IQZZ B O 00 s BIOLOGY LIBRARY On the Question of the Occur- rence of Creatinine and Creatine in Blood By JEANETTE ALLEN BEHRE and STANLEY R. BENEDICT Reprinted from an article based upon a thesis presented to the faculty of the Graduate School of Cornell University for the degree of Doctor of Philosophy by Jeanette Allen Behre. . The typewritten copy of the thesis upon which this article is based is on file in the Library of Cornell University. On the Question of the Occurrence of Creatinine and Creatine in Blood A THESIS PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF CORNELL UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY fcY JEANETTE ALLEN BEHRE i! Reprinted from JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. LIT, No. 1, MAY, 1922. BIOLOGY LIBRARY G Reprinted from THE JOURNAL OF BIOLOGICAL. CHEMISTRY Vol. LII, No. 1, May, 1922 STUDIES IN CREATINE AND CREATININE METABOLISM. IV. ON THE QUESTION OF THE OCCURRENCE OF CREATININE AND CREATINE IN BLOOD. BY JEANETTE ALLEN BEHRE AND STANLEY R. BENEDICT. (From the W. A. Clark Special Research Fund and the Department of Chemistry, Cornell University Medical College, New York City.) (Received for publication, February 14,1922.) The very rapid advances in analytical technique applied to biological tissues and fluids made during the past 10 years have resulted in a rather anomolous situation. We are constantly determining substances whose existence in the tissues or fluids analyzed has never been proved, and on the basis of a single, non- specific color reaction reports are made of the quantity of a sub- stance in a given tissue or fluid, although none of this substance has ever been separated as such from the material analyzed. The modern color reactions are very attractive playthings, but the facility with which they can be employed should not lead to neg- lect of the more fundamental work of seeking definitely to prove exactly what these color reactions may signify. It is not intended, in this connection, to decry the use of modern analytical technique for blood and tissues such studies should be pursued as far as they seem to promise results of value from any angle, but we should keep in mind the necessity of continued in- vestigation of the tissues and fluids of the organism from the qualitative standpoint. Over a year ago one of us encountered very anomolous results during an investigation of the question of the probable interference of creatinine when using the picrate method for determination of blood sugar. These results, some of which are cited below, were so at variance with known reactions of creatinine that they ap- peared to demonstrate that in some bloods at any rate, creatinine could not be present in anything like the quantity indicated by the colorimetric method of Folin for determining this substance. We were therefore led to take up a more detailed study of this question, and to include a study of the creatine content of blood. 11 507831 12 Creatinine and Creatine in Blood The question of the existence of creatinine in blood has already been raised by others. Hunter and Campbell (1) in 1917 reported a careful study of the question, based chiefly upon comparing the rate of color development of creatinine in alkaline picrate solution with color development in blood filtrates under corresponding conditions. Curves were constructed which showed the velocity of color development in this reaction, the amount of color being plotted against the time. With pure creatinine solutions it was found that the curve was typical and that solutions of different concentrations show proportionate color production. Various blood filtrates were compared with creatinine solutions, with the result that Hunter and Campbell concluded that Folin's picric acid method can be used to determine the creatinine in the plasma with accuracy, but that there is an additional substance in whole blood (present in the corpuscles) which increases the color of the reaction due to creatinine by about 50 per cent, while autoclaved filtrates from blood and plasma contain a still greater amount of a substance, not creatinine, which contributes 50 to 75 per cent of the color usually attributed to creatinine. These authors did not question the presence of a certain amount of creatinine in the blood but they suggested that a considerable part of the color was probably not due to creatinine. Green wald and McGuire (2) have* also studied certain factors bearing on the question of the true creatinine and creatine content of the blood. These authors did not draw definite conclusions as to the probable occurrence of these substances in blood, but a reading of their paper convinces one that they held serious doubts as to the true nature of the " creatinine" in blood. Their attempts to isolate creatinine from blood failed in every instance, and their final opinion is quite suggestive of suspicion of the whole subject. Thus they state: "For an investigation of normal creatine and creatinine metabolism, the methods are probably not satisfactory. Until it can be shown that the chromogenic substance is really creatinine, investigations in this field would seem to be of doubtful value." The available evidence that creatinine exists in blood may be briefly summarized as follows: (1) Creatinine occurs in the urine, hence it is probably present in the blood and (2) the rate of color J. A. Behre and S. R. Benedict 13 development in alkaline picrate solutions due to the blood com- ponent, closely approximates that found for pure creatinine solu- tions. Obviously neither of these arguments for the presence of t creatinine in blood, in the amounts indicated by present quantita- tive methods is at all conclusive. Concerning the first argument it may be pointed out that creatinine may be produced by the kidney, or the kidney may be able to concentrate this substance from dilutions in the blood very much greater than is commonly assumed. The point established by Hunter and Campbell also fails to demonstrate the existence of creatinine in blood. An unknown blood constituent might duplicate the rate of color pro- duction, and the possible presence of catalysts affecting the reac- tion in blood nitrates, as suggested by Green wald (2), must be taken into consideration. We are therefore of the opinion that * no results so far available offer definite evidence of the existence of creatinine in blood. Our method of studying the question has developed along several lines. We have applied certain reactions to both blood filtrates and pure creatinine solutions, and to creatinine added to blood, and compared