boric acid, or borax, gives less than
10 per cent of the usual reduction, and that glycocoll likewise has a
very depressing effect. Enough examples have been given of salt effects
 FACTORS
INFLUENCING THE REDUCTION,
&c. 177
of considerable magnitude, and uf different effects with different methods,
to warrant the statement that this factor is one which must always
be kept in mind as a possible source of error.
The effect of the concentration of sugar has been considered by
many authors, usually under the heading of proportionality. It is dis-
cussed under that heading in connection with fig. 1 and will be menti-
oned again in connection with table 2. In general the effect of this
factor is small in the less alkaline reagents. These have “good pro-
portionality” that is they have a constant copper number over con-
siderable ranges of sugar concentration. This factor is not likely to
be a source of error as it is always allowed fur by the use of standards
or corrected by the use of tables or curves. When the proportionality
is poor the solutions with less glucose, as far as the authors are aware,
always have a lower copper number.
So far the factors affecting sugar reduction have been discussed
separately. h’ow their effects upon each other will be considered. The
relation between titratable acidity and copper number is affected by
XaC1. At 5 minutes boiling time a solution containing 10 gms. NaCl
and 10 mg. of glucose in 100 c. c. gives a curve resembling the curve
in fig. 1 for 40 mg. of glucose (the 400 mg. per cent standard). NaCl
diminishes the rise in reduction produced by decreasing the alkalinity.
The relation between concentration of sugar and copper number (pro-
portionality) is affected by halogen salts, but the effects are different with
different sugar methods. NaCl has little effect upon the proportionality
obtained with the revised Folin-Wu method; K I on the other hand
has a very large effect upon the proportionality obtained with Shaffer-
Somogyi’s “Reagent 50”, the addition of 5 gms. of this salt per liter
making it mediocre for the range above the 100 mg. per cent sugar
standard and Yery poor at lower concentrations of glucose. With
1 gm. I<I the proportionality is good for all glucose concentrations.
The relation between titratable acidity and copper number is affected
by the concentration of sugar in the manner shown by figs. 1, 4 and 5
for the different reagents.
Up to this point we have discussed the effect of changes in the
solution whose sugar content is going to be determined. Changes in
the method have not been considered. The remainder of the paper
contains a brief discussion of the characteristics of the reduction, studied
by means of observing the results of altering the methods in various
respects. This introduces new variables; the duration of the heating
period, the temperature of the mixture of the unknown and the reagent
during the heating period, the composition of the reagent, and its pH.
The first, third and fourth of these variables are extensively discussed
Skandinav. Archiv. 70. 12
1i8 W. H. FORBES
AXII A. ASDREEN-SVEDBERG:

