sugar activation by alkali. it is well known that in alkaline

SUGAR ACTIVATION BY ALKALI.

I. FORMATION OF LACTIC AND SACCHARINIC ACIDS.

BY PHILIP A. SHAFFER AND THEODORE E. FRIEDEMANN.*

(From the Laboratory of Biological Chemistry, Washington University School of Medicine, St. Louis.)

(Received for publication, December 20, 1929.)

It is well known that in alkaline solution reducing sugars exhibit an instability and behavior which resembles in some important respects the changes accompanying the utilization of sugars by living cells. They develop strong reducing intensity and become autooxidizable, are to a considerable extent interconvertible, are polymerized and depolymerized, and in the absence of oxidizing agents are converted in part into lactic acid with the intermediate formation of methylglyoxal. The fact that each of these phenomena has its counterpart in carbohydrate metabolism, gives to the evident activation of sugars by alkali a special biochemical interest, besides the fascination of the reactions on purely chemical grounds.

The numerous investigations of the complex transformations which sugars undergo under the influence of cells or tissue extracts on the one hand and of alkali on the other have been frequently interpreted and reviewed (14) with the object of deciphering the path and sequence of the reactions involved. In both fields a central problem has been the search for the “active” forms of the sugars to which the characteristic instability may be attributed. The trend of recent conceptions is toward the choice of hypothetical ring structures as the “active” sugars. See Levene’s review (2).

A survey of the subject seems to us however to point to the

* The experimental data are taken in part from a dissertation presented by T. E. Friedemann in partial fulfilment of the requirement for the degree of Doctor of Philosophy, Washington University, 1923.

345

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Sugar Activation by Alkali. I

conclusion that in alkaline solution the instability of sugars is primarily the consequence of the formation of salts of the sugars acting as weak acids, a view advocated by Mathews (5) in connection with sugar oxidation and emphasized particularly by Nef (6, 7). Monobasic salts of glucose have long been known (8), and the acidic dissociation constants of various sugars have been determined (9). The first “active” forms of the sugars appear to be sugar ions, and their formation is probably the enabling act so to speak in each of several types of sugar transformation under the influence of alkali. The phenomena of mutarotation, involving isomerization of carbon atom 1 is due according to Lowry (10) to ionization of the sugar hydrate, while the data of Euler and associates (11) appear to show that the catalytic effect of acids and bases is due to salt formation and consequent increase in concentration of sugar ions. Somewhat similarly, the rate of loss of optical activity resulting from the Lobry de Bruyn aldose-ketose transformation, which involves isomerization of carbon atoms 2 and 3, is shown by the work of Groot (12) with glucose to be proportional not to pH (Michaelis and Rona (13)) but rather to the fraction of the sugar present in the form of alkali salt. Although having widely different ,velocities, both these transformations appear to be the spontaneous rearrangements of unstable sugar ions.

In this paper we present data concerning the relation of degree of alkalinity to a third type of non-oxidative transformation of sugars; namely, saccharinic acid and in particular lactic acid formation. We have attempted to correlate these reactions also with salt formation. The data considered here represent final yields of total saccharinic and lactic acids; in another paper the rates of reaction will be reported. After our experiments were undertaken and in large part completed, a series of papers by Evans and associates (14-18), covering’ somewhat similar experimental ground, appeared. Their results will be cited in relation to our own. Evans has recently published a summary of (19) this work. See also the recent studies of Bernhauer and associates (20-23) and Fischler and coworkers (24). We shall first review briefly the main facts and theories concerning the production of saccharinic acids.


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P. A. Shaffer and T. E. Friedemann 347

Formation of Xaccharinic Acids.

From the investigations by Kiliani (25) and others, especially Nef and his pupils (6, 7, 26-29), there have been isolated and identified as the chief products of the anaerobic destruction of hexoses by strong alkali, besides sugar resins and racemic lactic acid, c&l’, 3-dihydroxybutyric acid, and a series of optically active 6-carbon acids, the meta-, iso-, and parasaccharins. The main products are thus C,-, Cq, and Cs acids of the same empirical formule as the sugars (CH20),, the first two being racemic. In 8 N alkali about the same amounts of lactic acid are formed from glucose and fructose but less from galactose, which gives relatively more of the 4- and 6-carbon acids, the latter being different in the case of galactose from the 6-carbon acids of glucose and fructose. From the pentoses were obtained smaller amounts of lactic acid, the same dl-1 ,&dihydroxybutyric acids, and optically active 1,3,4-trihydroxyvalerianic acids. With more dilute caustic alkali Nef stated that there is formed in all cases “ein furchtbares Gemisch aller denkbaren Saccharinsauren, mit drei, vier, funf und sechs Kohlenstoffatomen” ((6) p. 9). He noted that the particular saccharinic acids formed depend upon the strength of alkali (see also Upson (28)).

Based on structural theory, on the sugars formed in weak alkali (the Lobry de Bruyn transformations), on the structure of the various saccharinic acids formed in strong alkali, and the products ,of oxidation, it will be recalled that Nef formulated a detailed general theory of the reactions, as proceeding through a series of intermediates, the 1,2-, 2,3-, and 3,4-dienols. Diagram 1, repre- senting the relations and sequence of events, will aid in the following discussion.

The idea as to the formation of intermediate dienols, an exten- sion of explanations advanced by Lobry de Bruyn and van Eken- stein (30) by Fischer (31) and by Wohl and Neuberg (32), supplies the means of passing from aldoses to 2- and 3-ketoses. In alkali carbonate or quite dilute hydroxide with large excess of sugar the reactions are practically limited to these transformations, involving isomerization of carbon atoms 2 and 3 without much saccharinic acid formation. With sufficiently dilute alkali and low temperature they may be almost wholly confined to carbon atom 2 with aldoses predominating. See the recent results of Wolfrom and


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Lewis (33), and Greene and Lewis (34). At similar low alkalinity slow polymerization of lower to higher sugars also occurs. With higher alkalinity or temperature the aldoses decrease in proportion and non-fermentable sugars of low optical activity appear, among which are a second Zketose and two 3-ketoses (from the glucose series d-pseudofructose, (Y-, and P-d-glutoses). Although to a considerable degree reversible as regards aldoses and 2-ketose (glucose-fructose-mannose), the 3-ketose formation appears to be irreversible; at sufficiently high alkalinity it passes readily to saccharinic acid (Lobry de Bruyn and Nef; see also Benedict, Dakin, and West (35) and Spoehr and Wilbur (36)).

