THE EFFECT OF DISODIUM PHOSPHATE ON d-GLUCOSE AND d-FRUCTOSE.

BY H. li. SpoEHR AND plio-L c. bvILf!iUR.

(From the Coastal Laboratory of the Carnegie Institution of Washington,

Carmel-by-the-Sea, California.)

(Received for publication, April 20, 1926.)

The extensive investigations of the influence of disodium phosphate on alcoholic fermentation and on aerobic and anaerobic respiration have demonstrated that phosphates play an important rBle in carbohydrate metabolism. In most of these investiga- tions the interest has centered about the function of the hexose phosphoric esters. It has also been found that disodium phos- phate accelerates the rate of oxidation of some hexoses by means of hydrogen peroxide or with air in the presence of certain catal- ysts. Thus Witzemann (1) has shown that glucose is oxidized quantitatively to carbon dioxide with hydrogen peroxide in the presence of disodium phosphate; in the absence of the latter no oxidation occurs or only very slowly. Warburg and Yabusoe (2) found that fructose in the presence of disodium phosphate is oxidized by atmospheric oxygen, while glucose is not thus affected. Spoehr (3-5) has also shown that in the presence of a catalyst, sodium ferropyrophosphate, and of disodium phosphate or neutral mixtures of this salt and potassium dihydrogen phosphate, hexoses, sucrose, trehalose, polyhydric alcohols, and some hydroxy acids are oxidized by air. Many other examples can be cited of the profound influence which phosphates, more especially disodium phosphate, exert upon the hexose monosaccharides as made evident by reactions in living cells as well as in vitro. As yet the role played by the phosphates in these reactions has not been definitely established.

It is not our object here to enter upon a discussion of the theories which have been advanced to describe the role of phosphates in

421

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422 d-Glucose and d-Fructose

these reactions. A widely accepted view is that the phosphates make the carbohydrates more accessible to oxidation through the formation of intermediate compounds and subsequent disintegra- tion of these. In this connection it is worthy of note that fruc- tose is generally more reactive than glucose or mannose: it is more easily oxidized (6), esterified, and split (7).

In order to gain more information of the manner in which phos- phates may bring about the reactions mentioned, a series of experiments has been carried out to determine the effect of phos- phates on d-glucose and d-fructose. In these experiments data on the following points were sought. (1) Whether the hexose sugars in solutions containing disodium phosphate or neutral mixtures of phosphates undergo the Lobry de Bruyn transfor- mation. Lobry de Bruyn and Van Ekenstein (S-10) and Nef (11) have stated that when either d-glucose, d-mannose, or d-fructose are treated with solutions of caustic alkalies, lead hydroxide, calcium hydroxide, or sodium carbonate, a mixture of all of these sugars besides pseudofructose and a- and p-d-glutose is formed. We desired to determine, for example, whether d-glucose was partially converted into d-fructose in a solution containing di- sodium phosphate. (2) Is the Lobry de Bruyn conversion a true equilibrium, or does the hexose molecule undergo splitting in the presence of phosphate ? (3) Whether there are any sac- charinic acids formed from the hexoses by the action of phos- phates, as occurs through the action of the stronger alkalies. (4) Whether any di- or polysaccharides are formed under these circumstances, as has been claimed by Nef to occur through the action of sodium carbonate on hexoses. (5) It seemed de- sirable to determine again whether any evidence could be gained of the formation of a hexose phosphoric acid compound under these conditions, i.e. without the influence of a living organism.

EXPERIMENTAL.

General Procedure.

