STUDIES IN KETONE BODY EXCRETION

I. DAILY VARIATIONS IN THE KETONE BODIES OF NORMAL URINE AND THE KBTONURIA OF SHORT FASTS, WITH

A NOTE ON DIABETIC KETONURIA DURING INSULIN TREATMENT

BY JEANETTE ALLEN BEHRE

(From the Union Central Life Insurance Company, Cincinnati)

(Received for pubhation, June 25, 1931)

By means of a technique, to be described later in this paper, which permits the determination of the ketone bodies in such con- centrations as are found in normal urine, a study has been made of the total excretion of these substances by normal individuals and of the variations which take place in this excretion during various periods of the day. The increase in the amount of ketone bodies excreted by a normal individual in the early stages of fasting has also been investigated and preliminary studies have been made of the ketonuria of diabetics under insulin treatment.

1. Normal Excretion of Ketone Bodies

Definite figures for the amount of the ketone bodies eliminated by normal individuals are not numerous in the literature. Ac- cording to Peters and Van Slyke (l), the total acetone body excre- tion, expressed as ,!%hydroxybutyric acid, is usually less than 0.5 gm. in 24 hours and probably rarely exceeds 1 gm. Shaffer (2) con- sidered any amount over 0.1 gm. of total acetone per day as dis- tinctly abnormal. Hubbard and Noback (3) found a variation of from 0.00 to 3.8 mg. per 100 cc. of urine in the normal excretion of total acetone, the acetone and diacetic acid varying from 0.00 to 1.2 mg. with an average of 0.19 mg., and the @-hydroxybutyric acid (as acetone) from 0.00 to 2.6 mg., averaging 0.46 mg. per 100 cc. of urine. For general purposes urine samples which give nega- tive reactions with nitroprusside have been considered normal with regard to acetone and diacetic acid. As has been pointed out

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elsewhere (4), the degree of acetonuria necessary to give a positive nitroprusside test varies considerably with variations in the method and in the ratio of acetone to diacetic acid present. It is probably equivalent to not less than between 3 and 5 mg. of diacetic acid per 100 cc. of urine.

The present experiments have covered the acetone and diacetic acid excreted by twelve normal individuals during the 24 hours, the results representing thirty-two daily periods. Determinations were also made of the P-hydroxybutyric acid excretion of eight individuals during fourteen daily periods. The results may be summarized as follows:

Ketone Bodies Excreted in 84 Hours (as Acetone)

From acetone and diacetic acid. 1.2 to 4.5 mg., average 2.9 mg. I‘ p-hydroxybutyrio acid.. 12.2 “ 20.5 “ “ 16.2 “ “ total acetone bodies., . . . -14.5 ” 23.5 “ “ 19.4 ‘(

Ketone Bodies per 100 Cc. Urine in 24 Hour Samples (as Acetone)

From acetone and diacetic acid. 0.10 to 0.66 mg., average 0.25 mg. ” &hydroxybutyric acid.. 0.79 ” 2.20 “ ” 1.30 IL “ total acetone bodies.. 0.91 “ 2.70 “ I‘ 1.50 “

A study of large numbers of unselected urine samples (such as come into this laboratory for routine analysis) by means of a sensi- tive qualitative test for acetone and diacetic acid (4) also indicates that the normal concentration of the urinary acetone and diacetic acid is below 0.5 mg. per 100 cc. of urine.

2. Variations in Normal Ketone Body Excretion Throughout the Day

Almost nothing is to be found in the literature regarding the variation in normal ketone body excretion during the 24 hours, although there have been reports on the daily variations under dietary conditions which produced marked ketonuria. The daily curves for ketonuria under the different conditions which have been reported are not identical, but in each case the variations during the day seem to bear a relation to certain specific factors. The highest excretion followed ketogenic meals and periods of exercise (Hubbard and Wright (5), McClellan and Toscani (6)) and the lowest excretion occurred at night (Hubbard and Wright) or in the morning (McQuarrie and Keith (7), McClellan and Toscani). A summation effect from two meals, or a lag in the appearance of

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ketone bodies, was sometimes not,ed (Hubbard and Wright, Mc- Clellan and Toscani). Hubbard and Wright also present one series of figures for an arthritic subject on a normal diet, which shows almost no variation throughout the day, but they believe these figures to be below the limits of accuracy of the method.

