THE

AMERICAN JOURNAL OF THE MEDICAL SCIENCES.

MAY, 1906.

SPECIAL ARTICLE.

THE CLINICAL SIGNIFICANCE OF THE URINARY NITROGEN.

By James Ewrnea, M.D., AND C. G. L. Worr, M.D.

(From the Departments of Chemistry and Pathology, Cornell University Medical College.)

TECHNIQUE, SOURCES, KELATIONS, AND NORMAL STANDARDS OF THE NITROGENOUS COMPOUNDS.

‘THE urine as an index of metabolism in health and disease has been the subject of numberless investigations for many decades, and divergent opinions have constantly existed regarding the validity of the results for diagnosis. It must, however, be conceded that with improved methods of analysis, and with a rational basis for dis- cussion, the results, although by no means absolute, are throwing more and more light on obscure metabolic disturbances. It is the object of this study to ascertain how far one is justified in drawing conclusions as to deep-seated changes which may be taking place from an examination of the nitrogen partition. Owing to the tech-. nical difficulties of estimating accurately the nitrogenous compounds of the urine, this type of analysis has been limited chiefly to labor- atories of physiological chemistry, in which the features of disturbed metabolism in many diseases have. been extensively studied. It would seem, however, that with the increasing facilities of chemical laboratories and the larger demands for accurate knowledge of the character of morbid processes, this type of urinary analysis should

vou. 131, No. 5.—may, 1906.

752 EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN ;

become more extensively employed in the routine study of disease by the clinician.

Outside of the inorganic salts, with which this study will not deal, practically al] the compounds excreted by the urine in normal metab- olism contain nitrogen. ‘The exceptions, if they may be so called, are the carbohydrates, the aceton compounds, some of the aromatic oxyacids, and the cresols.' If, therefore, all nitrogen-containing compounds are estimated as completely as possible, one has a cri- terion of all the organic metabolism as far as it is indicated by the urine. The difficulty which has heretofore’prevented an accurate analysis of the larger part of the nitrogenous compounds of the urine has arisen from several sources:

First. Until recently it has not been recognized that a complete picture of the urinary excretion was of any importance. ‘The tend- ency has been rather to the close examination of one feature of the output, such as the urea or the uric acid, and in so doing the general ensemble of the picture has been lost or distorted.

Second. ‘The methods used have been such that an approxi- mately complete examination could not be made with a twenty-four- hour specimen, and the operations were so time-consuming as to make the examinations nearly useless for clinical purposes. Even then they were not of the accuracy requisite for a nice perception of small differences.

Third. The difficulties in the way of procuring a twenty-four- hour specimen, and the failure on the part of the clinician and the chemist to realize the paramount importance of studying the entire excretion of the twenty-four hours, have rendered of no value much that might have been valuable material. And so it is that, despite the large amount of urinary analyses to be found in the literature of disease, in but a few instances only *’ is it possible to find a disease of which the urinary picture is sufficiently complete to serve as a stand- ard for future investigations. As a matter of fact, this does not apply to abnormal metabolism only. ‘Till within very recent times we have had no standards for normal metabolism, and the investigations of Folin“ are of fundamental importance in this respect.

Of the nitrogenous compounds excreted by the urine by far the largest amount is eliminated as urea. It was once thought that with normal conditions, irrespective of diet, 90 per cent. of the total nitro- gen would be eliminated in this way, and consequently a disturbed metabolism was indicated when the urea fell below this factor.” ‘This view, however, owing very largely to the work of Camerer“ and Folin, has been shown to be completely fallacious. The percentage of the total nitrogen eliminated as urea is independent on the total amount

1 We wish to express our thanks to Mr. W. McKim Marriott and to Dr. J.C. Roper for much help in this work. Our thanks are also especially due to Dr. B. J. Dryfuss for te careful manner in which he has performed the greater part of the analyses on which this study is based.

EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN 753

of nitrogen eliminated in the twenty-four hours. Hence, a urea ratio which would be highly abnormal for a nitrogen execretion of 20 grams per day is entirely normal when the subject has an output of 10 grams. It is from this point of view that ratios must be studied, and an absolute scale must in every case give place to a sliding one, in which the determining factor is the nitrogen elimination in the twenty-four hours. This point cannot be too strongly empha- sized, for the literature is replete with examples of clinical indications based on the estimation of the amount of a single substance excreted by the urine, or on the relation of one substance to another irrespec- tive of whether the patient is excreting 10 or 20 grams of nitrogen.

