BY FRANCIS P. CHINARD WITH THE TECHNICAL ASSISTANCE OF DORA M. NEWELL. (Received for publication, July 28, 1948)

Transcription

1 USE OF THE HYPOBROMITE REACTION FOR THE ESTIMATION OF AMMONIA PLUS UREA NITROGEN IN URINES CON- TAINING LARGE AMOUNTS OF PROTEIN; THE REAC- TION OF ALKALINE HYPOBROMITE WITH PROTEINS BY FRANCIS P. CHINARD WITH THE TECHNICAL ASSISTANCE OF DORA M. NEWELL (From the Hospital of The Rockefeller Institute for Medical Research, New York) (Received for publication, July 28, 1948) Alkaline hypohalites have been in use for nearly a century for the estimation of urea or of urea and ammonia nitrogen in blood and urine. Alkaline hypochlorite, first used by Davy (1) in 1854, was replaced a few years later by alkaline hypobromite as a result of Knop s work (2). The Nz evolved in the following reactions was measured volumetrically. (1) d = Br- ) Nz + CO2 + 3Br- + 2H20 I NH2 (2) 2NHd+ + 40Br- - Nz + 4Br- + 4HzO After Knop s work there was a spate of modifications of the method, modifications having to do with concentrations of the reagents, length of reaction time, design of apparatus, elimination, and occasionally identification of interfering substances. These modifications finally culminated in the manometric procedure of Van Slyke (3) as modified by Van Slyke and Kugel (4). This method has been extensively used both in this laboratory and elsewhere for the estimation of the blood and urine urea nitrogen from which, with knowledge of the urine flow, the urea clearance can be calculated. While reasonably satisfactory when only approximate results are required, the hypobromite methods all suffer from the fact that the hypobromite reaction is neither specific nor quantitative. Many other substances besides urea and ammonia evolve nitrogen; some, such as guanidine and mono-substituted guanidines, give off nitrogen in considerable amounts; others, such as amino acids, amines, and peptides, in smaller amounts. Proteins evolve N in amounts determined chiefly by their arginine content. In addition, glucose in the high concentrat,ions occasionally present in the urine of diabetics can cause error (5, 6). 1449

2 1450 HYPOBROMITE REACTION FOR URINE NH3 AND UREA N Under the conditions of blood or plasma analyses, the hypobromite liberates approximately 98 per cent of the urea nitrogen; under the conditions of the urine analyses, hypobromite liberates approximately 95 per cent of the urea and ammonia nitrogen (3, 4). In the case of blood or plasma, the presence of non-urea N-evolving substances in the filtrates requires that a subtractive correction be applied to the calculated results; in the case of urine, one depends on the evolution of nitrogen from nonurea or ammonia substances to compensate approximately for the deficit of 5 per cent in the nitrogen evolved from the urea and ammonia (3). In some cases, however, and especially when protein is present, the evolution of nitrogen instead of approximately compensating may introduce a positive error as high as 30 per cent. While it is possible to correct approximately for the protein error if the protein concentration in the urine is known, it is preferable to remove the proteins before doing the analyses. It is the purpose of the work reported here to present some additional data on possible sources of error inherent in the hypobromite procedure as at present applied (3,4) and to describe an obvious means of circumventing the protein error. In addition, the reaction of alkaline hypobromite with certain proteins and compounds will be described briefly. Removal 0.f Proteins from Urine The zinc hydroxide procedure of Somogyi (7) is used because it has been found to be effective in removing some of the interfering substances of blood as well as proteins (4). Reagents Acid zinc sulfate solution gm. of ZnS01.7Hz0 are dissolved in 125 ml. of 0.25 N HzS04 and diluted to 1 liter. This solution is used for the precipitation of proteins from whole blood. For urine and plasma 100 ml. of this solution are diluted to 212 ml. with water ~ NaOH. When 50 ml. of the zinc sulfate solution for whole blood are titrated with 0.75 N NaOH, with phenolphthalein as indicator, 6.7 to 6.8 ml. of the alkali should be required. The solution is shaken vigorously during the titration. Procedure For urine containing less than 80 gm. of protein per liter, 1 ml. of urine is pipetted into a centrifuge tube, and 8.5 ml. of the zinc sulfate diluted for urine or plasma are added, followed by 0.5 ml. of the 0.75 N NaOH. The tube is stoppered, shaken vigorously, and allowed to stand for 10 minutes. It is then centrifuged for 10 minutes at 2000 R.P.M. or more (18 cm. radius). The supernatant solution is then filtered through 3, pledget