We studied the daily variations in urinary output of ketone bodies of ten normal individuals, the observations covering thirty- six daily periods. Each voiding was collected and analyzed sepa- rately. For comparative purposes we have grouped the figures for the separate voidings into four periods for each day, as shown in Table I. As nearly as possible the morning period represents in each case the time between waking and taking lunch, the afternoon period, the time between lunch and dinner, the evening period, from dinner to bed time.

The subjects were all apparently normal individuals, under normal conditi0ns.l The figures for sugar in all of the samples (with the exception of one sample) were normal. Seven of the subjects were women, three men.

Six typical examples of these 24 hour periods are shown in Table I, and the range of variation and average figures for all of the re- sults of each period are also shown.

The variations in ketone body excretion during the day are very slight in most cases. There is, however, a distinct tendency for the amount of ketone bodies excreted per hour to be lower at night than during the day, while the percentage concentration tends to be lower at night than during the afternoon and evening, but lowest of all during the morning.

When the variations in hourly excretion of ketone bodies are compared with those of urinary volume for the same periods, it is found that the two curves almost always vary in the same direc- tion, though not to the same degree. This is somewhat more noticeable in the P-hydroxybutyric acid than in the acetone-diace- tic acid fraction, although in general the two ketone body fractions vary in the same direction. The relationship between hourly

1 One of the subjects was a possible exception, having suffered several years earlier from a nephritic condition from which there had been an apparent recovery. No abnormalities were found in the urine of this individual except that one sample, excreted just after lunch. showed 0.5 per cent sugar,

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excretion of ketone bodies and urinary volume is particularly striking when shorter periods than those shown in Table I are ob- served. Chart 1 illustrates one of the many cases in which a large volume of urine excreted during a short period is paralleled by a great increase in ketone body excretion, unaccounted for by other conditions.

This correlation between hourly excretion of ketone bodies and urinary volume, independent of the time of day, was further shown by the experiment illustrated in Chart 2.

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The subject of this experiment usually drank coffee and water for breakfast, which resulted in a marked diuresis during the morn- ing. Much less liquid was usually taken at night. The first period shown in Chart 2 illustrates one of the characteristic daily curves for this individual, with a low hourly volume of urine and low acetone figures during the night, and an increase in both during the morning. In the second period the usual custom was reversed and the subject drank large amounts of water at 9.30

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p.m. and again at 12.30 a.m., and following this took almost no liquid for breakfast. The result was an increase in urinary volume during the night, accompanied by a greater excretion of ketone bodies than usual during this period. From 5.30 a.m. when the subject rose, to 10.30 a.m., there was a decrease in ketone bodies excreted, parallel with the decrease in u&nary volume. At 10.30 a.m. 300 cc. of water were taken, which resulted in a diuresis and increase in ketone body excretion similar to the condition which usually occurred during the first period of the morning.

It is evident that the factor of urinary volume should be taken into account in any consideration of variations in the amount of ketone bodies excreted. An apparent lag in excretion may be due to the washing out, by large volumes of fluid, of ketone bodiM formed during an earlier period.

That urinary volume is not the only factor determining varia- tions in the normal excretion of these bodies is apparent from the fact that the percentage concentration also varies.

In so far as the actual amount of a substance excreted per unit time varies in a direct relation to urinary volume, the implication is that the substance is simply washed out by the kidneys, and that variations in the actual amount excreted are not in themselves significant of the time or the conditions of formation of the sub- stance. From this point of view variations in percentage figures may have a particular signscance as representing changes in the amount of the substance washed out per unit volume. More ex- tensive studies of all urinary constituents on this basis would be of interest.

