ON THE METABOLISM OF PHENYLALANINE AND TYROSINE BY AARON BUNSEN LERNER* (From the Department of Biochemistry, School of Medicine, Western Reserve University, Cleveland) (Received for publication, July 1, 1949) The metabolism of phenylalanine and tyrosine has been the subject of investigation for many years. Much evidence has been produced to show that in mammalian tissue phenylalanine is converted to tyrosine (l-5). Tyrosine can be oxidized to p-hydroxyphenylpyruvic acid, which can in turn form homogentisic acid (5-13). Embden, Salomon, and Schmidt (14) showed that phenylalanine, tyrosine, and homogentisic acid yield aceto- acetic acid when perfused through a surviving liver. It has long been known that these amino acids have ketogenic activity in tivo (15-17). Recently, Winnick, Friedberg, and Greenberg (18) and Weinhouse and Millington (19) working with C14-p-labeled DL- and L-tyrosine, respectively, showedthat tyrosine is at least in part converted to acetoacetic acid in the rat and in rat liver slices. Radioactive acetoacetic acid was isolated after feeding the rat p-labeled nn-tyrosine and after incubating rat liver slices with p-labeled n-tyrosine. Schepartz and Gurin (20) incubated Cl4 ring- labeled nn-phenylalanine with rat liver slices and showed for the first time that carbon atoms from the benzene ring are incorporated into acetoacetic acid. These workers also proved that if homogentisic acid is formed from phenylalanine the carbon side chain must shift on the benzene ring. Weinhouse and Millington (19) and Schepartz and Gurin (20) concluded from the unequal distribution of isotopic carbon in the acetyl and acetate fractions of acetoacetate that ketone bodies are formed as intact 4-carbon units in the metabolism of phenylalanine and tyrosine. We considered it desirable to obtain additional evidence for this belief and to determine the nature of substances other than ketone bodies which might arise from the metabolism of the aromatic nucleus of these amino acids. To this end labeled optically active phenylalanine was synthesized with Cl4 in the benzene ring and Cl3 in the a-carbon atom. Labeled, optically active tyrosine with Cl4 in the p position was also used. The results of a study of the metabolism of these labeled amino acids in rat liver slices,summarized in Diagram 1, indicate that phenylalanine and tyrosine are metabolized to ketone bodies and malic acid (or its precursor). The experimental evi- dence for these findings is given in this paper. * Fellow of the American Cancer Society. Present address, Department of Der- matology, University of Michigan Hospital, Ann Arbor. 281 by guest on December 1, 2018 http://www.jbc.org/ Downloaded from by guest on December 1, 2018 http://www.jbc.org/ Downloaded from by guest on December 1, 2018 http://www.jbc.org/ Downloaded from
Transcript
ON THE METABOLISM OF PHENYLALANINE AND TYROSINE
BY AARON BUNSEN LERNER*
(From the Department of Biochemistry, School of Medicine, Western Reserve University, Cleveland)
(Received for publication, July 1, 1949)
The metabolism of phenylalanine and tyrosine has been the subject of investigation for many years. Much evidence has been produced to show that in mammalian tissue phenylalanine is converted to tyrosine (l-5). Tyrosine can be oxidized to p-hydroxyphenylpyruvic acid, which can in turn form homogentisic acid (5-13). Embden, Salomon, and Schmidt (14) showed that phenylalanine, tyrosine, and homogentisic acid yield aceto- acetic acid when perfused through a surviving liver. It has long been known that these amino acids have ketogenic activity in tivo (15-17). Recently, Winnick, Friedberg, and Greenberg (18) and Weinhouse and Millington (19) working with C14-p-labeled DL- and L-tyrosine, respectively, showed that tyrosine is at least in part converted to acetoacetic acid in the rat and in rat liver slices. Radioactive acetoacetic acid was isolated after feeding the rat p-labeled nn-tyrosine and after incubating rat liver slices with p-labeled n-tyrosine. Schepartz and Gurin (20) incubated Cl4 ring- labeled nn-phenylalanine with rat liver slices and showed for the first time that carbon atoms from the benzene ring are incorporated into acetoacetic acid. These workers also proved that if homogentisic acid is formed from phenylalanine the carbon side chain must shift on the benzene ring.