The principal substances excreted by the kidney may be repre- sented in the following schema, and with them, for a given diet, are the total amounts in the twenty-four hours, and the percentages they form of the total nitrogen. The first line gives the absolute amount of the substance excreted; the second the absolute amount of nitro- gen in the substance; the third the percentage of the total nitrogen which the nitrogen of the substance forms.

Total Nitrogen 16.5 grams. Urea and Undeter- Urea. Ammonia. Kreatinin. Uric acid. ammonia. mined. Grams en 1.49 0.66

Grams, nitrogen . 14.1 0.50 0.55 0.22 ote 0.7

Nitrogen, percent. 87.5 3.2 3.4 3.4 90.0 4.5 This picture of the amounts and relationships of the nitrogen excretion changes entirely when the same subject, instead of elim-

inating 16 grams of nitrogen, excretes only one-fourth that amount.

Total Nitrogen 4.2 grams. Urea and Undeter- Urea. Ammonia. Kreatinin. Uric acid. ammonia. mined. Grams ie | 0.60 1.60 0.32 Grams, nitrogen oe 0.49 0.60 0.11 apres! 0.4 Nitrogen, percent. 61.8 11.5 14.0 2.6 73.3 10.0

It will be seen that the relative amounts of the various compounds of the urine have totally altered. There is of necessity an absolute decrease in the urea output, and at the same time the percentage relation to the total nitrogen has diminished. The absolute amount of ammonia is practically the same, but the relative amount has increased over 300 per cent., the value rising from 3.2 per cent. to 11.5 per cent.

The same may be said for kreatinin. The absolute elimination of this substance is practically constant throughout the whole range of diet, and, consequently, the percentage referred to the total nitrogen varies from 4 per cent. to over 15 per cent.“ The uric acid, also, to a lesser degree, tends to remain constant in an absolute sense. But this, with the purin-free diets on which these figures were obtained, is not by any means invariable. It will therefore be seen

754 EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN

how difficult it would be to establish any ratio such as uric acid to urea with two compounds both of which fluctuate markedly and often in no very uniform manner, under changes of diet alone, and how much more difficult when the diet is not absolutely free from nuclein. ‘The so-called undetermined nitrogen also varies extremely, amounting to 0.4 to 0.7 grams per diem, and forming from 3 to 15

r cent. of the total nitrogen.

It should be noted that the above results were obtained on a meat- free diet, consisting of eggs, cream, starch, and sugars. While this diet conforms in the main to the ordinary light diet of the hospital, it is probable that other diets containing the same amount of nitro- gen, and the same calorific value, will yield other if not radically different results

Tue Mernops or DETERMINING THE NITROGENOUS CoMPOUNDS AND THEIR SIGNIFICANCE. Since small differences may be the issue, only the most accurate methods are available in determining the nitrogen partition in the urine. The so-called clinical tests, in- cluding the hypobromite method and the various sedimentation methods of one kind and another, are not applicable to the present

otal Nitrogen. 'The total nitrogen was determined by the Kjeldahl-Argutinsky method, adding copper sulphate as a catalyst.

Ammonia. ‘Two methods were used. In the earlier part of the work the method proposed by Boussingault and modified by Shaffer” was employed. This consists in distilling the urine made alkaline with sodium carbonate, and mixed with methyl alcohol and sodium chlo- ride in a vacuum, into decinormal acid. The method is satisfactory, except that in some urines containing diamins one of us, with Mar- riott,** has observed somewhat high results. For this reason we have used more lately the method suggested by Folin.”

This consists in driving a rapid current of air through the urine made alkaline with sodium fom ath The ammonia removed by the air is caught in decinormal acid. This method has solved in a thoroughly satisfactory manner the problem of accurately deter- mining ammonia in metabolic experiments. It also has the great advantage that one may make six or more determinations in series without any attention being paid during the process. It is difficult to ascertain why investigators continue to use the Schlésing method” for this determination. The process is not easily carried out, the determination consumes much time, and the results are not accurate.

The significance of the ammonia nitrogen of the urine is a much debated subject. This urinary constituent is subject to wide vari- ations, both relative and absolute, and the variations may or may not be associated with other signs of metabolic disturbance.