In their study of the ketonuria produced in arthritic subjects by ketogenic diets, Hubbard and Wright (5) found the rate of excretion of ketone bodies to be independent of urinary volume, and the figures of McClellan and Toscani indicate that the same is true of the ketonuria of men on meat diets. It is possible that this is indicative of a difference in mechanism involved in normal and in increased excretion of ketone bodies.

To summarize, our figures indicate that in the normal subject the highest excretion of ketone bodies occurs during the afternoon and evening, that during the night there is a decrease both in percent- age and in absolute amount excreted, and that during the morn- ing there is a still great,er decrease in percentage concentration

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although the actual amount excreted per hour is greater than at night, the increase accompanying the increase in urinary volume, and representing, perhaps, a washing out of substances formed dur- ing the night. These variations are, in general, in accord with those which have been noted in the ketonuria produced by ketogenic diets, cited above.

3. Ketonuria in the Early Stages of Fasting

Starvation ketonuria is generally thought of as a rather sudden phenomenon, appearing on the 2nd or 3rd day of fasting,presumably when the carbohydrate reserves of the body have become depleted. Benedict (8) reported 0.5 gm. of ,&hydroxybutyric acid on the 2nd day of fasting. Folin and Denis (9), in their experiments on fasting in obesity, found 120 mg. of acetone and diacetic acid, but no p-hy- droxybutyric acid, in the urine of one obese woman on the 1st day of fast, but their second subject, still more obese, excreted none of the acetone bodies until the 3rd day. Goldblatt (10) points out that the time at which acetonuria begins in fasting depends upon the diet previous to the commencement of the fast. In his experiments the time at which ketosis appeared, as indicated by the nitroprusside reaction, was 20 hours after the beginning of the fast, when the previous diet had been low in carbohydrates, and 36 hours after the last food, when an average mixed diet had preceded. Goldblatt speaks of the appearance of ketosis as an acute phenomenon, the concentration of acetone bodies rising from 3 or 4 mg. per cent (still negative to the reaction with nitroprusside) to 20 or 30 mg. per cent within 15 minutes.

Using the normal daily variations in ketone body excretion as a basis, we have studied the earliest stages of an increase in this excretion due to lack of food in a normal individual. Some of the results are shown in Charts 3 to 5.

The subject of these experiments was a woman, 38 years old, 5 feet, 7 inches in height, weighing 54 kilos, whose normal diet was not much in excess of the maintenance requirement and contained relatively little protein. The subject was in good health and ac- tive, showing no tendency towards glycosuria and giving normal acetone curves.

In Chart 3 a control period is shown with normal diet and nor- mal ketone excretion. On the following day lunch was omitted.

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A slight rise in the acetone and diacetic acid excretion occurred from 5.5 to 7.5 hours after breakfast, and the output of these sub- stances increased to 10 mg. per cent, or 3 mg. per hour, during the period from 9.5 to 11.5 hours after breakfast. After dinner the figures gradually returned to normal. On the following day a rise

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again occurred before lunch (which was not eaten until 2 p.m.) with a return to normal by 4.30 p.m. These changes occurred independent of urinary volume.

Chart 4 shows a similar experiment on the same subject and includes figures for P-hydroxybutyric acid. During the period between 6 and 12 hours after breakfast, on the day when lunch

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688 Ketone Body Excretion. I

was omitted, both the acetone-diacetic acid and the P-hydroxy- butyric acid fractions showed an increase relative to the urinary volume (2 mg. per cent of acetone in each fraction) and the acetone and diacetic acid excreted per hour were also slightly above normal. During the following period, from 6 to 12 hours after breakfast,

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although dinner was eaten in the meantime, both fractions were increased per hour, the increase being paralleled by an increase in urinary volume. On the following day three rather light meals were eaten and the p-hydroxybutyric acid remained within normal limits but the acetone-diacetic acid fraction showed a tendency to rise again in the afternoon and evening.

