THE EFFECT OF SODIUM BENZOATE INGESTION UPON THE COMPOSITION OF THE BLOOD AND URINE WITH ESPECIAL REFERENCE TO THE POSSIBLE SYNTHESIS OF GLYCINE IN THE BODY. PRELIMINARY PAPER. BY W. W. SWANSO (Prom the Biochemical Laboratory, Department of Physiology, University of Minnesota, Minneapolis.) (Received for publication, June 26, 1924.) Since the work of Wiechowski (1) in 1906 several investigators have confirmed the fact that the excretion of urea is decreased after ingestion of sodium benzoate. In 1916 Lewis and Karr (2) found that uric acid was also markedly decreased in the urine under similar conditions. From these findings it was suggested that, where there is an abnormal demand for glycine, this amino acid is synthesized, in part, from the endproducts of protein metabolism. Recent experiments by Gibson and Doisy (3) indicate that some organic acids such as lactic and benzoic have the tendency to decrease the excretion of uric acid and to cause a slight increase of the same in the blood. However, figures showing the effect of the ingestion of sodium benzoate were not included in their paper. It is the aim in this paper to present values for the nonprotein nitrogen constituents in the blood and urine obtained by simultaneous analyses after ingestion of sodium benzoate; further, to investigate what bearing these values may have, if any, on the synthetic ability of the human body to produce glycine for conjugation with the benzoic acid. Analytical Methods. All the experiments were done on young healthy individuals. The blood was drawn from an arm vein and immediately transferred to a testtube containing 20 mg. of potassium oxalate per 565
566 Sodium Benzoate on Blood and Urine 10 of blood to prevent clotting. Filtrates were always prepared a few hours after being drawn and in every instance analysis of the blood was completed during the ensuing 24 hours. Drawn blood as well as filtrates were always kept in the refrigerator to minimize changes in composition. The preparation of proteinfree blood filtrates and determination of total nonprotein nitrogen, urea, and sugar were done according to directions found in Folin s Manual (4). Duplicate analyses were always made and, if these did not check, a second determination was completed. The uric acid in the blood was determined by the new method of Benedict (5). The amino acid nitrogen determination was carried out on the same filtrates using Folin s (6) calorimetric method, and the plasma proteins by the calorimetric method of Wu (7). In order that the data presented should be more complete, hourly specimens of urine were taken during the experimental period. A 4 hour morning specimen was generally used as control. The urines were preserved by adding 2 of a 2 per cent nitric acid solution and a few drops of a 10 per cent solution of %hymolchloroform. The amino acid nitrogen determination in urine was made as described by Folin (8) on hourly specimens. The determination of hippuric acid was carried out by the method of Kingsbury and the author (9), uric acid by the method of Benedict and Franke (lo), and urea nitrogen by the method described in Folin s Manual (4). To insure uniformity and obviate the influence of food, a slice of toast and a cup of coffee were taken in the morning, but no further food during the time of the experiment. There was no restriction placed upon the water intake, and in none of the experiments did the intake exceed the ordinary amount for the individual. EXPERIMENTAL. Tables I and II are submitted as controls for the work to follow. The constituents listed for blood and urine are well within the normal range when compared with values reported in the literature. In Tables III, IV, and V are shown the values for blood and urine after ingestion of 3 to 4 gm. of sodium benzoate. Some
W. W. Swanson 567 TABLE I.* Subject, W. W. S. Weight, 76 kilos. Whole blood. Plasma. A$;(y ur non: Uric protan acid. SUgar. Aa;jy Urea non Uric N prot;in acid. SUSW. ~~ ~ nlg.pe7 mg.per m7,per?ng.per mg.pnr mg.per mg.per mg.per no.wr %?.PW IOOCC. ioo 1oo IOOCC. 1oo ioo 10O 100ec. 10O IOOCC. 12.20 8.3 17.0 37 3.0 90 6.4 16.6 23 3.6 97 6.15 7.0 16.6 33 3.0 83 6.0 16.6 22 3.4 91 * Average of a series of five experiments. TABLE II. * Subject, W. W. 8. Weight, 76 kilos. Analysis on 1 hr. specimens of urine. Volume a.?% 6.40 to 11.30 54 12.30 58 1.40 60 3.30 38 4.30 36 * Average of a series of three experiments. TABLE Subject, W. W. S. Weight, 76 kilos. a:m. Whole blood. Plasma. mg. per mg. per mg.per mg.per n&g.pel 1oo 1oo 1oo loom. ioo 7 20to 11.oo* 6.6 19 33 5.5 16 7 12.00 6.4 14 30 5.5 15.0 1.00 6.4 15 32 5.9 13.0 2.00 5.7 15 30 5.9 14.0 4.30 5.7 15 30 6.2 15.0 III. rota1 nonrotein I VOIper ume hr. 30 46 27 71 28 253 30 77 27 46 acid Urine WY. 4.2 4.7 3.7 3.2 3.2 9. i gm. gm. 6.7 0.084 0.035 5.9 IO.994 0.44 5.1 1.85 0.77 3.9 0.73 0.31 3.5 / 0.18 0.07 * Took 3.34 gm. of sodium benzoate.