Nef was apparently of the opinion that enolization may occur &thou2 salt formation ((7) p. 225 and 241), being due to loss of water by splitting hydroxyl from the aldehyde or ketone hydrate and H from an adjacent carbon. But since alkalinity induces enolization, perhaps the enol is a salt. As pictured by Nef’s pupils, Spoehr ( (3) p. 18) and Woodyatt ((4) p. 659), the salt is that of the hydrate hydroxyl which loses OM and H (as OH and H above) to form the dienols. Wolfrom and Lewis (33) disagree with the view that water (or MOH) is lost (and added in the reverse direction) on the ground that tetramethylglucose and tetra- methyhnannose would thus form hemiacetals which should lose CH,OH and yield 3,4,6-trimethylfructose, which they find not to occur. Interconversion of the two methylated aldoses does take place, and an intermediate is formed. They picture enolization therefore as simple migration of H (as H+ ion) from carbon atom 2, which amounts to attributing to it some acidic property. Fischer (31) regarded sodium glucosate as a salt of 1,2-dienol. Evans writes ( (19) p. 309) the dienols as dibasic acids.

The limited equilibrium existing among the aldoses, ketoses, and intermediate dienols, is disturbed at higher alkalinity by two other reactions, both of which lead to saccharinic acids. Ac- cording to Nef’s theory a different acid is derived from each aldose and ketose present, as a result of salt formation and rearrangement first into respective orthodiketo forms, the “ortho osones,” the best known example of which is methylglyoxal. He supposed that the metal (M) takes the place of H of the hydroxyl group adjacent to the carbonyl group. By loss of MOH the salt becomes


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350 Sugar Activation by Alkali. I

I ii -CHOH-C-C-H

I

which rearranges to

73 -CHrC-C-H,

which under the influence of alkali adds MOH to give -CHs- CHOH-COOM. The meta-, iso-, and parasaccharinic acids are thus derived through benzylic acid rearrangements from aldoses, 2-, and 3-ketoses respectively ( (27) p. 301). A somewhat different derivation of the saccharinic acids is proposed by Evans and Benoy (cited by Evans and associates (14) p. 2675), who relate them directly to the 1,2-, 1,3-, and 3 ,bdienols, which thus become the immediate precursors of both cleavage fragments and ortho osones. This relation is incorporated in Diagram 1.

The other reaction is the spontaneous dissociation (theoreti- cally reversible) of each of the dienols into hydroxymethylene fragments, nascent aldoses (R-C-OH) which, having properties of

free radicals, are readily oxid&d, or in the absence of oxidizing agents, are again enolized and converted to corresponding saccharinic acids or are polymerized. Lactic acid is the metasac- charinic acid derived by dissociation of 3,4-dienol (of the hexoses) and conversion of the aldo triose via its ortho osone, methylglyoxal. The reaction dienol to ortho osone is also apparently reversible, in view of the recent observations by Bernhauer and Gijrlich of the formation of triose and hexose from methylglyoxal (22) at low alkalinity. The 1,3-dihydroxybutyric acids are similarly derived from. tetrose, formed by dissociation of hexose 2,3-dienol. Nef found no evidence for dissociation of hexose 1 ,2-dienol except on oxidation. The liability of the dienols to rupture increases in the order 1,2-, 2,3-, 3 ,4-, the last disrupting rapidly, The dissociation of the dienols Nef thought to occur spontaneously, independent of salt formation. It seems more probable that the enols exist mainly in the form of salt or ion, possibly of dibasic acids as sug- gested by Evans. Fischler (24) regards the splitting of hexose to triose as the rupture of a salt of the hydroxyl of carbon atom 4.


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From the above account it will be noted that ionization or salt formation has been demonstrated or hypothecated in connection with (1) mutarotation, (2) enolization with aldose-ketose conversion, (3) polymerization of low to higher sugars and dissociation of sugars or their dienols, (4) rearrangement to ortho osones and saccharinic acids, and (5) sugar oxidation. In these several reactions the mobility or dissociation of different H atoms (protons) appear to be involved, a fact which suggests that’the sugars may behave as di- or polybasic acids.

In the experiments here reported it was desired to learn the extent of variations in the &al amounts of the several saccharinic acids formed from various sugars at different levels of alkalinity. Because of the impossibility at present of separate determination of individual acids we resorted to the simple plan of determining the total by titration of the base bound, and of determining lactic acid by a rapid oxidation method (37) which was at first thought to be fairly specific for that substance. The amount of total acids less lactic acid gives approximately the sum of all others, the Cd and Cs acids.

Since the validity of the data depends upon the reliability of the method for determination of lactic acid in the presence of sugars and of other products of their destruction, this point was sepa- rately investigated. According to the results of that work (38), we conclude that the lactic acid figures here reported for glucose, fructose, mannose, and trioses are correct within about 5 per cent. Some of the results may be to this extent higher than the true values, because of plus errors due mainly to 1 ,3-dihydroxybutyric acid, which upon analysis by the method used is found to be equivalent to about one-tenth as much lactic acid. Lactic acid ac- companied by an equal amount of this acid would therefore give results about 10 per cent too high. But pure lactic acid gives results about 2 to 4 per cent too low, which thus tends to balance plus errors from moderate amounts of the l,&hydroxybutyric acid. Interference by other saccharinic acids is believed to be negligible. Residual reducing sugars, resins, and much of the higher acids are removed by copper-calcium hydroxide precipitation or are left behind by ether extraction. With glucose and fructose we have been able to isolate after ether extraction amounts


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of analyzed zinc lactate which agree fairly well with results by the oxidation method.

With galactose, arabinose, and xylose, however, the yield of zinc salt is lower than corresponds to results by the oxidation method, and it is probable that the latter are too high with these sugars because of the much larger amounts of 1,3-dihydroxybutyric acid formed from them (Nef). On the other hand the results by isolation of zinc salt are undoubtedly too low, because of incomplete crystallization in the presence of much other acids. Our “lactic acid” results from these three sugars are so designated in order to indicate their uncertainty. A series of experiments was conducted also with glycolaldehyde, which after decomposition by alkali followed by ether extraction or copper-lime precipitation yields by oxidation moderate amounts of apparent lactic acid. We did not succeed, however, in isolating zinc lactate from the solutions. Nef (and more recently Evans (19) ) failed to isolate lactic acid from the decomposition of this sugar, but did find 1,3- dihydroxybutyric acid. It is therefore doubtless this substance which gives acetaldehyde on oxidation, and thus appears to be lactic acid by this method. The data from this sugar are therefore omitted.

Procedure.