by Foliii (4) and Shaffer and Somogyi (5)and it is on their papers, as

me11 as on our own observations, that the following description is based.
The most striking characteristic of the reaction between glucose
and copper is that the more rapid is the process of reduction, the less
is the final amount of’ reduction. The reduction may be speeded up
by increasing the alkalinity; and may be slowed down b r decreasing
the alkalinit-j-, or by decreasing the temperature during the heating
period, or by the addition of certain salts. The following observatiuns
illustrate this behavior. The pH’s of the revised Folin-Wu and the
Shaffer-Somogyi reagents mixed with equal volumes of glucose solution
were found to be 10.3 and 9.8, respectively, and the times of heating
necessary to reach the maximum value of the reduction, 51/z and
15 minutes. The time recommended for these methods is 8 and
15 minutes, respectively. One would predict therefore that one could
add proportionally more acid in the revised Folin-Wu method (time
of boiling longer than necessary to reach maximum), than in the
Shaffer-Somogyi method, before getting lower values due to in-
complete reduction. A comparison of figs. 1 and 4 shows this to be
the case. The differences would be even more marked if “Reagent 50”
were not a little more buffered than the Folin-Wu. If the time of
heating with the revised Folin-Wu method is reduced to 5l/, minutes,
the experiments illustrated in fig. 1 give curves resembling those in
fig. 4 (for the 100 and 400 mg. per cent standards). If the heating
lasts only for 3 minutes, the maxima of the curves lie on the alkaline
side of pH 10.3 and are low in copper number. They resemble to a
certain extent the curves in fig. 5. If, on the other hand, the time
of heating is lengthened to 12 minutes, the curves have their maxima
further to the acid side and higher in copper number than those in fig.1.
A few experiments were done in which the temperature during
the heating period was below 99O C. At 90°, with the revised Folin-
W u reagent, we obtained the following values, giren as per cent of
a standard heated in the usual way i. e. put in boiling water for
8 minutes; for heating times of 3, 6, 12, and 20 minutes, values of
35, 79, 104, and 106 per cent, respectively. At 80°, 10 minutea gives
about 40 per cent, and 20 minutes, about 70 per cent. At 60° there is
scarcely any reduction in 10 minutes and about 10 per cent in 20 minutes.
The 20 minute values are probably a little low on account of reoxidation.
As to changing the reagent, Shaffer and Somogyi report slower
reduction and higher final ralues when the total carbonate content is
increased (keeping the ratio of Na,CO, to NaHCO, constant), which
they attribute to increased salt content. It may equally well be
attributed to a decrease in pH since the ps of carbonate solutions
 FACTORS
ISFLUENCISG THE REDUCTION,
Bc. 179
decreases as the cuncentration increases in the region of half molar
solutions. Changing the ratio of Xa,CO, tu SaHCO, in a reagent
differs in nu essential may from using the method with a sugar solution
containing H,SO, (the small decrease in total carbonate and formation
of a little Sa,SO, being without appreciable effect). However a change
such as introducing alanine and NaHSO, into a reagent has a very
great effect. The Benedict 1931 reagent has very nearly the same
pFr as the revised F u l i n - W u but behaves quite differently as is shown
by the difference between figs. 1 and 5.
In urder to obtain true glucose values for blood one or both of
two lines of appruach must be chusen; either one must use a selectii-e
reagent, or une must make a filtrate which contains very little reducing
sulistance other than glucose. Of the four sugar methods mentioned
in this paper the Folin 1926 (4) and the Benedict 1931 (6) make
use of the former and the revised Folin-Wu (4)and the Shaffer-
Somogyi (5) of the latter way (Shaffer-Somogyi combines the two
to some extent). Benedict obtains selectivity by using alanine and
SaSHO,, Folin by lowering the alkalinity as much as possible.
The following table gives a qualitative summary of this paper,
and attempts to give a general description of such alkaline copper
reagents as contain principally sodium carbonate, bicarbonate and
tartrate. The data on which it is based are to be found in papers
quoted above, largely in (4) and (5), and in this paper. We have
arbitrarily divided the pH range of copper reagents into three parts,
numbered I, I1 and 111, and given an idea of the heating time (in
boiling water) and of the number of mg. of copper reduced by 10 mg.
of glucose characteristic of each pH range. Since different methods
hare different times of heating both absolutely and compared with the
time taken to reach the maximum reduction for the reagent, the time
of immersion of the sugar tubes in boiling water has also been arbi-
trarily divided into three periods A, B and C. Methods are thus roughly
divided into 9 groups and under each group a description is given of
its selectivity, reproducibility and proportionality. Under the heading
of “effects of” is given information useful in estimating what might
be loosely described as the ‘‘stability” of the reagent.
. Selectivity means the tendency of the method to give values approxi-
mating the glucose content in blood filtrates and to be insensitive to
the other reducing substances found in them. It means also that the
method will be more sensitive to glucose than to the more slowly
reducing sugars, mannose, galactose etc. The selective method, however,
will not distinguish glucose from a more rapidly reducing sugar such
as fructose (5).
12*
Table 2. A qualitative description of the danges in some of the ckaracteristics of alkaline copper reagents with changes
in p ~ in, time of heating, and in some secondary cariables. F
00
0
__ - ~ - . -
Description of reagent Removal of tube from boiling water-bath
rime of heating Copper
needed to reach lumber at A. B. C.
PIX 38 per cent of ad percent Before completion of reduc- At or near completion, Heating continued after
of naximnm reduc of tion, (50-90 per cent (above 90 per cent 99 per cent of maximum
reagent ion values of r e maximum of maximum) of maximum) values has been reached
.gent in question reduction
Selectivity good Selectivity fair Selectivity mediocrc
Reproducibility poor Reproducibility fair Reproducibility fiLir
Proportionality fair Proportionality good Proportionality good
9.0-9.5 Ovcr 15 min. High, Effect of: Effect of: Effect o f
(over 33) Titr. acidity very large Titr. acidity large Titr. acidity medium
Time of boiling large Time of boiling medinm Time of boiling small
Salts largc Salts medium Salts mall
Reoxidation small Reoxidation medium Reoxidation large
Selectivity fair Selectivity- mediocre Selectivity poor
Repruducibility poor Reproducibility fair Reproducibility good
Proportionality mediocre Proportionality gocld Proportionality good
9.5-9 * 9 6-15 min. Medium Effect of: Effect of: Effect of:
Titr. acidity medium Titr. acidity small Titr. acidity small
Time of boiling large Time of boiling small Timc of boiling very small
Salts large Salts medium Salts small
Reoxidation very small Reoxidation small Reoxidation medium
____ _ _ ~ ~