the relative behavior of true creatinine and of the blood creatinine, both before and after conversion of pos- sible creatine into creatinine. We have also made studies based upon the use of adsorptive reagents which will remove true crea- tinine from pure solution or from blood filtrates. In most of the creatinine determinations we have employed the original picric acid method of Folin (3), in which the blood is ' diluted to five times its volume with saturated picric acid, and the total solution saturated with picric acid. For carrying out this saturation with dry picric acid we have placed the mixtures in & f shaking machine for from 5 to 10 minutes. Our studies in connection with this method have shown that not more than 4 or 5 mg. of creatinine added to 100 cc. of blood can be recovered quantitatively by this technique. Doubling the volume of dilution permits the satisfactory recovery of the larger quantities of creatinine. We have also employed precipitation by means of ** heat coagulation, trichloroacetic acid, and tungstic acid (Folin and Wu, 4) , followed by saturation of the filtrate (after exact neutrali- zation in the case of the trichloroacetic acid precipitation) with picric acid. All of the picric acid employed has at least fulfilled 14 Creatinine and Creatine in Blood the requirements of purity indicated by Folin and Doisy (5) as necessary for creatinine determination in blood. Results by the various methods of precipitation indicated that for normal bloods the various precipitants yield parallel figures, while for bloods high in color-reacting substance the Folin- Wu precipitation with tungstic acid usually yields much higher figures than does the picric acid precipitation. Heat coagulation filtrates show still higher figures (Table VI) . As mentioned earlier in this paper, our attention was first directed to the question of the occurrence of creatinine in blood by the results obtained by one of us in a study of the possible interference by creatinine in the picrate method for blood sugar determination. It was found that creatinine added to blood caused an increase in the apparent blood sugar equivalent, in terms of glucose, to about three times the quantity of creatinine added, provided that this was more than about 3 mg. It was also found that purified bone-black, when used in quantities of about 1.5 gm. for the 25 cc. of blood picrate mixture, after a few minutes shaking would remove up to 25 mg. of creatinine per 100 cc. of blood. When the bone-black procedure was applied to bloods containing high non-protein nitrogen it was found that while such bloods very commonly showed a marked drop in sugar content after treatment with bone-black, in many instances this drop bore no relationship to the creatinine content of the blood. Furthermore, some bloods which gave a high creatinine content by the regular method, showed no drop whatever in the sugar content when treated with bone-black. 1 A few typical figures in this connection are given in Table I. ^ An inspection of Table I shows that there is little relationship between the fall in the sugar content of the blood after treatment with bone-black and the creatinine content of the blood. The results obtained with Samples 7, 12, and 13 render it very im- probable, or indeed impossible, that creatinine should have been 1 It may be noted here that most bloods showing a high non-protein nitrogen content show a marked drop in sugar content as determined by the Folin-Wu method after treatment with bone-black, and a still greater drop with Lloyd's reagent. The Folin-Wu procedure cannot be influenced by creatinine, or any other known constituent of the blood except glucose. It is hoped that this work in connection with blood sugar determination can be reported upon in the near future. J. A. Behre and S. R. Benedict 15 present in these samples in the quantities indicated by the rec- ognized method for the determination of this substance. Thus Sample 12, with a creatinine content of 8 mg. per 100 cc. which should be equivalent to about 24 mg. of glucose in the picrate method, shows no appreciable drop after treatment with bone- black, which removes pure creatinine. TABLE I. The Effect of Bone-Black Treatment on Blood Sugar Determinations in Relation to Blood Creatinine. Sample No. Sugar without treatment with bone-black per 100 cc. of blood. Sugar after treatment with bone-black per 100 cc. of blood. Difference per 100 cc. of blood. Creatinine per 100 cc. of blood. mg. mg. mg. mg. 1 96 88 8 1.2 2 112 93 19 1.2 3 150 132 18 2.0 4 280 220 60 6.0 5 186 179 7 4.2 6 125 110 15 3.1 7 116 112 4 5.0 8* 119 101 18 1.6 9 124 111 13 1.7 10 137 122 15 1.4 11 108 81 27 1.0 12 107 104 3 8.3 13 142 142 4.6 14 117 118 1 1.4 15 120 104 16 1.4 * Figures on Samples 8 to 15 were obtained by Dr. Gertrude F. McCann in the laboratory of Dr. Frederick M. Allen. We are indebted to Dr. McCann and Dr. Allen for permission to use this material. Obviously such results as are reported in Table I cannot do more than show that any particular sample of blood does not con- tain the creatinine ascribed to it by another and more direct method of determination* Nevertheless, such results do throw doubt upon the validity of the general assumption that the picric acid reaction, as applied for creatinine, really determines that compound in blood. 16 Creatinine and Creatine in Blood Hence we were led to study the question further. A peculiarity of picric acid filtrates from blood which we have repeatedly noticed, and which has probably impressed others, is that such filtrates are very frequently definitely darker in color than pure picric acid solutions, without addition of any alkali. Thus a colorimetric reading often showed such filtrates to have twice as much color as a saturated aqueous solution of picric acid. This observation indicated a tendency on the part of some .blood con- stituent to reduce picric acid in acid solution. Thus we were led to try the effect on color development in picrate solution when sodium carbonate is used as the alkali instead of sodium hydroxide, and to compare the results thus obtained with those yielded by * pure creatinine under similar conditions. As an arbitrary stand- ard for color comparison in this connection we employed a pure aturated picric acid solution, adding 0.5 cc. of 20 per cent sodium carbonate to each 10 cc. of solution, and making the readings after 10 minutes. When sodium carbonate is added to picric acid the depth of color in the solution increases slightly. If creatinine is present, in such quantities as we assume in blood fil- trates, this