Known amounts of the sugars, dissolved in air-free water, were added to known amounts of KOH solution (previously boiled and cooled) in volumetric flasks, the contents being then diluted to the mark with freshly boiled and cooled water. After rapid mixing exposure to air was minimized by covering the solution with a layer of mineral oil or by replacing air over the liquid by nitrogen. This precaution is very important in order to avoid oxidation of the strongly reducing intermediates. The tightly stoppered flasks were allowed to. stand for days or months until it was thought, from other data, that the reactions had come about to an end. Control exper%ments containing the same concentrations of alkali without sugar were treated in the same way as blanks for titration of total base.

The amounts of sugar and of alkali were so chosen that the alkali would always be present in large excess over that required to


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P. A. Shaffer and T. E. Friedemann

neutralize the acids formed, in order that the reduction in alkalinity due both to the sugar’ and acids formed would not intro- duce too large a variable during the course of the reactions. With the more dilute alkali solutions the concentration of sugar was also small. The final alkalinity in each of the experiments presented may be learned by subtracting the product of total acid times mllr of sugar taken, from the initial alkalinity. For example, (Table I) starting with 0.02 N KOH and 5 mM glucose had at the end 20 - (5 X 1.59) = 12.05 or 0.012 N KOH. In others the reduction of alkalinity was much less: from 0.04 to 0.031 N, from 0.10 to 0.083 N, from 0.25 to 0.232 N, and so on.

At the conclusion of the reaction period 5 to 20 cc. aliquots of the solution were measured into 100 cc. of cold water and titrated with 0.1 N HCI to phenolphthalein for decrease of free base (total acid formed). Other aliquots were neutralized and after appropriate dilution were analyzed for residual reducing sugar by the Shaffer-Hartmann method (39), and for lactic acid by oxidation to acetaldehyde (37) after ether extraction or after precipitation by copper and lime, or both.

The sugars used were the following: glucose, Bureau of Stand- ards and Eastman; fructose, Special Chemicals Company, and

1 The following simple experiment demonstrates the base-binding power of sugars. To each of three test-tubes add the same amount of water (10 cc.) and of a dilute solution of indigo carmine (acid-base indicator in the range pH 12 to 14); in one tube dissolve about 0.5 gm. of glucose, and to this and one other tube add 1 cc. of N NaOH. In 0.1 N NaOH (pH about 13) the indigo assumes a yellow-green color, while in the tube containing the same concentration of NaOH in the presence of about 0.3 M glucose, the color remains blue; the base is neutralized by the acidic glucose to the extent that the solution has a pH below the zone of color change of the indicator. The same experiment gives a like result with stronger solutions of alkali if the sugar concentration be likewise increased. After a few minutes the color of the sugar tube changes from blue to carmine to yellow, due to the reduction of the dye, a phenomenon which follows, but is quite distinct from the instalitaneous change of color due to the formation of the sugar salt. The reduction of the dye indicates activation of the sugar as a reducing agent. I f an indicator such as phenolphthalein, changing color at lower pH, be substituted and the solution made just alkaline to that indicator, the addition of glucose gives no indication of its acidic property, showing that a greater alkalinity is required to call forth a perceptible dissociation.


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Kahlbaum; mannose and galactose, Special Chemicals Com-

pany; xylose and arabinose, Kahlbaum; dihydroxyacetone2

TABLE I.

Saccharinic Acid Formation from Hexoses at SY-40”.

Results are expressed as mols per mol of hexose.

Initial concentration. Total saccharinio acids. Lactic acid. Other acids by

difference.

. 8 d

d aj d d g $ g

; a; z .

ai d

KOH a 2

‘:ag”‘;z%%J $ 8 3 g z 8 2

G 0 $7 s &jggpgg;$

___ _- - --- 24 ?nM

~NrtlCOa 40 1.29 0.17 1.12 0.02 5 1.59 1.63 1.581.490.550.570.540.401.041.061.041.09 0.04 5 1.69 1.75 1.751.650.720.720.700.460.971.031.051.19 0.10 10 1.69 1.69 1.691.490.840.860.860.520.850.830.830.97 0.25 10 1.83 1.82 1.861.561.041.041.050.530.790.780.811.03 0.50 50 1.67 1.70 1.411.06 1.060.480.61 0.640.93 1.0 50 1.71 1.761.421.13 1.160.440.58 0.60,0.98 2.0 50 1.64 1.681.32 1.14 1.200.430.50 0.480.89 5.0 50 1.05 1.110.43

9.2-10.0 50 1.081.011.130.57

TABLE II.

Effect of Alkali Concentration on Total Acid and Lactic Acid Formation jrom Anaerobic Destruction of Pentoses at SY-42”.

Initial sugar concentration 60 mM per liter.

‘i%?

Mols per mol of sugar.

concentra- Total acid. Lactic acid. Other acids. tion.

Xylose. Arabinose. Xylose. Arabinose. Xylose. Arabinose. .___

M

0.5 1.27 1.26 0.49 0.45 0.78 0.81 1.0 1.29 1.29 0.54 0.47 0.75 0.82 2.0 1.30 1.30 0.51 0.48 0.79 0.82 5.0 0.48 0.51 9.0 0.55 0.77

The oxidation method by which the lactic acid data were determined is of doubtful reliability with products of pentoses.

(“oxantin”), Farbwerke and Hoechst ; glyceraldehyde, prepared by the Wohl and Lange method as modified by Witeemann (40).

2 The “oxantin” used in these experiments was generously supplied to us by the Mallinckrodt Chemicrtl Company.


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The purity of the sugars was checked when possible by optical rotation, moisture, and ash; trioses, by the acid formed on oxidation by HeOz in alkaline solution.3

Data showing the amounts of total acid and of lactic acid formed from the anaerobic destruction of glucose, fructose, mannose, and galactose in from 0.02 to 10 N KOH at about 40” are given in Table I. Data for arabinose and xylose are given in Table II and for glyceraldehyde and dihydroxyacetone in Table III. The results represent approximately maximum yields, the solutions having stood for periods of 2 months or longer. When analyzed, the reducing power had almost disappeared (less than 4 per cent remained), and it is assumed that the reactions had come to an end. Controls in some cases standing for much longer periods showed little or no additional change. A slight residual reduction is always found, even after many months. On longer standing exposed to air the reducing power and coincidentally the brown color of the solution decrease or disappear, but without any demonstrable further increase of lactic acid.

E$ect of KOH Concentration.