The solutions of the various sugars and those of the phosphates were sterilized separately in an autoclave at 15 pounds pressure for 20 minutes. After cooling, the sugar solution was poured into the flask containing the solution of phosphate. The flask was

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H. A. Spoehr and P. C. Wilbur 423

tightly stoppered with a rubber stopper and placed in the ther- mostat. Experiments were run with different concentrations of sugar and at 38” and 70-75”. After 1 hour the first sample was removed for analysis. This was done by carefully removing 15 cc. with a sterilized pipette and diluting to 100 cc.; the latter solution was again diluted for the sugar analysis; the concentra- tion of this second solution was such that it contained not more than 4 mg. per cc. As the exact volume of the original solution was not known, the analyses are reported in terms of gm. per cc. of this solution. Also the sample to be analyzed was measured at the temperature of the thermostat, usually 38”. This natur- ally would cause an error in the analyses, if absolute amounts were desired. In view of the fact that all the results are comparative and that the manipulation was exactly the same in each case, it is simpler to give the results as found rather than correct for the volume of the solution at 3F. The experiments were run for 50 to 150 days and analyses were made at different intervals, from 5 to 14 days, depending upon the rate of the reaction. In every case the solutions became red-brown in color. This caused some difficulty in determining the alkalinity and optical rotation in the last analyses of a series and for this reason these results have not quite the same accuracy as the others of the series.

Analytical Methods.

The sugar-phosphate mixtures were analyzed for (1) total re- ducing power, (2) total reducing power after hydrolysis, (3) aldoses, (4) alkalinity of the solution, (5) optical rotation of the solution before and after hydrolysis, and (6) phosphates.

The total reducing pawer was determined with a Benedict’s reagent (17.3 gm. CuS04.5HZ0, 100 gm. NazCOs, and 173 gm.

sodium citrate in 1000 cc. of solution). The method previously described by Spoehr (12) was used. Benedict’s solution was standardized with pure d-glucose (U. S. Bureau of Standards) and results of the analyses are reported in terms of gm. of glucose per cc. of solution. The latter fact must be taken into considera- tion in the experiments with d-fructose. It is highly probable that in all these experiments, d-glucose, d-fructose, d-mannose, pseudo- fructose, and cy- and /3-d-glutose are formed, just as in the Lobry

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424 d-Glucose and d-Fructose

de Bruyn transformations. Each of these sugars has a slightly different reducing power, but as it was not possible to determine quantitatively the ratios of these sugars, the corrections for the reducing power of each could not be applied. It seemed simplest, therefore, to report the analyses in terms of d-glucose, especially as the differences found are far greater than could be accounted for by the differences in reducing power of the various sugars concerned.

The hydrolysis of the sugar mixture was carried out by heating the dilute solution, containing 1 per cent hydrocholoric acid, to 70” for 3 hours, and neutralizing with sodium bicarbonate. For this analysis Benedict’s solution was standardized by treating the pure glucose solution in the same manner with acid, etc. It was found that after hydrolysis the reducing power of the mix- tures was slightly less than before, indicating the absence of polysaccharides and possible slight decomposition of one of the hexoses (fructose or glutose) due to the action of the acid.

The aldoses were determined by means of oxidation with iodine with the method described by Cajori (13). Iodine oxidizes al- doses to the corresponding monobasic hexonic acid, but does not effect ketoses. The results are also given in terms of gm. of glu- cose per 1 cc. of solution.

The alkalinity of the solutions was determined by titration with 0.1 normal hydrochloric acid, methyl orange as indicator. The alkalinity is reported as gm. of hydrochloric acid required to neutralize 1 cc. of solution.

The optical rotation was determined in a 2 dm. tube at 16”. As these results can have only a comparative value, the observed readings are reported.

The phosphates were determined by precipitation as magnesium ammonium phosphate in the usual manner. The phosphate of the hexose phosphoric esters is not precipitated in this way and, it was found the presence of hexose sugars does not interfere with the precipitation. The analyses are reported as gm. of N&HP04 per 1 cc. of solution.

Experimental Results.

Experiment A. 23 Per Cent d-Glucose in 2 Molal Disodium Phosphate; 75 Gm. of d-Glucose and 62.5 Gm. of NaSHPOd in 250

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H. A. Spoehr and P. C. Wilbur 425

Cc. of Water.-The solution was kept at 38” for 147 days. The analytical results are given in Table I.

