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THE EXCRETION OF CREATIN IN DIABETES. By R. A. KRAUSE, Crichton Research Scholar. (From the Chemical Laboratory of the Physiology Department, Edinburgh University.') (Received for publication 3rd August 1910.) SINCE the publication in 19062 of Folin's method for the estimation of creatin and creatinin, a large number of cases suffering from various diseases have been examined by different workers3 with the object of correlating the excretion of creatin in the urine with definite pathological conditions. These investigations have, however, not yielded any very definite results, since creatin was found to occur irregularly in a great many different pathological conditions. Only two conditions, viz., progressive muscular atrophy and cancer of the liver, have been found to be constantly associated with the appearance of creatin in the urine. The atrophy of muscular tissue offers an obvious explanation of the creatinuria. The excretion of creatin in cases of cancer of the liver is not readily explained, and requires further elucidation. My experience agrees with that of previous observers. In five cases of progressive muscular atrophy creatin was found to be present in every case. I have found further that diabetes mellitus is always associated with the appearance of creatin in the urine. So far as I am aware, no systematic investigations on this point have as yet been made. My conclusions are based on nine cases; these varied in severity, but none of them was of the simple type (alimentary glycosuria). Two cases terminated fatally, while several improved. No case, however, showed marked acidosis during these investigations. In order to determine the endogenous creatin excreted, the patients were put for a period on a creatin-free diet: this usually consisted of porridge and milk, toast and butter, milk puddings, etc. The observations on the first eight cases are given in tabular form in Table I. The observations for each case were made on consecutive days. 1 The observations published in this paper formed part of an essay which was submitted to the Medical Faculty of the University of Edinburgh, and was awarded the Ellis Prize in Physiology. 2 Folin, Zeitschr. f. physiol. Chemie, Bd. xli., 1904, 223. 3 Hoogenhuyze and Verploegh, Zeitschr. f. physiol. Chem., Bd. lvii., 1908, 161. Benedict and Myers, Amer. Journ. of Physiol., vol. xviii., 1907, 377. Shaffer, Amer. Journ. of Physiol., vol. xxiii., 1908-9, 1. Mellanby, Journ. of Physiol., vol. xxxvi., 1907-8, 447. Leffmann, Zeitschr. f. physiol. Chemie, vol. lvii., 1908, 476. Levene and Kristeller, Amer. Journ. of Physiol., vol. xxiv., 1909, 45.

290 Krause The creatinin coefficient is calculated according to ShafferI as the amount of creatinin-nitrogen expressed in milligrams per kilo of body weight. TABLE I. Urine. Creatin (as creatinin). Diet. Creatinin coeff. Creatin + creatinin coeff. Case 1. Woman, 41 years, weight 54-6 kgs. 3182 c.c. 3921 3694 3654 *795 gm. 1-117.443-706 *238 gm. *337-109 *142 5,2 7-6 2,9 3.4 6-8 9,8 3-6 4.3 Case 2. Woman, 38 years, weight 58-3 kgs. Case 3. Woman, weight 68-1 kgs. Case 4. (C.) Man, weight 53,9 kgs. Case 5. (M. G.) Woman, aged 20, weight 50 8 kgs. Case 6. (S.) Man, aged 45, weight 6 kgs. Case 7. (J. T.) Middle-aged man, weight 53,2 kgs. Case 8. (M.) Man, aged 22, weight 51-8 kgs. 2273 2273 2387 4404 1-792 1-266 -811-920 5115 1-012 6194 2-156 4830 *454 2855 1-048 2955 1-573 2728 1-096 2955, *564 2614-732 3012 *844 3182 *792 7 weeks later 4546-663 4092 630 3410 1-079 2671 940 3410-723 2784 1-256 3636-483 4204 *744 3124 *474 4887-723 4460-551 4602 1-196 1-115.454-561.745-644 *483 *391-604 -642 *842 IP06 *397 *107 *423 *181.433 *218-197 *229 207-306 -487-481 -504 *436.739 Meat Fish Half neat Half meat Full meat Fast Meat 7.9 5-0 4.9 14-6 310 40 5,2 6-0 6-1 5.3 7,1 3.3 5-1 3,2 50 3-8 10-7 8-5 8.8 17-8 5,6 11-6 7.4 6-7 7.3 6-4 5.4 8-2 53 8-4 6-5 8-5 6,8 As will be seen from the foregoing details, the period during which a creatin-free diet was given was not lengthy, and it might be urged that creatin had been retained in the body from the previous meat diet, and was only slowly being excreted. This view is negatived by the following case, in which the patient was kept on a creatin-free diet for five weeks, and at no time was creatin absent in the urine. 1 op. cit.