1, The ammonia nitrogen is increased in conditions of dyspnoea and insufficient aération of the blood, where its excess has been referred to the excess of CO, in the blood, which requires ammonia

EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN 755

for its neutralization. In such conditions the ammonia nitrogen has exceeded 30 per cent. of the total nitrogen. (Michaelis.™)

2. The character of the diet has a marked influence on the per- centage of ammonia nitrogen.‘ Pfaundler® refers the high ammonia nitrogen of the urine of infants to their milk diet, and by an exclusive milk diet he was able to raise the ammonia nitrogen in adults to over 10 per cent. The Breslau school refers the high ammonia nitrogen of marasmic infants suffering from gastroenteritis to the absorption of fatty acids from the food. A diet rich in either meats or fats considerably increases the excretion of ammonia. (Gerhardt and Schlesinger.”)

3. The doctrine of acid-intoxication is founded upon the appear- ance of excess of ammonia nitrogen in the urine, together with aceton and aliphatic acids. According to Walter“ and Hallervorden,™ ammonia serves to neutralize the acids absorbed from the food or produced in the body, and thus prevents these acids from absorbing the fixed alkalies of the tissues. Yet Limbeck” has shown that the administration of lactic or hydrochloric acids in man, while increasing the ammonia nitrogen 8.21 per cent., raises the excretion of sodium and potassium 38 to 40 per cent.; while Beckman® and Stadelmann™ found that after the excessive administration of alkali there always remains some ammonia in the urine which has nothing to do with the neutralization of acids. Hence, the urinary ammonia must have some other function besides that of neutral- izing acids.

4. According to Pfaundler,” excess of ammonia nitrogen in the urine is chiefly referable to deficient oxidation in the organism, principally in the liver, which results in a failure of the synthesis of ammonia and amido acids into urea, and the increased excretion of ammonia. In such states the aceton bodies, and, according to Zweifel,” lactic acid are often found in excess. The patient suffers from a variety of symptoms of intoxication, which Pfaundler refers chiefly to deficient oxidation and not specifically to the presence of poisonous acids.

5. During starvation all the above urinary signs of acid intoxi- cation are present, but here a different theory of their origin has been rather slowly demonstrated.” Studies of the metabolism of starvation by Luc ani,” Brugsch” and others have shown that high proportions of ammonia nitrogen and aceton bodies are found in the urine when symptoms of intoxication are absent. In Brugsch’s observations on the faster Succi, the proportion of urea nitrogen was low, the ammonia was high, and aceton and aliphatic acids were abundant during a period of twenty-nine days, when the sub- ject’s general condition was excellent, while under similiar con- ditions observed by Luciani, Succi was able to fence and run actively. Moreover, large amounts of aceton and aliphatic acids

may appear in the urine on the first day in healthy and fasting sub-

756 EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN

jects. (Waldvogel.”) Likewise, in diabetes, similar signs of acid- osis without other symptoms may occur, as in Van Noorden’s case, in which the patient during two and a half years of observation excreted at least 2.5 per cent. of S-oxybutyric acid daily, and yet, considering his disease, remained in good health or at least without symptoms of intoxication.

The true significance of the occurrence of ammonia, aceton, and ali- phatic acids in the urine under these conditions appears to be found in the observations on the oxidation of the depot fat when the organ- ism is unprotected by sufficientfood.” Inthestudies of F. Miiller™ on the faster Cetti, and of Brugsch on Succi, and in a case reported by Hirschfeld,” the urine contained large amounts of ammonia, aceton, and aliphatic acids, while in some cases of starvation studied by Waldvogel and Brugsch these compounds were not increased, and Brugsch points out that this occurs when the subjects are emaciated, and have little depot fat, in which case energy is supplied by the consumption of proteids. It thus appears that the occurrence of aceton, aliphatic acids, and ammonia in excess in the urine, when the food supply is deficient, means that the patient is burning his own fats and not necessarily that he is suffering from acid intoxi- cation.

It will be noticed that many of those conditions in which the pre- sence of the above urinary signs has led to the diagnosis of acid intoxication are really conditions of more or less marked inanition. This is true of pernicious vomiting, and the conditions sometimes following chloroform narcosis. In the studies of Greven™ and of Baldwin’ on the urinary changes following narcosis, no refer- ence appears to have been made to the food supply or to the presence of vomiting or other disturbances of digestion. Until these factors are known, no definite conclusions can be drawn from the urinary analysis. Waldvogel’* found that only 75 per cent. of his cases of narcosis gave acetonuria; which was absent or inconsiderable when the patient was fully nourished before and after the operation, even when the narcosis was severe enough to produce glycosuria.

Moreover, Satta and others have shown that by the administra- tion of alkalies or glucose the excretion of ammonia and aceton bodies may be largely suppressed,without apparent effect on the symptoms.