From the results given in Table I it is apparent that the di- sodium phosphate has a profound effect upon the glucose. The total reducing power of the mixture has decreased by about 6.5 per cent. Most striking perhaps is the decrease in the concen- tration of the aldose sugars as made evident by the amount of iodine used for oxidation. There has been a decrease in aldoses

TABLE I

Days.

0 21 44 69

147

Days.

0 22 42 74

140

Total reducing

power. Glucose per 00.

Pm.

0.2400

0.2356 0.2332 0.2245

Total reducing

power. Glucose per cc.

Aldoses (iodine). Glucose per cc.

gm. elm. gm.

0.2768 0.2702 0.0084 0.2347 0.2270 0.0574 0.2263 0.2191 0.0796 0.2060 0.2089 0.0902 0.1942 0.1992 0.1042

gm.

0.2229 0.2272 0.2250

Aldoses (iodine). Glucose per cc.

gm. !3m.

0.2408 0.2082 0.2102 0.2080 0.1920 0.2077 0.1823 0.2067 0.1625 0.2050

Optical rotation.

Id,

Alkalinity. HCl per co.

gm. 0.0536 0.0533 0.0528 0.0526 0.0522

TABLE II.

Phosphate. NanHPOa

per 00.

gm.

0.2079 0.2067 0.2067 0.2078 0.2037

Optical rotation.

I&

-0.54" -o.2fi" -0.20" -0.02" $0.02"

Alkalinity. HCl per cc.

gm.

0.0534 0.0529 0.0526 0.0525 0.0516

of 32.5 per cent.. The amount of free phosphate has remained virtually unchanged. Separate experiments to determine whether the small decrease in the amount of free phosphate, about 1.5 per cent, could be ascribed to hexose phosphoric ester formation, did not give positive results. The optical rotation also showed a decided decrease, finally becoming only slightly dextrorotatory. The alkalinity of the solution also changed very slightly, indica- ting (1) that only very slight saccharinic acid formation occurred,

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426 d-Glucose and d-Fructose

or (2) that only exceedingly little oxidation took place, which would result in the formation of acid products.

Experiment B. 23 Per Cent d-Fructose in 2 Molal Disodium Phosphate; 75 Gm. of d-Fructose in 62.5 Gm. of Na2HP04 in 250 Cc. of Water.-The solution was kept at 38” for 140 days. The analytical results are given in Table II.

The effect of disodium phosphate on d-fructose is more pro- nounced than on d-glucose. In the 140 days the total reducing power of the mixture originally containing d-fructose, is decreased by about 30 per cent. This is considerably greater than in the case of d-glucose. It is possible that the great decrease in re-

TABLE III.

Experi- ment C.

Experi- ment D.

Days.

0 22 41 84

148

0 0.1507 25 0.1247 43 0.1203 83 0.1094

146 0.1057

Total reducing

power. Glucose per cc.

gm.

0.1320 0.1285 0.1245 0.1232 0.1186

Total reducing

power af@ h&$$w~.

per cc.

gm. 0.1296 0.1260 0.1211 0.1240(?: 0.1207

0.1449 0.1205 0.1149

0.1062

--

I

_ -

-

Aldoses (iodine). GlUCOSe per cc.

gm. 0.1296 0.1113 0.1031 0.0936 0.0854

0.0050 0.0298 0.0487 0.0524 0.0588

Phos- phate.

NrtlHPOl per cc.

gm. 0.2208 0.2203 0.2201 0.2208 0.2179

0.2156

0.2143

T

Optical rotation.

MD

Alka- linity. HCl

per cc.

$0.30" $0.22" +o .ov $0.06" +o .09"

gm. 0.0572 0.0566 0.0561 0.0562 0.0551

-0.39" 0.0528 -0.27" 0.0546 -0.10" 0.0546 -0.02" 0.0548 -0.05" 0.0533

ducing power of the d-fructose solution may be accounted for by the difference in the reducing power of the different sugars. If it is assumed that these hexoses undergo the Lobry de Bruyn transformation, for which there appears to be some justification and if d-fructose is converted into d-glucose and d-mannose, the reducing power of the mixture will be lower, because the latter two sugars have a lower reducing power than d-fructose. Also, d-glutose has about half the reducing power of d-glucose. As is shown later, this sugar (or sugar mixture) is formed by the action of disodium phosphate. In the case of Experiment A, the total reducing power of the d-glucose mixture does not decrease as

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H. A. Spoehr and P. C. Wilbur 427

much as in the d-fructose mixture, probably because in the former case some d-fructose (with a higher reducing power) is formed and this would tend to mitigate the effect of the low reducing power of the d-glutose.