The Excretion of Creatin in Diabetes 291 TABLE I I. Date Urine. CreatiCin. (reatin Creatinin Creatin + Case. 1910. UieCrann. (as Notes. coeff. coeff.ni creatinin). cef Case 9. Feb. 5 2387 c.c. 977 gm. *294 gm. Total N. 14-97 gm. 4-0 5'1 (C.) Ammonia N. 0-1 Age 45, 6 2671 1-374 667 5,6 8,2 weight 7 2273 1-416 585 Sugar 137 53 7-6 89 kgs. 8 2216 1136 *311 144 4,6 5.8 9 2230 1P374, 375 5-6 7-1 10 2729 1674, 390 102., 6-8 8-3 11 2955 1:300 360 68 5-2 6-5 Mar. 2 2102 *944, 273 3,8 4-9 3 2216 1.(90 S. 14,9 4-2, 5 1647 1,-1e43 *267 Total N. 18-44 4-6 56 6 1300 703 095,, 7478328 Ur 6N. e Urea N. 6'743, Sugar nil. Ammonia N. 409 7 908 8 *681 *118 Only trace of sugar 2.7 3-7 8 1192 1X149 083 4X6 4X9 9 994 936, 058 3-8 3-8 * Fast from 8 p.m. of 4th March to 8 a.m. of 6th March. N.B.-A little acetone was present in the first specimen, but none beyond a trace in the other specimens. A record of the same case at a later date with different diets is given in Table III., which contains also the relative amounts of creatinin-nitrogen and creatin-nitrogen expressed as percentages of the total nitrogen. The ammonia-nitrogen was determined on several days, and was found to be within normal limits. Table III. is of special interest. It indicates a diminution or practical disappearance of sugar after a fast of thirty-six hours (April 18th). On a porridge and buttermilk diet a similar result was obtained (April 26th). There is no absolute parallelism between the excretion of sugar and that of creatin. But with the reduction in the sugar excretion which was brought about by the fast on 18th April there is also a reduction in the creatin excretion, and the latter remained on a lower level afterwards even during the days (April 23rd to 25th) when the sugar excretion showed a transitory rise to almost its former level. The statement by some older observers 1 who used the Neubaur method for the estimation of creatin, that the excretion of creatinin is increased in diabetes, need hardly be considered. These results are obviously due to the large amount of meat given in the diabetic diet, which was not taken into account. Senator observed in a few cases a decrease of urinary 1 R. L. Maly, Wiener med. Wochenschr., 1862, No. 20. Senator, Ziemssen's spez. Path. u. Ther., vol. xiii. 157,1875.

292 Krause TABLE IIT. Creatin. Creatin Date. Urine. Total Per- Per- Sugar. Diet. Creatinin + N. Abso- centage Abso- centqage coeff. creatinin lute of cre- lute of coeff. amount. atinin- amount. creatinnitrogen. nitrogen. Apr. 5 10 11 12 13 14 15 16 17 18 19 20 21 22 738 111-881 23 24 25 26 27 28 29 30 May 1 2 3 4 c-c. 2273 2427 2273 2386 2386 2841 2841 2784 2557 2727 2614 509 624 1533 1932 1932 2046 2841 2273 1363 1705 1590 1705 2484 1932 gms.... 19-03 22-119 23-864 24-091 21-047 18-819 21-761 35-498 9-364 12-966 14-548 13-601 13-57'7 12-244 10-910 10,151 13-505 20-8 21-994 30-379 21-406 gms. 1-736 -895... 1-123 1-098 1-307 1-343, 1-149 1-020 1-162 2-640 -808-835 -790-970 -909-948 -845-913 1-036 -862-987 1-341 1-381 1-584 1-167 per cent. 2X09 1-8 1-99 2-0 1X9 1-97 - 1-94 2-7 3-1' 2-3 2-4 2-4 2-4 2-2 2-6 3.4 3-06 2-6 2-2 2-2 1-9 1-9 gim. -482-432 *517 *458 *755-827 -777 *573-647 *775-351 -201-174' -183-228 -192-190 -237 *114-292 -256-331 -345-556 -429 Patient's weight on 7th April - 29th )I 3rd May 4th per cent. *.. *... -86-66 1-1 1-09 1-3 1-1 1P08 *79 1P36-56.53.45-61 *51-38 1X04-6 *57.57-66 -72 -~~~~~ gms. 98-4 145 138 133 139 126 121 132 158 28' 2'1 11 31-3 105 120 93 59 21 13 4.5 12 22-1 5-1 43.4 43 Boiled,extracted meat Boiled, extracted meat Broth Fast (36 hours) Cream, ij pints Cream, butter, cheese, buttermilk Cream, butter, cheese, buttermilk and toast Porridge and buttermilk diet Porridge and buttermilk diet Fast (30 hours). Milk Meat Milk and porridge Milk and porridge Milk and porridge 88-8 kgs. 87-4. 89-8. 91-1 it 7-2 3-6 4-6 4-5 5.3 5.5 4-7 4-7 10-8 3.3 3-4 3 1 3-9 3.7 3-8 3-4 3.9 3-5 5-7 5-7 6-4 4-6 8-9 6-4 6-1 8'4 8-4 7-9 6-4 7-3 15-1 4-7 4-2 3-8 4-6 4-6 4.5 4-8 4.5 3-6 6-9 7-1 7-1 8-6 6-2

