METABOLIC INVESTIGATIONS ON A CASE OF PHENYLPYRUVIC OLIGOPHRENIA* BY GEORGE A. JERVIS (From the Research Department, Letchworth Village, New York State Department of Mental Hygiene, Thiells) (Received for publication, April 20, 1938) An apparently new type of metabolic abnormality in man has been described recently by Foiling (1). This author reports having found phenylpyruvic acid in the urine of ten mentally defective patients. Shortly thereafter, similar cases were discovered by Penrose (2) in England, and by the writer (3) in the Unit,ed States. The term phenylpyruvic oligophrenia has been proposed for this condition. The present paper records the effects of feeding various compounds to a patient afflicted with this peculiarity. The subject, B. J., was a well developed and otherwise healthy male imbecile, aged 28 years. During the experiments he received two basal diets. The first (Diet A) was nitrogenfree. It consisted of 300 of cornstarch and 150 of sugar a day. The daily urinary excretion of nitrogen was thus reduced to 2 to 3, and the phenylpyruvic acid output to 250 to 350 mg. The second diet (Diet B) consisted of Cream of Wheat 100, bread 130, and milk 400 for breakfast; and two eggs, potatoes 225 to 230, bread 150, milk 400, and two oranges for both lunch and supper. The daily excretion of nitrogen and phenylpyruvic acid was constant within a range which was considered sufficiently satisfactory, the value of nitrogen ranging from 14 to 15, and the value of phenylpyruvic acid from 1.8 to 2.1 Methods All determinations were performed on 24 hour specimens of urine. Total nitrogen was determined by the usual Kjeldahl * Aided by a grant from Child Neurology Research (Friedsam Foundation). 305
306 Phenylpyruvic Oligophrenia method, urea according to the urease method of Van Slyke and Cullen, and ammonia with Folin s aeration procedure. The ph, amino acid nitrogen, and creatinine were determined colorimetritally. The figures for urea, ammonia, preformed creatinine, and ph showed nothing of significance, and have been omitted from the tables. For the quantitative determination of phenylpyruvic acid, the calorimetric method of Penrose and Quastel (4) was used. In addition, a gravimetric technique was worked out, based on the capacity of 2,4dinitrophenylhydrazine to form insoluble hydrazones with keto acids (Neuberg and Kobel (5)). To 100 cc. of filtered urine, 300 mg. of 2,4dinitrophenylhydrazine dissolved in 5 cc. of hot 2 N HCl were added. A precipitate of dinitrophenylhydrazone formed immediately. The mixture, after standing for several hours at room temperature, was extracted with ether. After evaporation of the ether, the hydrazone was dissolved in a small amount of saturated sodium carbonate and reprecipitated by acidification. The precipitate was removed by filtration through a Jena crucible, washed with 200 cc. of warm 2 N HCI in order to remove residua of hydrazine, and then with 200 cc. of water. It was dried overnight in an oven at 80 and weighed. A considerably easier method, based upon the capacity of phenylpyruvic acid to combine, molecule for molecule, with sodium bisulfite (Hemmer14 (6)), was also used in all determinations, but was not so accurate as the