THE QUALITATIVE TESTS FOR ACETONE BODIES ; THEIR SIGNIFICANCE AND VALUE.

BY E. J. BIGWOOD AND W. S. LADD.

(From the Chemical Division, Medical Clinic of the Johns Hopkins University and Hospital, Baltimore, and from the Department of Medicine, College

of Physicians and Surgeons, Columbia University and the Presbyterian Hospital, New York.)

(Received for publication, August 22, 1923.)

The qualitative tests most commonly used for acetone bodies in the urine are the sodium nitroprusside test and the ferric chloride test. These are usually regarded as selective tests for acetone and diacetic acids, respectively. In fact, few text-books on methods of analysis mention the sodium nitroprusside test as a test for diacetic acid, while some workers-Harding and Ruttan (1) and Hunter (2)Lregard it as probably selective for diacetic acid alone. Occasionally, in case reports these qualitative tests have been allotted an unwarranted quantitative significance. Both tests are known to be interfered with or rendered positive by other substances.

For these reasons and because there is contradictory evidence in the literature on the subject, we took the opportunity afforded in a metabolic study of a series of cases of diabetes to make both qualitative and quantitative analyses on the same samples of many specimens of urine. In addition we have carried out certain experiments devised to throw light upon the selectivity, sensitivity, and quantitative significance of these tests, in some instances repeating the work of others.

In 1882, Legal found that sodium nitroprusside in the presence of sodium or potassium hydroxide produces a ruby-red color in a solution containing acetone or creatinine. Coincidently, le Nobel (3) observed that if acetic acid is added to the solution before the*base, the color changes to purple (sometimes spoken of as violet or a permanganate tint), and that this occurs only when the solution contains acetone; it is not produced by creatinine.

347

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348 Qualitative Tests for Acetone Bodies

In 1908, Rothera (4) pointed out that ammonium hydroxide is more satisfactory than sodium or potassium hydroxide, and that the test is more sensitive in the presence of ammonium sulfate.

Harding and Ruttan (1) investigated the sodium nitroprusside reaction and conclude that it detects diacetic acid rather than acetone; that since an aqueous solution of free acetone shows a less marked Legal reaction than does diabetic urine containing the same amount of acetone, there must be some other substance in diabetic urine which favors the appearance of the purple ring; that diabetic urine saturated with NaCl, and freed from acetone by aeration gives a positive sodium nitroprusside test to dilutions of 1 to 30,000 and a positive ferric chloride test to dilutions of 1 to 7,000. Hydrolyzed diacetic ester gives a positive nitroprusside test to a dilution of 1 to 80,000.

EXPERIMENTAL.

The following is the technique used. Sodium Nitroprusde Pest.- Transfer about 10 cc. of urine to

a test-tube and add 20 drops of the following solution. Glacial acetic acid, 10 cc., + 10 cc. of a 10 per cent solution of Na nitro- prusside. (This solution will keep well for at least 2 weeks in a brown glass bottle.) Shake the test-tube until the specimen is well mixed, and then layer on this 1 to 2 cc. of concentrated NH,OH solution. At the contact of the fluids, a purple ring appears. After standing 2 minutes, the reading is made and recorded. In order to standardize these readings roughly, we record them as follows:

Trace.. . . . . . . . . . . . . . . . . The ring is very faint.

+Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Small, but definite ring. . . . . . . . . . . . . . , . . . . . . . . . . . .Large ring.

+++ . . . . . . . . . . . . . . . . . . . . . . . . .Very large ring. ++++ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Heavy purple mass.

Ferric Chloride Test.-10 cc. of a 10 per cent solution of ferric chloride (FeCh) are added to 10 cc. of urine, more is added, if necessary, to clear the precipitate. The color appears instan- taneously and the reading can be immediately made. For approx- imate comparison, the readings are recorded as follows:

Trace........................................Lightbrown. + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dark brown.

++. . . . . . . . . . . . . . . , . . . . . . . . . . . .Light burgundy. ++ + . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . Dark burgundy.

++++. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . .Black.

