THE DETERMINATION OF ACETONE BODIES IN BLOOD AND URINE.

Transcription

1 THE DETERMINATION OF ACETONE BODIES IN BLOOD AND URINE. REPLY TO CRITICISMS BY E. C. SMITH. BY DONALD D. VAN SLYKE. (From the Hospital of The Rockefeller Institute jar Medical Research, New York.) (Received for publication, June 8, 1929.) Methods for determination of the acetone bodies, acetone, acetoacetic acid, acid, have been published by the writer (Van Slyke, 1917), based upon Deniges procedure for precipitating acetone as a basic mercuric sulfate compound weighing approximately 20 times as much as the acetone it contains. Hydroxybutyric acid was oxidized to acetone by the chromate oxidation introduced by Shaffer (190309). Oxidation and precipitation were carried out simultaneously, so that the technique for determining the total acetone bodies consisted merely in boiling properly prepared urine or blood filtrate (Van Slyke and Fits, 1917,1919) witha specified mixture of sulfuric acid, mercuric sulfate, and dichromate under a reflux condenser, the resultant precipitate being either weighed or redissolved and determined by titration of its mercury according to Personne s method. Acetone and acetoacetic acid, without the hydroxybutyric acid, could be determined separately by omitting the chromate from the reaction mixture. Glucose and most interfering substances occurring in normal or pathological urine were removed by a preliminary precipitation with cupric sulfate and calcium hydroxide.,4 trace was found to remain of nonacetone substances which precipitate with the mercuric sulfate. For these substances a correction was described; the acetone was distilled off from the clarified urine and the residual solution was boiled with mercuric sulfate without chromate. In a series of twentythree normal urines the total acetone bodies determined without subtracting the correction for these interfering substances was in all cases below 0.05 per cent, calculated as acetone. Corrected for the nonacetone precipitate the total acetone bodies in all cases were below 0.03 per cent. Ketonefree diabetic urines yielded similar values. In a series of ketonecontaining urines the method for hydroxybutyric acid was compared with that of Black (190809), in which the acidified urine is mixed with plaster of Paris, the hydroxybutyric acid is extracted with ether, and estimated polarimetrically by its levorotation. Black s method gave 85 to 100 per cent as much hydroxybutyric 415

2 416 Acetone Bodies Determination acid as the writer s, Considering that, as previously pointed out by Shaffer and Marriott (191314), the polarimetric results are likely to be somewhat low, because of adsorption of hydroxybutyric acid by the charcoal used in preliminary clearing of the urine, because of slight racemization of the acid during extraction, and because of incomplete extraction, it was considered that the agreement was satisfactory. From the comparison with Black s method in ketonecontaining urines and from the nearly negative results with ketonefree urines, it appeared that the new method could be trusted to give complete yields of the acetone bodies present, and to be free from significant error due to interference by nonketone urinary constituents. Since its appearance the method has received fairly extensive use over periods of years in a number of laboratories, and has apparently met these requirements. Smith (1926), however, has recently criticized the procedure, for both blood and urine, on the following grounds. 1. In blood used for perfusion experiments he found that the amount of lactic acid present (of the order of magnitude of 100 per 100 cc.) could increase significantly the yield of acetonemercuricsulfate precipitate obtained in determination of the total acetone bodies. Smith ignores the fact that the behavior of lactic acid was pointed out with quantitative data, on p. 486 of the writer s original paper (1917). It was there shown that lactic acid when boiled with chromate under the conditions of the analysis yields some product which precipitates with mercuric sulfate. The amount of precipitate formed per of lactic acid was found, however, to be only about onetwentieth of that formed per of acetone. Blood freshly drawn from the circulation has not enough lactic acid to cause a significant error, except in comparatively rare specimens, such as are drawn immediately after severe exercise or asphyxia. As shown by Evans (1922), however, if drawn blood is allowed to incubate for some hours its glucose is in large part converted into lactic acid. This conversion appears to be especially rapid with dog blood, compared with human and horse bloods. The effect of the amount of lactic acid in the blood used in Smith s perfusion experiments could have been foretold from data in the writer s original paper. In ordinary blood analyses the effect is insignificant, as shdwn by the fact that only 1 or 2 of precipitate are obtained from the filtrate of 5 cc. of normal blood (Van Slyke and Fitz, 1917). To yield an amount of precipitate equal to that from acetone bodies