In the experiment with d-fructose the aldoses showed a decided increase, presumably a conversion of d-fructose into d-glucose and d-mannose. The optical rotation changed from strongly negative to close to zero. The amount of free phosphate and the alkalinity exhibited but slight changes. In both Experi- ments A and B, hydrolysis produced no increase in the reducing power of the mixture, indicating the absence of polysaccharides.

Experiments C and D. i5 Per Cent d-Glucose and d-Fructose.- Very similar results were obtained with lower concentrations of

TABLE IV.

Days.

Experiment E. 0 17 74

Experiment F. 0 16 69

Tots1 reducing power.

Glucose per cc

Optical rotation.

bl,

gm. gm. 0-L

0.2570 0.2593 0.2603 0.2605 0.2595 0.2546 0.2639 0.2646 0.2367

$0.33" +0.28" $0.24"

0.2262 0.2280 0.0043 -0.43" 0.2196 0.2222 0.0193 -0.32" 0.2131 0.2146 0.0495 -0.19"

-

d-glucose and d-fructose; in these cases the only difference was that the rate of change was less. The results are summarized in Table III. Experiment C = 15 per cent glucose in 2 molal Na2HP04 and Experiment D = 15 per cent d-fructose in 2 molal Na2HP04. Both experiments were run at 38”.

Experiment E. 23 Per Cent d-Glucose in Neutral Phosphate Mixture, 30 Gm. of d-Glucose, 25 Gm. of Neutral Phosphate Mix- ture, and 100 Cc. of Water.-The phosphate mixture was made from 1 part of KH2P04 and 4 parts of Na2HP04. The hydrogen ion concentration of the phosphate mixture was not determined, but test with litmus showed that this was very nearly neutral. The experiment was run for 74 days at 38”.

Experiment F. 23 Per Cent d-Fructose in Neutral Phosphate

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428 d-Glucose and d-Fructose

Mixture, 30 Gm. of d-Fructose, 25 Gm. of Neutral Phosphate Mix- ture, and 100 Cc. of Water.-The phosphate mixture was the same as that employed in Experiment E, and the experiment was run for 69 days at 38”. The results of Experiments E and F are summarized in Table IV.

From Experiments E and F it is apparent that the action of neutral phosphate mixtures of d-glucose and d-fructose is similar to that of the alkaline disodium phosphate. It is worthy of note that the solution of d-glucose shows a slight increase in the total reducing power. This is indicative of the formation of d-fruc- tose, as both the aldose content and the optical rotation decrease. The solution of d-fructose increases decidedly in aldose content, with a decrease in the negative optical rotation of the solution. This would indicate the formation of d-glucose and d-mannose.

TABLE V.

Days.

0 4

20

Total reducing power. Glucose per cc.

Total reducing power after

hydrolysis. Glucose per cc.

Aldose (iodine). Glucose per cc.

Qm. gm. gm. 0.2924 0.2938 0.2985 0.2202 0.2143 0.1559 0.1982 0.1921 0.1403

Experiment G. 23 Per Cent d-Glucose in 2 Molal Disodium Phos- phate at 70-75” in an Atmosphere of Hydrogen.-Although in the experiments already described no indication of bacteria or moulds could be detected and the possibility of oxidation was largely excluded by keeping the flasks containing the sugar- phosphate mixtures tightly stoppered, it seemed desirable to establish these points in a more direct manner. An aqueous solution of 220 cc. containing 66 gm. of d-glucose and 55 gm. of Na2HP04 was kept at 70-75” for 20 days, and a slow stream of hydrogen was passed through the flask continuously. The anal- yses are given in Table V.