two procedures mentioned above. The oxidation method described in a previous paper (3) yielded constantly higher figures, since hippuric and other chloroformsoluble acids cannot be completely eliminated. Administration of Phenykdanine The racemic and the two optically active forms of phenylalanine (HoffmannLa Roche) were repeatedly fed, the patient being kept on both Diets A and B. After each feeding, 100 cc. of urine, from which phenylpyruvic acid had been removed by precipitation as the hydrazone, were extracted with butyl alcohol. The alcohol was evaporated, and the dry residuum was tested for phenylalanine according to the calorimetric method of Kappeler Adler (7). In no case was phenylalanine detected. As seen in Table I, a rise in the urinary output of phenyl
G. A. Jervis 307 pyruvic acid constantly followed the ingestion of phenylalanine. This observation, which was originally reported by Fijlling (l), appears of basic importance. There is convincing evidence, derived from feeding experiments (Kotake (8), Chandler and Lewis (9)) and from oxidation studies in vitro with tissue slices (Krebs (lo), Neber (ll), Lieben and Kretschmayer (12)) in support of the assumption that phenylpyruvic acid is an inter Form of oom p%d (5 pm.1 T Feeding Date Total N May 1617 (I 18 ( 19 20 June 1617 ( 18 l1 19 20 24 25 26 Dec. 13 14 1516 17 l 18 I 19 20 I 21 TABLE of Phenylalanine Amino aoid N w. 15.5 0.24 1.37 1.96 16.1 0.31 1.31 3.28 14.9 0.18 1.39 2.25 14.3 0.16 1.27 1.77 13.9 0.16 1.44 1.70 14.0 0.29 1.42 2.40 14.6 0.16 1.49 2.41 15.5 0.16 1.39 2.04 13.9 0.17 1.39 1.79 16.4 0.29 1.40 3.04 15.0 0.19 1.43 2.16 4.1 0.13 1.62 0.63 3.6 0.18 1.17 1.30 3.6 0.10 1.32 0.52 3.9 0.11 1.50 0.91 3.3 0.13 1.50 0.78 3.7 0.12 1.55 0.50 3.7 0.16 1.28 1.Ol 3.8 0.19 1.38 0.63 I Total creatinine E henylpyruvic a&d I: Colorimetria Hydrahe 2.07 2.96 2.14 1.78 1.71 2.34 2.37 2.00 1.97 2.84 0.88 1.37 0.75 1.28 0.81 0.76 1.06 0.74 I 12.9 19.3 14.7 12.4 12.3 16.9 16.3 13.0 13.5 17.8 14.4 18.4 37.l 17.6 2s.l 24.1 17.0 27.a 18.0 mediary step in the metabolism of phenylalanine. It is, therefore, reasonable to assume that the phenylpyruvic acid present in the urine of the patient was derived from the incomplete oxidation of the amino acid. Following the ingestion of other pure amino acids, including glycine 20, alanine 10, valine 10, leucine 10, cystine 10, and tryptophane 4, no increased excretion of the ketonic acid was observed (Table II).
308 Phenylpyruvic Oligophrenia That phenylalanine is the source of the phenylpyruvic acid appears also from the following experiments. Proteins containing various amounts of phenylalanine were fed, the content of phenylalanine of each having been determined by the KappelerAdler method (7) : gelatin, casein, and edestin 100 each, representing 0.4, 4.4, and 3.8 of phenylalanine respectively. As shown in Table III, the higher the phenylalanine content of the protein, the greater the increase in output of phenylpyruvic acid. Compound Glycyldlphenylalanine, 3.5 fed Phenyl serine, 5 Ureido, 5 Glycine, Alanine, 20 10 Valine, 10 Leucine, 10 Cystine, 10 Tryptophane, 4 TABLE II Feeding of Amino Mar. 6 7 roi1 P. 14.3 15.1 I 8 16.0 Jan. 10 3.5 11 4.6 12 4.0 Sept. 28 16.5 29 18.6 Oct. 5 15.7 