3 D. D. Van Slyke 417 in a concentration of 1 millimol per liter of blood, about 110 of lactic acid per 100 cc. of blood would be required. Normal, diabetic, and most pathological blood drawn during rest or ordinary activity contains only 10 to 40 per 100 cc. (Clausen, 1922; Ronzoni and WallenLawrence, 1927). After severe exercise it may rise to over 100, likewise in cardiac decompensation sufficient to result in severe anoxemia (Meakins and Long, 1927). In these conditions the lactic acid may cause 5 to 10 of precipitate in the total acetone bodies determination in blood, enough to simulate a slight ketosis, sufficient to be detectable, but not sufficient to be important in the acidbase balance of the blood. 2. Smith states that with diabetic urine the writer s method for total acetone bodies (acetone plus acetoacetic acid plus Phydroxybutyric acid) yields results which are markedly too high. The basis for this conclusion was that when the precipitate was redissolved in 10 per cent hydrochloric acid, and the acetone was distilled therefrom and titrated with iodine, only 72 per cent as much acetone was indicated by the titration as was expected on the assumption that the precipitate consisted of 5 per cent by weight of acetone. When Smith applied the same procedure to the mercury precipitate from a pure acetone solution, the titration indicated the amount of acetone estimated from the weight of the precipitate. In reporting the low yield of acetone obtained by distillation from the urine precipitate Smith gives no details: it is impossible to tell whether he gives the result of a single observation or the mean of a number, whether the urine analyzed contained much or little acetone, and whether correction was made, as directed in the writer s original paper, for the slight weight of precipitate formed by the mercuric sulfate with nonacetone urinary constituents. The precipitate from such substances is only 1 or 2 per cent of the total precipitate obtained in analyses of urines from subjects with severe or moderate ketosis, but is more important if the ketosis is slight, and in normal urine may exceed the precipitate yielded by the acetone bodies themselves. These facts are illustrated by the series of analyses of diabetic and normal urines published in the original paper (Van Slyke, 1917). Smith limited his control analyses to pure acetone solutions. He reports none on pure /%hydroxybutyric acid, although in diabetic

4 418 Acetone Bodies Determination ketosis that substance constitutes about fourfifths of the total acetone bodies. We have repeated Smith s distillation procedure with mercury precipitates obtained in determinations of acetone plus acetoacetic acid and of total acetone bodies in a number of diabetic urines, and have controlled t,he results by performing similar determinations with solutions containing known amounts of both acetone and /3hydroxybutyric acid. Proper correction was made, as described in our original paper, for the mercury precipitate yielded by nonvolatile substances in the urine filtrates. The results fail to confirm Smith s criticisms; in fact the data constitute additional proof that the writer s method is extremely specific for acetone, acetoacetic acid, acid among the constituents of human urine. When pure acetone solutions were precipitated and the acetone from the redissolved precipitate was distilled and titrated, the yield per gram of precipitate depended somewhat on the amount of precipitate. When the latter was in the neighborhood of 500, (representing 25 of acetone) distillation yielded 97 to 98 per cent of the expected 5 per cent of the precipitate s weight of acetone. When the precipitate was a third as great the yield of titrated acetone from it was only 90 per cent of the expected (see Table I). Precipitates from the acetone plus acetoacetic acid of diabetic urines, when the prescribed correction was deducted for the small portion due to nonacetone substances, yielded, within 1 or 2 per cent, the same proportion of distilled acetone as precipitates of similar weight formed from solutions of pure acetone (compare Tables I and III). Precipitates from pure hydroxybutyric acid yielded, as shown by Table II, about 10 per cent less distilled acetone than precipitates of the same weight from acetone solutions. It appears accordingly that when /%hydroxybutyric acid is boiled with sulfuric acid, chromate, and mercuric sulfate, under the conditions prescribed for determining Bhydroxybutyric acid or total acetone bodies, about 10 per cent of the precipitate formed is from an oxidation product or products other than acetone. As seen by comparison of Tables II and III, precipitates from diabetic urines obtained in determination of the total acetone bodies yielded the

5 D. D. Van Slyke 419 same proportion of distilled acetone as precipitates of the same weight from pure hydroxybutyric acid. This fact adds to the evidence, quoted above from the writer s original paper (1917), that no significant part of the precipitate obtained in the determination of hydroxybutyric acid or total acetone bodies in ketonecontaining urines originates from substances other than the acetone bodies sought. The factors, by which hydroxybutyric acid or total acetone bodies are calculated from the weight of the precipitate obtained by the writer s method, were determined empirically by analyses of known solutions of pure acetone and /3hydroxybutyric acid (Van Slyke, 1917). These factors express the directly determined relation between the weight of precipitate and the amount of acetone or hydroxybutyric acid in the fluid analyzed, and involve no assumption concerning the amount of acetone recoverable from the precipitate. Hence the results in the present paper do not necessitate changing any of the factors used in calculating blood or urine acetone bodies content from the amount of precipitate obtained. EXPERIMENTAL. The acetone used in the experiments recorded in Table I was prepared by redistilling Kahlbaum s preparation made from the sulfite compound. Stock solutions containing approximately 1 per cc. were made by pipetting portions of the acetone into weighed measuring flasks partly filled with water, the acetone being measured by the increase in weight of the flask. These stock solutions when titrated for acetone with 0.1 N iodine solution and thiosulfate gave theoretical values. The calciumzinc salt of hydroxybutyric acid used for the experiments recorded in Table II was from the same lot of which the preparation (by the method of Shaffer and Marriott), analysis, and rotation are described in the original paper (Van Slyke, 1917). The precipitates were all formed under conditions with regard to volume of solution, reagents, time of boiling, etc., which conform to those prescribed in the original paper (Van Slyke, 1917), for determination of acetone plus acetoacetic acid or of total acetone bodies.