From this brief review of some of the factors influencing the excre- tion of ammonia it is evident that the correct interpretation of an excess of ammonia in the urine is a very complex subject, requiring careful analysis of all the possible conditions concerned.

We believe, therefore, that too much stress may be laid on the finding of these substances in the urine, especially when associated with vomiting, diarrhoea, or other disturbances of digestion. In the course of our studies we have encountered cases in which the urine gave as much as 40 per cent. of ammonia nitrogen, and considerable amounts of aceton and 8-oxybutyric acid, and yet the patients exhib-

‘EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN 757

ited no symptoms of intoxication. In the present state of our knowl- edge we believe that one cannot safely rely upon the presence of these compounds in the urine, either as an index or as a measure of autointoxication. On the other hand, since these urinary changes are so frequently found in cases suffering from autointoxication, they may, perhaps, be properly interpreted as a sign of danger, possibly to some extent from failure of adequate elimination of such compounds. Waldvogel suggests that some distinct injury of the body cells may, perhaps, be concerned in the production of aceton- uria following narcosis, and histological studies of the viscera give ample basis for such a view.

Urea. ‘There are two accurate methods for the determination of urea—the Morner-Sjovist® and that of Folin.” Both are about equal in point of accuracy. The latter is the one which has been used throughout this work. ‘The principle consists in heating urea to a high temperature with hydrochloric acid. Urea is decomposed quantitatively by this treatment into carbon dioxide and ammonia. The ammonia is then distilled off into standard acid as in the Kjel- dahl method. From the amount of ammonia so found, that belonging to the preformed ammonium salts is subtracted. The method is a most accurate one, as has been proven by the work of Folin himself and by Mérner.” Recently, Noel Paton® and W. H. Thompson™ have stated that they were unable to obtain concordant results by the method. We can only say that, in the course of this and other metabolic work, we have performed considerably over a thousand separate determinations by this method, all in duplicate. We have never known the method to fail us, except when we could distinctly trace our error to technique. We conclude, therefore, that Paton and Thompson have not followed out the very careful direc- tions for this determination as laid down by Folin.

Kreatinin and Kreatin. The colorimetric estimation of Folin® has been employed, using, however, the wedge instrument of Gallen- kamp, which is not quite so satisfactory as the Duboscq apparatus. It is very necessary in this determination to work within definite limits of concentration, as Folin has done, or to go over the entire scale with standard solutions of kreatinin and plot out the results in co-ordinate paper. The trend of the color-concentration curve is not represented by a straight line, but has the form of an hyperbola. Our results with the method, both with human and canine urines, have been satisfactory.

It is worthy of mention that the constancy of the kreatinin elim- ination in dogs is like that which Folin observed in man. It is quite likely that the determination of kreatinin, and perhaps of kreatin, will be of value in metabolic experiments as an index of the amount of endogenous nitrogenous metabolism which is going on. The work which has so far been done in this connection is insufficient to war- rant any precise statements. In some experiments which one of us

758 EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN

has performed,” it has been seen that in starving animals, under the influence of brombenzol, one may have the nitrogen excretion in- creased 200 to 300 per cent., with no parallel rise in the kreatinin. ‘From this one may infer that in many cases of increased nitrogenous elimination, even in the absence of food, one may affect the exog- enous metabolism severely, without touching the processes which are intimately connected with the bioplasm. If this be the case, we may have in kreatinin a valuable index of the more intimate processes which we are accustomed to speak of as endogenous. It has been shown, in certain conditions associated with hyperpyrexia, and with increased nitrogenous excretion, that the absolute excretion of krea- tinin is increased.” Here we would associate increased nitrogen output with distinct intracellular disturbances, while in the case of poisoning above referred to the process would be exogenous.

Uric Acid. ‘The determinations of uric acid were made by Folin and Shaffer’s“ modification of Hopkin’s method. The uric acid is precipitated as ammonium urate in the presence of ammonium sulphate and ammonium hydroxide. The method is accurate, and can be done without loss of time if one be dealing with a large num- ber of specimens. It suffers, in common with all determinations of purin compounds, from the length of time required for the precipi- tation of the ammonium salt.

Apart from the necessity of determining the uric acid as.an inte- gral part of the nitrogen, we do not attach a great deai of importance to its estimation. The prevalent view with regard to the elimination of uric acid is that it is derived from two sources: one, strictly endog- enous, and, even more than this, intranuclear; the other exogenous, and even less than exogenous in its strictest sense, viz., derived from the food.”