i li 568 Sodium Benzoate on Blood and Urine TABLE Subjm ec t, H. H. J. Weight, 86 kilos. IV. a.m. 11.30* 12.30 1.30 2.30 3.30 5.00 hint acid 7.9 8.5 8.2 Whole, _. 1 blood. nonrotein aid 7.0 7.6 8.4 *Took 3.65 gm. of sodium benzoate. Subject, B. B. Weight, 76 kilos. a.m. acid % r 6.40 to 11.15* 11.0 p.n. 12.15 8.3 1.15 1.20 8.7 3.15 8.7 Plasma. Urine non prg? TABLE V. VOlUme acid mg. 60 6.0 140 5.4 405 63 88 4.5 50 5.0 26 3.8 Whole blood. Urine Urea ;86 CT *Took 3.5 gm. oj i sodium Volume acid ml. 12.5 36 86 8.2 15.7 34 106 6.3 95 4.8 12.0 31 16.0 32 41 3.4 benzoate. Hip t; % gm. Glycine.!am. 1.56 0.652 1.89 0.794 0.62 0.262 Glycine. gm. 0.553 0.658 TABLE VI. Subject, A. G. M. Weight, 65 kilos. Whole blood. Plasma proteins. I nonarid Urea protein Fibrin. Globulin. Albumin. ;nogd :T,?% K % ET 12.20* 8.2 16.6 48.4 2.10 7.4 10.8 31.2 3.30 9.3 12.5 43.2 *Took 3.30 gm. of sodium benzoatc. 0.440 per cent per cent per cent 0.22 2.1 5.2 0.23 2.0 5.1 0.22 2.0 5.2
W. W. Swanson 569 variability appears to exist in the values obtained for the different individuals. These variations are, however, too small to draw any definite conclusions from, but may indicate to some extent the effect of the benzoate upon normal values in blood and urine. TABLE Subject, W. W. S. Weight, 76 kilos. Whole blood. I Plasma. Plasma proteins. 7% r 12.00* 7.2 2.00* 7.0 4.00 59 6.00 5.5 lz.m. 7.00 to 11.50 12.00* 12.50 1.50 2.00* 2.50 3.50 5.00 6.30 i? mg. per 100 protan nony mg.per IOOCC. 23.0 46 17.5 42 16.5 34 17.6 34 acid VII. %i %T 7% Urea p;;;;n iy % :LY 6.4 19.4 28.4 4.5 18.7 26.0 4.9 18.0 25.0 5.0 20.0 25.0 Fibrin. per cent Urine I acid mg. m7. 60 7.5 540 Globulin. per cent 0.32 2.2 0.33 2.3 0.33 2.3 0.34 2.3 Albumin. per cent 4.7 4.4 4.4 4.7 Urea Uric acid. Glycine. 198 7.2 166 13 1.03 0.43 215 5.5 150 11 1.25 0.52 170 4.9 136 8 1.44 0.60 172 5.3 136 7 1.56 0.65 134 4.6 134 9 2.86 1.20 90 4.3 135 15 1.13 0.47 * At 12.00 n. took 3.35 gm. and at 2.00, 6.70 gm. of sodium benzoate. mg. 62 cl, In Tables VI and VII is shown the stability of plasma proteins under varying quantities of ingested sodium benzoate. The blood values in Table VII with a trebled dose of sodium benzoate differ in no particular from those listed in Tables III, IV, and V. A more complete analysis of the urine in Table VII confirms again the established facts that urea and uric acid are markedly decreased after ingestion of benzoate.