Weinhouse and Millington (19) and Schepartz and Gurin (20) concluded from the unequal distribution of isotopic carbon in the acetyl and acetate fractions of acetoacetate that ketone bodies are formed as intact 4-carbon units in the metabolism of phenylalanine and tyrosine. We considered it desirable to obtain additional evidence for this belief and to determine the nature of substances other than ketone bodies which might arise from the metabolism of the aromatic nucleus of these amino acids. To this end labeled optically active phenylalanine was synthesized with Cl4 in the benzene ring and Cl3 in the a-carbon atom. Labeled, optically active tyrosine with Cl4 in the p position was also used. The results of a study of the metabolism of these labeled amino acids in rat liver slices, summarized in Diagram 1, indicate that phenylalanine and tyrosine are metabolized to ketone bodies and malic acid (or its precursor). The experimental evi- dence for these findings is given in this paper.
* Fellow of the American Cancer Society. Present address, Department of Der- matology, University of Michigan Hospital, Ann Arbor.
The steps in the synthesis of phenylalanine labeled with Cl4 in the benzene ring and Cl3 in the 01 position are given in Diagram 2.
Hippuric Acid-5.20 gm. of Cl3-labeled methyl iodide were converted to CP-methyl-labeled sodium acetate in 90 per cent yield through a nitrile synthesis and alkaline hydrolysis.’ 3.5 gm. of the labeled sodium acetate were fused and then treated with bromine and phosphorus pentabromide to give bromoacetyl bromide (21). Water (in 10 per cent excess) was added to the bromoacetyl bromide reaction mixture to give monobromo- acetic acid. The solution was treated with ammonia and benzoyl chloride
OH OEI OH
jf)- Q;H2;ooH CH*;ooH
/
OH
&+OCH2c'oOFl
+ + 4#- ** C02t HOOCCH22F;p
DIAGRAM 1. Metabolism of Cl* ring-labeled, Cl3 a-labeled phenylalanine. ? de- notes CP; c denotes Cl*.
(22) to give hippuric acid. 3.5 gm. (46 per cent of theory) of hippuric acid were obtained.
13enxaldehyde-Benzene was converted to benzaldehyde through a Gatter- mann reaction as described by Reformatsky (23). 3.187 gm. of benzene (Tracerlab) labeled with Cl4 in a single position and having a total activity of 0.5 mc. were diluted with 10 ml. of anhydrous cyclohexane in a 50 ml. centrifuge tube. The tube was cooled and kept in an ice bath throughout the entire reaction. 0.6 gm. of anhydrous cuprous chloride and 11.5 gm. of anhydrous aluminum bromide were added to the reaction. The mixture was stirred vigorously, and the anhydrous gases carbon monoxide and hy- drogen chloride (in a 2 : 1 ratio) (24-25) were bubbled through the mixture for 30 minutes. The contents of the reaction tube were then slowly poured onto chips of ice. Benzaldehyde was extracted from the aqueous layer
twice with 10 ml. portions of a benzene-cyclohexane (1:3) solution. The organic phase was shaken with an excess of alcoholic sodium bisulfite (26) and the resulting benzaldehyde-bisulfite complex was removed by filtration through a sintered glass funnel. The precipitate was washed with benzene and air-dried. 3.4 gm. (40 per cent of theory) of benzaldehyde-bisulfite
Hippuric acid
_ -.I kEOC,H,
AlBr,,Cu2Cla ,._ 1-1 cH3cooNa,(cB,co)ao 6~7% f-U-f?. V-Y I
I ‘Ii -C CeH,
azlactone of a benzoylmino- cinnamic acid
P
CE,C'HCOOE 1 nia
Phenylalanine
DIAGRAM 2. Synthesis of Odring-labeled, C’J a-labeled phenylalanine. 6 denotes
Or; d denotes CP.
complex were obt,ained. Control runs with non-isotopic benzene usually gave 60 per cent of theory.
The benzene-cyclohexane layer, after removal of the alcoholic bisulfite layer, was dried over anhydrous sodium sulfate and distilled. The fraction distilling at 70”, which contained some unreactive radioactive benzene, was reworked to give an additional amount of benzaldehyde of lower activ- ity. The two benzaldehyde preparations were converted to phenylalanine separately.