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These results were practically duplicated in another experiment which is not reported here. In still another experiment no signifi- cant increase in either fraction occurred on the day when lunch was omitted, but on the following day, a normal dinner and breakfast haying been eaten in the meantime, both fractions increased dur-

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CHART 5. 1st day, 7 a.m. : normal breakfast; 12.10 p.m. : lettuce, mayon- naise, 2 cups of black coffee, 1 glass of water; 3.30 p.m.: 90 cc. of wine; 7.30 1 stearo cube in water, 2 p.m.: gluten biscuits, 1 glass of water; 11.00 p.m.: 1 stearo cube in water, 1 glass of water, 2nd day, 7.30 a.m.: rice flakes with cream, 2 pieces of toast, butter, 2 cups of coffee, 2 lumps of sugar; 1.30 a.m.: 100 cc. of water; 3 p.m.: 15 co. of whisky in 1 glass of water; 6.30 : normal dinner. p.m.

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ing the period from 5 to 7 hours after breakfast (the acetone and diacetic acid to 0.9 mg. and the p-hydroxybutyric acid to 1.07 mg. per hour), returning to normal after lunch at 1.30 p.m.

In another experiment this subject ate almost nothing (practi- cally no carbohydrates) for 30 hours, as follows: 1st day: 7 a.m., normal breakfast; 1.15 p.m., 1 cup of pea soup, lettuce, mayon- naise, 1 cup of black coffee; 7 p.m., 2 cups of bouillon, lettuce, mayonnaise, 5 strips of bacon, 1 gluten biscuit; 2nd day: 7 a.m., 1 orange, 1 gluten biscuit, 2 cups of black coffee; 1.30 p.m., 1 slice of bread, Cole-slaw, 2 slices of canned pineapple, coffee with sugar; 7 p.m., normal dinner. On the 1st day the acetone and diacetic acid rose to 0.5 mg. per hour (2 mg. per cent) from 6 to 12 hours after breakfast and to 4 mg. per hour (15 mg. per cent) from 12 to 16 hours after breakfast. During the night the figures dropped to normal but on the following day rose to 2.5 mg. per hour (10 mg. per cent) between 10 a.m. and noon, and to 3.4 mg. per hour (20 mg. per cent) between noon and 1 p.m. During the 2 hours following the light carbohydrate lunch the acetone and diacetic acid fell to 1.75 mg. per hour (3 mg. per cent) and during the next 2 hours to 0.16 mg. per hour (1 mg. per cent). From 4 to 6 hours after the lunch they rose again to 2.8 mg. per hour (12 mg. per cent) and did not return to normal until 4 hours after the normal dinner.

Chart 5 illustrates an experiment on the same individual when practically no food was taken for periods of first 24 and then 12 hours. The experiment also shows the quick, temporary, anti- ketogenic effect from alcohol on this type of ketonuria. The food intake is recorded in the chart legend. The acetone and diacetic acid increased slightly from 7.5 to 8.5 hours after breakfast on the 1st day, and fell to normal during the 2 hours which followed the intake of 90 cc. of wine (about 8 per cent alcohol), independent of urinary volume. This was followed by a gradual increase in ace- tone and diacetic acid, which was only slightly diminished during the night. On the following day there was a still greater increase, which amounted to 10 mg. per hour (40 mg. per cent) at 3 p.m., when 15 cc. of whisky (about 40 per cent alcohol) were taken. During the following 3 hours there was a gradual decrease to 2.4 mg. of acetone per hour. Normal figures were not obtained, how- ever, until after dinner.