increase in color is slightly greater, but is scarcely more than detectable, and is not definitely proportional to the creatinine present. In fact, increasing the creatinine five times yields a scarcely detectable increase in color when carbonate is used as the alkali. When carbonate is added to the picric acid filtrate from blood, there is a marked increase in color, and this color is far greater than could possibly be due to the creatinine content of the blood, as indicated by the regular determination. In other words, there is in blood a substance other than creatinine which reduces % picric acid in the presence of sodium carbonate. It is fair to assume that this substance contributes to the reaction when hydroxide is employed as the alkali. Whether the reaction obtained with hydroxide is wholly due to the same substance which reacts (incompletely?) with carbonate cannot be answered. The in- crease of color in the blood filtrates with carbonate is usually particularly great in bloods which showed an abnormally high "creatinine" content, but in many other cases the increase due to carbonate is not proportional to that obtained with hydroxide. This may be due, as it is in the case of creatinine, to the fact that the reaction given by the blood compound is very incomplete J. A. Behre and S. R. Benedict 17 in presence of carbonate. We are inclined to believe that this explanation is correct, and that the "creatinine" in blood differs (among other particulars) from creatinine in that it reacts more strongly with picric acid in presence of carbonate. The evidence in this connection is, we admit, inconclusive beyond showing that results with hydroxide cannot be assumed to represent the true creatinine content of the blood. More definite evidence that the "creatinine" of blood is not creatinine is furnished by experiments based upon the fact that creatinine is destroyed by heating in alkaline solution. We have found that if solutions containing up to 4.5 mg. actual concentra- tion of creatinine per 100 cc. (corresponding to 22.5 mg. of crea- tinine per 100 cc. of blood where a 1 to 5 dilution has been em- ployed) to which have been added 2 per cent of sodium hydroxide, are heated for 1 hour, the creatinine has been completely destroyed so far as its power of yielding color in alkaline picrate solution is concerned. When filtrates from normal blood precipitated by tungstic acid according to Folin and Wu (4) are heated with alkali under similar conditions, the blood " creatinine" remains practi-' cally unchanged. When similar filtrates from blood to which creatinine has been added were treated in a similar way, the added creatinine was lost, while the blood creatinine was unchanged. We will cite two examples in this connection. The tungstic acid filtrate from a sample of beef blood showed a " creatinine" content for the original blood of 1.94 mg. per 100 cc. 20 cc. of this filtrate were treated with 5 cc. of 10 per cent sodium hydroxide, and the tube was heated in the water bath for 1 hour. The solution was then neutralized exactly with hydrochloric acid, saturated with picric acid, and the creatinine determined as usual. The color obtained corresponded to 1.82 mg. of creatinine per 100 cc. of blood, against the 1.94 mg. found without the treatment with alkali. When 2 mg. of creatinine per 100 cc. were added to this same blood (and recovered quantitatively in the filtrate), the reading after heating with the alkali showed 2.0 mg. per 100 cc. against 1.9 mg. in the original blood, and 4.0 mg. in the blood with the added creatinine. In another blood sample the filtrate showed 1.96 mg. of creatinine before heating with alkali and 2.1 after such heating. Addition of creatinine to this same blood so that the 18 Creatinine and Creatine in Blood filtrate showed a total of 8.60 mg. was then made. After heating with alkali the "creatinine" content had fallen to 2.50 mg., against 1.96 mg. in the original blood. In other words, more than 6 mg. of added creatinine per 100 cc. of blood were destroyed, while the original blood " creatinine" was practically unaffected. Additional experiments with alkali gave similar results, but they also led to the discovery that glucose heated under similar condi- tions with alkali may give rise to products which simulate crea- tinine in the picric acid reaction. If the glucose concentration in the original blood be not greater than 0.2 per cent there is no inter- ference due to the split-products, but with higher glucose concen- trations the split-products may replace part of the creatinine lost through the action of the alkali. The results cited above were obtained with bloods of low sugar concentration, and thus furnish quite direct evidence that blood " creatinine" is not creatinine. Our finding that high concentrations of glucose would interfere with the alkali study of the creatinine question on many bloods, led us to abandon this line of work until we had looked further for a method of attack less open to possible objection. The ques- tion of the occurrence of creatinine in blood is of such importance that definite conclusions with regard to it should not be based upon a line of study open to possible objection along any line. Our next study was concerned with the question of the removal of creatinine, and of the creatinine-reacting substance, from solu- tions by means of kaolin. Greenwald and McGuire (2) have reported that kaolin removes creatinine quantitatively from dilute solutions, and based a method for determination of creatinine and creatine in blood upon this fact. In their study Greenwald and McGuire employed blood filtrates obtained by heat coagulation of the blood proteins in presence of dilute acetic acid, and assumed, upon the basis of their results with pure creatinine, that the creatinine-reacting substance in blood was also removed by treatment with kaolin. They ap- parently failed to make creatinine determinations directly upon filtrates after treatment with kaolin. Had they not neglected this point, Greenwald and McGuire could hardly have escaped the conclusion that there is no creatinine in blood. Using the heat coagulation filtrate under the conditions pre- scribed by Greenwald and McGuire, we have found that true J. A. Behre and S. R. Benedict 19 creatinine is removed from pure solution or from blood filtrates quantitatively up to amounts corresponding to about 4 mg. of creatinine per 100 cc. of original blood. With higher amounts of creatinine the removal by kaolin is not quantitative. Employing the heat coagulation filtrates, the removal of the creatinine- reacting substance of the blood by kaolin is irregular and uncer- tain, even when only small quantities of the substance are present. In Table II are cited typical results in this connection upon one species of blood (beef). TABLE II. Showing Results of Kaolin Extraction of Filtrate Obtained by Heat Coagulation of Beef Blood, with and without Addition of Creatinine. 