Selectivity mediocre Selectivity poor selectivity very poor

Reproducibility poor Reproducibility fair Reproducibility good
I’roportionality poor Proportionality fair Proportionality fair
Over 9 9 - Under 6 min. Low, Effect of: Effect of: Effect of:
(under 28) Titr. acidity large Titr. acidity medium Titr. acidity small
Time of boiling very large Timc of boiling medinm Timc of boiling nil
Salts large 8:dts medinin Salts YII1:111
Reoxidation nil Reoxidation vcry Y I I I : L ~ ~ Reoxidation sni:ill
 INFLUENCING THE REDUCTION,
FACTORS Bc. 181
Reproducibilit!l refers to the ease with which the same copper
number can be obtained in different laboratories. It does not refer
to the obtaining of the same glucose values for filtrates, as this is
usuilll\. accomplished by means of comparison with a standard, and is
therefore independent of the copper number.
Propwtionality means the constancy of the copper number or of the
rtttio copper reduced/sugar present as the concentration of sugar is changed.
Titratable acidity refers to the solution whose sugar content is to
be measured and not to the reagent.
Tinw ofboiling means time of immersion in boiling water and is
the factor previously recorded for each prI range, but the differences
here considered are small accidental ones of 15 or 20 seconds such as
ciin occur in the ordinar? use of the method. Small differences in the
temperature of the tube depending on how vigorously the water boils
or how thick the glass of the tube is, have the same effect as differ-
ences in the time of immersion and are included under this heading.
Salts refers only to those salts which are relatively inactive: NaC1,
?u’a2S0,, ZnSO, etc. and not to NaHSO, or NaCN.
Reoxidation refers t o reoxidation of the reduced copper by the
atmospheric oxygen.
When the effect of a factor is referred t o as “very large” or “large”
it must always be considered, “medium” it may or many not be im-
portant, “small” it can probably safely be neglected in the ordinary
use of the method. “Nil” means that in the range considered it has
no effect measurable by the method in question. As an example take
%me of boiling nil” in group III C. With the revised Folin-Wu method
which falls in this group, ralues constant within experimental errors are
obtained between 6 and 12 minutes heating time. Thus if the time of
heating is 8 minutes variations of 15 seconds are said to have no effect.
The limitations of this table are many. In the first place it covers
only such reagents as the F o l i n , Folin-Wu, S h a f f e r - H a r t m a n n etc.
which contain chiefly sodium carbonate, bicarbonate, and Na-tartrate or
Rochelle salt. It does not apply t o the B e n e d i c t 1931 reagent con-
taining alanine and NaHSO,. In the second place it can give no
information concerning the relative importance of the various factors
as this depends on the use to which the method is to be put. For
example selectivity is very important when determinations are made
upon laked blood filtrates made with tungstic acid but of no importance
in work upon pure glucose solutions. In the third place the table
gives tendencies rather than exact information about specific methods.
Thus the general tendency of the proportionality to be better with the
less alkaline reagents (4) and to be better near completion of the
182 R. H. FORBES
ASD A. AXDREEN-SVEDBERG:

reduction than either considerabl-j before or after, is espreshed 1))

designating the proportionality in group I A as fair and that in
group I B as good etc. The F o l i n reagent (4) although falling in
group I A has good proportionality, but this was achieved only b!
careful revision of the method. It would be easier to get good pro-
portionality in group I B. Similarly two reagents described by Shaffer
and Somogyi (5) as ‘“0. 50 with 1 gm. KI”, and “No. 50 with
5 gms. KI”, both fall in group I1 B, ‘Proportionality good’. As can be
seen from table I1 in their paper the proportionality for the first is
very good, and for the second very poor. One more example. Group TI B
has ‘Reproducibility fair’. With “Reagent 50 with 5 gms. ICY, we mere
eventually able to get within 1 per cent of Shaffer and Somog)-i’s
values, which is good reproducibility, but this was only attained after
following in minute detail their method. With a water bath different
from theirs we obtained different values. Reproducibility can be made
good but it is difficult to do so; consequently it is classed as fair.
It is interesting t o note that the F o l i n 1926, Shaffer-Somogj i
and revised Folin-Wu methods fall in groups I A, I1 B and 111 C
respectively. The first secures selectivity at the expense of reliabilitj-
and stability, the second combines moderate selectirity with moderate
stability, the third attains speed, stability and reliability at the expense
of selectivity.
Summary and Conclusions.
Four variables in the composition of blood filtrates are considered
with respect to their influence on the sugar values obtained with alkaline
copper reagents. The variables are : pH, titratable acidity, salt content,
and sugar concentration. Their influence upon each other’s influence
is also dicussed. Three reagents are studied, the revised Folin-Wu,
the Shaffer-Somogyi, and the Benedict 1931.
The pH of the filtrates is of no consequence.
Solutions containing equal amounts of glucose but differing from
each other in titratable acidity to pH 9.5 by 0.008 N per c. c. were
found to differ by about 1 per cent in the reduction they produced. This
is true for each of the three methods studied. The titratable acidities
of six kinds of whole blood and plasma filtrates were found to be
between 0.001 and 0.008 N per C.C. Consequently in comparisons of
the sugar values of filtrates made by these methods the error due to
-
the differences in titratable acidity commonly ranges from 0 1 per cent
to 1 per cent. The largest difference in titratable acidity found was
between a Somogyi plasma and a Somogyi whole blood filtrate.
The presence of NaCl or of Na,SO, may be disregarded in ordinary
 FACTORS
IKFLUESCIXG THE REDUCTION,
&c. 183
work if its concentration is less than 1 per cent. ZnSO,, XaHSO,, and
XaCS can not be disregarded. See table 1.
Soine general charncterist$ics of the reduction of copper b ~ glucose
*
in alkaline solutions are discussed, and then summarized in table 2.