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E. J. Big-wood and W. S. Ladd 349

Data Concerning the Selectivity of the Qualitative Tests.

1. Acetoacetic ethyl ester gives a positive ferric chloride test and a negative sodium nitroprusside test.

2. These two tests could not be made on butyric ethyl ester because it does not mix with water. Attempts to test the sub- stance when water was added and the mixture shaken gave negative results with each test.

3. When applied to free acetone solutions, the ferric chloride test is negative, even with chemically pure acetone (not acid to litmus). The nitroprusside test is positive.

In order to be certain that this acetone solution contained no other acetone bodies, i.e. diacetic acid, we determined quantita- tively the content of acetone by Folin’s method for free acetone alone, and by Van Slyke’s method for total acetone bodies, expressed in grams of acetone. These determinations were made on a 1 per cent by volume solution. The results follow.

om. By Van Slyke’s method.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.674

0.716

Average................................................. 0.695

By Folin’smethod........................................ 0.698 0.700

Average . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.699

A 0.1 per cent solution by volume represents 1 cc. of acetone per liter. As 0.79 is the specific gravity of acetone, and the experi- ment was made at 25”C., 0.7 gm. of acetone is within the limits of error. Since the determinations of acetone by the two methods agree, it is obvious that no other acetone bodies, such as diacetic acid, were present in the solution.

A 10 per cent solution by volume of acetone was kept for 2 weeks in the ice box in a glass stoppered flask. The above qualita- tive tests were then repeated with the same results as before.

4. Diacetic acid was prepared by hydrolyzing diacetic ethyl ester according to the method of Harding and Ruttan (1). A little HCl was then added to the solution, to bring its pH approxi- mately to that of the average reaction of urine. This solution

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350 Qualitative Tests for Acetone Bodies

was kept at room temperature in a closed flask and analyzed at intervals of a few days.

The results of the analyses were as follows:

1st day. Ferric chloridetest....................................++++ Nitroprussidetest..................................... +++

Diacetic acid + acetone expressed as grams of acetone.. . . . . 5.56 Free acetone............................................... 0.29

Diacetic acid (by difference of the two). . . . . . . . . . 5.27

All determinations were made in duplicate and checked within the limits of error of the methods. As 64 is the molecular weight of acetone, and 142, that of the ester, 13 gm. of ester expressed

in acetone will be equal to 13 X g2 or 5.85 gm. Since the

diacetic ester was roughly weighed, 5.56 checks well with the expected 5.85.

Hydrolysis of ester was probably completed by the procedure of the Van Slyke method.

On the 2nd, 3rd, 4th, and 5th days, the qualitative tests re- mained the same. On the 5th day, a sample was withdrawn and treated by Folin’s method for free acetone determinations; that is, 10 drops of a 1 per cent solution of HsP04, excess of NaCI, and a little kerosene were added, and the mixture was aerated for 40 minutes. The qualitative tests on this solution were as follows:

Ferric chloride test.. . . . . . . + (or considerably decreased). Nitroprusside test.. . . . . . . . . ++++ (or increased).

The solution was again aerated, but this time through a known amount of 0.1 N iodine solution, in order to test whether the first aeration period had been continued long enough to carry off all the free acetone. Some of the iodine was converted into iodoform, showing that some acetone still remained. The period of aeration was therefore repeated and iodine titration proved the absence of all free acetone. The qualitative tests were applied and found to be + for ferric chloride and ++++ for Na nitroprusside. The diacetic acid by quantitative analysis proved to be 2.75 gm. per liter, the original concentration having been 5.56 gm. per liter.

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E. J. Bigwood and W. S. Ladd 351

Thus we have a solution containing less than half the original amount of acetoacetic acid and no free acetone, and yet the qualitative test with sodium nitroprusside is stronger than in the original solution. This suggests that the nitroprusside test is posi- tive when diacetic acid alone is present. But why there should be a more intense reaction with half as much diacetic acid present, was difficult to say. (See discussion under sensitivity of the tests.)