TABLE Subject, A. G. M. Weight, 65 kilos. VIII. Whole blood. Urea 1 Uricacid. 1 igy 1 Sugar. Urea Uric acid. 12.40* 2.40 4.40 Urine Volume Urea w. 8.00a.m. to 12.40* 40 420 1.40 51 216 2.40 65 140 3.40 163 195 4.55 40 150 * Took 5 gm. of sodium benzoate. TABLE Subject, W. W. S. Weight, 76 kilos. Urea Uric acid. 7% ::r % r 12.40% 19.0 3.6 2.40 15.4 4.0 4.40 15.0 4.0 5.40 14.0 4.4 IX. Whole blood. Plasma. nonprotein %!F 33.0 31.0 36.0 30.0 Urea N Uric a& Uric % r %?i :ct 17.0 4.2 16.8 4.5 16.0 4.5 acid mg. 28 9 8 9 12 1,, nonprotem ~ mg. per 100 25 22 22 brine Volume 7.30a.m.,to12.40* 35 1.45 90 2.50 85 3.50 200 4.50 345 5.50 66 Urea Uricaoid w. w?. 580 24 332 6 280 4 380 7 345 9 228 14 * Took 10 gm. of sodium benzoate. 570
W. W. Swanson 571 To show just what happens to the blood uric acid Tables VIII, IX, X, and XI are submitted. There is a definite rise in the uric acid content of both the whole blood and plasma, while the urine analyzed simultaneously shows the marked decrease. Subject, Wm. K. Weight, 70 kilos. TABLE X. a.m. I Uric acid. I Whole blood. mg. per 100 7.15 to 8.15 F3.00* 4.3 8.35 4.3 10.45 p.?n. 1.30 4.3 3.30 4.7 * Took 10 gm. of sodium benzoate. Plasma. nag. per 100 5.3 5.3 TABLE XI. Subject, Wm. K. Weight, 70 kilos. Volume 22 52 5.3 52 5.7 I 37 Uric acid. I Whole blood. Volume a.m. 7.00 to 8.00 8.30* 10.30 12.00 2.30 mg. per 100 CC. mg. per 100 CC. 4.0 5.0 4.2 5.3 4.7 5.2 24 Urine Urine Uric Uric T7. 18 3 3 4 acid. acid. m0. 18.0 60 2.7 52 2.0 33 2.5 * Took 10 gm. of sodium benzoate. DISCUSSIO From the experimental findings there may be some support for the supposition that the constituents, which during ordinary metabolism enter into the composition of urea, may in part be synthesized into glycine. If it were merely a retardation of excretion, then there should be an increase of urea nitrogen in the blood. This, however, is not the condition, for in most experiments the blood urea nitrogen is slightly decreased.
572 Sodium Benzoate on Blood and Urine The values obtained for uric acid in blood lead to a different conclusion. While the experiments are few in number, there appears to be a gradual increase in uric acid in the blood and plasma after benzoate ingestion. This fact seems to contradict the suggestion of Lewis and Karr (2) who thought that a decrease of the uric acid in the urine might mean a possible hydrolysis of this acid and synthesis of the resulting products into glycine. It is more probable that the excretion of uric acid is slightly retarded whenever large amounts of hippuric acid are being eliminated. It was hoped that a study of the blood and urine amino acid content, after sodium benzoate ingestion might throw some light on the nature of the circulating amino acids; that is, if the total amount of these was moderately lessened at the time of rapid hippuric acid formation, it would be reasonable to believe that t,he circulating amino acids were largely composed of glycine. Experiments recorded in Tables III, IV, and VII, showing little or no appreciable difference in the plasma amino acid after relatively large doses of sodium benzoate, indicate that this is not the case. The urine amino acid per hour shows a decrease after benzoate ingestion in all the experiments; but this is also noted in the experiment shown in Table II in which no benzoate had been taken and is due merely to inanition. SUMMARY AND CONCLUSIO After ingestion of sodium benzoate the simultaneous analyses of blood and urine of normal individuals show : (1) no increase in the urea nitrogen content of the whole blood and plasma, but a decrease in the urea nitrogen of the urine; (2) an increase of the uric acid content of the whole blood and plasma, and a marked decrease in the uric acid content of the urine; (3) the glycine portion of the blood amino acids and of those which are normally excreted while fasting appears to be only a small percentage, if any, of the total; and (4) the experimental findings suggest a probable synthesis of glycine from the constituents which normally are converted into urea, but it appears improbable that uric acid supplies any glycine.
W. W. Swanson 573 BIBLIOGRAPHY. 1. Wiechowski, W., Beitr. them. Physiol. u. Path., 1906, vii, 204. 2. Lewis, H. B., and Karr, W. G., J. Biol. Chem., 1916, xxv, 13. 3. Gibson, H. V., and Doisy, E. A., J. Biol. Chem., 1923, Iv, 605. 4. Folin, O., Laboratory manual of biological chemistry, New York and London, 1922, 3rd edition, 229291. 5. Benedict, S. R., J. Biol. Chem., 1922, li, 187. 6. Folin, O., J. Biol. Chem., 1922, Ii, 377. 7. Wu, H., J. Biol. Chem., 1922, li, 33. 8. Folin, O., J. Biol. Chem., 1922, li, 393. 9. Kingsbury, F. B., and Swanson, W. W., J. Biol. Chem., 1921, xlviii, 13. 10. Benedict, S. R., and Franke, E., J. Biol. Chem., 1922, lii, 387.
THE EFFECT OF SODIUM BENZOATE INGESTION UPON THE COMPOSITION OF THE BLOOD AND URINE WITH ESPECIAL REFERENCE TO THE POSSIBLE SYNTHESIS OF GLYCINE IN THE BODY: PRELIMINARY PAPER W. W. Swanson J. Biol. Chem. 1925, 62:565573. Access the most updated version of this article at http://www.jbc.org/content/62/3/565.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's email alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/62/3/565.citation.full.ht ml#reflist1