3 F. P. CHINARD 1451 of washed cotton placed in the stem of a funnel, and aliquots are taken for analysis. A blank analysis is run on 0.9 per cent NaCl to correct for the non-urea nitrogen-liberating substances in the cotton and the reagents. For urine containing 80 or more gm. of protein per liter, 0.5 ml. samples of urine are taken, and 0.5 ml. of distilled water is added to each. The diluted urines are then treated as above. Details concerning manometric determination of urea in the filtrates and the factors used in calculations are given in the original publications (3, 4). TABLE I Comparison of Nitrogen Evolved from Urines Containing Protein and Protein-Free Urines - Urine No. - - N before 1 pot& removal (0) - gm. per Filtrates of Same (0) - 0) gm. per 1. gm. per (a) as per cent of (b) Protein concentration gm. per Comparison of N Evolved from Urine Containing Protein and from Protein- Free Filtrates of Same Urines Table I shows the differences in N evolved from urines containing protein and protein-free filtrates of the same urines. The urines were obtained from patients with the nephrotic syndrome, some of whom were receiving large doses of human serum albumin intravenously. Generally speaking, unless the urines are from patients receiving serum albumin or plasma

4 1452 HYPOBROMITE REACTION FOR URINE NH3 AND UREA N intravenously, urine protein concentrations will not exceed 25 to 30 gm. per liter, provided the urine flow is reasonably high (1 or more ml. per minute). If, however, the patient s urine flow is small (less than 1 ml. per minute), then the urine protein concentration may rise to 40 or 50 gm. per liter. The urine protein concentrations were calculated from nitrogen determinations done by the micro- or macro-kjeldahl procedures of Hiller, Plazin, and Van Slyke (8). The total nitrogen was determined directly, the non-protein nitrogen was determined after removal of the proteins by I I I I I I zr R Urine protein concentration in gram5 pep liter FIG. 1. Relationship of concentration of urine protein to difference in N evolved from urines and protein-free filtrates. precipitation with equal volumes of 10 per cent trichloroacetic acid, and the protein nitrogen was calculated from the difference. It will be noted that the errors due to protein are all positive and vary from +1.4 per cent to per cent. The magnitude of the percentage error is determined by the ratio of the protein N evolved by hypobromite to the N evolved from the urea plus ammonia, not by the absolute amount of protein present. Fig. 1, in which protein concentration is plotted against the difference in N evolved before and after removal of the proteins, shows that there is considerable scattering. A correction made on the basis of the data of Table I would be approximate, though adequate for clinical purposes. Fig. 1 is to be compared with Fig. 2 which shows, from analyses of pure

5 F. P. CHINARD 1453 albumin solutions, the relatively close proportionality of N evolved to albumin present under the conditions of routine urine analyses when other N-evolving substances are absent. An additional point is that, in proteinfree urine from normal individuals, treatment of the urine with the Somogyi reagents resulted in lower values for urea plus ammonia N than in untreated urine; this effect is presumably due to precipitation of interfering substances other than protein. It may be noted here that the reaction of alkaline hypobromite with proteins and amino acids is not restricted to the guanidino group of the arginine; tyrosine is brominated, free amino groups react, and there is also reaction with peptide groups (see, for example, Goldschmidt et ae. (9, 10)). These reactions occur with little or no evolution of N. The result is that much more hypobromite is used up in reaction with proteins than would be expected from the N liberated. This fact becomes of importance when the protein concentrations are very high, because the reaction with protein may not leave enough hypobromite to give the expected N yield from urea. This effect has been found in a few cases in which the urine was not sufficiently diluted before analysis; less N was evolved per aliquot of urine than after greater dilution. If protein-free filtrates are used, error from this effect of excess protein is avoided. Evolution of N from Various Proteins The evolution of N from human serum albumin was studied in some detail. Under the conditions of the routine urine analyses (3, 4) (2.5 minutes reaction time measured from the moment of addition of the alkaline hypobromite to the moment the solution was brought to the 2.0 ml. mark of the gas chamber for reading the volume), a reasonably stoichiometric relationship was found between mg. of N evolved and mg. of protein present, as illustrated in Fig. 2. It was found, however, that if the reaction time was prolonged beyond 2.5 minutes more nitrogen was evolved, though at a much slower rate than during the first 2.5 minutes. An example of the time course of the reaction is given in Fig. 3 for 20.0 mg. of human serum albumin (Curve A), and for mg. of bovine r-globulin (Curve B). Similar curves were obtained with edestin and gelatin. Because arginine is the only guanidine derivative reported in proteins in appreciable amounts, it was thought that the N evolved from intact proteins by alkaline hypobromite might give a measure of the arginine content of those proteins. It was found, however, in the few proteins examined that less N was evolved than was calculated from the arginine contents. While the results were quite reproducible provided the reaction times were the same, in no case was the calculated amount of N evolved. The data in Table II were obtained from proteins in aqueous solutions of