In the ordinary class of acute hospital cases with which we deal it has been impossible to assure ourselves that we were examining a patient who was on strictly purin-free diet. Under these circum- stances it would seem inadvisable to make any very pronounced statements regarding the relation of uric acid to other constituents of the nitrogenous metabolism.

It is notable that uric acid has been the basis of almost countless investigations, and its importance as a factor in the causation and diagnosis of disease has waxed and waned with rhythmic regularity.” As far as the urine is concerned, it would appear that the present ten- dency is not to assign it a cardinal role.

The Undetermined Nitrogen and the Amino Acids. he start- ing point of the present investigation was intimately connected with this compound of the nitrogen partition. Our original object was to determine to some extent the clinical significance of the occurrence in the urine of leucin and tyrosin, which were supposed to be pre- sent in grave pathological conditions only. The findings were, how-

EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN 759

ever, so involved that it has been necessary to go into the matter in some detail.

The undetermined nitrogen is one of the most difficult, and cer- tainly one of the most interesting problems in the analysis of the urine. Closely associated with it is the question of the amino acids, and, in fact, so intimate is the relation that the undetermined nitro- gen and the amino acid fraction of Pfaundler have been regarded as nearly synonymous. As a matter of fact, if the estimation be properly performed a very close agreement between the two will be found. As the term “undetermined nitrogen” implies, it is that fraction which remains after the nitrogen estimated as ammonia, urea, kreatinin, and uric acid is subtracted from the total nitrogen. The term amino acid fraction arises from the method which had its origin in the work of several investigators, Pfliiger, Bohland, Bleibtreu,* Gumlich,” Pfaundler® and others. The method is in effect the same which Hausmann,” Giimbel,” Osborne® and others have applied to the partition of the nitrogen in proteid substances. The principle of the method is the following: If one precipitate a urine with a solution of phosphotungstic acid in the presence of either hydrochloric or sulphuric acid, practically all the organic constituents of the urine are precipitated, with the exception of urea and substances which are known, for want of any better term, as the amino acids. If, therefore, one determines the total nitrogen and the urea in the filtrate, and subtract the latter from the former, one should have the nitrogen due to the so-called amino acids. Cer- tain compounds, however, are partly precipitated by phosphotung- stic acid, which are chiefly present in the undetermined nitrogen remainder. ‘These are the diamin and the xanthin bases. As both these classes of compounds are present in very small amount, the error in comparing the two is not large. Furthermore, it has been ‘said that certain compounds are incompletely precipitated by phos- photungstic acid.“ This applies especially to kreatinin. We have satisfied ourselves that kreatinin is present in the amino acid filtrate after precipitation with phosphotungstic acid, but in such small amount that it has been impossible to estimate it. ‘The error, there- fore, is a small one.

With regard to the precipitation of monoamino acids by phos- photungstic acid we have not made any direct experiments. We have, however, in a large number of cases, determined all the con- stituents directly, and at the same time have determined the mono- amino fraction. On adding the two together the result is always a very close approximation to 100 per cent. The following example will illustrate this point:

760 | EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN

Specimen I. Specimen IJ. Volume - ° ; 4 r - > 8. 700 c.c. Specific gravity ; : F 5 , ; ; } 1032 Total nitrogen per cent. .65 Total nitrogen per day 55 Urea nitrogen per cent. 32 Urea nitrogen per day 22 Percentage total 3 0 Ammonia nitrogen per cent. 72 Ammonia nitrogen per day 51 Percentage total = 4 Utea and ammonia nitrogen per osnt. Urea and ammonia per day Percentage total Uric acid nitrogen per cent. Uric acid nitrogen per day Percentage total A Kreatinin nitrogen per cent. Kreatinin nitrogen per day Percentage total ; Undetermined nitrogen per > abl “ened ites ge 4 Undetermined nitrogen per day Percentage total * Amino acid nitrogen per oe: Pfaundler cian Amino acid nitrogen ner day : Percentage total

> . —_ iS

SHme Woe oa a

Noe ~ B ok No w

73 4

wooBuoace

bo

a ow

09 5 159 11 6

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= a @ x = CH OOHSOHROSOHOOROH ROOS OR Ee

em omoouos Aomn or on

=

We have discussed the Pfaundler method at some length, as we have employed it extensively, for the reason that in some important cases we have not been able to secure a twenty-four-hour specimen, nor enough of the urine to complete the determination of uric acid. In such cases we have estimated the ammonia and the urea

in the whole urine. The estimation of the urea in the phospho- tungstic acid filtrate has been done separately from the other, for the purpose of subtraction only. When this was done, we have noted that in many instances the urea, as found in the filtrate, was appreciably less than that in the whole urine. The observation which has been made that ph OF gr 30 acid some

of the urea is thus confirm Apparently this takes place in quite an arbitrary manner, for in many instances the urea deter- minations are in perfect agreement.