From the results of Experiment G it is apparent that the reaction proceeds very much faster at the higher temperature. The trend of the reaction is the same as in the experiments carried out at 38”: a decrease in the total reducing power of the mixture and a decided decrease in the aldoses present. The higher tem-

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H. A. Spoehr and P. C. Wilbur 429

perature and the absence of oxygen preclude the possibility that the decrease in the total reducing power of the solution is due to the action of microorganisms or to oxidation.

Glutose.

Lobry de Bruyn and Van Ekenstein (8, 9) have shown that when either d-glucose, d-mannose, or cl-fructose is treated with a solution of a weak alkali (e.g., lead hydroxide) a mixture of all these sugars results. They also endeavored to show (10) that in this mixtu’re, besides the sugars mentioned, there was contained a 3-ketohexose, d-glutose. The latter sugar is characterized by the following facts: It is optically inactive, does not ferment with yeast, its reducing power for Fehling’s solution is about one-half that of d-glucose, and it forms a phenylosazone melting at 165”. So far as we know, the constitution of d-glutose has never been definitely established, though Nef (11) has assumed that the substance obtained by Lobry de Bruyn and Van Ekenstein is a mixture of a! and fl-d-glutose:

H 0 OH II

h I CHzOH- - - c- -CHzOH

OH OH H

HH 0 H

CH,OH--/--/ -!+---CH~OH

dH dH CjH

We wished to determine whether any so called d-glutose had been formed in the various hexose-phosphate mixtures already mentioned. Accordingly, the solutions were evaporated to dryness at reduced pressure, the sugars extracted from the residue with alcohol, the latter distilled off at reduced pressure, and the residue dissolved in water. These aqueous solutions were fer- mented with yeast. After the first fermentation the solutions were filtered, evaporated almost to dryness, dissolved in water, and a second portion of yeast was added to this solution to insure complete fermentation. This solution was treat.ed in the manner

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430 d-Glucose and d-Fructose

just described and evaporated to dryness. From each of the experiments described above there was thus obtained a non- fermentable residue. By repeated extraction with 99 per cent alcohol the residue was obtained free from all but traces of inor- ganic material. The d-glucose solutions yielded about 10 per cent, the d-fructose solutions about 15 per cent of this non-fer- mentable residue. This had a reducing power for Fehling’s solution one-half that of glucose and had an exceedingly slight positive optical rotation. Phenylosazones were prepared from all of the material; the melting points of these, after recrystalli- zation, ranged from 1633166’. It seems highly probable, there- fore. that the non-fermentable material was the d-glutose of Lobry de Bruyn and Van Ekenstein.

TABI,E VI.

lhYS. I Total reducing power. Aldose (iodine).

Glucose per co. Gluoose per cc.

gm. !7m. 0 0 1362 0.0675

37 0.1230 0.0695

Optica; ;otation. =D

$0.04" $0.03"

If the Lobry de Bruyn-Van Ekenstein reaction of the hexoses represents a state of true equilibrium, as was maintained by Nef (11)) it would be expected that d-glutose could be converted into a mixture of d-glucose, d-fructose, and d-mannose. No in- dication of this could be obtained. d-Glutose was prepared according to the method of Lobry de Bruyn and Van Ekenstein from d-fructose and treated with disodium phosphate in the manner described in the experiments with d-glucose and d-fruc- tose. A 23 per cent solution of d-glutose in 2 molal disodium phosphate gave the results shown in Table VI.

From the results given in Table VI it is evident that with d-glutose the total reducing power also decreases on standing with disodium phosphate. It was a matter of some surprise that solutions of d-glutose should take up as much iodine as they did, for presumably ketoses are not oxidized with iodine. The slight increase in the amount of iodine used for oxidation after 37 days is not very significant. These results give no indication of a conversion of d-glutose into d-glucose and d-fructose.

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H. A. Spoehr and P. C. Wilbur 431

Preparation of d-Glutose.