A series of observations were also made on another subject, a

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man 5 feet, 8 inches in height, weighing 91 kilos, whose normal diet was very liberal. He was active and in apparently good health. This subject habitually omitted luncbon alternate days, but the other meals on these days were large. The control periods on these days showed no abnormal excretion of ketone bodies. During two periods, however, when, after omitting lunch, this subject ate a large dinner, containing only green vegetables and fats, and again omitted lunch on the following day, there was a slight rise in both ketone body fractions during the evening and night of the 1st day, and an earlier rise on the following day. These figures are not reported in detail because the conditions of the experiments made no clear cut interpretation of the results possible. They seemed to show, however, that even in a well nourished individual a slight increase in ketone body excretion above the normal may occur very shortly after a decrease in food (especially carbohydrate) intake.

Certain points in connection with these experiments call for comment.

1. The first stages of abnormal ketonuria may be observed in some individuals earlier in fasting than was noted by Goldblatt, even as early as from 6 to 8 hours after food ingestion. An in- crease above the normal is noticeable before the ketone body ex- cretion reaches the point at which a positive nitroprusside test is given. Goldblatt (10) evidently considered this point equiva- lent to not less than 3 or 4 mg. of acetone per 100 cc., which is considerably above the average normal figure. We are inclined to believe that although the acetonuria of fasting may increase in very rapid strides it is not a sudden phenomenon but may be traced as a development from the normal level.

2. It is difficult to correlate an increase in ketone body excretion so early in fasting with any extensive depletion of carbohydrate reserves. Other evidence may be adduced to show that abnormal ketonuria may occur before a maximum depletion of these reserves has taken place. The fasting subject of Benedict (8) excreted considerable amounts of /3-hydroxybutyric acid on the 2nd day of fasting although carbohydrate was still being extensively utilized on the 3rd day. The carbohydrate catabolism decreased suddenly on the 4th day, which evidently marked the reduction of the carbohydrate reserves to their fasting minimum, but during the

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692 Ketone Body Excretion. I

first 3 days of the fast there was a slight decrease in carbohydrate catabolism although fat and protein catabolism did not decrease. It seems probable that even during the earliest hours of fasting there is a gradually increasing ketogenic-antiketogenic ratio in the foodstuffs metabolized, which results in an increased production of ketone bodies, at least in some part of the body.

3. The time at which an increase in ketone body excretion becomes noticeable in fasting is evidently correlated to some extent with the nutritional condition of the subject (Goldblatt (10)). The subject of these experiments was probably closer to the border- line of fasting than one whose normal diet was more in excess of a maintenance requirement. In this individual the intake of small amounts of food, low in carbohydrates, delayed the increase in ketone body excretion several hours. A reduction of food on one day led to an earlier increase on the following day. The other subject, whose normal diet was much greater, developed fasting ketonuria more slowly. Variations in basal metabolism and in energy expended during the experiments must also be important factors in determining the speed with which the increase in ketone body excretion becomes noticeable. The tendency toward a decrease in ketone body excretion during the night in these experi- ments seems to be in line with this.

4. It has been stated that the early appearance of fasting keto- nuria is favored by a restriction of fluid intake (Goldblatt). This would seem to indicate a difference in mechanism between normal and fasting ketonuria. No attempt was made to study this point in our experiments and some water was taken during all of them. The acetone and diacetic acid tended to rise steadily dur- ing the day of fasting irrespective of urinary volume. Of the periods studied the one in which the rise appeared earliest was not the one in which the urinary volume per hour was lowest (Chart 3). The highest figures which were obtained, however, occurred when the urinary volume was unusually low (Chart 5), and an unex- pectedly low acetone figure was sometimes found in connecCion with a high urinary volume.

5. In these early stages of fasting the /3-hydroxybutyric acid of this individual tended to increase somewhat more slowly than the acetone and diacetic acid, and variation in its excretion seemed to be somewhat more closely correlated with variations in urinary volume.