6 fc V 'ft 1 Blood without added creatinine.* Blood with added creatinine. Before extraction with kaolin. After extraction with kaolin. Amount removed by kaolin Before extraction with kaolin. After extraction with kaolin. Amount of added creatinine. Total amount re- moved by kaolin. mg. mg. mg. mg. mg. mg. mg. 1 2.32 1.50 0.82 3.24 2.18 0.92 1.06 2 2.32 1.50 0.82 4.75 1.72 2.43 3.03 3 2.14 1.05 1.07 5.04 2.16 2.90 2.88 4 2.32 1.50 0.82 6.12 3.36 3.80 2.76 5 2.37 1.05 1.32 6.10 1.94 3.75 4.16 6 3.06 2.14 0.92 8.05 2.40 5.00 5.65 7 3.51 1.83 1.68 8.61 3.52 5.10 5.09 8 3.21 1.87 1.34 8.61 4.02 5.40 4.59 * All figures are in terms of milligrams of creatinine per 100 cc. of blood. It will be noted that the quantity of the blood " creatinine" extracted by the kaolin would average from about 30 to 50 per cent. The same results hold true for the blood of other species. These results may perhaps explain the very irregular creatinine values reported by Greenwald and McGuire, using their method, as compared with figures obtained for the same samples by the Folin method. As a result of our work with kaolin upon the heat coagulation filtrates we soon gained the impression that the amount of the blood chromogenic substance removed by the kaolin from these filtrates depended upon variable factors, which could not be well 20 Creatinine and Creatine in Blood controlled. Hence we set out to find conditions yielding more constant results in connection with the use of kaolin. Experiments showed that in solutions completely freed from protein, kaolin, 2 used in a quantity of 2 gm. for 25 cc. of solution will remove creatinine quantitatively from solutions containing up to about 0.7 mg. per 100 cc. actual concentration, which cor- responds to 3.5 mg. of creatinine per 100 cc. of blood. The mix- ture with kaolin was shaken in a shaking machine for about 10 minutes. The solutions we employed were acid with picric, tri- chloroacetic, or hydrochloric acids. Such conditions are safest for extraction of creatinine. Pure creatinine is removed by kaolin from neutral solution, but the presence of even the minutest amount of alkali prevents this. Even calcium carbonate will interfere with removal of creatinine from a solution by means of kaolin. Employing kaolin as above indicated upon blood filtrates ob- tained after a 1 to 5 dilution and saturation with picric acid as in the Folin method for creatinine determination, our results appear to show conclusively that normal beef, dog, or human bloods con- tain no creatinine within the limit of accuracy of the method as we employed it. Creatinine in pure picric acid solution, or added to blood, is practically quantitatively removed up to about 3.5 mg. per 100 cc. of blood, while the creatinine chromogenic substance in the blood is unaffected. A hundred or more analyses have been carried out along this line. In no instance has there been an appreciable amount of the chromogenic substance of normal blood removed by kaolin, nor have we ever failed to remove added creatinine satisfactorily by the use of kaolin. Typical results in connection with this study are given in Table III. In connection with the results reported in Table III it should be remembered that creatinine in pure saturated picric acid in con- centrations equal to those reported for the bloods, is removed so completely by the kaolin treatment that such filtrates show only We have employed several different samples of kaolin with identical results. Eimer and Amend's "Kaolin, Acid Washed" was the one we used chiefly. We have also used Eimer and Amend's "Kaolin" which we our- selves washed with hydrochloric acid. Dr. Greenwald kindly sent us a sample of the kaolin employed by him. This~ preparation gave similar results to the other products we used. J. A.|BehreJandlS. R. Benedict 21 TABLE III. Showing Removal by Kaolin from Picric Acid Filtrates of Creatinine Added to Blood, and That the Chromogenic Substance Is Unaffected by Treatment with Kaolin. Source of blood. Blood without added oreatinine.* Blood with added creatinine. Before extraction with kaolin. After extraction with kaolin. Before extraction with kaolin. After extraction with kaolin. Amount of creatinine added. Amount removed by kaolin. mg. mg. mg. mg. mg. mg. Beef. .86 1.80 2.91 2.01 1.05 0.90 14 .50 1.35 2.40 1.45 0.90 0.95 tt .29 .33 2.24 1.29 0.95 0.95 Dog. .27 .18 1.92 1.33 0.65 0.59 Beef. .29 .33 3.45 1.50 2.16 1.95 tt .86 .80 4.45 2.26 2.59 2.19 .29 .33 4.05 1.56 2.76 2.49 <{ .90 .96 5.04 2.16 3.14 2.88 tt .50 .35 4.70 1.78 3.20 2.92 It .86 .80 5.25 2.00 3.39 3.25 Dog. .27 .18 4.85 1.47 3.58 3.38 Beef .40 .18 6.40 2.37 4.00 4.03 .29 .33 5.30 1.86 4.01 3.44 Human. .84 .71 4.55 2.00 2.71 2.55 3.36 .60 6.06 3.90 2.70 2.16 u 3.60 .27 5.76 3.81 2.16 1.95 ti 1.57 .26 it l.OQ .15 it 1.23 .02 it 1.96 2.12 tt 1.09 1.25 M 1.44 1.18 Beef. 2.28 2.28 u 2.15 2.35 2.34 2.22 Dog. 1.21 1.23 1.27 1.18 t< 1.88 1.82 * All figures are in terms of milligrams of creatinine per 100 cc. of blood. about 0.35 mg. of creatinine, which is the amount usually obtained with a blank against a "0.5 mg. standard" creatinine solution. The figures reported in Table III are, as stated above, typical 22 Creatinine and Creatine in Blood of the results obtained in more than a hundred analyses. Plasma yielded results similar to those obtained from whole blood. We have included experiments showing maximal removal of the blood "creatinine," as well as those in which removal of added creatinine was least satisfactory. It appears that the results reported in Table III furnish convincing evidence that the bloods studied do not contain creatinine, or at least do not contain this substance in excess of a few hundredths of a milligram per 100 cc. Exceptions to this statement might be taken, based upon occasional results where kaolin treatment lowers the creatinine value of the blood by a few tenths of a milligram per 100 cc. But conversely it may be noted that instances also occur where the chromogenic value of the blood is increased by a few tenths of a milligram after treatment with kaolin. The method of study employed has its limits of accuracy. The probable error for a large number of analyses would not, we believe, exceed about 0.05 mg. of creatinine per 