PHENYLALANINE METABOLISM IN THE PHENYLPYRUVIC CONDITION. I. DISTRIBUTION, POOL SIZE, AND TURNOVER RATE IN HUMAN PHENYLKETONURIA Hanns-Dieter Grümer, … , Hans Koblet, Carol Woodard J Clin Invest. 1961;40(9):1758-1765. https://doi.org/10.1172/JCI104399. Research Article Find the latest version: https://jci.me/104399/pdf PHENYLALANINE METABOLISM IN THE PHENYLPYRUVIC CONDITION. I. DISTRIBUTION, POOL SIZE, AND TURNOVER RATE IN HUMAN PHENYLKETONURIA * By HANNS-DIETER GRUMER, HANS KOBLET AND CAROL WOODARD (From the Biochemical Laboratory, Pineland Hospital and Training Center, Pownal, Maine; and the Arthur G. Rotch Research Laboratories, The Boston Dispensary, Boston, Mass.) (Submitted for publication March 25, 1960; accepted May 19, 1961) The inborn metabolic error in phenylpyruvic demonstrate a defined metabolic variation from oligophrenia consists of the inability to hydroxy- the norm, they also provide the opportunity to late phenylalanine to tryrosine in any significant obtain further information that is not readily avail- amount. Recent work has focused mainly on the able in normal subjects. For example, pool size investigation of this hydroxylating system, its determinations of amino acids have rarely been purification and mode of action (1-5), the inhibi- performed and have mostly proved to be unsatis- tory effect of phenylalanine and its derivatives on factory, bceause they are based on experiments enzymes (6-9), and the prevention of mental re- with N'5-labeled amino acids and many assump- tardation by a diet low in phenylalanine (10, 11). tions have to be made [see Wu, Sendroy and With this diet the free phenylalanine of plasma Bishop (14, 15) and Tschudy and co-workers and total body fluid can be adjusted to any value (16)]. Experiments similar to ours with S35_ that might be required to study the effect of labeled methionine in lnonphenylketonuric individ- phenylalanine concentration on metabolic proc- uals have been published by Maurer (17). To esses in vivo. Bickis, Kennedy and Quastel (12), our knowledge no studies on human beings have for example, demonstrated that phenylalanine, at been made, so far, with C14-labeled amino acids, concentrations similar to those observed in the except by Gutman and co-workers (18), who esti- blood of phenylketonuric patients, inhibited the mated the pool size of glycine. The method used enzymatic degradation of tyrosine in vitro. We, by them, however, was entirely different from ours. on the other hand (13), were unable to find inhi- The use of C14-labeled amino acids eliminates bition of the breakdown of orally administered the necessity of assuming, as did Sprinson, Ritten- p-hydroxyphenylpyruvic acid in patients with berg and San Pietro (19, 20), that the rate of plasma concentrations of phenylalanine as high nitrogen transfer is faster than other metabolic as 50 mg per 100 ml. This discrepancy could be processes involved here. It will be shown later explained, for instance, by a diminished rate of that we have to make a similar assumption; p-hydroxyphenylpyruvic acid formation from tyro- namely, that equilibration of the labeled phenyl- sine in the in vitro experiments, or by a lower alanine within the body is faster than other proc- intracellular phenylalanine concentration in our esses. If it be assumed that the conversion of in vivo study than one would have expected from phenylalanine to tyrosine is negligible in the the plasma phenylalanine levels. Since the dis- penylketonuric patient, then phenylalanine can tribution coefficient of phenylalanine between tis- only be stored, incorporated into proteins, ex- sue and plasma has not yet been determined in creted as such or as its metabolites-phenylpyruvic vivo, the intracellular phenylalanine concentra- acid and phenylacetic acid. tions in our experiments were not known. In That this assumption is correct will be shown order to evaluate properly the significance of inl a second paper (21). In addition, no apprecia- metabolic experiments with phenylalanine, some ble amount of phenylpyruvic acid or phenylacetic estimate must be made of the distribution of the acid has been found in the circulating plasma of phenylalanine between plasma and tissue. phenylpyruvic patients in comparison with the Since patients with phenylpyruvic oligophrenia total amount of free phenylalanine (22). There- * This work was supported by Research Grants 2729 fore, the pool size, the turnover time, and the and 3961 from the National Institute of Mental Health. absolute turnover rate of free phenylalanine pool 1758 PHENYLALANINE METABOLISM IN THE PHENYLPYRUVIC CONDITION 1759 can be estimated by means of C14-labeled phenyl- heparinized blood samples were centrifuged at 700 RCF alanine without isolating free phenylalanine from for 10 minutes. In order to determine the radioactivity of the free L-phenylalanine in deproteinized plasma, 1.0 the plasma. The