Total Acids.-From the results it appears that the amount of total acids formed from glucose, fructose, and mannose is practically the same and is to a considerable degree independent of the initial alkalinity (from 0.02 to 2.0 N KOH), provided the alkali hydroxide is present in excess, as was the case in these experiments. At the lower alkalinity, M Na2C03, the total acid formed from glucose was distinctly lower.4 In another paper it will be shown that great differences exist in the rates of total acid formation from these three sugars at the same alkalinity, but thesedifferences are not apparent in the jinal yields. The observed variations in amounts of acid are not regular and are perhaps within experimental errors.

3 Method and results to appear in a later paper. 4 In this experiment, 0.04 M glucose in M Na&ZOa, the residual reduction

after 4 days at 65”, corresponded to 5.3 mM of glucose. The calculations are based on the assumption that 34.7 mM of sugar were destroyed. If the re- maining sugar were 2-ketoses of lower reducing power (35) toward the copper reagent used, the yields of acid per mol of sugar destroyed would be correspondingly increased.


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TABLE III.

Trioses. Effect of Alkali Concentration on Yield of Total Acid and Lactic Acid from Anaerobic Destruction at 37-.@“.

Ezf2~:~~::. ( EEZE. / Total acid. M; zc,;;;:““; Other acids.

Dihydroxyacetone.

af 11161 per 2.

0.02 10 0.78 0.25 0.04 10 0.95 0.32 0.10 10 0.75 0.40 0.25 10 0.71 0.53 0.50 10 0.67 1.00 10 0.82 2.00 10 0.90 5.00 10 0.93

10.00 10 0.95

0.53 0.63 0.35 0.18

0.025 20 0.050 20 0.10 20 0.25 20 0.50 20 1.00 20 2.50 20 5.0 20

10.0 20

0.25 40 0.50 4-o 1.00 40 1.00 40

1.00 80

0.50 100 1.00 100 2.00 100 5.00 100

10.00 100

1.00 160

0.17 0.25 0.30 0.42 0.52 0.65 0.81 0.86 0.82

0.823 0.328 0.855 0.43 0.95 0.60

0.593

0.514

0.865 0.435 0.913 0.513 0.91 0.657

0.827 0.810

0.472

Glyceraldehyde.

0.495 0.425 0.35

0.43 0.40 0.253

0.02 9.4 0.82 0.24 0.58 0.04 9.4 0.93 0.31 0.62 0.10 18.7 0.89 0.39 0.50 0.25 18.7 0.94 0.48 0.46

10.00 32.7 0.74

356


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The amount of total acid formed from glucose, mannose, and fructose (1.6 to 1.8 cc. of N per mM) and the somewhat smaller amount from galactose (1.3 to 1.6 cc. of N per rn@ show that a portion of the sugar molecules, but not all, are ruptured to give more than 1 molecule of acid, even in 0.02 N KOH. The amount from pentoses is slightly less per mol than from galactose, indicating less rupture of the chain into two acid-forming fragments than occurs in the glucose series. The total acid formed from the two trioses is substantially the same, and, within the range of

n 7 A .6 .a 1.0 2 4 6 0 I .- INITI~ MO~AR%JNC~NT~~~ION OF KOH.

FIG. 1. The effect of the alkali concentration on the yield of lactic acid from glucose, fructose, mannose, and galactose at 37.5”.

alkalinity up to 0.5 N, the yield from 2 mols of triose equals lhat from 1 mol of glucose. This fact accords with the view, to be mentioned presently, that in the lower range of alkalinity the trioses are polymerized to hexose before being transformed to saccharinic acid.

Lactic A&-In marked contrast with the approximate constancy of the total acids, their distribution between lactic and other saccharinic acids varies with the alkalinity. With low alkali concentration the amount of lactic acid formed is small and the proportion of other acids is correspondingly great. As the


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alkalinity is increased the proportion appearing as lactic acid increases, the ratio changing for glucose, mannose, and fructose from about 1 part lactic to 2 of other acids at 0.02 N alkali to about 2 : 1 at N alkali or above, the maximum yield of lactic acid being slightly over 1 mol per mol of hexose. With galactose, the rise of “lactic acid” (see p. 352) with alkalinity is less marked, reaching a maximum of about 0.5 mol per mol of hexose, which again indicates that the galactose chain (dienols) is less readily

TABLE IV.

Relation of Alkalinity to Y,ield of Lactic Acid.

Initial KOH.

(1)

M

0.02 0.025 0.04 0.05 0.10 0.25 0.50 1.0 2.0 2.5 5.0

10.0

Log (OH),

(activity 25”).

-

/ Log (OH),

:conduct- tnce 18”).

(2) (3)

-1.76 -1.72 -1.67 -1.63 -1.48 -1.43 -1.39 -1.34 -1.11 -1.04 -0.74 -0.66 -0.44 -0.38 -0.13 -0.101 $0.23 +0.13 $0.37: +0.19r

($0.87: $0.35 (f1.30: +0.30

- I Final yield of lactic acid per mol of hexose or

2 mols of triose.

Glucose series

a”.

(4)

T- 10 nlM.

(5)

M

0.55

M

0.50

0.72 0.64

0.86 0.80 1.04 1.06 1.06 1.34 1.14 1.64 1.17 1.80

1.08 1.07

1.86 1.90

Dihydroxyacetone.

-

M

0.34

0.50 0.60 0.84 1.04 1.30

1.62 1.72 1.64

-

M

0.65 0.86 1.20

M

0.87 1.03 1 31

1.65 1.62

Glycer- aldehyde.

(9)

M

0.48*

0.62*

0.78t 0.961

1.481

* 9 mM triose concentration. t 19 mM triose concentration. t: 33 mM triose concentration.

broken than in the case of the three other hexoses. The results from pentoses are similar to those from galactose.

The yield of lactic acid (as well as total acids) at any single alkalinity is, within the limits of experimental error, the same for each of the three hexoses, glucose, fructose, and mannose, but quite different for galactose (Fig. 1). The similarity of the results with the first three sugars is in keeping with their relationship through the 1 ,Bdienol, and confirms the observation by Nef ((6) p. 89)


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and Evans ( (19) p. 299).6 It may be noted that 1 equivalent of KOH is not sufficient for maximum conversion to lactic acid.

The maximum yield of lactic acid from the hexoses, determined by the oxidation method, is somewhat greater than the amounts isolated by others as zinc salt6

The trioses, glyceraldehyde and dihydroxyacetone, yield under similar conditions very nearly the same amounts of lactic acid, in confirmation of Evans and Cornthwaite (16). (The quantitative discrepancies between the results of Evans and our own with trioses are noted below.)

On comparing the lactic acid production from the trioses with that from glucose (or fructose or mannose) we encounter a remark- able relation. The data are given in Table IV and in Fig. 2.