Bi bliographie.
~

A n d r e e n - S v ed b e r g , A., 1naug.-Diss. Stockholm 1933.

1.
S o m o g y i , M., Proc. Soe. exp. Biol. Med. 1929. Vol. 26. p. 353.
2.
F o l i n , O., J. Biol. Chem. 1930. Vol. 86. p. 173.
3.
F o l i n . O., Ibidem. 1929. Vol. 52. p. 83.
4.
S h a f f e r , P. A., and S o m o g y i , M., Ibidem. 1933. Vol. 100. p. 695.
5.
B e n e d i c t , S. R., Ibidem. 1931. Vol. 92. p. 141.
6.
F o l i n , O., and W u , H., Ibidem. 1919. Vol. 38. 11. 81.
7.
5. W u , H., Ibidem. 1922. Vol. 51. p. 21.
9. S o m o g y i , M., Ibidem. 1931. Vol. 90. p. 725.
10. E v e r e t t , M.R., S h o e m a k e r , H. A., and S h e p p a r d , F., Ibidem.
1927. vol. 74. p. 739.
11. S o m o g y i , M., Ibidem. 1926. Vol. 70. p. 599.
12. E v e r e t t , M. R , Ibidem. 1929. Vol. 82. p. 369.
13. L e v y , RI., and D o i s y , E. A , Ibidem. 1928. Vol. 77. p. '733.
14. L i t n d i n , H., Biochem. 2. 1929. Vol. 207. p. 107.
15. M a c K a y , J. Biol. Chenz. 1932. Vol. 97. p. 655.

q ST4NDAR3S
146-
Fig. 1.
120-
The effect of the titra-
100 - table acidity of sugar
solutions upon the va-
80- lues obtained with the
revised F o l i n - W u
60 -
method at different con-
8 centrations of sugar.
40 -
7;medheatinj rninutr,

10 -
I I I I I I I I
0 o
05 04 03 02 a/ 0.1 0.2 03
 W. H. FORBES
AND A. ANDREEN-SVEDBERG:

7 i f r d o n Curvc d &vised
hhn-hu Reogenf Ddukd
HuH-tMf w/fh Wohr

Y3 C c % HzSOC a&d fo 4cc

Fig. 2.
c r & NOOH to 4 c c

i I I I I I I I l l l l l l l l i l l I I I I I i
az OJ o ai a7 LU w 05 06 07 08 09 10 u
cc%vQ ad&dfolcc otfi/h& cc $=NuOH

Fig. 3.
The titration curves of six common blood filtrates.
 FACTORS
ISFLUENCING THE R-c.
REDUCTION, 185
df
9.4
I f

Fig. 4.
The effect of the titra-
table acidity of sugar
solutions upon the va-

t
lues obtained with the
Shsffcr-Somogyi
method at different con-
60 / centrations of sugar.

OJ I I I 1 I I I
0.5 0.1 0 d/
7 i L r a t a b l r acrdrty y' It' of/ritrate ropn 9.6'" rerms of
9 acid or n ( t a &

Fig. 5.
/ 2 0 - f The effect of the titra-
table acidity of sugar
120 -
' solutions upon the va-
I00 - lues obtained with the
Benedict 1931 method
-
80 at different concentra-
tions of sugar.
60 -

40 -
20-
alka(c'

0 0.2 01 0 a!
Ttrutablt aridity $ / u d/flfratt topH 9501t ( m J o / C K % a d oralid1