6 420 Acetone Bodies Determination The following procedure was used for distilling, and titrating by Messinger s method, the acetone from the mercury precipitates. Each precipitate was collected, dried, and weighed in a Gooch crucible. It was then moistened with water, and the precipitate, together with the asbestos in the crucible, was transferred with the aid of a rod and wash water to a 500 cc. Kjeldahl flask. The volume of water in the flask was brought up to about 300 cc., and Acetone present. Weight of precipitate obtained. TABLE I. Results with Solutions of Acetone. 0.1 N iodine to titrate rtcetone distilled from precipitate. Cd (b) Cc) ma cc bxtone C&l lated from might of prf :1p1tate = 2( (b ketone calculated from titration of distillate = (c). (4 (e) Ratio of distilled acetone 0 acetone calculated from precipitate = (4 Gi ; cc. of concentrated hydrochloric acid were added, dissolving the precipitate. The flask was immediately closed with a stopper bearing a Kjeldahl trap which was connected with a glass condenser. The outlet tube at the bottom of the condenser dipped below the surface of 150 cc. of water in a 500 cc. flask, which was cooled in an ice bath. The distillation was continued for 25 to 30 minutes. Longer distillation did not increase the yield.

7 D. D. Van Slyke 421 To the distillate.lo cc. of 40 per cent KOH were added, and an excess of 0.1 N iodine solution. The flask was covered with a watchglass and permitted to stand at room temperature for 20 minutes. Then sufficient concentrated hydrochloric acid (5.5 cc.) was added to neutralize the KOH and provide an excess of 0.2 cc. of acid. The solution was gently shaken to mix the reagents and the excess iodine was titrated with thiosulfate until the brown color had nearly disappeared. Then a few cc. of 5 per cent starch Hydroxybutyric present. Calciumsin0 a& added TABLE Results with Solutions of /3Hydroxybutric Acid. acid I P L&one cd ( Ll N iodine c ulated fron 1A L&one cd Weight of to titrate weight of c ulated fror 1 precipitate. a&one dis precipitate 1 titration 01 tilled from precipitate. (b) iistills.te = (c). =20 w (6) Cc) nag. cc II. Cd) mv. ( : pa Ratio of distilled B&one to eetone calulated from 1 precipitate =@ ( solution were added and the titration was continued until the blue color disappeared. Blank determinations were performed in which asbestos mats from control Gooch crucibles were washed into Kjeldahl flasks, acidified, distilled, and the distillates were titrated in the same manner. The iodine used in the blank, about 0.4 cc. of 0.1 N solution, was subtracted from that used in the titration of the acetone distillates.

8 422 Acetone Bodies Determination TABLE III. Results with KetoneContaining Urines. Determination. Acetone + acetoacetic acid. Total bodies. acetone d : 2.a 0 d (4 m m!?. cc. nw C : C t E OO.C O.OC Acetone + aceto f Of acetic acid Total acetone f OE bodies C E Acetone + aceto i54.a acetic acid Total bodies. acetone Acetone + aceto acetic acid a ; C C Total acetone bodies Acetone + aceto 51.8 acetic acid rota1 bodies. acetone , Qcetone + aceto 18.2 acetic acid. IQ.0 rota1 acetone!2.4 bodies L

9 D. D. Van Slyke 423 Stock solutions of acetone distilled and titrated in this manner gave theoretical results. SUMMARY. The basic mercuric salt precipitates, obtained by applying to,diabetic urines the writer s methods for determination of acetone plus acetoacetic acid and of total acetone bodies (acetone plus acetoacetic acid plus Phydroxybutyric acid) yield when dissolved and distilled, the same proportions of acetone as precipitates of equal weight obtained from solutions of pure acetone and Phydroxybutyric acid respectively. This fact affords added evidence that no significant amount of the precipitate obtained from diabetic urines originates from substances other than the acetone bodies. Smith s cont rary conclusion is attributable to his failure to carry out control analyses with pure hydroxybutyric acid. The analyses reported above were performed by Mr. John Plaein. BIBLIOGRAPHY. Black, 0. F., J. Biol. Chem., 6,207 (190809). Clausen, S. W., J. Biol. Chem., 62, 263 (1922). Evans, C. L., J. Physiol., 66, 146 (1922). Meakins, J., and Long, C. N. H., J. Clin. Inz~., 9,273 (1927). Ronzoni, E., and WallenLawrence, Z., J. BioLChem., 74,363 (1927). Shaffer, P. A., J. Biol. Chem., 6,211 (190809). Shaffer, P. A., and Marriott, W. McK., J. Biol. Chem., 16, 265 (191314). Smith, E. C., Biochem. J., 2Q, 1024 (1926). Van Slyke, D. D., J. Biol. Chem., 32, 455 (1917). Van Slyke, D. D., and Fitz, F., J. Biol. Chew&., 32,495 (1917); 39,23 (1919).

10 THE DETERMINATION OF ACETONE BODIES IN BLOOD AND URINE: REPLY TO CRITICISMS BY E. C. SMITH Donald D. Van Slyke J. Biol. Chem. 1929, 83: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#reflist1