100 cc. Our results as a whole indicate that the true creatinine con- tent of blood cannot exceed this figure, and we feel that it must probably be well below it. Having demonstrated the absence of creatinine in detectable amounts from normal blood, it was considered important to de- termine whether creatinine accumulates in the blood in conditions of renal insufficiency or of complete ablation of renal function. Results here should indicate whether the creatinine in the urine is secreted by a process of concentration from undetectable traces which occur in blood, or whether the kidney itself produces creatinine. In this connection we have studied bloods obtained from human cases where the kidney function was impaired, and also bloods obtained from dogs 42 to 72 hours after extirpation of the kidneys or after ligating the ureters. The results in connection with the high bloods are not conclu- sive on the question of the existence of true creatinine in these bloods. Human bloods yielding figures up to about 4 mg. of creatinine per 100 cc. show no loss of the chromogenic substance after treatment with kaolin. Higher bloods show a loss, following treatment with kaolin, amounting to from 20 to 50 per cent, pro- vMing the initial dilution of these bloods is great enough so that the actual concentration of reacting substance is about the same as in normal bloods. Such filtrates may also lose a considerable J. A. Behre and S. R. Benedict 23 percentage of color-yielding power (70 to 80 per cent) after heating with alkali, as detailed earlier in this paper. Very similar results were obtained with the dog bloods after ablation of the kidney function. We should be inclined to the view that true creatinine accumu- lates in the blood after impairment of the kidney function, were it not for the following considerations: After the demonstration that creatinine does not occur in normal blood in detectable amounts, more definite evidence is needed to prove its accumulation in abnormal blood than one or two non- specific reactions. Failure of removal by kaolin or of destruction by alkali may well demonstrate that a compound is not creatinine, but reverse findings do not demonstrate that a substance is crea- tinine. We are especially cautious in subscribing to the view that creatinine accumulates in the blood under the special conditions above cited because of the failure of an isolation experiment to demonstrate the presence of this compound. By the use of Lloyd's reagent a technique was developed which permits isolation of minute amounts of creatinine from large volumes of solution. So far only a single isolation experiment has been carried out in this connection, upon blood obtained from a dog after ligation of the ureters. The result was wholly negative for the isolation of creatinine. Though the final solution showed colorimetrically a creatinine content of about 9 mg. in 5 cc. of solution, creatinine- zinc chloride failed to separate from the solution after standing 2 days. After the addition of 5 mg. of creatinine to this same solution the added creatinine was recovered almost exactly as the zinc salt within a period of 3 hours. It is this result which makes us feel that more data are needed before any conclusion can be drawn concerning the true creatinine content of the "high creatinine" bloods. In the near future we shall report further data upon this problem together with full details of our isolation experiments. Studies on the muscle crea- tinine are also contemplated. After our investigation of the creatinine content of the blood we were led to study the behavior of the blood "creatine" and to attempt to find out whether all or part of this represents true creatine. The study has been complicated by the lack of an adequate method for conversion of creatine into creatinine in the 24 Creatinine and Creatine in Blood concentration found in the blood, which can be relied upon not to yield decomposition or other products which seriously interfere with the process. A review of the literature on methods of creatine determination in blood cannot but leave one with the impression that each inves- tigator, using a new technique, and one seemingly accurate, is able to get figures quite different from those, obtained by any previous method. The original method for conversion of creatine into creatinine in blood filtrates was proposed by Folin (3) and consisted in heat- ing the filtrates with picric acid. Wilson and Plass (6) and Hunter and Campbell (1) have raised the question of the accuracy of creatine determination by this procedure. Folin and Wu (4) subsequently recognized the fact that such a procedure led to high results for the " total creatinine" and suggested that traces of hydrogen sulfide were formed during such heating. Folin and Wu, however, did not seem to give much weight to their own sugges- tion, for their new method for blood creatine included autoclaving blood filtrates, and it is not apparent why picric acid filtrates should evolve hydrogen sulfide any more easily than should any other blood filtrate. Our experiments in connection with the picric acid filtrates obtained from blood have shown that blood contains some sub- stance other than creatine, which rapidly reacts with picric acid in hot solution to give a product yielding color on addition of alkali. After heating the picric acid filtrates from normal bloods in the water bath for 1 hour the " creatinine" value is usually doubled, and after 3 hours heating the value is about trebled. In a case of human nephritic blood we have obtained an increase in color-yielding power equivalent to 6 mg. of creatinine after 15 minutes heating of the picric acid filtrate in boiling water. The increases due to such heating are out of all proportion to possible conversion of pure creatine into creatinine under identical condi- tions. Similar results are not obtained if other acids (acetic, trichloroacetic, hydrochloric) are substituted for the picric acid. Hence the reaction is not a change in the blood constituent due to heating with dilute acid, but is a specific reaction between the blood compound and picric acid in hot solution. Apparently the compound giving rise to this reaction is not the same as that re- J. A. Behre and S. R. Benedict 25 sponsible for the creatine reaction of the blood filtrate when heated with acid in the absence of picric acid, for after treatment which will completely convert creatine into creatinine there is still an increase if the solutions are heated with picric acid prior to addition of alkali, but the increase in color is not as great as