absolute turnover rate in the ml of plasma was added to 2.0 ml of 10 g per 100 ml of phenylketonuric organism is, therefore, only the an aqueous solution of trichloroacetic acid (TCA). sum of incorporation into proteins, excretion, and The resulting precipitate was centrifuged at 1,400 RCF conversion to compounds other than tyrosine. for 20 minutes and a duplicate 1.0 ml of the supernatant However, a small, nonspecific conversion to tyro- was plated on plastic planchets, 7.1 cm' in area, and dried at room temperature for counting. The samples were sine might be included. In respect to many counted in a gas-flow end-window counter with a Micro- metabolic functions, phenylketonuric patients seem mil window and an efficiency of about 22 per cent. All to behave like normal individuals; but from a values were corrected for background and self-absorption metabolic point of view they may be considered and given in counts per minute for 1.0 ml of plasma. human mutants. Thus, it might be possible to The latter values were plotted on semilogarithmic paper against time, and the resulting first straight line carry out studies at normal phenylalanine levels, was extrapolated to zero time (to). This zero time taking advantage of the existence of the metabolic value was used to calculate the distribution volume by block, to derive data applicable to persons other the formula: than phenylketonurics. cpm injected cpm per 1.0 ml protein-free plasma at to [1] MATERIALS AND METHODS The pool size of the free phenylalanine is the product of the distribution volume and the free phenylalanine plasma Two male and two female patients suffering from concentration in milliliters. phenylpyruvic oligophrenia were studied. Three (J.I., The slope of the first straight line, so obtained, per- N.C. and K.H.) had been on a diet low in phenylalanine mitted the estimation of the half-life time and the turn- for at least 3 weeks prior to the experiments. High over time of the pool. The turnover time, as used here, protein foods were replaced by a casein hydrolysate from is the reciprocal of the slope constant. The absolute turn- which phenylalanine had been removed (Lofenalac, Mead over rate is given by the quotient of milligrams of free Johnson Co.). The plasma phenylalanine was adjusted phenylalanine pool to turnover time in minutes. The to levels of 2.3 mg in J.I., 7.0 mg in N.C., and 10.5 and average concentration in 1.0 ml extravascular antipyrine 17.0 mg per 100 ml in K.H., and K.H., (the experiments space was derived from the following equation by Dost in K.H. were carried out on two different occasions). No phenylpyruvic acid was excreted in the urine as (24): amount injected - aplasma X checked by the ferric chloride test. Patient R.S. was Vplasma [2] maintained on a normal diet, excreted large amounts of Vantip. - Vplasma phenylpyruvic acid, and had a plasma phenylalanine con- where atis8ue = average apparent phenylalanine concentra- centration of 47 mg per 100 ml plasma. The experiments tion in cpm X ml-' extravascular antipyrine space at to; were carried out on 3 consecutive days, in the morning. aplasma = apparent phenylalanine level in cpm X ml-1 Breakfast was omitted. plasma at to; Vplasma = Evans blue space in ml; V..tip. First day. Determination of antipyrine space was done = antipyrine space in ml; and the distribution quotient according to Brodie, Axelrod, Soberman and Levy (23). is given by: Second day. A needle with a three-way stopcock was atissue [3] inserted into the cubital vein and a blood sample was aplasma taken for the determination of the free phenylalanine Third day. Plasma volume determinations were carried plasma level, total plasma proteins, hematocrit, and hemo- out by injecting 5.0 ml of 0.5 per cent aqueous solution globin. About 8 X 106 cpm to 11 X 106 cpm of uniformly of Evans blue (Warner-Chilcott). labeled T,-phenylalanine-C141 (specific activity 3.2 X 10' Blood samples were taken at 10, 20, and 30 minutcs, cpmi per mg), dissolved in 30 ml of physiological saline, as well as before injecting the dye. The heparinized was injected through the needle and the syringe was blood was centrifuged at 700 RCF and the clear, non- washed three times with about 10 ml of saline from an hemolyzed supernatant was measured at 600 m/i in the attached infusion bottle. A constant saline infusion was Beckman DU spectrophotometer. The resulting optical started for 5 to 6 hours (the duration of the experi- densities were plotted on linear graph paper and extrap- ments), the total amounting to not more than 350 ml. olated to zero time. From the value so obtained the Heparinized blood samples were taken at short inter- dilution of the dye was calculated. vals for 5.5 hours; in Patient K.H., for 48 hours. The In Patient N.C. the plasma volume was estimated to 1 Nuclear Chicago. This corporation claims that their be 1/22 of the body weight.