6 The lactic acid yields reported by Evans and coworkers from these hexoses are, however, quantitatively very different from our own, especially at the lower range of alkalinity. In 0.5 tir KOH at 50” they find, for example, only 0.08 mol per mol of fructose (17) and 0.24 mol per mol of mannose and glucose (18) compared with our yield of about 1.04 mol in 0.5M KOH. At lower alkalinity the discrepancy is still greater, at higher alkalinity less. The difference is due in large part to the high sugar concentration (0.5 M)

used by Evans and the consequent initial decrease of alkalinity through binding of base by sugar. This point will be referred to also in connection with the results by these workers with trioses.

6 Nef states ((6) p. 89) that from 100 gm. of glucose, fructose, or mannose there are formed in strong alkali 40 to 45 gm. of lactic acid. This is the equivalent of 0.8 to 0.9 mol per mol of hexose. The amounts recorded in his protocols are, however, less, from 40 gm. of glucose and fructose 16.4 and 17 gm. of zinc lactate ((26) p.326) or about 0.5 mol. Meisenheimer (41) and Oppenheimer (42) obtained from glucose and fructose from 0.6 to 1.0 mol of lactic acid per mol of hexose. Friedemann ((38) p. 80) obtained from glucose (1.1 mol in 3.5 N NaOH) 0.66 to 0.85 mol isolated as zinc salt, while the oxidation method on the same solution gave 0.86 mol per mol of hexose; Evans, Edgar, and Hoff (14) record a maximum of about 40 per cent conversion of glucose to lactic aid in 7 N KOH at 50”-or about 0.8 mol (isolated as zinc salt). Evans and O’Donnell (18) found approximately the same amounts of lactic acid from glucose, fructose, and mannose decomposed at 75” in KOH, the yield rising to a maximum of 35 per cent or 0.7 mol with rising alkalinity. At 50” .glucose yielded 1 mol of lactic acid in 7 M KOH. From the decomposition of galactose Nef (25) and Meisenheimer (41) isolated about 15 per cent of lactic acid (about 0.3 mol), and much more 1,3- dihydroxybutyric acid (Nef) than from glucose; while Evans, Edgar, and Hoff obtained less than 2 per cent of lactic acid. Wolf (23) reports yields of lactic acid amounting to 70 per cent or more of theory from sucrose by heating with 1 or 2 mols of CaO to 200” in an autoclave.


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Lactic acid yields, expressed in mols from 1 mol of hexose or from the equivalent 2 mols of triose, are plotted against logs of initial millimolar KOH concentration. In the lower range of alkalinity up to about 0.1 to 1.0 N KOH, depending on triose concentration, the trioses yield somewhat less lactic acid than does glucose, but with greater alkalinity the trioses overtake the hexoses and

w 1.8

B z 1.7 I-

? 1.6

s N ‘5

E -I+

1.4

1.9

8 1.3

t;: 0 1.2

5 = 1.1

ti E 1.0

E a . 9

; 8

F Q 7

Y E .6

2 .5 -I

‘41.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0

LOG MM KOH CONCENTRATION

FIG. 2. Relation of initial alkali (KOH) concentration to yieid of lactic acid from glucose and trioses. Curve 1 is for glucose; Curve 2, dihydroxyacetone 10 mM; Curve 3, glyceraldehyde; Curves 4, 5, and 6, dihydroxyacetone 20, 40, and 100 rnM respectively,

approach quantitative conversion into lactic acid. The conversion is more nearly complete the higher the alkali concentration (though note that the curves dip downward at 10 N KOH) and the lower the triose concentration. It will be noted that the curve repre- senting lactic acid yield from the series of 10 mrvr dihydroxyacetone


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(and glyceraldehyde, 9 mM), parallels closely the glucose curve up to about 0.1 N KOH. Within this zone the yield of total acids is also substantially the same from the trioses and hexoses. These facts suggest that i?~ this range of alkalinity the triose is converted more or less completely to hexose, and by this round about process yields approximately the same small portion of lactic acid as the hexoses (together with larger amounts of the G and Ca saccharinic acids). That triose is condensed to hexose in dilute alkali was shown by Fischer and Tafel (43). Above an alkalinity of 0.1 N KOH the 10 mM curve turns sharply upward and reaches 95 per cent of the theoretical lactic acid in 10 N KOH. At alkalinities corresponding to the rising slope of the curve hexose formation is suppressed, doubtless by a more rapid direct conversion of the triose to lactic acid. Although reversible equilibria are doubtless involved and influenced by -alkalinity, it cannot be assumed that the high lactic acid yield from trioses is due merely to a displacement of the equilibrium, hexoses ++ trioses, to the right by high alkali, because the yield much exceeds the maximum obtainable from hexoses. When once the (j-carbon chain is formed, its rearrangement to CG acids (without splitting) and rupture of the 2,3-dienol both intervene to reduce the yield of lactic acid. ’ The curves for 20, 40, and 100 mM dihydroxyacetone are less complete, but seem to fall in the same family and to differ by having the upward inflection spread out over wider zones, at progressively higher alkalinity, and by reaching progressively lower maxima.

Injluence of Sugar Concentration.

To illustrate the influence of sugar concentration Fig. 3 has been prepared by plotting the data on the yield of lactic acid (per mol of triose) against concentrations of dihydroxyacetone. The curves show that at any given alkalinity the lactic acid yield tends to approach the theoretical at zero sugar concentration.

A similar effect of sugar concentration is shown for glucose by the following experiments. Varying quantities of glucose were added to air-free NaOH solutions of 3 to 6 N final concentration. In all cases the concentration of alkali was greatly in excess of the sugar, and the effect of change of alkali concentration may perhaps


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be ignored. After allowing sufficient time for approximately complete destruction, the lactic acid content and the relative amount of color were determined, the latter by dilution to equal initial sugar concentration and reading in a calorimeter. The results are shown in Table V. The data show a decline of lactic acid with increasing sugar concentration, and a reverse relation to color production, the effects becoming marked when the sugar concentration is molar and above.

The same effect may be demonstrated also with alkali-earth hydroxide. Varying amounts of glucose were placed in 250 cc. volumetric flasks, and the volume was brought to the mark by a 20 per cent suspension (previously boiled and cooled) of Ca(OH)l.

p .3 , I I I

I 0.1 H KOH

.2 IO 20 30 40 50 60 70 80

MM DIHYDROXYACETONE PER LITER

FIG. 3. Relation of triose (dihydroxyacetone) concentration to yield of lactic acid.