6 1454 RYPOBROMITE REACTION FOR URINE NH3 AND UREA N Human serum albumin concentoation jn grams pep liter FIG. 2. Evolution of N from various amounts of human serum albumin in 2.5 minutes $ 0.14 a $ t$ I I I I I I I III lime in minute5 FIG. 3. Evolution of N from 20.0 mg. of human serum albumin (Curve A) and from mg. of bovine r-globulin (Curve B).

7 F. P. CHINARD 1455 approximately 1 per cent concentration, and the results were calculated on the basis of total protein N as determined by macro-kjeldahl analysis (8). The values for arginine N content were taken from tables published by Chibnall (11) and Brand (12) and were not determined by analysis of the protein preparations used in these experiments; for this reason the data should be considered as preliminary. TABLE Evolution of Nitrogen from Various Proteins by Alkaline Hypobromite II Protein Calculated Calculated N N evolved by N e,olved argmme N evolvab!e by hypobromite in sample hypobromite* in 2.5 min. as per cent of (cl (6) (a) w. w. Human serum albumin Gelatin......I EZ ~ EEE; 1 Et& ~ glef% 1 iii Edestin..., * Calculated il; corrected for 5 per cent deficit in N evolved from urea and arginine under the conditions used for the analysis. Calculated arginine K is based on data of Brand (12) for human serum albumin. For gelatin and edestin, calculat,ions are based on data assembled by Chibnall (11). Evolution of N by Hypobromite Reaction from Guanidine, Guanidine Derivatives, Amino Acids, and Other Compounds Under the conditions of the routine urinalysis, many substances besides proteins mere found to evolve N when they reacted with alkaline hypobromite. In particular, guanidine and its derivatives of the type R~Rz- N-C( = NH)-NH2 (where RI and R2 may be H, alkyl, or aryl groups) give off nearly two-thirds of their guanidine nitrogen. Of such a type are, for example, arginine, methylguanidine, creatine, and guanidoacetic acid, and of a similar type are dicyandiamide and guanylurea. The evolution of N from the guanidino groups is quite rapid with these compounds and is complete or nearly complete in the 2.5 minutes required for urine analysis. Another group of substances, aliphatic diamines, evolve N at a slower but still appreciable rate. Pentamethylene- and hexamethylenediamine, ornithine and lysine evolve about 3 per cent of their total nitrogen in 2.5 minutes, and about 10 per cent of their total nitrogen in 10 minutes. This evolution of nitrogen may stem from nitrile formation, ring closure to form an amidine, and reaction of this latter group with more hypobromite. In contrast, monoamino-, mono-, or dicarboxylic acids, asparagine, glutamine, and creatine evolve practically no N in the routine reaction time. Glutathione evolves slightly less N per mole than do the