before treatment with acid, due probably to the conversion of the creatine. If filtrates from plasma instead of whole blood be heated after saturation with picric acid the curve of increase in color-yield after addition of alkali is similar to that for whole blood, but the actual increase is only about one-half as great (cf. also Wilson and Plass, 6). We have no clue as to the nature of the substance reacting in hot picric acid solution to yield the increased color. Neither glucose nor other known constituents of the blood can apparently be responsible for the reaction. The substance is partially, but not completely, removed by treatment with kaolin. Although blood contains this substance simulating creatine when heated with picric acid, it appears probable that the creatine figures obtained for normal, and for some pathological bloods, represent real creatine. For our creatine determinations we have adopted the procedure of precipitating the blood proteins by dilu- tion of the blood with 4 volumes of 5 per cent trichloroacetic acid. After filtration a portion of the filtrate is treated with hydrochloric acid (5 cc. of 1.0 N acid to 25 cc. of filtrate) and boiled down to a small volume. A little granulated or powdered metallic lead is then added, and the solution taken to dryness and heated on the water bath to expel the hydrochloric acid. 3 The residue is dis- solved in water and treated with sodium hydroxide, drop by drop, until a permanent precipitate (of lead hydroxide) is produced. The solution is then made up to a definite volume and filtered. A portion of the filtrate is saturated with dry picric acid and the creatinine determined as usual after filtration. The 10 per cent sodium hydroxide employed here should contain 10 per cent of Rochelle salt. This procedure gives a satisfactory conversion of pure creatine and is, we believe, reasonably accurate for the creatine determina- tion in normal bloods. While satisfactory for some pathological 1 This method is similar to the one recommended by one of us for the determination of creatine in urine (Benedict, S. R., J. Biol. Chem., 1914, xviii, 191.) 26 Creatinine and Creatine in Blood bloods, we are inclined to the view that in others no method of creatine determination so far developed can yield results which represent the true creatine content very closely. When we applied the procedures of heating with alkali and treatment with kaolin as described earlier in this paper, to blood filtrates after heating with acid, and compared the " total creat- inine" before and after such procedures, we found that for normal bloods the color-producing substance which resulted from the action of acid was destroyed by alkali and removed by treatment with kaolin exactly as is pure creatinine. Creatine added to blood behaved in a similar manner. These findings hold for beef and for dog blood. We have not yet had sufficient normal human bloods to warrant conclusions concerning them, but we believe that creatine actually exists in such blood. The findings in connection with the creatine content of blood are of interest also in relation to the question of the nature of the creatinine chromogenic substance in the blood. If this latter substance represents a loosely combined form of creatinine, or some similar compound, we might expect that boiling with acid should convert it into true creatinine. Yet after treatment to convert creatine into creatinine, it is possible by means of the action of alkali or of kaolin, to separate practically quantitatively the creat- inine derived from creatine from the chromogenic substance originally present in the blood. Typical results in this connection are presented in Tables IV and V. The results recorded in Tables IV and V serve to show how dif- ferently the creatine of normal blood behaves after conversion to creatinine as compared with the chromogenic substance originally present in the blood. The remarkably sharp separations effected by means of alkali or of kaolin are very striking, and serve to further substantiate our earlier conclusion that preformed crea- tinine does not exist in blood. The view that creatine occurs in blood in very appreciable amounts under certain conditions finds confirmation in an isola- tion experiment, in which creatine was isolated as creatinine-zinc chloride. In this experiment the blood obtained from a dog 46 hours after ligation of both ureters was employed. The total non- protein nitrogen of this blood was 186 mg. per 100 cc., and the blood showed a preformed "creatinine" content of 12 mg., and a J. A. Behre and S. R. Benedict 27 TABLE IV. Showing the Removal by Kaolin of Blood Creatine and of Creatine Added to Blood After Treatment for Conversion of Creatine to Creatinine, and the Non-Removal of the Original "Creatinine" in the Blood by means of Kaolin after the Boiling with Acid. Source of sample. "Preformed creatinine."* "Total creatinine." 1 w 1 3 ofl 111 Amount of ad- ded creatine removed. Before kaolin treat- ment. After kaolin treat- ment Before kaolin treat- ment . After kaolin treat- ment. mg. mg. mg. mg. mg. mg. mg. mg. Beef. 2.67 2.40 4.35 2.22 1.68 1.68+ 2.92 2.72 " 2.75 3.75 2.10 0.93 0.93+ 3.55 3.75 a 2.22 1.96 3.48 2.04 1.00 1.00 it 2.22 1.96 3.21 1.70 1.00 1.00 " 1.21 1.23 3.30 1.59 2.09 1.71 "\rirr 2.67 2.40 4.35 2.22 1.68 1.68 LJOg (ureters ligated). ~)f)fT 6.35 2.96 15.30 2.30 8.95 8.95 L^Ug (ureters ligated). 4.52 2.02 14.30 1.86 9.78 9.78+ * All figures refer to milligrams per 100 cc. of blood, in terms of creatinine. TABLE V. Showing the Destruction by Alkali of the Blood Creatine and of Creatine Added to Blood, after Treatment for Its Conversion to Creatinine, and the Failure of Alkali to Destroy the Original "Creatinine." Blood 1 ' t before creatine 43 a fjj conversion.* J"2 9 8S Source of sample. o g Before After T3 gta 73 B o> In alkaline alkaline 8 o v o-o 1 TJO^ T3^ heating. heating. PQ 3 <1 mg. mg. mg. mg. mg. mg. Beef. 3.15 3.15 2.60 2.60 3.4 2.00 2.18 2.00 2.02 1.40 7.7 5.40 it 2.30 2.30 2.80 2.75 4.58 4.36 {( 2.26 2.38 3.09 2.75 Dog with ligated ureters. 4.08 0.72 5.62 5.41 * All figures refer to milligrams per 100 cc. of blood, in terms of creatinine. 