Azluctone of a-Benxoylaminocinnamic Acid-3.4 gm. of benzaldehyde- bisulfite complex, 2.91 gm. of hippuric acid, 1.35 gm. of freshly fused sodium acetate, 9.2 ml. of acetic anhydride, and 1.0 ml. of acetic acid were added to 20 ml. of anhydrous benzene. The mixture was refluxed for 6 hours. The azlactone of cr-benzoylaminocinnamic acid which crystallized on cool- ing was filtered and washed with a few ml. of boiling water. 2.19 gm. of azlactone were obtained.
Phenylalanine-The azlactone was treated with red phosphorus and hydriodic acid in the usual manner (27) and gave 0.760 gm. of pure DL-
phenylalanine. The yield of phenylalanine was 11.3 per cent based on the initial amount of benzene and 11.7 per cent based on the initial amount of methyl iodide.
Resolution of the nn-phenylalanine to the optically active forms was car- ried out by the procedure of du Vigneaud and Meyer (28), in which the brucine salts of the formyl derivatives of phenylalanine are separated. After two recrystallizations from water and alcohol, 86.0 mg. of pure L-phenylalanine and 185.0 mg. of pure n-phenylalanine were obtained. Both preparations of phenylalanine decomposed at 284-288” and gave the expected amount of carbon dioxide when oxidized with chromic acid. The specific rotations were not determined on the isotopic phenylalanine prep- arations, but control non-isotopic preparations made in the same manner with the same reagents were resolved completely. Incubation of the radio- active L-phenylalanine with n-amino acid oxidase2 prepared from sheep kidney showed no oxygen uptake, thus indicating that the L isomer of phenylalanine was not contaminated with the D isomer. The phenylalanine preparations had an activity of 3.30 X lo6 counts per minute per mM of phenylalanine and contained 9.45 per cent Cl3 in the cr position.
Tyrosine--5.0 mg. of C14-p-labeled nn-tyrosine3 and 27.5 mg. of pure non- isotopic L-tyrosine were dissolved in 7.5 ml. of boiling water. On cooling, L-tyrosine precipitated and was separated from the mixture by filtration through a sintered glass funnel. The precipitate was washed with a few drops of cold water and dried at 110” for 1 hour. 14.1 mg. of L-tyrosine were obtained.
The filtrate was evaporated to dryness and 18.5 mg. of tyrosine were ob- tained. This fraction of tyrosine contained a mixture of n-tyrosine, having a high specific activity (47.8 X lo6 counts per minute per mM), and n-tyro- sine having a lower specific activity. To determine the efficiency of the resolution procedure, the radioactivity in each of the two preparations was determined. The L-tyrosine preparation had an activity of 4.26 X lo6
2 I am indebted to Dr. E. Kearney and Dr. T. P. Singer for the purified n-amino acid oxidase preparation.
3 Obtained through the kindness of Dr. Melvin Calvin, University of California.
counts per minute per mM of tyrosine and the mixed tyrosine fraction (fil- trate residue) contained 9.65 X lo6 counts of tyrosine. The expected activity of L-tyrosine is 3.98 X lo6 counts per minute per mM of tyrosine (determined by calculating the total activity present in the 32.5 mg. of tyrosine from the above values). The value of 3.98 X lo6 is in good agree- ment with that of 4.26 X lo6 actually found and indicates that practically pure n-tyrosine was obtained.
Experiments in Vitro and Results
Liver slices from adult, male albino rats were suspended in Krebs’ calcium-free 0.01 M phosphate buffer at pH 7.4 (29) with the appropriate substrate and shaken for 2 hours at 38”. Oxygen was passed over the reaction mixture continuously, and the exit gases were passed through a bubbler with 2.5 N sodium hydroxide to collect the respiratory COZ.