6 14.3 7 15.8 8 15.5 9 16.0 10 16.3 21 3.0 22 2.9 Acids 0.11 0.12 0.14 0.15 0.19 0.17 0.26 0.28 0.36 0.48 0.22 0.07 Total I: matinine :&Ximetric 3ydW rim 1.35 1.74 1.70 1.34 2.33 2.42 I Ratio, P henyla,k?f 12.0 15.7 1.40 2.00 2.00 12.5 1.40 0.38 0.47 12.1 1.40 0.46 0.51 10.5 1.33 0.35 0.40 9.4 1.39 1.98 2.03 12.2 1.39 2.05 2.02 10.9 1.34 1.90 2.00 12.4 1.30 1.90 1.92 13.4 1.52 1.88 2.12 12.7 1.41 1.98 1.88 12.5 1.50 2.14 1.90 12.6 1.53 1.69 2.13 11.7 1.34 0.36 0.37 12.2 1.41 0.40 0.41 14.0 When the deamination of phenylalanine is blocked, no increase in the urinary output of phenylpyruvic acid results, as is shown in feeding experiments with the ureido derivative of phenylalanine (Table II). Negative results were also obtained following the ingestion of @hydroxyphenylalanine in which the presence of the phydroxyl group prevents OL oxidation (Dakin (13)). On the other hand, the ingestion of glycyldlphenylalanine is followed by an increased output of phenylpyruvic acid. It will be noted that the output of phenylpyruvic acid was not
G. A. Jervis 309 equivalent to the total amount of phenylalanine fed. It appears, therefore, that part of the amino acid was completely oxidized, since none was recovered as such in the urine. This finding may be explained either by an alternative path in the oxidation of phenylalanine through tyrosine (Embden and Baldes (14), Kotake (8), Medes (15), Edson (16)), or by the assumption that the metabolic error in our subject was not complete. The study of the behavior of the two optically active forms of phenylalanine appears of some interest. It will br noted (Table I) that on both diets the excretion of phenylpyruvic acid was greater after feeding the d form than after feeding the 1 form. These results appear to indicate that the naturally occurring Lphenylalanine is utilized more readily than its optical isomer, as has been Compound fed (100 ) Date Gelatin Casein Edestin TABLE Feeding of Various Proteins Mar. 9 10 17 18 21 22 _ III T?l Amino acid N 14.9 0.17 21.5 0.16 15.9 0.18 24.6 0.25 14.8 0.14 21.5 0.24 Total reatinine Phenzp Colorimetric P. Pm 1.37 1.95 1.40 2.06 1.42 2.05 1.52 2.67 1.35 1.81 1.43 2.35 Ratio, phenyl &mine Hydra to N iine I_ 9. 1.94 13.1 2.09 9.2 1.84 12.2 2.75 11.0 1.68 11.8 2.27 10.8 shown to be the case in the normal human organism by Kotake et al. (17), and in animals by Chandler and Lewis (9). The formyl and acetyl derivat.ives of both forms of phenylalanine, prepared according to the directions of du Vigneaud and Meyer (18), were also fed to the patient. As is shown in Table IV, the formyl and acetyl derivatives of dphenylalanine are not oxidized to the stage of phenylpyruvic acid, whereas the derivatives of the natural enantiomorph undergo oxidation in the organism, since they yield a small but constant increase in the output of phenylpyruvic acid. These results appear in agreement with evidence obtained in growth experiments (du Vigneaud et al. (19), Jackson and Block (20)), indicating that the hydrolytic