In this connection may be mentioned a convenient method of preparing the substance which is identical with that to which Lobry de Bruyn and Van Ekenstein have given the name d- glutose. In the method described by the authors just mentioned, considerable difficulty is encountered in freeing the solution com- pletely from lead, and the latter substance may interfere in the complete fermentation of the d-glucose and d-fructose.

To 500 gm. of commercial cane sugar in 2000 cc. of water 2 cc. of an active invertin preparation are added and kept at 25” for 48 hours. To this solution are then added 284 gm. of disodium phosphate, and it is heated to 70-75” for 24 hours. The solution is then concentrated to one-half its original volume by evaporat- ing the water at reduced pressure and 60”. To the concentrated solution three times its volume of alcohol is added, which precipi- tates most of the sodium phosphate; this is removed by filtration and extracted with 95 per cent alcohol. The alcoholic solution is evaporated to dryness in vacuum and dissolved in water to make a 10 per cent solution. This is fermented with yeast for 3 days, filtered, concentrated to half its volume and an equal volume of water added, and fermented again for 3 days. The solution is then heated for 15 minutes to 80”, filtered, and evapo- rated in vacuum at 60”. The residue is dissolved in the smallest possible amount of water, and alcohol is added to make the latter 92 per cent. This gives a solution with which the remaining sugar can be extracted free from phosphate. The combined alcoholic solutions are evaporated in vacuum to dryness, the

residue dissolved in water, treated with charcoal, and again evaporated in vacuum to dryness. The resulting pale yellow gum has all the properties of d-glutose, described by Lobry de Bruyn and Van Ekenstein and represents about 50 per cent of the origi- nal weight of the cane sugar.

DISCUSSION.

The experiments described indicate that in the presence of disodium phosphate the aldose sugars are converted into ketoses and vice zjersa. With neutral phosphate mixtures the same re- action occurs, though more slowly. Disodium phosphate con-

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432 d-Glucose and d-Fructose

verts both d-glucose and d-fructose into a non-fermentable sub- stance having properties corresponding to those of Lobry de Bruyn and Van Ekenstein’s d-glutose (10). It appears, therefore, that disodium phosphate is capable of inducing the same trans- formations of hexose sugars which these authors as well as Nef (11) found occurred through the action of caustic alkalies, lead hydroxide, calcium hydroxide, and sodium carbonate.

The fact that there were only very small changes in the amount of reserve alkali in the experiments with disodium phosphate shows that (1) there was no saccharinic acid formation, and (2) no oxidation products, which would be acids, were formed. Also, in an atmosphere of hydrogen and under conditions in which all possibility of the existence of microorganisms is excluded, as at 75”, the same reaction occurs.

The action of disodium phosphate on d-glucose and d-fructose results in a decided decrease in the total reducing power of the solution. That this is not due to the formation of non-reducing polysaccharides is demonstrated by the fact that the reducing power of the solution does not increase on hydrolysis. The decrease in the reducing power of the sugar-phosphate mixture can in part be ascribed to the formation of d-glutose, which has about half the reducing power of d-glucose.

Lobry de Bruyn and Van Ekenstein as well as Nef have con- sidered that d-glutose is a 3-ketohexose. This still remains to be definitely established, a problem upon which we are still at work. The question may fairly be asked whether d-glutose is actually a 3-ketohexose of definite constitution or whether it is a mixture of the nature of formose, acrose, and the condensa- tion product of glyceric aldehyde and dihydroxyacetone. Too much importance cannot be attached to the fact that it gives a phenylosazone of constant melting point, for this may be the osazone of a constant component of the mixture.

The following facts may also be of significance in this connec- tion. Solutions of both d-glucose and d-fructose, in the presence of disodium phosphate, in time become colored through the formation of tar. Dakin and Dudley (14) have found indication of the split- ting of glucose under the influence of disodium phosphate into methyl glyoxal. Probably the simplest conception of the formation of the latter compound is by the splitting of the aldohexose (i.e. its

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H. A. Spoehr and P. C. Wilbur

34 endiol) into two molecules of glyceric aldehyde and subsequent rearrangement according to the views of Nef (15). The glyceric aldehyde, once formed, is capable of various reactions: formation of methyl glyoxal and dihydroxyacetone, as well as the condensa- tion of the latter with glyceric aldehyde. In the reactions of glucose and fructose with disodium phosphate special experi- ments were carried out to determine whether any lactic acid was formed, but this was found not to be the case.