28 Creatinine and Creatine in Blood creatine content of 16.8 mg. per 100 cc. The preformed creatinine chromogenic substance was practically completely removed by the use of Lloyd's reagent, while the creatine was unaffected. The filtrate was then boiled with acid and the creatinine content of a portion of the solution determined. The main portion was treated with Lloyd's reagent, which removed the chromogenic substance practically quantitatively. Thus two portions of Lloyd's reagent were obtained, one containing the preformed chromogenic sub- stance of the blood, the second containing the chromogenic sub- stance resulting from boiling with acid. Both' portions of the Lloyd's reagent were treated in similar manner to liberate the chromogenic substance, and portions of the solutions thus obtained were analyzed colorimetrically and each was found to contain a chromogenic compound. The solutions were boiled down to very small volumes (about 5 cc.) and washed into small centrifuge tubes with the help of alcohol. The total volume in each tube was about 8 cc.; a few drops of zinc chloride solution were added to each tube, together with a few drops of a mixture of acetic acid and sodium acetate. Within half an hour typical creatinine-zinc chlo- ride crystals began to separate from the tube containing creatinine derived from the creatine in the blood. After standing over night these crystals were centrifuged. washed with alcohol, and dissolved in a few drops of hydrochloric acid, and the solution thus obtained was diluted to a definite volume and analyzed for creatinine colori- metrically. It was found that 10.2 mg. of creatinine had been recovered as the zinc salt, against a theoretical value of 13.6 mg. as determined in a portion of the blood filtrate after hydrolysis. As mentioned earlier in this paper, none of the zinc salt was ob- tained during 3 days from the tube containing the preformed chromogenic substance in the blood, though addition of a small amount of pure creatinine to the final solution promptly resulted in the separation of typical crystals of the zinc compound. Full details of these isolation experiments will be reported later. In regard to the question of the occurrence of creatine in blood of patients with renal insufficiency we should state that our results have been so contradictory that we are led to believe that some of these bloods contain large quantities of one or more interfering substances in the creatine determination, while in others the crea- tine figures may be fairly exact. Some nephritic bloods show J. A. Behre and S. R. Benedict 29 removal of the creatinine derived from creatine by means of kaolin, while others behave very differently. In one case of bichloride poisoning with a very high apparent creatine we failed to effect practically any removal of the chromogenic substance resulting from the action of acid, by means of kaolin treatment. Our results indicate that cases may fall into definite groups in regard to the true creatine content and the study of the question is being continued. DISCUSSION. Our finding that creatinine does not exist in blood in detectable quantities need not, of course, raise any question as to the value of the determination of the chromogenic substance for clinical or other purposes. In connection with such determination our work has brought out some points which may be of interest. In the first place there is the question of complete saturation of the solu- tions with picric acid prior to the creatinine determination. This point has been emphasized by Greenwald and McGuire, but we believe that it has not received sufficient recognition. For any method of creatinine determination in blood, except the procedure advocated by Folin and Wu, it is necessary to saturate a solution or mixture with solid picric acid. The Folin- Wu procedure, while perhaps theoretically preferable to the earlier methods from cer- tain standpoints, yields such weak colors that we question the general usefulness of the method. Few analysts can read such colors with even approximate accuracy unless the bloods are very high in chromogenic substance. We therefore believe that satura- tion with picric acid is preferable in all ordinary work. Such saturation may, of course, be applied to any filtrates (such as the Folin- Wu tungstic acid filtrate) as well as to the original blood. In any case, we believe it essential that laboratories where attempted saturation with picric acid is a routine procedure should be equipped with shaking machines (a rotary type is satisfactory), and that all solutions to be saturated with picric acid should be placed in such machines for from 5 to 10 minutes, prior to filtra- tion. When numerous bloods are handled it is probable that in no other way can even approximate saturation with picric acid be secured. Where Myers' method, which involves preliminary dilu- tion of the blood with distilled water, is employed, especial care 30 Creatinine and Creatine in Blood has to be taken in regard to saturation with the picric acid. In this method the preliminary stirring as recommended by Myers should be followed by from 5 to 10 minutes in a shaking machine. While it is possible to saturate a blood as recommended by Myers, our experience shows that this is rarely accomplished in practice, and that where many bloods are handled simultaneously, it is very difficult to secure saturation by means of stirring. The necessity for full saturation is clearly brought out by Greenwald and McGuire. A second point of interest in connection with the technique for the chromogenic substance in blood is in connection with the pro- tein precipitant employed and the amount of dilution at the time of the protein precipitation. As noted earlier in this paper, the original method of Folin, employing a 1 to 5 dilution with saturated picric acid when applied to bloods with high "creatinine" values, results in a loss of a very considerable quantity of the chromogenic substance. This loss may amount to 5 mg. or more, per 100 cc. The tungstic acid precipitation, carried out at a 1 to 10 dilution as proposed by Folin and Wu gives a much better recovery of the blood chromogenic substance in the abnormal bloods than does the picric acid method at the dilution of 1 to 5. The figures ob- tained with this filtrate agree closely with those given by a precipi- tation with trichloroacetic acid in a 1 to 5 dilution. Figures by these methods are usually appreciably lower than where heat coagulation is employed in a 1 to 5 dilution. The difference, how- ever, is not great, and on account of convenience we should at present recommend use of the Folin- Wu precipitation with tung- stic acid, followed by saturation of a portion of the filtrate with dry picric acid in a shaking machine for from 5 to 10 minutes. After filtering from the excess of picric acid the chromogenic sub- stance is determined as in the original Folin method, using stan- dard creatinine solutions in saturated picric acid. Table VI shows some comparative figures