The Excretion of Creatin in Diabetes 293 creatinin, although a meat diet was given. This he ascribed to a fallacy in the method of estimating creatinin (Neubaur method). Several authors 1 state that creatinin is decreased in the urine of diabetics, but none of them could give definite figures, as the sugar present in the urine seems to interfere with the proper deposition of creatinin as the zinc chloride salt. By none of these observers nor by any other has the presence of creatin been determined in diabetic urine. In all of the foregoing cases of diabetes mellitus examined by me there was a diminution of creatinin. Even the creatin + creatinin coefficient is generally below the normal, since the creatinin - coefficient of healthy persons lies, according to Shaffer,2 between 11@7 and 54, the average being 8-1. Shaffer believes "that creatinin - coefficients below 7 are normal only for' elderly, inactive, poorly developed or excessively fat subjects, and, strictly speaking, none of these conditions is normal." It was of interest to investigate whether acetone, diacetic acid, and fl-oxybutyric acid interfere with the estimation of creatinin and creatin, ince many of the conditions in which creatin is excreted may also be associated with the appearance in the urine of acetone and its allies.3 Jaffe, in his original paper4 on the colour reaction on which the estimation of creatin is based, states that only acetone shows a faint reddish-yellow colour after the addition of picric acid and caustic soda solutions. Folin 5 recognised that, besides acetone, aceto-acetic acid and aceto-acetic ether disturb the reaction, but he does not mention in what manner and to what extent these substances affect the estimation of creatinin and creatin. Klercker6 states that the presence of acetone makes it impossible to take any readings, owing to the rapid fading, and advises that the acetone be first driven away by heating. This, of course, would introduce another fallacy, namely, that some of the creatin might be converted into creatinin. Hoogenhuyze and Verploegh7 found that the colour reaction which is obtained with acetone appears more quickly and also disappears more rapidly than the colour reaction obtained with creatinin. They hold that if a sufficient number of successive readings are taken no error will arise in colorimetric work. I have taken a number of readings from urines to which small measured quantities of acetone, aceto-acetic acid and ether, and 8B-oxybutyric acid 1 K. B. Hofmann, Virchow's Archiv, vol. xlviii. 358, 1869. Winogradoff, ibid., vol. xxvii. 533, 1863. Stopczanski and Iahtgens, cit. by Senator in Ziemssen's spez. Path. u. Ther., vol. xiii. 157, 1875. 2 Shaf fer, op. cit. 3 Krause and Cramer, Proc. Physiol. Soc., 9th July 1910. Journ. of Physiol., vol. xl. 4 J affe, Zeitschr. f. physiol. Chem., vol. x., 1886, 400. 6 Folin, op. cit. 6 Klercker, Biochem. Zeitschr., vol. iii., 1907, 51. 7 Hoogenhuyze and Verploegh, Zeitschr. f. physiol. Chem., vol. lvii., 1908, 165.