8 1456 HYPOBROMITE REACTION FOR URINE NH3 AND UREA N diamines. Mono-N-substituted ureas evolve nearly one-half their total urea nitrogen. In addition to the above nitrogen compounds, glucose also evolves gas which is measured as N in the routine analyses. A 1 per cent solution of glucose evolves an amount of gas equivalent to approximately gm. of N per liter. Hence the hypobromite method should not be employed for analyses of urine from diabetics. DISCUSSION The sources of error and their significance are apparent from the above paragraphs. Of ancillary interest is the finding that not all of the arginine guanidino groups in proteins appear to be available for reaction with alkaline hypobromite under the condition used; this suggests that some of these groups may be involved in linkages and are therefore not free. (There is little likelihood that this effect is due to exhaustion of the OEW because of the proportionality of the results with different amounts of protein. The expected N is evolved from protein-urea mixtures.) Similar observations have been made in the case of egg albumin by Goldschmidt et al. (9); in this protein even after a 4 hour exposure to hypobromite, there was still some intact arginine which could be demonstrated, suggesting that the arginine linkages in proteins were not all identical and that some of the guanidino groups were protected. Of similar import are the observations of Roche and his collaborators (see, for example, (13)) who have used the Sakaguchi reaction on intact proteins and found less color developed than would be expected from the total arginine content of the proteins examined. In addition, Simms has suggested, from the evidence offered by his titration curves of certain proteins, that some of the arginine guanidino groups are somehow linked to other portions of the protein molecule (14). Further work is under way at present on the reaction of hypobromite with proteins and guanidine derivatives; preliminary experiments suggest that the hypobromite reaction may be of some use for rapid semimicroestima- Con of arginine in protein hydrolysates (cf. (15)). SUMMARY Under the conditions used for gasometric determination of urea and ammonia by the hypobromite reaction, the greater part of the guanidino groups in proteins reacts with evolution of nitrogen gas. In urine with high protein concentration (e.g. 30 or more gm. per liter) the nitrogen gas evolved from the proteins may cause a plus error of as much as 30 per cent in the urea determination. This error can be prevented by preliminary removal of the proteins by Somogyi s zinc hydroxide precipitation. The reaction of hypobromite with human serum albumin, bovine y-

9 F. P. CHINARD 1457 globulin, edestin, and gelatin has been studied with regard to its time course and evolution of total nitrogen gas. The final amounts of nitrogen gas evolved were from 64 to 93 per cent of that which would be evolved by the guanidino groups of arginine in the amounts reported to be present in these proteins. It appears that some of the guanidino groups in the protein molecules are not free to react with alkaline hypobromite. The reactions of other substances with hypobromite have been reviewed. Glucose, if present in more than 2 per cent concentration, will cause evolution of enough gas to produce a significant positive error in determination of urine urea. BIBLIOGRAPHY 1. Davy, E. W., Phil. Mug., 7, 385 (1854). 2. Knop, W., and Wolf, W., Chem. Zent., 6, 257 (1860). 3. Van Slyke, D. D., J. Biol. Chem., 83,449 (1929). 4. Van Slyke, D. D., and Kugel, V. H., J. Biol. Chem., 102,489 (1933). 5. Esbach, G., Compt. rend. Acad., 89, 417 (1879). 6. Krogh, Rf., 2. physiol. Chem., 84, 379 (1913). 7. Somogyi, M., J. Biol. Chem., 86,655 (1930). 8. Hiller, A., Plazin, J., and Van Slyke, D. D., J. BioZ. Chem., 176, 1401 (1948). 9. Goldschmidt, S., Wolff, R. R., Engel, L., and Gerisch, E., Z. physiol. Chem., 189, 193 (1930). 10. Goldschmidt, S., Wiberg, E., Nagel, F., and Martin, R., Ann. Chem., 466,l (1927). 11. Chibnall, A. C., J. Znternat. Sot. Leather Trades Chem., 30, 1 (1946). 12. Brand, E., Ann. New York Acad. SC., 47,187 (1946). 13. Roche, J., and Blanc-Jean, G., Compt. rend. Acad., 210,681 (1940). 14. Simms, H. S., J. Gen. Physiol., 14, 87 (1930). 15. Tsuverkalov, D. A., Biokhimyia, 9, 101 (1944).

10 USE OF THE HYPOBROMITE REACTION FOR THE ESTIMATION OF AMMONIA PLUS UREA NITROGEN IN URINES CONTAINING LARGE AMOUNTS OF PROTEIN; THE REACTION OF ALKALINE HYPOBROMITE WITH PROTEINS Francis P. Chinard and With the technical assistance of Dora M. Newell J. Biol. Chem. 1948, 176: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at html#ref-list-1