Formation of Acetoacetic Acid-The procedure followed in most of the experiments is exemplified by the following description. 2.0 mg. of Cl4 ring-labeled L-phenylalanine were incubated with approximately 10 gm. (wet weight) of rat liver slices in 20 ml. of phosphate buffer as described above. After 2 hours the reaction was stopped. 35.7 mg. of respiratory carbon dioxide were found in the alkali. 2.5 ml. of an aqueous solution containing 105.1 mg. of acetoacetic acid were added as carrier. The mix- ture was diluted with 30 ml. of water and 20 ml. of 25 per cent cupric sul- fate solution. Sufficient solid calcium hydroxide was mixed into the solu- tion to make the mixture alkaline to litmus. After standing at room tem- perature for 45 minutes, the mixture was centrifuged at 3000 R.P.M. for 10 minutes and then filtered. 39 ml. of filtrate were obtained, which was brought to pH 2 with concentrated sulfuric acid. To remove any radio- active carbon dioxide present in the solution tank carbon dioxide and then nitrogen were bubbled through the solution at a rapid rate for periods of 5 minutes. Acetoacetic acid was converted to acetone and carbon dioxide according to the method of Van Slyke (30) as follows: After adding 35 ml. of 10 per cent mercuric sulfate and 10 ml. of 50 per cent sulfuric acid, the mixture was refluxed for 45 minutes. The acetone formed precipitated as Deniges’ mercury complex, and the carbon dioxide derived from the car- boxy1 group was collected in a bubbler containing 3 ml. of 2.5 N sodium hydroxide. 18.1 mg. of carbon dioxide were obtained. The mercury- acetone complex was filtered off, washed with cold water, and dried at 110” for 1 hour. 487.4 mg. of mercury-acetone complex representing approxi- mately 24.4 mg. of acetone were obtained. This value is in accord with that expected when 18.1 mg. of carbon dioxide come from the carboxyl group of acetoacetic acid.
The mercury-acetone complex was divided into two portions. One por-
tion (121.5 mg.) was oxidized quantitatively with chromic acid (31) to carbon dioxide. The second portion, representing 356 mg. of the mercury- acetone complex, was dissolved in 15 ml. of 6 N hydrochloric acid and the acetone was distilled directly into a cooled mixture of 30 ml. of 0.1 N iodine and 15 ml. of 17 N sodium hydroxide. The cooled receiving flask was shaken continuously during the distillation of acetone. By this procedure acetone was degraded to acetaldehyde and iodoform. The Cl4 activity of iodoform represents the mean activity of the two methyl groups of acetone. The iodoform, after being allowed to stand overnight, was filtered onto an asbestos filter and then dried over calcium chloride for 48 hours in a small desiccator. In this manner 59.9 mg. of iodoform (50 per cent of theory) were obtained. The iodoform was oxidized to carbon dioxide (4.7 mg., 70 per cent of theory) with chromic acid.4 A brief r&urn4 of the degradation of acetoacetic acid is as follows:
0 7 Q,S,Y *//
*CH,C-CH~OOH *//O /
-+ ?~c--cH~ + do, 7 B a 7sa\
h* CHa*CHO + &I,
“,Y P a,7
The various carbon dioxide fractions obtained from acetoacetate by the above procedures are carboxyl as CO 2, LY, & and y as acetone, and an aver- age of (Y and y as iodoform. The Cl4 activity was determined with a Geiger counter on barium carbonate precipitated and filtered on paper disks. Control experiments with 2.0 mg. of non-isotopic L-phenylalanine yielded less than 1 mg. of acetoacetic acid. Since this amount is small compared with the total weight of carrier added in the above experiment (105.1 mg.), the activities calculated for 105.1 mg. of acetoacetic acid very nearly repre- sent the total of isotopic acetoacetic acid present in the reaction mixture at the end of the experiment.
Results of the above experiment are shown in Table I and are in accord with the composition of acetoacetate as given in Diagram 1. It can be seen that the carboxyl group of acetoacetic acid has relatively little activity and that the remaining 3-carbon unit has much activity. If the acetoacetic acid is formed from phenylalanine, as illustrated in Diagram 1, it would be expected that all the radioactivity would be in the p- and y-carbons and none in the LY- and carboxyl carbons. Calculation of the specific activity
* This particular batch of chromic acid was prepared from concentrated sulfuric acid instead of fuming sulfuric acid. Oxidation of iodoform with this preparation gives a 70 per cent yield of carbon dioxide.
of the & and y-carbons from the specific activities of the acetone gives 161.8 (107.9 X 3/2) and from the iodoform 160.8 (80.4 X 2/l). These results are thus in agreement with the proposed scheme. Experimental evidence supporting this view is found in the work of Weinhouse and Mil- lington (19) who showed that acetoacetic acid was produced with Cl4 only in the a-carbon atom when CY4+?-labeled n-tyrosine was incubated with liver slices. The radioactive phenylalanine used in our experiments had no activity in the p-carbon atom, and hence no activity would be expected in the a-carbon atom of acetoacetic acid. Since there is good evidence that in the liver phenylalanine is converted to tyrosine, the ketone bodies pro- duced from phenylalanine and from tyrosine should arise by identical mechanisms. The present results support this view.