294 Krause had been added. These urines were treated in exactly the same way as the normal urine, which served as a control. In the case of the urines to which acetone had been added, successive readings were taken, as recommended by Hoogenhuyze and Verploegh. In Tables IV. and V. I have given some of the actual readings obtained in each case with the colorimeter. It may be mentioned here that the conversion of creatin into creatinin was carried out on the water bath. TABLE IV.-EFFECT OF ACETONE AND OXYBUTYRIC ACID ON CREATININ AND CREATIN ESTIMATIONS. Normal urine, 10 c.c. Same urine+ acetone (2 per cent.). Same urine+ oxybutyric I (about 1 per cent.). ~~~~~~~~~~~~acid Creatinin Creatin (after Time in Creatin (after Creatin. Creatin (after heating). minutes. m.heating). heating). 7-8 7-8 2-6,o 7 0 7 85 7-75 7-9 7,8 06-2 70 7,9 7-6 8,0 78 6-9 7-8 7,8 7-8 8,0 4-6.75 (7-3 7-8 7 8 7 85 8,0 685 7.3 7-85 7-85 7.9 779 6-754 f765 7.9 7.7 - ~~7-8 7-5 7-8 -- Av. 7 A87 8-8-l 787 Av. 7-85 Av. 7-74 Av. 7787 { {7.7 79 10-7s 785 (7 85 From the foregoing table it will be seen that oxybutyric acid has no effect on the colour reaction. Acetone, however, causes at first lower readings (i.e. increased values for creatinin), which, owing to the fading of the acetone reaction, gradually rise until they stop at the ordinary creatinin readings. This, of course, will also begin to fade if left long enough (after fifteen minutes). These observations are, therefore, in agreement with those of Hoogenhuyze and Verploegh in showing that the fallacy due to the presence of acetone can be eliminated by taking a sufficient number of successive readings. In any case, any effect which acetone may produce is in the direction of increasing the values of creatinin and diminishing those for creatin. The following table contains the readings obtained with different samples of urines to which small quantities of aceto-acetic acid and ether had been added:

The Excretion of Creatin in Diabetes 295 TABLE V.-EFFECT OF AcETO-ACETIc ACID AND ETHER ON CREATININ AND CREATIN ESTIMATIONS. Normal urine (10 c.c.) Same urine + aceto-acetic ether ( 5 per cent.). Creatin (after heating). Normal urine (10 c.c.) Same urine+ acetoacetic acid (1 per cent.). 8,35 8-2 8-25 8,3 8&25 Av. 8-27 7.5 7.5 7.5 7.55 7.5 Av. 7-51 8-2 8-3 8-4 8-25 8'3 Av. 8-29 10'25 102 10-2 102 10-15 1025 Av. 10 21 10*1 10-2 10'15 101 10-2 10-15 10.1 Av. 1014 Normal urine. Same urine+ aceto-acetic acid (1 per cent.). Creatin (after Creatin (after heating). heating). 7.9 7V85 6,8 7-85 7.95 7.9 6-8 7.75 7-85 7.9 6,75 7-8 7-85 7.9 6-75 7-85 7.95 7-85 6,8 7 8 7-85 Av. 7-88 Av. 7-88 Av. 6-78 Av. 7-81 It will be seen that aceto-acetic acid in the quantities given in the table interferes with the quantitative estimation of creatinin in a more pronounced way than acetone. It diminishes the readings for creatinin (i.e. increases the values for creatinin), but its effect does not pass off as is the case with acetone. This is what one would expect, since the colour obtained by adding caustic soda and picric acid to aceto-acetic ether or acid persists, whilst in the case of acetone it fades rapidly. On the readings for creatin, i.e. after heating the urine with hydrochloric acid for three hours, acetoacetic acid has practically no effect. The results indicate, therefore, that aceto-acetic acid interferes with the estimation of creatinin in such a way as to increase the values for creatinin. It does not interfere with the actual readings for creatin, but owing to the increased values obtained for creatinin it diminishes the values for creatin, which are calculated from the difference between the readings. With urines containing aceto-acetic acid and creatin one would, therefore, obtain values for creatinin which are too high, and values for creatin which are too low.

296 The Excretion of Creatin in Diabetes CONCLUSIONS. 1. Typical diabetes mellitus is associated with the excretion of creatin in the urine. This conclusion is confirmed by the observations of Krause and Cramer' on the appearance of creatin in the urine in phloridzin diabetes. It is in agreement with the observations of Cathcart2 on the creatin excretion of normal persons living on a diet free from carbohydrates. 2. In each of the cases examined the excretion of creatinin was found to be below the normal. 3. Observations are recorded regarding the fallacies which are introduced in the estimation of creatin and creatinin by the presence of small quantities of acetone and its allies. This work was carried out at the suggestion of Dr Cramer, to whom I desire to express my indebtedness for advice and assistance. I also wish to thank Professor Greenfield, Dr Gibson, and Dr Russell for their kindness in giving me facilities for the study of cases in their wards. vol. 1 Krause and Cramer, Proc. Physiol. Soc., 9thJuly xl. 2 Cathcart, Journ. of Physiol., vol. xxxix., 1909-10, 311. 1910. Journ. of Physiol.,

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