In order to test further the selectivity of sodium nitroprusside for diacetic acid, we carried out the following experiments.

Experiment 1.-A 100 cc. specimen of solution of hydrolyzed ester, withdrawn from the stock solution as it reached the 5th day of hydrolysis (see Table III), was boiled again under reflux condenser. The flask was not removed from the reflux condenser until it had cooled. The volume of the fluid remained the same. Both qualitative tests were again negative. The solution con- tained acetone only, because Van Slyke’s method for diacetic acid + acetone showed 0.280 gm. per liter, and Folin’s method for free acetone showed 0.293 gm. per liter (that is, the same result in both determinations); hence all the diacetic acid had dis- appeared. If it was converted into acetone, most of this had disappeared too, because the concentration of free acetone re- mained unchanged, in spite of which fact the sodium nitroprusside test was strongly positive (+ ++) before boiling, and negative after boiling. ph. zs can only be explained by the disappearance of the diacetic acid, since the amount of free acetone left in solu- tion is too dilute to be capable of producing a positive nitro- prusside test. (See Table II.)

Experiment 2.-A 50 cc. sample (accurately withdrawn with a pipette) of the specimen corresponding to the 5th day of hydrolysis was aerated for 40 minutes after adding 10 drops of a 10 per cent solution of H3P04, excess of NaCI, and some kerosene in order to free the specimen from acetone (Folin’s procedure). 10 cc. were then pipetted for qualitative tests.

Ferric chloride.. . . . . . . . . . . .O (it was positive before aeration). Nitroprusside.. . . . . . ++++ (it was only +++ before aeration).

The 40 cc. left were aerated again for 20 minutes, through 10 cc. of 0.1 N iodine solution. The excess of iodine was titrated with

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352 Qualitative Tests for Acetone Bodies

0.1 N Na thiosulfate in the presence of starch, and showed no free acetone in the solution after the first aeration period since exactly 10 cc. of iodine were recovered. The total acetone bodies in the remaining solution were determined with Van Slyke’s method (5). The result was a concentration of 0.138 gm. per liter, which evidently represented nothing but diacetic acid (initial concentration of diacetic acid before aeration being 0.530 gm. per liter). Aeration has then removed much of the diacetic acid in addition to the acetone. What happens to the former, we do not know. However, the nitroprusside test is increased, at least not decreased, even when diacetic acid is decreased.

That sodium nitroprusside detects diacetic acid is obvious from the fact that it remains positive, although quantitatively there is no free acetone left in solution.

The purple ring in this solution, saturated with NaCl, instead of fading on long standing, as is usual,-changes readily and steadily into a heavy, nearly black mass, as if the substance responsible for the positive test were gradually accumulating around the level of the ring in the test-tube. This, however, is not the case, because after standing 1 hour, the clear, colorless fluid at the bottom of the test-tube, beneath the level of the purple ring, on being pipetted out, poured into a fresh tube, and tested again with nitroprusside, showed a new ring, just as increasingly heavy as the first one.

We were not satisfied that these experiments showed that nitroprusside in contaot with diacetic acid formed a purple ring in the absence of any trace of acetone; since we had added other substances to the solution, any one of several of which might clearly influence the test. In order to prove our assumption it was necessary, therefore, to repeat our experiment of this removal of all free acetone without the presence of any additional substances.

The solution used was that of the 8th day of hydrolysis (see Table III). 25 cc. of this solution plus 25 cc. of Hz0 (both accurately pipetted) were aerated for 4 hours. The total volume remained 50 cc. at the end of the period of aeration. The ferric chloride test on this mixture gave a negative result (there was a trace before aerating) while the nitroprusside test gave a + reading (it was + ++ before aerating).

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E. J. Bigwood and IV. S. Ladd 353

*wt. per zitm

Diacetic acid + acetone ................................... 0.056 Free acetone (Folin’s method). ............................ 0 Diacetic acid ............................................... 0.056

Hence, a 4 hour period of aeration is sufficient to remove all free acetone from a dilute solution in the absence of NaCl (less than 4 hours did not remove all free acetone). However, the nitro- prusside test remains positive, and must be due to the small amount of diacetic acid present in the mixture (0.056 gm. per liter).