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6. The antiketogenic effect of alcohol upon fasting ketonuria, shown in these experiments, is in line with the finding that alcohol can replace fat in its sparing action on protein in normal subjects (Neumann (ll), Rosemann (12), Atwater and Benedict (13)) and that it is selected by the body for oxidation in preference to carbohydrate and fat when it is fed with them (Cushny (14)). It is not in itself ketogenic. Diabetic ketosis is evidently little, if at all, affected by alcohol (Allen and Wishart (15), Joslin (16)). Evidently diabetic and fasting ketonuria are not comparable in this respect.

4. Insulin and Diabetic Ketonuria

In addition to the preceding discussion of normal ketonuria we would like to mention briefly certain points which have been brought out by a study of the urine of two diabetics during insulin treatment.

It has been well established that in practically all cases the excretion of ketone bodies by diabetics is reduced by insulin treat- ment, but the extent to which ketonuria becomes truly normal, and the time relationship between the lowering of sugar and of ketone bodies in the urine, after insulin, are points which might well be further investigated. The fact that has been of particular interest to clinicians is the reduction of ketone bodies in blood and urine to amounts below the level of severe acidosis. Most of the reports in the literature on this subject are based upon the results of qualit#ative tests. Examination of the reports in which quanti- tative methods have been used shows that the lowest figures reached after insulin treatment are still in most cases above the normal (Campbell (17), Fonseca (US), Chaikoff et al. (19), Killian (20), Davies et al. (21), Fitz, cf. Joslin (16) p. 59). There is also some indication that ketone bodies may decrease more slowly and reappear in the urine more rapidly than glucose, after insulin treatment, although this has not always been corroborated.

The two cases which we wish to discuss briefly at the present time were both hospitalized diabetic women who were receiving insulin three times a day before meals and whose urine we analyzed over extended periods.2 In both cases the daily curves for the

2 We are indebted for these samples to Mrs. Horning, of the laboratory of the Bethesda Hospital.

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percentage concentration of urinary acetone and diacetic acid were remarkably similar from day to day, and were in contrast to the normal concentration curves, showing an increase during the night to a maximum in the morning, averaging 8.4 mg. per cent in one case and 11.5 mg. per cent in the other, and a gradual de- crease during the day to their lowest point (0.1 mg. per cent in one case, and 2 mg. per cent, in the other) during the 6 hours which followed the last insulin treatment of the day. There was an ap- parent summation effect from the three insulin treatments. In one of t,he cases, in which the rate of acetone and diacetic acid excretion per hour could also be calculated, a similar tendency was found in the hourly excretion, although other variations occurred, which were evidently correlated with variations in uri- nary volume.

Of particular interest was the fact that in one case the glycosuria was completely controlled by the insulin treatments, the figures for urinary sugar never rising above the normal, but that never- theless the acetone and diacetic acid iigures approached the nor- mal range only during a brief period late in the afternoon or even- ing, and at all other times were distinctly above the normal. In the other case the urinary sugar was not controlled by the insulin, showing only slight variations, between 0.7 and 1.2 per cent, which had no apparent relation to the insulin treatments or to the variations in urinary acetone and diacetic acid.

Such cases illustrate the variations in ketone body excretion which may occur under continued insulin treatment. We plan to continue this type of study.

5. Methods Used for the Determinations

Ketone bodies were determined as acetone in distillates from urine by a method which involves the reaction of acetone with salicylic aldehyde in strongly alkaline solution (22).

Acetone and diacetic acid were determined by a single distilla- tion from one portion of urine which had been acidified with sul- furic acid. The volume of urine used and the volume of distillate collected were varied according to the amount of acetone present. It was found that the most accurate results are obtained when the original volume in the distilling flask does not exceed the volume

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of distillate to be collected by more than 10 or 15 cc. In order to obtain a sufficient concentration of acetone in the distillate for the determination it was sometimes necessary, however, to distil a volume of only 10 cc. from an original volume of as much as 50 cc. of urine, no water being added to the contents of the distilling flask. In such cases slight traces of acetone remain undistilled, but the error involved is so small that it seemed justifiable to use this procedure as a means of obtaining normal urinary figures which are very nearly, if not quite, accurate, and which have a relative, if not an absolute, value. The limits of accuracy of the method may be considered approximately do.03 mg. per 100 cc. of urine.