The question which now confronts one is the ultimate composi- tion of this undetermined nitrogen part, which forms no small amount of the nitrogen excreted in the twenty-four hours.

Of the amino acids which are found on the hydrolysis of pro- teins, the most notable are leucin and tyrosin, and with the classical statement of the presence of these substances in the urine in grave derangements of metabolism, the inquiry may be made: Is an in- crease in the amino acid fraction to be referred to the presence of these two compounds?

Leucin and Tyrosin. Special interest has always attached to leucin, ever since its demonstration by Frerichs and Stiideler*’ in 1858, in the urine, blood, and liver of acute yellow atrophy. From numerous observations, frequently verified, the presence of leucin

EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN 76]

and its associated aromatic amido acid tyrosin, came to be regarded as virtually pathognomonic of acute yellow atrophy and phosphorus poisoning. As the search for these substances became more extended, it appeared that they occur in smaller amounts in many other con- ditions, so that current text-books of clinical pathology commonly state that they are present in the urine of most infectious diseases, in cirrhosis of the liver, gout, hysteria, and many other conditions. Pouchet,” in i880, claimed to have demonstrated traces of leucin and tyrosin in normal urine, by methods which we have not been able to ascertain. The views of various authors regarding the occur- rence and significance of leucin and tyrosin do not agree. Minkow- ski®™ finds them present in the urine only when there is destruction of liver tissue. Throughout the literature there are constantly recorded cases of acute yellow atrophy and phosphorus poisoning, in which leucin and tyrosin were stated to be absent from the urine. In many of the cases in which they were reported as present, the identification of these compounds appears to have been based upon the microscopic appearance of certain crystals appearing in the urinary sediment, or in the concentrated filtrate after treatment with basic lead acetate. It seems probable that the somewhat conflicting reports have resulted, in part, from the readiness of one observer to accept as leucin certain crystals which resemble this substance, while others did not trust to the microscopic appearance, and failed to demonstrate leucin by more exacting chemical tests.

The clinical method commonly employed for the demonstration of leucin and tyrosin is known as the lead-acetate method, and was originally devised by Frerichs and Stiideler. The urine, freed from albumin, is precipitated with basic lead acetate, which removes much of the inorganic salts, and is filtered; the lead is removed by a current of hydrogen sulphide, and the filtrate evaporated on a water-bath to a syrup, and set aside to crystallize more slowly. In the residue, leucin is said to appear as “characteristic yellow balls with radial striations,” or as “bushy balls with radial projections,” or as “oily globules.” Various confirmatory tests ave recommended, such as the production of an oily globule when the crystals are heated on a platinum foil, or heating with mercurous nitrate which yields a deposit of metallic mercury.

Kirkbride” has given details of the partial isolation of leucin and its identification by the blue solution which leucin copper produces.

From the beginning of our observations on the urinary nitrogen, we employed the lead method for the demonstration of leucin and tyrosin. Crystals resembling leucin were often encountered in typhoid fever, in the toxemia of pregnancy, especially in yellow atrophy, in fatal alcoholism, in phosphorus poisoning, in fact in the typical conditions in which they have previously been found, and for a time we regarded these crystals as probably leucin. This impression was also strengthened by the success obtained in repro-

762 EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN

ducing exactly similar yellow, striated, conglomerate crystals from ere leucin, obtained from the digestion of casein. Dr. P. A.

vene kindly furnished us with a supply of this leucin, and we wish to express to him our sincere thanks.

However, a doubt always existed in our minds regarding the true nature of these urinary crystals, and when they were finally obtained from the urine of a comparatively healthy man, this doubt was strengthened, and led to a systematic study of the crystals and an effort to determine their true nature.