It has also been found that the tar formation, already alluded to, does not take place at all if there is present in the mixture an oxidizing or reducing agent. In the oxidation of these hexoses with air in the presence of an iron catalyst no tar whatsoever is formed (4). Similarly, when a glucose-disodium phosphate mixture is reduced with aluminium amalgam no tar is formed; under these conditions acetone was formed as a product of reduc- tion. This is evidence of the splitting of the hexose into a mole- cule containing three carbon atoms, presumably glyceric aldehyde which is converted into dihydroxyacetone, or acetal; either of the latter two compounds yields acetone on reduction. It there- fore seems highly probable that the catalytic effect of disodium phosphate on the oxidation of hexoses is due to the dissociating influence of this salt, and that, to a considerable measure at least, the reaction of the oxidizing agent is with the splitting products rather than with the undissociated sugar. This is supported by the fact that the rate of oxidation is increased when the glucose has been previously treated with disodium phosphate, as was found to be the case by Witzemann (1) and Spoehr (3). In the absence of an oxidizing or reducing agent, the splitting products apparently polymerize, resulting in tar formation, or are condensed to an optically inactive mixture. Whether d-glutose is such a mixture, or actually a 3-ketohexose remains to be established.

For the further elucidation of the reactions brought about by the action of disodium phosphate on hexose sugars it will be necessary to study more closely the properties of glyceric alde- hyde and of dihydroxyacetone; to determine the conditions of equilibrium between these two substances in solution, if such an equilibrium actually exists; to establish the nature of the con- densation products and the substances obtained through the action of oxidizing and reducing agents. Experiments on these problems are now in progress.

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434 d-Glucose and d-Fructose

BIBLIOGRAPHY.

1. Witzemann, E. J., J. Biol. Chem., 1920-21, xiv, 1. 2. Warburg, O., and Yabusoe, M., Biochem. Z., 1924, cxlvi, 380. 3. Spoehr, H. A., J. Am. Chem. Sot., 1924, xlvi, 1494. 4. Spoehr, H. A., and Smith, J. H. C., J. Am. Chem. Sot., 1926, xlviii, 236. 5. Smith, J. H. C., and Spoehr, H. A., J. Am. Chem. Xoc., 1926, xlviii, 107. 6. Kuhn, R., and Wagner-Jauregg, T., Ber. them. Ges., 1925, lviii, 1441. 7. Euler, H., and Nilsson, R., 2. physiol. Chem., 1925, cxlv, 184. 8. Lobry de Bruyn, C. A.? and Van Ekenstein, A., Rec. trav. Chim. Pay-

Bas, 1895, xiv, 203. 9. Lobry de Bruyn, C. A., and Van Ekenstein, A., Rec. trav. Chim. Pay-

Bas, 1896, xv, 92. 10. Lobry de Bruyn, C. A., and Van Ekenstein, A., Rec. Irav. Chim. Pay-

Bas, 1897, xvi, 257. 11. Nef, J. U., Ann. Chem., 1914, cdiii, 204. 12. Spoehr, H. A., Carnegie Institution of Washington, Pub. No. $87, 1919,

61. 13. Cajori, F. A., J. Biol. Chem., 1922, liv, 617. 14. Dakin, H. D., and Dudley, II. W., J. Biol. Chem., 1913, xv, 127. 15. Nef, J. U., Ann. Chem., 1910, ccclxxvi, 3.

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H. A. Spoehr and Paul C. Wilbur-FRUCTOSE

d-GLUCOSE AND dPHOSPHATE ON THE EFFECT OF DISODIUM

1926, 69:421-434.J. Biol. Chem. 

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