In order to rule out the possibility that the procedure of the sodium nitroprusside test might decompose some diacetic acid into acetone sufficiently to render the latter responsible for the appearance of the purple ring, the following experiment was performed.

A solution containing diacetic acid and acetone was saturated with NaCl and aerated through a known amount of iodine. The concentration of free acetone proved to be O.4O2 gm. per liter. The same titration was repeated on a second sample of the solu- tion, but Na nitroprusside, acetic acid, and NH3 were added in the same proportion as those for Legal’s test. The color of the mixture became dark purple. It was left standing for 4 hour under kerosene, and then aerated through iodine. The titration showed a concentration of 0.395 gm. per liter. Hence, we con- clude that no free acetone is liberated from a diacetic acid solu- tion, when tested for ketone bodies with Na nitroprusside, acetic acid, and ammonia. Therefore, there is no doubt that diacetic acid alone may give a positive nitroprusside test.

Data Regarding the Sensitivity of the Qualitative Tests.

The intensity of the reaction of the ferric chloride test to dif- ferent dilutions of the diacetic ethyl ester, is given in Table I.

The intensity of the sodium nitroprusside tests, obtained with various concentrations of pure acetone solutions, is presented in Table II.

We conclude from this that a pure aqueous solution of acetone, free from diacetic acid, gives a nitroprusside test to dilutions of 1: 1,000.

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354 Qualitative Tests for Acetone Bodies

With diacetic acid solution, the sodium nitroprusside test showed a positive reaction to a dilution of 1: 20,000. At this grade of dilution, the ferric chloride test is not positive.

It is interesting, moreover, to compare this with the findings on acetone solutions of Table II, which show that at the corre- sponding grade of dilution of acetone, the test is not positive. It is also serviceable to compare these figures with those found in a fresh specimen of urine, which contained no acetone, and some

TABLE 1.

Concentration of acetoacetic ethyl ester. Color reaction with FeCL. Symbol.

per cent

0.1 0.2 0.25 0.3 0.5 1.0

More than 1.0 per cent by volume.

Yellow. Light brown. Dark “

“ “

Light burgundy. Dark “ Black.

0 Trace.

+

+f+ +++

++++

D;;il$neby

per c‘mt 0.0001 0.001 0.01 0.1 1

10

TABLE II.

Solvent.

0 0 0

Trace. +

++

Normal urine.

0 Very faint trace.

Trace.

2t +++

Diabetic urine sugar-free and both acetone bodies

tested negative.

0 Very faint trace ?

Trace. +

A++

diacetic acid (0.076 gm. per liter). Both qualitative tests were slightly positive. This would suggest that the limit of dilution for which a nitroprusside test is still positive would be about 1 to 15,000, whereas, in our solution of pure diacetic acid, acetone- free, the dilution of which was greater (1 .to 20,000), the nitro- prusside test was still definitely positive. Apparently, then, the Na nitroprusside test is less sensitive for acetone than for diacetic acid.

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E. J. Bigwood and W. S. Ladd 355

It was noted on page 351 that we had a solution “containing less than half the original amount of acetoacetic acid and no free acetone, and yet the qualitative test with sodium nitroprusside is stronger than in the original solution.” In the hope of learning the reason for this discrepancy, we repeated the experi- ment, using less diacetic ethyl ester, in order to obtain proportions of ketone bodies which correspond to the usual amounts found in

TABLE III.

Repeated Analysis, at d or d Day Intervals, of a Mixture of Diacetic Acid and Acetone Obtained by Hydrolysis of Ethyl Diacetate.

+++

+ Dark

brown. Trace.

Light brown.

Trace.

G 1

1 g

++ t+-i

t+-t

t+t

-

-.

-

“%?;1,r 0.720

0.750

0.80

0.81

gm.

0.0365 0.0352 0.0380 0.0372

0.0398 0.0406

0.0406 0.0405

0.270

0.270

gm.