TABLE I Distribution of Cl4 Activity in Acetoacetic Acid
2.0 mg. ofC14 ring-labeled L-phenylalsnine (3.30 X 106 counts per minute per mM) were incubated with approximately 10 gm. of rat liver slices for 2 hours at pH 7.4 and 38”. Inactive acetoacetic acid carrier (105 mg.) was added to the system at the end of the reaction.
Carbon atoms in scetoacetic acid Specific activity, counts per min.
per mg. c Total Cl” activity,
counts per min.
Carboxyl ................................... 19.6 242 a-,&, y- ................................... 107.9 4040 Average of LY- and y-. ...................... 80.4 1980
The question arises as to how Cl4 activity, even though relatively small, was found for the carboxyl group of acetoacetate. It will be seen shortly that radioactive malate is formed in the metabolism of ring-labeled phenyl- alanine. This means that small amounts of carboxyl-labeled acetoacetate could arise from 2-carbon units derived from the metabolism of labeled malate. Another explanation for finding some activity for the carboxyl group of acetoacetate is that it represents contamination from other readily decarboxylated radioactive substances (e.g. oxalacetate) that may be pres- ent in the reaction mixture. Activity in the carboxyl group does not ap- pear to result from a randomization produced by cleavage of acetoacetate to acetate and then resynthesis of acetoacetate. It will be shown that the carboxyl group had much Cl3 activity, while the a-, fl-, and y-carbon atoms had very little Cl3 activity, thus indicating lit’tle randomization.
If ketone bodies (p-hydroxybutyric and acetoacetic acids) are derived from phenylalanine as intact 4-carbon units, the Cl3 and CL4 of the phenyl- alanine should be diluted equally on conversion to acetoacet’ic acid, and the labeling should not be randomized in the acetoacetate. Since in the
experiments described above the excess Cl3 found in the carboxyl group of acetoacetic acid was insufficient for accurate measurement, similar ex- periments were carried out in triplicate, and the reaction mixtures from the three flasks were mixed with a t,otal of 39.5 mg. of acetoacetic acid added as carrier. In this way dilution was reduced. The mixture was degraded as described above.
The results are shown in Table II. Carbon dioxide from the carboxyl group was found to contain 0.11 atom per cent excess CY3. The original phenylalanine had 9.45 atom per cent excess Cl3 in the a-carbon atom. Hence, there was a 1:85.9 dilution of the Cl3 from the original phenylalanine to the acetoacetic acid which was isolated with the carrier.
Cl* activity of the acetone fragment of acetoacetic acid was determined after combustion of .the mercury-acetone complex to carbon dioxide.
TABLE II
Comparison of 04 and Cl3 Activities of Phenylalanine and Acetoacetic Acid
6.0 mg. of 04 ring-labeled n-phenylalanine (3.30 X l@ counts per minute per mrd) were incubated with approximately 30 gm. of rat liver slices for 2 hours at pH 7.4 and 38’. Inactive acetoacetic acid carrier (39.5 mg.) was added to the system at the end of the reaction.
Compound 04 activity of labeled carbon Cl2 activity of labeled atoms, counts per min. per mg. C carbon atom, per cent axcass
Phenylalanine .................... Ring carbons 45,918 Acetoacetic acid .................. p- and y- Dilution ..........................
1 1 “;::;I :::z / 1;:
Assuming that Cl4 activity is present in only 2 of the 3 carbon atoms of acetone, i.e. only the /L and y-carbons of acetoacetic acid, it was found that the labeled carbon atoms had 613.8 counts per minute per mg. of carbon. The benzene carbons of the initial phenylalanine had 45,918 counts per minute per mg. of carbon. Hence, Cl4 activity was diluted 74.8 times, and this dilution is identical, within experimental limits, with the dilution of Cl3 activity. Only a trace (less than 0.02 atom per cent excess) of Cl3 was found in the carbon dioxide from the acetone fragment. These findings indicate that the 4 carbon atoms of acetoacetic acid are split off as a unit when phenylalanine is metabolized to ketone bodies.