First were employed the tests recommended in text-books, and the crystals from acute yellow atrophy, which proved practically insoluble in water, failed to produce an oily globule on platinum foil, and did not sublime. But we did not succeed in these tests when pure leucin was directly added to the sediment, after the lead treat- ment, and we do not believe them to be of any value as clinical methods. ‘The accurate application of the tests for leucin, such as sublimation, in a mixture so complex as that resulting from the treatment of a urine with basic lead acetate and hydrogen sul- phide, is practically impossible. Next, pure leucin was added in con- siderable quantities to normal urine and the lead method applied, but no crystals resembling leucin appeared in the sediment. When, however, a larger quantity of pure leucin was added to 50 c.c. of normal urine, we did find certain large homogeneous crystals, soluble in water, appearing slowly after the sediment had stood for forty-eight hours. These we believe to have been leucin, but we have not ob- served such crystals in the urine of phosphorus poisoning and acute yellow atrophy. From the concentrated filtrates of urines, contain- ing the suspected crystals in large quantities, attempts were made to obtain the characteristic crystals of the copper salt of leucin, but without success. Finally, urines yielding the crystals in abundance were treated in large volumes by two of the newer reagents for the precipitation of leucin, a-naphthyl-isocyanate and /-naphthalin- sulphochlorid, but no leucin was demonstrated by these methods.

any of the forms of the crystals and their insolubility in water suggested that they were urates. When ammonium urate was added to normal alkalized urine, with subsequent neutralization by acetic acid, the crystals appeared in considerable numbers, and the murexid test for uric acid was obtained from them, showing that urates pass through in the lead method. Finally, the crystals obtained in phos- phorus poisoning of dogs, and from typhoid fever, were secured in quantity, and these gave a distinct murexid test for uric acid, and characteristic whetstone forms of uric acid when treated with hydro- chloric acid.

From these results it must be concluded that the demonstration of leucin by the microscopic identification of crystals after the lead method is: unreliable, and that most of the crystals, especially the yellow, bushy, and striated balls found in the urine of typhoid fever,

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acute yellow atrophy, and phosphorus poisoning in dogs, after the lead method, are not leucin, but urates, or possibly urea. That some of these crystals may be leucin need not be denied, but that any of them can be positively identified as leucin by the microscope must be doubted. Even the homogeneous oily globules of leucin may be closely simulated by urates, although the homogeneous urate balls are refractive while globules of leucin are almost transparent and non-refractive. When pure leucin crystallizes in bushy or radially striated balls, the striz are much more opaque than those of urate crystals, but we have not been able to identify such opaque striz in the crystals from the above conditions. We have also endeavored to distinguish the crystals of leucin from those of other substances by means of the polarizing microscope, but without success. Although these observations seem, to throw doubt on the value of many of the clinical reports of the discovery of leucin in the urine by the lead method, it seems possible that leucin crystals may really be obtained by this method. Leucin crystals, such as we have prepared, are very soluble in water; while urates are comparatively insoluble. In the sediment obtained from typhoid urine, on the addition of water to a specimen under the microscope, some of the crystals we have found to be rapidly dissolved. It is possible that these were leucin, but their structure seemed to be identical with that of the other crystals which remained undissolved. As a result of this work we have abandoned the attempt to detect leucin in the urine by the lead method, and feel that many previous observations on the occurrence of leucin in the urine are open to doubt. In some cases, e. g., that of W. G. Smith,” a “large amount of sediment” spontaneously apeestins in the urine has been taken for leucin. ‘The correctness of this observation we strongly doubt. Extremely small amounts of impurity in leucin are sufficient to increase its solubility to such an extent as to make its crystallization impossible, except in concen- trated solution. Anyone who has worked with the hydrolysis of proteins will confirm this statement. We have, ourselves, suffered from the influence of the current views regarding the nature of crystals obtained after the lead method, and in a brief review, pub- lished* by one of us, on the “'Toxeemia of Pregnancy,” and in a con- tribution on the same subject by Stone,” for whose chemical analyses we are responsible, prominence was given to the probable identity of such crystals with leucin.

The uncertainties inherent in the lead method and the micro- scopic identification of leucin have, doubtless, been the chief incen- tive to the search for other more reliable means of detecting leucin in the urine. Embden and Reese“ have found in normal urine a substance precipitated by 8-naphthalin-sulphochlorid, which had the microscopic appearance of leucin, and gave a solution with copper having the blue color of leucin copper. Thus, the statement made over twenty-five years ago by Pouchet, regarding the presence of

Vou. 131, no. 5.—mar, 1906. 50

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leucin in normal urine, receives a confirmation, and the great impor- tance hitherto attached to the presence of this principle in the urine may be seriously reduced.

With regard to the microscopic identification of tyrosin the same doubt is present, but the case is not quite so indistinct.