0.10 0.615 0.11 0.187 0.575 0.163

Duplicated 0.53 check within 1 drop of O.lN thio- sulfate.

0.25 0.54 0.29

diacetic urine, and also to detect definite changes in the intensity of the nitroprusside tests, which were always very intense (large purple ring) with the more concentrated solution just used. Exactly the same technique was followed for hydrolysis of the diacetic ester, but only about 1.5 gm. were used instead of 13 gm. After standing (slightly acidified because diacetic acid is more easily converted into acetone in an acid medium (6)) at room

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356 Qualitative Tests for Acetone Bodies

temperature for 24 hours, the characteristic odor of the ester had disappeared. The solution was allowed to stand for about 48 hours and analyzed from time to time. The results are given in Table III.

The content of free acetone has nearly tripled but the amount of diacetic acid has decreased, in spite of the fact that the total ketone bodies have increased in concentration. After the 5th day of hydrolysis, an equilibrium seems to have been reached, and as hydrolysis has proceeded, the ferric chloride test has diminished, and the nitroprusside test increased, a phenomenon commonly observed in diabetic urine. For example see Table IV.

TABLE IV.

Time.

Specimen of urine 15 min. after voided. . . . . . . . .

Same specimen 24 hrs. later.. .

5 a i 8 2

+

++

-

I .-

-

.-

-

om.

0.012, 0.012:

0.011: 0.010

0.11

0.125

id a.0 8% &.a s$ $Q P ag 9s n

0.10 0.12

0.13 0.12

7

/

r

;

I :

-

-

om.

0.14

0.095

When the hydrolyzed solution of diacetic ester had reached the stage corresponding to the 3rd day of our chart (see Table III) a 100 cc. specimen was boiled under reflux condenser for 40 minutes. This on being tested, when sufficiently cool, showed negative qualitative tests. Quantitative determinations showed 0.24 gm. of diacetic acid per liter. Boiling had reduced the amount of diacetic acid to less than one-half, and it had also reduced the amount of acetone to about one-quarter of its original amount (notwithstanding the use of a reflux condenser), a result in direct disagreement with Harding and Ruttan’s assumption that the acetone is increased after boiling. This decrease in the amount of acetone present may account in a large part for the disappearance of the sodium nitroprusside test.

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E. J. Bigwood and W. S. Ladd 357

The disappearance of diacetic acid such as occurred in Experiment 2 on page 351 when its solution is aerated and satu- rated with NaCl, is rather puzzling. Since it exists only in the form of its esters, the properties of this compound are unknown. The literature gives scant information on the subject. The first hypothesis which we considered was that diacetic acid might be volatile like acetone and carried off by the current of air. There- fore, we saturated with NaCl a mixture containing 0.315 gm. of acetone per liter and 2.63 gm. of diacetic acid, and aerated it through 10 cc. of 0.1 N NaOH and finally through iodine. The titration of the iodine solution showed that the concentration of free acetone was 0.298 gm. per liter and the 10 cc. of 0.1 N NaOH were exactly recovered. The amount of diacetic acid left was 1.23 gm. per liter. This experiment was repeated with the same result. It shows that diacetic acid is not carried off as such with the current of air, nor in form of a volatile acid into which it might possibly have been converted, in which case it would have neu- tralized part of the 0.1 N alkali. This procedure did not interfere with the quantitative determination of free acetone, which was all recovered except a trace. Nor can diacetic acid have been carried out in the form of acetone, because the free acetone quanti- tative determinations showed no increase.

We have aerated a pure solution of acetic acid through a known amount of alkali, for a period corresponding to those of our experi- ments, in order to test whether acetic acid could be carried over by the current of air in appreciable amounts. It showed very definitely to be the case. If, then, acetic acid could have been formed from diacetic acid during the aeration process, it would have been caught by the 0.1 N NaOH solution, which in the experiment above showed no change.

In order to ascertain whether NaCl, H3P04, or kerosene had any effect on the diacetic acid, we performed the following experiment.