The tightly stoppered flasks were placed in a water bath at 37.5” and were frequently shaken during the first 12 hours. (The flasks were very nearly filled.) To insure complete reaction the flasks were allowed to react 18 days at 37.5”, and were then main- tained at 65” for 1 week, with occasional shaking. The results, given in Table V, show a progressive decrease of lactic acid with increasing sugar concentration. Here the effect is in part due to reduction of initial alkalinity by added sugar, though excess Ca(OH)z was present in all flasks.

Nef obtained only very small amounts of lactic acid from glucose destroyed by Ca(OH)z. His observation was due to the high


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sugar concentration used as well as to incomplete separation of zinc lactate. With low sugar concentration, saturated Ca(OH)z (about 0.03 N) forms nearly as much lactic acid from glucose as does an equal alkalinity of alkali hydroxide.

There are doubtless several factors concerned in this effect of sugar concentration. One is the decrease of initial alkalinity due to base bound by sugar (before saccharinic acid formation). This decrease of alkalinity is, of course, greater with increasing

TABLE V.

Influence of Sugar Concentration on Lactic Acid Yield. Glucose.

Temperature 37-65”.

Initial glucose Initial NaOH concentration. concentration.

?nM

50 100

M

3.0 5.0

M

1.1 1.05

550 5.0 1.01 1110 3.0 0.83 2000 6.0 0.79 2600 6.0 0.74 3400* 17.0+ 0.46

Lactic acid yield per mol

of glucose.

Relative color of solutions per mol

of sugar.

M

1.0

1.4

5.4 22.0

Excess Ca(OH),.

Lactic acid yield per mol

glucose.

7n.w M

20 0.83 50 0.67

100 0.45 250 0.31 500 0.22

‘* 50 gm. of glucose, 60 gm. of powdered KOH, and 20 cc. of 18 M KOH were stirred. The reaction began immediately and was very violent. The volume of liquid at the end of the reaction was 82 cc. The temperature of the reaction was about 145”. It is included in this table to show the relation between the lactic acid yield and the formation of colored resin and products.

sugar concentration; but it is unlikely that this factor is very important in our experiments, in which the KOH was in large excess. Another factor is the greater probability, with increasing sugar concentration, of active molecules or ions condensing to higher sugars, resins, or other products and thereby being diverted from saccharinic acid formation.

It may be noted here that these two factors doubtless account for the large quantitative discrepancies between the results of Evans and coworkers and our own. Their results with hexoses


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were noted above (p. 359). From the trioses Evans and Cornth- Waite (16) obtained very much lower yields of lactic acid than those here reported, finding in N KOH at 50’ only about 20 per cent (or 0.4 mols per 2 mols of triose compared to our 1.2 to 1.65), with maxima of 40 to 45 per cent in 4 N KOH (0.8 to 0.9 mol per 2 mols of triose, compared to our maxima of 1.6 to 1.9). They used 1 M solutions of triose, which neutralize at the start up to equivalent amounts of added alkali, leaving the initial alkalinity far less than corresponds to the added hydroxide. This fact was

overlooked by these workers and to a large extent invalidates the relation of alkalinity to lactic acid and other products in their experiments. The fact is however of interest that 1 or even 2 equivalents of KOH (shown also in our data) are insufficient to bring about maximum conversion to lactic acid. The high sugar concentration used by Evans also directly influences the products formed, as noted above.

In$uence of Temperature.

Table VI gives data on the lactic acid yield from glucose in 0.5 M KOH at different temperatures, from slightly below 0” to that of a boiling water bath. Although the destruction is much slower at low temperature, the total lactic acid ultimately formed is much greater, due presumably to a lower rate of side reactions and polymerizations leading to resins and other complex products. At low temperature the color of the solutions is less, being almost absent at 0”. An opposite effect of temperature is indicated by the recent report by Wolf (23) of 74 per cent lactic acid from cane sugar heated with 3 mols of CaO in an autoclave to 197”.

Table VII gives similar data for galactose and two pentoses, within higher temperature range. With the pentoses also the lactic acid yield decreases with rise of temperature; while with galactose the effect of temperature is slight.

Before passing to our calculations of salt fractions at the various alkalinities, we may consider briefly the facts already noted in relation to the reactions indicated in Diagram 1. It is evident that at low alkalinity (and temperature) (alkali carbonate and bicarbonate) the reactions must be largely limited to the reversible interconversions among the sugars and dienols across the center of the diagram without saccharinic acid formation or rupture.


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Within this range polymerization of lower sugars to higher also occurs, with the conversion of methylglyoxal to triose and of triose to hexoses (20-23, 43). The equilibrium is toward the center and to the left of the diagram, the products converging toward aldohexoses. At the higher alkalinity of KOH (or higher

TABLE VI.

Injiuence of Temperature on Yield of Lactic Acid. Glucose, in 1.6 M KOH.

Temperature.

-

_ _ Reaction time.

-

_- GlUCO%S

destroyed.

‘C. hs ?ndl ?nM

-3 to -1 215 38.7 29.7 25-26 12 55.6 48.7 25-26 24 57.0 50.0 37-38 29 54.9 50.0 60-70 1 39.4 40.0 70-95 2 41.0 50.0 68-73 3 45.7 50.0 99 3 39.9 50.0

TABLE VII.

Lactic acid per mol of sugar.

2.f

1.30 1.14 1.14 1.10 0.98 0.82 0.91 0.80

Influence of Temperature on Yield of Lactic Acid. Sugars in 0.6 dd KOH.

Sugar.

d-Galactose.

I-Xylose.

I-Arabinose.

-7

remperature

“C.

37-38 71 99 37-38 71 99 37-38 71 99

Reaction time.

aau.3

29 3 3

29 3 3

29 3 3

24.9 24.1 24.3 30.0 24.5 23.7 28.1 26.2 25.3

Sllgar destroyed.

7n.M

50 50 50 60 60 60 60 60 60

- Lactic acid per mol of

sugar.

M

0.498 0.482 0.487 0.500 0.408 0.395 0.468 0.437 0.422

temperature) the etiols begin to rearrange into ortho osones and saccharinic acids and to separate into smaller fragments, thus destroying the sugars. In the lower region of this range the acids are mainly from 1,2- and 2,3-dienols. With further rise of alkalinity, although the total acids formed are not much increased, the proportion as lactic acid (from trioses and the glucose series)


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Sugar Activation by Alkali. I

rises to a maximum, indicating a diversion from the other reactions, presumably because of a relative increase in the concentration of the 3,4-dienol or its salt, at t.he expense mainly, it seems, of the 2,3-dienol. These relations could be understood if it might be supposed that there is associated with each dienol an additional acidic group (or groups) decreasing in individual strength but increasing in their sum of base-binding ability, in the order 1,2-, 2,3-, and 3,4-dienol. This would require that the sugars behave as polybasic acids, which should be susceptible to test by experiment. An attempt to decide this point is now under way.