310 Phenylpyruvic Oligophrenia reaction which occurs before the utilization of an amino acid is extremely specific with respect to spatial configuration. Effects of Phenylpyruvic Acid and Certain Related Compounds Phenylpyruvic acid, prepared according to the directions of Hemmerle (6), was fed repeatedly. A rapid rise in the urinary output of the acid, and a marked increase in the ratio of phenylpyruvic acid to nitrogen, constantly followed (Table V). It seems confirmed, therefore, that this acid was broken down with difficulty in the organism of the patient. An increase in the out TABLE Feeding of Formyl and Acetylphenylalanine Compound fed (6 ) Date Formyldphenylalanine FormylZphenylalanine AcetylZphenylalanine Acetyldphenylalanine Aug. 2 Mar. 3 IV T??l Cm. 2.7 2.8 I 4 2.7 Feb. 5 2.6 6 2.9 7 2.4 8 2.4 9 2.4 l 10 2.5 11 2.6 P Lmina acid N. _ 0.05 0.07?hcnylpyruvic acid Morinetric IJidra zinc am.!jm. 1.27 0.33 0.31 1.24 0.29 0.32 1.32 0.32 1.05 0.43 1.09 0.41 1.08 0.33 1.10 0.47 0.94 0.48 1.15 0.35 1.06 0.31 0.33 0.44 0.29 0.49 0.40 0.43 0.36 Ratio, hmyl, ; o z 11.9 10.9 12.0 18.6 14.7 12.9 a0.o 18.9 15.6 12.9 put of phenylpyruvic acid occurred also following the ingestion of phenyllactic acid (5 ), whereas no increase was obtained with the phenyl derivatives of other fatty acids containing 3 carbon atoms in the aliphatic chain (Table V). Influence of Tyrosine and Tyrosine Derivatives Tyrosine (HoffmannLa Roche) in doses of 10 and 25 was without influence upon the urinary excretion of phenylpyruvic acid. Nor were increases observed following the administration of diiodotyrosine 4, glycyltyrosine 4, dihydroxyphenylalanine 3, homogentisic acid 3, and phydroxyphenylpyruvie acid 3 (Table VI). In each of these experiments the urine
Tyrosine, TABLE V Feeding of Phenylpyruvic Acid and Other Compounds Acid fed (5 em.) Phenylpyruvic Phenylpyruvic Phenyllactic Phenylglyceric Phenylpropionic Cinnamic Phenylacetic T Feeding Date Jan. 12 ( 13 14 15 Sept. 13 14 15 16 Jan. 19 20 21 8 9 I 10 Apr. 30 May 1 2 June 28 29 30 Apr. 19 ( 20 21 P 4.0 3.8 3.2 3.2 15.6 16.5 17.1 17.1 3.5 3.6 2.9 3.6 3.7 3.5 16.5 16.6 15.0 15.9 16.1 15.6 15.0 17.2 16.5 Compound fed Date 25 Homogentisic acid, 3 Dihydroxyphenylalanine, 3 Diiodotyrosine, 4 Glycyltyrosine, 4 Hydroxyphenylpyruvic 3 acid, 0.19 0.16 0.16 0.20 0.14 0.15 0.15 0.15 0.10 0.10 0.07 0.10 0.11 0.18 0.17 0.14 0.22 0.21 0.14 0.18 0.20 0.23 Total aeatinine l?3 1.23 1.15 1.17 1.37 1.36 1.43 1.58 1.29 1.17 1.23 1.33 1.48 1.40 1.55 1.44 1.43 1.36 1.28 1.57 1.50 1.58 1.48 TABLE VI of T1 yrosine and Derivatives July 27 28 Oct. 18 ( 19 I 20 c7*. 16.0 17.0 4.1 4.2 3.8 22 2.8 23 2.5 Mar. 3 13.4 4 15.4 5 311 14.3 elm. 0.09 0.10 0.11 0.07 1.06 hhenylpyruvio acid C blori I netric am. 0.35 1.62 0.54 0.45 1.89 3.63 2.20 2.00 0.36 0.98 0.52 0.42 0.46 0.36 1.82 2.00 1.93 1.75 1.73 1.94 2.13 1.84 2.12 Total seatinine Sydra eine P. 0.42 1.49 0.61 1.86 3.92 1.90 1.95 0.34 0.79 0.32 0.62 0.63 2.13 2.01 1.93 1.80 2.09 1.83 2.00 i Ratio, P henyla &mine to N 1?henylpyruvic acid >olori..i netrio I 1.60 1.85 1.58 1.90 1.10 0.36 1.40 0.42 1.42 0.42 1.41 0.40 1.38 3.35 1.26 1.76 1.69 2.17 1.36 1.82 9.6 39.8 18.0 14.0 12.0 22.6 11.9 11.5 10.0 24 A 14.5 14.4 14.7 10.3 11.9 12.1 12.9 11.0 10.9 12.9 14.2 10.6 12.5 Iydrazinc am. 2.03 1.95 0.35 0.44 0.40 0.40 ( 1.42 11.74 : 1.10