A solution, containing 2.10 gm. of diacetic acid and 0.40 gm. of acetone, was saturated with NaCl; to this were added 10 gm. of a 10 per cent solution of HaPOd and a little kerosene; the whole was left standing for + hour (with an occasional shaking). This mixture was not aerated; quantitative estimation showed that there remained 2.10 gm. of total acetone bodies in solution which corresponded to the initial diacetic acid; no free acetone

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358 Qualitative Tests for Acetone Bodies

remained. From this it will be seen that the reagents added do not destroy diacetic acid. But subsequent aeration caused roughly one-half of the initial amount of diacetic acid to disappear. This is not carried off as diacetic acid nor acetic acid.

In order to prove that it does not enter into a new compound, the following experiment was carried out.

A sample of a solution of diacetic acid, acetone, and some ethyl diacetate containing 2.864 gm. per liter of total acetone bodies was saturated with NaCl, HaPOd and kerosene were added in the usual way, and the mixture was aerated for 13 hours. The total acetone bodies were now reduced to 0.945 gm. per liter. The solution was hydrolyzed for 24 hours in the pres- ence of 0.7 per cent KOH. The concentration of total acetone bodies was again determined, and found unchanged.

Further information is needed to explain the disappearance of diacetic acid under the conditions of the experiment.

It became evident from our experiments, particularly those in which aeration was used, that the sensitiveness of the nitro- prusside test was influenced by some of the substances used in the Folin free acetone determination, as well as by certain sub- stances present in urine, whether normal or pathological. The following experiment was performed in order to determine the responsible agent.

Four test-tubes, each containing the same amount of a 1 per cent solu- tion of acetone (see page 349), were treated respectively as follows:

To Tube I no additional substances were added. To Tube II a few drops of a 10 per cent solution of H,POa were added. To Tube III some kerosene was added and the tube shaken. To Tube IV sufficient NaCl was added to saturate it. Then the nitroprusside test was performed in the four tubes, which were

subsequently allowed to stand for some time; the results are presented in Table V.

It is obvious that whereas H3P04 had no effect at all on the the reaction, kerosene had slight influence, while the NaCl had an immediate and very pronounced effect. The ring changed immediately into a heavy purple mass. The fluid at the bottom of Tube IV was pipetted out, retested with nitroprusside, and showed a ring with the same intensity. NaCl tested alone in solution with the nitroprusside reagent gave an absolutely nega- tive result.

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E. J. Bigwood and W. S. Ladd 359

We finally diluted the 1 per cent solution of acetone with dis- tilled water and with a 10 per cent solution of NaCI, and applied the nitroprusside test. Our results are given in Table VI.

When compared with the data of Table II, it becomes evident that NaCl intensifies the color, but does not produce a positive reaction at greater dilutions than those obtained with distilled water. If we remember that when the solvent is urine, the test stays positive until dilutions of 1 to 100,000 (by volume) it be-

TABLE V.

Tube IV. Time. Tube I. Tube II. Tube III.

___- . . Immediately after the test was per-

formed............................ + + + thr.later........................... + + ++ 3

“ “ . . . . . . . . . . . . . . . . . . . . . . . . . . . Trace. Trace. +++ 1 “ “ . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0 +++

-

+++ ++++ ++++ ++++

TABLE VI.

Dilution. Solvent is water

The solvent is.a 10

(see page 348). per ce*n~at.d~tlo”

1 per cent (by volume). . . . . . . . . . + +++ 0.1 “ “ CL6 “ ) . . . . . . * . . . . . . . . Trace. + 0.01 “ “ (“ “ ) . . . . . . . . . . . . . . 0 0 0.001 “ “ (“ “ ) . . . . . . . . . . . . . 0 0

TABLE VII.

Influence of Various Salts on the Sensitiveness of the Nitroprusside Test.

Test-tuba.. . . . . . . . Control. I II III IV V

---

Salt.. . . . . None. N&l (NHah301 Mek-304 CHaCOOK K oxalate. ~-

qitroprusside test.. + +++ +++ +++ +++ +++

comes clear that other substances than NaCl are present in urine, which increase the sensitiveness of the test or even produce the test.