Starting with the trioses, the reactions seem somewhat more decipherable, The results show that in sufficiently dilute solutions of the sugar and at very high alkalinity almost theoretical conversion to lactic acid may take place. The same is true of methylglyoxal, even at somewhat lower alkalinity (44). In dilute alkali, however, a portion of both triose (43) and methylglyoxal (20-23) is diverted in the opposite direction, apparently to hexoses, from which a small fraction may again pass, presumably via triose and methylglyoxal, to lactic acid, the balance giving resins and other saccharinic acids. In neutral or acid solutions methylglyoxal and the trioses are stable except at high temperatures. We may picture the relations as given in the form&e somewhat as follows: Starting at methylglyoxal (I), we suppose that it enolizes in two ways, one to give (2) which by adding water (H+ and OH-) gives lactic acid (3), and the other (4) which by adding water gives glyceraldehyde (5). The last is in equilibrium (?) with (a) 3,4-hexose dienol and (b) with the 1,2-triose dienol. High alkalinity evidently favors the first type of enolization, as is presumably the case also with the other ortho osones. The reverse of these changes would represent the formation of the ortho osone from the aldose. It is possible to describe this behavior somewhat acceptably in terms of electronic structure if it be permissible to regard glyoxals and dienols as dibasic acids. Justification for such views must await further work.

Relation of Lactic Acid Yield to Salt Fraction.

It would seem reasonably certain that the influence of increasing concentrations of KOH on the yield of lactic acid must be an effect


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II .?

C-

OH

I ?

Hexo

se

3,4-

dieno

l. e

H-C-

OH

High

alk

alinit

y --f

E

I +-

Lo

w alk

alinit

y HO

-C-H

&

I g

i7

E

H-C-

OH

H-C-

OH

HC=O

H-

-C==

0 HC

=O

I II

c=o

HO-C

=0

I I

I II

2 I

c=o

z$

‘---- :

C-

OH

z$

H-C-

OH

= ~

H,-C

-OH

S c=

o f

C-OH

+

H-C-

OH

I I

I ;

/ II

m

I I

I HO

-C-H

HO

-C-H

HO

-C-H

!?

!I?;-C

-H

H-C-

H H-

C-H

H-C-

I? s

I I

I I

I I

I zi

;’ H

H H

H H

H H

Dihy

drox

y- 5?

Tr

iose

Gl

ycer

- M

ethylg

lyoxa

l M

ethylg

lyoxa

l. M

ethylg

lyoxa

l La

ctic

acid

. 9

acet

one.

1,

2-die

nol.

aldeh

yde.

en

ol.

enol.

E

(5)

(4)

(1)

(2)

(3)

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of hydroxyl ions in determining the proportion of sugar present as salt or ions, possibly of second or even of third acidic groups. It was therefore of interest to attempt closer correlation of these quantities. There are unfortunately a number of difficulties which prevent satisfactory treatment. More serious than errors in the analytical results are uncertainties due to the “abnormal” behavior of strong solutions of electrolytes, such as the KOH solutions in which the formation of saccharinic acids takes place. Assumptions as to constancy of apparent hexose dissociation constants and of water activity must be made which are open to question, while the constants for the trioses are unknown. For these and other reasons the calculations here given can be regarded only as trial approximations, which nevertheless are suggestive and we believe worth recording.

We have calculated the initial hydroxyl ion concentration and activity, of the solutions as follows: concentration p(OH),, from conductance data for KOH at 18” (International Critical Tables,

6) (

2 X M = [OH],, in which M is the initial molarity of KOH,

[OH],ihe hydroxyl ion concentration, A, and A0 (=235 by extrapolation) their conventional meaning). Activity p(OH). was calculated from Scatchard’s (45) activity coefficients for KOH at 25’ (yrn X M = (OH),). The difference in temperature between that for which [OH], and (OH), are calculated and that of the lactic acid formation (3740”) was ignored. When the molar lactic acid yields are plotted against p(OH) values thus calculated, we obtain a series of curves similar to those of Fig. 1, which give little additional information, except that in the highest alkalinity the activity data give a smoother curve than do those from conductance. In this region the activity coefficients are uncertain, though much greater than dissociation factors by equivalent conductance. (The curves are not reproduced.)

Calculation of the salt fraction of the sugars from the equation

[Al ~H-pK=log~

requires evaluation of the pH of the solutions and pK1 of the sugar, For calculation of pH we take the value 13.50 for pK, at 37.5”,


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and, ignoring both temperature differences and the possibility of change of pK, in concentrated solutions, assume

13.50-p(OH) (activity at 25” or conductance 18”) = pH (37.5”)

For pKi of glucose we chose the value 12.00 at 37.5” (Landolt and Bornstein give 9.8 X lo-l3 at 40”). The antilog of (log 1-W R

-) =R,and- IHAl Rfl

= salt fraction of sugar (as the monobasic

salt of the acid whose pKi is 12.00 at 37.5”). We thus calculate

TABLE VIII.

Calculated Salt Fraction and Lactic Acid Yield. Glucose Series.

Hl?X. me

con- ce*- tra-

tion.

11)

??Lu

5 5

10 10 50 50 50 50 17.5

I$% (OH), on?X- activity. ration.

(2) (3) __-

M M

0.02 0.017, 0.04 0.033, 0.10 0.077 0.25 0.183 0.50 0.361 1.00 0.743 2.00 1.70 5.00 (7.50)

10.00 (20.00)

,

--

4 4 2

(

(4) (5) --

x

0.013 11.74 0.029 12.02 0.069 12.39 0.176 12.76 0.325 13.06 0.706 13.37 1.66 13.73

(7.42) (14.381 :20.00) (14.80)

?H cm. rected (37.5”).

(N

11.61 0.36 11.96 0.51 12.34 0.71 12.75 0.85 13.01 0.92 13.35 0.96 13.72 0.98 14.37 0.99) 14.80 1.00:

salt raetiol

%ior

(7)

-

Salt ractio

COP rected

(8)

0.29 0.48 0.69 0.85 0.91 0.96 0.98 0.99 1.60

Lactic acid yield.

Per m01 hexose.