&Zarbon Unit from Benzene Ring-Relatively little has been reported on the fate of the 4 ring carbon atoms of phenylalanine and tyrosine which are not involved in ketone body formation. It has been shown that these amino acids promote formation of liver glycogen in the fasting rat (32, 33). This suggests that the glycogen may be derived from this part of the mole- cule. The only evidence opposed to this view is that phlorhizinized ani-
mals excrete ketone bodies and no glucose when given phenylalanine or tyrosine (15). Actually, these data do not offer conclusive evidence against the formation of glycogenic substances, because such substances could be rapidly metabolized, whereas ketone bodies, which are metabolized rela- tively slowly, could be excreted in the urine.
In all of the experimentIs described above much Cl” activity but no Cl3 activity was found in the respiratory carbon dioxide. It must be pointed out, however, that little Cl3 activity would be detectable because of the great dilution. These results indicated that carbon dioxide was formed from the ring carbons that were not involved in ketone body formation. Additional evidence for this belief is given by experiments described later in the paper, which show that little activity is present in respiratory car- bon dioxide but much activity in ketone bodies when CY4-p-labeled tyrosine is metabolized by liver slices. The results from the acetoacetic acid derived from CY4- and CY3-labeled phenylalanine indicated that 2 carbons of the benzene ring (plus 2 carbons of the aliphatic side chain) form ketone bodies. The question naturally arises as to how the 4 remaining carbon atoms of the ring are separated from the rest of the molecule. It is unlikely that 2-carbon fragments are formed, because if such a reaction occurred ketone bodies formed from the 2-carbon units would be labeled in all 4 carbons. This was not found to be the case. The formation of a single carbon unit, e.g. carbon dioxide, also is unlikely because the total activity in the respira- tory carbon dioxide was always appreciably less than the total activity in the ketone bodies. This indicates that the formation of carbon dioxide is an indirect process and that many of its radioactive precursors remain in solution. It was, therefore, logical to assume that a 4-carbon unit (such as fumarate or malate) is formed which eventually is oxidized to carbon dioxide. Several years ago Neubauer (34) suggested that fumaric acid might be formed from phenylalanine and tyrosine. Since malate and fumarate are in equilibrium with each other in the presence of liver tissue and since this equilibrium is greatly in favor of malate, malic acid was used to test our hypothesis.
Experiments were set up as described above and 300 mg. of malic acid were added as carrier at the beginning of the reaction. Sufficient dilute sodium hydroxide was added to adjust the pII to 7.4. After the reaction had proceeded for 2 hours, 2.5 ml. of concentrated sulfuric acid were added and the entire reaction mixture was mixed with 100 gm. of calcium sulfate. Organic acids were separated from the calcium sulfate-tissue mixture by extraction with ether.6 The ether was evaporated and 10 ml. of cold water were added. Insoluble fats were removed by filtration and the filtrate neutralized with a known quantity of dilute sodium hydroxide. The solu-
2.0 mg. of Cl4 ring-labeled n-phenylalanine (3.30 X 106 counts per minute per mM) were metabolized by approximately 10 gm. of rat liver slices for 2 hours at pH 7.4 and 38”. Inactive malic acid carrier (300 mg.) was added to the system at the begin- ning of the reaction. 69.6 mg. of malio acid were isolated at the end of the experi- ment.
Carbon atoms in malic acid Cl’ activity, counts per min. FW.C
--.-
290 PHESYLALhNINE AND TYROSINE
tion was evaporated to dryness, and 50 per cent sulfuric acid (in 5 per cent excess of the sodium hydroxide previously used) was added to the residue. Approximately 100 mg. of anhydrous sodium sulfate and two portions of 3 ml. of solvent mixture (‘TO per cent chloroform and 30 per cent butanol saturated with 0.5 N sulfuric acid) were added. The two dry extracts were passed through a 15 gm. column of silica gel. After approximately 150 ml. of solvent had run through the column, the malic acid fraction ap- peared. Approximately 70 ml. of additional solvent were used to elute the malic acid. 69.6 mg. of malic acid were extracted from chloroform- butanol with water. By neutralizing the malic acid with sodium hydroxide and evaporating the solution to dryness, solid sodium malate was obtained.