for the chromogenic substance in dog bloods rich in this substance where different protein precipitants were employed. The question naturally arises as to the possible bearing upon theories of creatine and creatinine metabolism of the findings reported in this paper. While we feel that the present data do not warrant detailed discussion, there are certain points brought up by the present work which may be briefly considered. J. A. Behre and S. R. Benedict 31 If creatinine cannot be demonstrated in the blood, there are two possible sources for its presence in the urine. It may be present in the blood in traces, and the kidney may be able to concentrate creatinine from this exceedingly dilute solution. If this view is correct, we should, as pointed out earlier in this paper, be able to demonstrate creatinine in blood after impairment or ablation of the kidney function. No demonstration short of isolation will suffice in this connection. In view of the failure of isolation experi- ments reported so far, it would seem of interest to discuss the possible origin of the urinary creatinine if creatinine does not occur TABLE VI. Apparent Creatinine by the Different Methods for Blood of Dogs with Ureters Ligated. Source f blood. Hours between operation and withdrawal of blood. Apparent creatinine in mg. per 100 cc. of blood. Picric acid method 1:5 dilution. Sodium tungatate precipitation 1:10 dilution. Trichloro- acetic acid precipitation 1:10 dilution. Heat coagulation method 1:5 dilution. hrs. Dogl " 2 48 72 4.85 6.55 6.96 8.58 " 3 42 7.35 11.6 11.1 12.0 " 4 46 6.37 8.10 9.04 10.3 " 5 46 11.10 12.24 12.24 14.52 in the blood. Taking this latter view, we must assume that the kidney itself produces creatinine from some precursor substance in the blood. The most probable precursor, from the chemical stand- point, would, of course, be creatine. It is in line with this hypoth- esis that we find real evidence for the existence of creatine in blood, and it is reasonably certain that under normal conditions the creatine content of the blood is very appreciable. What then is the fate of this creatine? Is it a waste product, or is it to be built up into body tissue? The positive result of our isolation experiment on the creatine of the blood of a dog after ablation of the excretory function of the kidney, where we were able to demon- strate an accumulation of creatine in this blood to an extent ex- ceeding 13 mg. per 100 cc., would seem to show that the creatine of the blood is a waste product, to be eliminated by the kidney. 32 Creatinine and Creatine in Blood All five of our dogs in which kidney function was ablated showed high creatine figures as follows: 8.9, 8.1, 9.3, 18.5, and 16.8 mg. per 100 cc. This creatine behaves as true creatine, and was iso- lated (as creatinine) in the only attempt we have made. Hence we may conclude that creatine accumulates in the blood when the excretory function of the kidney is defective, and we may further conclude that creatine in the blood is a waste product. Creatine is not normally eliminated as such in the urine, hence it would seem that the kidney must eliminate creatine as creatinine or as some other product perhaps urea. An alternative view that there is some organ which cannot act in destroying creatine except when the kidney is performing its excretory function would seem too extreme a view to deserve consideration. It would seem then that the kidney normally eliminates creatine as creatinine or as some other product. Elimination as creatinine seems far more probable. There are, of course, difficulties in the way of accepting this view. The first which suggests itself is the fact that ingested creatine does not appear in the urine as creatinine except in traces. Most observers except Folin, are agreed that administration of creatine is followed by an appreciable, though very slight increase in the urinary creatinine. The observations of Folin and Denis (7) would explain how it would be possible for ingested creatine to fail to be eliminated in considerable amounts either as creatine or creatinine, even though normally creatine is the source of the urinary creatinine. These investigators have shown that mus- cular tissue appears to have a marked affinity for creatine, and that this substance rapidly disappears from the blood and is found increased in the muscles. Assuming even an equal affinity for creatine between muscular and kidney tissue, we can readily see why, on account of the relative bulk of the muscular tissue, nearly all of the creatine ingested could get into the muscles as creatine, instead of into the urine as creatinine, even though normally the kidney eliminates the creatine as creatinine. Furthermore, it is quite possible that the formation of creatinine in the kidney represents some definite form of kidney metabolism associated with a portion of its excretory function. Thus crea- tinine might even represent a measure of a special type of kidney metabolism. It is also possible that the creatine in the circulation differs from ordinary creatine, and is in a combination which cao readily be converted into creatinine. J. A. Behre and S. R. Benedict 33 The question of the occurrence of creatine in the urine need not be considered until isolation experiments have shown whether the so called creatine in urine is really that compound. We are study- ing the question by means of a method which we feel will demon- strate whether creatine occurs in urine. In the meantime we feel that any theory of creatine and crea- tinine metabolism must take into account the fact that creatine circulating in the blood appears to be essentially a waste product. The view above proposed attempts to do this, and is the only view so far put forward which takes account of the increased blood creatine after impaired kidney function. BIBLIOGRAPHY. 1. Hunter, A., and Campbell, W. R., /. Biol. Chem., 1917, xxxii, 195. 2. Greenwald, I., and McGuire, G., /. Biol. Chem., 1918, xxxiv, 103. 3. Folin, O., J. Biol. Chem., 1914, xvii, 475. 4. Folin, O., and Wu, H., J. Biol. Chem., 1919, xxxviii, 81. 5. Folin, O., and Doisy, E. A., J. Biol. Chem., 1916-17, xxviii, 349. 6. Wilson, D. W., and Plass, E. D., J. Biol. Chem., 1917, xxix, 413. 7. Folin, O., and Denis, W., J. Biol. Chem., 1914, xvii, 493. . . THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW AN INITIAL FINE OF 25 CENTS WILL BE ASSESSED FOR FAILURE TO RETURN THIS BOOK ON THE DATE DUE. THE PENALTY WILL INCREASE TO SO CENTS ON THE FOURTH DAY AND TO $1.OO ON THE SEVENTH DAY OVERDUE. AUG 4- LD 21-95m-7,'37 507H.;.. UNIVERSITY OF CALIFORNIA LIBRARY