/3-Hydroxybutyric acid was determined in another portion of urine after the removal of interfering substances by treatment with calcium hydroxide and copper sulfate as described in the regular method (22). In order to keep the volume as low as possible, equal volumes of urine and 40 per cent copper sulfate, without additional dilution, were made alkaline with solid calcium hydrox- ide. It was found that the mixing of these substances is made easier if an approximately correct amount of the hydroxide is mixed with the urine first and the copper solution then added with shaking. More calcium hydroxide may then be added if neces- sary, to give the mixture a bright blue color and an alkaline reaction to litmus. 30 cc. of urine, with an equal volume of 40 per cent copper sulfate, require about 5 gm. of calcium hydroxide, 100 cc., about 15 gm. The mixture was allowed to stand, with occasional shaking, for about 40 minutes and was then filtered with a little suction. The use of suction results in the loss of from 20 to 30 per cent of the acetone, so that it was always necessary to deter- mine the acetone and diacetic acid in a separate portion of urine, which required less time than filtering the mixture without suction. A measured volume of the filtrate was then distilled with acid and bichromate as described in the regular method (22), the volume of the distillate being kept as low as possible (to between 40 and 60 CC.). If necessary because of the low concentration this distillate was then acidified and redistilled to a smaller volume (from 25 to 35 cc.).

The determination of acetone in the final distillates was made either as described in the regular method, or by the shorter method

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described more recently, which involves the use of permanent color standards (23) .3

Sugar determinations were made by Benedict’s clinical quanti- tative test (24).

SUMMARY

Figures are presented for the normal excretion of ketone bodies and for the variation in this excretion during the day. The highest excretion occurred during the afternoon and evening. To a cer- tain extent the excretion of ketone bodies per hour was correlated with urinary volume per hour.

The appearance of increased excretion of ketone bodies in the early stages of fasting is discussed and experiments are described which show that a slight increase above the normal may be noticed in an apparently normal individual as early as from 6 to 12 hours after the last meal.

The variations in urinary acetone and diacetic acid of two hos- pitalized diabetics receiving insulin are briefly described. The con- centration of these substances showed characteristic daily curves with figures above the normal even when the urinary sugar was maintained within normal limits.

We wish to express our sincere appreciation to Dr. William Muhlberg for his cooperation and interest throughout this work.

3 The constancy of the color given in the acetone reaction by many different samples of Eimer and Amend’s Acid Salicylous, Synthetic, ob- tained throughout a period of several years, made it seem possible to recommend the use of permanent color standards in this method. Since the completion of the work described in this paper, Eimer and Amend have discontinued the manufacture of this product and the imported product which they are now putting out under the same label gives slightly less color with acetone solutions than the former product. The difference is apparently negligible for practical purposes but it renders the advisability of the use of permanent color standards somewhat doubtful. This difh- culty is not encountered when acetone solutions are used for standards as described in the method of Behre and Benedict (22). The new Eimer and Amend product has been found unsatisfactory for use in the qualitative test (4) probably due to the fact that it forms a less soluble mixture with sodium hydroxide than the former product. It can be used in this test with potas- sium hydroxide. The technical salicylic aldehyde of the Eastman Kodak Company has been found entirely satisfactory for use in the qualitative test, and is recommended for this.

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J. A. Behre 697

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Jeanette Allen BehreINSULIN TREATMENT

DIABETIC KETONURIA DURINGSHORT FASTS, WITH A NOTE ON URINE AND THE KETONURIA OF

THE KETONE BODIES OF NORMAL INEXCRETION: I. DAILY VARIATIONS

STUDIES IN KETONE BODY

1931, 92:679-697.J. Biol. Chem. 

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