The two carboxyl groups of malic acid were converted quantitatively to carbon dioxide by means of permanganate oxidation (35). In this oxida-
tion the 2 internal carbon atoms appeared as acetaldehyde, which was collected in a bisulfite bubbler. The theoretical amount of acetaldehyde was obtained. The acetaldehyde was oxidized quantitatively to carbon dioxide with persulfate (36).
The results presented in Table III show that radioactive malic acid is formed from phenylalanine and that the activity is equally distributed be- tween the carboxyl and internal carbon atoms. This finding is consistent with the formation of a 4-carbon unit from the benzene ring.
The results presented in Table IV indicate that when ring-labeled L-
phenylalanine is the substrate much activity is found in malic acid, but that when p-labeled L-tyrosine is the substrate almost no activity is found. 21.5 per cent of the activity of 4 ring carbon atoms from phenylalanine ap- peared as carbon dioxide or malic acid. This value is probably low be- cause malic acid was not extracted quantitatively from anhydrous sodium sulfate prior to chromatographic analysis. From this observation one can
conclude that the formation of malic acid represents a major metabolic pathway in the oxidation of n-phenylalanine and tyrosine.
Yield of Ketone BodiesIf phenylalanine and tyrosine are metabolized to two 4-carbon fragments, one being malic acid (or its precursor) and the other a ketone body, the amount of ket.one bodies produced from these amino acids should be comparable to the amount of malic acid produced. This was found to be the case.
Quantitative measurements of the total amount of radioactivity in aceto- acetic acid and P-hydroxybutyric acid were carried out as follows: Separat,c experiments with 2.0 mg. of n-phenylalanine and 2.0 mg. of L-tyrosine were carried out in the usual manner. After acetoacetic acid was decsrboxylat cd
TABLE IV Conversion of Phenylalanine and Tyrosine to &1alic Acid and Carbon Dioxide
2.0 mg. of C*” ring-labeled L-phenylalsnine (3.30 .X 106 counts per minute per mM) and 2.0 mg. of Cl4-p-labeled n-tyrosine (4.26 X 106 counts) were incubated with ap- proximately 10 gm. of rat liver slices for 2 hours at pH 7.4 and 38”. Inactive malic acid carrier (300 mg.) was added to the system at the beginning of the reaction. At the end of the phenylalanine and tyrosine experiments 69.6 and 66.6 mg. of malic acid were isolated, rer
* See the text for the methods used to calculate these values.
and the mercury-acetone complex separated from the solution, additional acetoacetic acid carrier was added to the filtrate. The mixture was re- fluxed with dilute chromic acid (30) to decompose P-hydroxybutyric acid and carrier acetoacetic acid to acetone and carbon dioxide. Acetone was isolated as DenigBs’ mercury complex. The mercury complexes mere oxidized to carbon dioxide, which was then precipitated as barium car- bonate and 04 was measured. The results are shown in Table V. In genera; the fi-hydroxybutyric acid fraction had approximately twice the activity of acetoacetic acid. 44.4 per cent of the Cl4 act’ivity originally present in tyrosine was found in ketone bodies after tyrosine had been in- cubated with liver slices for 2 hours. Relatively little activity was present in the respiratory carbon dioxide. In calculating the percentage conversion of phenylalanine to ketone bodies it has been assumed that only 2 of the
6 ring carbons were used in ketone body formation and the remaining 4 carbons in the benzene ring were used in the formation of radioactive re- spiratory carbon dioxide. Thus in the metabolism of P-labeled tyrosinc little activity would be expected in the respiratory carbon dioxide. This was the case and indicated that little respiratory carbon dioxide came from ketone bodies. The results indicate that the formation of ketone bodies from phenylalanine and tyrosine in rat liver slices represents a major meta- bolic pathway of these amino acids, and, as expected, the ketone body for- mation is similar from each amino acid.