312 Phenylpyruvic Oligophrenia was tested for the presence of tyrosine and its catabolites by the Millon method, as described by Fiirth and Scholl (21). No significant results were obtained in either the ethersoluble or the etherinsoluble fractions. The findings indicate, as would be expected theoretically, that tyrosine does not yield phenylpyruvic acid. Moreover, since no catabolites of tyrosine were recovered, it seems likely that the patient experienced no difficulty in utilizing this amino acid. SUMMARY 1. Feeding experiments to a patient with phenylpyruvic oligophrenia are reported. The following pure amino acids were fed: phenylalanine, tyrosine, tryptophane, phenylserine, dihydroxyphenylalanine, alanine, leucine, cystine, valine, and glycine. Of these only phenylalanine increases the urinary output of phenylpyruvic acid. 2. The dextro form of phenylalanine induces a greater increase in phenylpyruvic acid than the levo form. Formyl and acetyldphenylalanine do not yield phenylpyruvic acid, whereas the corresponding derivatives of Zphenylalanine increase the excretion of phenylpyruvic acid. 3. The ingestion of phenylpyruvic and phenyllactic acids leads to increased excretion of phenylpyruvic acid. Phenylpropionic, phenylglyceric, cinnamic, phydroxyphenylpyruvic, and homogentisic acids fail to augment the output of phenylpyruvic acid. 4. The significance of these findings is briefly discussed. It is concluded that the disease is characterized biochemically by an inhibition in the metabolism of phenylalanine at the stage of phenylpyruvic acid, the subject being unable to oxidize this keto acid at a normal rate. The author is indebted to Professor H. B. Lewis of the University of Michigan, who revised the manuscript and offered valuable suggestions. BIBLIOGRAPHY 1. Fdling, A., 2. physiol. Chem., 327, 169 (1934). 2. Penrose, L., Lancet, 1, 23 (1935). 3. Jervis, G. A., Arch. Neurol. and Psychiat., 33, 944 (1937).
G. A. Jervis 313 4. Penrose, L., and Quastel, J. H., Biochem. J., 31, 266 (1937). 5. Neuberg, C., and Kobel, M., Biochem. Z., 203, 463 (1928). 6. Hemmerle, R., Ann. chim. et physiq., 7, 226 (1917). 7. KappelerAdler, R., Biochem. Z., 262, 185 (1932). 8. Kotake, Y., 2. physiol. Chem., 132, 241 (1922). 9. Chandler, J. P., and Lewis, H. B., J. Biol. Chem., 96, 619 (1932). 10. Krebs, H., 2. physiol. Chem., 217, 191 (1933). 11. Neber, M., Z. physiol. Chem., 240, 59 (1936). 12. Lieben, F., and Kretschmayer, R., Enzymologia, 3, 21 (1937). 13. Dakin, H. D., J. BioZ. Chem., 6, 235 (1909). 14. Embden, G., and Baldes, K., Biochem. Z., 66, 301 (1913). 15. Medes, G., Biochem. J., 26, 917 (1932). 16. Edson, N. L., Biochem. J., 29, 2498 (1935). 17. Kotake, Y., Masai, Y., and Mori, Y., 2. physiol. Chem., 122, 195 (1922). 18. du Vigneaud, V., and Meyer, C. E., J. BioZ. Chem., 98,295 (1932). 19. du Vigneaud, V., Sealock, R. R., and Van Etten, C., J. BioZ. Chem., 98, 565 (1932). 20. Jackson, R. W., and Block, R. J., J. BioZ. Chem., 122, 425 (193738). 21. Ftirth, O., and Scholl, R., Biochem. Z., 243,274 (1931).
METABOLIC INVESTIGATIONS ON A CASE OF PHENYLPYRUVIC OLIGOPHRENIA George A. Jervis J. Biol. Chem. 1938, 126:305313. Access the most updated version of this article at http://www.jbc.org/content/126/1/305.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/126/1/305.citation.full.h tml#reflist1