As it was probable that the peculiar behavior of NaCl in regard to the nitroprusside test might be similar to the influence of (NH&SO+ which had been noticed long ago by Rothera, we felt

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360 Qualitative Tests for Acetone Bodies

it would be of interest to test various neutral salts in the same way as sodium chloride and ammonium sulfate had been tested. Six test-tubes, each of 20 cc. capacity, containing, respectively, 4 to 5 gm. of various dry salts, were filled with, a like amount (about 15 cc.) of the same specimen of diabetic urine. The nitroprusside test was performed on each specimen after time had been allowed for the various salts to dissolve. The intensity of the tests is given on Table VII.

It can easily be shown that the influence of the salt is propor- tional to its concentration, with a maximum effect at saturation.

The Quantitative SigniJicance of the Qualitative Tests.

Sodium Nitroprusside Test.-Where the technique given on page 348 is followed, no electrolytes being used to intensify the color reaction, the following approximations have been made from the data of qualitative and quantitative findings on 260 specimens of diabetic urine. When the concentration of total acetone bodies expressed as acetone is less than 25 mg. per liter, the test is negative; from 25 to 50 mg. the test may be positive or negative. When it is positive, it is either “Trace” or “+.” It is often negative for 50 mg. per liter and positive for 25 mg. per liter. The test is irregularly “Trace,” “+,” or “++” for concen- trations, varying from 50 mg. to 0.5 gm. per liter.

For concentrations of 0.5 to several grams per liter, absolutely no rule can be ascertained. The test may be “+,” “++,” “+++, ” “++++,” with no definite relation at all to con- centration of total acetone bodies. This is evidently due to the fact that the proportion of diacetic acid plus acetone to the total acetone bodies is very variable, and also that many conditions, especially salt concentrations, influence the sensitiveness of the teat.

Ferric Chloride Test.-Using the technique given on page 348 on the same specimens of diabetic urine, for concentrations of less than 0.10 gm. of total acetone bodies per liter, the test is always negative. It is irregularly positive (slightly) or negative for concentrations varying from 0.10 to 0.40 gm. per liter of total acetone bodies. It is constantly, but slightly, positive (brown- mahogany) from 0.4 to 1.0 gm. of total acetone bodies per liter. It is detiitely positive from 1 to 2 gm. per liter, and for still higher concentrations, it becomes almost black.

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E. J. Big-wood and W. S. Ladd 361

CONCLUSIONS.

1. A solution of pure acetone gives a color reaction with sodium nitroprusside.

2. A solution of diacetic acid, as free as possible from acetone, also gives a color reaction with sodium nitroprusside.

3. Such a solution of diacetic acid gives a color reaction with ferric chloride; acetone alone does not.

4. Electrolytes present in urine, especially NaCl, tend to in- tensify the color of the ring in the sodium nitroprusside test.

5. Quantitatively, because of the many interfering substances, the tests as routinely done serve as only crudest approximations in indicating the amounts of acetone and diacetic acid present. The ferric chloride test appears to give results somewhat less eccentric than the nitroprusside test.

BIBLIOGRAPHY.

1. Harding, V. J., and Ruttan, R. F., Biochem. J., 1912, vi, 445. 2. Hunter, A., Quart. J. Exp. Physiol., 1914-15, viii, 13. 3. le Nobel, C., Arch. exp. Path. u. Pharmakol., 1884, xviii, 6. 4. Rothera, A. C. H., J. Physiol., 1908, xxxvii, 491. 5. Van Slyke, D. D., J. Biol. Chem., 1917, xxxii, 455. 6. Folin, O., J. Biot. Chem., 1907, iii, 177.

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E. J. Bigwood and W. S. LaddSIGNIFICANCE AND VALUEACETONE BODIES; THEIR

THE QUALITATIVE TESTS FOR

1923, 58:347-361.J. Biol. Chem. 

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