Abderhalden and Barker’ have shown conclusively the presence of tyrosin in phosphorus poisoning in dogs. Abderhalden and Schittenhelm*® were able to isolate tyrosin in abundance on con- centrating the urine of a patient suffering from cystinuria. As no mention is made in this paper of any severe symptoms associated with the condition, it may be inferred that the patient was excreting conspicuous quantities of tyrosin without being in any very imminent

ril.

It is also interesting to note that a residue obtained by Embden and Reese from normal urine contained a substance giving an intense Millon reaction. This they ascribe to the possible presence of tyrosin.

As far as reliable methods for the isolation of individual amino acids in the urine are concerned, two only have so far been used with success. ‘These are the §-naphthalin-sulphochlorid method proposed by Fischer and Bergell,** and the esterification method of Fischer used by Abderhalden and Barker.*

Both of these methods, especially the modification of the former, used by Embden and his co-workers, are quite without the range of clinical application.

The Other Constituents of the Amino Acid Filtrate. Unquestion- ably most of the compounds contained in the amino acid filtrate, if one excludes urea, are amino acids in the strict sense; they contain both a carboxyl and an amino group. With a few exceptions, no definite information is at hand regarding their exact compo- sition.

It has been stated, only very recently, that even by working up fifty litres of normal urine no trace of. the ordinary amino acids— such as glycocoll, alanin, phenylalanin, leucin, and tyrosin, ete.— were present. But a few months after the appearance of this state- ment Embden™ announced that by a modification of the Fischer- Bergell naphthalin-sulphochlorid method, the same method on which the previous statement was based, he had been able to isolate notable quantities of glycocoll from normal urine, and in three subse- quent papers by the same observer and his co-workers this has been confirmed. Ignatowski” by a less active modification of the method has found this compound in the urine of patients suffering from gout, and gave it as his opinion that the glycocoll output is increased in this affection. Most recently Lipstein™ has repeated the work under Embden’s direction, and has shown that at no period of the disease is the glycocoll elimination different from that in the

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normal individual. Here also we have an example of a distinct amino acid being isolated from the urine, where the significance was supposed to have been of great importance. Further work shows that its significance per se is by no means so momentous a was at one time supposed,

For the isolation of glycocoll and amino acids of this. type, one other reagent may be of value. This is the a-naphthyl isocyanate proposed by Neuberg.® We have controlled the action of this compound with that of §-naphthalin-sulphochlorid, and have found both reagents give practically the same results when amino acids are added to the urine. We have not, however, been able to detect these compounds in normal urine with the latter reagent. Embden and Reese” state that with their modification of the Fischer-Bergell method it is possible that the glycocoll which they find is in combina- tion with some other substance which prevents its detection in the ordinary way. When, however, the solution is strongly alkaline this compound is hydrolyzed, yielding free glycocoll. Some facts speak in favor of this view. Ignatowski,” using the old method, was able to isolate eighty per cent. of the glycocoll added to the urine, but was unable to obtain this acid in the urine itself. We have repeated his work with similar results.

If now the glycocoll present in normal urine is only to be obtained in the presence of strong alkali, and by long-continued shaking, forty-eight to ninety-six hours, it may well be that the glycocoll is present in the urine in the combined condition.

Of the other constituents of the amino acid fraction probably most are sulphur containing amino acids, not of the type of cystin, but much more complex. To them belong the oxyproteic acid of Bondzynski and Gottlieb,*® and the alloxyproteic and antoxypro- teic acids of Bondzynski and Panek.’ Bondzynski says that the nitrogen of these acids constitutes over one per cent. of the total nitrogen output of the urine. This can be only on approximation, but gives an idea of the nitrogenous excretion referable to such compounds, It is quite possible that these acids may come into importance in future discussions on the clinical side.

Zweifel” in his studies of eclampsia has pointed out what he believes to be a relationship between an increase in the neutral sulphur and the onset of the attack. A start in this direction is made by the assumption that one of the causative factors in eclampsia is a decrease in the oxydative power of the organism, which he believes to be expressed by an increase in the ratio of the neutral sulphur at the expense of the sulphates—alkaline and ethereal. It would seem that he has here fallen into the same error as have other observers, when dealing either with the nitrogen elimination or with the excretion of aceton compounds. He has not fully realized that the relationship of the compounds depends, to a very decided extent, on the sufficient or insufficient compensation of heat losses of the body

766 EWING, WOLF: SIGNIFICANCE OF THE URINARY NITROGEN

and on the nitrogen content of