Opening of Benzene Ring-It is likely that the benzene ring is first broken at only one point and that an g-carbon dicarboxylic acid is formed. Such a mechanism would be similar to the oxidation of phenol to a-ketoadipic
TABLE V
Conversion of Phenylalanine a& Tyrosine to Ketone Bodies and Carbon Dioxide 2.0 mg. of 0’ ring-labeled L-phenylalanine (3.30 X l@ counts per minute per m&f)
and 2.0 mg. of C”-b-labeled L-tyrosine (4.26 X l@ counts) were incubated with ap- proximately 10 gm. of rat liver slices for 2 hours at pH 7.4 and 38”. Inactive aceto- acetic acid carrier was added to the system at the end of the reaction.
Initial substrate Substance isolated at end of reaction Per cent of ori inal activity found in fina product* P
Phenylalanine Respiratory CO1 Ketone bodies
Tyrosine Respiratory CO* Ketone bodies
* See the text for the method used to calculate these values.
24.4 56.0 2.2
44.4
acid by a strain of Vibrio (37). Further speculation on the details of the mechanism by which two 4-carbon units are split from homogentisic acid is not justified at the present time.
Metabolism oj D-Phenykdanine-When n-phenylalanine was used in the above experiments, it was found that the activity in the respiratory carbon dioxide and in the ketone bodies was approximately half that found for an equivalent quantity of L-phenylalanine.
Alanine from Tyrosine (P)-Recently Felix and Zorn (38) claimed, on the basis of their experiments, that alanine is formed directly from tyrosine in quantitative yields when tyrosine is metabolized by a liver mash. If this view were correct, one would expect CY4-p-labeled tyrosine to be con- verted to C14-/Mabeled alanine. To test this theory, 1.3 mg. of the mixed D and L radioactive tyrosine preparation described earlier in this paper were incubated wit.h 5 gm. of rat liver slices. Small aliquots of solution were removed from the reaction mixture at 0, 10, 20, 40, 60, 90, and 120
minutes for filter paper chromatography. Tyrosine and alanine can be separated effectively by means of filter paper chromatography with a sol- vent consisting of 6 parts of 2,6-lutidine, 1 part of benzyl alcohol, and 3 parts of water. No activity could be detected on the filter paper at the spot where alanine should appear. On the other hand, activity over the tyrosine spot gradually decreased throughout the experiment, and at the end of 2 hours two-thirds of the original activity had disappeared. From this one can conclude that alanine does not accumulate in significant amounts from tyrosine, as suggested by Felix and Zorn.
SUMMARY
The work presented in this paper shows that a major pathway in the metabolism of L-phenylalanine and n-tyrosine by rat liver slices results in the formation of two intact 4-carbon units. One unit is a ketone body, and the other malic acid (or its precursor). 2 carbon atoms of the benzene ring, together with 2 carbon atoms of the aliphatic side chain of these amino acids, are converted to ketone bodies. The remaining 4 carbon atoms of the benzene ring form malic acid.
A single experiment with n-phenylalanine showed that this amino acid was metabolized at approximately one-half the rate of the natural isomer.
The belief of Felix and Zorn that tyrosine is metabolized quantitatively to alanine could not be confirmed with tracer studies.
The synthesis of benzaldehyde labeled with CL4 in the ring and of phenyl- alanine labeled with Cl4 in the ring and Cl3 in the LY position is reported.
I should like to express my thanks to Dr. H. G. Wood for his contribu- tions of time and advice to this study. I wish to express my appreciation to Mr. J. D. Bordeaux for his aid in the synthesis of labeled phenylalanine. To Dr. J. Meyer I should like to acknowledge my gratefulness for the details of the method for extraction and separation of organic acids from tissues.
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In the articles beginning on pages 1,23,33, and 43, Vol. 168, No. 1, April, 1947, and in the article beginning on page 11, Vol. 179, No. 1, May, 1949, read Lactobacillus brevis (8287) for Lactobacillus brevis (8267).
In Vol. 180, No. 3, October, 1949, on page 1154, legend to Fig. 6, read Sample V for Sample VI; on page 1155, legend to Fig. 7, read Sample VI for Sample VIZ; on page 1156, second paragraph, read fasting 18 hours and 4 hours for fasting 4 hours and 18 hours.
In Vol. 181, No. 1, November, 1949, on page 232, third structure,