(9)

Frac- 1 !%c- >ion of tlOn Of maxi- theory

mum. mc%,

(10) (11) --

M

0.5! 0.47 0.27 0.71 0.61 0.35 0.8: 0.72 0.42 1.01 0.89 0.52 l.O( 0.91 0.53 1.1: 0.98 0.57 1.1; 1.0 0.58 1.08 0.92 0.54 1.U 0.94 0.55

Figures in parentheses are less reliable values obtained by extrapolation.

the data given in Table VIII. Only the data calculated from activity coefficients are given, since these appear to be more con- sistent in the stronger solutions. In the lower ranges there is no significant difference between results calculated by the two methods.

In Column 5 the pH and in Column 7 (Table VIII) the salt fraction is calculated without taking account of base bound by sugar or by acids formed, while in Columns 6 and 8 the same values are calculated on the assumption that the added sugar neutralized and removed 1 equivalent of base. The point of


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interest is the comparison of the calculated salt fractions with lactic acid yield. In the two lower concentrations of KOH (0.02 and 0.04 M) the lactic acid yield (in fraction of maximum) exceeds the salt fraction, and in the two strongest solutions a smaller discrepancy appears in the opposite direction. Otherwise the agreement is good. It does not seem probable that the fair agreement is fortuitous. We are disposed to interpret the relation to mean that the final amount of lactic acid formed from these sugars is within the limits given proportional to the fraction of the sugar

TABLE IX.

Calculated Salt Fraction for Assumed Dissociation Constants. Dihydroxyacetone, 10 mM.

Initial KOH pHa L hit fraction corrected. P K1 = 12.OC

(1) (2) (3)

M

0.02 11.44 0.22 0.04 11.90 0.44 0.10 12.34 0.69 0.25 12.75 0.85 0.50 13.05 0.92 1.0 13.37 0.96 2.0 13.75 0.98 5.0 114.37) 0.99

10.0 (14.80) 1.0

ft,

_

-

Salt fraction ,K, = 12.8

Lactic acid yield per

mol triose.

T Lactic acid -,

Salt fraotmn

(4)

f:

. - (5)

pK 12.00

(6)

pK 12.80

(7) _-

zd

0.03 0.25 1.14 8.3 0.11 0.32 0.73 3.0 0.26 0.40 0.58 1.5 0.47 0.53 0.62 1.12 0.64 0.67 0.73 1.05 0.79 0.82 0.85 1.04 0.90 0.90 0.92 1.0 0.97 0.93 0.94 0.96 0.99 0.95 0.95 0.96

- Figures in parentheses are less reliable values obtained by extrapolation.

present as salt or ions. The results indicate that the ion is that of the primary acidic group, which is contrary to our expectation. (The rate of formation shows a different relation.) We have no explanation why the lactic acid formed is higher than expected at low pH, unless it be that the lactic acid results in this range are erroneously high due to presence of much 1,3-dihydroxybutyric acid. Another point should be noted: the dissociation constants of fructose and mannose are higher than that of glucose (9), and if the above conclusion be valid, it might be expected that they would yield somewhat more lactic acid at a given (lower) pH than glucose. The results give no evidence of such differences in yield. It may be thought that at such high alkalinity each of


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these sugars is converted into substantially the same mixture, though in a later paper it will be shown that the rates of acid formation are different for each sugar.

We have attempted to calculate the salt fractions similarly for the trioses. Here the calculation becomes more venturesome, since besides the difficulties already noted, the dissociation constants of the sugars are unknown. We may however try various hypothetical values for pKi and infer that the one yielding data corresponding to lactic acid may approach the correct one. Table IX gives data for the salt fraction calculated for values of pKi of 12.00 and 12.8 for dihydroxyacetone (10 mM as to triose). The values of pH and salt fraction are calculated on the assumption that the KOH concentration is reduced by 1 equivalent of the triose added. Otherwise the calculations are based on the same assumptions as with the glucose series. Except in 0.02 M KOH the results are not greatly different when no correction is made for base bound by sugar. On comparing the calculated salt fractions with the yield of lactic acid expressed as fraction of the maximum theoretical value (1 mol per mol of triose), we find only at the lowest alkalinity (0.02 M KOH) good agreement for a pKi value of 12.00, the same used for hexose; at higher alkalinity the lactic acid yield falls far short of the calculated salt fraction. But with increasing alkalinity there is an approach to good agreement with the salt fractions calculated for a pK value of 12.8 (Columns 4, 5, and 7). This would seem to suggest that the direct conversion of dihydroxyacetone into lactic acid under the influence of alkali is proportional to the fraction of the sugar present as salt (ions), the sugar having an apparent acidic dissociation con- stant (perhaps the second) of about 1.6 X lo-13.

In the lower range of alkalinity where this relation does not hold there is reason to believe that the deviation is due to partial conversion of the triose to hexose. This conversion is known to occur at low alkalinity (Fischer and Tafel (43)). Furthermore comparison of the yields of lactic acid from triose (2 mols) with that from the glucose series of hexoses at the same alkalinity shows them to be very similar in the low range of alkalinity 0.02 to 0.10 M KOH at which the above deviation occurs.

It should be noted that since saccharinic acid formation is a slow process it might be expected that alkalinity would affect the


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rate, but it is not equally evident why alkalinity should influence qualitatively the acids formed. It is this qualitative difference which is involved in the relation considered above. The only explanation of this influence appears to be that the degree of alkalinity determines the proportions of several different salts (or different dienols). In another paper somewhat similar though quantitatively different relations between rates of reaction and alkalinity will be discussed.

SUMMARY AND CONCLUSIONS.

Data are reported on the amounts of total saccharinic acids and of lactic acid formed from hexoses, pentoses, and trioses under the influence of varying concentrations of KOH. The influences of hydroxyl ion concentration, sugar concentration, and temperature are considered. High alkalinity favors, while high Oemperature and high sugar concentration lower the, yield of lactic acid. Within limits the yield of lactic acid from glucose appears to be a function of the fraction of the sugar present in the form of salt. At quite high alkalinity and low sugar co&en- tration dihydroxyacetone is almost quantitatively converted to lactic acid, the yield being apparently a function of a salt fraction of the triose, calculated for an assumed pK1 value of 12.80. It is concluded that alkalinity and temperature “activate” the sugars by salt formation and consequent ionization, and that saccharinic acid formation may be regarded as the spontaneous rearrangement of unstable sugar ions. The ratio of particular acids formed probably depends upon the proportions of di$erent salts or ions.

BIBLIOGRAPHY.

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Philip A. Shaffer and Theodore E. FriedemannSACCHARINIC ACIDS

FORMATION OF LACTIC AND SUGAR ACTIVATION BY ALKALI: I.

1930, 86:345-374.J. Biol. Chem.

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sugar activation by alkali. it is well known that in alkaline

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